Porcine oviduct sperm binding glycoprotein and its deleterious effect on sperm: a mechanism for negative selection of sperm?

In their journey through the oviduct some subpopulations of sperm are preserved in a reservoir, while others are negatively selected. Sperm binding glycoprotein (SBG) is a pig oviductal epithelial cell glycoprotein that produces, under capacitating conditions, acrosome alteration, p97 tyrosine-phosphorylation and reduction of the motility of sperm. In this paper, we show that SBG is accessible at the extracellular surface of the oviductal epithelial cells, supporting a sperm interaction biological role in situ. We analyze the possible dependence of the tyrosine-phosphorylation of p97 on the PKA mechanism, finding that apparently it is not PKA dependent. Also, after SBG treatment the phosphorylated proteins locate mainly at the detached periacrosomal region and at the tail of sperm; the latter may be related to SBG's motility reduction effect. The study of the time course effect of SBG on sperm as detected by chlortetracycline (CTC) staining and of its binding to sperm by immunodetection in conjunction with CTC, shows results in agreement with the hypothesis that this glycoprotein is involved in the alteration of acrosomes in a specific sperm subpopulation. The results suggest that SBG may be part of a mechanism for negative selection of sperm.

Porcine oviduct sperm binding glycoprotein and its deleterious effect on sperm: a mechanism for negative selection of sperm?

In their journey through the oviduct some subpopulations of sperm are preserved in a reservoir, while others are negatively selected. Sperm binding glycoprotein (SBG) is a pig oviductal epithelial cell glycoprotein that produces, under capacitating conditions, acrosome alteration, p97 tyrosine-phosphorylation and reduction of the motility of sperm. In this paper, we show that SBG is accessible at the extracellular surface of the oviductal epithelial cells, supporting a sperm interaction biological role in situ. We analyze the possible dependence of the tyrosine-phosphorylation of p97 on the PKA mechanism, finding that apparently it is not PKA dependent. Also, after SBG treatment the phosphorylated proteins locate mainly at the detached periacrosomal region and at the tail of sperm; the latter may be related to SBG's motility reduction effect. The study of the time course effect of SBG on sperm as detected by chlortetracycline (CTC) staining and of its binding to sperm by immunodetection in conjunction with CTC, shows results in agreement with the hypothesis that this glycoprotein is involved in the alteration of acrosomes in a specific sperm subpopulation. The results suggest that SBG may be part of a mechanism for negative selection of sperm.

Dynamics of heparin-binding proteins on boar sperm.

The presence, topology and dynamics of heparin-binding proteins (HBP) on boar sperm were evaluated. HBP distribution was analyzed by subcellular parting, using biotinylated heparin followed by colorimetric detection. HBP were detected as peripherical and integral periacrosomal membrane proteins. Indirect fluorescence microscopy of sperm incubated with biotinylated heparin was used to evidence heparin binding on sperm at different physiological stages. Two different fluorescent patterns (A and B) were found, which probably correspond to non-capacitated and capacitated sperm as assessed by the ability to undergo acrosome reaction with calcium ionophore A23187 and by the increase of p32 phosphorylated protein. In A pattern, corresponding to untreated sperm, fluorescence located mostly on the post-acrosomal region; in B pattern, corresponding to incubated sperm, on the acrosomal region. Upon incubation under capacitating conditions (TALP), sperm having the B pattern was augmented compared with non-incubated sperm (p<0.001). Differences in the HBP patterns (p<0.0001) were observed in sperm incubated under non-capacitating conditions in relation to sperm incubated in TALP, indicating that the modification of HBP patterns is probably related to capacitation. No difference was observed when untreated sperm were permeabilized prior to staining, suggesting that HBP are present on the sperm surface. The effect of heparin on capacitation dependent protein tyrosine phosphorylation was also analyzed, finding a decrease in p32 phosphorylation in the presence of heparin. This suggests that the capacitation enhancement mediated by this glycosaminoglycan involves an alternative intracellular pathway. The finding that heparin binds to sperm differently according to its physiological state, is a new evidence of the remodelling of sperm membrane surface upon capacitation and may provide a useful and relatively simple method to evaluate in vitro modification of boar sperm physiological state.

Effect of heparin on in vitro capacitation of boar sperm.

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.

Effect of heparin on in vitro capacitation of boar sperm

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 µM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 µg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.