Function of RUNX transcription factors in COCs

描述（由申请人提供）：本提案的总体目标是确定参与卵丘细胞复合物（COCs）成熟过程的分子/细胞机制。COC的适当成熟对体内和体外生殖成功至关重要。我们对这一成熟过程缺乏全面的了解，限制了我们管理体内生育能力的能力，并阻碍了诸如体外受精和体外成熟等抗逆转录病毒治疗的实施。我们的实验室和其他人最近的研究已经开始揭示一个小的核转录因子家族，RUNX1和RUNX2，作为参与COC扩增的关键转录调节因子。我们的初步数据表明，Runx1和Runx2在卵泡积云细胞中被高度诱导表达。我们还发现RUNX1和RUNX2调控Ptgs2（前列腺素生物合成中的限速酶）和Hapln1（积云基质的稳定剂）在壁粒细胞和/或积云细胞中的表达。已知这两个基因产物在COC扩增中起关键作用。最重要的是，我们的初步研究表明，在积云细胞中，显性阴性RUNX的过度表达阻断了COC的扩增。基于这些新发现，我们假设RUNX1/2是促性腺激素激增诱导的COC扩增所必需的转录调节因子。这一假设将通过首先证明Runx1/2在积云细胞中表达的功能意义来验证（目的1）。其次，我们将确定RUNX1和RUNX2对其靶基因的调控作用（Aim #2）。接下来，我们将确定Runx1和Runx2在积云细胞中的表达调控机制（Aim #3）。这3个目标将用大鼠COCs进行研究。为了确保从大鼠研究中获得的数据可翻译为人类，RUNX1/2及其靶基因表达谱也将使用整个排卵期收集的人类积云细胞进行验证。我们还将利用体外受精妇女获得的COCs，研究RUNX1/2及其靶基因的积云表达与卵母细胞质量、受精和胚胎发育的相关性（Aim #4）。这些人体研究将为未来的转化/临床应用奠定基础。从拟议的研究中获得的信息不仅将促进我们对COC成熟机制的理解，而且对未来的转化/临床应用也有帮助，从而改善体内和体外生育管理。

DESCRIPTION (provided by applicant): The overall goal of the present proposal is to determine the molecular/cellular mechanism(s) involved in the maturation process of cumulus oocyte complexes (COCs). Proper maturation of the COC is critical to reproductive success in vivo and in vitro. Our lack of a complete understanding of this maturation process restricts our ability to manage fertility in vivo and hinders implementation of ART such as in vitro fertilization and in vitro maturation. Recent studies by our laboratory and others have begun to shed light on a small family of nuclear transcription factors, RUNX1 and RUNX2, as key transcriptional regulators involved in COC expansion. Our preliminary data demonstrated that Runx1 and Runx2 expression is highly induced in cumulus cells of periovulatory follicles. We also found that RUNX1 and RUNX2 regulate the expression of Ptgs2 (a rate-limiting enzyme in the biosynthesis of prostaglandins) and Hapln1 (a stabilizer of cumulus matrix) in mural granulosa cells and/or cumulus cells. Both gene products are known to play crucial roles in COC expansion. Most importantly, our pilot study demonstrated that over-expression of dominant negative RUNX in cumulus cells blocked COC expansion. Based on these novel findings, we hypothesized that RUNX1/2 are essential transcriptional regulators necessary for the gonadotropin surge-induced COC expansion. This hypothesis will be tested by first demonstrating the functional significance of Runx1/2 expression in cumulus cells (Aim #1). Secondly, we will determine the regulation action(s) of RUNX1 and RUNX2 on their target genes (Aim #2). Next, we will determine the regulatory mechanism(s) of Runx1 and Runx2 expression in cumulus cells (Aim #3). These 3 Aims will be studied using rat COCs. To insure that the data obtained from rat studies is translatable to humans, RUNX1/2 and their target gene expression profile will be also verified using human cumulus cells collected throughout the periovulatory period. We will also examine the correlation of the cumulus expression of RUNX1/2 and their target genes to oocyte quality, fertilization, and embryo development using COCs obtained from women undergoing IVF (Aim #4). These human studies will serve as a foundation for future translational/clinical application. The information obtained from the proposed studies will not only advance our understanding of the mechanism involved in COC maturation, but also be instrumental for future translational/clinical application(s), thus leading to improved management of fertility in vivo and in vitro. PUBLIC HEALTH RELEVANCE: Proper maturation of COCs is requisite for successful ovulation and fertilization. Appropriate COC expansion induced by the preovulatory gonadotropin surge is directly linked to oocyte quality and developmental competence. There are still huge gaps in our understanding of how the preovulatory gonadotropin surge induces the maturation process in COCs. Previous studies and our preliminary data suggest that RUNX transcription factor(s) plays a critical role in cumulus expansion by regulating the transcription of specific periovulatory cumulus genes. Building upon these findings, the present proposal will determine the molecular/cellular mechanism(s) involved in COC expansion by elucidating the functional role(s) of RUNX transcription factors during maturation of COCs. Information derived from the present proposal will provide new insight into the mechanisms involved in COC maturation. Such knowledge can be applied for promoting and inhibiting this critical facet of ovarian physiology, thereby allowing us to better manage fertility in vivo and in vitro.