ISOLATING NA+K+ATPASE IN BOAR (Sus scrofa) SPERMATOZOA MEMBRANES

Abstract

Na+K+ATPase exists in the membranes of bull spermatozoa, concentrated in the head plasma membrane. In sperm, Na+K+ATPase actively transports ions contributing to the intracellular pH change in capacitation, and has very recently been implicated as a direct signalling molecule leading to tyrosine phosphorylation in capacitating bull sperm. The characteristics of Na+K+ATPase in boar sperm are unknown, and were the focus of this study. To determine the existence of Na+K+ATPase, and any preferential location of it within boar sperm, Western blot analyses were performed on whole sperm and purified boar sperm head plasma membrane (HPM). The HPM was isolated (n=8 ejaculates) by nitrogen cavitation and differential centrifugation. Its purity was confirmed by its significant enrichment in two plasma membrane marker enzymes (alkaline phosphatase 224 ± 18%, Mean ± SE; acid phosphatase 248 ± 65% vs activity in homogenised whole sperm); and by the significant diminution of activity of marker enzymes associated with the acrosome (acetylglucosaminidase 41.9 ± 8%) and mitochondria (succinate dehydrogenase; 3.21 ± 1%). Equal protein amounts from whole sperm (WS), HPM and sperm body membranes (SB; whole sperm minus HPM) from 3 ejaculates were analyzed by Western blotting for the alpha (isoforms 1 and 3), and beta (isoforms 1,2 and 3) subunits of Na+K+ATPase. Alpha 1 and 3, and beta 1 were less prevalent in HPM than in WS or SB, while alpha 2 and beta 3 were equally prominent in all fractions, based on densitometry of equal protein concentrations. Monoclonal antibodies for each subunit bound at the kD of the rat brain positive control, and several additional bands for each subunit; these are being investigated as multimers, glycosylated residues, and/or breakdown products. Immunofluorescence of the same monoclonal antibodies then delineated specific subunit locations on the sperm. Fresh sperm (n=4 ejaculates) on poly-LLysine slides at 4°C were permeabilized with Methanol or left intact, and incubated at 37°C with antibodies for each enzyme subunit, and compared to binding that occurred when all steps were at room temperature. The α3 subunit bound post-equatorially regardless of temperature; all other isoforms bound only at room temperature, indicating a high degree of temperature sensitivity. The antibodies to all beta subunits bound over the anterior acrosomes of permeabilised cells only, suggesting the antigens are on internal surface, while the alpha 1 bound to the anterior acrosome of intact cells, indicating an extracellular antigen. These results indicate that Na+K+ATPase exists in specific regions of boar spermatozoa membranes, and its configuration is temperature-sensitive. Funding from NSERC, OMAFRA, Bioniche and Semex Canada gratefully acknowledged. (poster)