title: 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.
authors.affiliation: Division of Animal Biochemistry, National Dairy Research Institute, Karnal, 132001, Haryana, India, sivaram3@gmail.com.
authors.forename: Sivaram
authors.initials: S
authors.lastname: Vedantam
authors.forename: Rita
authors.initials: R
authors.lastname: Rani
authors.forename: Monica
authors.initials: M
authors.lastname: Garg
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Atreja
mesh_terms: D000818:Animals; D002020:Buffaloes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D008297:Male; D021381:Protein Transport; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D013075:Sperm Capacitation / Q000502:physiology; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism*
chemical_list: D044140:Receptor, Angiotensin, Type 1; D000242:Cyclic AMP; D002118:Calcium