Localization of angiotensin-II type 1(AT1) receptors on buffalo spermatozoa: AT1 receptor activation during capacitation triggers rise in cyclic AMP and calcium

Abstract

The purpose of this study was to determine the role of Ang-II in buffalo spermatozoa; localize angiotensin type 1 (AT1) receptors on the sperm surface and understand the signaling mechanisms involved therein. Immunoblotting and immunocytochemistry using polyclonal Rabbit anti-AT1 (N-10) IgG were performed to confirm the presence of AT1 receptors. Intracellular levels of cyclic adenosine monophosphate (cAMP) were determined by non-radioactive enzyme immunoassay, while that of Calcium [Ca(2+)] were estimated by fluorimetry using Fura2AM dye. The results obtained showed that AT1 receptors were found on the post-acrosomal region, neck and tail regions. Immunoblotting revealed a single protein band with molecular weight of 40 kDa. Ang-II treated cells produced significantly higher level of cAMP compared to untreated cells (22.66 ± 2.4 vs. 10.8 ± 0.98 pmol/10(8) cells, p < 0.01). The mean levels of Ca(2+) were also higher in Ang-II treated cells compared to control (117.4 ± 6.1 vs. 61.15 ± 4.2 nmol/10(8) cells; p < 0.01). The stimulatory effect of Ang-II in both the cases was significantly inhibited in the presence of Losartan (AT1 antagonist; p < 0.05) indicating the involvement of AT1 receptors. Further, presence of neomycin (protein kinase C inhibitor) inhibited significantly the Ang-II mediated rise in Ca(2+) indicating the involvement of PKC pathway. These findings confirm the presence of AT1 receptors in buffalo spermatozoa and that Ang-II mediates its actions via the activation of these receptors. Ang-II stimulates the rise in intracellular levels of cAMP and Ca(2+) during capacitation.