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WO2014131891A1 - Use of fetuin-b for culture of oocytes - Google Patents

Use of fetuin-b for culture of oocytes Download PDF

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Publication number
WO2014131891A1
WO2014131891A1 PCT/EP2014/053960 EP2014053960W WO2014131891A1 WO 2014131891 A1 WO2014131891 A1 WO 2014131891A1 EP 2014053960 W EP2014053960 W EP 2014053960W WO 2014131891 A1 WO2014131891 A1 WO 2014131891A1
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WIPO (PCT)
Prior art keywords
fetuin
oocyte
oocytes
ovary tissue
serum
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/EP2014/053960
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French (fr)
Inventor
Wilhelm Jahnen-Dechent
Eileen DIETZEL
Julia FLOEHR
Joseph NEULEN
Benjamin RÖSING
Walter STÖCKER
Irene YIALLOUROS
André HILDEBRAND
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Johannes Gutenberg Universitaet Mainz
Rheinisch Westlische Technische Hochschuke RWTH
Original Assignee
Johannes Gutenberg Universitaet Mainz
Rheinisch Westlische Technische Hochschuke RWTH
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Publication of WO2014131891A1 publication Critical patent/WO2014131891A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to the field of culture of an ovary tissue comprising an oocyte and in particular to a method of culture of the ovary tissue comprising adding the ovary tissue to a medium comprising fetuin-B immediately after its isolation from a female body.
  • Infertility affects about 15% of couples of reproductive age worldwide and thus has a major public health impact. The causes of infertility for most patients vary widely and the basic mechanisms regulating fertility are still poorly understood. About 25% of clinical infertility cases are idiopathic. Assisted reproductive technologies like in vitro fertilisation (IVF) are common practice to achieve pregnancy when natural fertilisation fails. However, up to 15%) of IVF also fails, mostly because of defective sperm-oocyte interaction.
  • IVVF in vitro fertilisation
  • ZP zona pellucida
  • ZPH is caused by proteolytic processing of ZP glycoproteins, especially the cleavage of ZP2 (120 kDa) to ZP2f (90 kDa) (Bleil et al, 1981). Cleavage is performed by proteases, which are released by the fertilised oocyte during the cortical reaction (Szollosi, 1967; Barros and Yanagimachi, 1971). Recently, the metalloprotease ovastacin was shown to be critical for ZP2 cleavage and definitive ZP hardening (Burkart et al, 2012).
  • ZPH occurs much faster, resulting in a decreased fertilisation success (Ducibella et al, 1990; Schiewe et al, 1995).
  • ZPH can be prevented by adding serum, especially fetal calf serum (FCS), to IVF medium (Downs et al, 1986), suggesting that both follicular fluid and serum may contain factors that inhibit premature ZPH.
  • FCS fetal calf serum
  • the fetuin protein family comprises two members: fetuin-A and fetuin-B (Olivier et al, 1999, 2000). Like fetuin-A, fetuin-B is a liver-derived plasma protein (Denecke et al, 2003) with serum concentrations of -0.01 g/L and -0.3 g/L in human and mouse, respectively. Fetuins belong to the cystatin superfamily, comprising structurally related protease inhibitors.
  • fetuin-A originally designated as fetuin
  • fetuin-A deficient mice are fully fertile, arguing against a role for fetuin-A in fertilisation. The role of fetuin-B has not been elucidated so far.
  • Maintaining fertility of an oocyte after isolation from a female body and during preservation of said oocyte in the fertilisable stage for future processing and use such as in vitro maturation and fertilisation or re-transplantation would be an approach to remedy fertility problems in women who cannot generally become pregnant naturally or who must not become pregnant at a specific time due to medical reasons e.g. cancer treatment.
  • the present inventors have found, by generating fetuin-B deficient mice, that fetuin-B plays an important role in the maturation of oocytes.
  • the present inventors have found that fetuin-B prevents premature hardening of the zona pellucida of oocytes after their isolation from a female body. Consequently, the above problem is solved by the provision of the claims.
  • the present invention provides a method for culturing an ovary tissue comprising an oocyte, comprising contacting the ovary tissue with a medium comprising fetuin-B immediately after isolation of the ovary tissue from a female body.
  • the present invention provides the above method for stabilising the oocyte.
  • the present invention provides a use of fetuin-B for stabilising an ovary tissue comprising an oocyte.
  • the present invention provides the above method or use for maintaining the fertilisable state of the ovary tissue.
  • the present invention provides the above method or use for preventing hardening of the zona pellucida of the oocyte.
  • the present invention provides the above method or use for inhibiting the activity of ovastacin.
  • the present invention provides the above method or use, wherein the oocyte is an immature oocyte.
  • the present invention provides the above method or use, wherein the ovary tissue is an oocyte, a follicle, a cortex of an ovary or an ovary.
  • the present invention provides the above method or use, wherein fetuin-B is recombinant fetuin-B.
  • the present invention provides the above method or use, wherein fetuin-B is human or mouse fetuin-B.
  • the present invention provides the above methods and uses, wherein the ovary tissue comprising an oocyte has been isolated from a female individual, wherein the serum fetuin-B level in the female individual is enhanced due to the treatment of the female individual with a substance which is capable of enhancing the fetuin-B level in the female individual.
  • the substance is a hormone which is used for stimulation of maturation of oocytes for use in in vitro fertilization, an estrogen or an estrogen agonist.
  • the hormone used for stimulation may be FSH (follicle stimulating hormone) or a derivative thereof, or may be hMG (human menopausal gonadotropin) or a derivative thereof.
  • the estrogen or estrogen agonist may be estrone, estradiol, estriol, estetrol (E4), ethinylestradiol, mestranol, 11 ⁇ - methyl-ethinylestradiol, turisteron, moxestrol (1 ⁇ -methoxy-ethinylestradiol), 6- dehydroestrone, 17-deoxyestradiol, 2-hydroxyestradiol, isoestradiol (8a-estradiol), 2- methylestradiol, 4-methylestradiol, polyestradiol-phosphate, promestriene, 2- chloroestradiol, 1,1 ⁇ -ehanoestradiol, diethylstilbestrol, dienestrol, dimestrol, chlorotrianisene, stilbestrol-monobenzyl-ether, fosfestrol or homoestradiol.
  • the estrogen agonist may be a derivative of estrogen having the hormonal function of estrogen or may be any substance that increases the activity of an estrogen
  • the present invention provides a substance which is capable of enhancing the serum fetuin-B level in a female individual for use in a method for enhancing fertility of an oocyte, the method comprising,
  • the substance is defined, as above.
  • the present invention furthermore refers to isolated ovary tissue cultivated in a medium comprising fetuin-B.
  • fetuin-B affects oocyte development.
  • fetuin-B affects the hardening of the ZP.
  • the inventors could show that the ZP of ovulated oocytes of fetuin-B deficient mice was hardened before fertilisation.
  • the inventors could show that the cortical granule protease ovastacin is inhibited by fetuin- B.
  • fetuin-B which is produced by the liver, is present in the follicular fluid and prevents hardening of the ZP of oocytes in the immature or mature state.
  • Low level degranulation of the cortical granules of the oocyte and low level cortical granule release of ovastacin occurs during the maturation of an oocyte within the body.
  • Ovastacin which effects hardening of the ZP of the oocyte by cleavage of ZP2 to ZP2f, is inhibited by fetuin-B present in the follicular fluid so that hardening of the ZP of the oocyte is prevented.
  • a medium comprising fetuin-B corresponds to the medium surrounding the oocytes in their natural environment. Addition of fetuin-B to isolated oocytes inhibits ovastacin and thus prevents premature ZPH of oocytes. Thus, isolated oocytes in a medium comprising fetuin-B are maintained or preserved in a fertilisable state.
  • the term "culturing an ovary tissue comprising an oocyte” means that the ovary tissue is held or stored in a medium comprising fetuin-B. In an aspect of the invention, the term “culturing” means "stabilising".
  • the term "stabilising an ovary tissue comprising an oocyte” or “stabilising an oocyte” or similar terms refer to the ability of fetuin-B to maintain or preserve the fertilisable state of an oocyte after isolation.
  • “culturing” or “stabilising” means that hardening of ZP of the oocyte is prevented. Most preferred, this may be effected by the inhibition by fetuin-B of the activity of ovastacin, which is released by premature degranulation of the cortical granules of the oocyte.
  • Inhibition of the activity of ovastacin means inhibition of the proteolytic activity processing ZP glycoproteins, which are present in the zona pellucida of oocytes, especially inhibition of cleavage of ZP2 to ZP2f.
  • ZP2 cleavage into ZP2f is a hallmark of hardening of ZP.
  • stabilising may also include that the ovary tissue comprising the oocyte survives and remains viable.
  • the term "maintain the fertilisable state of an oocyte” or similar terms mean that the oocyte does not loose the ability or vice versa maintains the ability to be fertilised, e.g. under suitable in vitro conditions or after transplantation in vivo.
  • An immature oocyte maintains the fertilisable state if that oocyte was fertilisable at the time of isolation.
  • an immature oocyte has been isolated from a female body and has been kept in a medium comprising fetuin-B and has then been matured, the oocyte will be able to become fertilised by a sperm cell.
  • a mature oocyte maintains its mature developmental state and can be fertilised by a sperm cell.
  • the term "stabilising" may include that the oocyte in the medium comprising fetuin-B does not loose its ability for further development or vice versa maintains its ability to proceed further with development, if transferred into the respective conditions in vitro or after transplantation into a female body.
  • the oocyte does not loose its ability to proceed further with maturation.
  • an immature oocyte which is isolated from a female body and is treated with fetuin-B, may proceed with its development, e.g. maturation, within the medium comprising fetuin-B or may proceed with its development, if it is removed from the medium comprising fetuin-B and brought under appropriate conditions.
  • a mature oocyte maintains the mature developmental state and remains fertilisable, i.e. it can be fertilised. However, hardening of ZP is prevented.
  • ovastacin which is released by degranulation of the cortical granules of the immature or mature oocyte, is inhibited, so that ZP2 cannot be cleaved into ZP2f.
  • Stabilisation of the ovary tissue is achieved by applying fetuin-B to the isolated ovary tissue during or at the latest immediately after isolation from a female body. If the ovary tissue is removed from the female body in the absence of fetuin-B, the release of ovastacin is triggered resulting in the immediate beginning of ZP hardening. In order to prevent hardening and to maintain the fertilisable state of the oocyte, the immediate treatment of the oocyte with fetuin-B is necessary.
  • the term “immediate” or “immediately” in the context “immediately after isolation of the ovary tissue from the female body” or similar terms mean that the tissue is isolated from a female body and the tissue and fetuin-B are immediately brought into contact with each other, for example, the tissue is immediately added to a medium comprising fetuin-B.
  • the tissue is taken from the female body and fetuin-B is applied within the next few seconds or minutes, such as 0, 5, 10, 20, 30 or 60 seconds to 10 minutes, preferably, 0, 5, 10, 20, 30 or 60 seconds to 5 minutes, more preferably 0, 5, 10, 20, 30 or 60 seconds to 2 minutes, whereby the limiting factor is the time which is needed for handling the isolated ovary tissue and contacting it with a medium comprising fetuin-B.
  • the medium comprising fetuin-B is already used for flushing and/or washing the ovary tissue during the process of isolating the ovary tissue from the female ' s body.
  • the ovary tissue is contacted with a medium comprising fetuin-B.
  • the tissue may be processed, if this is deemed necessary.
  • processing refers to procedures where handling or manipulating the ovary tissue is necessary, such as examination of the ovary tissue to determine the developmental state of the oocyte, isolation of the oocyte from the ovary tissue, removal of surrounding cells such as cumulus cells etc. After culture, the ovary tissue may be further used.
  • the term “further use” refers to procedures such as maturation, fertilisation or transplantation into the female body.
  • GenBank accession number As available from the NCBI (National Centre for Biotechnology Information; National Library of Medicine 38A, Bethesda, MD20894; USA; www.ncbi.nih.gov). The sequences are indicated in the following.
  • polynucleotide sequence of human fetuin-B is available under the GenBank Accession No. NM 014375.2.
  • mouse fetuin-B The polynucleotide and amino acid sequences of mouse fetuin-B have been published under the following GenBank accession number as available from the NCBI (National Centre for Biotechnology Information; National Library of Medicine 38A, Bethesda, MD20894; USA; www.ncbi.nih.gov). The sequences are indicated in the following.
  • mouse fetuin-B (fetuin-B isoform 1 precursor form) is available under the GenBank Accession No. NM_021564.2.
  • mouse fetuin-B The amino acid sequence of mouse fetuin-B is available under the GenBank Accession No. NP 067539.1
  • Fetuin-B as used in the present invention may be provided by any methods known in the art. Fetuin-B is a liver-derived plasma protein. Thus, it can be isolated from plasma. However, more convenient is the provision of recombinant fetuin-B, which is obtained from a cell transformed with a polynucleotide encoding fetuin-B.
  • the polynucleotide encoding fetuin-B may be any polynucleotide encoding a fetuin-B polypeptide.
  • the polynucleotide encoding fetuin-B may be from human, mouse, cow, horse, dog, cat, pig, goat, sheep, camel, or zoo animals, such as panda bear, large cat etc.
  • the fetuin-B polynucleotide is from human, mouse, cow, horse or camel and more preferably from human.
  • a polynucleotide encoding a fetuin-B polypeptide is a polynucleotide comprising or consisting of SEQ ID NO: 1 or 3 coding for fetuin-B having the amino acid sequence of SEQ ID NO: 2 or 4, respectively.
  • the term "polynucleotide encoding fetuin- B" or “fetuin-B polynucleotide” or “polynucleotide” as used herein encompasses polynucleotides which are defined by SEQ ID NO: 1 or 3 as well as naturally occurring or non-naturally occurring variants or fragments thereof (as defined herein).
  • the most common (and therefore preferred) polynucleotides are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • the polynucleotide can be a DNA molecule such as a genomic DNA molecule or a cDNA molecule, which can be single- or double-stranded as well as a synthetic DNA such as a synthesized single-stranded polynucleotide.
  • the polynucleotide of the present invention can also be an RNA molecule.
  • the term also relates to non-coding regions of a gene, wherein these sections are of a relevant size in order to be specific for that gene. Examples of those regions are regulatory elements such as a promoter.
  • polynucleotide relates to gene, open reading frame (ORF), promoter, DNA, cDNA or mRNA.
  • the polynucleotide encoding the desired genetic information, preferably DNA may comprise the gene of interest, a promoter region, a start codon and a stop codon and possibly further regions which may be used for regulation of expression of the gene.
  • the polynucleotide may encode functionally active variants of the polypeptide of SEQ ID NO: 2 or 4.
  • Such functionally active variants have a sequence identity with SEQ ID NOs: 2 or 4 of more than 50%, of more than 60%, preferably more than 70%, more preferably of more than 80%, still more preferably more than 85%, even more preferably more than 90%, even more preferably more than 95%, most preferably more than 97% and/or have an activity of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97% or more than 100 % , e.g.
  • the polynucleotides encoding such variants may contain deletions, insertions, substitutions and/or additions within and/or at the 5 " and/or 3 " termini of SEQ ID NO: 1 or 3 and show an identity to the polynucleotide of SEQ ID NO: 1 or 3 of more than 50%, more than 60%, more than 70%, preferably more than 80%, more preferably more than 85%, even more preferably more than 90%, even more preferably more than 95%, most preferably more than 97%.
  • a variant polynucleotide encoding a functionally active variant of the polypeptide of SEQ ID NO: 2 or 4 means a sequence encoding a variant polypeptide which has the same activity as the polypeptide of SEQ ID NO: 2 or 4.
  • the polynucleotide fragments may encode functionally active fragments of the polypeptide of SEQ ID NO: 2 or 4, respectively.
  • This may include fragmental polypeptides with short internal and/or C- and/or N-terminal deletions whereby the activity of the resulting polypeptides as identified herein is maintained to an extent of more than 50 %, more than 60 %, more than 70 %, more than 80 %, more than 90 %, more than 95 %, more than 97 % or more than 100 %, e.g. more than 150 %, 200 %, 300 % 400 %> or 500 %>, of the activity of the wild-type polypeptide as encoded by SEQ ID NO: 1 or 3.
  • a fragmental polynucleotide encoding a functionally active fragment of the polypeptide of SEQ ID NOs: 2 or 4 means a sequence encoding a fragment which has the same activity as the polypeptide of SEQ ID NO: 2 or 4. It is noted that the above mentioned modifications may be combined.
  • a polynucleotide as comprised by the present invention may be a fragment comprising one or more variations.
  • fragments or variants include fragments or variants, as defined herein, of promoter or regulatory sequences with which the polynucleotide of SEQ ID NO: 1 or 3 or fragments or variants thereof are associated in nature. These variants are functionally active in that they regulate the transcription or translation of the genes associated therewith.
  • the fetuin-B polynucleotide encodes a fetuin-B polypeptide of SEQ ID NO: 2 or 4. Due to the degeneracy of the genetic code, the polynucleotide sequence of SEQ ID NO: 1 or 3 may be modified by replacing nucleotides in such a way that the same amino acids are encoded.
  • the fetuin-B polynucleotide hybridizes under stringent conditions, preferably highly stringent conditions, to the homologous polynucleotide of SEQ ID NO: 1 or 3 or a functionally active variant or fragment thereof.
  • stringent conditions preferably highly stringent conditions
  • Such conditions include conditions under which a complementary strand of a highly identical nucleic acid, namely a DNA composed of a nucleotide sequence having 70 % or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more and even more preferably 95% or more identity with the polynucleotide of SEQ ID NO: 1 or 3, hybridizes, while a less complementary strand of a nucleic acid less identical than the above does not hybridize.
  • a filter e.g. a nitrocellulose filter
  • a hybridization solution e.g. containing 50% formamide, high salt (either 5x SSC [20x: 3M NaCl/0.3M trisodium citrate] or 5x SSPE [20x: 3.6M NaCl/0.2M NaH 2 PO 4 /0.02M EDTA, pH 7.7]), 5x Denhardt's solution, 1% SDS, and 100 ⁇ denatured salmon sperm DNA. This is followed by several washes with buffer, e.g.
  • the hybridizing sequence is the complementary non-coding strand of a putative hybridizing fetuin-B polynucleotide which hybridizes to the coding strand of SEQ ID NO: 1 or 3.
  • the polynucleotide is complementary to a polynucleotide of SEQ ID NO: 1 or 3 or a polynucleotide encoding a polypeptide of SEQ ID NO: 2 or 4 or functionally active fragments or variants thereof.
  • the polynucleotide of the present invention may be provided by any methods known in the art. Using the sequence information provided herein, primers suitable for amplification/isolation of the polynucleotide of SEQ ID NO: 1 or 3 can be determined according to standard methods well known to those of skill in the art. Primers suitable for amplification/isolation of the polynucleotide of SEQ ID NO: 1 or 3 are designed according to the nucleotide sequence information provided in the sequence listing. The procedure is as follows: a primer is selected which may consist of 10 to 40, preferably 15 to 25 nucleotides.
  • primers containing C and G nucleotides in a proportion sufficient to ensure efficient hybridization; i.e., an amount of C and G nucleotides of at least 40%, preferably 50% of the total nucleotide content.
  • a standard PCR reaction will be performed which typically contains 0.5 to 5 Units of Taq DNA polymerase per 100 ⁇ , 20 to 200 ⁇ deoxynucleotide each, preferably at equivalent concentrations, 0.5 to 2.5 mM magnesium over the total deoxynucleotide concentration, 105 to 106 target molecules, and about 20 pmol of each primer. About 25 to 50 PCR cycles are performed.
  • a more stringent annealing temperature improves discrimination against incorrectly annealed primers and reduces incorporation of incorrect nucleotides at the 3' end of primers.
  • a denaturation temperature of 95°C to 97°C is typical, although higher temperatures may be appropriate for denaturation of G+C-rich targets. The number of cycles performed depends on the starting concentration of target molecules, though typically more than 40 cycles are not recommended as non-specific background products tend to accumulate.
  • An alternative method for retrieving polynucleotides encoding variant polypeptides as defined herein is by hybridization screening of a DNA or RNA library using the primers and probes as defined herein. Hybridization procedures are well-known and are described in the art and herein.
  • the polynucleotide may be provided by cloning and thereby introducing it into and amplifying it in a cell.
  • the procedure of introducing a gene into a recipient cell is called transformation.
  • the genes can be introduced into the cells by a variety of means known in the art and adapted to each cell type.
  • the term "cell” refers to the cell in which the gene is expressed irrespective of whether it is a prokaryotic cell or a eukaryotic cell and of whether the cell naturally expresses the respective genes or not.
  • Recombinant DNA cloning techniques well known in the art for introducing and expressing a nucleic acid molecule can be used to introduce and express the gene which is either endogenous if the cell harbors the respective gene or is heterologous if the gene is not endogenous to the cell.
  • Cells can be transformed using any appropriate means, including viral or bacteriophage based vectors, chemical agents, electroporation, calcium phosphate co -precipitation or direct diffusion of DNA.
  • the regulatory regions for regulating expression of the fetuin-B polynucleotide in a vector are promoters and possibly enhancers or other regulatory sequences. They may be heterologous to the respective gene or may be associated therewith in nature.
  • the genetic information may be expressed permanently or under the control of a repressor and/or a promoter region in a cell into which the nucleic acid of the present invention is introduced.
  • the obtained cells may be either used directly or used for tissue cultures or the cells may be harvested and samples comprising the respective polypeptide are obtained by disrupting the cells.
  • DNA or RNA may be used either in cells or in cell-free expression systems such as, e.g., microarray systems in which the DNA or RNA is immobilized and is translated and/or transcribed by the addition of functional cell lysate, comprising the factors required for transcription and/or translation (enzymes, ribosomes, tRNA, amino acids, nucleotides, ATP etc.). Also included are artificial nucleic acids which include peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule.
  • PNA peptide nucleic acid
  • LNA morpholino and locked nucleic acid
  • GAA glycol nucleic acid
  • TAA threose nucleic acid
  • a medium comprising fetuin-B may be a medium that contains fetuin-B and additional functionally active components or a medium that only comprises fetuin-B as functionally active component. This does not affect the presence of medium ingredients such as nutrients as referred to in the chapters directed to "medium”.
  • heterologous as it relates to polynucleotide sequences or polypeptides denotes sequences that are normally not associated with a region of a recombinant construct and/or a particular cell.
  • a "heterologous" region is an identifiable segment of a polynucleotide within or attached to another polynucleotide that is not found in association with the other molecule in nature.
  • a heterologous region of a construct could be a regulatory region not found to be associated with a gene as identified herein in nature.
  • a heterologous sequence could be a coding sequence which is itself not found in nature as it contains e.g. synthetic sequences with codons different from the native gene.
  • a cell transformed with a construct, which is not normally present in the cell would be considered heterologous for the purposes of the present invention.
  • a homologous polynucleotide sequence is a variant sequence as defined herein.
  • the term “homologous” may be used interchangeably with variant.
  • the term “homologous” may also refer to a polynucleotide or polypeptide that is from another species and that has the same physiological function in said other species.
  • the term “homologous” may also refer to an identical sequence.
  • Vectors are agents that transport an endogenous or heterologous gene into the cell.
  • Vectors typically consist of a number of genetic components, including but not limited to regulatory elements such as promoters, leaders, introns, and terminator sequences. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the "promoter".
  • Vectors can be a plasmid, a cosmid, a virus (e. g. bacteriophage) a phagemide, an artificial chromosome or a retro-viral vector or others as known in the art. Vectors are able to autonomously replicate in a host cell or can be incorporated into chromosomal DNA.
  • vectors includes those that function primarily for insertion of a polynucleotide into a cell, those that function primarily for replication of a polynucleotide (replication vector) in a cell of a photosynthetic eukaryote or those that function primarily for transcription and/or translation of DNA or RNA in a cell.
  • the promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription into mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA.
  • transformation of a cell or tissue with a polynucleotide is the introduction of a polynucleotide into the cell or tissue by any means.
  • fetuin-B or "fetuin-B polypeptide” as comprised by the present invention encompasses any fetuin-B polypeptide, in particular human or mouse fetuin-B, more preferably human fetuin-B and still more preferably the polypeptide of SEQ ID NO: 2 or 4, as well as functionally active variants or fragments of SEQ ID NO: 2 or 4, as defined above, which may be natural or non-natural.
  • the variants or fragments differ from the sequence of SEQ ID NO: 2 or 4 e.g.
  • amino acids by addition, deletion, substitution and/or insertion of amino acids and have a sequence identity with SEQ ID NO: 2 or 4 of more than 50%, of more than 60%, more than 70%, preferably of more than 80%, more preferably more than 85%, even more preferably more than 90%, even more preferably more than 95%, most preferably more than 97% and/or have an activity of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97% or more than 100 % , e.g. more than 150 %, 200 %, 300 % 400 % or 500 % of the activity of the polypeptide of SEQ ID NO: 2 or 4.
  • the variant of the polypeptide of SEQ ID NO: 2 or 4 as comprised by the present invention is a functionally active polypeptide in that it maintains the biological function of the reference polypeptide of SEQ ID NO: 2 or 4.
  • Non-naturally occurring variants or naturally occurring variants of the polypeptide of SEQ ID NO: 2 or 4 may comprise a limited number of amino acid deletions, insertions and/or substitutions, particularly deletions, insertions and/or substitutions of, e.g., at most 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid(s) thereby obtaining a sequence identity or activity of the respective wild-type proteins, e.g. with respect to SEQ ID NO: 2 or 4, as mentioned above.
  • Non-naturally occurring fragments or naturally occurring fragments of the polypeptide of SEQ ID NO: 2 or 4 may comprise a limited number of amino acid deletions of, e.g., at most 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid(s).
  • the activity of the respective wild-type proteins, e.g. with respect to SEQ ID NO: 2 or 4, is maintained, as mentioned above.
  • the variant may be a modified protein or a modified protein variant which comprises a further component.
  • the variant may be a fusion protein comprising (i) a polypeptide of SEQ ID NO: 2 or 4 or functionally active variant and (ii) a further protein or peptide component.
  • the polypeptide may be coupled to a marker, such as a tag used for purification purposes (e.g. 6 His (or HexaHis) tag, Strep tag, HA tag, c-myc tag or glutathione S-transferase (GST) tag).
  • a marker such as 6 His (or HexaHis) tag, Strep tag, HA tag, c-myc tag or glutathione S-transferase (GST) tag.
  • a marker such as 6 His (or HexaHis) tag, Strep tag, HA tag, c-myc tag or glutathione S-transferase (GST) tag.
  • Examples of such double or tandem tags are the GST-His-tag (glutathione- S-transferase fused to a polyhistidine-tag), the 6xHis-Strep-tag (6 histidine residues fused to a Strep-tag), the 6xHis-tagl00-tag (6 histidine residues fused to a 12-amino-acid protein of mammalian MAP -kinase 2), 8xHis-HA-tag (8 histidine residues fused to a hemagglutinin-epitope-tag), His-MBP (His-tag fused to a maltose-binding protein, FLAG- HA-tag (FLAG-tag fused to a hemagglutinin-epitope-tag), and the FLAG-Strep-tag.
  • GST-His-tag glutlutathione- S-transferase fused to a polyhistidine-tag
  • the marker could be used in order to detect the tagged protein, wherein specific antibodies could be used. Suitable antibodies include anti-HA (such as 12CA5 or 3F10), anti-6 His, anti-c-myc and anti-GST.
  • the protein could be linked to a marker of a different category, such as a fluorescence marker such as green fluorescent protein, to a binding protein such as steptavidin, one or more small molecular dyes such as Cy dye, or a radioactive marker, which allows for the detection of a protein as comprised by the present invention.
  • the polypeptide of SEQ ID NO: 2 or 4 could be part of a fusion protein, wherein the second part could be used for detection, such as a protein component having enzymatic activity.
  • polypeptide of SEQ ID NO: 2 or 4 or variants or fragments thereof as described above may comprise one or more amino acid substitution(s).
  • amino acid substitution(s) may comprise one or more amino acid substitution(s).
  • semi-conservative and especially conservative amino acid substitutions wherein an amino acid is substituted with a chemically related amino acid are preferred.
  • Typical substitutions are among the aliphatic amino acids, among the amino acids having aliphatic hydroxyl side chain, among the amino acids having acidic residues, among the amide derivatives, among the amino acids with basic residues, or the amino acids having aromatic residues.
  • fetuin-B also comprises fetuin-B agonists, which mimic the functional activity of fetuin-B or fetuin-B surrogates, which are substitutes of fetuin-B and have the same activity as fetuin-B.
  • fetuin-B inhibits ovastacin, as shown in the present invention, the term “fetuin-B” also includes inhibitors of ovastacin.
  • the term "activity" of fetuin-B refers to the ability of fetuin-B to maintain or preserve the fertilisable state of an oocyte in a medium comprising fetuin-B.
  • stabilising may also include that the oocyte in the medium comprising fetuin-B does not loose its ability for further development.
  • the term "activity" of fetuin-B corresponds to the definition given above with respect to “stabilising”.
  • a mature oocyte has been isolated, the mature developmental state is maintained and the oocyte remains fertilisable, i.e. is able to become fertilised by a sperm.
  • activity refers to the prevention of the hardening of ZP of the oocyte, most preferably by inhibiting the activity of ovastacin, which is released by degranulation of the cortical granules of the oocyte, such as to proteolytically process ZP glycoproteins, especially to cleave ZP2 to ZP2f.
  • the activity of fetuin-B may cause that the oocyte does not loose its ability to proceed further with maturation.
  • the oocyte may proceed with maturation within the medium comprising fetuin-B, or if removed from the medium comprising fetuin-B and brought under appropriate conditions.
  • the "activity" of fetuin-B may be determined visually such as microscopically in that it is observed whether the status of the ovary tissue is changed or maintained in the medium comprising fetuin-B. In the presence of fetuin-B, the outer appearance of the ovary tissue is not changed, whereas in the absence of fetuin-B the outer appearance is changed, for example, a hardened ZP is generated. This may be determined by any method known in the art, preferably by visual inspection or by determining the protease resistance of the zona pellucida, which is determined by measuring the time required to digest the zona pellucida by chymotrypsin.
  • Digestion of the zona pellucida may be scored by observing the oocyte with an inverted microscope in a medium containing oocyte culture medium and chymotrypsin.
  • the time required to digest the ZP in half of the oocytes (t 50 ) is much longer for fetuin-B deficient oocytes such as oocytes which have been isolated from a female body and have not been treated with fetuin-B, so that hardening of ZP occurs, or fertilised zygotes or two-cell embryos with a hardened ZP than for fertilisable oocytes, wherein the ZP is not hardened, such as oocytes which have been isolated from a female body and have been treated with fetuin-B immediately after isolation.
  • t 50 in fetuin-B deficient oocytes e.g. 31.5 min ⁇ 4.7 min
  • two-cell embryos e.g. 38.5 min ⁇ 6.0 min
  • Activity of fetuin-B may also preferably be determined by the presence or amount of ZP2f, which appears if ovastacin is active and cleaves ZP2 into ZP2f.
  • At most 10%, at most 5%, at most 3%, at most 2%, at most 1% or 0% of ZP2 present at the time of isolation is cleaved into ZP2f during storage.
  • Detection may be performed by using an antibody against ZP2 and ZP2f (Rankin et al, 2003), e.g. in an immunological detection method such as Western Blotting, whereby ZP2 shows a signal at 120 kDa and ZP2f shows a signal at 90 kDa.
  • the time interval of storage of the ovary tissue in the medium comprising fetuin-B may be several minutes such as 10, 20, 30 minutes to several hours such as 4, 5, 6 hours.
  • the oocyte may be fertilized within these time intervals.
  • the medium comprising fetuin-B and the ovary tissue may be frozen to extend the time interval of the storage to several days such as 2, 3, 5, 8 days to several weeks or months. Freezing methods are known to those skilled in the art and may be flash freezing also known as vitrification or controlled freezing in the presence of anti-freeze protectants, preferably vitrification.
  • the term "ovary tissue comprising an oocyte” is any part of an ovary which can be isolated from a female and which comprises an oocyte.
  • the oocyte can be at any stage, be it an immature or a mature oocyte.
  • the oocyte is an immature oocyte.
  • the ovary tissue may be a follicle.
  • the follicle is an immature follicle comprising an immature oocyte.
  • the follicle may also be a mature follicle comprising a mature oocyte.
  • the ovary tissue may be an oocyte, preferably an immature oocyte, but may also be a mature oocyte.
  • the ovary tissue may be the cortex of the ovary comprising an immature follicle with an immature oocyte.
  • the ovary tissue may be a whole ovary.
  • oocytes surrounded by follicular somatic cells are maintained in a prolonged stage of the first meiotic prophase.
  • the oocytes become developmentally arrested in the ovaries at the germinal vesicle stage in prophase of the first meiotic division (G2/M transition).
  • the oocytes are called primary oocytes (primordial follicles).
  • follicle stimulating hormone FSH
  • LH luteinizing hormone
  • FSH induces the formation of LH receptors on the granulosa cells.
  • oocytes of preovulatory follicles resume meiosis.
  • the first meiotic division is completed with the extrusion of a haploid set of chromosomes into the first polar body while the other haploid set of chromosomes remains within the cytoplasm of the oocyte.
  • secondary oocytes then proceed to the second meiotic division, where the oocyte becomes arrested at metaphase ("Met-II").
  • Met-II oocytes are mature, and can be ovulated and fertilised. Without fertilisation, the mature oocyte will not continue with meiosis.
  • meiosis II completes, forming a second polar body.
  • immature oocyte means an oocyte that has not yet reached metaphase-II (M-II).
  • Immature oocytes used in the present invention exist within follicles which pass through the following distinct stages including primordial, primary, secondary, and antral stages including early antral, later antral, and pre-ovulatory stages.
  • Immature oocytes used in the present invention may be present in prophase I (germinal vesicle), metaphase I, anaphase I or telophase I.
  • Immature oocytes preferably used in the invention are at the germinal vesicle (GV) or metaphase-I (M-I) stage.
  • GV germinal vesicle
  • M-I metaphase-I
  • immature oocytes of M-I stage will be readily recognised as oocytes with a tight cumulus mass, no polar bodies or germinal vesicles visible.
  • an immature oocyte is present in an ovarian follicle with a diameter of 8 to 12 mm.
  • the advantage of such small follicles is that they are present in substantial numbers without severe hormonal treatment and they can be seen by ultrasound.
  • Follicles preferably used in the present invention are present in the primordial, primary, secondary, and antral stages.
  • transvaginal ultrasonographically-guided oocyte collection is done involving needle piercing of the vaginal wall.
  • the follicles, oocytes and follicular fluids may be aspirated and immediately stored in a medium comprising fetuin-B. If the follicle is further processed in order to isolate the oocyte, all processing steps are performed in the medium comprising fetuin-B.
  • the oocytes may be retrieved from the ovarian follicle by e.g.
  • the oocytes are aspirated into a medium comprising fetuin-B.
  • the oocytes may be inspected microscopically.
  • the outer layer of the ovary may be isolated from a female.
  • preantral follicles exist in the relatively avascular cortex of the ovary. Isolation of ovary tissue comprises ovary tissue collection, immediate tissue treatment with medium comprising fetuin-B and separation of the cortical layer from the medullar part.
  • the cortex is sliced into strips 1 millimeter thick, which remain in the medium comprising fetuin-B. After culture, the slices are implanted back into the abdomen, close to the fallopian tubes near the uterus.
  • the cortex tissue strips contain immature follicles. They start to produce hormones and oocytes like a normal ovary, when they are implanted back into the body.
  • mature oocyte means an oocyte at the metaphase-II stage. Metaphase-II is typically detected by visual identification under a microscope. Processes for retrieving mature oocytes from a female body are known in the art.
  • the skilled person knows how the status of an ovary tissue isolated from a female ' s body can be determined. For example, the developmental stage of a follicle can be determined microscopically.
  • immature oocytes there is substantial interest in the art to isolate immature oocytes from a female and to preserve the oocytes in a culture medium for later use.
  • immature cultivated oocytes may be re-transplanted into the female, where they can mature to a mature, fertilisable oocyte.
  • the method of the present invention may also be used in techniques wherein the immature oocytes may be used at a time after collection for in vitro maturation of oocytes for in vitro fertilisation.
  • a premature hardening of the zona pellucida should be prevented in order to ensure that the oocyte can be fertilised at a time after isolation, either in vitro or in vivo after transplantation. Hardening of the zona pellucida would otherwise occur, if the oocyte is removed from its natural environment and not brought into a medium comprising fetuin- B.
  • immature oocytes may be collected during IVF.
  • Current IVF (in vitro fertilisation) treatment requires that multiple oocytes be harvested. Therefore, women are normally pre-treated with hormones, and oocytes, which have matured to the M-II stage are harvested. Usually, at least 10-15% of the developing oocytes are immature and are discarded at most IVF clinics.
  • Such immature oocytes may be preserved by the method of the present invention and are available for future applications, e.g. if IVF (in vitro fertilisation) has failed and the immature oocytes are brought to maturity, in vitro, for subsequent fertilisation.
  • immature oocytes may be collected from unstimulated ovaries and may be matured to the M-II stage by way of in vitro maturation (IVM), in order to avoid the entire regimen of treatment of women with GnRHa and stimulation of ovaries with gonadotropin during IVF.
  • IVM in vitro maturation
  • fetuin-B in a culture medium comprising fetuin-B may be useful for maintaining the fertilisable status of the oocyte, if such fertilisable status is present at the time of isolation.
  • Another group of patients, for which it may be desirable to collect ovary tissue and preserve it, are cancer patients. Women undergoing chemotherapy or radiotherapy during cancer treatment have associated morbidity of their reproductive organs resulting in post- therapy infertility. Ovary tissue from these patients can be harvested and preserved before the start of cancer therapy. Ovary tissue can be re-transplanted when the woman is ready to have children.
  • an ovary is surgically removed and preserved according to the present invention. Part or whole of the ovary may later be used for the woman. The removal of ovary tissue or the ovary does not require a significant delay in cancer treatment beyond one to two days of recovery. Additionally, children and infants as young as 6 months can undergo this procedure since neither reaching puberty nor fertilisation are required.
  • ovarian tissue or ovary can be dissected out and preserved in a culture medium comprising fetuin-B prior to initiation of treatment that might cause sterility as cytostatic or radiation treatment often does. Immature oocytes can immediately or later be extracted from the ovarian tissue or ovary and finally be matured and fertilised.
  • the present invention offers a method of ovary tissue preservation for transplantation into female patients with malignant neoplasms of reproductive system organs.
  • the method comprises the collection of ovary tissue, the addition of the isolated ovary tissue into medium comprising fetuin-B and the storage in the medium comprising fetuin-B for future transplantation.
  • the recovery of immature oocytes and culturing them in a medium comprising fetuin-B may be a useful for women with polycystic ovarian syndrome (PCOS) related infertility.
  • PCOS polycystic ovarian syndrome
  • Immature oocytes may be collected from these women and cultivated in a medium comprising fetuin-B and may be used at a later time point, for example, for in vitro maturation techniques or for transplantation.
  • the ovary tissue may be isolated from a wide variety of animal species, particularly mammalian species. Examples are humans, cows, horses, camels, dogs, cats, pigs, goats, sheep, or zoo animals, such as panda bears, large cats etc. Humans are of a very particular interest, however, also animals with high commercial value such as high efficiency cows, race horses or race camels are of particular interest. Animal models, particularly small mammals, e.g. mice, etc., are of interest for experimental investigations. Most preferably, the ovary tissue is isolated from humans.
  • the present invention involves the provision and use of a culture medium comprising fetuin-B for culturing ovary tissue.
  • the invention comprises holding the ovary tissue in the culture medium comprising fetuin-B for a period following isolation of the ovary tissue, to ensure that the oocytes do not loose the ability to mature and to be fertilised at a later time.
  • the period of culturing the ovary tissue may be any period, until it is desired to use the ovary tissue for in vitro maturation and possibly fertilisation processes or for implantation into the female for fertilisation. The period may be several minutes to hours, days, months or years.
  • Culture conditions for ovary tissue in a medium comprising fetuin-B are not critical to the invention, as long as the culture conditions support the activity of fetuin-B, as referred to above.
  • the culture conditions should be selected such that the status of the ovary tissue is maintained at the status present when isolating the ovary tissue.
  • the conditions should preserve the survival of the ovary tissue and in particular of the oocytes comprised by the ovary tissue and should maintain the ability of the oocytes for maturation and fertilisation.
  • Suitable culture conditions include e.g.
  • culturing the ovary tissue under cold or ambient temperature such as from 0 to 37°C, preferably in the cold, such as in a refrigerator, from 0 to 10°C such as 4 to 6°C in an atmosphere of 95% air and 5% C0 2 at high humidity, e.g. 100% humidity.
  • a "triple gas" atmosphere of 5% 0 2 , 5% C0 2 and 90% N 2 may also be used.
  • Mineral oil may be overlaid on the medium to control evaporation and/or temperature.
  • Isolated oocytes are typically cultured in a well containing 1 ml of culture medium comprising fetuin-B or more, or may be cultured in 10 ⁇ of culture medium comprising fetuin-B or less in a droplet in a culture dish.
  • the present invention provides a culture medium for culturing ovary tissue including fetuin-B.
  • the term “medium”, “culture medium” or a similar term is a medium for culturing ovary tissue, which does not include fetuin-B. If fetuin-B is present, this is indicated.
  • the culture medium comprising fetuin-B is used as a substitute for the follicular fluid surrounding the oocytes in the natural state.
  • the kind of medium is not critical to the present invention, as long as the medium is suitable to support or not to interfere with the activity of fetuin-B to maintain the status of the ovary tissue.
  • the activity of fetuin-B to preserve the capability of the oocytes for maturation and fertilisation should be supported by the medium or at least the medium should not interfere with this activity of fetuin-B. If medium is selected which interferes with the activity of fetuin-B, then the medium comprising fetuin-B should in totality show the activity of fetuin-B. This may, e.g. be achieved by increasing the concentration of fetuin-B. The skilled person is capable of elucidating media which in the presence of fetuin-B show the above features.
  • the medium useful for the method of the present invention contains an energy source, inorganic salts, essential and/or non-essential amino acids and possibly other factors.
  • the energy source, inorganic salts, and essential and/or non-essential amino acids in the medium are not critical to the invention, as long as the requirements as described above with respect to the medium are met.
  • the energy source may be e.g. glucose, sodium pyruvate, lactate, or a mixture of some or all of these energy sources.
  • the inorganic salts are inter alia provided to buffer the pH of the medium within a range preferably of about 5 to 9, preferably 6 to 8 and more preferably 7.2-7.4 and to maintain correct osmolarity of the medium with the ovary tissue.
  • Typical inorganic salts include CaCl 2 , KC1, MgS0 4 , NaCl, NaHC0 3 , NaH 2 P0 4 , Fe(N0 3 ) 3 , KH 2 P0 4 , Na acetate, Na 2 H 2 P0 4 , etc.
  • the medium contains at least one amino acid or a source thereof.
  • at least the essential amino acids, or sources thereof, are included in the medium.
  • "Essential" amino acids are those amino acids not synthesized in the oocyte and that are essential for protein synthesis.
  • the essential amino acids are generally considered to be iso leucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.
  • the medium of the invention comprises alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • the medium preferably contains vitamins as are known in the art for inclusion in culture medium.
  • Vitamins that may be included in the medium include, without limitation, vitamins Al (retinol), A2 (an alternative form of retinol), Bl (thiamine), B2 (riboflavin), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamin), B17, C (ascorbic acid), D, D2 (calciferol), D3 (cholecalciferol), E (tocopherol), H (biotin), K, Kl (phylloquinone), K2, K3 (menadione), P, etc.
  • a particularly preferred combination of vitamins comprises biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyroxidal-HCl, riboflavin, and thiamine-HCl.
  • Buffers for controlling the pH of the medium may be added, such as HEPES, as may be pH indicators such as phenol red.
  • Antibiotics such as penicillin or streptomycin may be added to the medium to prevent contamination.
  • Synthetic Serum Supplement (SSS®) may be included in the medium as a source of protein for the oocyte and to prevent cells from adhering to glassware during in vitro culture. SSS has the advantage of having received regulatory approval for human IVF, and is commercially available.
  • Suitable media may be commercial media currently approved for and in use for IVF/ ART (assisted reproductive technologies) and for cryopreservation of tissues and cells destined for IVF/ART.
  • IVF media are comprised of a cocktail of physiological inorganic salts, energy sources, amino acids and proteins, and there is a range of different formulations commercially available.
  • Media can include broad spectrum antibiotics like gentamicin sulphate to prevent microbial contamination of the media.
  • Others include human serum albumin to act as a chelating agent for deleterious contaminants including heavy metals that may be present in minute quantities in the micro-environment, and also as a anti- adhesive to facilitate oocyte manipulation by preventing them from sticking to glass or tissue culture ware.
  • Examples of media include, but are not limited to, GM501 Wash (Gynemed Medizin expend GmbH, Lensahn) and Sydney IVF Gamete buffer (Cook Medical Europe, Ireland) for collecting oocytes, and GM501 Culture (Gynemed) and Sydney IVF Fertilization Medium (Cook Medical) for insemination, GM501 Culture and Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium for zygote and embryo culture.
  • Further media approved and in use for consecutive use in IVF/ART include, but are not limited to, Early Cleavage Medium (Irvine Scientific), Multiblast (Irvine Scientific), G-l Plus (Vitro life), G-2 Plus (Vitrolife), innovative Sequential Medium (ISM-1, Origio), innovative Sequential Medium (ISM-2, Origio).
  • IVF/ART Further commercial complete media approved and in use for IVF/ART include, but are not limited to, Fertikult (Fertipro), G3 (Fertipro), Gynedmed gentamycin, Gynemed501 pen/strep (Gynedmed), HTF Medium (Irvine Scientific), Blastassist (Origio), Quinn advantage cleavage (Sage), Single step Medium (Irvine Scientific), Universal medium (Origio), Global medium (IVFonline).
  • cryopreservation Commercial media currently approved for and in use for cryopreservation include, but are not limited to, EmbryoFreeze, FertiPro, and RapidVit Blast, Vitrolife for freezing and flash freezing (vitrification), respectively, and EmbryoThaw, FertiPro, Thawing Kit, Rapid Warm Cleave and Vitrolife, for thawing.
  • the medium useful for the method of the present invention may resemble a medium known in the art as Human Tubal Fluid medium HTF, and may contain as components in mM concentrations: Sodium Chloride 97.8, Potassium Chloride 4.69, Magnesium Sulfate Anhydrous 0.20, Potassium Phosphate Monobasic 0.37, Calcium Chloride Anhydrous 2.04, Sodium Bicarbonate 25.0, Glucose 2.7, Sodium Pyruvate 0.33, Sodium Lactate 21.4, Gentamicin 10 ⁇ g/mL, Phenol Red 5 mg/L.
  • HTF Human Tubal Fluid medium
  • the medium useful for the method of the present invention may further be enhanced with a serum substitute and may contain as components in mM concentrations: Sodium Chloride 91.44, Potassium Chloride 4.22, Magnesium Sulfate Anhydrous 0.18, Potassium Phosphate 0.33, Calcium Chloride 1.84, Sodium Bicarbonate 22.5, Glucose 2.5, Sodium Pyruvate 0.3, Sodium Lactate 19.26, Phenol Red 4.5 mg/L, Gentamicin 9 ⁇ g/mL, Human Serum Albumin 5 mg/L, Globulins 1 mg/L.
  • a further modification of the medium useful for the method of the present invention may contain as components in mM concentrations: Sodium Chloride 97.8, Potassium Chloride 4.69, Magnesium Sulfate Anhydrous 0.2, Potassium Phosphate Monobasic 0.37, Calcium Chloride Anhydrous 2.04, Sodium Bicarbonate 4.0, HEPES buffer 21, Glucose 2.78, Sodium Pyruvate 0.33, Sodium Lactate 21.4, Gentamicin 10 ⁇ g/L, Phenol Red 5 mg/L.
  • the concentration of fetuin-B in the medium may be from 0.005 to 3 g/L, preferably 0.005 to 1 g/L and more preferably 0.005 to 0.5 g/L.
  • the concentration is similar or equal to the serum concentration of fetuin-B being about 0.01 g/L in human or 0.3 g/L in mouse. Consequently, in a particularly preferred embodiment of the present invention, the concentration of fetuin-B in the medium is 0.005 to 0.03 g/L, more preferably 0.007 to 0.02 g/L and most preferably about 0.01 g/L.
  • Additional preferred embodiments comprise concentrations of fetuin-B from 0.01 to 0.5 g/L, more preferably from 0.05 to 0.5 g/L and still more preferably about 0.1 g/L.
  • fetuin-B for stabilising an ovary tissue comprising an oocyte is an in vitro use.
  • the present inventors have furthermore found a correlation between the level of fertility of an oocyte in an IVF procedure and the serum fetuin-B level of the female individual from which the oocyte has been removed.
  • An increased serum fetuin-B level of an individual induced by a substance which is capable of increasing the fetuin-B level in a female individual enhances the fertility rate in IVF.
  • fetuin-B is effective in maintaining and/or increasing the fertility of oocytes in vivo, such that they can be successfully used in an IVF after isolation.
  • the present invention also shows that fetuin-B is effective in maintaining fertility in vitro after isolation of the oocytes if the isolated oocytes are immediately contacted with a medium comprising fetuin-B.
  • a successful IVF can be performed if the oocytes are under the influence of enhanced levels of fetuin-B during their production in the female body and have immediate and constant contact with fetuin-B in in vitro culturing after isolation until the time of fertilization.
  • a constant contact of oocytes with enhanced levels of fetuin-B within the body and an immediate and constant contact of oocytes with fetuin-B outside the body are necessary to generate and maintain high fertility of oocytes, resulting in fertilization during IVF.
  • serum fetuin-B levels remained constant.
  • the concentration of serum fetuin-B under normal conditions has been newly determined to be about 0.004 g/L in human, but differs from individual to individual. This concentration is lower than the concentration originally determined.
  • This level of fetuin-B in serum can be enhanced by a substance which is capable of increasing the fetuin-B level in a female individual.
  • the substance may be any substance which is capable of enhancing serum fetuin-B levels in a female individual.
  • the substance may be a hormone.
  • An effective hormone for the purpose of the present invention may be estrogens.
  • Estrogens may be endogenously produced by the application of hormones such as FSH or hMG which are administered for inducing the production or maturation of oocytes for use in IVF. Estrogens may also be administered to the female individual.
  • hormones such as FSH or hMG which are administered for inducing the production or maturation of oocytes for use in IVF. Estrogens may also be administered to the female individual.
  • the present inventors have detected the usefulness of estrogens for increasing serum fetuin-B levels in women receiving a contraceptive treatment of ethinylestradiol with or without combination with gestagen.
  • the present inventors have also detected that enhanced serum fetuin-B levels during the period of oocytes maturation, e.g. as a result of hormone treatment for maturing oocytes for use in IVF, in a female body result in fertilization in IVF.
  • the amounts of estrogen to be administered in order to result in enhanced fetuin-B levels are within the amounts which are administered to woman to inhibit ovulation and thus conception.
  • the amounts are preferably 0.02 to 0.05 mg in total per cycle, e.g. 0.03 mg per menstrual cycle.
  • the amounts of hormones such as FSH for inducing endogenous estrogen production are those which are usually administered to a woman for maturation of oocytes for use in IVF and are preferably from 40 to 90 IU/L per injection, more preferably 50 to 75 IU/L per injection and in total 1650 to 4200 IU/L.
  • the amount of estrogen present in the female serum which is suitable to increase fetuin-B level is dependent on the female individual. Measured values were between 5 and 25000 pmol/L. Usually, the concentration of fetuin-B remains constant during maturation of oocytes or increases.
  • the level of fetuin-B which is produced in the female individual depends on the individual. There may be a sharp rise of the level of fetuin-B in response to estrogen. There may be only a slight increase of the level of fetuin-B. Decisive is not the extent of increase of the level of fetuin-B. Decisive is that there is an increase of the level of fetuin-B in response to substance treatment, as compared to the level of fetuin-B in the absence of substance treatment. Nevertheless, the increase of fetuin-B reaches serum fetuin-B levels which are above the basal serum fetuin-B level.
  • the amount of serum fetuin-B in women with substance treatment rises to levels which are 1.5 to 4 times of the basal level, e.g. about 1,5; 1 ,6; 1,7; etc. to 4 times per female individual, more preferably, the amount rises by about twice of the basal level.
  • Exemplary enhanced serum concentrations of fetuin-B are 5 to 20 mg/L, e.g. about 7, 8, 9, etc. or 15 mg/L per female individual.
  • the female individuals that are treated by the use of a substance for enhancing serum fetuin-B levels are the women as indicated above.
  • the female individuals are individuals who want to perform an IVF and take hormones for induction of maturation of oocytes.
  • FIG. 1 Fetuin-B gene deletion strategy and phenotyping
  • A The genomic fetuin locus depicting fetuin-A (Ahsg) exon E7 and fetuin-B (Fetub) exons El to E8. Relevant features of the gene targeting vector (B), the deletion control vector (C), and the resulting recombinant genomic Fetub locus (D).
  • the translation start codon is located in exon 2, which was replaced by the neomycin cassette by homologous recombination of the gene targeting vector and the genomic sequence resulting in deletion of exons E2 and E3.
  • the genomic Fetub locus was conserved except for the 235 bp deletion. Exons are depicted by black boxes.
  • (A) Breeding performance. The mean litter sizes of Fetub-/- and Fetub+I- females were compared with the litter size of Fetub+/+ females and the respective males by pairwise two-sided t test. ***p ⁇ 0.001, ns not significant.
  • Human fetuin-B antibody was used to probe for fetuin-B in sera of wildtype, Fetub-/- and Fetua-I- mice, as well as various commercial bovine fetuin preparations: Sigma- Aldrich F2379 (bov fetuinl), Sigma-Aldrich F3004 (bov fetuin2), Sigma-Aldrich F2379 (bov fetuin3) further purified by gel filtration, AppliChem A2783 (bov fetuin4); Dade-Behring human plasma fetuin- AJ Ahsg (hu fetuin-A), bovine serum albumin (BSA), human serum albumin (HSA), human serum and human follicular fluid.
  • C ZP digestion times of wildtype oocytes (+/+), fetuin-B deficient oocytes (-/-) and wildtype 2- cell stage embryos.
  • the ZP digestion time t 50 equals the time required for 50% of the oocytes to become zona-free following a-chymotrypsin treatment. Every dot represents one assay performed with at least 20 oocytes harvested from one mouse.
  • D In vitro sperm binding to wildtype and fetuin-B deficient oocytes. 2-cell embryos were used as wash controls.
  • E Immune detection of ZP2 protein of wildtype oocytes, fetuin-B deficient oocytes and wildtype 2-cell embryos.
  • Fetuin-B deficient oocytes were further isolated pre- and postovulatory.
  • Active recombinant ovastacin was inhibited by recombinant mouse fetuin-B (circles; concentration range: 0.6 nM - 4.5 ⁇ ) with an IC 50 of 76.4 nM ⁇ 3.35 nM.
  • recombinant fetuin-A did not inhibit ovastacin (squares; concentration range: 0.6 nM - 11 ⁇ ).
  • Pairwise two-sided t test **p ⁇ 0.01, ***p ⁇ 0.001, ns not significant.
  • the horizontal lines depict the mean ⁇ SD (A, B, D) or the mean (C). Background strain C57BL/6, corresponding data for DBA/2 mice are shown in figure 8. Figure 5. Putative mechanism for the interaction of fetuin-B and ovastacin
  • FIG. 6 Female infertility in fetuin-B deficient DBA/2 mice, similar to C57BL/6 mice shown in figure 2
  • B Wildtype (+/+) cumulus- oocyte complex (COC) and COC isolated from a Fetub-/- mouse at day 0.5 after mating.
  • C Oocyte isolated from a wildtype female or a Fetub-/- female at day 0.5 after mating. 89 oocytes recovered from 10 female Fetub-/- mice were unfertilised. In contrast 17 of 20 observed oocytes were fertilised in a total of 3 Fetub+/+ mice.
  • C ZP digestion time of wildtype oocytes (+/+), fetuin-B deficient oocytes (-/-) and wildtype two- cell embryos (2-cell stage).
  • the ZP digestion time was defined as the time required for 50% of the oocytes to become zona-free (tso) following a-chymotrypsin treatment. Every dot represents one assay performed with at least 20 oocytes harvested from one mouse per genotype.
  • fetuin-B 0.1 mg/ml fetuin-B ( ⁇ ) to the medium into which the oocytes isolated from mice have been placed increases the fertilization success compared to the IVF without fetuin-B ( ⁇ ).
  • the x axis indicates the time point of fertilization after isolation of the oocytes.
  • the y axis indicates the percent of fertilized oocytes.
  • the figure shows a positive correlation of serum fetuin-B levels with estradiol levels during hormone treatment with FSH.
  • the x axis shows the concentration of estradiol in the body and the y axis shows the plasma fetuin-B level in the body.
  • Each black dot shows the estradiol and associated fetuin-B level of one measuring sample.
  • the figure shows the correlation between FSH treatment and serum fetuin-B level.
  • A and (B)
  • the x axis shows the day of the hormone treatment for producing oocytes for use in IVF.
  • the y axis shows the serum fetuin-B level. Circles and quadrats indicate the mean values of all the measured values, available at a specific treatment day; linear regression), grey lines reflect the fetuin-B levels of one patient over time, black lines reflect the mean fetuin-B levels.
  • A shows the results for patients with successful IVF.
  • B shows the results for patients with IVF failure.
  • Serum fetuin-B is stimulated by the synthetic estrogen, ethinyl estradiol
  • the figure shows the influence of estrogens on the serum level of fetuin-B.
  • the x axis indicates the cycle day of the woman and the y axis indicates the serum fetuin-B level.
  • the shaded grey area shows the contraceptive treatment using a combined gestagen / ethinylestradiol (EE2) formulation.
  • the curve indicates the level of serum fetuin-B dependent on the day of the cycle.
  • B The same applies for figure B, except that the woman received a different contraceptive treatment regime, namely gestagen alone (day -6 to 0) and gestagen plus EE2 (shaded grey area).
  • the 5' and 3' homologous sequences were amplified by PCR using these primers: 5R3s 5'-CCG CTC GAG ATG TTG GCT ACT GGT TTG CTG T-3' (SEQ ID NO: 5) and 5Rlas 5'-AGC TTT GTT TAA ACC TCT AGT GAG TGG AAC ACA ACT TCT-3' (SEQ ID NO: 6) for the 5' homologous region and 3Rls 5 '-TTG GCG CGC CTG GTT TAG TGA TAC GGG CCA-3' (SEQ ID NO: 7) and 3Rlas 5 '-ACG CGT CGA CAA GCT GCT AGG TAG ATA TTT CCC AT-3' (SEQ ID NO: 8) for the 3' homologous region.
  • a targeting control vector was cloned, resembling the targeted Fetub genomic sequence.
  • the 3' homologous sequence was expanded using primer 3R2as 5 '-ACG CGT CGA CTG TTG AAC TTT GCA AGC GAC T-3' (SEQ ID NO: 9) instead of 3Rlas as shown in figure 1A.
  • the PCR products were ligated into the gene targeting vector pGKneo-TV.
  • the gene targeting vector was transfected into the ES cell line Knut 1 (129/SvJ x C57BL/6) by electroporation.
  • the transfected cells were selected by G418 (250 ⁇ g/ml active substance) treatment. Resistant colonies were analyzed by PCR and Southern blot.
  • mice heterozygous for the fetuin-B deletion were back-crossed for at least 10 generations to inbred mice of strains C57BL/6 and DBA/2 obtained from a commercial breeder.
  • the mouse strains were named B6-Fetub tmlWja and D2- Fetub tmlWja according to Institute of Laboratory Animal Resources (ILAR, http://dels.nas.edu/ilar/) terminology.
  • Fetuin-B and fetuin-A Western blot (murine). Blood was collected by cardiac puncture of adult mice after an overdose of isofluran. The blood was applied to a serum tube and incubated for 1 hour at room temperature. Then, the serum tube was centrifuged at 1000 x g for 10 minutes and the serum supernatant was transferred to a reaction tube. Serum samples were diluted 1 : 100 in SDS-PAGE loading buffer and 15 ⁇ were loaded on a 10% polyacrylamide gel. The proteins were blotted onto a nitrocellulose membrane (Protran, Whatman, Schleicher und Schuell) using a semidry blotting device (BioRad).
  • the membrane was blocked at 4°C over night with blocking solution (PBS + 0.05% Tween 20 + 5% nonfat dried milk powder).
  • the primary antisera (rabbit anti-mouse) were diluted in blocking solution 1 :4000.
  • the secondary antibody (swine anti-rabbit IgG/HRP, Dako) was diluted 1 :5000. All antisera were incubated for 1 hour at 37°C. After each antibody incubation the blot was rinsed 3 x 5 minutes in washing solution (PBS + 0.05% Tween 20). Protein detection was achieved by chemiluminescence using a Fuji LAS Luminescent Image Analyser 4000.
  • oocytes and two-cell embryos Follicular growth of 5-10 week old mice was stimulated by injection of 5 IU PMSG. For the collection of preovulatory oocytes, the mice were sacrificed. Using a stereo microscope and a micropipette, antral follicles were punctured to recover oocytes. For the collection of post-ovulatory oocytes, 5 IU human chorionic gonadotropin (hCG) were injected 48 hours post PMSG to stimulate ovulation. 13 hours post-hCG the female mice were sacrificed by cervical dislocation. The oocytes were isolated from the ampulla and the proximal oviduct.
  • hCG human chorionic gonadotropin
  • Oocytes were liberated from surrounding cumulus cells by digestions in M2 medium containing 0.3 mg/ml hyaluronidase (22.5 - 45 IU/ml, Sigma) for 2-3 minutes. A comprehensive protocol is found in (Hogan et al, 1994). Two-cell embryos were isolated at day 1.5 post ovulation. Ovary transplantation. Ovaries of C57BL/6 mice were dissected from the bursa bilaterally of each female. Dissections were performed on two females simultaneously (one fetuin-B deficient and one wildtype). Eleven females of each genotype of similar age from the same mouse colony were used. Explanted ovaries were kept in PBS supplemented with 10% FCS until required for transplantation.
  • Genomic DNA of tail biopsies of ear marked individuals was isolated by Proteinase K digestion. Genotyping was performed using the following primers: P4as 5'-GCT TGA ACG ATG GGA TAG GC-3 ' (SEQ ID NO: 10), P6s 5'-CAA GTT CTA ATT CCA TCA GAA GC-3' (SEQ ID NO: 11) and P8s 5'-GGG CCT GCT CAG TGT CTA CC-3' (SEQ ID NO: 12).
  • Fetuin-B Western blot (human). The assay was done like described above for mouse. Commercial bovine fetuin preparations (7.5 ⁇ g) from various sources were separated by SDS PAGE (10% acrylamide). Primary antiserum rabbit anti-human fetuin-B was diluted 1 : 1000 and swine anti-rabbit IgG/HRP (Dako) as secondary antibody was diluted 1 :2000. In vitro fertilisation and embryo transfer. Oocytes were collected after superovulation as described in the paragraph "collection of oocytes". Sperm was collected from the cauda epididymis and vas deferens of 14-16 week old males and incubated for 60 minutes at 37°C.
  • Oocyte diameter and ZP thickness Oocyte diameter as well as ZP thickness was determined using a microscope and DISKUS image acquisition and analysis software (Carl Hilgers, Konigs winter).
  • Oocytes were isolated as described. ZP digestion was done like reported before using a 0.2 mg/ml alpha-chymotrypsin (10 IU/ml) solution (Gulyas and Yuan, 1985). The percentage of zona free oocytes was plotted against the time. Data were plotted as a sigmoidal time curve with variable slope using GraphPad Prism statistics software. T50 the time point at which 50% of oocytes had become zona free was calculated using a four parameter logistic regression.
  • ZP2 Western blot 20-30 oocytes from wildtype and Fetub-/- mice as well as two-cell embryos were isolated, dissolved and separated by a 8% polyacrylamide SDS PAGE to detect cleavage of ZP2 as described before (Rankin et al., 2003).
  • Mouse full-length fetuin-A cDNA (Yang et al, 1992) was amplified using the primers (sense) 5 '- AGA ATT CGA GCA ACC ATG AAG TCC CTG -3 ' (SEQ ID NO: 13) and (antisense) 5'- TAC CGG TGC GTC CCT CAA TGA TTT TGA AGT GTC TGA TCC TCC -3' (SEQ ID NO: 14) to introduce a 5' EcoRI restriction site and a 3' Agel restriction site.
  • fetuin- B cDNA was amplified by PCR using the mouse clone MGC:25848 (BCO 18341) and the primers (sense) 5'- AGA ATT CTT ACA GTG GAA TGG GCC -3' (SEQ ID NO: 15) and (antisense) 5'- ATG ACC GGT GGG TGG GAG AAC CAG -3' (SEQ ID NO: 16) to introduce a 5' EcoRI restriction site and a 3' Agel restriction site. Both constructs were first subcloned into pGEM-T vector (Promega) and transfected into high efficiency JM109 competent cells (Promega).
  • the media was changed to a serum-free culture and incubated for another 24 hours to obtain serum-free cell culture supernatant containing fetuin-A or fetuin-B.
  • the cell culture supernatant was filtered through a 0.45 ⁇ filter and the His-tagged recombinant fetuin-A or fetuin-B was purified using HisTrap affinity columns and an AKTA liquid chromatography system (GE Healthcare).
  • the fractions containing the purified proteins were pooled and dialyzed against PBS. Recombinant proteins were identified by Western blot, the purity was judged by SDS-PAGE and Coomassie Blue staining.
  • Mouse full-length ovastacin cDNA (AJ537599.2 at NCBI) was amplified from the EST clone J0247F11 (ATCG, LGC Promochem, Wesel) using the primers (sense) 5'- ATA CTG GCA TGC GTC GAC ACT AGT CCA CCA TGG GTA TCA TGG GAA GCC TG -3' (SEQ ID NO: 17) and (antisense) 5'-TAT GTT AAG CTT ATT ATT TTT CGA ACT GCG GGT GGC TCC AAG ATC TGT CTC TGG GCA CCT CTC TAA T -3 ' (SEQ ID NO: 18) (Carl Roth, Düsseldorf) to introduce a 5 ' Sphl restriction site and a 3' strep-tag-coding region followed Bglll and Hindlll sites.
  • the construct was transferred into pFastBacl (Bac-to-Bac system, Life Technologies, Darmstadt) using the Sphl and Hindlll sites, verified by sequencing (SeqLab, Gottingen) and transposed into baculo virus.
  • the resulting bacmids were used to infect Sf9 insect cells for virus amplification.
  • Protein was expressed in 400 ml suspension cultures of infected Hi5 insect cells at 27°C for 72 hours in a shaking incubator (INFORS, Basel). Cells and debris were removed by centrifugation and the proteins precipitated from the supernatant in 50 mM Tris/HCl, pH 7.4 containing 60% (w/w) ammonium sulfate for 12 hours at 4°C.
  • Protein pellet was obtained by centrifugation (10.000 x g, 2 hours, 4°C), dissolved in 1/10 volume of 50 mM Tris/HCl, pH7.4, 150 mM NaCl, dialyzed vs. the same buffer, loaded onto a strep-tactin sepharose column (IB A, Gottingen) and the purified ovastacin eluted by adding 2.5 mM desthiobiotin (IBA, Gottingen).
  • Mouse full-length ovastacin cDNA deposited under the accession number AJ537599.2 is disclosed in the sequence listing as SEQ ID NO: 19.
  • Oocytes were collected after superovulation as described in the paragraph "collection of oocytes and two-cell embryos".
  • the oocytes were liberated from surrounding cumulus cells by digestion in human tubular fluid (HTF) medium containing 0.3 mg/ml hyaluronidase (22.5 - 45 IU/ml, Sigma), unsupplemented, or supplemented with 0.1 mg/ml fetuin-B, for 2-3 minutes at 37°C.
  • HEF human tubular fluid
  • oocytes were kept in 200 ⁇ HTF with or without fetuin-B, and were fertilized after 0 h, 1 h, 3 h, 5 h, 7 h, 9 h, 12 h, 14 h, 17 h, and 25 h.
  • sperm for fertilization was collected from the cauda epididymis and vas deferens of 14-16 week old males in 90 ⁇ TYH medium and incubated for 60 minutes at 37°C. Fresh sperm was isolated for each time point of fertilization of oocytes. 10 ⁇ diluted sperm were added to 200 ⁇ of HTF with oocytes. Following 4 hours of incubation, the zygotes were washed four times to remove excess sperm and any residual debris. 24 hours later the embryos were visually inspected using a microscope and the percentage of fertilized oocytes (2-cell stage embryos) was determined.
  • human serum (diluted 1 :900 - 1 : 1800 in blocking buffer, regression line with recombinant human fetuin-B (R&D) from 8,0 - 0,125 ng/ml) was added at ⁇ /well and incubated for 2h at RT.
  • detection antibody (R&D) with 2 ⁇ g/ml in blocking buffer, ⁇ /well, was incubated for 2 h at RT.
  • streptavidin/HRP (1 :200 in blocking buffer
  • substrate solution 1% TMB in citrate/acetate buffer + H 2 0 2
  • Stop solution was added and measurement was performed with absorbance at 450nm and 570nm.
  • Figure 2C illustrates one of 14 oocytes recovered from 2 wildtype mice that were all fertilised showing a second polar body and a well-developed perivitelline space. In contrast, no oocyte out of 54 oocytes recovered from 9 female Fetub-/- mice showed signs of fertilisation ( Figure 2C). Thus, an early block to fertilisation most likely caused the infertility in female Fetub-I- mice.
  • zygotes developed into two-cell embryos in vitro, and further into fetuses when transferred to wildtype foster mothers as shown in figure 3G-L.
  • fetuin-B deficient oocytes could only be fertilised after overcoming the ZP barrier by laser perforation, while further fetal development was unaffected.
  • ZPH is associated with reduced susceptibility to proteolytic degradation (Inoue and Wolf, 1974).
  • chymotrypsin-mediated ZP digestion time in unfertilised fetuin- B deficient and wildtype oocytes, as well as two-cell embryos, which represent the physiologically hardened ZP phenotype (for each condition n > 20; 4 independent experiments).
  • Figure 4C shows that tso, the time required to digest the ZP in half of the oocytes in each matching series, was 5 times longer in fetuin-B deficient oocytes (31.5 min ⁇ 4.7 min) than in unfertilised wildtype oocytes (6.4 min ⁇ 0.9 min).
  • the ZP digestion time of fetuin-B deficient oocytes was comparable to that of wildtype two-cell embryos (38.5 min ⁇ 6.0 min), indicating that the ZP structure of fetuin-B deficient oocytes resembled a hardened phenotype.
  • Fetuin-B Inhibits Ovastacin and Thus Zona Pellucida Hardening
  • FIG. 4F shows that activated ovastacin was inhibited to background activity by recombinant fetuin-B (IC 50 76.4 nM ⁇ 3.35 nM), but not by recombinant fetuin-A. This finding suggests that premature ZPH triggered by spontaneous cortical granula release of ovastacin should be entirely prevented by the micromolar concentrations of fetuin-B present in plasma and follicular fluid.
  • the present inventors have shown that the rate of successful IVF of oocytes is enhanced if the oocytes are immediately contacted with fetuin-B after their isolation from the female body. This is shown in Figure 9. It can be seen that the immediate contact with fetuin-B directly after isolation results in a higher rate of fertilized embryos over a long incubation period, as compared to oocytes which have not been contacted with fetuin-B after isolation. Not until about 17 hours incubation, the percentages of fertilization equalized and tended towards zero. The results indicate that the immediate contact of oocytes after their isolation with culture medium comprising fetuin-B maintain the fertilization success at a higher level, compared to medium without added fetuin-B.
  • Fetuin-B serum level is associated with serum estradiol
  • Serum samples from patients undergoing hormone stimulation for IVF were analyzed for estradiol and fetuin-B (sandwich ELISA). The data were pooled from >20 patients with serum samples taken at various time points during hormone treatment.
  • Figure 10 shows an enhancement of estradiol over time in view of FSH treatment.
  • Serum fetuin-B correlates with IVF success
  • the serum fetuin-B level was measured at different time points in serum samples of patients undergoing a hormonal treatment with FSH for stimulation of production of oocytes for use in IFV.
  • the data were collected with regard to IVF success or IVF failure.
  • the plasma fetuin-B level increased during the hormone therapy (p ⁇ 0.0001), as shown in figure 11 A.
  • fetuin-B levels remained constant ( Figure 11 B).
  • Serum fetuin-B was measured in a woman on contraceptive treatment using a combined gestagen / ethinyl estradiol (EE2) formulation. From day 5 of hormonal contraception, serum fetuin-B increased up to twice, as can be seen in figure 12 A. In a woman on a different contraceptive treatment regime, medication was changed from gestagen alone (day -6 to 0) to gestagen plus EE2, showing that EE2, but not gestagen increased serum fetuin-B (figure 12 B). Collectively, these results show that fetuin-B is produced constitutively, and that normal fluctuations in serum estrogen do not affect serum fetuin-B.
  • EE2 gestagen / ethinyl estradiol
  • figures 10 to 12 show that an enhancement of serum fetuin-B levels over basal levels of a female individual induced by treatment with a substance which is capable of enhancing serum fetuin-B level in a female individual results in IVF success with a high fertilization rate.
  • Infertility affects about 15% of couples who wish to have children worldwide. A great number of hormones and growth factors have been shown to affect female fertility. Crosstalk between somatic cells and oocytes, as well as endocrine signaling are necessary for normal folliculogenesis and ovulation. For example, in the absence of the oocyte secreted growth and differentiation factor-9 (GDF-9) an arrest at the primary follicle stage is observed. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are synthetized by the anterior pituitary, regulate follicle growth during the preovulatory stage and initiate COC ovulation, respectively.
  • FSH Follicle stimulating hormone
  • LH luteinizing hormone
  • Granulosa cell-derived growth factors such as activins, inhibins, and stem cell factor (SCF/Kit-ligand) are also necessary for follicle development.
  • SCF/Kit-ligand stem cell factor
  • fetuin-B inhibits premature zona pellucida hardening (ZPH).
  • ZPH zona pellucida hardening
  • the ZP of Fetub-/- mice was more resistant to enzyme digestion than wildtype oocytes, comparable to the physiologically hardened ZP of two-cell embryos, and the sperm oocyte binding was blocked.
  • ZP2f cleaved form of ZP2
  • ZP2f cleaved form of ZP2
  • fetuin-B To elucidate the molecular mechanism of ZPH inhibition by fetuin-B, we turned to proteases, because fetuins belong to the cystatin superfamily, a group of thiol protease inhibitors (Lee et al., 2009). Fetuin-A is known to interact with several proteases, including trypsin and the astacin-type metalloprotease meprin. Because fetuin-B shares with fetuin-A cystatin-like proteinase-inhibition domains we hypothesized that fetuin-B might inhibit proteases involved in oocyte maturation and fertilisation.
  • fetuin-B of 55 kDa can freely diffuse through the ZP, which is permeable for molecules up to 170 kDa (Legge, 1995).
  • fetuin-B can readily antagonize the proteolytic action of prematurely released ovastacin as shown in figure 5.
  • the amount of ovastacin will overwhelm the inhibition capacity of fetuin-B, which is in steady state with plasma fetuin-B, but does not increase upon fertilisation.
  • fetuin-B to fertilisation media for IVF procedures safeguards against ZPH and against unsuccessful fertilisation even in oocytes deprived of fetuin-B.
  • addition of fetuin-B to immature oocytes prevents premature degranulation of cortical granule and premature hardening of the ZP.
  • the Fetub gene is well conserved in mammals. Because spontaneous ZPH is also reported in humans (Schiewe et al, 1995), alterations in the FETUB gene could also lead to human female infertility.
  • fetuin-B represents a novel target for fertilisation biology.
  • a network of protease (ovastacin), protease inhibitor (fetuin-B) and hardening substrates (ZP proteins) is oddly pronounced of blood coagulation, an equally important and medically relevant biological cascade, which is just celebrating the introduction of proteinase inhibitors as so- called 2 nd generation blood clotting inhibitors.
  • ZP proteins hardening substrates
  • a female individual Treatment of a female individual with a substance which is capable of enhancing serum fetuin-B level in a female individual such as estrogens or a hormone such as FSH for stimulation of oocyte production in the female individual for use in IVF whereby the hormone attains an increase of endogenous estrogen levels results in enhanced serum fetuin-B.
  • Enhanced serum fetuin-B levels effect the production or maturation of oocytes with a high rate of fertility, resulting in an enhanced fertilization of the oocytes in a subsequent IVF.

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Abstract

The present invention relates to the field of culture of ovary tissue comprising an oocyte and in particular to a method of stabilisation of the ovary tissue comprising contacting the ovary tissue with a medium comprising fetuin-B immediately after its isolation from a female body. By this contacting, fetuin-B maintains the fertilisable state of the oocyte by preventing the premature hardening of the zona pellucida of the oocyte by inhibiting the activity of ovastacin to cleave ZP2 to ZP2f. The present invention further relates to the culture of ovary tissue comprising an oocyte whereby the ovary tissue has been isolated from a female individual, wherein the serum fetuin-B level is enhanced in the female individual.

Description

Use of fetuin-B for culture of oocytes
The present invention relates to the field of culture of an ovary tissue comprising an oocyte and in particular to a method of culture of the ovary tissue comprising adding the ovary tissue to a medium comprising fetuin-B immediately after its isolation from a female body. Infertility affects about 15% of couples of reproductive age worldwide and thus has a major public health impact. The causes of infertility for most patients vary widely and the basic mechanisms regulating fertility are still poorly understood. About 25% of clinical infertility cases are idiopathic. Assisted reproductive technologies like in vitro fertilisation (IVF) are common practice to achieve pregnancy when natural fertilisation fails. However, up to 15%) of IVF also fails, mostly because of defective sperm-oocyte interaction.
Human as well as other mammalian oocytes are surrounded by a glycoprotein matrix called the zona pellucida (ZP). Genetic studies in mice revealed that a functional ZP is critical for follicular development and fertilisation, and for the protection of the preimplantation embryo. Prior to gamete fusion, sperm are able to bind and penetrate the ZP. Fertilisation, however, induces rapid changes in the ZP, which transforms into a physical barrier that prevents further sperm from binding. This process is called ZP hardening (ZPH), conferring resistance to proteolytic digestion and mechanical stiffening. ZPH is caused by proteolytic processing of ZP glycoproteins, especially the cleavage of ZP2 (120 kDa) to ZP2f (90 kDa) (Bleil et al, 1981). Cleavage is performed by proteases, which are released by the fertilised oocyte during the cortical reaction (Szollosi, 1967; Barros and Yanagimachi, 1971). Recently, the metalloprotease ovastacin was shown to be critical for ZP2 cleavage and definitive ZP hardening (Burkart et al, 2012). Low level cortical granule release occurs already before fertilisation during meiotic maturation and ovulation (Rousseau et al, 1977; Ducibella et al, 1988). In mice, germinal vesicle intact oocytes have about 5400 cortical granules, of which about 1300 are released during germinal vesicle breakdown (Ducibella et al, 1988). Partial degranulation, however, does not normally trigger ZPH. Physiological ZPH starts about 30 hours after ovulation, limiting the time window for oocyte fertilisation (Dodson et al., 1989). In vitro, ZPH occurs much faster, resulting in a decreased fertilisation success (Ducibella et al, 1990; Schiewe et al, 1995). ZPH can be prevented by adding serum, especially fetal calf serum (FCS), to IVF medium (Downs et al, 1986), suggesting that both follicular fluid and serum may contain factors that inhibit premature ZPH.
The fetuin protein family comprises two members: fetuin-A and fetuin-B (Olivier et al, 1999, 2000). Like fetuin-A, fetuin-B is a liver-derived plasma protein (Denecke et al, 2003) with serum concentrations of -0.01 g/L and -0.3 g/L in human and mouse, respectively. Fetuins belong to the cystatin superfamily, comprising structurally related protease inhibitors. Several studies suggest that fetuin-A (originally designated as fetuin) prevented ZPH in vitro. However, fetuin-A deficient mice are fully fertile, arguing against a role for fetuin-A in fertilisation. The role of fetuin-B has not been elucidated so far.
Maintaining fertility of an oocyte after isolation from a female body and during preservation of said oocyte in the fertilisable stage for future processing and use such as in vitro maturation and fertilisation or re-transplantation would be an approach to remedy fertility problems in women who cannot generally become pregnant naturally or who must not become pregnant at a specific time due to medical reasons e.g. cancer treatment. However, so far maintaining the fertilisable state of an isolated oocyte has been difficult. The present inventors have found, by generating fetuin-B deficient mice, that fetuin-B plays an important role in the maturation of oocytes. In particular, the present inventors have found that fetuin-B prevents premature hardening of the zona pellucida of oocytes after their isolation from a female body. Consequently, the above problem is solved by the provision of the claims.
In a first aspect, the present invention provides a method for culturing an ovary tissue comprising an oocyte, comprising contacting the ovary tissue with a medium comprising fetuin-B immediately after isolation of the ovary tissue from a female body.In a second aspect, the present invention provides the above method for stabilising the oocyte.
In a third aspect, the present invention provides a use of fetuin-B for stabilising an ovary tissue comprising an oocyte.
In a fourth aspect, the present invention provides the above method or use for maintaining the fertilisable state of the ovary tissue.
In a fifth aspect, the present invention provides the above method or use for preventing hardening of the zona pellucida of the oocyte.
In a sixth aspect, the present invention provides the above method or use for inhibiting the activity of ovastacin.
In a seventh aspect, the present invention provides the above method or use, wherein the oocyte is an immature oocyte. In an eighth aspect, the present invention provides the above method or use, wherein the ovary tissue is an oocyte, a follicle, a cortex of an ovary or an ovary.
In a ninth aspect, the present invention provides the above method or use, wherein fetuin-B is recombinant fetuin-B.
In a tenth aspect, the present invention provides the above method or use, wherein fetuin-B is human or mouse fetuin-B.
In further aspects of the present invention, the present invention provides the above methods and uses, wherein the ovary tissue comprising an oocyte has been isolated from a female individual, wherein the serum fetuin-B level in the female individual is enhanced due to the treatment of the female individual with a substance which is capable of enhancing the fetuin-B level in the female individual. In a preferred embodiment, the substance is a hormone which is used for stimulation of maturation of oocytes for use in in vitro fertilization, an estrogen or an estrogen agonist. For example, the hormone used for stimulation may be FSH (follicle stimulating hormone) or a derivative thereof, or may be hMG (human menopausal gonadotropin) or a derivative thereof. The estrogen or estrogen agonist may be estrone, estradiol, estriol, estetrol (E4), ethinylestradiol, mestranol, 11β- methyl-ethinylestradiol, turisteron, moxestrol (1 Ιβ-methoxy-ethinylestradiol), 6- dehydroestrone, 17-deoxyestradiol, 2-hydroxyestradiol, isoestradiol (8a-estradiol), 2- methylestradiol, 4-methylestradiol, polyestradiol-phosphate, promestriene, 2- chloroestradiol, 1,1 Ιβ-ehanoestradiol, diethylstilbestrol, dienestrol, dimestrol, chlorotrianisene, stilbestrol-monobenzyl-ether, fosfestrol or homoestradiol. The estrogen agonist may be a derivative of estrogen having the hormonal function of estrogen or may be any substance that increases the activity of an estrogen. The function of the estrogen may be the enhancement of serum fetuin-B in a female individual.
In further aspects of the present invention, the present invention provides a substance which is capable of enhancing the serum fetuin-B level in a female individual for use in a method for enhancing fertility of an oocyte, the method comprising,
(a) administering to a female individual a substance which is capable of enhancing the serum fetuin-B level in a female individual;
(b) isolating ovary tissue comprising an oocyte; and
(c) contacting the ovary tissue with a medium comprising fetuin-B immediately after isolation of the ovary tissue from the female body.
The substance is defined, as above. The present invention furthermore refers to isolated ovary tissue cultivated in a medium comprising fetuin-B.
So far, no function has been assigned to fetuin-B. In order to elucidate the role of fetuin-B, the present inventors generated fetuin-B deficient mice to study its physiological role. It could be shown that fetuin-B affects oocyte development. In particular, it could be shown that fetuin-B affects the hardening of the ZP. The inventors could show that the ZP of ovulated oocytes of fetuin-B deficient mice was hardened before fertilisation. Moreover, the inventors could show that the cortical granule protease ovastacin is inhibited by fetuin- B.
Within the female body, fetuin-B, which is produced by the liver, is present in the follicular fluid and prevents hardening of the ZP of oocytes in the immature or mature state. Low level degranulation of the cortical granules of the oocyte and low level cortical granule release of ovastacin occurs during the maturation of an oocyte within the body. Ovastacin, which effects hardening of the ZP of the oocyte by cleavage of ZP2 to ZP2f, is inhibited by fetuin-B present in the follicular fluid so that hardening of the ZP of the oocyte is prevented. If the oocyte is removed from the female body and in the absence of fetuin-B, the immediate release of ovastacin is triggered resulting in ZP hardening. Oocytes with a hardened ZP cannot proceed with maturation and cannot be fertilised. In order to prevent hardening and to maintain the fertilisable state of the oocyte, treatment of the oocyte with fetuin-B is necessary.
A medium comprising fetuin-B corresponds to the medium surrounding the oocytes in their natural environment. Addition of fetuin-B to isolated oocytes inhibits ovastacin and thus prevents premature ZPH of oocytes. Thus, isolated oocytes in a medium comprising fetuin-B are maintained or preserved in a fertilisable state.
As used in the present invention, the term "culturing an ovary tissue comprising an oocyte" means that the ovary tissue is held or stored in a medium comprising fetuin-B. In an aspect of the invention, the term "culturing" means "stabilising".
As used in the present invention, the term "stabilising an ovary tissue comprising an oocyte" or "stabilising an oocyte" or similar terms refer to the ability of fetuin-B to maintain or preserve the fertilisable state of an oocyte after isolation. In an aspect of the present invention and particularly preferred, "culturing" or "stabilising" means that hardening of ZP of the oocyte is prevented. Most preferred, this may be effected by the inhibition by fetuin-B of the activity of ovastacin, which is released by premature degranulation of the cortical granules of the oocyte. Inhibition of the activity of ovastacin means inhibition of the proteolytic activity processing ZP glycoproteins, which are present in the zona pellucida of oocytes, especially inhibition of cleavage of ZP2 to ZP2f. ZP2 cleavage into ZP2f is a hallmark of hardening of ZP. The term "stabilising" may also include that the ovary tissue comprising the oocyte survives and remains viable.
In the context of the present invention, the term "maintain the fertilisable state of an oocyte" or similar terms mean that the oocyte does not loose the ability or vice versa maintains the ability to be fertilised, e.g. under suitable in vitro conditions or after transplantation in vivo. An immature oocyte maintains the fertilisable state if that oocyte was fertilisable at the time of isolation. Thus, if an immature oocyte has been isolated from a female body and has been kept in a medium comprising fetuin-B and has then been matured, the oocyte will be able to become fertilised by a sperm cell. A mature oocyte maintains its mature developmental state and can be fertilised by a sperm cell.
Moreover, the term "stabilising" may include that the oocyte in the medium comprising fetuin-B does not loose its ability for further development or vice versa maintains its ability to proceed further with development, if transferred into the respective conditions in vitro or after transplantation into a female body. For example, the oocyte does not loose its ability to proceed further with maturation. For example, an immature oocyte, which is isolated from a female body and is treated with fetuin-B, may proceed with its development, e.g. maturation, within the medium comprising fetuin-B or may proceed with its development, if it is removed from the medium comprising fetuin-B and brought under appropriate conditions. A mature oocyte maintains the mature developmental state and remains fertilisable, i.e. it can be fertilised. However, hardening of ZP is prevented. Preferably, ovastacin, which is released by degranulation of the cortical granules of the immature or mature oocyte, is inhibited, so that ZP2 cannot be cleaved into ZP2f.
Stabilisation of the ovary tissue is achieved by applying fetuin-B to the isolated ovary tissue during or at the latest immediately after isolation from a female body. If the ovary tissue is removed from the female body in the absence of fetuin-B, the release of ovastacin is triggered resulting in the immediate beginning of ZP hardening. In order to prevent hardening and to maintain the fertilisable state of the oocyte, the immediate treatment of the oocyte with fetuin-B is necessary. In the context of the present invention, the term "immediate" or "immediately" in the context "immediately after isolation of the ovary tissue from the female body" or similar terms mean that the tissue is isolated from a female body and the tissue and fetuin-B are immediately brought into contact with each other, for example, the tissue is immediately added to a medium comprising fetuin-B. By the term "immediate" or "immediately" in the context of the present invention, it is understood that the tissue is taken from the female body and fetuin-B is applied within the next few seconds or minutes, such as 0, 5, 10, 20, 30 or 60 seconds to 10 minutes, preferably, 0, 5, 10, 20, 30 or 60 seconds to 5 minutes, more preferably 0, 5, 10, 20, 30 or 60 seconds to 2 minutes, whereby the limiting factor is the time which is needed for handling the isolated ovary tissue and contacting it with a medium comprising fetuin-B. Preferably the medium comprising fetuin-B is already used for flushing and/or washing the ovary tissue during the process of isolating the ovary tissue from the female's body. Immediately after isolation, the ovary tissue is contacted with a medium comprising fetuin-B. In this medium, the tissue may be processed, if this is deemed necessary. The term "processing" refers to procedures where handling or manipulating the ovary tissue is necessary, such as examination of the ovary tissue to determine the developmental state of the oocyte, isolation of the oocyte from the ovary tissue, removal of surrounding cells such as cumulus cells etc. After culture, the ovary tissue may be further used. The term "further use" refers to procedures such as maturation, fertilisation or transplantation into the female body. The polynucleotide and amino acid sequences of human fetuin-B have been published under the following GenBank accession number as available from the NCBI (National Centre for Biotechnology Information; National Library of Medicine 38A, Bethesda, MD20894; USA; www.ncbi.nih.gov). The sequences are indicated in the following.
The polynucleotide sequence of human fetuin-B is available under the GenBank Accession No. NM 014375.2.
GTTGTAACAA AACCGCTCAA GTCTGCCTTA AAGAGCCTTA CAAGCCAGCC AGTCCCTGCA
GCTCCACAAA CTGACCCATC CTGGGCCTTG TTCTCCACAG AATGGGTCTG CTCCTTCCCC
TGGCACTCTG CATCCTAGTC CTGTGCTGCG GAGCAATGTC TCCACCCCAG CTGGCCCTCA
ACCCCTCGGC TCTGCTCTCC CGGGGCTGCA ATGACTCAGA TGTGCTGGCA GTTGCAGGCT
TTGCCCTGCG GGATATTAAC AAAGACAGAA AGGATGGCTA TGTGCTGAGA CTCAACCGAG
TGAACGACGC CCAGGAATAC AGACGGGGTG GCCTGGGATC TCTGTTCTAT CTTACACTGG
ATGTGCTAGA GACTGACTGC CATGTGCTCA GAAAGAAGGC ATGGCAAGAC TGTGGAATGA
GGATATTTTT TGAATCAGTT TATGGTCAAT GCAAAGCAAT ATTTTATATG AACAACCCAA
GTAGAGTTCT CTATTTAGCT GCTTATAACT GTACTCTTCG CCCAGTTTCA AAAAAAAAGA
TTTACATGAC GTGCCCTGAC TGCCCAAGCT CCATACCCAC TGACTCTTCC AATCACCAAG
TGCTGGAGGC TGCCACCGAG TCTCTTGCGA AATACAACAA TGAGAACACA TCCAAGCAGT
ATTCTCTCTT CAAAGTCACC AGGGCTTCTA GCCAGTGGGT GGTCGGCCCT TCTTACTTTG
TGGAATACTT AATTAAAGAA TCACCATGTA CTAAATCCCA GGCCAGCAGC TGTTCACTTC
AGTCCTCCGA CTCTGTGCCT GTTGGTCTTT GCAAAGGTTC TCTGACTCGA ACACACTGGG AAAAGTTTGT CTCTGTGACT TGTGACTTCT TTGAATCACA GGCTCCAGCC ACTGGAAGTG
AAAACTCTGC TGTTAACCAG AAACCTACAA ACCTTCCCAA GGTGGAAGAA TCCCAGCAGA
AAAACACCCC CCCAACAGAC TCCCCCTCCA AAGCTGGGCC AAGAGGATCT GTCCAATATC
TTCCTGACTT GG AT GAT AAA AATTCCCAGG AAAAGGGCCC TCAGGAGGCC TTTCCTGTGC
ATCTGGACCT AACCACGAAT CCCCAGGGAG AAACCCTGGA TATTTCCTTC CTCTTCCTGG
AGCCTATGGA GGAGAAGCTG GTGGTCCTGC CTTTCCCCAA A G A A A A A G C A CGCACTGCTG
AGTGCCCAGG GCCAGCCCAG AATGCCAGCC CTCTTGTCCT TCCGCCATGA GAATCACACA
GAGTCTTCTG TAGGGGTATG GTGCGCCGCA TGACATGGGA GGCGATGGGG ACGATGGACA
GAGACAGAGC GTGCACACGT AGAGTGGCTA GTGAAGGACG CCTTTTTGAC TCTTCTTGGT
CTCAGCATGT TGACTGGGAT TGGAAATAAT GAGACTGAGC CCTCGGCTTG GGCTGCACTC
TACCCTGTAC ACTGCCTTGT ACCCTGAGCT GCATCACCTC CTAAACTGAG CAGTCTCATA
CCATGGAGAG ATGCCTCTCT TATGTCTTCA GCCACTCACT TATAAAGATA CTTATCTTTT
CAGCAGTATA TATGTGCTGA AATCTCAGCA TGAAAGCATT GCATGAGTAA AGATACTTTC
CCTAACAAAA AAAAAAAAAA
(SEQ ID NO: 1)
The amino acid sequence of human fetuin-B is available under the GenBank Accession
No.NP_055190.2.
MGLLLPLALCILVLCCGAMSPPQLALNPSALLSRGCNDSDVLAVAGFALRDINKDRKDGYV LRLNRVNDAQEYRRGGLGSLFYLTLDVLETDCHVLRKKAWQDCGMRIFFESVYGQCKAIFY MNNPSRVLYLAAYNCTLRPVSKKKI YMTCPDCPSS I P DSSNHQVLEAATESLAKYNNENT SKQYSLFKVTRASSQWVVGPSYFVEYLIKESPCTKSQASSCSLQSSDSVPVGLCKGSLTRT HWEKFVSVTCDFFESQAPATGSENSAVNQKPTNLPKVEESQQKNTPPTDSPSKAGPRGSVQ YLPDLDDKNSQEKGPQEAFPVHLDL NPQGE LDI SFLFLEPMEEKLVVLPFPKEKAR A ECPGPAQNASPLVLPP
(SEQ ID NO: 2)
The polynucleotide and amino acid sequences of mouse fetuin-B have been published under the following GenBank accession number as available from the NCBI (National Centre for Biotechnology Information; National Library of Medicine 38A, Bethesda, MD20894; USA; www.ncbi.nih.gov). The sequences are indicated in the following.
The polynucleotide sequence of mouse fetuin-B (fetuin-B isoform 1 precursor form) is available under the GenBank Accession No. NM_021564.2.
TTAATCTCTG TAAAGGCGCC ACCTCTCCAC AGGTTTCATA AGCGTTCGTT TTCTGAGCTT TGGGAACGTG TTCAAAGCAC ACCAGACTTG CAAAGCTCTT GATTCCGGAC AGCTTACCTT GATTCCTGAC ATTGTGCAGT GGCTCAGCCA GAACCTGTGA GTCACAGGTA GCTGCTGTGT TACACAACAT CTAACAACGT CTAGCCTTCT GCGATTTCTG GTGCAGGGTC ATGATTAGTG CATTTGACAG ATTTGGCAGA GTGGCTTACA GTGGAATGGG CCTGCTCCGA CTTCTGGTGC TCTGCACCCT GGCTGCATGC TGCATGGCCC GCTCTCCCCC AGCCCCACCC CTGCCACAAC
GCCCCCTCTC ACCTCTGCAT CCTCTGGGCT GTAACGACTC TGAAGTGCTG GCAGTTGCAG
GATTTGCCCT GCAGAACATC AACAGAGACC AAAAGGATGG CTATATGTTG AGCCTTAACA
GAGTGCATGA TGTTCGGGAG CACTACCAGG AAGACATGGG ATCTCTGTTC TACCTCACAT
TGGATGTCTT AGAGACTGAC TGCCACGTGC TCAGCAGGAA GGCACAGAAG GACTGCAAAC
CGAGGATGTT CTATGAGTCG GTTTATGGCC AGTGCAAAGC AATGTTTCAC ATTAACAAGC
CAAGAAGAGT TCTCTACTTA CCTGCTTATA ACTGTACACT TCGCCCAGTT TCCAAAAGAA
AGACTCATAC AACGTGCCCT GACTGCCCTA GCCCCATTGA CTTGTCAAAC CCCAGTGCTC
TGGAAGCTGC CACGGAGTCG CTTGCAAAGT TCAACAGTAA GAGCCCCTCA AAAAAGTATG
AACTCGTCAA AGTCACCAAG GCTATGAACC AGTGGGTGTC TGGCCCTGCT TACTATGTGG
AATATTTGAT CAAAGAGGCA CCATGTACCA AATCCCAGGC CAGCTGTTCG CTCCAGCACT
CTGACTCTGA GCCCGTTGGT ATTTGCCAAG GTTCTACGGT CCAAAGTTCT CTGAGGCACG
TTCCTCTGAT CCAACCCGTA GAAAAGTCTG TCACTGTGAC TTGTGAGTTC TTCGAATCTC
AGGCTCAGGT CCCTGGAGAT GAGAACCCTG CTGTTACCCA GGGCCCTCAG AAACTCCCTC
AGAAAAACAC GGCCCCTACC AGCTCCCCCT CCGTAACTGC ACCAAGAGGA TCCATCCAGC
ACCTCCCCGA ACTGGATGAT GAGAAGCCCG AGGAGTCCAA GGGAGGGAGC CCTGAGGAAG
CCTTTCCTGT GCAGCTGGAT CTAACCACCA ATCCCCAGGG GGACACACTA GATGTCTCCT
TCCTGTACCT GGAGCCTGGG GACAAGAAGC TGGTGGTGCT GCCTTTCCCT GGAAAGGAAC
AGCGCTCCGC CGAGTGCCCA GGGCCCGAGA AGGAGAACAA CCCTCTGGTT CTCCCACCCT
GAGACTCCCT AGCAGGGTTT CATAGGGCTA TGGTCCCCAG CACTAAATGG GAGGTGGTGG
GGATTGGGAA GGACACAGAC AATGAAATGT AGACAGGCTA ATAAAGTGTG TCCTTTTGAT
GCTTCTTGGC TTCAAAAAAA AAAAAAAAAA AAAAAAAAAA
(SEQ ID NO: 3)
The amino acid sequence of mouse fetuin-B is available under the GenBank Accession No. NP 067539.1
MGLLRLLVLCTLAACCMARSPPAPPLPQRPLSPLHPLGCNDSEVLAVAGFALQNINRDQKD GYMLSLNRVHDVREHYQEDMGSLFYLTLDVLETDCHVLSRKAQKDCKPRMFYESVYGQCKA MFHINKPRRVLYLPAYNCTLRPVSKRKTHTTCPDCPSPIDLSNPSALEAATESLAKFNSKS PSKKYELVKVTKAMNQWVSGPAYYVEYLIKEAPCTKSQASCSLQHSDSEPVGICQGSTVQS SLRHVPLIQPVEKSVTVTCEFFESQAQVPGDENPAVTQGPQKLPQKNTAPTSSPSVTAPRG SIQHLPELDDEKPEESKGGSPEEAFPVQLDLTTNPQGDTLDVSFLYLEPGDKKLVVLPFPG KEQRSAECPGPEKENNPLVLPP
(SEQ ID NO: 4)
Fetuin-B as used in the present invention may be provided by any methods known in the art. Fetuin-B is a liver-derived plasma protein. Thus, it can be isolated from plasma. However, more convenient is the provision of recombinant fetuin-B, which is obtained from a cell transformed with a polynucleotide encoding fetuin-B.
The polynucleotide encoding fetuin-B, also designated fetuin-B polynucleotide, may be any polynucleotide encoding a fetuin-B polypeptide. The polynucleotide encoding fetuin-B may be from human, mouse, cow, horse, dog, cat, pig, goat, sheep, camel, or zoo animals, such as panda bear, large cat etc. Preferably, the fetuin-B polynucleotide is from human, mouse, cow, horse or camel and more preferably from human. In the context of the present invention, a polynucleotide encoding a fetuin-B polypeptide is a polynucleotide comprising or consisting of SEQ ID NO: 1 or 3 coding for fetuin-B having the amino acid sequence of SEQ ID NO: 2 or 4, respectively. The term "polynucleotide encoding fetuin- B" or "fetuin-B polynucleotide" or "polynucleotide" as used herein encompasses polynucleotides which are defined by SEQ ID NO: 1 or 3 as well as naturally occurring or non-naturally occurring variants or fragments thereof (as defined herein).
The most common (and therefore preferred) polynucleotides are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The polynucleotide can be a DNA molecule such as a genomic DNA molecule or a cDNA molecule, which can be single- or double-stranded as well as a synthetic DNA such as a synthesized single-stranded polynucleotide. The polynucleotide of the present invention can also be an RNA molecule. Preferably, the term also relates to non-coding regions of a gene, wherein these sections are of a relevant size in order to be specific for that gene. Examples of those regions are regulatory elements such as a promoter. Most preferably, the term "polynucleotide" relates to gene, open reading frame (ORF), promoter, DNA, cDNA or mRNA. The polynucleotide encoding the desired genetic information, preferably DNA, may comprise the gene of interest, a promoter region, a start codon and a stop codon and possibly further regions which may be used for regulation of expression of the gene.
The polynucleotide, as referred to above, may encode functionally active variants of the polypeptide of SEQ ID NO: 2 or 4. Such functionally active variants have a sequence identity with SEQ ID NOs: 2 or 4 of more than 50%, of more than 60%, preferably more than 70%, more preferably of more than 80%, still more preferably more than 85%, even more preferably more than 90%, even more preferably more than 95%, most preferably more than 97% and/or have an activity of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97% or more than 100 % , e.g. more than 150 %, 200 %, 300 % 400 % or 500 % of the activity of the polypeptide of SEQ ID NO: 2 or 4. Consequently, the polynucleotides encoding such variants may contain deletions, insertions, substitutions and/or additions within and/or at the 5" and/or 3" termini of SEQ ID NO: 1 or 3 and show an identity to the polynucleotide of SEQ ID NO: 1 or 3 of more than 50%, more than 60%, more than 70%, preferably more than 80%, more preferably more than 85%, even more preferably more than 90%, even more preferably more than 95%, most preferably more than 97%. In the context of the present invention, a variant polynucleotide encoding a functionally active variant of the polypeptide of SEQ ID NO: 2 or 4 means a sequence encoding a variant polypeptide which has the same activity as the polypeptide of SEQ ID NO: 2 or 4.
The polynucleotide fragments, as referred to above, may encode functionally active fragments of the polypeptide of SEQ ID NO: 2 or 4, respectively. This may include fragmental polypeptides with short internal and/or C- and/or N-terminal deletions whereby the activity of the resulting polypeptides as identified herein is maintained to an extent of more than 50 %, more than 60 %, more than 70 %, more than 80 %, more than 90 %, more than 95 %, more than 97 % or more than 100 %, e.g. more than 150 %, 200 %, 300 % 400 %> or 500 %>, of the activity of the wild-type polypeptide as encoded by SEQ ID NO: 1 or 3. Consequently, the respective polynucleotide encoding such fragments contains deletions within and/or at the 5" and/or 3" termini resulting in the polypeptide of SEQ ID NO: 2 or 4 with, e. g., deletions of at the most 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10 %, 5%, or less. In the context of the present invention, a fragmental polynucleotide encoding a functionally active fragment of the polypeptide of SEQ ID NOs: 2 or 4 means a sequence encoding a fragment which has the same activity as the polypeptide of SEQ ID NO: 2 or 4. It is noted that the above mentioned modifications may be combined. For example, a polynucleotide as comprised by the present invention may be a fragment comprising one or more variations. It should also be noted that fragments or variants include fragments or variants, as defined herein, of promoter or regulatory sequences with which the polynucleotide of SEQ ID NO: 1 or 3 or fragments or variants thereof are associated in nature. These variants are functionally active in that they regulate the transcription or translation of the genes associated therewith.
In a further embodiment, the fetuin-B polynucleotide encodes a fetuin-B polypeptide of SEQ ID NO: 2 or 4. Due to the degeneracy of the genetic code, the polynucleotide sequence of SEQ ID NO: 1 or 3 may be modified by replacing nucleotides in such a way that the same amino acids are encoded.
In a further embodiment, the fetuin-B polynucleotide hybridizes under stringent conditions, preferably highly stringent conditions, to the homologous polynucleotide of SEQ ID NO: 1 or 3 or a functionally active variant or fragment thereof. Such conditions include conditions under which a complementary strand of a highly identical nucleic acid, namely a DNA composed of a nucleotide sequence having 70 % or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more and even more preferably 95% or more identity with the polynucleotide of SEQ ID NO: 1 or 3, hybridizes, while a less complementary strand of a nucleic acid less identical than the above does not hybridize. Examples of stringency hybridization conditions can be found e.g. in Ausubel et al, (1989). In a particular example, a filter, e.g. a nitrocellulose filter, is incubated overnight at 68°C with a probe in a hybridization solution e.g. containing 50% formamide, high salt (either 5x SSC [20x: 3M NaCl/0.3M trisodium citrate] or 5x SSPE [20x: 3.6M NaCl/0.2M NaH2PO4/0.02M EDTA, pH 7.7]), 5x Denhardt's solution, 1% SDS, and 100 μ^ιηΐ denatured salmon sperm DNA. This is followed by several washes with buffer, e.g. in 0.2x SSC/0.1%) SDS at a temperature selected based on the desired stringency and the melting temperature (Tm) of the DNA hybrid. For example, 68°C are appropriate for high stringency hybridization. It should be noted that the hybridizing sequence is the complementary non-coding strand of a putative hybridizing fetuin-B polynucleotide which hybridizes to the coding strand of SEQ ID NO: 1 or 3.
In a further embodiment, the polynucleotide is complementary to a polynucleotide of SEQ ID NO: 1 or 3 or a polynucleotide encoding a polypeptide of SEQ ID NO: 2 or 4 or functionally active fragments or variants thereof.
The polynucleotide of the present invention may be provided by any methods known in the art. Using the sequence information provided herein, primers suitable for amplification/isolation of the polynucleotide of SEQ ID NO: 1 or 3 can be determined according to standard methods well known to those of skill in the art. Primers suitable for amplification/isolation of the polynucleotide of SEQ ID NO: 1 or 3 are designed according to the nucleotide sequence information provided in the sequence listing. The procedure is as follows: a primer is selected which may consist of 10 to 40, preferably 15 to 25 nucleotides. It is advantageous to select primers containing C and G nucleotides in a proportion sufficient to ensure efficient hybridization; i.e., an amount of C and G nucleotides of at least 40%, preferably 50% of the total nucleotide content. A standard PCR reaction will be performed which typically contains 0.5 to 5 Units of Taq DNA polymerase per 100 μΐ, 20 to 200 μΜ deoxynucleotide each, preferably at equivalent concentrations, 0.5 to 2.5 mM magnesium over the total deoxynucleotide concentration, 105 to 106 target molecules, and about 20 pmol of each primer. About 25 to 50 PCR cycles are performed. A more stringent annealing temperature improves discrimination against incorrectly annealed primers and reduces incorporation of incorrect nucleotides at the 3' end of primers. A denaturation temperature of 95°C to 97°C is typical, although higher temperatures may be appropriate for denaturation of G+C-rich targets. The number of cycles performed depends on the starting concentration of target molecules, though typically more than 40 cycles are not recommended as non-specific background products tend to accumulate. An alternative method for retrieving polynucleotides encoding variant polypeptides as defined herein is by hybridization screening of a DNA or RNA library using the primers and probes as defined herein. Hybridization procedures are well-known and are described in the art and herein.
Alternatively or additionally to the above, the polynucleotide may be provided by cloning and thereby introducing it into and amplifying it in a cell. The procedure of introducing a gene into a recipient cell is called transformation. The genes can be introduced into the cells by a variety of means known in the art and adapted to each cell type. The term "cell" refers to the cell in which the gene is expressed irrespective of whether it is a prokaryotic cell or a eukaryotic cell and of whether the cell naturally expresses the respective genes or not. Recombinant DNA cloning techniques well known in the art for introducing and expressing a nucleic acid molecule can be used to introduce and express the gene which is either endogenous if the cell harbors the respective gene or is heterologous if the gene is not endogenous to the cell. Cells can be transformed using any appropriate means, including viral or bacteriophage based vectors, chemical agents, electroporation, calcium phosphate co -precipitation or direct diffusion of DNA.
The regulatory regions for regulating expression of the fetuin-B polynucleotide in a vector are promoters and possibly enhancers or other regulatory sequences. They may be heterologous to the respective gene or may be associated therewith in nature. The genetic information may be expressed permanently or under the control of a repressor and/or a promoter region in a cell into which the nucleic acid of the present invention is introduced. The obtained cells may be either used directly or used for tissue cultures or the cells may be harvested and samples comprising the respective polypeptide are obtained by disrupting the cells. Alternatively, DNA or RNA may be used either in cells or in cell-free expression systems such as, e.g., microarray systems in which the DNA or RNA is immobilized and is translated and/or transcribed by the addition of functional cell lysate, comprising the factors required for transcription and/or translation (enzymes, ribosomes, tRNA, amino acids, nucleotides, ATP etc.). Also included are artificial nucleic acids which include peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule.
The term "comprising" as used herein is meant to "include or encompass" the desired feature and further features which must not be specifically mentioned. The term "comprising" also means "consisting of the desired feature as the sole functionally active component and does not include further features except the desired feature. For example, "a medium comprising fetuin-B" may be a medium that contains fetuin-B and additional functionally active components or a medium that only comprises fetuin-B as functionally active component. This does not affect the presence of medium ingredients such as nutrients as referred to in the chapters directed to "medium".
The term "heterologous", as it relates to polynucleotide sequences or polypeptides denotes sequences that are normally not associated with a region of a recombinant construct and/or a particular cell. A "heterologous" region is an identifiable segment of a polynucleotide within or attached to another polynucleotide that is not found in association with the other molecule in nature. For example, a heterologous region of a construct could be a regulatory region not found to be associated with a gene as identified herein in nature. Similarly, a heterologous sequence could be a coding sequence which is itself not found in nature as it contains e.g. synthetic sequences with codons different from the native gene. Moreover, a cell transformed with a construct, which is not normally present in the cell would be considered heterologous for the purposes of the present invention.
A homologous polynucleotide sequence is a variant sequence as defined herein. The term "homologous" may be used interchangeably with variant. The term "homologous" may also refer to a polynucleotide or polypeptide that is from another species and that has the same physiological function in said other species. The term "homologous" may also refer to an identical sequence.
Vectors are agents that transport an endogenous or heterologous gene into the cell. Vectors typically consist of a number of genetic components, including but not limited to regulatory elements such as promoters, leaders, introns, and terminator sequences. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the "promoter". Vectors can be a plasmid, a cosmid, a virus (e. g. bacteriophage) a phagemide, an artificial chromosome or a retro-viral vector or others as known in the art. Vectors are able to autonomously replicate in a host cell or can be incorporated into chromosomal DNA. The term "vectors" includes those that function primarily for insertion of a polynucleotide into a cell, those that function primarily for replication of a polynucleotide (replication vector) in a cell of a photosynthetic eukaryote or those that function primarily for transcription and/or translation of DNA or RNA in a cell.
The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription into mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA.
As generally used herein, transformation of a cell or tissue with a polynucleotide is the introduction of a polynucleotide into the cell or tissue by any means.
The term "fetuin-B" or "fetuin-B polypeptide" as comprised by the present invention encompasses any fetuin-B polypeptide, in particular human or mouse fetuin-B, more preferably human fetuin-B and still more preferably the polypeptide of SEQ ID NO: 2 or 4, as well as functionally active variants or fragments of SEQ ID NO: 2 or 4, as defined above, which may be natural or non-natural. Preferably, the variants or fragments differ from the sequence of SEQ ID NO: 2 or 4 e.g. by addition, deletion, substitution and/or insertion of amino acids and have a sequence identity with SEQ ID NO: 2 or 4 of more than 50%, of more than 60%, more than 70%, preferably of more than 80%, more preferably more than 85%, even more preferably more than 90%, even more preferably more than 95%, most preferably more than 97% and/or have an activity of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97% or more than 100 % , e.g. more than 150 %, 200 %, 300 % 400 % or 500 % of the activity of the polypeptide of SEQ ID NO: 2 or 4.
In the context of the present invention, the variant of the polypeptide of SEQ ID NO: 2 or 4 as comprised by the present invention is a functionally active polypeptide in that it maintains the biological function of the reference polypeptide of SEQ ID NO: 2 or 4.
Non-naturally occurring variants or naturally occurring variants of the polypeptide of SEQ ID NO: 2 or 4 may comprise a limited number of amino acid deletions, insertions and/or substitutions, particularly deletions, insertions and/or substitutions of, e.g., at most 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid(s) thereby obtaining a sequence identity or activity of the respective wild-type proteins, e.g. with respect to SEQ ID NO: 2 or 4, as mentioned above.
Non-naturally occurring fragments or naturally occurring fragments of the polypeptide of SEQ ID NO: 2 or 4 may comprise a limited number of amino acid deletions of, e.g., at most 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid(s). The activity of the respective wild-type proteins, e.g. with respect to SEQ ID NO: 2 or 4, is maintained, as mentioned above. The variant may be a modified protein or a modified protein variant which comprises a further component. In one embodiment, the variant may be a fusion protein comprising (i) a polypeptide of SEQ ID NO: 2 or 4 or functionally active variant and (ii) a further protein or peptide component. For example, the polypeptide may be coupled to a marker, such as a tag used for purification purposes (e.g. 6 His (or HexaHis) tag, Strep tag, HA tag, c-myc tag or glutathione S-transferase (GST) tag). If e.g. a highly purified polypeptide of SEQ ID NOs: 5 to 8 or variant should be required, double or multiple markers (e.g. combinations of the above markers or tags) may be used. In this case the proteins are purified in two or more separation chromatography steps, in each case utilizing the affinity of a first and then of a second tag. Examples of such double or tandem tags are the GST-His-tag (glutathione- S-transferase fused to a polyhistidine-tag), the 6xHis-Strep-tag (6 histidine residues fused to a Strep-tag), the 6xHis-tagl00-tag (6 histidine residues fused to a 12-amino-acid protein of mammalian MAP -kinase 2), 8xHis-HA-tag (8 histidine residues fused to a hemagglutinin-epitope-tag), His-MBP (His-tag fused to a maltose-binding protein, FLAG- HA-tag (FLAG-tag fused to a hemagglutinin-epitope-tag), and the FLAG-Strep-tag. The marker could be used in order to detect the tagged protein, wherein specific antibodies could be used. Suitable antibodies include anti-HA (such as 12CA5 or 3F10), anti-6 His, anti-c-myc and anti-GST. Furthermore, the protein could be linked to a marker of a different category, such as a fluorescence marker such as green fluorescent protein, to a binding protein such as steptavidin, one or more small molecular dyes such as Cy dye, or a radioactive marker, which allows for the detection of a protein as comprised by the present invention. In a further embodiment, the polypeptide of SEQ ID NO: 2 or 4 could be part of a fusion protein, wherein the second part could be used for detection, such as a protein component having enzymatic activity.
Alternatively or additionally, the polypeptide of SEQ ID NO: 2 or 4 or variants or fragments thereof as described above may comprise one or more amino acid substitution(s). However, semi-conservative and especially conservative amino acid substitutions, wherein an amino acid is substituted with a chemically related amino acid are preferred. Typical substitutions are among the aliphatic amino acids, among the amino acids having aliphatic hydroxyl side chain, among the amino acids having acidic residues, among the amide derivatives, among the amino acids with basic residues, or the amino acids having aromatic residues.
The term "fetuin-B" also comprises fetuin-B agonists, which mimic the functional activity of fetuin-B or fetuin-B surrogates, which are substitutes of fetuin-B and have the same activity as fetuin-B. As fetuin-B inhibits ovastacin, as shown in the present invention, the term "fetuin-B" also includes inhibitors of ovastacin.
The term "activity" of fetuin-B refers to the ability of fetuin-B to maintain or preserve the fertilisable state of an oocyte in a medium comprising fetuin-B. The term "stabilising" may also include that the oocyte in the medium comprising fetuin-B does not loose its ability for further development. In principle, the term "activity" of fetuin-B corresponds to the definition given above with respect to "stabilising". Thus, if an immature oocyte has been isolated and treated with fetuin-B, the immature oocyte will still be able to become fertilised by a sperm cell, e.g. after it has been matured in vitro. If a mature oocyte has been isolated, the mature developmental state is maintained and the oocyte remains fertilisable, i.e. is able to become fertilised by a sperm. In an aspect of the present invention, "activity" refers to the prevention of the hardening of ZP of the oocyte, most preferably by inhibiting the activity of ovastacin, which is released by degranulation of the cortical granules of the oocyte, such as to proteolytically process ZP glycoproteins, especially to cleave ZP2 to ZP2f. Moreover, the activity of fetuin-B may cause that the oocyte does not loose its ability to proceed further with maturation. The oocyte may proceed with maturation within the medium comprising fetuin-B, or if removed from the medium comprising fetuin-B and brought under appropriate conditions.
The "activity" of fetuin-B may be determined visually such as microscopically in that it is observed whether the status of the ovary tissue is changed or maintained in the medium comprising fetuin-B. In the presence of fetuin-B, the outer appearance of the ovary tissue is not changed, whereas in the absence of fetuin-B the outer appearance is changed, for example, a hardened ZP is generated. This may be determined by any method known in the art, preferably by visual inspection or by determining the protease resistance of the zona pellucida, which is determined by measuring the time required to digest the zona pellucida by chymotrypsin. Digestion of the zona pellucida may be scored by observing the oocyte with an inverted microscope in a medium containing oocyte culture medium and chymotrypsin. The time required to digest the ZP in half of the oocytes (t50) is much longer for fetuin-B deficient oocytes such as oocytes which have been isolated from a female body and have not been treated with fetuin-B, so that hardening of ZP occurs, or fertilised zygotes or two-cell embryos with a hardened ZP than for fertilisable oocytes, wherein the ZP is not hardened, such as oocytes which have been isolated from a female body and have been treated with fetuin-B immediately after isolation. Thus, in the presence of 0.2 mg/mL of chymotrypsin, t50 in fetuin-B deficient oocytes (e.g. 31.5 min ± 4.7 min) or two-cell embryos (e.g. 38.5 min ± 6.0 min) is about 5 to 6 times longer than in wildtype oocytes (e.g. 6.4 min ± 0.9 min), if same conditions of chymotrypsin digestion are applied. "Activity" of fetuin-B may also preferably be determined by the presence or amount of ZP2f, which appears if ovastacin is active and cleaves ZP2 into ZP2f. In a preferred embodiment, at most 10%, at most 5%, at most 3%, at most 2%, at most 1% or 0% of ZP2 present at the time of isolation is cleaved into ZP2f during storage. Detection may be performed by using an antibody against ZP2 and ZP2f (Rankin et al, 2003), e.g. in an immunological detection method such as Western Blotting, whereby ZP2 shows a signal at 120 kDa and ZP2f shows a signal at 90 kDa.
The time interval of storage of the ovary tissue in the medium comprising fetuin-B may be several minutes such as 10, 20, 30 minutes to several hours such as 4, 5, 6 hours. In a preferred embodiment, the oocyte may be fertilized within these time intervals. Alternatively, the medium comprising fetuin-B and the ovary tissue may be frozen to extend the time interval of the storage to several days such as 2, 3, 5, 8 days to several weeks or months. Freezing methods are known to those skilled in the art and may be flash freezing also known as vitrification or controlled freezing in the presence of anti-freeze protectants, preferably vitrification.
As used herein, the term "ovary tissue comprising an oocyte" is any part of an ovary which can be isolated from a female and which comprises an oocyte. The oocyte can be at any stage, be it an immature or a mature oocyte. In a preferred embodiment, the oocyte is an immature oocyte. The ovary tissue may be a follicle. Preferably, the follicle is an immature follicle comprising an immature oocyte. However, the follicle may also be a mature follicle comprising a mature oocyte. The ovary tissue may be an oocyte, preferably an immature oocyte, but may also be a mature oocyte. The ovary tissue may be the cortex of the ovary comprising an immature follicle with an immature oocyte. The ovary tissue may be a whole ovary.
During the early stages of development of oocytes, oocytes surrounded by follicular somatic cells are maintained in a prolonged stage of the first meiotic prophase. The oocytes become developmentally arrested in the ovaries at the germinal vesicle stage in prophase of the first meiotic division (G2/M transition). At this stage, the oocytes are called primary oocytes (primordial follicles). The growth and maturation of mammalian follicle cells and oocytes are dependent upon and intricately controlled by hormones, including gonadotropins, such as follicle stimulating hormone (FSH) and luteinizing hormone (LH) secreted by the anterior pituitary, and other local paracrine factors secreted by the surrounding follicular somatic cells. Periodically, a group of primordial follicles enters a stage of follicular growth. During this time, the oocyte undergoes a large increase in volume and the number of surrounding follicular granulosa cells increases. In early stages of the cycle, maturing follicles respond to FSH with further growth and cellular proliferation. In later stages, FSH induces the formation of LH receptors on the granulosa cells. In response to the mid-cycle LH surge, oocytes of preovulatory follicles resume meiosis. The first meiotic division is completed with the extrusion of a haploid set of chromosomes into the first polar body while the other haploid set of chromosomes remains within the cytoplasm of the oocyte. These oocytes, called secondary oocytes, then proceed to the second meiotic division, where the oocyte becomes arrested at metaphase ("Met-II"). Met-II oocytes are mature, and can be ovulated and fertilised. Without fertilisation, the mature oocyte will not continue with meiosis. At fertilisation meiosis II completes, forming a second polar body.
The normal ovulating woman will recruit approx. 300 immature oocytes for each menstrual cycle. This recruitment takes place before the actual cycle. At the day of menstruation, around 20 to 30 immature oocytes will still be present. Normally, during a process of apoptosis all but one oocyte will die before ovulation. At day 5 to 10 approx. 10 to 15 immature oocytes will be present in their small follicles being 10 to 12 mm in diameter. Some are still growing and some are starting to undergo an apoptotic process. As used herein, the term "immature oocyte" means an oocyte that has not yet reached metaphase-II (M-II). Immature oocytes used in the present invention exist within follicles which pass through the following distinct stages including primordial, primary, secondary, and antral stages including early antral, later antral, and pre-ovulatory stages. Immature oocytes used in the present invention may be present in prophase I (germinal vesicle), metaphase I, anaphase I or telophase I. Immature oocytes preferably used in the invention are at the germinal vesicle (GV) or metaphase-I (M-I) stage. For example, immature oocytes of M-I stage will be readily recognised as oocytes with a tight cumulus mass, no polar bodies or germinal vesicles visible. In another preferred embodiment, an immature oocyte is present in an ovarian follicle with a diameter of 8 to 12 mm. The advantage of such small follicles is that they are present in substantial numbers without severe hormonal treatment and they can be seen by ultrasound. Follicles preferably used in the present invention are present in the primordial, primary, secondary, and antral stages.
The processes for retrieving immature oocytes of each month transvaginally is known in the art. In one embodiment, transvaginal ultrasonographically-guided oocyte collection is done involving needle piercing of the vaginal wall. The follicles, oocytes and follicular fluids may be aspirated and immediately stored in a medium comprising fetuin-B. If the follicle is further processed in order to isolate the oocyte, all processing steps are performed in the medium comprising fetuin-B. The oocytes may be retrieved from the ovarian follicle by e.g. placing a special needle into the ovarian follicle and aspirating the fluid that contains the oocyte, using known techniques. The oocytes are aspirated into a medium comprising fetuin-B. The oocytes may be inspected microscopically.
Alternatively, the outer layer of the ovary, called the cortex, may be isolated from a female. In vivo, preantral follicles exist in the relatively avascular cortex of the ovary. Isolation of ovary tissue comprises ovary tissue collection, immediate tissue treatment with medium comprising fetuin-B and separation of the cortical layer from the medullar part. The cortex is sliced into strips 1 millimeter thick, which remain in the medium comprising fetuin-B. After culture, the slices are implanted back into the abdomen, close to the fallopian tubes near the uterus. The cortex tissue strips contain immature follicles. They start to produce hormones and oocytes like a normal ovary, when they are implanted back into the body.
As used herein, the term "mature oocyte" means an oocyte at the metaphase-II stage. Metaphase-II is typically detected by visual identification under a microscope. Processes for retrieving mature oocytes from a female body are known in the art.
The skilled person knows how the status of an ovary tissue isolated from a female's body can be determined. For example, the developmental stage of a follicle can be determined microscopically.
There is substantial interest in the art to isolate immature oocytes from a female and to preserve the oocytes in a culture medium for later use. For example, immature cultivated oocytes may be re-transplanted into the female, where they can mature to a mature, fertilisable oocyte. The method of the present invention may also be used in techniques wherein the immature oocytes may be used at a time after collection for in vitro maturation of oocytes for in vitro fertilisation.
While the culture of an immature oocyte is a preferred embodiment of the present invention, there is also a substantial interest in the art to isolate a mature oocyte and maintain the mature oocyte in a status, which is present at the time of isolation. In particular, a premature hardening of the zona pellucida should be prevented in order to ensure that the oocyte can be fertilised at a time after isolation, either in vitro or in vivo after transplantation. Hardening of the zona pellucida would otherwise occur, if the oocyte is removed from its natural environment and not brought into a medium comprising fetuin- B.
A group of females for whom it may be desirable to collect ovary tissue and preserve it are woman undergoing IVF. Therefore, immature oocytes may be collected during IVF. Current IVF (in vitro fertilisation) treatment requires that multiple oocytes be harvested. Therefore, women are normally pre-treated with hormones, and oocytes, which have matured to the M-II stage are harvested. Usually, at least 10-15% of the developing oocytes are immature and are discarded at most IVF clinics. Such immature oocytes may be preserved by the method of the present invention and are available for future applications, e.g. if IVF (in vitro fertilisation) has failed and the immature oocytes are brought to maturity, in vitro, for subsequent fertilisation.
Alternatively, immature oocytes may be collected from unstimulated ovaries and may be matured to the M-II stage by way of in vitro maturation (IVM), in order to avoid the entire regimen of treatment of women with GnRHa and stimulation of ovaries with gonadotropin during IVF. The use of fetuin-B in a culture medium comprising fetuin-B may be useful for maintaining the fertilisable status of the oocyte, if such fertilisable status is present at the time of isolation.
Another group of patients, for which it may be desirable to collect ovary tissue and preserve it, are cancer patients. Women undergoing chemotherapy or radiotherapy during cancer treatment have associated morbidity of their reproductive organs resulting in post- therapy infertility. Ovary tissue from these patients can be harvested and preserved before the start of cancer therapy. Ovary tissue can be re-transplanted when the woman is ready to have children. In an embodiment of this invention, an ovary is surgically removed and preserved according to the present invention. Part or whole of the ovary may later be used for the woman. The removal of ovary tissue or the ovary does not require a significant delay in cancer treatment beyond one to two days of recovery. Additionally, children and infants as young as 6 months can undergo this procedure since neither reaching puberty nor fertilisation are required. If a patient is diagnosed with cancer, ovarian tissue or ovary can be dissected out and preserved in a culture medium comprising fetuin-B prior to initiation of treatment that might cause sterility as cytostatic or radiation treatment often does. Immature oocytes can immediately or later be extracted from the ovarian tissue or ovary and finally be matured and fertilised.
In another embodiment, the present invention offers a method of ovary tissue preservation for transplantation into female patients with malignant neoplasms of reproductive system organs. The method comprises the collection of ovary tissue, the addition of the isolated ovary tissue into medium comprising fetuin-B and the storage in the medium comprising fetuin-B for future transplantation.
In a further embodiment, the recovery of immature oocytes and culturing them in a medium comprising fetuin-B may be a useful for women with polycystic ovarian syndrome (PCOS) related infertility. PCOS is one of the most common reproductive disorders in women of childbearing age. It has a heterogeneous presentation, which is clinically characterized by anovulation and hyperandrogenism, and on pelvic ultrasound examination shows numerous antral follicles within the ovaries. Immature oocytes may be collected from these women and cultivated in a medium comprising fetuin-B and may be used at a later time point, for example, for in vitro maturation techniques or for transplantation. The ovary tissue may be isolated from a wide variety of animal species, particularly mammalian species. Examples are humans, cows, horses, camels, dogs, cats, pigs, goats, sheep, or zoo animals, such as panda bears, large cats etc. Humans are of a very particular interest, however, also animals with high commercial value such as high efficiency cows, race horses or race camels are of particular interest. Animal models, particularly small mammals, e.g. mice, etc., are of interest for experimental investigations. Most preferably, the ovary tissue is isolated from humans.
The present invention involves the provision and use of a culture medium comprising fetuin-B for culturing ovary tissue. The invention comprises holding the ovary tissue in the culture medium comprising fetuin-B for a period following isolation of the ovary tissue, to ensure that the oocytes do not loose the ability to mature and to be fertilised at a later time. The period of culturing the ovary tissue may be any period, until it is desired to use the ovary tissue for in vitro maturation and possibly fertilisation processes or for implantation into the female for fertilisation. The period may be several minutes to hours, days, months or years.
Culture conditions for ovary tissue in a medium comprising fetuin-B are not critical to the invention, as long as the culture conditions support the activity of fetuin-B, as referred to above. Thus, the culture conditions should be selected such that the status of the ovary tissue is maintained at the status present when isolating the ovary tissue. Moreover, the conditions should preserve the survival of the ovary tissue and in particular of the oocytes comprised by the ovary tissue and should maintain the ability of the oocytes for maturation and fertilisation. Suitable culture conditions include e.g. culturing the ovary tissue under cold or ambient temperature such as from 0 to 37°C, preferably in the cold, such as in a refrigerator, from 0 to 10°C such as 4 to 6°C in an atmosphere of 95% air and 5% C02 at high humidity, e.g. 100% humidity. A "triple gas" atmosphere of 5% 02, 5% C02 and 90% N2 may also be used. Mineral oil may be overlaid on the medium to control evaporation and/or temperature. Isolated oocytes are typically cultured in a well containing 1 ml of culture medium comprising fetuin-B or more, or may be cultured in 10 μΐ of culture medium comprising fetuin-B or less in a droplet in a culture dish.
The present invention provides a culture medium for culturing ovary tissue including fetuin-B. As used herein, the term "medium", "culture medium" or a similar term is a medium for culturing ovary tissue, which does not include fetuin-B. If fetuin-B is present, this is indicated. The culture medium comprising fetuin-B is used as a substitute for the follicular fluid surrounding the oocytes in the natural state. The kind of medium is not critical to the present invention, as long as the medium is suitable to support or not to interfere with the activity of fetuin-B to maintain the status of the ovary tissue. In particular, the activity of fetuin-B to preserve the capability of the oocytes for maturation and fertilisation should be supported by the medium or at least the medium should not interfere with this activity of fetuin-B. If medium is selected which interferes with the activity of fetuin-B, then the medium comprising fetuin-B should in totality show the activity of fetuin-B. This may, e.g. be achieved by increasing the concentration of fetuin-B. The skilled person is capable of elucidating media which in the presence of fetuin-B show the above features.
The medium useful for the method of the present invention contains an energy source, inorganic salts, essential and/or non-essential amino acids and possibly other factors. The energy source, inorganic salts, and essential and/or non-essential amino acids in the medium are not critical to the invention, as long as the requirements as described above with respect to the medium are met. The energy source may be e.g. glucose, sodium pyruvate, lactate, or a mixture of some or all of these energy sources. The inorganic salts are inter alia provided to buffer the pH of the medium within a range preferably of about 5 to 9, preferably 6 to 8 and more preferably 7.2-7.4 and to maintain correct osmolarity of the medium with the ovary tissue. Typical inorganic salts include CaCl2, KC1, MgS04, NaCl, NaHC03, NaH2P04, Fe(N03)3, KH2P04, Na acetate, Na2H2P04, etc. The medium contains at least one amino acid or a source thereof. Preferably, at least the essential amino acids, or sources thereof, are included in the medium. "Essential" amino acids are those amino acids not synthesized in the oocyte and that are essential for protein synthesis. The essential amino acids are generally considered to be iso leucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. A commercial amino acid mixture, containing the eight essential amino acids as well as some or all of the remaining nonessential amino acids may conveniently be used. Non-naturally occurring amino acids or amino acid derivatives as are known in the art may also be included in the medium. In a particularly preferred embodiment, the medium of the invention comprises alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The medium preferably contains vitamins as are known in the art for inclusion in culture medium. Vitamins that may be included in the medium include, without limitation, vitamins Al (retinol), A2 (an alternative form of retinol), Bl (thiamine), B2 (riboflavin), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamin), B17, C (ascorbic acid), D, D2 (calciferol), D3 (cholecalciferol), E (tocopherol), H (biotin), K, Kl (phylloquinone), K2, K3 (menadione), P, etc. A particularly preferred combination of vitamins comprises biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositol, nicotinamide, pyroxidal-HCl, riboflavin, and thiamine-HCl. Buffers for controlling the pH of the medium may be added, such as HEPES, as may be pH indicators such as phenol red. Antibiotics such as penicillin or streptomycin may be added to the medium to prevent contamination. Synthetic Serum Supplement (SSS®) may be included in the medium as a source of protein for the oocyte and to prevent cells from adhering to glassware during in vitro culture. SSS has the advantage of having received regulatory approval for human IVF, and is commercially available.
Suitable media may be commercial media currently approved for and in use for IVF/ ART (assisted reproductive technologies) and for cryopreservation of tissues and cells destined for IVF/ART. IVF media are comprised of a cocktail of physiological inorganic salts, energy sources, amino acids and proteins, and there is a range of different formulations commercially available. Media can include broad spectrum antibiotics like gentamicin sulphate to prevent microbial contamination of the media. Others include human serum albumin to act as a chelating agent for deleterious contaminants including heavy metals that may be present in minute quantities in the micro-environment, and also as a anti- adhesive to facilitate oocyte manipulation by preventing them from sticking to glass or tissue culture ware. Examples of media include, but are not limited to, GM501 Wash (Gynemed Medizinprodukte GmbH, Lensahn) and Sydney IVF Gamete buffer (Cook Medical Europe, Ireland) for collecting oocytes, and GM501 Culture (Gynemed) and Sydney IVF Fertilization Medium (Cook Medical) for insemination, GM501 Culture and Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium for zygote and embryo culture. Further media approved and in use for consecutive use in IVF/ART include, but are not limited to, Early Cleavage Medium (Irvine Scientific), Multiblast (Irvine Scientific), G-l Plus (Vitro life), G-2 Plus (Vitrolife), Innovative Sequential Medium (ISM-1, Origio), Innovative Sequential Medium (ISM-2, Origio). Further commercial complete media approved and in use for IVF/ART include, but are not limited to, Fertikult (Fertipro), G3 (Fertipro), Gynedmed gentamycin, Gynemed501 pen/strep (Gynedmed), HTF Medium (Irvine Scientific), Blastassist (Origio), Quinn advantage cleavage (Sage), Single step Medium (Irvine Scientific), Universal medium (Origio), Global medium (IVFonline). Commercial media currently approved for and in use for cryopreservation include, but are not limited to, EmbryoFreeze, FertiPro, and RapidVit Blast, Vitrolife for freezing and flash freezing (vitrification), respectively, and EmbryoThaw, FertiPro, Thawing Kit, Rapid Warm Cleave and Vitrolife, for thawing. The medium useful for the method of the present invention may resemble a medium known in the art as Human Tubal Fluid medium HTF, and may contain as components in mM concentrations: Sodium Chloride 97.8, Potassium Chloride 4.69, Magnesium Sulfate Anhydrous 0.20, Potassium Phosphate Monobasic 0.37, Calcium Chloride Anhydrous 2.04, Sodium Bicarbonate 25.0, Glucose 2.7, Sodium Pyruvate 0.33, Sodium Lactate 21.4, Gentamicin 10 μg/mL, Phenol Red 5 mg/L. The medium useful for the method of the present invention may further be enhanced with a serum substitute and may contain as components in mM concentrations: Sodium Chloride 91.44, Potassium Chloride 4.22, Magnesium Sulfate Anhydrous 0.18, Potassium Phosphate 0.33, Calcium Chloride 1.84, Sodium Bicarbonate 22.5, Glucose 2.5, Sodium Pyruvate 0.3, Sodium Lactate 19.26, Phenol Red 4.5 mg/L, Gentamicin 9 μg/mL, Human Serum Albumin 5 mg/L, Globulins 1 mg/L. A further modification of the medium useful for the method of the present invention may contain as components in mM concentrations: Sodium Chloride 97.8, Potassium Chloride 4.69, Magnesium Sulfate Anhydrous 0.2, Potassium Phosphate Monobasic 0.37, Calcium Chloride Anhydrous 2.04, Sodium Bicarbonate 4.0, HEPES buffer 21, Glucose 2.78, Sodium Pyruvate 0.33, Sodium Lactate 21.4, Gentamicin 10 μg/L, Phenol Red 5 mg/L.
The concentration of fetuin-B in the medium may be from 0.005 to 3 g/L, preferably 0.005 to 1 g/L and more preferably 0.005 to 0.5 g/L. In a preferred embodiment, the concentration is similar or equal to the serum concentration of fetuin-B being about 0.01 g/L in human or 0.3 g/L in mouse. Consequently, in a particularly preferred embodiment of the present invention, the concentration of fetuin-B in the medium is 0.005 to 0.03 g/L, more preferably 0.007 to 0.02 g/L and most preferably about 0.01 g/L. Additional preferred embodiments comprise concentrations of fetuin-B from 0.01 to 0.5 g/L, more preferably from 0.05 to 0.5 g/L and still more preferably about 0.1 g/L.
The use of fetuin-B for stabilising an ovary tissue comprising an oocyte is an in vitro use. The present inventors have furthermore found a correlation between the level of fertility of an oocyte in an IVF procedure and the serum fetuin-B level of the female individual from which the oocyte has been removed. An increased serum fetuin-B level of an individual induced by a substance which is capable of increasing the fetuin-B level in a female individual enhances the fertility rate in IVF. Thereby, "increased" or "enhanced" serum fetuin-B level or similar terms have as reference the serum- fetuin-B level in the absence of an inducing substance, also termed "basal" serum fetuin-B level herein. This means that fetuin-B is effective in maintaining and/or increasing the fertility of oocytes in vivo, such that they can be successfully used in an IVF after isolation. Moreover, the present invention also shows that fetuin-B is effective in maintaining fertility in vitro after isolation of the oocytes if the isolated oocytes are immediately contacted with a medium comprising fetuin-B. Consequently, a successful IVF can be performed if the oocytes are under the influence of enhanced levels of fetuin-B during their production in the female body and have immediate and constant contact with fetuin-B in in vitro culturing after isolation until the time of fertilization. Thus, a constant contact of oocytes with enhanced levels of fetuin-B within the body and an immediate and constant contact of oocytes with fetuin-B outside the body are necessary to generate and maintain high fertility of oocytes, resulting in fertilization during IVF. In patients with IVF failure, serum fetuin-B levels remained constant.
The concentration of serum fetuin-B under normal conditions (i.e. without treatment for enhancing fertuin-B level) has been newly determined to be about 0.004 g/L in human, but differs from individual to individual. This concentration is lower than the concentration originally determined. This level of fetuin-B in serum can be enhanced by a substance which is capable of increasing the fetuin-B level in a female individual. The substance may be any substance which is capable of enhancing serum fetuin-B levels in a female individual. In a preferred embodiment, the substance may be a hormone. An effective hormone for the purpose of the present invention may be estrogens. Estrogens may be endogenously produced by the application of hormones such as FSH or hMG which are administered for inducing the production or maturation of oocytes for use in IVF. Estrogens may also be administered to the female individual. The present inventors have detected the usefulness of estrogens for increasing serum fetuin-B levels in women receiving a contraceptive treatment of ethinylestradiol with or without combination with gestagen. The present inventors have also detected that enhanced serum fetuin-B levels during the period of oocytes maturation, e.g. as a result of hormone treatment for maturing oocytes for use in IVF, in a female body result in fertilization in IVF. The amounts of estrogen to be administered in order to result in enhanced fetuin-B levels are within the amounts which are administered to woman to inhibit ovulation and thus conception. The amounts are preferably 0.02 to 0.05 mg in total per cycle, e.g. 0.03 mg per menstrual cycle. The amounts of hormones such as FSH for inducing endogenous estrogen production are those which are usually administered to a woman for maturation of oocytes for use in IVF and are preferably from 40 to 90 IU/L per injection, more preferably 50 to 75 IU/L per injection and in total 1650 to 4200 IU/L. The amount of estrogen present in the female serum which is suitable to increase fetuin-B level is dependent on the female individual. Measured values were between 5 and 25000 pmol/L. Usually, the concentration of fetuin-B remains constant during maturation of oocytes or increases.
The level of fetuin-B which is produced in the female individual depends on the individual. There may be a sharp rise of the level of fetuin-B in response to estrogen. There may be only a slight increase of the level of fetuin-B. Decisive is not the extent of increase of the level of fetuin-B. Decisive is that there is an increase of the level of fetuin-B in response to substance treatment, as compared to the level of fetuin-B in the absence of substance treatment. Nevertheless, the increase of fetuin-B reaches serum fetuin-B levels which are above the basal serum fetuin-B level. Preferably, the amount of serum fetuin-B in women with substance treatment rises to levels which are 1.5 to 4 times of the basal level, e.g. about 1,5; 1 ,6; 1,7; etc. to 4 times per female individual, more preferably, the amount rises by about twice of the basal level. Exemplary enhanced serum concentrations of fetuin-B are 5 to 20 mg/L, e.g. about 7, 8, 9, etc. or 15 mg/L per female individual.
The female individuals that are treated by the use of a substance for enhancing serum fetuin-B levels are the women as indicated above. Preferably, the female individuals are individuals who want to perform an IVF and take hormones for induction of maturation of oocytes.
FIGURES
Figure 1. Fetuin-B gene deletion strategy and phenotyping (A) The genomic fetuin locus depicting fetuin-A (Ahsg) exon E7 and fetuin-B (Fetub) exons El to E8. Relevant features of the gene targeting vector (B), the deletion control vector (C), and the resulting recombinant genomic Fetub locus (D). The translation start codon is located in exon 2, which was replaced by the neomycin cassette by homologous recombination of the gene targeting vector and the genomic sequence resulting in deletion of exons E2 and E3. The genomic Fetub locus was conserved except for the 235 bp deletion. Exons are depicted by black boxes. The homologous sequences are highlighted as grey bars. Grey arrows denote the PCR primers used for amplifying the homologous sequences of the gene targeting and the knockout control vector. TK: thymidine kinase; neo: neomycin resistance. (E) Immunodetection of fetuin-B and (F) of fetuin-A in sera of wildtype (+/+), hemizygous (+/-) and fetuin-B deficient (-/-) mice. Molecular mass is indicated at the right. Background strain was C57BL/6.
Figure 2. Female infertility in fetuin-B deficient C57BL/6 mice
(A) Breeding performance. The mean litter sizes of Fetub-/- and Fetub+I- females were compared with the litter size of Fetub+/+ females and the respective males by pairwise two-sided t test. ***p < 0.001, ns = not significant. (B) Wildtype (+/+) cumulus-oocyte complex (COC) and COC isolated from a Fetub-/- (-/-) mouse at day 0.5 after mating. (C) Oocyte isolated from a wildtype female or a Fetub-/- female at day 0.5 after mating. Corresponding data for DBA/2 mice are shown in figure 6.
Figure 3. Successful fertilisation of fetuin-B deficient oocytes following ovary transplantation and laser-assisted in vitro fertilisation
(A) Transplantation of the ovaries of fetuin-B deficient mice (white symbol) into ovariectomized wildtype recipients (black symbols). The transplanted mice were mated with proven potent wildtype males. The offspring was genotyped by PCR heterozygous for fetuin-B deficiency (grey symbols). Wildtype (WT); Fetub-/- (KO). (B) Commercial fetuin preparations with zona pellucida hardening inhibition activity contain fetuin-B. Human fetuin-B antibody was used to probe for fetuin-B in sera of wildtype, Fetub-/- and Fetua-I- mice, as well as various commercial bovine fetuin preparations: Sigma- Aldrich F2379 (bov fetuinl), Sigma-Aldrich F3004 (bov fetuin2), Sigma-Aldrich F2379 (bov fetuin3) further purified by gel filtration, AppliChem A2783 (bov fetuin4); Dade-Behring human plasma fetuin- AJ Ahsg (hu fetuin-A), bovine serum albumin (BSA), human serum albumin (HSA), human serum and human follicular fluid. Molecular weight is indicated at the right. Images of IVF of (C) wildtype (+/+) and (D) fetuin-B deficient (-/-) oocytes following conventional IVF, or laser-assisted IVF (LAIVF) of (E) wildtype (+/+) and (F) fetuin-B deficient (-/-) oocytes. Arrows point to the laser lesions. The micrographs were taken at day 1.5 post IVF and LA-IVF. Numbers of oocytes employed for IVF and LA-IVF and the respective fertilisation rates are given below the respective photographs. (G-L) Fetuses dissected from wildtype foster mothers at day 16 following embryo transfer of LA-IVF fertilised oocytes. Background strain was C57BL/6 in all cases. Corresponding data for DBA/2 mice are shown in figure 7.
Figure 4. Premature zona pellucida hardening of fetuin-B deficient oocytes lacking ovastacin protease inhibition
(A) Diameter of unfertilised wildtype oocytes (69.25 ± 4.19 μιη, n = 137) and Fetub-/- derived oocytes (68.96 ± 4.16 μιη, n = 196). (B) ZP thickness of unfertilised wildtype oocytes (7.58 ± 0.96 μιη, n = 137), Fetub-/- derived oocytes (6.54 ± 0.95 μιη, n = 196) and wildtype 2-cell stages (6.95 ± 0.70 μιη, n = 108). Every dot represents one oocyte. (C) ZP digestion times of wildtype oocytes (+/+), fetuin-B deficient oocytes (-/-) and wildtype 2- cell stage embryos. The ZP digestion time t50 equals the time required for 50% of the oocytes to become zona-free following a-chymotrypsin treatment. Every dot represents one assay performed with at least 20 oocytes harvested from one mouse. (D) In vitro sperm binding to wildtype and fetuin-B deficient oocytes. 2-cell embryos were used as wash controls. (E) Immune detection of ZP2 protein of wildtype oocytes, fetuin-B deficient oocytes and wildtype 2-cell embryos. Fetuin-B deficient oocytes were further isolated pre- and postovulatory. (F) Active recombinant ovastacin was inhibited by recombinant mouse fetuin-B (circles; concentration range: 0.6 nM - 4.5 μΜ) with an IC50 of 76.4 nM ± 3.35 nM. In contrast, recombinant fetuin-A did not inhibit ovastacin (squares; concentration range: 0.6 nM - 11 μΜ). Pairwise two-sided t test **p < 0.01, ***p < 0.001, ns = not significant. The horizontal lines depict the mean ± SD (A, B, D) or the mean (C). Background strain C57BL/6, corresponding data for DBA/2 mice are shown in figure 8. Figure 5. Putative mechanism for the interaction of fetuin-B and ovastacin
(A) In healthy wildtype oocytes, fetuin-B inhibits ovastacin activity liberated by spontaneous cortical release and thus prevents premature ZP hardening. (B) After fertilisation and cortical degranulation the concentration of ovastacin becomes too high to be blocked by fetuin-B. As a consequence, ZP2 is cleaved into ZP2f and the ZP is hardened (indicated by a thinner, more compact ZP). (C) In the absence of fetuin-B premature ZP hardening leads to infertility.
Figure 6. Female infertility in fetuin-B deficient DBA/2 mice, similar to C57BL/6 mice shown in figure 2 (A) Breeding performance. The mean litter sizes of Fetub-/- and Fetub+/- females were compared with the litter size of Fetub +/+ females and the respective males by pairwise two-sided t test. *p < 0.05, ***p <0.001, ns = not significant. (B) Wildtype (+/+) cumulus- oocyte complex (COC) and COC isolated from a Fetub-/- mouse at day 0.5 after mating. (C) Oocyte isolated from a wildtype female or a Fetub-/- female at day 0.5 after mating. 89 oocytes recovered from 10 female Fetub-/- mice were unfertilised. In contrast 17 of 20 observed oocytes were fertilised in a total of 3 Fetub+/+ mice.
Figure 7. Successful fertilisation of fetuin-B deficient oocytes following laser assisted in vitro fertilisation (LA-IVF), related to figure 3
Images of IVF of (A) wildtype (+/+) and (B) fetuin-B deficient (-/-) oocytes following conventional IVF, or LA-IVF of (C) wildtype (+/+) and (D) fetuin-B deficient (-/-) oocytes. Arrows indicate the laser lesions. The micrographs were taken at day 1.5 post IVF and LA-IVF. Numbers of oocytes employed for IVF and LA-IVF and the respective fertilisation rates are given below. Background strain DBA/2.
Figure 8. Premature zona pellucida (ZP) hardening of fetuin-B deficient oocytes, related to figure 4
(A) Diameter of unfertilised wildtype oocytes (72.16 ± 1.90 μιη, n = 100) and Fetub-/- derived oocytes (71.83 ± 2.01 μιη, n = 100). (B) ZP thickness of unfertilised wildtype oocytes (7.70 ± 0.70 μιη, n = 100), Fetub-/- derived oocytes (6.19 ± 0.71 μιη, n = 100) and wildtype 2-cell stages (6.57 ± 0.58 μιη, n = 54). Every dot represents one oocyte. (C) ZP digestion time of wildtype oocytes (+/+), fetuin-B deficient oocytes (-/-) and wildtype two- cell embryos (2-cell stage). The ZP digestion time was defined as the time required for 50% of the oocytes to become zona-free (tso) following a-chymotrypsin treatment. Every dot represents one assay performed with at least 20 oocytes harvested from one mouse per genotype. (D) Immune detection of ZP2 protein of wildtype oocytes, fetuin-B deficient oocytes and wildtype 2-cell embryos. Fetuin-B deficient oocytes were further isolated pre and postovulatory. Pairwise two-sided t test **p < 0,01, ***p < 0.001, ns = not significant. The horizontal lines depicts the mean ± SD (A, B) or the mean (C). Background strain DBA/2.
Figure 9. In vitro fertilization (IVF) success of oocytes that had been kept in culture for the indicated times
Addition of 0.1 mg/ml fetuin-B (·) to the medium into which the oocytes isolated from mice have been placed increases the fertilization success compared to the IVF without fetuin-B (■). The x axis indicates the time point of fertilization after isolation of the oocytes. The y axis indicates the percent of fertilized oocytes.
Figure 10. Positive correlation of serum fetuin-B with estradiol
The figure shows a positive correlation of serum fetuin-B levels with estradiol levels during hormone treatment with FSH. The x axis shows the concentration of estradiol in the body and the y axis shows the plasma fetuin-B level in the body. Each black dot shows the estradiol and associated fetuin-B level of one measuring sample. There is a significant positive correlation of the two parameters estradiol level and fetuin-B level according to the Pearson correlation test. This means for an individual sample that the fetuin-B level increases with increase of estradiol.
Figure 11. Serum fetuin-B during the hormone therapy in IVF cycles
The figure shows the correlation between FSH treatment and serum fetuin-B level. (A) and (B) The x axis shows the day of the hormone treatment for producing oocytes for use in IVF. The y axis shows the serum fetuin-B level. Circles and quadrats indicate the mean values of all the measured values, available at a specific treatment day; linear regression), grey lines reflect the fetuin-B levels of one patient over time, black lines reflect the mean fetuin-B levels. (A) shows the results for patients with successful IVF. (B) shows the results for patients with IVF failure.
Figure 12. Serum fetuin-B is stimulated by the synthetic estrogen, ethinyl estradiol The figure shows the influence of estrogens on the serum level of fetuin-B. (A) The x axis indicates the cycle day of the woman and the y axis indicates the serum fetuin-B level. The shaded grey area shows the contraceptive treatment using a combined gestagen / ethinylestradiol (EE2) formulation. The curve indicates the level of serum fetuin-B dependent on the day of the cycle. (B) The same applies for figure B, except that the woman received a different contraceptive treatment regime, namely gestagen alone (day -6 to 0) and gestagen plus EE2 (shaded grey area).
EXAMPLES
Experimental procedures
Animal experiments. The animal welfare committee of the Landesamt fur Natur-, Umwelt- und Verbraucherschutz (LANUV) of the state of North Rhine Westfalia approved our animal study protocol. Animal maintenance, handling and anesthesia were performed according to the Federation for Laboratory Animal Science Associations FELASA recommendations. Animals were sacrificed by isoflurane overdosing. Mutant mouse lines. The genomic Fetub sequence used for the cloning of the gene targeting vector was taken from the Pac clone RPCIP711A0948Q2, library RPCI21. The gene targeting strategy is shown in figure 1A-D. The 5' and 3' homologous sequences were amplified by PCR using these primers: 5R3s 5'-CCG CTC GAG ATG TTG GCT ACT GGT TTG CTG T-3' (SEQ ID NO: 5) and 5Rlas 5'-AGC TTT GTT TAA ACC TCT AGT GAG TGG AAC ACA ACT TCT-3' (SEQ ID NO: 6) for the 5' homologous region and 3Rls 5 '-TTG GCG CGC CTG GTT TAG TGA TAC GGG CCA-3' (SEQ ID NO: 7) and 3Rlas 5 '-ACG CGT CGA CAA GCT GCT AGG TAG ATA TTT CCC AT-3' (SEQ ID NO: 8) for the 3' homologous region. Additionally a targeting control vector was cloned, resembling the targeted Fetub genomic sequence. The 3' homologous sequence was expanded using primer 3R2as 5 '-ACG CGT CGA CTG TTG AAC TTT GCA AGC GAC T-3' (SEQ ID NO: 9) instead of 3Rlas as shown in figure 1A. The PCR products were ligated into the gene targeting vector pGKneo-TV. The gene targeting vector was transfected into the ES cell line Knut 1 (129/SvJ x C57BL/6) by electroporation. The transfected cells were selected by G418 (250 μg/ml active substance) treatment. Resistant colonies were analyzed by PCR and Southern blot. Following established protocols (Hogan et al, 1994) homologous recombined ES cell clones were further cultivated (4th passage) and then injected into blastocysts which were collected from superovulated Balb/c mice. The resulting chimeras were mated with C57BL/6 wildtype males and the offspring was genotyped. To obtain mutant mouse strains with defined genetic backgrounds, mice heterozygous for the fetuin-B deletion (Nl) were back-crossed for at least 10 generations to inbred mice of strains C57BL/6 and DBA/2 obtained from a commercial breeder. The mouse strains were named B6-FetubtmlWja and D2- FetubtmlWja according to Institute of Laboratory Animal Resources (ILAR, http://dels.nas.edu/ilar/) terminology.
Fetuin-B and fetuin-A Western blot (murine). Blood was collected by cardiac puncture of adult mice after an overdose of isofluran. The blood was applied to a serum tube and incubated for 1 hour at room temperature. Then, the serum tube was centrifuged at 1000 x g for 10 minutes and the serum supernatant was transferred to a reaction tube. Serum samples were diluted 1 : 100 in SDS-PAGE loading buffer and 15 μΐ were loaded on a 10% polyacrylamide gel. The proteins were blotted onto a nitrocellulose membrane (Protran, Whatman, Schleicher und Schuell) using a semidry blotting device (BioRad). The membrane was blocked at 4°C over night with blocking solution (PBS + 0.05% Tween 20 + 5% nonfat dried milk powder). The primary antisera (rabbit anti-mouse) were diluted in blocking solution 1 :4000. The secondary antibody (swine anti-rabbit IgG/HRP, Dako) was diluted 1 :5000. All antisera were incubated for 1 hour at 37°C. After each antibody incubation the blot was rinsed 3 x 5 minutes in washing solution (PBS + 0.05% Tween 20). Protein detection was achieved by chemiluminescence using a Fuji LAS Luminescent Image Analyser 4000.
Breeding performance. Females were mated at 10-25 weeks of age. Copulation success was scored by the presence of a vaginal plug.
Collection of oocytes and two-cell embryos. Follicular growth of 5-10 week old mice was stimulated by injection of 5 IU PMSG. For the collection of preovulatory oocytes, the mice were sacrificed. Using a stereo microscope and a micropipette, antral follicles were punctured to recover oocytes. For the collection of post-ovulatory oocytes, 5 IU human chorionic gonadotropin (hCG) were injected 48 hours post PMSG to stimulate ovulation. 13 hours post-hCG the female mice were sacrificed by cervical dislocation. The oocytes were isolated from the ampulla and the proximal oviduct. Oocytes were liberated from surrounding cumulus cells by digestions in M2 medium containing 0.3 mg/ml hyaluronidase (22.5 - 45 IU/ml, Sigma) for 2-3 minutes. A comprehensive protocol is found in (Hogan et al, 1994). Two-cell embryos were isolated at day 1.5 post ovulation. Ovary transplantation. Ovaries of C57BL/6 mice were dissected from the bursa bilaterally of each female. Dissections were performed on two females simultaneously (one fetuin-B deficient and one wildtype). Eleven females of each genotype of similar age from the same mouse colony were used. Explanted ovaries were kept in PBS supplemented with 10% FCS until required for transplantation. After 18 days the females were caged with wildtype males to test their fertility and the offspring was genotyped. Genomic DNA of tail biopsies of ear marked individuals was isolated by Proteinase K digestion. Genotyping was performed using the following primers: P4as 5'-GCT TGA ACG ATG GGA TAG GC-3 ' (SEQ ID NO: 10), P6s 5'-CAA GTT CTA ATT CCA TCA GAA GC-3' (SEQ ID NO: 11) and P8s 5'-GGG CCT GCT CAG TGT CTA CC-3' (SEQ ID NO: 12).
Fetuin-B Western blot (human). The assay was done like described above for mouse. Commercial bovine fetuin preparations (7.5 μg) from various sources were separated by SDS PAGE (10% acrylamide). Primary antiserum rabbit anti-human fetuin-B was diluted 1 : 1000 and swine anti-rabbit IgG/HRP (Dako) as secondary antibody was diluted 1 :2000. In vitro fertilisation and embryo transfer. Oocytes were collected after superovulation as described in the paragraph "collection of oocytes". Sperm was collected from the cauda epididymis and vas deferens of 14-16 week old males and incubated for 60 minutes at 37°C. Sperm was counted and a final concentration of 1- 2.5xl06 sperm per ml was mixed with 2-5 COCs. Following 4 hours of incubation the zygotes were washed four times to remove excess sperm and any residual debris. 24 hours later the embryos were evaluated and counted. In another group of oocytes the zona pellucida was twice perforated using a laser before the addition of sperm. All incubations were done at 37°C in gassed (5% C02, 5% 02, 90% N2) Cook MVF medium covered with mineral oil. CD-I females 6 weeks of age were mated with vasectomized males to induce pseudopregnancy. 1.5 days post mating the embryos derived from laser-assisted in vitro fertilisation were transferred into the pseudopregnant mice following established protocols (Hogan et al, 1994). 15 wildtype and Fetub-/- derived embryos were implanted each per mouse. Fetuses were dissected at embryonic day 16.
Oocyte diameter and ZP thickness. Oocyte diameter as well as ZP thickness was determined using a microscope and DISKUS image acquisition and analysis software (Carl Hilgers, Konigs winter).
Zona pellucida digestion. Oocytes were isolated as described. ZP digestion was done like reported before using a 0.2 mg/ml alpha-chymotrypsin (10 IU/ml) solution (Gulyas and Yuan, 1985). The percentage of zona free oocytes was plotted against the time. Data were plotted as a sigmoidal time curve with variable slope using GraphPad Prism statistics software. T50 the time point at which 50% of oocytes had become zona free was calculated using a four parameter logistic regression.
Sperm binding. Oocytes and 2-cell embryos were collected as described in the paragraph "collection of oocytes". Sperm was collected and capacitated as described in the paragraph "in vitro fertilisation". Sperm was counted and a final concentration of lxl 06 sperm per ml was mixed with 30-50 oocytes or 2-cell embryos, respectively. After 1 hour of incubation oocytes were washed up to 10 times employing 0.1 ml medium in each washing step to remove all but two to six sperm on normal two-cell embryos (negative control). Oocytes were fixed in 2% para-formaldehyde, and sperm binding was quantified by a microscope and DISKUS image acquisition and analysis software (Carl Hilgers, Konigswinter).
ZP2 Western blot. 20-30 oocytes from wildtype and Fetub-/- mice as well as two-cell embryos were isolated, dissolved and separated by a 8% polyacrylamide SDS PAGE to detect cleavage of ZP2 as described before (Rankin et al., 2003).
Recombinant fetuin-A and fetuin-B production. Mouse full-length fetuin-A cDNA (Yang et al, 1992) was amplified using the primers (sense) 5 '- AGA ATT CGA GCA ACC ATG AAG TCC CTG -3 ' (SEQ ID NO: 13) and (antisense) 5'- TAC CGG TGC GTC CCT CAA TGA TTT TGA AGT GTC TGA TCC TCC -3' (SEQ ID NO: 14) to introduce a 5' EcoRI restriction site and a 3' Agel restriction site. Correspondingly, fetuin- B cDNA was amplified by PCR using the mouse clone MGC:25848 (BCO 18341) and the primers (sense) 5'- AGA ATT CTT ACA GTG GAA TGG GCC -3' (SEQ ID NO: 15) and (antisense) 5'- ATG ACC GGT GGG TGG GAG AAC CAG -3' (SEQ ID NO: 16) to introduce a 5' EcoRI restriction site and a 3' Agel restriction site. Both constructs were first subcloned into pGEM-T vector (Promega) and transfected into high efficiency JM109 competent cells (Promega). An EcoRI and Agel fragment containing the complete fetuin-A or fetuin-B coding sequence was subcloned into the eukaryotic expression vector pcDNA3.1-V5-His (Invitrogen) to introduce a C-terminal His-tag. Fragments were verified by sequencing, ligated into the adenoviral shuttle vector pAElsplA and transposed into adenovirus (Ad5) as described (Weiskirchen et al, 2000). The resulting viruses were used to infect COS-7 cells. After an overnight incubation period the media was changed to a serum-free culture and incubated for another 24 hours to obtain serum-free cell culture supernatant containing fetuin-A or fetuin-B. The cell culture supernatant was filtered through a 0.45 μιη filter and the His-tagged recombinant fetuin-A or fetuin-B was purified using HisTrap affinity columns and an AKTA liquid chromatography system (GE Healthcare). The fractions containing the purified proteins were pooled and dialyzed against PBS. Recombinant proteins were identified by Western blot, the purity was judged by SDS-PAGE and Coomassie Blue staining.
Ovastacin expression. Mouse full-length ovastacin cDNA (AJ537599.2 at NCBI) was amplified from the EST clone J0247F11 (ATCG, LGC Promochem, Wesel) using the primers (sense) 5'- ATA CTG GCA TGC GTC GAC ACT AGT CCA CCA TGG GTA TCA TGG GAA GCC TG -3' (SEQ ID NO: 17) and (antisense) 5'-TAT GTT AAG CTT ATT ATT TTT CGA ACT GCG GGT GGC TCC AAG ATC TGT CTC TGG GCA CCT CTC TAA T -3 ' (SEQ ID NO: 18) (Carl Roth, Karlsruhe) to introduce a 5 ' Sphl restriction site and a 3' strep-tag-coding region followed Bglll and Hindlll sites. The construct was transferred into pFastBacl (Bac-to-Bac system, Life Technologies, Darmstadt) using the Sphl and Hindlll sites, verified by sequencing (SeqLab, Gottingen) and transposed into baculo virus. The resulting bacmids were used to infect Sf9 insect cells for virus amplification. Protein was expressed in 400 ml suspension cultures of infected Hi5 insect cells at 27°C for 72 hours in a shaking incubator (INFORS, Basel). Cells and debris were removed by centrifugation and the proteins precipitated from the supernatant in 50 mM Tris/HCl, pH 7.4 containing 60% (w/w) ammonium sulfate for 12 hours at 4°C. Protein pellet was obtained by centrifugation (10.000 x g, 2 hours, 4°C), dissolved in 1/10 volume of 50 mM Tris/HCl, pH7.4, 150 mM NaCl, dialyzed vs. the same buffer, loaded onto a strep-tactin sepharose column (IB A, Gottingen) and the purified ovastacin eluted by adding 2.5 mM desthiobiotin (IBA, Gottingen). Mouse full-length ovastacin cDNA deposited under the accession number AJ537599.2 is disclosed in the sequence listing as SEQ ID NO: 19.
Ovastacin inhibition by fetuin-B. Ovastacin activity with or without added recombinant fetuin-B was monitored at 37°C in 50 mM Tris/HCl, pH 7.4, 150 mM NaCl using a thermostatted Varioscan fluorescence plate reader (Thermo Scientific) in FRET (fluorescence resonance energy transfer) mode, and the synthetic peptide substrate Ac- RE(EDANS)-DRnLVGDDPY-K(DABCYL)-NH2 at 10 μΜ (Biosyntan, Berlin; nL = norleucine). IC50 analysis was performed using GRAFIT (Erithacus Software, Wilmington House, UK). Initial rates were normalized to complete substrate turnover subsequently obtained by proteinase K (Sigma- Aldrich) treatment.
Statistics. Data analysis was performed using GraphPad Prism software and the statistical testing methods detailed in the figure legends.
IVF success of in vitro cultured mouse oocytes using recombinant fetuin-B
Oocytes were collected after superovulation as described in the paragraph "collection of oocytes and two-cell embryos". The oocytes were liberated from surrounding cumulus cells by digestion in human tubular fluid (HTF) medium containing 0.3 mg/ml hyaluronidase (22.5 - 45 IU/ml, Sigma), unsupplemented, or supplemented with 0.1 mg/ml fetuin-B, for 2-3 minutes at 37°C. For in vitro culture, 25-30 oocytes were kept in 200 μΐ HTF with or without fetuin-B, and were fertilized after 0 h, 1 h, 3 h, 5 h, 7 h, 9 h, 12 h, 14 h, 17 h, and 25 h.
Sperm for fertilization was collected from the cauda epididymis and vas deferens of 14-16 week old males in 90 μΐ TYH medium and incubated for 60 minutes at 37°C. Fresh sperm was isolated for each time point of fertilization of oocytes. 10 μΐ diluted sperm were added to 200 μΐ of HTF with oocytes. Following 4 hours of incubation, the zygotes were washed four times to remove excess sperm and any residual debris. 24 hours later the embryos were visually inspected using a microscope and the percentage of fertilized oocytes (2-cell stage embryos) was determined.
All incubations were done at 37°C in gassed (5% C02, 5% 02, 90% N2) medium covered with mineral oil.
Human fetuin-B ELISA A 96 well-microtiterplate (Nunc maxisorp) was captured with 4 μ /ηι1 capture antibody (R&D) in PBS (0,2 μΜ sterile filtered), ΙΟΟμΙ/well. After incubation overnight by room temperature (RT), the wells were washed 4 times and blocked with 1% Probumin (bovine serum albumin diagnostic grade, Millipore) in PBS (0,2 μΜ sterile filtered). After washing, human serum (diluted 1 :900 - 1 : 1800 in blocking buffer, regression line with recombinant human fetuin-B (R&D) from 8,0 - 0,125 ng/ml) was added at ΙΟΟμΙ/well and incubated for 2h at RT. After washing, detection antibody (R&D) with 2 μg/ml in blocking buffer, ΙΟΟμΙ/well, was incubated for 2 h at RT. After washing, streptavidin/HRP (1 :200 in blocking buffer), ΙΟΟμΙ/well, was incubated for 20min at RT. After washing, substrate solution (1% TMB in citrate/acetate buffer + H202), ΙΟΟμΙ/well, was added for 20 min. Stop solution was added and measurement was performed with absorbance at 450nm and 570nm.
Results
Fetuin-B Deficient Female Mice are Infertile Due to an Early Block to Fertilisation
We inactivated fetuin-B by targeted Fetub gene deletion in mice (Figure 1 A-D) following established protocols (Hogan et al, 1994). Hemizygous Fetub+/- mice had half the serum fetuin-B of wildtype Fetub +/+ littermates, and homozygous Fetub-I- mice had no detectable serum fetuin-B (Figure IE). Serum fetuin-A, which is encoded by Ahsg/Fetua, the immediate neighboring gene located 19 KB upstream of the Fetub gene on chromosome 16 in mice (chromosome 3 in humans) was not affected (Figure IF). We found no differences in fetuin-B deficient mice compared to wildtype mice in body weight, systolic and diastolic blood pressure, pulse rate and blood chemistry (data not shown), but females were completely infertile. The infertility was observed exclusively in Fetub-/- female mice (Figure 2A). Heterozygous females remained fertile and had mean litter sizes comparable to wildtype females. Males of either genotype were fertile, too. The infertility of Fetub-I- female mice was independent of their genetic background and occurred in both C57BL/6 and DBA/2 inbred mouse strains (Figure 6).
To determine the time of infertility manifestation in Fetub-I- females, we harvested oocytes at various times following natural mating. One and a half days post mating, we routinely recovered 2-cell embryos from oviducts of wildtype females, but never from Fetub-I- females (n = 6, not shown). This finding suggested that infertility was caused by an event prior to the formation of 2-cell embryos. To study earlier events in fertilisation we recovered oocytes from naturally mated mice at day 0.5 post mating. Figure 2B shows that both wildtype and Fetub-I- females had well developed cumulus oocyte complexes (COCs) suggesting that ovulation had occurred in both genotypes. Figure 2C illustrates one of 14 oocytes recovered from 2 wildtype mice that were all fertilised showing a second polar body and a well-developed perivitelline space. In contrast, no oocyte out of 54 oocytes recovered from 9 female Fetub-/- mice showed signs of fertilisation (Figure 2C). Thus, an early block to fertilisation most likely caused the infertility in female Fetub-I- mice.
Female Infertility is Rescued by Liver-Derived Fetuin-B
We analyzed reproductive tract morphology, follicle development, ovulation rates, hormonal status as well as sperm motility and chemoattraction, and detected no difference between Fetub-I- and wildtype animals (data not shown). In vivo, fertility was restored by transplanting postpubertal Fetub-/- ovaries into ovariectomized isogenic wildtype recipients (Figure 3A). After subsequent mating of the recipients with wildtype males, 7 of 11 transplanted female mice produced a total of 37 offspring, indicating that plasma fetuin- B was necessary and sufficient to restore the fertility of the Fetub-I- ovaries.
Next, we asked how fetuin-B reached the oocytes despite the virtual absence of ovarian expression compared to liver expression (Denecke et al, 2003). Western blotting detected fetuin-B in human follicular fluid in amounts similar to human serum suggesting transfer of fetuin-B from blood into follicular fluid (Figure 3B).
Fetuin-B Deficient Oocytes Undergo Premature Zona Pellucida Hardening
Earlier work (George and Johnson, 1993; Kalab et al, 1991, 1993; Schroeder et al, 1990; Xu et al, 2011) had demonstrated that commercial fetuin preparations partially prevent ZPH. The authors could not discriminate between fetuin-A and fetuin-B at the time, because the existence of two distinct fetuin proteins was unknown until the year 2000. Considering that fetuin-A deficient mice have a severe phenotype of soft tissue calcification, yet are fully fertile (Jahnen-Dechent et al, 1997; Schafer et al, 2003) we reasoned that the infertility of the Fetub-/- mice was due to defective ZP maturation in the absence of fetuin-B and that fetuin- AJ Ahsg preparations that had prevented ZPH in vitro, had in fact contained fetuin-B. Western blot analysis of bovine fetuin preparations indeed scored positive for both fetuin-A and fetuin-B (Figure 3B). Thus the demonstrated ZPH inhibition effected by bovine fetuin preparations was likely due to their fetuin-B content. Therefore, we reasoned that the defective ZP observed in fetuin-B deficient oocytes was due to premature ZPH. We performed IVF with and without laser perforation of the ZP to test this hypothesis. Conventional IVF of fetuin-B deficient oocytes using fertile sperm remained unsuccessful, whereas wildtype oocytes became fertilised as shown in figure 3C and D. In contrast, laser beam-induced perforation of the ZP followed by IVF was met with success (Figures 3E, F). The zygotes developed into two-cell embryos in vitro, and further into fetuses when transferred to wildtype foster mothers as shown in figure 3G-L. Thus, fetuin-B deficient oocytes could only be fertilised after overcoming the ZP barrier by laser perforation, while further fetal development was unaffected.
To study potential oocyte defects, we first analyzed general oocyte morphology. The diameter of fetuin-B deficient oocytes was comparable to wildtype oocytes (Figure 4A). However, ZP thickness of fetuin-B deficient oocytes (6.54 μιη ± 0.95 μιη) was significantly reduced compared to unfertilised wildtype oocytes (7.58 μιη ± 0.96 μιη), but was indistinguishable from fertilised two-cell embryos (6.95 μιη ± 0.70 μιη), which have a physiologically hardened ZP (Figure 4B).
ZPH is associated with reduced susceptibility to proteolytic degradation (Inoue and Wolf, 1974). Thus we measured chymotrypsin-mediated ZP digestion time in unfertilised fetuin- B deficient and wildtype oocytes, as well as two-cell embryos, which represent the physiologically hardened ZP phenotype (for each condition n > 20; 4 independent experiments). Figure 4C shows that tso, the time required to digest the ZP in half of the oocytes in each matching series, was 5 times longer in fetuin-B deficient oocytes (31.5 min ± 4.7 min) than in unfertilised wildtype oocytes (6.4 min ± 0.9 min). Furthermore, the ZP digestion time of fetuin-B deficient oocytes was comparable to that of wildtype two-cell embryos (38.5 min ±6.0 min), indicating that the ZP structure of fetuin-B deficient oocytes resembled a hardened phenotype.
Post fertilisation oocyte ZPH results in a block to further sperm binding thus preventing polyspermy. Figure 4D shows that oocytes obtained from superovulated FetuB-/- mice indeed had strongly reduced sperm binding compared to wildtype oocytes, supporting the view that oocytes from FetuB-/- had undergone ZPH even without fertilisation.
Proteolytic cleavage of ZP2, a hallmark of ZPH (Bleil et al, 1981), was analyzed using monoclonal antibody directed against ZP2 (Rankin et al, 2003). Western blots of extracts prepared from unfertilised wildtype oocytes showed a signal at 120 kDa that corresponds to uncleaved ZP2 protein (Figure 4E). Two-cell embryos showed an additionial signal at 90 kDa that corresponds to the cleaved ZP2f protein as expected. Postovulatory fetuin-B deficient oocytes also showed the cleaved ZP2f protein band. In contrast, preovulatory fetuin-B deficient oocytes only showed the uncleaved ZP2 protein suggesting that ZPH started at the time of ovulation. These results indicate that the ZP of ovulated fetuin-B deficient oocytes had indeed hardened prematurely and, accordingly, that infertility in Fetub-/- females was due to premature ZPH.
Fetuin-B Inhibits Ovastacin and Thus Zona Pellucida Hardening
A recent publication demonstrated that the cortical granula protease ovastacin is critically involved in definitive ZPH by cleaving ZP2 (Burkart et al, 2012). Because other astacin metalloproteases such as meprins are effectively inhibited by cystatinlike protease inhibitors including fetuin-A (Hedrich et al, 2010), we tested if recombinant mouse fetuin- B inhibited mouse ovastacin activity. To this end, we cloned mouse fetuin-A and fetuin-B into adenoviral expression vectors and purified His-tagged fusion proteins from virus infected COS cell supernatants. In addition, we expressed recombinant ovastacin in insect cells. Figure 4F shows that activated ovastacin was inhibited to background activity by recombinant fetuin-B (IC50 76.4 nM ± 3.35 nM), but not by recombinant fetuin-A. This finding suggests that premature ZPH triggered by spontaneous cortical granula release of ovastacin should be entirely prevented by the micromolar concentrations of fetuin-B present in plasma and follicular fluid.
Recombinant murine fetuin-B improves IVF success of in vitro cultured mouse oocytes
The present inventors have shown that the rate of successful IVF of oocytes is enhanced if the oocytes are immediately contacted with fetuin-B after their isolation from the female body. This is shown in Figure 9. It can be seen that the immediate contact with fetuin-B directly after isolation results in a higher rate of fertilized embryos over a long incubation period, as compared to oocytes which have not been contacted with fetuin-B after isolation. Not until about 17 hours incubation, the percentages of fertilization equalized and tended towards zero. The results indicate that the immediate contact of oocytes after their isolation with culture medium comprising fetuin-B maintain the fertilization success at a higher level, compared to medium without added fetuin-B.
Fetuin-B serum level is associated with serum estradiol
Serum samples from patients undergoing hormone stimulation for IVF (FSH) were analyzed for estradiol and fetuin-B (sandwich ELISA). The data were pooled from >20 patients with serum samples taken at various time points during hormone treatment. Figure 10 shows an enhancement of estradiol over time in view of FSH treatment. Figure 10 also shows a clear association between increasing serum estradiol levels and increase of serum fetuin-B levels (Pearson correlation test, n=102, r=0.1906, p<0.0001). These results indicate that enhancement of serum estrogen levels due to FSH treatment results in enhanced fetuin-B levels.
Serum fetuin-B correlates with IVF success
The serum fetuin-B level was measured at different time points in serum samples of patients undergoing a hormonal treatment with FSH for stimulation of production of oocytes for use in IFV. The data were collected with regard to IVF success or IVF failure. In patients with successful IVF, the plasma fetuin-B level increased during the hormone therapy (p < 0.0001), as shown in figure 11 A. In patients with IVF failure, fetuin-B levels remained constant (Figure 11 B). These results indicate that in patients with IVF success, FSH treatment is associated with enhancement of serum fetuin-B level. In contrast thereto, where FSH does not result in an enhancement of serum fetuin-B level, IVF failure results. Ethinyl estradiol increases serum fetuin-B
Serum fetuin-B was measured in a woman on contraceptive treatment using a combined gestagen / ethinyl estradiol (EE2) formulation. From day 5 of hormonal contraception, serum fetuin-B increased up to twice, as can be seen in figure 12 A. In a woman on a different contraceptive treatment regime, medication was changed from gestagen alone (day -6 to 0) to gestagen plus EE2, showing that EE2, but not gestagen increased serum fetuin-B (figure 12 B). Collectively, these results show that fetuin-B is produced constitutively, and that normal fluctuations in serum estrogen do not affect serum fetuin-B. Treatment with the estrogen agonist EE2 or endogenous estrogen levels, like the ones attained upon FSH stimulation for IVF, can, however, further enhance serum fetuin-B. In summary, figures 10 to 12 show that an enhancement of serum fetuin-B levels over basal levels of a female individual induced by treatment with a substance which is capable of enhancing serum fetuin-B level in a female individual results in IVF success with a high fertilization rate.
Discussion
Infertility affects about 15% of couples who wish to have children worldwide. A great number of hormones and growth factors have been shown to affect female fertility. Crosstalk between somatic cells and oocytes, as well as endocrine signaling are necessary for normal folliculogenesis and ovulation. For example, in the absence of the oocyte secreted growth and differentiation factor-9 (GDF-9) an arrest at the primary follicle stage is observed. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are synthetized by the anterior pituitary, regulate follicle growth during the preovulatory stage and initiate COC ovulation, respectively. Granulosa cell-derived growth factors such as activins, inhibins, and stem cell factor (SCF/Kit-ligand) are also necessary for follicle development. Among 200 subfertile or infertile genetic mouse models reported, about 50 showed complete female infertility, mostly due to sex hormone disturbance, developmental arrest or implantation defects. Fetuin-B deficient mice add to this group, and to our knowledge, fetuin-B is the first liver-derived non-hormonal plasma protein, which is essential for female fertility.
We demonstrate that fetuin-B inhibits premature zona pellucida hardening (ZPH). Physiological ZPH occurs after fertilisation triggered by the release of cortical granule proteases blocking further sperm binding and penetration. The phenotype of fetuin-B deficient female mice suggests that the absence of fetuin-B causes premature ZPH. First, fetuin-B deficient oocytes showed a thin ZP reminiscent of naturally hardened ZP of two- cell embryos. Second, infertility of fetuin-B deficient oocytes was rescued by ZP laser perforation. Third, the ZP of Fetub-/- mice was more resistant to enzyme digestion than wildtype oocytes, comparable to the physiologically hardened ZP of two-cell embryos, and the sperm oocyte binding was blocked. In addition, we showed that the ZP of Fetub-/- mice contained the cleaved form of ZP2 (ZP2f) immediately after ovulation, whereas normally, ZP2f only forms after fertilisation and ZPH. The fact that preovulatory fetuin-B deficient oocytes only showed the uncleaved ZP2 protein suggested that ZPH was triggered by ovulation. Thus, fetuin-B is critical for fertilisation because it prevents premature ZPH.
To elucidate the molecular mechanism of ZPH inhibition by fetuin-B, we turned to proteases, because fetuins belong to the cystatin superfamily, a group of thiol protease inhibitors (Lee et al., 2009). Fetuin-A is known to interact with several proteases, including trypsin and the astacin-type metalloprotease meprin. Because fetuin-B shares with fetuin-A cystatin-like proteinase-inhibition domains we hypothesized that fetuin-B might inhibit proteases involved in oocyte maturation and fertilisation. Early studies employing golden hamster oocytes showed that ZPH is triggered by the release of cortical granule material including proteases (Barros and Yanagimachi, 1971). ZPH could be induced by several other factors as well. To our knowledge notably none of these factors has been found to interact with ZP2 processing, yet cleavage of ZP2, a hallmark of ZPH, occurred prematurely in Fetub-/- mice.
Prompted by a recent report, which showed that the cortical granula metalloproteinase ovastacin cleaves ZP2 triggering definitive ZPH (Burkart et al, 2012), we studied the molecular interaction of recombinant fetuin-B protein with active ovastacin protease. Indeed, recombinant fetuin-B inhibited recombinant ovastacin at concentrations fully compatible with the measured fetuin-B plasma and follicular fluid concentrations. As for the mode of action we suggest that fetuin-B of 55 kDa can freely diffuse through the ZP, which is permeable for molecules up to 170 kDa (Legge, 1995). Thus fetuin-B can readily antagonize the proteolytic action of prematurely released ovastacin as shown in figure 5. Following fertilisation and degranulation of the cortical granula, the amount of ovastacin will overwhelm the inhibition capacity of fetuin-B, which is in steady state with plasma fetuin-B, but does not increase upon fertilisation.
Successful fertilisation and live birth of Fetub+/- litters after transfer of Fetub-/- ovaries into wildtype recipient mice demonstrated that fetuin-B deficient oocytes are not defective per se. Fetub-/- oocytes, however, required fetuin-B supplied from the blood during oocyte maturation. The fact that embryos obtained by laser-assisted IVF properly developed into fetuses when transferred into wildtype foster mothers, further showed that fetuin-B was critically required before fertilisation, but may be dispensable during later stages of embryonic and fetal live. Thus, addition of fetuin-B to fertilisation media for IVF procedures safeguards against ZPH and against unsuccessful fertilisation even in oocytes deprived of fetuin-B. Moreover, addition of fetuin-B to immature oocytes prevents premature degranulation of cortical granule and premature hardening of the ZP.
The Fetub gene is well conserved in mammals. Because spontaneous ZPH is also reported in humans (Schiewe et al, 1995), alterations in the FETUB gene could also lead to human female infertility. The genome project lists multiple splice variants and more than 200 non- synonymous allelic variants for FETUB that might potentially affect the function of human fetuin-B protein (http://browser.lOOOgenomes.org/Homo_sapiens/GeneA^ariation_Gene/Table?db=core;g= ENSG00000090512;r=3: 186353758-186370930). Because of lack of knowledge about the structure-function relationship between fetuin-B and ovastacin, we do not know if any of these variants are functional. One would predict, however, that IVF should fail in the absence of functional fetuin-B because of blocked oocyte-sperm binding. A study of Liu and Baker showed that about 6% of failed IVF with low sperm-ZP interactions had normal sperm morphology (Liu and Baker, 2000), and thus could be due to defects of the ZP. In these patients intracytoplasmatic sperm injection (ICSI) is used to overcome the ZP barrier, both at higher costs and considerable loss of the number of implantable embryos (Liu et al, 2004). Addition of fetuin-B to oocytes at the earliest convenience might improve IVF success in humans with mutations in the FETUB gene. In conclusion, fetuin-B represents a novel target for fertilisation biology. A network of protease (ovastacin), protease inhibitor (fetuin-B) and hardening substrates (ZP proteins), is oddly reminiscent of blood coagulation, an equally important and medically relevant biological cascade, which is just celebrating the introduction of proteinase inhibitors as so- called 2nd generation blood clotting inhibitors. We envisage similar developments in reproductive biology may become possible.
Treatment of a female individual with a substance which is capable of enhancing serum fetuin-B level in a female individual such as estrogens or a hormone such as FSH for stimulation of oocyte production in the female individual for use in IVF whereby the hormone attains an increase of endogenous estrogen levels results in enhanced serum fetuin-B. Enhanced serum fetuin-B levels effect the production or maturation of oocytes with a high rate of fertility, resulting in an enhanced fertilization of the oocytes in a subsequent IVF. Therefore, especially IVF patients in whom serum fetuin-B levels remain unchanged or increase to a small extent only upon treatment with a hormone such as FSH for stimulation of oocyte maturation for use in IVF, the administration of estrogens or other substances which are capable of enhancing serum fetuin-B level is useful. Administration of such substances results in increase of fetuin-B levels which in turn enhance fertility of oocytes. The capability for high fertilization of such oocytes is maintained by placing the oocytes into a medium comprising fetuin-B immediately after isolation from the female individual.
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Claims

1. A method for culturing an ovary tissue comprising an oocyte, comprising contacting the ovary tissue with a medium comprising fetuin-B immediately after isolation of the ovary tissue from a female body.
2. The method of claim 1, wherein the ovary tissue comprising an oocyte has been isolated from a female individual, wherein the serum fetuin-B level in the female individual is enhanced due to the treatment with a substance which is capable of enhancing the serum fetuin-B level in the female individual.
3. The method according to claim 2, wherein the substance is a hormone which is used for stimulation of the production of oocytes for in vitro fertilization, an estrogen or an estrogen agonist.
4. The method according to claim 3, wherein the hormone used for stimulation is FSH (follicle stimulating hormone) or a derivative thereof, or
wherein the estrogen is estrone, estradiol, estriol, estetrol (E4), ethinylestradiol, mestranol, 1 Ιβ-methyl-ethinylestradiol, turisteron, moxestrol (1 Ιβ-methoxy- ethinylestradiol), 6-dehydroestrone, 17-deoxyestradiol, 2-hydroxyestradiol, isoestradiol (8a-estradiol), 2-methylestradiol, 4-methylestradiol, polyestradiol- phosphate, promestriene, 2-chloroestradiol, 1,1 Ιβ-ehanoestradiol, diethylstilbestrol, dienestrol, dimestrol, chlorotrianisene, stilbestrol-monobenzyl-ether, fosfestrol or homoestradiol.
5. The method according to any one of claims 1 to 4 for stabilising the ovary tissue.
6. Use of fetuin-B for stabilising an ovary tissue comprising an oocyte.
7. The use of claim 6, wherein the ovary tissue comprising an oocyte has been isolated from a female individual, wherein the serum fetuin-B level in the female individual is enhanced due to the treatment with a substance which is capable of enhancing the serum fetuin-B level in the female individual.
8. The use according to claim 7, wherein the substance is a hormone which is used for stimulation of the production of oocytes for in vitro fertilization, an estrogen or an estrogen agonist.
9. The use according to claim 8, wherein the hormone used for stimulation is FSH (follicle stimulating hormone) or a derivative thereof, or
wherein the estrogen is estrone, estradiol, estriol, estetrol (E4), ethinylestradiol, mestranol, 1 Ιβ-methyl-ethinylestradiol, turisteron, moxestrol (1 Ιβ-methoxy- ethinylestradiol), 6-dehydroestrone, 17-deoxyestradiol, 2-hydroxyestradiol, isoestradiol (8a-estradiol), 2-methylestradiol, 4-methylestradiol, polyestradiol- phosphate, promestriene, 2-chloroestradiol, 1,1 Ιβ-ehanoestradiol, diethylstilbestrol, dienestrol, dimestrol, chlorotrianisene, stilbestrol-monobenzyl-ether, fosfestrol or homoestradiol.
10. The method according to any one of claims 1 to 5 or the use according to any one of claims 6 to 9 for maintaining the fertilisable state of the ovary tissue.
11. The method according to any one of claims 1 to 5 or 10 or the use according to any one of claims 6 to 10 for preventing hardening of the zona pellucida of the oocyte.
12. The method according to any one of claims 1 to 5, 10 or 11 or the use according to any one of claims 6 to 11 for inhibiting the activity of ovastacin.
13. The method according to any one of claims 1 to 5 or 10 to 12 or the use according to any one of claims 6 to 12, wherein the oocyte is an immature oocyte.
14. The method according to any one of claims 1 to 5 or 10 to 13, or the use according to any one of claims 6 to 13, wherein the ovary tissue is an oocyte, a follicle, a cortex of an ovary or an ovary.
15. The method according to any one of claims 1 to 5 or 10 to 14, or the use according to any one of claims 6 to 14, wherein fetuin-B is recombinant fetuin-B.
16. The method according to any one of claims 1 to 5 or 10 to 15, or the use according to any one of claims 6 to 15, wherein fetuin-B is human or mouse fetuin-B.
17. A substance which is capable of enhancing the serum fetuin-B level in a female
individual for use in a method for enhancing fertility of an oocyte, the method comprising,
(a) administering to a female individual a substance which is capable of enhancing the serum fetuin-B level in a female individual;
(b) isolating ovary tissue comprising an oocyte; and
(c) contacting the ovary tissue with a medium comprising fetuin-B immediately after isolation of the ovary tissue from the female body.
18. The substance which is capable of enhancing the serum fetuin-B level in a female individual for use in a method for enhancing the fertility of an oocyte of claim 17, wherein the substance is as defined in claim 3 or 4.
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