Seasonal differences in ram seminal plasma revealed by partition in an aqueous two-phase system

摘要

Seminal plasma plays an important role in maturation of spermatozoa through hormonal,enzymatic and surface-modifying events.We have previously shown that adsorption of seminal plasma proteins (SSPs) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa,reproducing those of live cells.In the present report,we investigate the hypothesis that seasonal differences in seminal plasma could affect its ability to recover membrane integrity of cold-shocked sperm.The effect of seminal plasma proteins,obtained in breeding (bsSPPs) and non-breeding (nbsSPPs) season,on cold-shocked ram spermatozoa previously freed from seminal plasma,was analysed by centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system as well as membrane integrity determination by fluorescence markers.Cold-shock treatment greatly lowered cell viability in both breeding and non-breeding season spermatozoa.The cold-shocked sperm viability obtained was approximately 20%.The loss of heterogeneity and the decrease in viability revealed by CCCD analysis was reversed by the addition of increasing amounts of bsSPP,which induced restoration of the surface characteristics of viable-like spermatozoa,as well as an increase in the number of recovered viable sperm.However,this restoring effect was much lower when nbsSPPs were added,even in a sixfold higher concentration than used with bsSPPs.Incubation of cold-shocked cells with both kinds of proteins performed in both seasonal periods,showed that the recovering effect was related to the season when the plasma sample was obtained rather than to the semen season.The addition of bsSPPs to cold-shocked sperm accounted for a nearly 50% reversion for both studied breeding seasons.However,the reversion percentages obtained with nbsSPPs were significantly lower (p<0.05) than those found with bsSPPs in both studied seasonal periods.This different reversion capacity of bsSPPs and nbsSPPs was related to a different protein composition,as revealed by comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.The bands of 20,21,24,36 and 67kDa of the bsSP sample profile decreased in winter-spring SP,and were even less intensely stained in summer SP.Densitometric analysis of the stained gel patterns allows automatic comparison among the separated bands,and revealed an important decrease in the content of several bands.The 21.5 kDa band showed the highest decrease,lowering to 14% in June-August plasma with respect to the value obtained in September-December plasma.