Oviduct Binding and Elevated Environmental pH Induce Protein Tyrosine Phosphorylation in Stallion Spermatozoa1

Abstract

Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca 2+ , and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 3 10 6 spermatozoa/ml), during which it transpired that the highest density (per mm 2 ) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a timedependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviductbound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pH i ) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pH i , presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pH i changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm.

Validation of merocyanine 540 staining as a technique for assessing capacitation-related membrane destabilization of fresh dog sperm

“…While changes in the membrane fluidity and membrane destabilization of the sperm plasma membrane induced by bicarbonate have been shown to be immediate [5], it may be possible that dog sperm are more susceptible to prolonged periods of incubation, inducing membrane destabilization as detectable by M540-YP even in the absence of bicarbonate. Recent studies on stallion sperm have shown that an increase in the pH of the incubation medium induces capacitation (46). Unfortunately the pH of the media used in the current study was not monitored in order to detect a change therein.…”

The Role of Oviductal Cells in Activating Stallion Spermatozoa

“…Unfortunately, heparin-like glycosaminoglycans have no capacitation-inducing effects on stallion spermatozoa [40]. Using the oviduct explant model, however, we found that in vitro binding of stallion spermatozoa to oviduct epithelium was able to induce capacitation [38]. Oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response that was not observed in unbound spermatozoa or when spermatozoa were incubated in oviduct explant-conditioned medium.…”

“…oviduct binding, while a time-dependent gradual increase in intracellular pH was observed in oviduct-bound spermatozoa; unbound spermatozoa did not show intracellular pH changes [38].…”

“…As stated above, we observed that stallion spermatozoa bound to oviduct epithelial cells in vitro showed higher rates of protein tyrosine phosphorylation than unbound spermatozoa -with the proportion of protein tyrosine phosphorylated spermatozoa increasing in a time-dependent fashion [38]. Initially, we hypothesized that the interaction between the sperm cells and the oviduct was important to switch on the intracellular capacitation processes in vitro.…”

“…Since then, very little novel data has been reported on this topic. In order to develop in vitro oviduct models that more closely mimic the peri-ovulatory situation in vivo, we recently [38][39][40] used alternative approaches to study equine sperm-oviduct interaction, namely oviduct explant and apical plasma membrane models. The aim was to generate new insights into (1) the molecular basis of equine spermoviduct binding, (2) the capacitation-related mechanisms that induce sperm tail-associated protein tyrosine phosphorylation, hyperactivated motility and the acrosome reaction, (3) how hyperactivated, protein tyrosine phosphorylated spermatozoa are triggered to release from the oviduct epithelium, and (4) the fertilizing capacity of hyperactivated, protein tyrosine phosphorylated spermatozoa.…”