Content of testis-specific isoform of Na/K-ATPase (ATP1A4) is increased during bovine sperm capacitation through translation in mitochondrial ribosomes

Abstract

Capacitation comprises a series of structural and functional modifications of sperm that confer fertilizing ability. We previously reported that the testis-specific isoform of Na/K-ATPase (ATP1A4) regulated bovine sperm capacitation through signaling mechanisms involving kinases. During subsequent investigations to elucidate mechanisms by which ATP1A4 regulates sperm capacitation, we observed that ATP1A4 was localised in both raft and non-raft fractions of the sperm plasma membrane and that its total content was increased in both membrane fractions during capacitation. The objective of the present study was to investigate mechanism(s) of capacitation-associated increase in the content of ATP1A4. Despite the widely accepted dogma of transcriptional/translational quiescence, incubation of sperm with either ouabain (specific ligand for ATP1A4) or heparin increased ATP1A4 content in raft and non-raft sperm membrane fractions, total sperm protein extracts (immunoblotting) and fixed sperm (flow cytometry), with a concurrent increase in Na/K-ATPase enzyme activity. This capacitation-associated increase in ATP1A4 content was partially decreased by chloramphenicol (mitochondrial translation inhibitor) but not affected by actinomycin D (transcription inhibitor). To demonstrate de novo ATP1A4 synthesis, we evaluated incorporation of bodipy conjugated lysine in this protein during capacitation. A partial decrease in bodipy-lysine incorporation occurred in ATP1A4 from sperm capacitated in the presence of chloramphenicol. Therefore, increased ATP1A4 content during capacitation was attributed to mitochondrial translation of ATP1A4 mRNA present in ejaculated sperm, rather than gene transcription. To our knowledge, this is the first report demonstrating ATP1A4 synthesis during bovine sperm capacitation.

Na/K‐ATPase regulates bovine sperm capacitation through raft‐ and non‐raft‐mediated signaling mechanisms

“…We previously noted an increase in the content of ATP1A4 during capacitation, which was attributed to mitochondrial translation of ATP1A4 mRNA (Rajamanickam et al, 2017); a similar mechanism is likely responsible for ATP1A1 production during capacitation. Despite this increase in protein content in membrane fractions, ATP1A4 and ATP1A1 enzyme activity was lower in the raft fraction compared to the non-raft fraction.…”

“…We used a non‐detergent (sodium carbonate)‐based approach to separate raft and non‐raft domains, followed by biochemical characterization, as previously reported (Asano et al, ; Rajamanickam et al, ). The non‐raft marker α‐tubulin (Asano et al, ; Cross, ) confirmed the purity of the membrane fractions.…”

“…Raft and non‐raft membrane fractions were prepared from uncapacitated and capacitated sperm using a non‐detergent (sodium carbonate) lysis buffer, as previously described (Rajamanickam et al, ). Non‐detergent lysis buffer consisted of 0.5 M Na 2 CO 3 (pH 11), 1 mM EDTA, 1 mM Na 3 VO 4 , 1 mM NaF, and a cocktail of protease inhibitors (Roche, Laval, QC, Canada).…”

“…An isoform of Na/K‐ATPase (testis‐specific isoform of Na/K‐ATPase; ATP1A4) was previously identified as a regulator of sperm motility (Jimenez, McDermott, Sanchez, & Blanco, ; Jimenez, Sanchez, et al, ) and capacitation (Newton et al, ; Rajamanickam, Kastelic, & Thundathil, ; Thundathil, Anzar, & Buhr, ), although their underlying molecular mechanisms were not clearly elucidated. Na/K‐ATPase is a heterodimer of α and β subunits, and is responsible for maintenance of Na + and K + gradients between the cell and its environment.…”

The Na+ and K+ transport system of sperm (ATP1A4) is essential for male fertility and an attractive target for male contraception†

“…Since mature spermatozoa are thought to be transcriptionally and translationally silent, the increase in NKAα4 at the plasma membrane could be due to translocation of the protein from intracellular compartments to the sperm surface. Alternatively, in bull sperm, it has been shown that NKAα4 increase can take place by mitochondrial ribosome-associated translation [ 85 ]. We are currently performing additional experiments to ascertain the location and molecular mechanisms involved in the subcellular translocation of NKAα4 in mouse and human sperm.…”