The aims of the present study were to characterize a slow capacitation system that records initial changes in the sperm membrane state, and, using a canine model, to order the specific protein tyrosine phosphorylation signaling in the sequence of capacitational events and to associate them with hyperactivated motility. Dog sperm washed through Percoll were incubated in complete bicarbonate Tyrode medium for 6 hours in 5% CO(2). Capacitation was evaluated using chlortetracycline staining. Tyrosine phosphorylation patterns were assessed by immunocytochemistry. Parallel to this, a computer-assisted motility analysis was performed. Significant changes in the percentage of capacitated and acrosome-reacted cells were first observed after 90 minutes, increasing in a linear manner during further incubation (P <.05). Changes in the percentage of capacitated cells were accompanied by motility changes. During incubation, a strictly sequential phosphorylation of sperm tail (midpiece, principal piece, and end piece) and head proteins was observed. According to an analysis of kinetics, phosphorylation of head proteins occurred after the tail became completely phosphorylated. Changes in head phosphorylation progressed at the same rates as capacitation and acrosome reaction. Sperm motility, curvilinear velocity, average path velocity, straight line velocity, and lateral head displacement were correlated positively or negatively with phosphorylation of midpiece or end piece proteins, respectively. The bicarbonate-stimulated increases in cyclic adenosine monophosphate levels and changes in protein phosphatase activity may be involved in the signaling system that controls membrane changes and motility in dog sperm. Phosphorylation kinetics of sperm proteins are potentially useful for diagnostic purposes to characterize the response of individual males to fertilizing conditions.

No abstract

Progress of essential steps of the capacitation is coordinated in the oviductal isthmus, where sperm are stored in close contact with the epithelium. A crucial capacitational event is the phosphorylation of sperm membrane proteins. Regulation of the tyrosine phosphorylation by the oviduct has not been examined in dog sperm yet. The aim of this work was to study the effect of dog sperm binding to porcine oviductal epithelium on capacitation-induced cellular and molecular changes. Epithelial cells were stripped from the oviducts of post-puberal sows and cultured for 5-7 days at 39 degrees C and 5% CO2 on Biomatrix-covered Chamber slides. Sperm washed through Percoll was co-incubated with the oviductal epithelium cell cultures in a bicarbonate Tyrode's medium. During co-incubation, sperm membrane changes, the state of tyrosine phosphorylation and motility were determined after 3, 30, 90, 180, 240 and 360 min. Significant increases in the percentage of capacitated and dead cells were observed in unbound sperm, while bound sperm remained uncapacitated, live and motile. An increasing tyrosine phosphorylation of tail proteins in bound, unbound and control sperm suspensions and a subsequent phosphorylation of head proteins in unbound and control sperm suspensions were observed. A significant difference regarding head phosphorylation (p < 0.05) was found between sperm bound to oviductal epithelium and unbound sperm. Binding occurred mainly in sperm with non- phosphorylated heads, while higher proportions of phosphorylated cells were found in unbound populations. The head phosphorylation progressed significantly during incubation in unbound spermatozoa (p < 0.05); however, it was suppressed in population of sperm attached to oviductal epithelium. Significant correlations between motility parameters related to hyperactivation and tail phosphorylation were found in unbound sperm. These observations support the hypothesis that spermatozoa with non-phosphorylated heads preferentially attach to epithelial cells. It can be concluded that tyrosine phosphorylation of head membrane proteins and capacitation are delayed in canine spermatozoa being in closed contact with oviductal epithelium.