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

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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.

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Acknowledgments

G.D. Rajamanickam was supported through a studentship from the CIHR training program in Genetics, Child Development and Health, University of Calgary. We thank Alta Genetics Inc., Calgary for providing semen samples for the study. We also thank Laurie Kennedy (Centre for Advanced Technologies, University of Calgary) for assisting with flow cytometric analysis and Grace Kwong (Faculty of Veterinary Medicine, University of Calgary) for statistical consultation.

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  1. Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Heritage Medical Research Building RM 400, Calgary, AB, T2N4N1, Canada

    Gayathri D. Rajamanickam, John P. Kastelic & Jacob C. Thundathil

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Correspondence to Jacob C. Thundathil.

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This study received funding from the Natural Sciences and Engineering Research Council (NSERC) of Canada (Grant # RGPN-2014-04850 to JT).

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ESM 1

Fig. S1-S1''''' Sperm kinematic parameters in ouabain- and heparin-capacitated sperm. Motility parameters (total and progressive motility) were recorded using CASA and hyperactivation was monitored by three kinematic values including ALH, VCL and LIN under ouabain and heparin capacitating conditions for 4 h. Data shown were expressed as mean ± SEM (n = 5). Values without a common lowercase letter differed (P < 0.05). Fig. S2. Tyrosine phosphorylation patterns in ouabain- and heparin-capacitated sperm. For evaluation of tyrosine phosphorylation, total sperm homogenates were immunoblotted with anti-phosphotyrosine antibody (upper panel) and reprobed with β-tubulin (lower panel) for equal protein loading (n = 5). Fig. S3. Distribution of GM1 (raft marker indicated by red arrows) in raft and non-raft membrane fractions (GIF 91 kb)

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Rajamanickam, G.D., Kastelic, J.P. & Thundathil, J.C. Content of testis-specific isoform of Na/K-ATPase (ATP1A4) is increased during bovine sperm capacitation through translation in mitochondrial ribosomes. Cell Tissue Res 368, 187–200 (2017). https://doi.org/10.1007/s00441-016-2514-7

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