The hypothalamic neuropeptide orexin A- a possible regulator in glucose homeostasis and germ cell kinetics in adult mice testes.

Orexin A (OXA), a hypothalamic neuropeptide, regulates food intake, sleep-wake cycle and energy balance by binding to its receptor (OX1R). Apart from brain, OXA and OX1R are also present in peripheral organs including reproductive tissues. Mammalian reproduction depends on uptake and proper utilization of glucose in the testes. This study, therefore, examined role of OXA/OX1R system in regulation of glucose homeostasis in adult mouse testis under in vivo and ex vivo conditions. Binding of OXA to OX1R was blocked using an OX1R antagonist, SB-334867. Mice were given a single bilateral intratesticular injection of the antagonist at doses of 4 and 12µg/mouse and sacrificed 24 h post-injection. In order to understand the direct role of OXA in testes of adult mice, an ex vivo experiment was performed where binding of OXA to OX1R in the testis was blocked by using the same OX1R antagonist. The antagonist treatment affected testicular glucose and lactate concentration with concomitant down-regulation in the expression of glucose transporters 3 and 8. A decreased activity in lactate dehydrogenase enzyme and imbalance between germ cell survival and proliferation were also noted in testes in treated mice. The results of ex vivo study supported the results obtained from in vivo study. The findings thus suggest involvement of OXA/OX1R system in regulation of testicular glucose homeostasis and germ cell kinetics in adult mice.

Localization, expression and role of Orexin A and its receptor in testes of neonatal mice.

Orexin A (OXA), a hypothalamic neuropeptide, and its receptor (OX1R) are primarily expressed in lateral hypothalamus and are involved in the control of various biological functions. Expressions of OXA and OX1R have also been reported in peripheral organs like gastrointestinal and genital tracts. In the present study, expressions of OXA and OX1R have been observed in the testis of Parkes strain neonatal mice by semi-quantitative RT-PCR and western blot analyses. Immunohistochemical study also revealed their presence on spermatogonia, Sertoli cells and in the interstitium of the testis. In order to understand the role of OXA and OX1R in testicular development, an in vitro study was also performed. For this, binding of OXA to OX1R was blocked using OX1R specific antagonist, SB-334867. Eighteen mice were sacrificed and their testes were cultured in complete media containing vehicle and two doses (0.1 and 4.0µg/ml media) of SB-334867 for 72h in CO2 incubator at 37°C. At the end of culture period, testes were used for western blot and RT-PCR analyses to study the expression of various markers of gonadal development, such as steroidogenic factor 1 (SF-1), Wilms' tumor 1 (Wt1), Mullerian inhibiting substance (MIS) and stem cell factor (SCF). Further, expressions of OXA, OX1R and glucose transporter 3 (GLUT 3) were also studied. A marked increase in the expression of SF-1 and a decrease in the expression of Wt1 at both transcript and protein levels were noted, while there was a decrease in the expression of SCF and MIS at transcript level at both doses of the antagonist; this suggests that blockage of OXA binding to OX1R by SB-334867 affects testicular development. The decrease in expressions of OXA, OX1R and GLUT 3 in the test is in response to both doses of the antagonist points to their down-regulation causing inefficient uptake of glucose by the testicular cells, thereby affecting gonadal development. In conclusion, our results suggest that the binding of OXA to OX1R is important for the development of the testis.