Hostname: page-component-586b7cd67f-dsjbd Total loading time: 0 Render date: 2024-11-24T04:12:12.743Z Has data issue: false hasContentIssue false
  • English
  • Français

In vitro culture of oocytes with surrounding cumulus complexes and granulosa cells (COCGs) from bovine early antral follicles

Published online by Cambridge University Press:  18 August 2016

S. Saha ,
M. Shimizu ,
M. Geshi  and
Y. Izaike
S. Saha*
Affiliation:
Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Tsukuba Norindanchi, PO Box 5, Ibaraki 305-0901, Japan
M. Shimizu
Affiliation:
National Agricultural Research Centre for Tohoku Region, Morioka, Iwate, 020-0198, Japan
M. Geshi
Affiliation:
Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Tsukuba Norindanchi, PO Box 5, Ibaraki 305-0901, Japan
Y. Izaike*
Affiliation:
Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Tsukuba Norindanchi, PO Box 5, Ibaraki 305-0901, Japan
*
†Present address: Agricultural and Forestry Research Centre, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. E-mail:[email protected]
‡ Present address: National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
Get access

Abstract

Cumulus-oocyte complexes with surrounding granulosa cells (COCGs) in early antral follicles (0·5 to 0·7 mm in diameter) were surgically collected from sections of bovine ovarian cortex under a dissection microscope and subsequently cultured in vitro using follicle stimulating hormone (FSH), epidermal growth factor (EGF), insulin-transferrin-selenium (ITS) and hypoxanthine, singly or in combination, to obtain fully grown matured oocytes. Oocytes cultured in the presence of FSH + hypoxanthine increased (P < 0·05) in diameter from 93 µm on the day of commencement of culture to 106·37±0·34 µm on day 5. Oocytes cultured in the presence of FSH, hypoxanthine or hypoxanthine + ITS + FSH increased (P < 0·05) to mean diameters of 105·40 (s.e. 0·47) µm, 105·50 (s.e. 0·39) µm and 105·35 (s.e. 0·55) µm, respectively. By day 11 of culture, oocyte diameters 110·50 (s.e. 0·35) µm, 110·13 (s.e. 0·39) µm, 109·49 (s.e. 0·46) µm, 109·53 (s.e. 0·58) µm and 109·16 (s.e. 0·43) µm were recorded for treatments FSH + hypoxanthine, hypoxanthine + ITS + FSH, FSH, hypoxanthine and FSH + EGF + hypoxanthine + ITS, respectively. The proportions with COCGs which formed an antrum while cultured in vitro; were categorized as morphologically normal following recovery from the gel; matured in vitro; showed germinal vesicle break down and reached metaphase II were highest (P < 0·05) for the FSH + hypoxanthine treatment (49/60 (81·7%), 48/60 (80·0%), 47/60 (78·3%), 45/60 (75·0%) and 15/60 (25·0%), respectively, followed by hypoxanthine + ITS + FSH (47/60 (78·3%), 44/60 (73·3%), 41/60 (68·3%), 41/60 (68·3%) and 12/60 (20%), respectively), FSH (43/60 (71·7%), 42/60 (70%), 40/60 (66·7%), 39/60 (65·0%) and 9/60 (15%), respectively) and hypoxanthine (41/60 (68·3%), 38/60 (63·3%), 36/60 (60%), 35/60 (58·3%) and 8/60 (13·3%), respectively). In experiment II, the in vitro fertilization and cleavage rates of COCGs were highest (P < 0·05) for FSH + hypoxanthine treatment (17/60; 28·3%) followed by hypoxanthine + ITS + FSH (13/60; 21·6%), FSH (12/60; 20%) and hypoxanthine (11/60; 18·3%) treatments. The results of this study show that COCGs from early antral follicles can be isolated, cultured and grown in vitro. Furthermore, supplements like FSH and hypoxanthine can be used singly or in combination(s) in culture medium to enhance the growth of COCGs.

Keywords

cowsgraafian folliclesin vitro cultureoocytes
Type
Reproduction
Information
Animal Science , Volume 77 , Issue 1 , August 2003 , pp. 141 - 147
Copyright
Copyright © British Society of Animal Science 2003

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Brackett, B. G. and Oliphant, G. 1975. Capacitation of rabbit spermatozoa in vitro . Biology of Reproduction 12: 260–274.CrossRefGoogle ScholarPubMed
Coskun, S., Sanbuissho, A., Lin, Y. C. and Rikihisa, Y. 1991. Fertilizability and subsequent developmental ability of bovine oocytes matured in medium containing epidermal growth factor (EGF). Theriogenology 36: 485–495.CrossRefGoogle ScholarPubMed
Crozet, N., Ahmed, A. and Dubos, M. P. 1995. Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro . Journal of Reproduction and Fertility 105: 293–298.Google Scholar
Downs, S. M., Coleman, D. L., Ward-Bailey, P. F. and Eppig, J. J. 1985. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid. Proceedings of the National Academy of Sciences of the USA 82: 454–458.Google Scholar
Downs, S. M., Daniel, S. A. J., Bornslaeger, E. A., Hoppe, P. C. and Eppig, J. J. 1989. Maintenance of meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP phosphodiesterase activity. Gamete Research 23: 323–334.Google Scholar
Fair, T., Hyttel, P. and Greve, T. 1995. Bovine oocyte diameter in relation to maturational competence and transcriptional activity. Molecular Reproduction and Development 42: 437–442.Google Scholar
Gore-Langton, R. E. and Daniel, S. A. J. 1990. Follicle-stimulating hormone and estradiol regulate antrum-like reorganization of granulosa cells in rat preantral follicle cultures. Biology of Reproduction 43: 65–72.Google Scholar
Gutierrez, C. G., Ralph, J. H., Telfer, E. E., Wilmut I. and Webb, R. 2000. Growth and antrum formation of bovine preantral follicles in long-term culture in vitro . Biology of Reproduction 62: 1322–1328.Google Scholar
Harada, M., Miyano, T., Matsumura, K., Osaki, S., Miyake, M. and Kato, S. 1997. Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine. Theriogenology 48: 743–755.Google Scholar
Hirao, Y. N. 1994. In vitro growth and maturation of pig oocytes. Journal of Reproduction and Fertility 100: 333–339.Google Scholar
Hyttel, P., Fair, T., Callesen, H. and Greve, T. 1997. Oocyte growth, capacitation and final maturation in cattle. Theriogenology 47: 23–32.Google Scholar
Lonergan, P., Monaghan, P., Rizos, D., Boland, M. P. and Gordan, D. 1994. Effects of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization and culture in vitro . Molecular Reproduction and Development 37: 48–53.CrossRefGoogle ScholarPubMed
May, J. V. and Schomberg, D. W. 1981. Granulosa cell differentiation in vitro: effect of insulin on growth and functional integrity. Biology of Reproduction 25: 421–431.CrossRefGoogle ScholarPubMed
Miyano, T., Osaki, S., Yamamoto, K. and Otoi, T. 1998. In vitro growth of bovine oocytes. Proceedings of the fourth Asian symposium on animal biotechnology, Kamiina, Japan, pp. 67–74.Google Scholar
Motlik, J., Crozet, N. and Fulka, J. 1984. Meiotic competence in vitro of pig oocytes isolated from early antral follicles. Journal of Reproduction and Fertility 72: 323–328.Google Scholar
Nayudu, P. L. and Osborn, S. M. 1992. Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro . Journal of Reproduction and Fertility 95: 349–362.Google Scholar
Osaki, S., Matsumura, K., Yamamoto, K., Miyano, T., Miyake, M. and Kato, S. 1997. Fertilization of bovine oocytes grown in vitro . Reproduction, Fertility and Development 9: 781–787.Google Scholar
Pavlok, A., Lucas-Hahn, A. and Niemann, H. 1992. Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles. Molecular Reproduction and Development 31: 63–67.CrossRefGoogle ScholarPubMed
Roy, S. K. and Greenwald, G. S. 1989. Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture. Journal of Reproduction and Fertility 87: 103–114.Google Scholar
Saha, S., Shimizu, M. and Izaike, I. 1999. In vitro cultivation of oocytes from bovine early antral follicles. International workshop on embryogenesis and implantation, Hawaii, USA, February 1-4, p. 1.Google Scholar
Sato, E., Matsuo, M. and Miyamoto, H. 1990. Meiotic maturation of bovine oocytes in vitro: improvement of meiotic competence by dibutyryl cyclic adenosine 3’, 5’-monophosphate. Journal of Animal Science 68: 1182–1187.CrossRefGoogle ScholarPubMed
Savion, N., Lui, G. -M. and Laherty, R. 1981. Factors controlling proliferation and progesterone production by bovine granulosa cells in serum-free medium. Endocrinology 109: 409–420.CrossRefGoogle ScholarPubMed
Shen, X., Hirata, K., Miyano, T. and Kato, S. 1997. In vitro antrum formation of oocyte-cumulus-granulosa cell complexes from pig early antral follicles. Journal of Mammalian Ovarian Research 14: 183–190.Google Scholar
Statistical Analysis Systems Institute. 1989. SAS/STAT user’s guide, version 6, fourth edition. SAS Institute Inc., Cary, NC.Google Scholar
Telfer, E. 1996. The development of methods for isolation and culture of preantral follicles from bovine and porcine ovaries. Theriogenology 45: 101–110.Google Scholar
Wandji, S. A., Srsen, V., Voss, A. K., Eppig, J. J. and Fortune, J. E. 1996. Initiation of in vitro growth of bovine primordial follicles. Biology of Reproduction 55: 942–948.CrossRefGoogle ScholarPubMed