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

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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 [Ca2+] 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/108 cells, p < 0.01). The mean levels of Ca2+ were also higher in Ang-II treated cells compared to control (117.4 ± 6.1 vs. 61.15 ± 4.2 nmol/108 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 Ca2+ 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 Ca2+ during capacitation.

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Acknowledgments

The authors are grateful to the Indian Council of Agricultural Research (ICAR) for funding this project.

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Authors and Affiliations

  1. Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India

    Sivaram Vedantam, Rita Rani, Monica Garg & Suresh K. Atreja

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  1. Sivaram Vedantam
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  2. Rita Rani
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  3. Monica Garg
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  4. Suresh K. Atreja
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Correspondence to Sivaram Vedantam.

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Vedantam, S., Rani, R., Garg, M. et al. Localization of angiotensin-II type 1(AT1) receptors on buffalo spermatozoa: AT1 receptor activation during capacitation triggers rise in cyclic AMP and calcium. Mol Biol Rep 41, 1959–1965 (2014). https://doi.org/10.1007/s11033-014-3043-7

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