Mammalian follicles are dormant at the primordial/primary stage until recruited to grow by unknown mechanisms. Growth differentiation factor (GDF)-9, and its paralog, GDF-9B or BMP-15, are expressed mainly in the oocyte and represent paracrine germ cell factors capable of regulating the adjacent somatic follicular cells. Mice defective in the GDF-9 gene showed an arrest of early follicle development whereas treatment of neonatal rats with recombinant GDF-9 stimulates the development of early follicles. Recent studies of mutant sheep further demonstrated that GDF-9B and a putative GDF-9/-9B receptor (ALK6) are important infertility and fecundity genes. We have generated recombinant GDF-9 and demonstrated its ability to promote early follicle development in vitro and in vivo. Analysis of the structure-functional relationship of TGF-beta proteins allows the design of a GDF-9 antagonistic mutant with an extended C-trial that is capable of blocking GDF-9 stimulation of granulosa cell proliferation in vitro. Based on bioinformatic analysis of the sequenced human genome, we predicted several known genes as potential receptors for GDF-9/GDF-9B and demonstrated the ability of BMPRII to interact with GDF-9. In this application, we propose to extend studies on the GDF-9 functional antagonists and to investigate the role of BMPRII and related receptors (ALK6, ALK3, and ALK2) in GDF-9 signaling. We will test the direct binding of BDNF-9 to its putative receptors and the activation of reporter constructs in cells expressing genes of the GDNF-9 signaling pathway. We will also analyze the expression of putative GDNF and GDF-9B receptors in human ovarian cells. Based on the important role of GDF-9 and GDF-9B in initial follicle recruitment and our recent observation that GDF-9 treatment stimulates inhibit production by granulosa cells, we hypothesize that genes in the GDF-9/GDF-9B pathway could be mutated in families with genetic susceptibility for dizygotic twinning. We will test if gain-of-function mutations of ALK6, ALK3 or ALK2 as well as heterozygous loss-of-function mutations in GDF-9 or GDF-9B genes could be associated with dizygotic twins. The present study could provide a better understanding of the GDF-9/GDF-9B signaling pathway in the ovary as well as the role of this pathway in human fecundity and infertility.
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