No abstract

ABSTRACT:The signaling pathways that characterize the process of capacitation of human spermatozoa are still largely unknown. Modifications in the lipid architecture of the sperm plasma membrane have been described in spermatozoa from different species, including translocation of phosphatidylserine (PS) from the inner to the outer leaflet and increased phospholipid disorder in the membrane. In human spermatozoa, however, results of PS exposure are controversial. In the present study, we used flow cytometry to investigate both membrane PS exposure by Annexin V (Ann V) binding and lipid disorder by merocyanine 540 (M540) staining, in swimup-selected live spermatozoa after incubation in conditions leading to capacitation. Our results indicate that neither probe is able to detect capacitation-related membrane modifications. Investigation of the nature of PS exposure and M540-positive live cells was then carried out. We found that M540 stains elements devoid of nuclei are present in seminal plasma. Live PS-exposing cells were mainly represented by damaged spermatozoa as revealed by the occurrence of a negative correlation between PS exposure and normal morphology and motility in unselected samples. The same cells were also positive for M540. These results demonstrate that Ann V and M540 binding in human sperm samples mainly detects cells with early membrane degeneration as well as dead cells, which is in agreement with findings obtained for somatic cells in which the two probes recognize cells with a damaged membrane due to the apoptotic process.

Mammalian testicular spermatozoa are immotile, thus, to reach the oocyte, they need to acquire swimming ability under the control of different factors acting during the sperm transit through the epididymis and the female genital tract. Although bicarbonate is known to physiologically increase motility by stimulating soluble adenylate cyclase (sAC) activity of mammalian spermatozoa, no extensive studies in human sperm have been performed yet to elucidate the additional molecular mechanisms involved. In this light, we investigated the effect of in vitro addition of bicarbonate to human spermatozoa on the main intracellular signaling pathways involved in regulation of motility, namely, intracellular cAMP production and protein tyrosine phosphorylation. Bicarbonate effects were compared with those of the phosphatidyl-inositol-3 kinase inhibitor, LY294002, previously demonstrated to be a pharmacological stimulus for sperm motility. Bicarbonate addition to spermatozoa results in a significant increase in sperm motility as well as in several hyperactivation parameters. This stimulatory effect of bicarbonate and LY294002 is mediated by an increase in cAMP production and tyrosine phosphorylation of the A kinase anchoring protein, AKAP3. The specificity of bicarbonate effects was confirmed by inhibition with 4,4'-di-isothiocyanostilbene-2,2'-disulfonic acid. We remark that, in human spermatozoa, bicarbonate acts primarily through activation of sAC to stimulate tyrosine phosphorylation of AKAP3 and sperm motility because both effects are blunted by the sAC inhibitor 2OH-estradiol. In conclusion, our data provide the first evidence that bicarbonate stimulates human sperm motility and hyperactivation through activation of sAC and tyrosine phosphorylation of AKAP3, finally leading to an increased recruitment of PKA to AKAP3.