FSH and Retinoid Signaling in the Testis

描述（由申请人提供）：如果睾丸中没有功能性视黄酸受体α (RAR α)，则不会发生正常的精子发生。然而，这种受体在睾丸中的调节机制尚不清楚。发育中的睾丸以不同的时间和细胞模式表达RAR α，因此，睾丸提供了一个独特的生理系统来研究内源性受体是如何被调节的。在睾丸中，我们在类视黄酮生物学领域取得了一个重大的、令人惊讶的发现，包括RAR α在内的类视黄酮受体在睾丸发育过程中并不是组成性地存在于细胞核中，但它们的核运输可能受到调节。这一点很重要，因为RAR α的核定位增加与支持细胞中该受体转录活性的增加直接相关。此外，我们发现，虽然维甲酸是必需的，但最终控制支持细胞中受体活性的是促卵泡激素（FSH）等因子，它们通过蛋白激酶A （PKA）系统起作用。因此，即使在视黄酸（RAR α活性的正诱导剂）存在的情况下，FSH的作用也可以支配和抑制RAR α活性。FSH和RAR α的相互作用可能对睾丸有重要影响。FSH信号被认为在细胞增殖中起作用，我们假设，只有当FSH信号减弱时，RAR α可能在细胞分化中发挥作用。本研究旨在验证卵泡刺激素的作用是通过抑制视黄酸诱导的RAR α的核定位以及随后通过翻译后机制的转录活性来抑制睾丸中RAR α功能的假设。目的1是通过改变RAR α磷酸化模式来确定FSH是否抑制支持细胞RAR α核定位。目的2是确定FSH是否通过抑制细胞核中稳定转录复合物的形成来抑制RAR α核定位。视黄酸响应元件、异二聚体伴侣和共激活因子在形成转录复合体中的作用将被研究。在Aim 3中，FSH功能将被遗传和反义策略破坏，然后，RAR α翻译后机制的改变将在Sertoli细胞培养和睾丸器官培养中进行研究。结合质谱法和反义技术，包括siRNA方法，这两种新应用，来确定Sertoli细胞中RAR α和其他相互作用蛋白的翻译后机制的变化是非常令人兴奋的。三个目标现在紧密集中在提供睾丸中FSH和RAR α之间的关键联系。

DESCRIPTION (provided by applicant): Normal spermatogenesis does not occur without functional retinoic acid receptor alpha (RAR alpha) in the testis. However, how this receptor is regulated in the testis is not known. Developing testes express RAR alpha with varying temporal and cellular patterns, and thus, the testis provides a unique physiological system to study how the endogenous receptor is regulated. In the testis, we made a major, surprising finding for the retinoid biology field that retinoid receptors including RAR alpha are not constitutively found in the nucleus during testis development, but their nuclear trafficking may be regulated. This is important because an increased nuclear localization of RAR alpha is directly related to an increased transcriptional activity of this receptor in Sertoli cells. In addition, we found that, although retinoic acid is definitely required, it is factors such as follicle stimulating hormone (FSH) acting through the protein kinase A (PKA) system, which ultimately control the receptor activity in Sertoli cells. Thus, even in the presence of retinoic acid, a positive inducer for the RAR alpha activity, FSH action can dominate and inhibit the RARalpha activity. This FSH and RAR alpha interplay may have a major implication in the testis. FSH signaling is postulated to function in cell proliferation and, we postulate that, only when FSH signaling diminishes, RAR alpha may play a role in cell differentiation. This research is designed to test the hypothesis that a role of FSH is to inhibit RARalpha function in the testis by inhibiting retinoic acidinduced nuclear localization of RAR alpha and subsequently transcriptional activity by post-translational mechanisms. Aim 1 is to determine whether FSH inhibits RAR alpha nuclear localization in Sertoli cells by changing the phosphorylation pattern on RAR alpha. Aim 2 is to determine whether FSH inhibits RAR alpha nuclear localization by inhibiting the formation of a stable transcription complex in the nucleus. The role of retinoic acid responsive element, heterodimer partner, and co-activators in forming transcriptional complex will be examined. In Aim 3, FSH function will be disrupted by both genetic and antisense strategies, and then, alterations in the post-translational mechanisms of RAR alpha will be examined in Sertoli cell cultures and in testicular organ cultures. It is very exciting to combine mass spectrometry and antisense technologies, including siRNA method, both novel applications, to determine alterations in the post-translational mechanisms of RAR alpha and other interacting proteins in Sertoli cells. Three Aims are now tightly focused to provide a critical link between FSH and RAR alpha in the testis.