Vedantam Sivaram, Rani Rita, Garg Monica, Atreja Suresh K
Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India,
Mol Biol Rep. 2014;41(4):1959-65. doi: 10.1007/s11033-014-3043-7. Epub 2014 Jan 17.
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.
本研究的目的是确定血管紧张素II（Ang-II）在水牛精子中的作用；将1型血管紧张素（AT1）受体定位在精子表面，并了解其中涉及的信号传导机制。使用兔抗AT1（N-10）多克隆IgG进行免疫印迹和免疫细胞化学，以确认AT1受体的存在。通过非放射性酶免疫测定法测定细胞内环磷酸腺苷（cAMP）的水平，而使用Fura2AM染料通过荧光法估计钙[Ca(2+)]的水平。获得的结果表明，AT1受体存在于顶体后区域、颈部和尾部区域。免疫印迹显示一条分子量为40 kDa的单一蛋白带。与未处理的细胞相比，Ang-II处理的细胞产生的cAMP水平显著更高（22.66±2.4对10.8±0.98 pmol/10(8)个细胞，p<0.01）。与对照组相比，Ang-II处理的细胞中Ca(2+)的平均水平也更高（117.4±6.1对61.15±4.2 nmol/10(8)个细胞；p<0.01）。在氯沙坦（AT1拮抗剂；p<0.05）存在的情况下，Ang-II在这两种情况下的刺激作用均被显著抑制，表明AT1受体参与其中。此外，新霉素（蛋白激酶C抑制剂）的存在显著抑制了Ang-II介导的Ca(2+)升高，表明PKC途径参与其中。这些发现证实了水牛精子中存在AT1受体，并且Ang-II通过激活这些受体介导其作用。在获能过程中，Ang-II刺激细胞内cAMP和Ca(2+)水平升高。
