1. Effects of Intermittent Hypoxia on Testosterone Production in Leydig Cells Yu-Min Cho 1, S.-C. Cheng 1, C.-F. Fang 1, Chan-Hsun Hsu 1, Yung-Chiong Chow 1,2, Hsiao-Fung Pu 1, Paulus S. Wang 1 1 Department of Physiology, National Yang-Ming University & 2 Department of Urology, Mackay Memorial Hospital, Taipei, Taiwan, Republic of China Abstract It has been shown that hypoxia plays an important role in regulating physiological functions. However, the effects of hypoxia on the reproductive endocrine systems are unclear. The aim of this study was to explore the effect of intermittent hypoxia on the production of testosterone both in vivo and in vitro. Male rats were housed in a hypoxic chamber (12%O2 + 88%N2), 8 h daily for 14 days. Normoxic rats were used as control animals. In vivo, hypoxic and normoxic rats were challenged with a single intravenous (i.v.) injection of gonadotropin-releasing hormone (GnRH) or human chorionic gonadotropin (hCG). Blood samples were collected following i.v. injection. The concentrations of luteinizing hormone (LH) and testosterone in the plasma were measured by radioimmunoassay (RIA). In vitro, the rat Leydig cells of normoxic and hypoxic rats were challenged with hCG, forskolin (an adenylyl cyclase activator), or 8- bromo- adenosine 3’,5’- cyclic monophosphate (8-Br-cAMP, a membrane permeable analog of cAMP) in the presence or absence of 25- OH- hydroxycholesterol at 34 ℃ for 1 h. The concentration of testosterone and pregnenolone in the incubation media were measured by RIA. The effect of intermittent hypoxia on the protein expression of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc) was examined by Western blot. Intermittent hypoxia resulted in an increase of plasma testosterone levels in response to hCG. Intermittent hypoxia for 14 days was effective to increase the basal and forskolin stimulated release of testosterone and StAR protein expression as compared to the normoxic group. Angiogenesis were observed in rat testicular tissue following intermittent hypoxia. These results suggest that intermittent hypoxia increases production of testosterone by rat Leydig cells in part through the mechanisms involving the increase of the activites of cAMP pathway and StAR protein expression. Introduction It has been shown that hypoxia influences the reproductive endocrine systems. By hypothalamic- pituitary-adrenal axis, hypoxia alters the corticotrophs of the anterior pituitary and ACTH release (Gosney, 1984), or stimulates StAR protein expression directly to increase glucocorticoid secretion (Raff et al., 2003). Hypoxia also inhibits the biosynthesis of aldosterone (Raff et al., 1997). We previously reported that the mechanism of hypoxia or erythropoietin release from the kidneys in male rats are testosterone-dependent (Wang et al., 1996). However, the effects of hypoxia on the male reproductive endocrine systems are unclear. It has been well known that testosterone biosynthesis in Leydig cells is dependent on StAR and P450scc protein. It will be interesting to understand whether P450scc and StAR are involved in the regulation of testosterone secretion in rats by hypoxia. It is known that hCG stimulated the secretion of testosterone both in vivo and in vitro via mechanisms involving increased production of cAMP (Lin et al., 1998). The mechinasm by hypoxia modulates testosterone secretion in Leydig cells has not yet been well known. Purpose Fig.2 Administration of intermittent hypoxia resulted in a significant increase (p<0.05) of both basal and hCG-stimulated plasma testosterone secretion by 1.5~2.1-fold after a singal i.v. injection of hCG. * p<0.05, **p<0.01 compared to normoxic group. Each value represents mean ±SEM. Effect of Intermittent Hypoxia on the Concentration of Plasma Testosterone In Vivo Fig.7 P450scc (54 kDa) and StAR (30 kDa) protein expression under intermittent hypoxic treatment. Western blot analysis of cell extract subjected to SDS- PAGE and developed by ECL reaction. * p<0.05, **p<0.01 compared to normoxic group. Each value represents mean ± SEM. Effect of Intermittent Hypoxia on the Protein Expression Fig.6 Effects of intermittent hypoxia on 25-OH- cholesterol-stimulated pregnenlone release by rat Leydig cells. Effect of Intermittent Hypoxia on Cholesterol- Stimulated Pregnenolone Release In Vitro Fig.5 Effects of intermittent hypoxia on basal and hCG-, 8- Br-cAMP, and forskolin- stimulated testosterone release in rat Leydig cells. + p<0.05, ++p<0.01 compared to vehicle group. * p<0.05, **p<0.01 compared to normoxic group. Each value represents mean ±SEM. Effect of Intermittent Hypoxia on cAMP - Related Testosterone Secretion In Vitro Fig.4 The rat testes were removed after decapitation. Angiogenesis and vasodilation were observed in rat testicular tissues following intermittent hypoxia. Effect of Intermittent Hypoxia on Testis Morphology Intermittent hypoxia Leydig cells testosterum production gonadotropin –stimulated cAMP? Experiment design Rats hypoxia ( 12%O 2 +88%N 2 for 8h/day, 14days) GnRH(2μg/ml/kg) Single i.v. injection Leydig cell Blood collection 0, 15, 30, 60, 90, 120 min + hCG(0.012U/ml) + forskolin(10 -5 M) + 8-Br-cAMP (10 -4 M) + 25-OH-cholesterol (10 -5 M) + trilostane(10 -5 M) Plasma LH, Testosterone 34 ℃, 1h Media collection Testosterone Pregenenolone RIAs Western Blot of P450scc and StAR Normoxia Hypoxia Effect of Intermittent Hypoxia on the Concentration of Plasma LH In Vivo Results Fig.1 Effect of intermittent hypoxia on the concentration of plasma LH in male rats after an intravenous injection of GnRH. Each value represents mean ±SEM. Normoxia Hypoxia Conclusion In summary, these results demonstrated that intermittent hypoxia stimulated testosterone production in Leydig cells via an activation of cAMP pathway and an increase of StAR protein expression as well as an angiogenesis over testicular surface. in vivo in vitro Normoxia Hypoxia Time (min) Fig.3 As compared to normoxic group, administration of intermittent hypoxia for 14 days significantly increased the basal level of testosterone secretion. * p<0.05, **p<0.01 compared to normoxic group. Each value represents mean ±SEM.