Animal Reproduction (AR)
Animal Reproduction (AR)
Conference Paper

Na/K-ATPase and Regulation of Sperm Function

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

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A standard bull breeding soundness evaluation (BBSE) identifies bulls with semen that is grossly abnormal. Nonetheless, semen samples classified as satisfactory based on these traditional approaches differ in fertility; perhaps there are submicroscopic differences in sperm characteristics affecting fertility. Therefore, a better understanding of molecular regulation of sperm function could promote development of novel, evidence-based approaches to predict male fertility. Recently the α4 isoform of Na/K-ATPase (ATP1A4) has received considerable attention, due to its testis-specific expression in post-meiotic germ cells and mature sperm, in addition to its regulation of sperm motility and capacitation. Using fresh bull sperm, we determined that ATP1A4 resided in specialized microdomains (raft and non-raft) of the sperm plasma membrane and activated specific signaling (caveolin-1, EGFR, Src, ERK1/2) molecules during sperm capacitation. Furthermore, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in capacitated sperm. Despite the widely accepted dogma of transcriptional/translational quiescence, bovine sperm translated ATP1A4 mRNA on mitochondrial or mitochondrial-type ribosomes, increasing their content and activity during capacitation. Proteomic analysis of raft and non-raft fractions revealed a significant interaction between ATP1A4 and plakoglobin, a member of the β-catenin family of proteins involved in cell adhesion, in the equatorial segment of capacitated sperm, suggesting a potential role in sperm-oolemma fusion. In frozen-thawed sperm, ATP1A4 content and activity was greater in high- versus low-fertility bulls. Additionally, ATP1A4-induced increases in ROS, calcium, actin polymerization and tyrosine phosphorylation were also involved in regulating post-thaw sperm function in these bulls. Overall, results demonstrated that ATP1A4 had unique roles in controlling several aspects of sperm physiology, acting through well-established enzyme activity and signaling functions. Consequently, isoforms of Na/K-ATPase are potential biomarkers for male fertility.


bull sperm, Na/K-ATPase, ouabain.


Ahmed NA, Salem MH, El-Oksh HA, Pursel VG. 1984. Effect of incubation conditions, inhibitors and seminal plasma on protein synthesis in ram spermatozoa. J Reprod Fertil, 71:213-219

Amikura R, Kashikawa M, Nakamura A, Kobayashi S. 2001. Presence of mitochondria-type ribosomes outside mitochondria in germ plasm of Drosophila embryos. Proc Natl Acad Sci U S A, 98:9133-9138.

Amikura R, Sato K, Kobayashi S. 2005. Role of mitochondrial ribosome-dependent translation in germline formation in Drosophila embryos. Mech Dev, 122:1087-1093.

Anpalakan K. 2010. Sodium, potassium-ATPase signalling mechanism inducing capacitation in bull sperm. Thesis. University of Guelph,Ontario, Canada. pp.1-281.

Arystarkhova E, Sweadner KJ. 1997. Tissue-specific expression of the Na,K-ATPase beta3 subunit. The presence of beta3 in lung and liver addresses the problem of the missing subunit.  J Biol Chem, 272:22405-22408.

Asano A, Selvaraj V, Buttke DE, Nelson JL, Green KM, Evans JE, Travis AJ. 2009. Biochemical characterization of membrane fractions in murine sperm: identification of three distinct sub-types of membrane rafts. J Cell Physiol, 218:537-548.

Bailey JL, Bilodeau JF, Cormier N. 2000. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl, 21:1-7.

Bellin ME, Oyarzo JN, Hawkins HE, Zhang H, Smith RG, Forrest DW, Sprott LR, Ax RL. 1998. Fertility-associated antigen on bull sperm indicates fertility potential. J Anim Sci, 76:2032-2039.

Blanco G, Mercer RW. 1998. Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol, 275:F633-50.

Blanco G, Sanchez G, Melton RJ, Tourtellotte WG, Mercer RW. 2000. The alpha4 isoform of the Na,K-ATPase is expressed in the germ cells of the testes. J Histochem Cytochem, 48:1023-1032.

Bou Khalil M, Chakrabandhu K, Xu H, Weerachatyanukul W, Buhr M, Berger T, Carmona E, Vuong N, Kumarathasan P, Wong PT, Carrier D, Tanphaichitr N. 2006. Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid. Dev Biol, 290:220-235.

Bragg PW, Handel MA. 1979. Protein synthesis in mouse spermatozoa. Biol Reprod, 20:333-337

Carlier MF, Ressad F, Pantaloni D. 1999. Control of actin dynamics in cell motility. Role of ADF/cofilin. J Biol Chem, 274:33827-33830.

Christensen P, Hansen C, Liboriussen T, Lehn-Jensen H. 2005. Implementation of flow cytometry for quality control in four Danish bull studs. Anim Reprod Sci, 85:201-208.

Cohen G, Rubinstein S, Gur Y, Breitbart H. 2004. Crosstalk between protein kinase A and C regulates phospholipase D and F-actin formation during sperm capacitation. Dev Biol, 267:230-241.

Counillon L, Pouyssegur J. 2000. The expanding family of eucaryotic Na(+)/H(+) exchangers. J Biol Chem, 275:1-4.

Cross NL. 2004. Reorganization of lipid rafts during capacitation of human sperm. Biol Reprod, 71:1367-1373.

D'Amours O, Frenette G, Fortier M, Leclerc P, Sullivan R. 2010. Proteomic comparison of detergent-extracted sperm proteins from bulls with different fertility indexes. Reproduction, 139:545-556.

Daniel L, Etkovitz N, Weiss SR, Rubinstein S, Ickowicz D, Breitbart H. 2010. Regulation of the sperm EGF receptor by ouabain leads to initiation of the acrosome reaction. Dev Biol, 344:650-657.

Das PJ, McCarthy F, Vishnoi M, Paria N, Gresham C, Li G, Kachroo P, Sudderth AK, Teague S, Love CC, Varner DD, Chowdhary BP, Raudsepp T. 2013. Stallion sperm transcriptome comprises functionally coherent coding and regulatory RNAs as revealed by microarray analysis and RNA-seq. PloS one, 8:e56535.

de Lamirande E, Gagnon C. 1995. Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Reprod, 10:15-21.

de Lamirande E, O’Flaherty C. 2008. Sperm activation: role of reactive oxygen species and kinases. Bichim Biophys Acta, 1784 (1):106-15

De Vries WN, Evsikov AV, Haac BE, Fancher KS, Holbrook AE, Kemler R, Solter D, Knowles BB. 2004. Maternal beta-catenin and E-cadherin in mouse development. Development, 131:4435-4445.

Del Olmo E, Bisbal A, Garcia-Alvarez O, Maroto-Morales A, Ramon M, Jimenez-Rabadan P, Anel-Lopez L, Soler AJ, Garde JJ, Fernandez-Santos MR. 2015. Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility. Reprod Fertil Dev, 27(8):1187-96

Doris PA, Jenkins LA, Stocco DM. 1994. Is ouabain an authentic endogenous mammalian substance derived from the adrenal? Hypertension, 23:632-638

Geering K. 1991. The functional role of the beta-subunit in the maturation and intracellular transport of Na,K-ATPase. FEBS letters, 285:189-193.

Gilbert I, Bissonnette N, Boissonneault G, Vallee M, Robert C. 2007. A molecular analysis of the population of mRNA in bovine spermatozoa. Reproduction, 133:1073-1086.

Gur Y, Breitbart H. 2006. Mammalian sperm translate nuclear-encoded proteins by mitochondrial-type ribosomes. Genes Dev, 20:411-416.

Hansen GR. 2006. Managing Bull Fertility in Beef Cattle Herds. Animal Science Department. Florida Cooperative, Extension, Service, Institute of Food and Agricultural Sciences, University of Florida. Available on:

Hickey KD, Buhr MM. 2011. Lipid bilayer composition affects transmembrane protein orientation and function. J Lipids, 208457.

Hlivko JT, Chakraborty S, Hlivko TJ, Sengupta A, James PF. 2006. The human Na,K-ATPase alpha 4 isoform is a ouabain-sensitive alpha isoform that is expressed in sperm. Mol Reprod Dev, 73:101-115.

Homareda H, Kawakami K, Nagano K, Matsui H. 1989. Location of signal sequences for membrane insertion of the Na+, K + −ATPase alpha subunit. Mol Cell Biol, 9:5742-5745

Ho HC, Suarez SS. 2003. Characterization of the intracellular calcium store at the base of the sperm flagellum that regulates hyperactivated motility. Biol Reprod, 68:1590-1596

Jimenez T, Sanchez G, Wertheimer E, Blanco G. 2010. Activity of the Na,K-ATPase alpha4 isoform is important for membrane potential, intracellular Ca2+, and pH to maintain motility in rat spermatozoa. Reproduction, 139:835-845.

Jimenez T, McDermott JP, Sanchez G, Blanco G. 2011a. Na,K-ATPase alpha4 isoform is essential for sperm fertility. Proc Natl Acad Sci U S A, 108:644-649.

Jimenez T, Sanchez G, McDermott JP, Nguyen AN, Kumar TR, Blanco G. 2011b. Increased expression of the Na,K-ATPase alpha4 isoform enhances sperm motility in transgenic mice. Biol Reprod, 84:153-161.

Jimenez T, Sanchez G, Blanco G. 2012. Activity of the Na,K-ATPase alpha4 isoform is regulated during sperm capacitation to support sperm motility. J Androl, 33:1047-1057.

Jorgensen PL, Hakansson KO, Karlish SJ. 2003. Structure and mechanism of Na,K-ATPase: functional sites and their interactions. Annu Revf Physiol, 65:817-849.

Kasimanickam V, Kasimanickam R, Arangasamy A, Saberivand A, Stevenson JS, Kastelic JP. 2012. Association between mRNA abundance of functional sperm function proteins and fertility of Holstein bulls. Theriogenology, 78:2007-2019.e2.

Kastelic JP, Thundathil JC. 2008. Breeding soundness evaluation and semen analysis for predicting bull fertility. Reprod Domest Anim, 43(Suppl 2):368-373.

Kumar G, Patel D, Naz RK. 1993. c-MYC mRNA is present in human sperm cells. Cell Mol Biol Res, 39:111-117

Kuster CE, Hess RA, Althouse GC. 2004. Immunofluorescence reveals ubiquitination of retained distal cytoplasmic droplets on ejaculated porcine spermatozoa. J Androl, 25:340-347.

Liang M, Tian J, Liu L, Pierre S, Liu J, Shapiro J, Xie ZJ. 2007. Identification of a pool of non-pumping Na/K-ATPase. J Biol Chem, 282:10585-10593

Liu J, Tian J, Haas M, Shapiro JI, Askari A, Xie Z. 2000. Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations. J Biol Chem, 275:27838-27844.

Liu L, Mohammadi K, Aynafshar B, Wang H, Li D, Liu J, Ivanov AV, Xie Z, Askari A. 2003. Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase. Am J Physiol Cell Physiol, 284:C155s0-60.

Mobasheri A, Avila J, Cozar-Castellano I, Brownleader MD, Trevan M, Francis MJ, Lamb JF, Martin-Vasallo P. 2000. Na+, K+-ATPase isozyme diversity; comparative biochemistry and physiological implications of novel functional interactions. Biosci Rep, 20:51-91.

Moldovan L, Moldovan NI, Sohn RH, Parikh SA,Goldschmidt-Clermont PJ. 2000. Redox changes of cultured endothelial cells and actin dynamics. Circ Res, 86:549-557

Naz RK. 1998. Effect of actinomycin D and cycloheximide on human sperm function. Arch Androl, 41:135-142.

Newton LD, Kastelic JP, Wong B, van der Hoorn F, Thundathil J. 2009. Elevated testicular temperature modulates expression patterns of sperm proteins in Holstein bulls. Mol Reprod Dev, 76:109-118.

Newton LD, Krishnakumar S, Menon AG, Kastelic JP, van der Hoorn FA, Thundathil JC. 2010. Na+/K+ATPase regulates sperm capacitation through a mechanism involving kinases and redistribution of its testis-specific isoform. Mol Reprod Dev, 77:136-148.

Ninomiya Y, Ichinose S. 2007. Subcellular distribution of mitochondrial ribosomal RNA in the mouse oocyte and zygote. PloS one, 2(11):e1241.

Nixon B, Aitken RJ. 2009. The biological significance of detergent-resistant membranes in spermatozoa. J Reprod Immunol, 83:8-13.

O'Flaherty C, de Lamirande E, Gagnon C. 2006. Positive role of reactive oxygen species in mammalian sperm capacitation: triggering and modulation of phosphorylation events. Free Radic Biol Med, 41:528-540.

Ostermeier GC, Dix DJ, Miller D, Khatri P, Krawetz SA. 2002. Spermatozoal RNA profiles of normal fertile men. Lancet, 360:772-777.

Parent S, Lefièvre L, Brindle Y, Sullivan R. 1999. Bull subfertility is associated with low levels of a sperm membrane antigen. Mol Reprod Dev, 52:57-65.

Peddinti D, Nanduri B, Kaya A, Feugang JM, Burgess SC, Memili E. 2008. Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility. BMC Syst Biol, 2:19. Doi: 10.1186/1752-0509-2-19.

Purdy PH. 2008. Ubiquitination and its influence in boar sperm physiology and cryopreservation. Theriogenology, 70:818-826.

Rajamanickam GD, Kastelic JP, Thundathil JC. 2017a. Na/K-ATPase regulates bovine sperm capacitation through raft- and non-raft-mediated signaling mechanisms. Mol Reprod Dev, 84(11):1168-1182

Rajamanickam GD, Kroetsch T, Kastelic JP, Thundathil JC. 2017b. Testis-specific isoform of Na/K-ATPase (ATP1A4) regulates sperm function and fertility in dairy bulls through potential mechanisms involving reactive oxygen species, calcium and actin polymerization. Andrology, 5(4):814-823

Rajamanickam GD, Kastelic JP, Thundathil JC. 2017c. 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(1):187-200.

Rufas O, Fisch B, Ziv S, Shalgi R. 2000. Expression of cadherin adhesion molecules on human gametes. Mol Hum Reprod, 6:163-169.

Schiefelbein D. 1998. Back to the Basics: A Real World Strategy for Improving the Quality and Consistency of Beef. In: Proceedings of the Annual Mtg. 30th of Beef Improvement Federation, Calgary, AB, pp.74-89.

Sendler E, Johnson GD, Mao S, Goodrich RJ, Diamond MP, Hauser R, Krawetz SA. 2013. Stability, delivery and functions of human sperm RNAs at fertilization. Nucleic Acids Res, 41:4104-4117.

Shadan S, James PS, Howes EA, Jones R. 2004. Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa. Biol Reprod, 71:253-265.

Shahar S, Hillman P, Lubart R, Ickowicz D, Breitbart H. 2014. Activation of sperm EGFR by light irradiation is mediated by reactive oxygen species. Photochem Photobiol, 90:1077-1083.

Skou JC, Esmann M. 1992. Chapter One: The Na, K-ATPase. J Bioenerg Biomembr, 24(3):249-61.

Sleight SB, Miranda PV, Plaskett NW, Maier B, Lysiak J, Scrable H, Herr JC, Visconti PE. 2005. Isolation and proteomic analysis of mouse sperm detergent-resistant membrane fractions: evidence for dissociation of lipid rafts during capacitation. Biol Reprod, 73:721-729.

Sutovsky P, Terada Y, Schatten G. 2001. Ubiquitin-based sperm assay for the diagnosis of male factor infertility. Hum Reprod, 16:250-258.

Sutovsky P, Neuber E, Schatten G. 2002. Ubiquitin-dependent sperm quality control mechanism recognizes spermatozoa with DNA defects as revealed by dual ubiquitin-TUNEL assay. Mol Reprod Dev, 61:406-413.

Sutovsky P, Turner RM, Hameed S, Sutovsky M. 2003. Differential ubiquitination of stallion sperm proteins: possible implications for infertility and reproductive seasonality. Biol Reprod, 68:688-698.

Sutovsky P, Aarabi M, Miranda-Vizuete A, Oko R. 2015. Negative biomarker based male fertility evaluation: Sperm phenotypes associated with molecular-level anomalies. Asian J Androl, 17:554-560.

Takezawa Y, Yoshida K, Miyado K, Sato M, Nakamura A, Kawano N, Sakakibara K, Kondo T, Harada Y, Ohnami N, Kanai S, Miyado M, Saito H, Takahashi Y, Akutsu H, Umezawa A. 2011. Beta-Catenin is a Molecular Switch that Regulates Transition of Cell-Cell Adhesion to Fusion. Sci Rep, 1:68.

Thundathil JC, Anzar M, Buhr MM. 2006. Na+/K+ATPase as a signaling molecule during bovine sperm capacitation. Biol Reprod, 75:308-317.

Toshimori K. 2009. Dynamics of the sperm head: Modification and maturation events from spermatogenesis to egg activation. Adv Anat Embryol Cell Biol, 204:5-94.

van Gestel RA, Brewis IA, Ashton PR, Helms JB, Brouwers JF, Gadella BM. 2005. Capacitation-dependent concentration of lipid rafts in the apical ridge head area of porcine sperm cells. Mol Hum Reprod, 11: 583-590

van Gestel RA, Brewis IA, Ashton PR, Brouwers JF, Gadella BM. 2007. Multiple proteins present in purified porcine sperm apical plasma membranes interact with the zona pellucida of the oocyte. Mol Hum Reprod, 13:445-454

Villegas J, Araya P, Bustos-Obregon E, Burzio LO. 2002. Localization of the 16S mitochondrial rRNA in the nucleus of mammalian spermatogenic cells. Mol Hum Reprod, 8:977-983.

Wagoner K, Sanchez G, Nguyen AN, Enders GC, Blanco G. 2005. Different expression and activity of the alpha1 and alpha4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny. Reproduction, 130:627-641.

Wennemuth G, Babcock DF, Hille B. 2003. Calcium clearance mechanisms of mouse sperm. J Gen Physiol, 122:115-128.

Woo AL, James PF, Lingrel JB. 2000. Sperm motility is dependent on a unique isoform of the Na,K-ATPase. J Biol Chem, 275:20693-20699.

Woo AL, James PF, Lingrel JB. 2002. Roles of the Na,K-ATPase alpha4 isoform and the Na+/H+ exchanger in sperm motility. Mol Rprod Dev, 62:348-356.

Xie Z, Kometiani P, Liu J, Li J, Shapiro JI, Askari A. 1999. Intracellular reactive oxygen species mediate the linkage of Na+/K+-ATPase to hypertrophy and its marker genes in cardiac myocytes. J Biol Chem, 274:19323-19328.

Yagi A, Paranko J. 1995. Actin, alpha-actinin, and spectrin with specific associations with the postacrosomal and acrosomal domains of bovine spermatozoa. Anat Rec, 241:77-87.

Yamada S, Pokutta S, Drees F, Weis WI, Nelson WJ. 2005. Deconstructing the cadherin-catenin-actin complex. Cell, 123:889-901.

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