MOLECULAR REPRODUCTION AND DEVELOPMENT 70:11–19 (2004) Expression of Growth Differentiation Factor 9 (GDF9), Bone Morphogenetic Protein 15 (BMP15), and BMP Receptors in the Ovaries of Goats J.R.V. SILVA, 1,2 R. VAN DEN HURK, 1 * H.T.A. VAN TOL, 1 B.A.J. ROELEN, 1 AND J.R. FIGUEIREDO 2 1 Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 2 Faculty of Veterinary Medicine, PPGCV, State University of Ceara, Fortaleza, CE, Brazil ABSTRACT The process of ovarian folliculo- genesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. In goats, relatively little information is available on the local factors that regulate this process. We studied the pre- sence and distribution of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and BMP receptors types 2 (BMPR2), 1A (BMPR1A), and 1B (BMPR1B) in goat ovaries to find evidence for their possible roles in folliculogenesis. Ovaries of cyclic goats were collected and fixed in paraformaldehyde for immunohistochemical localization of GDF9 and BMP15 proteins or used to collect follicles and luteal tissue to study the mRNA expression of GDF9, BMP15, and BMP receptors using reverse transcrip- tase polymerase chain reaction (RT-PCR). GDF9 and BMP15 proteins were found in oocytes of all types of follicles and granulosa cells of primary, secondary, and antral but not primordial follicles. The mRNAs for GDF9, BMP15, BMPR2, BMPR1A, and BMPR1B were detected in primordial, primary, and secondary follicles as well as in oocyte and granulosa cells of antral follicles. Transcripts for BMPR2, BMPR1A, BMPR1B, and GDF9, and GDF9 protein were further- more found in corpora lutea. It is concluded that the mRNAs and proteins of GDF9 and BMP15 and the mRNAs of BMP receptors are expressed in goat ovarian follicles at all stages of their development, and that they form a complex intrafollicular regulatory system during folliculogenesis. Expression of all BMP receptor mRNAs and GDF9 mRNA and protein in luteal tissue additionally points to a role of GDF9 in corpus luteum function. Mol. Reprod. Dev. 70: 11–19, 2004. ß 2004 Wiley-Liss, Inc. Key Words: follicles; oocyte; cumulus; mural gran- ulosa cells; corpora lutea INTRODUCTION Ovarian folliculogenesis is a complex process con- sisting of follicular development through primordial, primary, secondary, and antral stages, after which ovulation occurs and the residual cells luteinize to form corpora lutea. Regulation of cell proliferation, differ- entiation, and atresia associated with folliculogenesis is brought about through complex interaction mainly between local growth factors and hypophyseal gonado- tropins from the pituitary. Since the actions of the gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) have been well documented, considerable recent research focuses on the regulatory intraovarian proteins. The transforming growth factor b (TGFb) superfamily consists of over 40 members of secreted growth factors grouped together upon structural homology (Chang et al., 2002). Recent studies on the expression patterns and biological functions of two recently identified members of this superfamily (growth differentiation factor 9 [GDF9] and bone morphogenetic protein 15 [BMP15]) have demonstrated that they play a critical role in ovarian function (Findlay et al., 2002; Juengel et al., 2002; Knight and Glister, 2003; Lin et al., 2003). Mice and sheep lacking GDF9 and BMP15, respectively, have folliculogenesis arrested at primary follicles and are sterile (Dong et al., 1996; Galloway et al., 2000). Within the ovary, the oocyte expresses both mRNA and protein for GDF9 and BMP15 in various staged follicles (rodents: McGrath et al., 1995; Elvin et al., 1999a, 2000a; Jaatinen et al., 1999; primates: Aaltonen et al., 1999; Gougeon and Busso, 2000). In primates (Sidis et al., 1998; Duffy, 2003), GDF9 mRNA and protein are also expressed in granulosa cells of antral follicles. In rodents (McGrath et al., 1995; Fitzpatrick et al., 1998) and human (Aaltonen et al., 1999), GDF9 is detectable in oocytes of follicles from the primary stage onward, but not in primordial follicles. In possum (Eckery et al., 2002), ovine and bovine (Bodensteiner et al., 1999) oocytes, however, expression of GDF9 mRNA begins at ß 2004 WILEY-LISS, INC. Address after January 2005: Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai, 980- 8575, Japan. E-mail: ymatsui@idac.tohoku.ac.jp Grant sponsor: Coordenac ¸a ˜o de Aperfeic ¸oamento de Pessoal de Nı ´vel Superior—CAPES, Brazil. *Correspondence to: R. van den Hurk, Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, PO Box 80151, 3584 CL, Yalelaan 7, Utrecht, The Netherlands. E-mail: r.vandenhurk@vet.uu.nl Received 1 April 2004; Accepted 6 May 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20127