Isolation of an Oocyte Stimulatory Peptide from the Ovarian Follicular Fluid of Water Buffalo (Bubalus bubalis)

  • Gupta, P.S.P. (National Institute of Animal Nutrition and Physiology) ;
  • Ravindra, J.P. (Production Enhancement Division, National Dairy Development Board) ;
  • Nandi, S. (Veterinary College, University of Agricultural Sciences) ;
  • Raghu, H.M. (Production Enhancement Division, National Dairy Development Board) ;
  • Ramesha, K.P. (Southern Campus, National Dairy Research Institute)
  • Received : 2004.09.26
  • Accepted : 2005.05.04
  • Published : 2005.11.01


Ovarian follicular fluid contains both stimulatory and inhibitory agents that influence the growth and maturation of oocyte. In the present study, an attempt was made to isolate and study the biological properties of ovarian follicular fluid peptide(s) in buffaloes. Bubaline ovarian follicular was made steroid- and cell-free. A protein fraction was obtained by saturation (30-35% level) of the follicular fluid with ammonium sulfate. The protein fraction was purified with Sephadex-G 50 gel filtration chromatography and a single peak was obtained in the eluant volume, which was lyophilized. SDS-PAGE of the lyophilized fraction revealed a single band and the molecular weight of the peptide was 26.6 kDa. The peptide stimulated the cumulus cell expansion and in vitro maturation rate of oocytes in buffaloes in a dose dependent manner when it was incorporated at different dose levels (0, 10, 25, 50, 100 and 1,000 ng $ml^{-1}$ of maturation medium). The basic culture medium consisted of TCM 199 with Bovine serum albumin (0.3%). The in vitro maturation rates were comparable to those obtained with a positive control medium (TCM 199+20 ng EGF $ml^{-1}$+steer serum (20%)). Further purification and biological assays may throw more light on the nature and functions of this peptide.


Supported by : Indian Council of Agricultural Research


  1. Ayoub, M. A. and A. G. Hunter. 1993. Inhibitory effect of bovine follicular fluid on in vitro maturation of bovine oocytes. J. Dairy Sci. 76:95-100.
  2. Beker, A. R. C. L., F. Izadyar, B. Colenbrander and M. M. Bevers. 2000. Effect of growth hormone releasing hormone (GHRH) and vasoactive intestinal peptide (VIP) on in vitro bovine oocyte maturation. Theriogenology 53:1771-1782.
  3. Bevers, M. M., H. T. M. Van Tol and F. A. M. De Loos. 1992. Protein patterns of immature bovine oocytes incubate with Smethionine in either medium or medium supplemented with dibutyryl-cyclic AMP or follicular fluid. 12th Int. Cong. Anim. Reprod. 1:312-314.
  4. Bruck, I., J. Bezard, G. Duchamp, M. Balsten, P. Dales and T. Greve. 2000. Pure preovulatory follicular fluid for in vitro maturation of equine oocytes: An alternative to conventional culture media? Theriogenology 53:450 (Abstr.).
  5. Chauhan, M. S., P. Palta, S. K. Das, P. K. Katiyar and M. L. Madan. 1997. Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects of maturation, fertilization and subsequent development of buffalo oocytes in vitro. Theriogenology 48:461-469.
  6. Coskun, S. and Y. C. Lin. 1994. Effects of transforming growth factors and activin A on in vitro porcine oocyte maturation. Mol. Reprod. Dev. 38:153-59.
  7. Dostal, J., A. Pavlok and J. E. Flechon. 1996. Isolation and characterization of maturation inhibiting compound in bovine follicular fluid. Reprod. Nutr. Develop. 36:681-690.
  8. Downs, S. M. 1989. Specificity of Epidermal growth factor action on maturation of the murine oocyte and cumulus oophorus in vitro. Biol. Reprod. 41:371-379.
  9. Eckert, J. and H. Niemann. 1996. Effects of platelet-derived growth factor (PDGF) on the in vitro production of bovine embryos in protein-free media. Theriogenology 46:307-320.
  10. Fujimori, K., R. M. Nakamura, S. A. Tonetta and G. S. Di Zerega. 1987. Cessation of transition-phase follicle growth in the Guinea pig by Follicle Regulatory Protein. Biol. Reprod. 37:812-822.
  11. Guler, A. N., P. Poulin, M. Mermillod, M. Terqui and Y. Cognie. 2000. Effect of growth factors, EGF and IGF-I, and Estradiol on in vitro maturation of sheep oocytes. Theriogenology 54:209-218.
  12. Grupen, C. G., H. Nagashima and M. B. Nottle. 1997. Role of epidermal growth factor and insulin-like growth factor-I on porcine oocyte maturation and embryonic development in vitro. Reprod. Fertil. Dev. 9:571-575.
  13. Gupta, P. S. P., S. Nandi, B. M. Ravindranatha and P. V. Sarma. 2001a. Effect of buffalo follicular alone and in combination with M199 and PMSG on buffalo oocyte maturation. Asian-Aust. J. Anim. Sci. 14:693-696.
  14. Gupta, P. S. P., S. Nandi, B. M. Ravindranatha and P. V. Sarma. 2001b. Effect of commercially available PMSG on maturation, fertilization and embryo development of buffalo oocytes in vitro. Reprod. Fertil. Dev. 13:355-360.
  15. Gupta, P. S. P., S. Nandi, B. M. Ravindranatha and P. V. Sarma. 2002. In vitro maturation of buffalo oocytes with Epidermal growth factor and Fibroblast growth factor. Indian J. Anim. Sci. 72:23-26.
  16. Gupta, P. S. P., J. P. Ravindra, V. Girishkumar, H. M. Raghu, S. Nandi. 2005.Stimulation of in vitro ovine oocyte maturation with a novel peptide isolated from follicular fluid of the buffalo (Bubalus bubalis). Small Rumin. Res. 59(1):33-40.
  17. Guraya, S. S. 1997. Follicular growth and its Regulation and Functions. In Ovarian Biology in Buffaloes and Cattle. 1st Edition, ICAR, New Delhi, India.
  18. Hynes, A. C., M. T. Kane and J. M. Sreenan. 1996. Partial purification from bovine follicular fluid of a factor of low molecular mass with inhibitory effects on the proliferation of bovine granulosa cells in vitro and on rat follicular development in vivo. J. Reprod. Fertil. 108:185-191.
  19. Harper, K. M. and B. G. Brackett. 1993. Bovine blastocyst development after in vitro maturation in a defined medium with epidermal growth factor and low concentrations of gonadotropins. Biol. Reprod. 48:409-416.
  20. Kobayashi, K., S. Yanagamachi and H. Hoshi. 1994: Influence of epidermic growth factor and transforming growth factor-alpha on in vitro maturation of cumulus enclosed bovine oocytes in defined medium. J. Reprod. Fertil. 100:439-446.
  21. Kumar, S. and H. C. Pant. 1990. Purification of inhibitor(s) from buffalo follicular fluid to compensatory ovarian hypertrophy in mice. Indian J. Anim. Reprod. 11:135-140.
  22. Laemeli, U. K. 1970. Cleavage of structural protein during the analysis of head of bacteriophage T4. Nature 227:680-685.
  23. Larocca, C., S. Kmaid and J. Calvo. 1993. Effect of follicular fluid and estrus cow serum on maturation, fertilization and development of the bovine oocyte in vitro. Theriogenology 39:253 (Abstr.).
  24. Mukesh Vinze, M. K. Sharma and Dheer Singh. 2004. Effect of follicular fluid proteins and gonadotropins on progesterone secretion by buffalo granulosa cells in vitro. Asian-Aust. J. Anim. Sci. 17:1496-1500.
  25. Nandi, S., B. M. Ravindranatha, P. S. P. Gupta and P. V. Sarma. 2002. Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes. Theriogenology 57:1151-1159.
  26. Ocana-Quero, J. M., M. Moreno-Millan, M. Pinedo-Merlin and A. Rodero-Franganillo. 1997. Influence of different follicular fractions on in vitro maturation of immature bovine oocytes. Arch. de Zootec. 46:51-59.
  27. Palta, P. and M. S. Chauhan. 1998. Laboratory production of buffalo (Bubalus bubalis) embryos. Reprod. Fertil. Dev. 10:379-91.
  28. Pawshe, C. H., K. B. C. Appa Rao and S. M. Totey. 1998. Effect of insulin-like growth factor I and its interaction with gonadotrophins on in vitro maturation and embryonic development, cell proliferation and biosynthetic activity of cumulus-oocyte complexes and granulosa cells in buffalo Mol. Reprod. Dev. 49:277-89.<277::AID-MRD8>3.0.CO;2-L
  29. Ramsoondar, J., W. Khalil and B. R. Downey. 1995. Partial purification and characterization of a protein in porcine follicular fluid which restricts sperm-egg interaction in vitro. Canadian J. Vet. Res. 59:8-14.
  30. Richards, J. S. 1994. Hormonal control of gene expression in the ovary. Endocr. Rev. 15:725-751.
  31. Riley, S. C., R. Thomassen, S. E. Bay, R. Leask, H. G. Pedersen and E. D. Watson. 2004. Matrix metalloproteinase-2 and -9 secretion by the equine ovary during follicular growth and prior to ovulation. Anim. Reprod. Sci. 81:329-339.
  32. Rivera, G. M. and J. E. Fortune. 2003. Selection of the dominant follicle and insulin-like growth factor (IGF)-binding proteins: evidence that pregnancy-associated plasma protein A contributes to proteolysis of IGF-binding protein 5 in bovine follicular fluid. Endocrinology 144:437-446.
  33. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning : A laboratory manual, 2nd edn. Cold Spring Harbor, New York.
  34. Srivastava, S. K. and H. C. Pant. 1999. Inhibitory activity in buffalo follicular fluid and its elution pattern during different season. Indian J. Exp. Biol. 37(4):370-373.
  35. Sun, F. J., P. Holm, B. Irvine and R. F. Seamark. 1994. Effect of follicular fluid during in vitro maturation of bovine oocytes on in vitro fertilization and early embryonic development. Biol. Reprod. 41:981-988.
  36. Tajik, P., S. Goorani-Nejad and H. Ghasemzadeh-Nava. 2000. In vitro maturation of water buffalo oocytes in different concentrations of bovine and buffalo follicular fluid. Theriogenology 53: 470 (Abstr.).
  37. Talevi, R., P. Bergamo, G. Sansone, A. Savarese, P. Venditti and P. Abrescia. 1994. Association of cumulus-oocyte complexes with the intrafollicular levels of a blood protein in Bubalus bubalis. Zygote 2:167-168.
  38. Xia, P., F. R. Tekpetey and D.T. Armstrong. 1994. Effect of IGF-I on pig oocyte maturation, fertilization, and early embryonic development in vitro, and on granulosa and cumulus cell biosynthetic activity. Mol. Reprod. Dev. 38:373-379.
  39. Xu-Ruo, J., Q. C. Doan, G. O. Regester and R. J. Xu. 1999. Detection and characterisation of transforming growth factorbeta in porcine colostrum. Biol. Neonate 75:59-64.
  40. Zak, L. J., X. Xu, R. T. Hardin and G. R. Foxcroft. 1997. Impact of different patterns of feed intake during lactation in the primiparous sow on follicular development and oocyte maturation. J. Reprod. Fertil. 110:99-106.
  41. Zhang, D. and J. Wang. 1994. In vitro maturation and fertilization of rabbit oocytes. Acta Agric. Shanghai 10:83-89.

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