FSH and Retinoid Signaling in the Testis

描述(申请人提供)：正常的精子发生没有功能性维甲酸受体α(RARα)在睾丸发生。然而，这种受体在睾丸中是如何调节的还不清楚。发育中的睾丸以不同的时间和细胞模式表达RARα，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调节的。在睾丸中，我们在维甲酸生物学领域有了一个重大的、令人惊讶的发现：在睾丸发育过程中，包括RARα在内的维甲酸受体并不是结构性地存在于细胞核中，但它们的核运输可能受到调控。这一点很重要，因为RARα的核定位增加与该受体在Sertoli细胞中转录活性的增加直接相关。此外，我们还发现，虽然维甲酸是必需的，但最终控制支持细胞受体活性的是卵泡刺激素(FSH)等通过蛋白激酶A(PKA)系统发挥作用的因素。因此，即使在RARα活性的正诱导剂维甲酸存在的情况下，FSH的作用也可以支配和抑制RARα的活性。这种FSH和RARα的相互作用可能在睾丸中有重要意义。FSH信号被认为在细胞增殖中起作用，我们推测，只有当FSH信号减弱时，RARα才可能在细胞分化中发挥作用。本研究旨在验证一种假设，即FSH的作用是通过抑制维甲酸诱导的RARα的核定位，从而通过翻译后机制抑制RARα的转录活性，从而抑制RARpha在睾丸中的功能。目的1确定FSH是否通过改变RARα的磷酸化模式抑制RARα在Sertoli细胞中的核定位。目的2确定FSH是否通过抑制核内稳定转录复合体的形成而抑制RARα的核定位。我们将研究维甲酸反应元件、异源二聚体伙伴和共激活剂在形成转录复合体中的作用。在目标3中，FSH功能将被遗传和反义策略扰乱，然后，将在支持细胞培养和睾丸器官培养中检测RARα翻译后机制的变化。结合质谱学和反义技术，包括siRNA方法，确定支持细胞中RARα和其他相互作用蛋白翻译后机制的变化是非常令人兴奋的，这两种方法都是新的应用。现在有三个目标被密切关注，以提供睾丸中FSH和RARα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.