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.
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