RESUMO
Caltrin is a small and basic protein of the seminal vesicle secretion that inhibits sperm calcium uptake. The influence of rat caltrin on sperm physiological processes related to fertilizing competence was studied by examining its effect on 1) spontaneous acrosomal exocytosis, 2) protein tyrosine phosphorylation, and 3) sperm-egg interaction. Results show that the presence of caltrin during in vitro capacitation both reduced the rate of spontaneous acrosomal exocytosis without altering the pattern of protein tyrosine phosphorylation, and enhanced the sperm ability to bind to the zona pellucida (ZP). The significantly higher proportion of sperm with intact acrosome observed in the presence of caltrin was accompanied by a strong inhibition in the acrosomal hyaluronidase release. Enhancement of sperm-ZP binding was evident by the increase in the percentage of eggs with bound spermatozoa as well as in the number of bound sperm per egg. Similar results were obtained when the assays were performed using spermatozoa preincubated with caltrin and then washed to remove the unbound protein, indicating that the sperm-bound caltrin was the one involved in both acrosomal exocytosis inhibition and sperm-ZP binding enhancement. Caltrin bound to the sperm head was partially released during the acrosomal exocytosis induced by Ca-ionophore A23187. Indirect immunofluorescence and immunoelectron microscopy studies revealed that caltrin molecules distributed on the dorsal sperm surface disappeared after ionophore exposure, whereas those on the ventral region remained in this localization after the treatment. The present data suggest that rat caltrin molecules bound to the sperm head during ejaculation prevent the occurrence of the spontaneous acrosomal exocytosis along the female reproductive tract. Consequently, more competent spermatozoa with intact and functional acrosome would be available in the oviduct to participate in fertilization.
Assuntos
Acrossomo/metabolismo , Exocitose/fisiologia , Proteínas Secretadas pela Vesícula Seminal/fisiologia , Capacitação Espermática/fisiologia , Acrossomo/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Animais , Exocitose/efeitos dos fármacos , Feminino , Masculino , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteínas Secretadas pela Vesícula Seminal/farmacologia , Capacitação Espermática/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Zona Pelúcida/metabolismoRESUMO
Epididymal protein CRISP1 participates in rat and mouse gamete fusion through its interaction with complementary sites on the egg surface. Based on in vivo observations, in the present study we investigated the possibility that CRISP1 plays an additional role in the sperm-zona pellucida (ZP) interaction that precedes gamete fusion. In vitro fertilization experiments using zona-intact rat and mouse eggs indicated that the presence of either an antibody against rat CRISP1 (anti-CRISP1) or rat native CRISP1 (rCRISP1) during gamete co-incubation produced a significant decrease in the percentage of fertilized eggs. However, differently to that expected for a protein involved in gamete fusion, no accumulation of perivitelline sperm was observed, suggesting that the inhibitions occurred at the sperm-ZP interaction level. Bacterially expressed recombinant CRISP1 (recCRISP1) also significantly inhibited egg fertilization. In this case, however, an increase in the number of perivitelline sperm was observed. Subsequent experiments evaluating the effect of anti-CRISP1 or rCRISP1 on the number of sperm bound per egg indicated that the protein is involved in the initial step of sperm-ZP binding. In agreement with these functional studies, indirect immunofluorescence experiments revealed that although rCRISP1 is capable of binding to both the ZP and the oolema, recCRISP1 only binds to the egg surface. The finding that deglycosylated rCRISP1 behaves as the untreated protein, whereas the heat-denatured rCRISP1 associated only with the oolema, indicates that the protein ZP-binding ability resides in the conformation rather than in the glycosydic portion of the molecule. The interaction between rCRISP1 and the ZP reproduces the sperm-ZP-binding behavior, as judged by the failure of the protein to interact with the ZP of fertilized eggs. Together, these results support the idea that CRISP1 participates not only in sperm-egg fusion but also in the prior stage of sperm-ZP interaction.