Date of Award

12-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular Therapeutics and Cell Signaling

Research Advisor

John D. Schuetz, Ph.D.

Committee

Linda Hendershot, Ph.D. Susan Senogles, Ph.D. Brian Sorrentino, MD. Beatriz Sosa-Pineda, Ph.D.

Abstract

The physiological role of multidrug resistance protein 4 (Mrp4) in the testes is unknown. It was discovered that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone biosynthesis. The role of Mrp4 was investigated in Leydig cells where testosterone production is regulated by cAMP, an intracellular second messenger formed when the luteinizing hormone (LH) receptor (Lhr) is activated. As Mrp4 regulates cAMP, we compared testosterone levels in our Mrp4 WT and KO mice. Prepubertal KO mice had significantly reduced testicular testosterone, impaired gametogenesis, and disrupted cAMP homeostasis, resulting in decreased expression of genes involved in testosterone biosynthesis. Testosterone production was also impaired in adult KO mice but testicular morphology was normal. In vitro culture of primary KO Leydig cells stimulated with LH revealed decreased intracellular cAMP concentrations and attenuated cAMP-response element binding protein (CREB) phosphorylation of downstream targets, notably several genes directly involved in testosterone biosynthesis. However, chemical inhibition of Mrp4 in WT Leydig cells revealed a substantial elevation in intracellular cAMP concentration but paradoxically reduced testosterone production. The reduced testosterone production was related to decreased immunoreactive StAR expression, the rate limiting step in testosterone biosynthesis. Future analysis will focus on the mechanism controlling cAMP concentrations under these conditions, with a primary focus on cAMP regulation by phosphodiesterases (PDEs). Continued assessment of our KO animals revealed pre-pubertal animals had reduced systemic testosterone concentrations while adult mice had normal circulating levels. The difference is pre-pubertal KO mice have increased Cyp2b10, a known testosterone metabolizing enzyme, expression and catalytic activity due to disrupted testicular testosterone production. Therefore, defective testicular testosterone production deregulates hepatic Cyp-mediated testosterone metabolism to disrupt gametogenesis. The findings presented here have important implications for understanding the side effects of therapeutics that disrupt Mrp4 function and can alter androgen production.

DOI

10.21007/etd.cghs.2012.0217

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