Identification and Quantification of Sperm Head Plasma Membrane Proteins Associated with Male Fertility

Abstract

The major objective was to characterize proteins in head plasma membrane (HPM) of sperm from animals of two species to identify species’ and proteins’ differences related to fertility. HPM’s sodium/potassium-ATPase (Na+⁄K+-ATPase) acts as a receptor, inducing capacitation when bound by its hormone ouabain. Na+/K+-ATPase is an α/β dimer, each with several isoforms (α1, α2, α3, α4, β1, β2, β3) whose exact relationship to in vivo fertility and capacitation is unknown. In the first study, specific Na+/K+-ATPase isoforms in sperm HPM of boars with different Direct Boar Effects (DBEs) for farrowing rate (FR) and litter size, differed between low and high fertility boars (LF, HF, n=6/each; DBE-based). SDS-PAGE and immunoblotting detected more α3 (P<0.05) in HF HPM, correlating with FR; immunocytochemistry identified differing α3 and α2 localizations in HF vs LF whole sperm. In second study, tandem mass spectrometry (spectra aligned to UniProtKB ‘mammals’) revealed that non-ATPase HPM proteins differ between bulls of HF and LF Bull Fertility Index (BFI, BFI > or <100; Semex evaluated). Statistical analysis identified 67 differential abundance proteins (DAPs) between HF and LF (n=3/group; P<0.05), which associated by meta-analysis to BFI. Gene ontology assigned 48 up-regulated HF proteins to sperm fertilization, and 19 down-regulated to catalytic and transporter activity. 38-up-regulated DAPs (HF and LF, n=16) correlated positively (r2=0.29 to 0.66; P≤0.05) and 6 down-regulated negatively (r2=0.26 to 0.44; P≤0.05) to BFI. The third study characterized HPM Na+/K+-ATPase in 16 bulls with differing BFI but similar sperm motility kinetics. Normalized Spectral Abundance Factor (NSAF) of α1 was significantly greater in 8 higher- vs 8 lower-fertility bulls. Linear regression positively correlated BFI to NSAF of α1 and β2 (r2=0.42 and 0.47, respectively; P≤0.05), and negatively correlated BFI to α4 (r2=0.37; P≤0.05), confirmed by bioinformatics predictions. These results suggest involvement of α1 and β2 in fertilization as potential fertility biomarkers. Overall, specific Na+/K+-ATPase isoforms identified in boar and bull sperm HPM significantly correlate with in vivo fertility, as do other specific bull HPM proteins. Elucidating potential fertility biomarkers in two species improves understanding of key proteins and their roles in various, complex mechanisms that enable successful sperm fertilization.