Hamster Sperm Possess Functional Na+/Ca2+-Exchanger 1: Its Implication in Hyperactivation

Previous studies demonstrated that hamster sperm hyperactivation is suppressed by extracellular Na+ by lowering intracellular Ca2+ levels, and Na+/Ca2+-exchanger (NCX) specific inhibitors canceled the suppressive effects of extracellular Na+. These results suggest the involvement of NCX in the regulation of hyperactivation. However, direct evidence of the presence and functionality of NCX in hamster spermatozoa is still lacking. This study aimed to reveal that NCX is present and is functional in hamster spermatozoa. First, NCX1 and NCX2 transcripts were detected via RNA-seq analyses of hamster testis mRNAs, but only the NCX1 protein was detected. Next, NCX activity was determined by measuring the Na+-dependent Ca2+ influx using the Ca2+ indicator Fura-2. The Na+-dependent Ca2+ influx was detected in hamster spermatozoa, notably in the tail region. The Na+-dependent Ca2+ influx was inhibited by the NCX inhibitor SEA0400 at NCX1-specific concentrations. NCX1 activity was reduced after 3 h of incubation in capacitating conditions. These results, together with authors’ previous study, showed that hamster spermatozoa possesses functional NCX1 and that its activity was downregulated upon capacitation to trigger hyperactivation. This is the first study to successfully reveal the presence of NCX1 and its physiological function as a hyperactivation brake.

[1]  Shukei Sugita,et al.  Decrease in Ca2+ Concentration in Quail Cardiomyocytes Is Faster than That in Rat Cardiomyocytes , 2022, Processes.

[2]  Gen L. Takei,et al.  Oviductal high concentration of K+ suppresses hyperpolarization but does not prevent hyperactivation, acrosome reaction and in vitro fertilization in hamsters , 2020, Zygote.

[3]  Keitaro Hayashi,et al.  Na+/K+-ATPase α4 regulates sperm hyperactivation while Na+/K+-ATPase α1 regulates basal motility in hamster spermatozoa. , 2020, Theriogenology.

[4]  A. Salicioni,et al.  Transient Sperm Starvation Improves the Outcome of Assisted Reproductive Technologies , 2019, Front. Cell Dev. Biol..

[5]  Yukari Arai,et al.  Identification of isoforms of calyculin A-sensitive protein phosphatases which suppress full-type hyperactivation in bull ejaculated spermatozoa. , 2019, Theriogenology.

[6]  P. Lishko,et al.  CatSper: A Unique Calcium Channel of the Sperm Flagellum. , 2018, Current opinion in physiology.

[7]  A. Salicioni,et al.  Transient exposure to calcium ionophore enables in vitro fertilization in sterile mouse models , 2016, Scientific Reports.

[8]  P. Greer,et al.  The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm , 2016, Development.

[9]  Gen L. Takei,et al.  Regulation of hamster sperm hyperactivation by extracellular Na. , 2016, Reproduction.

[10]  K. Naruse,et al.  Induced NCX1 overexpression attenuates pressure overload-induced pathological cardiac remodelling. , 2016, Cardiovascular research.

[11]  Gen L. Takei,et al.  Non-genomic regulation and disruption of spermatozoal in vitro hyperactivation by oviductal hormones , 2015, The Journal of Physiological Sciences.

[12]  D. Hilgemann,et al.  Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  M. Islam,et al.  A Transcriptome for the Study of Early Processes of Retinal Regeneration in the Adult Newt, Cynops pyrrhogaster , 2014, PloS one.

[14]  A. Darszon,et al.  Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways , 2013, Proceedings of the National Academy of Sciences.

[15]  T. Nishigaki,et al.  Calcium channels in the development, maturation, and function of spermatozoa. , 2011, Physiological reviews.

[16]  M. Morad,et al.  NCX1 phosphorylation dilemma: a little closer to resolution. Focus on "Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A". , 2011, American journal of physiology. Cell physiology.

[17]  Mitsuaki Suzuki,et al.  Regulation of hyperactivation by PPP2 in hamster spermatozoa. , 2010, Reproduction.

[18]  S. Suarez,et al.  CatSper-null mutant spermatozoa are unable to ascend beyond the oviductal reservoir. , 2009, Reproduction, fertility, and development.

[19]  S. Suarez,et al.  Bovine Sperm Hyperactivation Is Promoted by Alkaline-Stimulated Ca2+ Influx1 , 2007, Biology of reproduction.

[20]  D. Clapham,et al.  All four CatSper ion channel proteins are required for male fertility and sperm cell hyperactivated motility , 2007, Proceedings of the National Academy of Sciences.

[21]  T. Márián,et al.  Role of the Na+/Ca2+ exchanger in calcium homeostasis and human sperm motility regulation. , 2006, Cell motility and the cytoskeleton.

[22]  S. Suarez,et al.  Calcium/Calmodulin and Calmodulin Kinase II Stimulate Hyperactivation in Demembranated Bovine Sperm1 , 2005, Biology of reproduction.

[23]  M. Ensslin,et al.  Mammalian fertilization , 2004, Current Biology.

[24]  T. Doetschman,et al.  Targeted Ablation of Plasma Membrane Ca2+-ATPase (PMCA) 1 and 4 Indicates a Major Housekeeping Function for PMCA1 and a Critical Role in Hyperactivated Sperm Motility and Male Fertility for PMCA4* , 2004, Journal of Biological Chemistry.

[25]  K. Knobeloch,et al.  Plasma Membrane Ca2+ ATPase 4 Is Required for Sperm Motility and Male Fertility* , 2004, Journal of Biological Chemistry.

[26]  I. Imanaga,et al.  Molecular Determinants of Na+/Ca2+ Exchange (NCX1) Inhibition by SEA0400* , 2004, Journal of Biological Chemistry.

[27]  R. Hammer,et al.  Hyperactivated sperm motility driven by CatSper2 is required for fertilization , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[28]  B. Hille,et al.  Calcium Clearance Mechanisms of Mouse Sperm , 2003, The Journal of general physiology.

[29]  M. Morad,et al.  Molecular determinants of cAMP‐mediated regulation of the Na+–Ca2+ exchanger expressed in human cell lines , 2003, The Journal of physiology.

[30]  V. Vacquier,et al.  A flagellar K+-dependent Na+/Ca2+ exchanger keeps Ca2+ low in sea urchin spermatozoa , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Marco Perizzolo,et al.  Molecular Cloning of a Third Member of the Potassium-dependent Sodium-Calcium Exchanger Gene Family,NCKX3 * , 2001, The Journal of Biological Chemistry.

[32]  A. Tiwary,et al.  Effect of 2',4'-dichlorobenzamil hydrochloride, a Na(+)-Ca(2+) exchange inhibitor, on human spermatozoa. , 2001, European journal of pharmacology.

[33]  R. Yeoman,et al.  Evidence for nitric oxide regulation of hamster sperm hyperactivation. , 1998, Journal of andrology.

[34]  B. Quednau,et al.  Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat. , 1997, The American journal of physiology.

[35]  D. Hilgemann,et al.  Regulation of Cardiac Na+,Ca2+ Exchange and KATP Potassium Channels by PIP2 , 1996, Science.

[36]  T. Iwamoto,et al.  Phosphorylation-dependent Regulation of Cardiac Na+/Ca2+ Exchanger via Protein Kinase C* , 1996, The Journal of Biological Chemistry.

[37]  G S Kopf,et al.  Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. , 1995, Development.

[38]  G. Kopf,et al.  Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. , 1995, Development.

[39]  P. Renard,et al.  An in vitro promoting role for hydrogen peroxide in human sperm capacitation. , 1994, International journal of andrology.

[40]  A. Yu,et al.  Tissue-specific expression of Na(+)-Ca2+ exchanger isoforms. , 1994, The Journal of biological chemistry.

[41]  W. Lederer,et al.  Mutually exclusive and cassette exons underlie alternatively spliced isoforms of the Na/Ca exchanger. , 1994, The Journal of biological chemistry.

[42]  D. Nicoll,et al.  Mapping of the cardiac sodium-calcium exchanger with monoclonal antibodies. , 1993, The American journal of physiology.

[43]  S. Suarez,et al.  Intracellular calcium increases with hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[44]  S. Suarez,et al.  Hyperactivation enhances mouse sperm capacity for penetrating viscoelastic media. , 1992, Biology of reproduction.

[45]  D. Katz,et al.  Evidence for the function of hyperactivated motility in sperm. , 1991, Biology of reproduction.

[46]  K. Philipson,et al.  Purification of the cardiac Na+-Ca2+ exchange protein. , 1988, Biochimica et biophysica acta.

[47]  R. Yanagimachi.  The movement of golden hamster spermatozoa before and after capacitation. , 1970, Journal of reproduction and fertility.