Prostasomes of canine seminal plasma - zinc-binding ability and effects on motility characteristics and plasma membrane integrity of spermatozoa.

Abstract

Prostasomes are small lipid membrane-confined vesicles that are involved in various fertilization-related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross-bred dogs (n=50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301nm. The zinc-affinity chromatography on a Chelating Sepharose Fast Flow - Zn(2 +) showed that from 93 to 100% of the prostasome proteins bind zinc ions (P(+) Zn). SDS-PAGE revealed that canine P(+) Zn comprised four protein bands, with low molecular weights (10.2-12 kDa). We have also shown a positive effect of prostasomes (p<0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2h storage at 5°C (TMOT%, 44.75±5.18) and PMOT%, 12.42±1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30±1.41 and PMOT%, 1.70±0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold-stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.