The role of Zn-α2 glycoprotein in sperm motility is mediated by changes in cyclic AMP

Abstract

Sperm motility is essential for male reproduction or natural fertilization. The cyclic AMP (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway is generally recognized as one of the significant signaling pathways in the regulation of mammalian spermatozoan motility. Since Zn-a2-glycoprotein (ZAG) activity in mammalian adipose tissue is mediated via the b 3 -adrenoreceptor, with upregulation of the cAMP pathway, we hypothesize that ZAG may play the same role in sperm motility regulation, a new factor of regulation of sperm motility. Therefore, the gene encoding human ZAG was cloned and polyclonal antibodies were generated, and then laser scanning confocal microscopy and flow cytometry were employed to identify this protein in human spermatozoa. The results showed that ZAG protein was mostly localized on the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum of spermatozoa. Furthermore, using computer-assisted sperm analysis, we found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90-or 120-min incubation (P!0.05 and P!0.01 respectively), together with the decreasing of intracellular cAMP and PKA levels. In conclusion, these data suggest that ZAG is present in human spermatozoa and may be involved in the regulation of sperm motility via the cAMP/PKA signaling pathway.

RNASET2 in human spermatozoa and seminal plasma: a novel relevant indicator for asthenozoospermia

“…A segment of RNASET2 was amplified with the forward primer 5′-GCGGATCCCCCGATAAAAGTGAAGGA-3′ (containing BamHI site, underlined) and the reverse primer 5′-GCAAGCTTAG-GTGGGGGATAGAAGAC-3′ (containing HindIII site, underlined). Prokaryotic expression and purification of recombinant RNA-SET2 were performed as previously described (Qu et al, 2007). The new recombinant prokaryotic expression vector was named pET-28a(+)/RNASET2 and propagated in Escherichia coli BL21 (DE3) host cells cultured with 0.8 mM isopropyl-b-D-thiogalactopyranoside (IPTG) for 5 h at 37°C with gentle shaking.…”

“…Polyclonal anti-recombinant RNASET2 antibodies were produced by immunizing white New Zealand rabbits (~6 months old, body weight~2.5 kg; purchased from Shanghai SLAC Laboratory Animal Co., Shanghai, China), as previously described (Qu et al, 2007). The titre of rabbit anti-rRNASET2 sera was detected by ELISA at 450 nm in conjunction with an Anthos Zenyth 1100 multimode detector (Anthos Labtec Instruments GmbH, Wals, Austria) with a 5-s pre-read shaker.…”

“…Western blot was performed as previously described (Qu et al, 2007). Protein samples (30 lg) were separated by SDS-PAGE, then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) using a semi-dry transfer apparatus (Bio-Rad, Hercules, Calif, USA); membranes were blocked with 5% bovine serum albumin (BSA) in TBS containing 0.2% Tween-20.…”

“…To identify the localization of RNASET2 on human spermatozoa, high quality spermatozoa was obtained by a discontinuous 47/90% Percoll density gradient centrifugation at 800 g for 30 min at 4°C as described (Qu et al, 2007) and subsequently washed thrice with PBS. Acrosome reaction (AR) was induced using a calcium ionophore, 10 lM A23187 (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37°C.…”

Prevalence of zinc- 2-glycoprotein binding peptide among Omani blood donors

“…The effect of ZAG binding peptide in fertilization is focused in the role it plays in sperm motility, as sperms are independent on the metabolism of lipid to initiate and maintain the motility of their flagella. Moreover, ZAG binding peptide has showed an ability to bind to the sperm membrane and significantly increasing the cAMP concentration (Fei Qu, 2007). The cAMP-dependent phosphorylation is counted as one of the most important pathways for regulating sperm motility.…”