Molecular Basis of BMP-15 Function

描述(申请人提供)：骨形态发生蛋白-15(BMP-15)，a 是转化生长因子-β超家族的成员，是一种卵母细胞特异性生长因子。近期 对绵羊[Inverdale(FecX‘)]自然发生的突变的研究已经 提示BMP-15在卵泡发生和生育中起重要作用。在……里面 纯合子FecX‘女性，初级卵泡发育停滞 阶段，导致不孕不育。相比之下，杂合子FecX1雌性 排卵率增加，双胞胎和三胞胎出生增加。现在已经有了 已证明在第31位用天冬氨酸取代缬氨酸(V31D) 的BMP-15基因存在于FecX‘携带者中。因此，BMP-15突变 似乎与不孕不育和超级生育有关 剂量敏感方式。我们实验室最近生产出了重组 BMP-15，并确定颗粒细胞(GC)为该因子的靶细胞。 利用重组蛋白，我们发现BMP-15(I)对大鼠GC有刺激作用 增殖，(Ii)抑制FSH受体的表达，以及(Iii)刺激KIT 配基在GC中的表达。这些发现确立了新的概念，即 卵母细胞来源的BMP-15在功能上参与调节三种主要的 卵泡发育的几个方面，即GC增殖、GC 细胞分化和卵子发生。在这里，我们建议阐明分子 BMP-15从受体到靶基因作用的基础 骨形态发生蛋白-15受体和信号分子(AIM 1)及其分子机制 其中BMP-15调节FSH受体和KIT配体基因的表达(目的2)。我们 还发现，尽管缺乏亚基间二硫键，但 大多数二聚体转化生长因子-β超家族成员，BMP-15和密切相关的 相关的GDF-9是同源二聚体。此外，我们还获得了证据表明 BMP-15和GDF-9可以形成异二聚体，因此我们建议探索 BMP-15/GDF-9异源二聚体的生物学作用(目标3)。虽然V31D 在Inverdale绵羊中发现BMP15基因突变，其作用是 Inverdale绵羊BMP-15-V31D突变尚不清楚。我们将探索 BMP-15-V31D突变的生物学活性，并检验我们的假设 BMP-15-V31D可影响GDF-9的生物合成(目标4)。最后我们会 在小鼠身上产生相同的突变，并确定该基因的体内效应 BMP-15-V31D突变对卵泡发生和排卵的影响(目标5)。这些研究 应在理解赛事规则方面取得重大进展 对女性生育至关重要，并最终改进节育方法 并用于治疗性腺异常和不育症。

DESCRIPTION (provided by applicant): Bone Morphogenetic Protein-15 (BMP-15), a member of the TGF-beta superfamily, is an oocyte-specific growth factor. Recent studies of a naturally occurring mutation in sheep [Inverdale (FecX')] have suggested an essential role for BMP-15 in folliculogenesis and fertility. In the homozygous FecX' female, follicular development arrests at the primary stage, resulting in infertility. In contrast, the heterozygous FecX1 females exhibit increases in ovulation rate and in twin and triplet births. It has now been shown that a substitution of valine by aspartic acid at residue 31 (V31D) of the BMP-15 gene is present in FecX' carriers. Therefore, BMP-15 mutation appears to be associated with infertility and super fertility in a dosage-sensitive manner. Our laboratory has recently produced recombinant BMP-15 and identified granulosa cells (GCs) as target cells for this factor. Using the recombinant protein, we have found that BMP-15 (i) stimulates rat GC proliferation, (ii) inhibits FSH receptor expression, and (iii) stimulates kit ligand expression in the GCs. These findings establish the new concept that oocyte-derived BMP-15 is functionally involved in regulating three major aspects of follicle development, namely GC proliferation, GC cytodifferentiation, and oogenesis. Here, we propose to elucidate the molecular basis of BMP-15 action from the receptors to the target genes by characterizing BMP-15 receptor and signaling molecules (Aim 1) and the molecular mechanisms by which BMP-15 regulates FSH receptor and kit ligand gene expression (Aim 2). We have also found that, despite lacking the inter-subunit disulfide bonds seen in most of the dimeric TGF-beta superfamily members, BMP-15 and the closely related GDF-9 are homodimers. Furthermore, we have obtained evidence that BMP-15 and GDF-9 can form heterodimers, and thus we propose to explore the biological roles of BMP-15/GDF-9 heterodimers (Aim 3). Although the V31D mutation in the BMP15 gene was found in Inverdale sheep, the role of BMP-15-V31D mutation in Inverdale sheep is yet unclear. We will explore the biological activities of the BMP-15-V31D mutation and test our hypothesis that BMP-15-V31D could influence the biosynthesis of GDF-9 (Aim 4). Finally we will generate the same mutation in mice and determine the in vivo effect of the BMP-15-V31D mutation on folliculogenesis and ovulation (Aim 5). These studies should provide significant advances in understanding regulation of the events central to female fertility and ultimately improve approaches for birth control and for treating gonadal abnormalities and infertility.