Molecular Basis of BMP-15 Function

BMP-15 功能的分子基础

• 批准号: 7038282
• 负责人: SHUNICHI SHIMASAKI
• 金额: $ 33.4万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7038282
• 关键词: biological signal transduction; bone morphogenetic proteins; follicle stimulating hormone; gene expression; gene mutation; genetically modified animals; granulosa cell; growth factor receptors; hormone receptor; laboratory mouse; laboratory rat; oogenesis; peptide chemical synthesis; protein biosynthesis; protein purification; protein structure function; receptor binding; receptor expression; site directed mutagenesis; southern blotting; transfection /expression vector; transforming growth factors

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.

描述（由申请人提供）：骨形态发生蛋白-15（BMP-15），a TGF-β超家族成员，是卵母细胞特异性生长因子。最近 对绵羊中自然发生的突变[Inverdale（FecX '）]的研究表明， 提示BMP-15在卵泡发生和生育中的重要作用。在 纯合子FecX'雌性，卵泡发育在初级卵泡发育期停止， 阶段，导致不孕。相反，杂合子FecX 1女性 显示排卵率和双胞胎及三胞胎出生率增加。现已 已经表明，在残基31处缬氨酸被天冬氨酸取代（V31 D）， BMP-15基因存在于FecX载体中。因此，BMP-15突变 似乎与不孕症和超级生育力有关， 剂量敏感的方式。我们的实验室最近生产了重组 BMP-15，并确定颗粒细胞（GC）作为该因子的靶细胞。 使用重组蛋白，我们发现BMP-15（i）刺激大鼠GC 增殖，（ii）抑制FSH受体表达，和（iii）刺激kit GC中的配体表达。这些发现确立了新的概念， 卵母细胞衍生的BMP-15在功能上参与调节三种主要的 卵泡发育方面，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 通过表征BMP-15从受体到靶基因的作用的基础 BMP-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配体基因表达（Aim 2）。我们 他们还发现，尽管缺乏亚基间的二硫键， 大多数二聚体TGF-β超家族成员BMP-15和与之密切相关的 相关的GDF-9是同源二聚体。此外，我们有证据表明， BMP-15和GDF-9可以形成异源二聚体，因此我们建议探索BMP-15和GDF-9之间的相互作用。 BMP-15/GDF-9异源二聚体的生物学作用（目的3）。虽然V31 D 在Inverdale绵羊中发现了BMP 15基因的突变， BMP-15-V31 D在Inverdale绵羊中的突变尚不清楚。我们将探讨 BMP-15-V31 D突变的生物学活性，并验证我们的假设， BMP-15-V31 D可影响GDF-9的生物合成（目的4）。最后我们将 在小鼠中产生相同的突变，并确定 BMP-15-V31 D突变对卵泡发生和排卵的影响（目的5）。这些研究 应该提供了重大的进展，在理解规则的事件， 并最终改善生育控制方法 以及用于治疗性腺异常和不育症。