Prohibitin-1 Transgenic Mice Revealed an Important Role of Prohibitin in Testicular Steroidogenesis

Abstract

Testosterone, the male sex hormone, plays an important role in the sexual development and fertility. Consequently, its deficiency causes infertility, obesity, osteoporosis and cardiovascular diseases. Leydig cells (LCs) are the testicular interstitial cells responsible for the biosynthesis of testosterone in response to luteinizing hormone (LH) from the pituitary. Cholesterol is the essential substrate for steroidogenesis which is translocated from the cytosol to the mitochondria where it gets converted to pregnenolone (by P450 side chain cleavage enzyme). Subsequently, pregnenolone translocate to endoplasmic reticulum where action of various enzymes results in the biosynthesis of testosterone. Prohibitin-1 (PHB1) is an evolutionary conserved ubiquitously expressed protein with cell compartment and cell-type specific functions. Mitochondrial function of PHB1 has been widely studied but its role in testicular steroidogenesis is unexplored. Recently, we have reported two transgenic mice models of PHB1, PHB-Tg and mutant-PHB-Tg (mPHB-Tg), expressing PHB1 or Y114F (mutant PHB1) respectively under the control of Fabp-4 gene promoter. During phenotypic characterization of these mice models, we observed a drastic size/weight difference in the testis of PHB-Tg and mPHB-Tg when compared with wild type mice. The mPHB-Tg mice testis was significantly smaller than the PHB-Tg and wild type mice. Further analysis of mPHB-Tg testis revealed wider testicular interstitium with LC hyperplasia and elongated seminiferous tubules. Ultrastructure investigation revealed that LCs of mPHB-Tg mice have prominent nucleus with increased number of mitochondria and lipid droplets. In addition, electron microscopic images of mPHB-Tg mice LCs revealed a sign of lipophagy and mitophagy. This prompted us to measure testosterone levels in these mice; surprisingly mPHB-Tg mice showed significantly higher testosterone levels as compared to PHB-Tg and wild type mice. Furthermore, testicular lysates and primary LCs cell lysates from transgenic mice models revealed that overexpression of PHB/mPHB in LCs inversely effect expression levels of steroidogenic acute regulatory protein (StAR). Moreover, co-immunoprecipitation of PHB1 displayed an interaction with StAR, P450scc and LC3 further revealing a key role of PHB1 in cholesterol translocation, testicular steroidogenesis and autophagy. Taken together, this finding suggests that PHB1 plays a multifaceted role in testicular steroidogenesis from determining testis size to the translocation of cholesterol into the mitochondria, in maintaining lipid homeostasis and biosynthesis of testosterone. Implications of our findings are broad because cholesterol translocation to the mitochondria and its subsequent utilization for steroidogenesis is conserved in all steroidogenic tissues.