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WO1995016916A1 - Procedes et reactifs pour l'evaluation de sperme - Google Patents

Procedes et reactifs pour l'evaluation de sperme Download PDF

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Publication number
WO1995016916A1
WO1995016916A1 PCT/AU1994/000772 AU9400772W WO9516916A1 WO 1995016916 A1 WO1995016916 A1 WO 1995016916A1 AU 9400772 W AU9400772 W AU 9400772W WO 9516916 A1 WO9516916 A1 WO 9516916A1
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WO
WIPO (PCT)
Prior art keywords
clusterin
spermatozoa
irm
binding
acrosomal
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PCT/AU1994/000772
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English (en)
Inventor
Hugh William Gordon Baker
Brendan Francis Murphy
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University of Melbourne
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University of Melbourne
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Publication date
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Priority to AU12689/95A priority Critical patent/AU1268995A/en
Publication of WO1995016916A1 publication Critical patent/WO1995016916A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

Definitions

  • the present invention relates generally to methods of assessing spermatozoa morphology and to reagents and kits used in such methods.
  • spermatozoa in a sample.
  • a laboratory technician makes an assessment as to the normality of the. spermatozoa and likelihood of infertility.
  • a high proportion of morphologically abnormal spermatozoa is an indicator of infertility.
  • the proportion of sperm with intact acrosomes in a given sample is related to the likelihood of success of in vitro fertilization
  • acrosome status and sperm morphology provide very useful prognostic information for advising patients about their chances of achieving fertilization by standard in vitro fertilization procedures and for selection of patients likely to have poor results with assisted fertilization techniques such as intracytoplasmic sperm injection (Liu and Baker, 1992).
  • Current methods of assessment are necessarily subjective and labour intensive requiring at least two sperm preparations and a specialised epifluorescence microscope for assessing the human acrosome (WHO 1992, Liu and Baker, 1992).
  • Clusterin is an 80 kD glycoprotein dimer made of and ⁇ subunits. It is found at several sites in the human male reproductive tract (O'Brien et al, 1993). It has been suggested that clusterin has an array of functions ranging from inhibition of autologous complement attack (Murphy et al, 1989, Jenne and Tschopp, 1989), lipid transport (de Silva et al, 1990), programmed cell death (Buttyan et aL, 1989, Lee and Sensibar, 1987), cell maturation (Harding et al, 1991, O'Bryan et al, 1993A) and aggregation of cells in vitro (Fritz et al, 1983).
  • clusterin is a major product of Sertoli cells, epididymal principal cells and seminal vesicle principal cells (O'Bryan et al, 1993, Sylvester et al 1984, Hermo et al, 1991, Aulitzky et al, 1992).
  • morphologically normal spermatozoa and morphologically abnormal spermatozoa substantially have distinguishable forms of clusterin. Specifically the present inventors have discovered that morphologically abnormal spermatozoa display the 80 kD dimeric clusterin form over substantially the whole of the spermatozoa whereas morphologically normal spermatozoa have a different form of clusterin, "acrosomal clusterin", substantially associated with the acrosomal cap of the spermatozoa. Although not intending to be bound by theory at present this different form of clusterin is thought to be the ⁇ subunit or a portion or variant thereof.
  • the object of the present invention is to provide a more reliable method for investigating spermatozoa morphology and, where required, acrosomal status of spermatozoa.
  • the present invention provides a method of assessing spermatozoa morphology in a spermatozoa sample comprising contacting said sample with an immunologically reactive molecule (IRM) capable of binding to at least one form of clusterin under conditions and for a time sufficient to allow binding of said IRM and clusterin, and detecting binding of said IRM to said clusterin, wherein the distribution of said IRM binding to abnormal spermatozoa is different to the distribution of IRM binding to normal spermatozoa.
  • IRM immunologically reactive molecule
  • the present invention provides a method of assessing acrosomal status of spermatozoa comprising contacting a sample of spermatozoa with an IRM reactive with the acrosomal form of clusterin as described herein under conditions and for time sufficient to allow binding of said IRM and clusterin, and detecting binding of" said spermatozoa and IRM, wherein morphologically normal spermatozoa which have not undergone an acrosomal reaction will display IRM binding to the acrosomal region.
  • the present invention provides a kit for assessing spermatozoa morphology and/ or acrosomal status comprising a first part or a multiplicity of parts adapted to receive a spermatozoa sample or samples, a second part or multiplicity of parts adapted to contain an IRM labelled with a reporter as herein described and capable of binding to at least one form of clusterin, and optionally a third part or parts adapted to contain substrate, buffers and other reagents necessary for detecting binding of said IRM to spermatozoa.
  • FIGURES Fig ⁇ re 1 A photographic representation of human spermatozoa stained for clusterin using avidin-biotin amplified immunoperoxidase technique and the G7 and E5 mAbs. All fields are magnified 1000X except Figure 1 E (X2,000).
  • G The distribution of G7-reactive clusterin on spermatozoa prepared by the swim-up method. Only those remaining abnormal (Ab) spermatozoa were stained for clusterin using this mAb. Using spermatozoa prepared by the swim-up technique, spermatozoa with diffuse clusterin are infrequent.
  • H Swim-up preparation of human spermatozoa stained for clusterin using the E5 mAb after induction of the acrosome reaction with calcium ionophore. Spermatozoa which lack acrosomal clusterin staining (N) may have spontaneously undergone the acrosome reaction.
  • Figure 2 A graphic representation which compares pelleted spermatozoa and swim- up spermatozoa in the distribution of G7-reactive and E5-reactive clusterin.
  • Spermatozoa were prepared using each technique from halves of the same four seminal plasma samples.
  • Panels A and B show the reduction, after swim-up preparation, in the number of spermatozoa with diffuse clusterin staining using both antibodies.
  • Panel C shows that spermatozoa with acrosome E5-reactive clusterin are more likely to be selected by the swim-up method.
  • Panel D illustrates that the swim-up method selects for spermatozoa which do no-L have surface G-7 reactive clusterin.
  • the present invention provides a method of assessing spermatozoa morphology in a spermatozoa sample comprising contacting said sample with an immunologically reactive molecule (IRM) capable of binding to at least one form of clusterin under conditions and for a time sufficient to allow binding of said IRM and clusterin, and detecting binding of said IRM to said clusterin wherein the distribution of said IRM binding to abnormal spermatozoa is different to the distribution of IRM binding to normal spermatozoa.
  • IRM immunologically reactive molecule
  • the spermatozoa sample used in the method of the invention can be any spermatozoa sample in which the clusterin forms differ between morphologically normal and morphologically abnormal spermatozoa and where this difference is detectable with an IRM.
  • this inventors' discovery has been specifically observed in humans, the spermatozoa of other animals including domestic animals such as cattle, sheep, goats, alpacas, llamas and horses may also be assessable in the method of the present invention.
  • the term "IRM” means any molecule capable of participating in an immunological reaction and displaying differential binding ability.
  • the IRM is preferably an antibody and advantageously a monoclonal antibody (mAb).
  • antibody includes naturally occurring antibodies, recombinant antibodies, synthetic antibodies including fusions or chimers of antibodies, fragments of any of the foregoing such as Fab and F(ab') 2 and genetically engineered scantibodies (See Skerra A. Bacterial expression of immunoglobulin fragments Curr. Opin. Immunol. 1993; 5:
  • the molecule may be encoded by a naturally occurring or synthetic nucleotide sequence and expressed in any convenient expression system such as those disclosed in Sambrook et al (1989) and other Genetic Engineering Laboratory Manuals.
  • the molecule is synthetic, it is conveniently prepared by a stepwise addition of single amino acid groups or amino acid fragments of, for example antibodies.
  • the synthetic antibody may be a fusion or chimeric antibody comprising light or heavy chains derived from other antibodies.
  • clusterin used herein includes different forms of clusterin such as the 80 kD form, the ⁇ chain, the ⁇ chain, the acrosomal form of clusterin and mutants, variants, derivatives and parts thereof.
  • mutants and variants cover clusterin forms which display amino acid, glycosylation or other changes when compared to native clusterin, but that retain substantially the same immunological character as native clusterin or a part thereof.
  • part thereof means a portion or fragment of clusterin which retains at least some of the character of native clusterin.
  • derivatives means a polypeptide molecule derived from clusterin which retains at least a part of the native clusterin character.
  • the IRM must be specific for at least one form of clusterin the IRM may be capable of binding more than one form of clusterin.
  • the IRM may bind two forms of clusterin but have a higher affinity for one form than the other.
  • the IRM binds two forms of clusterin (viz the ⁇ subunit or acrosomal clusterin and the heterodimer) it will be understood that the differential clusterin distribution on abnormal and normal spermatozoa will enable these two categories of spermatozoa to be distinguished.
  • the IRM is labelled with a reporter providing, under suitable conditions, a detectable signal.
  • reporter may include molecules such as chemiluminescent molecules, bioluminescent molecules, radio-nucleotides, fluorescent molecules or enzymes amongst others.
  • enzymes include peroxidises such as horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others. Enzymes such as peroxidises maybe used in conjunction with avidin-biotin systems.
  • the reporter may consist of particles including colloidal gold, polyacrylamide or latex microbeads amongst others.
  • the phrase “distribution of antibody binding” refers to the physical distribution over the surface or exposed areas of the spermatozoa such as at the head, midpiece and tail and includes whether the binding is intense or diffuse.
  • Detecting of binding of said IRM can be achieved by any convenient detection means.
  • the detection means is one that provides a good visual signal detectable upon microscopic examination.
  • the IRM is labelled directly by a reporter as herein described, such as a reporter enzyme, or by reporter particles such as colloidal gold or biosensers.
  • the presence of antigen- antibody complex may be detected by an anti-immunoglobulin labelled with a label, reporter or other detector molecule capable of providing a detectable signal.
  • the anti-immunoglobulin binds to the bound clusterin reactive antibody and then the label, reporter or detector molecule is read.
  • Suitable reporters are as hereinbefore described.
  • Assessment of samples may be aided by automated means such as computer assisted methods for image analysis of IRM binding patterns on spermatozoa.
  • the IRM reacts with both the 80 kD form and the different form of clusterin ("acrosomal clusterin") described above so that morphologically abnormal spermatozoa can be distinguished from morphologically normal spermatozoa due to the binding pattern of the IRM to the spermatozoa.
  • the acrosomal region is bound by the IRM whereas in abnormal spermatozoa there is a generalised binding over the whole spermatozoa.
  • the IRM is an antibody, still more preferably a monoclonal antibody.
  • the IRM is mAbE5 or an antibody with the same reactivity or specificity (Murphy et al, J. Clin. Invest. SI: 1858-1864).
  • the advantage of using mAbE5, or an IRM with functionally similar characteristics is that the method of the invention provides an opportunity to assess the proportion of normal sperm with intact acrosomes as well as assess abnormal sperm status of a sample at one time.
  • one slide can be used to assess the two characteristics leads to substantial advantages such as reduced sample preparation time and lower costs.
  • the potential for use of automated computer assisted methods of image analysis reduces the subjectivity of present assessment methods.
  • the present invention provides a method of assessing acrosomal status of spermatozoa comprising contacting a sample of spermatozoa with an IRM reactive with the acrosomal form of clusterin under conditions and for a time sufficient to allow binding of said IRM and said clusterin, and detecting binding of said spermatozoa and IRM, wherein morphologically normal spermatozoa which have not undergone an acrosomal reaction will display IRM binding to the acrosomal region.
  • the spermatozoa sample is fixed or otherwise treated to expose the contents of the acrosome prior to contacting it with the IRM. Fixing may be carried out by standard methods known to those skilled in the art such as by acetone immersion. It will be understood that while the IRM must be reactive with at least the acrosomal form of clusterin it may bind other forms of clusterin. In the latter case the distribution pattern of clusterin on normal and abnormal spermatozoa will enable an assessment of the acrosomal status of the spermatozoa.
  • the IRM is an antibody, still more preferably a monoclonal antibody.
  • the IRM is mAbE5 or an antibody with equivalent reactivity (Murphy et al, J. Clin. Invest. 81: 1858-1864).
  • the spermatozoa may be prepared by an convenient technique such as those described above.
  • the present invention provides a method of assessing spermatozoa morphology comprising contacting a spermatozoa sample with an IRM under conditions and for a time sufficient to allow binding of such IRM and clusterin wherein said IRM substantially binds with one form of clusterin but does not substantially bind with another form of clusterin, and detecting binding of said IRM to said spermatozoa.
  • spermatozoa may be prepared by any convenient technique such as those described above.
  • the invention also extends to an isolated preparation of IRM when used in any one of the methods of the invention.
  • the IRM may be produced by standard methods. For example where the IRM is a monoclonal antibody the methods of Oi & Immunberg 1980) may be used. Where the IRM is a natural antibody the methods of O'Bryan et al 1994 may be utilized. The following methods for making IRMs - u - may be used. These may be in the form of recombinant, synthetic antibodies and scantibodies according to Skerra A and Pluckthun A. Assembly of a functional immunoglobulin FV fragment in E. coli, Science 1988, 240; 1038-1041, Skerra et al
  • the invention extends to an isolated preparation of an IRM specific for one form of clusterin as hereinbefore defined.
  • the IRM is an antibody more preferably a mAb labelled with a reporter as hereinbefore described.
  • the present invention provides a kit for assessing spermatozoa morphology comprising a first part or multiplicity of parts adapted to receive a spermatozoa sample or samples, a second part or multiplicity of parts adapted to contain an IRM labelled with a reporter and capable of binding to at least one form of clusterin and optionally a third part or parts adapted to contain substrate, buffers and other reagents necessary for detecting binding of said IRM to spermatozoa.
  • the reporter associated with the IRM provides, under suitable conditions, a detectable signal.
  • Such reporters may include molecules such as chemiluminescent molecules, bioluminescent molecules, radio-nucleotides, fluorescent molecules or enzymes amongst others. Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the reporter may consist of particles including colloidal gold, polyacrylamide or latex microbeads amongst others.
  • Detecting of binding of said IRM can be achieved by any convenient detection means.
  • the detection means is one that provides a good visual signal detectable upon microscopic examination such as those described above.
  • Clusterin was localised on human spermatozoa using the anti-human clusterin monoclonal antibodies E5 and G7 (Murphy et al, 1988). Western immunoblotting studies using native (heterodimeric) or separated clusterin ⁇ and ⁇ subunits indicate that the E5 mAb is directed against an epitope on the ⁇ -chain. E5 will therefore react with free ⁇ -chains and also with native clusterin heterodimer. The affinity of the E5 mAb however, is greater for free ⁇ -chains than for native heterodimeric clusterin.
  • the G7 mAb is strongly reactive with native heterodimeric clusterin but, does not react with either of the separated clusterin ⁇ and ⁇ subunits.
  • the epitope for which G7 is specific must therefore encompass a portion of both the ⁇ -chain and ⁇ -chain, or be conformationally dependent on the heterodimeric structure.
  • spermatozoa were separated from seminal plasma by centrifugation and washing twice in Tyrode's solution (670g/10min) (Commonwealth Serum Laboratories, Melbourne, Australia). From the remaining 11 semen samples, motile spermatozoa were preferentially isolated using the swim- up technique (Baker et al, 1993).spermatozoa were resuspended in 0.5 ml of Tyrode's solution and the concentrations determined in a counting chamber.spermatozoa were diluted to a concentration of approximately 1 x 10 * spermatozoa/ml in 0.1M phosphate buffered saline, pH 7.4 (PBS). To control for intersample variation, 4 additional semen samples were obtained and divided in half immediately after liquefaction. Spermatozoa were prepared from one half of these samples by centrifugation and from the other half of the samples using the swim-up technique.
  • spermatozoa suspension Fifty microlitres of each spermatozoa suspension were spotted onto glass microscope slides and allowed to air dry. Slides were then fixed by immersion in acetone for 10 min, stored at room temperature and used within 48 hrs of preparation. Prior to immunohistochemistry, smears were re-hydrated by immersion in PBS for 10 min. Clusterin was localised on spermatozoa using an avidin-biotin amplified immunoperoxidase technique. Non-specific antibody binding was blocked by pre- incubating slides in 10% non-immune rabbit serum (DAKOPATTS) for 20 min in a humid environment.
  • DAKOPATTS non-immune rabbit serum
  • the acrosomal region was positive, but the remainder of the spermatozoan was negative. 2. Equatorial.
  • the equatorial segment were positive, but the remainder of the spermatozoan was negative.
  • the acrosome reaction was induced in three human spermatozoa preparations using a calcium ionophore method (Liu and Baker 1990).
  • Spermatozoa prepared by the swim-up method
  • HVF human tubal fluid medium
  • human serum heat inactivated 56 °C, 30 min
  • Acrodisc filter 0.2 ⁇ m: Gelman Sciences Inc., Ann Arbor, ML, USA
  • spermatozoa were incubated with 10 ⁇ M A23187 (Sigma) in HTF for 1 hr at 37 °C in 5% C0 2 in air. After incubation, spermatozoa were pelleted, the supernatant removed, and resuspended in fresh HTF medium. Motility was assessed using phase-contrast microscopy. The percentage of acrosome reacted spermatozoa was determined using FITC conjugated pisum sativum agglutinin (FITC-PSA) (Sigma)(Liu and Baker, 1988) and slides were also prepared and stained for clusterin using the immunoperoxidase method outline above.
  • FITC-PSA conjugated pisum sativum agglutinin
  • spermatozoa As a negative control, two additional swim-up spermatozoa preparations, were treated in exactly the same manner with the exception that they were incubated in HTF without A23187. These control spermatozoa were also assessed for the percentage of acrosome reacted forms using the FITC-PSA, and the distribution of clusterin determined using the immunoperoxidase technique. It will be understood that in normal use of the present invention the acrosome reaction will not be artifically induced prior to treating the spermatozoa with the IRM.
  • Table 1 lists the parameters of semen analysis and the clusterin staining characteristics of the 38 spermatozoa preparations labelled with both the E5 and G7 mAbs. Of the 38 semen samples, 17 were classified as normal using the WHO criteria (WHO 1992). None of the patients had anti-spermatozoa antibodies. Leucocytes, immature germ cells and small spherical bodies were found in variable numbers in many of the samples.
  • E5-reactive acrosomal clusterin is likely to be an internal acrosomal component as no acrosomal region staining was seen in spermatozoa that were unfixed.
  • the acetone fixation used in this study appears to allow access of the E5 monoclonal antibody to the acrosome contents.
  • Example 2 Material and Methods weie as in Example 1 with the exception that the E5 antibody only was used for immunohistology and only washed pellet sperm preparations were used.
  • a further 25 sperm preparations were made from washed pellets of semen samples obtained from 25 additional donors or patients (Andrology laboratory, the Royal Women's Hospital).
  • An antibody against the clusterin ⁇ chain is produced to enhance the ability immunohistologically to detect abnormal sperm.
  • the anti- ⁇ chain reacts with the surface heterodimeric clusterin on abnormal sperm but not the apparently ⁇ chain restricted form of clusterin in the acrosome of normal sperm.
  • Heterodimeric clusterin is cleaved and the chains separated (Murphy 1988) and sheep immunized 3-4 times with ⁇ chain. Antiserum is harvested and tested for reactivity with ⁇ chain and any cross reactivity with ⁇ chain is absorbed using solid phase ⁇ chain. Alternatively mice are immunized with purified ⁇ chain and a series of murine monoclonal antibodies are produced against human clusterin ⁇ chain (Oi and Herzenberg 1980).
  • Example 3 The same methods are used as in Example 3 except an isolated ⁇ -chain of clusterin is employed to -produce a ⁇ -chain specific antibody.
  • the kit contains a first part of parts in the form of a support for microscopic examination such as a microscope slide or slides for the spermatozoa sample.
  • a second part or parts contains the IRM (preferably E5 or an equivalent antibody) coupled to a reporter (such as horseradish peroxidase) and is made available in a final working solution of buffer containing preservative and irrelevant protein for stablization (for example, Tris buffered saline containing 0.05% Na azide and 2% serum albumin).
  • the kit also contains a substrate solution provided as a buffer to which supplied tablets are added prior to use. Wash buffer may be provided as powder sachets to be dissolved in water prior to use. Additional information in the kit includes detailed instructions, photographic examples, cover slips and mounting material.
  • Table 1 Semi.. . analysis parameters of the 38 semen samples used in to prepare sperm for analysis of clusterin distribution.
  • Monoclonal antibody G7 Monoclonal antibody E5 (clusterin heterodimer) (clusterin ⁇ chain)
  • apolipoprotein designated ApoJ is a marker for subclasses human plasma high density lipoproteins. J. Biol. Chem. 265, 13240-13247.

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Abstract

La présente invention concerne principalement des procédés d'évaluation de la morphologie de spermatozoïdes au moyen d'une molécule immunologiquement réactive ainsi que des réactifs et des trousses utilisés selon ces procédés.
PCT/AU1994/000772 1993-12-17 1994-12-14 Procedes et reactifs pour l'evaluation de sperme Ceased WO1995016916A1 (fr)

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AU12689/95A AU1268995A (en) 1993-12-17 1994-12-14 Methods and reagents for sperm assessment

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AUPM3012A AUPM301293A0 (en) 1993-12-17 1993-12-17 Methods and reagents for sperm analysis

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6383808B1 (en) 2000-09-11 2002-05-07 Isis Pharmaceuticals, Inc. Antisense inhibition of clusterin expression
WO2023010101A1 (fr) * 2021-07-30 2023-02-02 Abs Global, Inc. Procédés d'amélioration de la qualité d'un échantillon de cellule reproductrice

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOLOGY OF REPRODUCTION, (1985), 33, pages 177-186, TUNG P.S. and FRITZ I.B., "Immunolocalization of Clusterin in the Ram Testis, Rete testis and Excurrent Ducts". *
BIOLOGY OF REPRODUCTION, (1994), 50(3), pages 502-509, O'BRYAN M.K. et al., "Immunohistological Localization of Clusterin in the Male Genital Tract in Humans and Marmosets". *
EUROPEAN JOURNAL OF BIOCHEMISTRY, (1994), 221(3), pages 917-925, WONG et al., "Molecular Characterization of Human TRPM-2/Clusterin, a Gene Associated With Sperm Maturation, Apoptosis and Neurodegeneration". *
THE JOURNAL OF CLINICAL INVESTIGATION, June 1988, 81, pages 1858-1864, MURPHY B F et al., "SP-40, 40 a Newly Identified Normal Human Serum Protein Found in the SC5b-9 Complex of Complement and in the Immune Deposits in Glomerulonenephritis". *
THE JOURNAL OF CLINICAL INVESTIGATION, May 1990, 85, pages 1477-1486, O'BRYAN et al., "Human Seminal Clusterin". *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6383808B1 (en) 2000-09-11 2002-05-07 Isis Pharmaceuticals, Inc. Antisense inhibition of clusterin expression
WO2023010101A1 (fr) * 2021-07-30 2023-02-02 Abs Global, Inc. Procédés d'amélioration de la qualité d'un échantillon de cellule reproductrice

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