Article Id: ARCC180 | Page : 472-478
H.N. Malik*, S.K. Dash1, S.K. Mohapatra2 and T. Nanda
Address : Animal Biotechnology Center, CCS Haryana Agricultrual University, Hisar-125 004, India


The present study was carried out on buffalo ovarian follicles. From these follicles cumulus oocyte complexes were aspirated and in vitro matured in TCM-199 supplemented 10% v/v fetal bovine serum, 1µg/ml estradiol, 10µg/ml LH, 5µg/ml FSH and 1% v/v gentamycin. In vitro matured cumulus oocyte complexes were investigated for the presence of GDF -9 gene transcript using RT-PCR analysis. After the RT-PCR analysis the amplified product was purified and cloned. The cloned product was further sequenced and analyzed to study the phylogenetic relationship of GDF-9 gene of buffalo with other species. Upon RT-PCR analysis it was revealed that GDF-9 transcripts were expressed exclusively in in vitro matured cumulus oocyte complexes. Nucleotide sequence analysis of GDF-9 gene of Bubalus bubalis revealed that it is 99% similar to that of Bos taurus and clustered together with Bos taurus and Rattus norvegicus shared a separate cluster.


Cumulus oocyte complexes Follicle In vitro maturation.


  1. Aaltonen, J., Laitnen, M.P., Vuojolainen, K., Jaatinen, R., Horalli-Kuitunen, N., Seppa, L., Louhio, H., Tuuri, T., Sjoberg, J., Butzow, R., Hovata, O., Dale, L. and Ritvos, O. (1999). Human growth differentiation factor-9 (GDF-9) and its novel homolog GDF-9â are expressed in oocytes during early folliculogenesis. J. Clin. Endocrinol. Metab. 84: 2744-2750.
  2. Bodensteiner, K.J., Clay, C.M., Moeller, C.L. and Sawyer, H.R. (1999). Molecular Cloning of the Ovine Growth Differentiation Factor-9 and Expression of Growth Differentiation Factor-9 in Ovine and Bovine Ovaries. Biol. Reprod. 62: 1479-1485.
  3. Chauhan, M.S., Singla, S.K., Palta, P., Manik, R.S. and Madan, M.L. (1998). In vitro maturation and fertilization and subsequent development of buffalo (Bubalus bubalis) embryos: effect of oocye quality and type of serum. Reprod. Fertil. Dev. 10: 173-177.
  4. Duranthon, V. and Renard, J.P. (2001). The developmental competence of mammalian oocytes: A convenient but biologically fuzzy concept. Therio. 55: 1277-1289.
  5. Elvin, J.A., Yan, C., Wang, P., Nishimori, K. and Matzuk, M.M. (1999). Molecular characterization of the follicle defects in the growth differentiation factor-9 deficient ovary. Mol. Endocrinol. 13: 1018-1034.
  6. Fair, T., Carter, F., Park, S., Evans, A.C. and Lonergan, P. (2007). Global gene expression analysis during bovine oocyte in vitro maturation. Therio. 68: 91-97.
  7. Fitzpatrick, S.L., Sindoni, D.M., Shughrue, P.J., Lane, M.V., Merchenthaler, I.J. and Frail, D.E. (1998). Expression of growth differentiation factor -9 messenger ribonucleic acid in ovarian and nonovarian rodent and human tissues. Endocrinol. 139: 2571-2578.
  8. Gittens, J.E., Barr, K.J., Vanderhyden, B.C. and Kidder, G.M. (2005). Interplay between paracrine signaling and gap junctional communication in ovarian follicles. J. Cell Sci. 118: 113-122.
  9. Gougeon, A. and Busso, D. (2000). Morphologic and functional determinants of primordial and primary follicles in the monkey ovary. Moll. Cell Endocrinol. 163: 33-41.
  10. Jaatinen, R., Laitinen, M.P., Vuojolainen, K., Aaltonen, J., Louhio, H., Heikinheimo, K., Lehtonen, E. and Ritvos, O. (1999). Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9 â. Mol. Cell Endocrinol. 156: 189-193.
  11. Jones, G.M., Cram, D.S., Song, B., Magli, M.C.,Gianaroli, L., Lacham Kaplan, O., Findlay, J.K., Jenkin, G. and Trounson, A.O. (2008). Gene expression profiling of human oocytes following in vivo or in vitro maturation. Hum. Reprod. 23: 1138-1144.
  12. Juengel, J.L., Hudson, N.L., Heath, D.A., Smith, P., Reader, K.L., Lawrence, S.B., O’Connell, A.R., Laitinen, M.P., Cranifield, M., Groome, N.P., Ritvos, O. and McNatty, K.P. (2002). Growth differentiation factor-9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep. Biol. Reprod. 67: 1777- 1789.
  13. Livestock census. (2007). Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture, Government of India.
  14. McGrath, S.A., Esquela, A.F. and Lee, S.J. (1995). Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9: 131-136.
  15. Raghu, H.M., Reddy, S.M. and Nandi, S. (2002). Effect of insulin, selenium and epidermal growth factor on development of buffalo oocytes to the blastocyst stage in in vitro with serum free and semi defined media. Vet. Rec. 151(9): 260-265.
  16. Sambrook, J. and Russel, D.W. (2001). Extraction, Purification and analysis of messenger RNA from eukaryotic cells. In: Molecular cloning: A laboratory manual. 3rd Edn. Cold Spring Harbor Laboratory Press, New York. pp. 1.1- 1.34.
  17. Sendai, Y., Itoh, T., Yamashita, S. and Hoshi, H. (2001). Molecular cloning of a cDNA encoding a Bovine Growth Differentiation Factor-9 and Expression of GDF-9 in Bovine Ovarian Oocytes and In Vitro Produced Embryos. Cloning 3: 3-10.
  18. Sidis, Y., Fujiwara, T., Leykin, L., Isaacson, K., Toth, T. and Schneyer, A. (1998). Characterization of inhibin/Activin subunit, activin receptor and follistatin messenger ribonucleic acid in human and mouse oocytes: evidence for activin’s paracrine signaling from granulosa cells to oocytes. Biol. Reprod. 59: 807-812.
  19. Su, Y.Q., Sugiura, K., Wigglesworth, K., O’Brien, M.J., Affourtit, J.P., Pangas, S.A., Matzuk, M.M. and Eppig, J.J. (2008). Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells. Development 135(1):111–121.
  20. Su, Y.Q., Sugiura, K., Woo, Y., Wigglesworth, K., Kamdar, S., Affourtit, J. and Eppig, J.J. 2007. Selective degradation of transcripts during meiotic maturation of mouse oocytes. Dev. Biol. 302: 104-117.
  21. Sugiura, K., Pendola, F.L. and Eppig, J.J. (2005). Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism. Dev. Biol. 279(1): 20–30.
  22. Teixeira, Filho FL., Baracat, E.C., Lee, T.H., Suh, C.S., Matsui, M., Chang, R.J., Shimasaki, S. and Erickson, G.F. (2002). Aberrant expression of growth differentiation factor-9 in oocytes of women with polycystic ovary syndrome. J. Clin .Endocrinol. Metab. 87: 1337–1344.
  23. Yamamoto, N., Christenson, L.K., McAllister, J.M. and Strauss III, J.F. (2002). Growth differentiation factor-9 inhibits 3,5-adenosine monophosphate stimulated steroidogenesis in human granulosa and theca cells. J. Clin. Endocrinol. Metab. 87(6): 2849-1856.

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