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WO1990013627A1 - Maturation in vitro d'ovocytes bovins - Google Patents

Maturation in vitro d'ovocytes bovins Download PDF

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Publication number
WO1990013627A1
WO1990013627A1 PCT/US1990/002365 US9002365W WO9013627A1 WO 1990013627 A1 WO1990013627 A1 WO 1990013627A1 US 9002365 W US9002365 W US 9002365W WO 9013627 A1 WO9013627 A1 WO 9013627A1
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WO
WIPO (PCT)
Prior art keywords
oocytes
bovine
oocyte
maturation
culture
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
Application number
PCT/US1990/002365
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English (en)
Inventor
Frank L. Barnes
Mark E. Westhusin
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Granada Biosciences Inc
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Granada Biosciences Inc
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Filing date
Publication date
Application filed by Granada Biosciences Inc filed Critical Granada Biosciences Inc
Publication of WO1990013627A1 publication Critical patent/WO1990013627A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/02—Breeding vertebrates
    • 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
    • C12N2501/30—Hormones
    • C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
    • 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
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer

Definitions

  • oocytes With techniques being developed for _in vitro fertilization as well as the more recent nuclear trans ⁇ fer techniques in bovine embryos, a source of readily available oocytes would compliment the procedures available for genetic selection and breeding. Immature ova have been collected from bovine ovaries at abba- toirs, and the follicles were aspirated to release cumulus-oocyte complexes. The oocytes have been tested for maturation in media containing hormones and granu- losa cells. The interaction of granulosa cells with cumulus complexes have been determined to contribute to developmental competence of bovine oocytes.
  • Activation is the term used for the initiation of the developmental program which normally occurs at the time of fertiliza ⁇ tion. Activation can be induced by electropulsation without fertilization of the oocyte. Control over activation is important in nuclear transfer to optimize the time to implant the transferred nuclear material which incorporates into and "reconditions" in the presence of recipient maturing ooplasms.
  • the subsequent development is intended to reflect the genetic make-up of the transferred nuclear material after insertion into an enucleated egg.
  • a suitable supply of immature bovine oocytes exists and is inexpensive.
  • a reliable in vitro maturation procedure would give synchronous and measurable oocyte development. Therefore, a mature supply of oocytes would be available for use for genetic selective breeding or cloning.
  • the present invention is a new method for _in vitro maturation of bovine oocytes.
  • the process can be used to produce oocytes for nuclear transfer, and pregnancies have been confirmed.
  • bovine oocyte _in vitro maturation with the co-culture feature is successful for use in the nuclear transfer process to produce cloned animals.
  • the use of unique recombinant gonadotropins is another feature of the new process to mature the bovine oocytes.
  • the bovine ovaries were collected at abattoirs and maintained and transported in Dulbeccos PBS at 32° to 39°C. The collection and transport time ranged from 1.5 to 3.75 hours past death.
  • the compact cumulus oocyte complexes (COC) were selected from the aspirate of antral follicles. Some follicles were dissected as discussed below.
  • the follicle size was investigated as to the best source of oocytes. The most useful range is 3-8 mm. The preferred follicle size determined to give the best oocytes for further developmental competence were 6-8mm. Although the l-2mm follicles yielded some oocyctes that would mature to develop to morula and blastocyst stage after in vitro fertilization, the development was at a lower frequency. The follicle size was examined in dissected ovaries because 2mm and 3-5mm follicles are difficult to distinguish when measured at the ovarian surface.
  • TCM Tissue Culture Medium
  • HFCS heat inactivated fetal calf serum
  • bFSH recombinant FSH
  • the NIADDK-oFSH is a purified natural FSH from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases (NIADDK) , Baltimore, Maryland.
  • the maturation media also contained lug/ml estradiol and 1% penicillin streptomycin solution.
  • the COC are placed in the maturation media for at least 20 hours.
  • a comparison of other alternative maturation media was performed by subjecting the oocytes from the dif ⁇ ferent media to jLn vitro fertilization and monitoring subsequent development.
  • the maturation media described above was supplemented with 0.01 units/ml bLH (recombi ⁇ nant LH from Integrated Genetics, Inc.) resuspended in BSA and mannitol solution which is equivalent to 0.01 units of NIADDK-oLH standard.
  • the NIADDK-oLH is a purified natural LH from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, Baltimore, Maryland.
  • Alternative commercially available FSH and LH such as pituitary FSH (pFSR) and pituitary LH (pLH) can be used.
  • the oocytes were matured in TCM 199, lug/ml estradiol and either 0.01 units/ml of bF ⁇ H or 0.01 and 0.012 units/ml of bFSH and bLH, respectively, supple ⁇ mented with 10% HTFCS.
  • the oocytes were cultured at 39°C 5% CO- and air.
  • the oocytes were then assayed for developmental competence by _in vitro fertilization followed by culture in ovine oviducts for six days.
  • the addition of bLH adversely affected developmental compe ⁇ tence of _in vitro matured oocytes although there was some development to the blastocysts and morula stage.
  • TABLE 1 is a summary of the in vitro fertilization results with use of the recombinant gonadotropins.
  • recombinant gonadotropins bFSH and bLH in maturation media was compared to the media with the natural gonadotropins oFSH and oLH supplied by NIADDK. Maturation was carried out in a maturation culture media TCM 199 with 10% HTFCS, 1% penicillin and streptomycin, 1 ug/ml estradiol, 0.01 units/ml of NIADDK-oFSH, 0.012 units/ml of NIADDK-oLH in one media preparation and the equivalent amounts of bFSH and bLH in another media preparation.
  • Bovine oocytes were matured as described above and subjected to in vitro fertilization. The fertilized oocytes were cultured in ovine oviducts and. a development to morula or blastocysts was comparable.
  • TABLE 2 shows the results of the NIADDK gonadotropins and the recombinant gonadotropins.
  • the recombinant gonadotropins compare favorably to the NIADDK standards.
  • TABLE 2 COMPARISON OF GONADOTROPINS
  • In vitro oocytes were placed in maturation culture containing both oFSH and oLH to study the ability to activate. Activation was defined as the completion of meiosis and the progression of a metaphase II oocyte to a pronuclear egg.
  • the jLn vitro matured oocytes were removed from the maturation culture media over intervals of 26 to 32 hours as shown in TABLE 3.
  • the oocytes were electropulsed under the same conditions as those used for fusion according to "Bovine Nuclear Transplanta ⁇ tion", cited below and incorporated by reference. The following data shows that oocytes 30 hours and older have increased activation potential. The results of the activation procedure are shown below in TABLE 3.
  • the in vitro oocyte maturation process which sustains nuclear transfer and embryonic development includes a further co-culture procedure.
  • the COC were removed from the maturation media after 20 to 24 hours, preferably 22 hours, and stripped of cumulus cells by vortexing (Vortex Genie 2; shake setting #8) for 2 minutes, 15 seconds in 2ml of Tyrodes-Hepes medium in a 15ml conical tube.
  • About 15 to 30 denuded oocytes of medium color with a polar body were selected and placed into 23ul microdrops and co-cultured with bovine oviduc ⁇ tal cells.
  • the polar bodies were visualized with a dissecting microscope 60-120X.
  • the oviductal cell co-culture consisted of 3ul of packed oviductal cells in 20ul of co-culture media TCM 199 with 10% HTFCS and 1% penicillin and streptomycin.
  • the bovine oviductal epithelial cells were collect ⁇ ed from oviductal flushings 36 to 48 hours, preferably 36 hours, after injection of HCG.
  • the preferred oviduc ⁇ tal cells for co-culture are predominantly elongated, ulticellular, ciliated clumps.
  • the oviductal cells were preconditioned in the co-culture media for 22 to 24 hours prior to the addition of the oocytes.
  • the hand ⁇ ling time is 1 to 2 hours prior to introducing the selected oocytes with the co-culture.
  • the oocyte-ovi- ductal cell co-cultures were then incubated at 39°C 5% C0 for about 3 to about 5 hours.
  • the co-culture is supplemented with an equal volume of fresh TCM 199 with HTFCS between 3 to 5 hours after initiating of the co-culture.
  • the co-culture with oocytes was incubated up to 19 hours. Some oocytes were withdrawn at earlier intervals.
  • Nuclear transfer was performed on oocytes that had been incubated in maturation culture with recombinant bFSH and bLH and co-culture.
  • the oocytes were matured in maturation media for 20 to 24 hours, preferably 22 hours, co-cultured for 3 hours prior to micro-manipula ⁇ tion and then cultured an additional 2 hours in TCM 199 with 10% HTFCS prior to fusion.
  • the preferred method of micro-manipulation in addition to the general technique disclosed in "Bovine Nuclear Transplantation” includes a staining procedure to visualize the female chromatin.
  • the preferred technique provides for removing about 1/2 of the ooplasm from an oocyte and placing the removed ooplasm into a foreign zona pellucida creating two egg halves each with a surrounding zonae. One half should include the female chromatin and one-half should not. With light micro ⁇ scopy, it is impossible to discern the enucleated half without chromatin which is the preferred recipient egg half for the donor nuclear material.
  • the iri vitro matured oocyte halves are placed in phosphate saline supplement ⁇ ed with 0.4% BSA, 1% antibiotic penicillin/streptomycin (MPBS) and 5ug/ml Hoechst stain (33342, bisbenzimide trihydrochloride) at 37°C for about 30 minutes.
  • the oocyte halves are then placed in fresh MPBS without stain several minutes and viewed under a fluorescent microscope with the appropriate excitation and barrier filters.
  • Each oocyte half is viewed at 200-400X magnification using white light. The white light is shut off and the oocyte half is exposed to UV light.
  • the chromatin fluoresces a bright blue and should be located as quickly as possible to reduce the exposure time to UV light.
  • the oocyte halves with chromatin are discarded.
  • the oocyte halves without chromatin are recipients of donor nuclear material.
  • each of the egg halves was stained. In the event the half stained contained the chromatin, the other enucleated half was not exposed to the Hoechst stain. However, the enucleated halves exposed to the Hoechst stain were viable and developed normally.
  • the method of this invention is a successful in vitro oocyte maturation process which can be used for nuclear transfer or other genetic manipulation or fertilization techniques.
  • the method offer an alterna ⁇ tive to use of in vivo oocytes.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Environmental Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de production d'ovocytes matures à partir d'une culture d'ovocytes immatures dans des milieux de maturation et une co-culture présentant une capacité de développement et pouvant être utilisé pour le transfert nucléaire. Les milieux de maturation peuvent contenir des gonadotrophines recombinantes. Ladite co-culture se compose de milieux comportant des cellules de l'oviducte bovin.
PCT/US1990/002365 1989-05-01 1990-04-27 Maturation in vitro d'ovocytes bovins Ceased WO1990013627A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US345,402 1982-02-03
US34540289A 1989-05-01 1989-05-01

Publications (1)

Publication Number Publication Date
WO1990013627A1 true WO1990013627A1 (fr) 1990-11-15

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Family Applications (1)

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PCT/US1990/002365 Ceased WO1990013627A1 (fr) 1989-05-01 1990-04-27 Maturation in vitro d'ovocytes bovins

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AU (1) AU5531090A (fr)
CA (1) CA2015707A1 (fr)
IL (1) IL94184A0 (fr)
WO (1) WO1990013627A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0521674A1 (fr) * 1991-07-01 1993-01-07 Tanja Dominko Maturation in vitro des oocytes de bovin dénudés
EP0559307A1 (fr) * 1992-03-04 1993-09-08 ABS Global, Inc. Procédé de maturation des oocytes receveurs pour la multiplication des embryons bovins
WO1994019455A1 (fr) * 1993-02-23 1994-09-01 Genentech, Inc. Maturation in vitro d'oocytes en presence d'inhibine, d'activine ou de combinaisons d'inhibine/activine
WO1996025496A1 (fr) * 1995-02-17 1996-08-22 Instituut Voor Dierhouderij En Diergezondheid (Id-Dlo) PRODUCTION DE FOLLICULOSTIMULINE BOVINE RECOMBINANTE (rec bFSH) A ACTIVITE BIOLOGIQUE DANS LE SYSTEME D'EXPRESSION DE BACULOVIRUS
US6107543A (en) * 1992-08-20 2000-08-22 Infigen, Inc. Culture of totipotent embryonic inner cells mass cells and production of bovine animals
US6906238B2 (en) 2000-03-15 2005-06-14 University Of Georgia Research Foundation, Inc. Effective nuclear reprogramming in mammals using CDK2 inhibitors
US7790459B2 (en) 2002-03-08 2010-09-07 Mcgill University In vitro maturation of immature human oocytes
CN105624100A (zh) * 2016-04-06 2016-06-01 云南中科胚胎工程生物技术有限公司 水牛卵母细胞体外成熟培养方法
WO2017143412A1 (fr) * 2016-02-24 2017-08-31 Universidade Estadual Paulista "Júlioo De Mesquita Filho" - Unesp Système folliculaire pour la maturation in vitro d'ovocytes et kit
CN113249304A (zh) * 2021-06-04 2021-08-13 中国农业科学院北京畜牧兽医研究所 萝卜硫素在制备牛卵母细胞体外成熟液中的应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IE881133L (en) * 1988-04-14 1989-10-14 Univ Dublin In-vitro culture of bovine embryos

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biology of Reproduction, Vol. 37, 1987, R.S. PRATHEr et al.: "Nuclear Transplantation in the Bovine Embryo: Assessment of Donor Nuclei and Recipient Oocyte", pages 859-866 *
Journal of Reproduction and Fertility, Vol. 81, No. 1, 1987, F. GANDOLFI et al.: "Stimulation of Early Embryonic Development in the Sheep by Co-Culture with Oviduct Epithelial Cells", pages 23-28 *
Theriogenology, Vol. 27, No. 1, January 1987, W.H. EYESTONE et al.: "Co-Culture of Early Bovine Embryos with Oviductal Epithelium", page 228 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0521674A1 (fr) * 1991-07-01 1993-01-07 Tanja Dominko Maturation in vitro des oocytes de bovin dénudés
EP0559307A1 (fr) * 1992-03-04 1993-09-08 ABS Global, Inc. Procédé de maturation des oocytes receveurs pour la multiplication des embryons bovins
US6107543A (en) * 1992-08-20 2000-08-22 Infigen, Inc. Culture of totipotent embryonic inner cells mass cells and production of bovine animals
WO1994019455A1 (fr) * 1993-02-23 1994-09-01 Genentech, Inc. Maturation in vitro d'oocytes en presence d'inhibine, d'activine ou de combinaisons d'inhibine/activine
US5563059A (en) * 1993-02-23 1996-10-08 Genentech, Inc. Use of human inhibin and human activin to increase the number of mature primate oocytes
US5693534A (en) * 1993-02-23 1997-12-02 Genentech, Inc. Enhancement of fertilization capability of oocytes
WO1996025496A1 (fr) * 1995-02-17 1996-08-22 Instituut Voor Dierhouderij En Diergezondheid (Id-Dlo) PRODUCTION DE FOLLICULOSTIMULINE BOVINE RECOMBINANTE (rec bFSH) A ACTIVITE BIOLOGIQUE DANS LE SYSTEME D'EXPRESSION DE BACULOVIRUS
US6906238B2 (en) 2000-03-15 2005-06-14 University Of Georgia Research Foundation, Inc. Effective nuclear reprogramming in mammals using CDK2 inhibitors
US7547818B2 (en) 2000-03-15 2009-06-16 The University Of Georgia Research Foundation, Inc. Metaphase donor cells for effective nuclear reprogramming in mammals
US7790459B2 (en) 2002-03-08 2010-09-07 Mcgill University In vitro maturation of immature human oocytes
WO2017143412A1 (fr) * 2016-02-24 2017-08-31 Universidade Estadual Paulista "Júlioo De Mesquita Filho" - Unesp Système folliculaire pour la maturation in vitro d'ovocytes et kit
CN105624100A (zh) * 2016-04-06 2016-06-01 云南中科胚胎工程生物技术有限公司 水牛卵母细胞体外成熟培养方法
CN113249304A (zh) * 2021-06-04 2021-08-13 中国农业科学院北京畜牧兽医研究所 萝卜硫素在制备牛卵母细胞体外成熟液中的应用

Also Published As

Publication number Publication date
CA2015707A1 (fr) 1990-11-01
AU5531090A (en) 1990-11-29
IL94184A0 (en) 1991-01-31

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