Plasma Membrane Ca2+ ATPase 4 Is Required for Sperm Motility and Male Fertility

Abstract

Calcium and Ca2+-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca2+-dependent pathways are incompletely understood. Here we show that homozygous male mice with a targeted gene deletion of isoform 4 of the plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA), which is highly enriched in the sperm tail, are infertile due to severely impaired sperm motility. Furthermore, the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester reduced sperm motility in wild-type animals, thus mimicking the effects of PMCA4 deficiency on sperm motility and supporting the hypothesis of a pivotal role of the PMCA4 on the regulation of sperm function and intracellular Ca2+ levels. Calcium and Ca2+-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca2+-dependent pathways are incompletely understood. Here we show that homozygous male mice with a targeted gene deletion of isoform 4 of the plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA), which is highly enriched in the sperm tail, are infertile due to severely impaired sperm motility. Furthermore, the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester reduced sperm motility in wild-type animals, thus mimicking the effects of PMCA4 deficiency on sperm motility and supporting the hypothesis of a pivotal role of the PMCA4 on the regulation of sperm function and intracellular Ca2+ levels. Successful fertilization requires the sperm to travel long distances and undergo capacitation prior to reaching the female egg. After reaching their target, the sperm must interact with the extracellular matrix of the egg, including proteins of the zona pellucida, and release acrosomal material. Calcium is considered to exert a function on most, if not all, of these processes. In this field, most of the work on Ca2+ signaling has focused on Ca2+ entry mechanisms, especially on the role of Ca2+ channels (1Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Crossref PubMed Scopus (280) Google Scholar, 2Publicover S.J. Barratt C.L. Hum. Reprod. (Oxf.). 1999; 14: 873-879Crossref PubMed Scopus (59) Google Scholar, 3Wassarman P.M. Jovine L. Litscher E.S. Nat. Cell Biol. 2001; 3: E59-E64Crossref PubMed Scopus (340) Google Scholar, 4O'Toole C.M. Arnoult C. Darszon A. Steinhardt R.A. Florman H.M. Mol. Biol. Cell. 2000; 11: 1571-1584Crossref PubMed Scopus (194) Google Scholar). For example, gene ablation of the cation channel of sperm (CatSper) leads to impaired sperm motility and male infertility (5Ren D. Navarro B. Perez G. Jackson A.C. Hsu S. Shi Q. Tilly J.L. Clapham D.E. Nature. 2001; 413: 603-609Crossref PubMed Scopus (712) Google Scholar), and mice lacking the mitochondrial voltage-dependent anion channel type 3 (VDAC3) are also infertile due to immotile sperm (6Sampson M.J. Decker W.K. Beaudet A.L. Ruitenbeek W. Armstrong D. Hicks M.J. Craigen W.J. J. Biol. Chem. 2001; 276: 39206-39212Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). These results show that tight regulation of ion entry by ion channels is critical to sperm function. Although there is little doubt as to the importance of calcium homeostasis in sperm motility and fertilization (7Fukami K. Yoshida M. Inoue T. Kurokawa M. Fissore R.A. Yoshida N. Mikoshiba K. Takenawa T. J. Cell Biol. 2003; 161: 79-88Crossref PubMed Scopus (129) Google Scholar, 8Jungnickel M.K. Marrero H. Birnbaumer L. Lemos J.R. Florman H.M. Nat. Cell Biol. 2001; 3: 499-502Crossref PubMed Scopus (279) Google Scholar, 9Weyand I. Godde M. Frings S. Weiner J. Muller F. Altenhofen W. Hatt H. Kaupp U.B. Nature. 1994; 368: 859-863Crossref PubMed Scopus (233) Google Scholar, 10Wiesner B. Weiner J. Middendorff R. Hagen V. Kaupp U.B. Weyand I. J. Cell Biol. 1998; 142: 473-484Crossref PubMed Scopus (166) Google Scholar, 11Arnoult C. Cardullo R.A. Lemos J.R. Florman H.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13004-13009Crossref PubMed Scopus (206) Google Scholar, 12Wood C.D. Darszon A. Whitaker M. J. Cell Biol. 2003; 161: 89-101Crossref PubMed Scopus (81) Google Scholar), the function of the plasma membrane Ca2+/calmodulin-dependent Ca2+ ATPase (PMCA) 1The abbreviations used are: PMCA, plasma membrane calcium/calmodulin-dependent calcium ATPase; CE, 5-(and-6)-carboxyeosin diacetate succinimidyl ester; RT, reverse transcriptase. during this process remained enigmatic. PMCA represents a family of enzymes that extrude calcium from the cytosol across the plasma membrane of eukaryotic cells. Since their initial identification in erythrocytes (13Schatzmann H.J. Experientia (Basel). 1966; 22: 364-365Crossref PubMed Scopus (280) Google Scholar), four different isoforms have been identified, and multiple splice forms of these isoforms have been described. The well defined tissue-specific expression pattern of different isoforms and splice variants of the pump in various mammalian tissues (14Brandt P. Neve R.L. Kammesheidt A. Rhoads R.E. Vanaman T.C. J. Biol. Chem. 1992; 267: 4376-4385Abstract Full Text PDF PubMed Google Scholar) and the regulated expression pattern during mouse development (15Zacharias D.A. Kappen C. Biochim. Biophys. Acta. 1999; 1428: 397-405Crossref PubMed Scopus (59) Google Scholar) strongly suggest a specific physiological function for each isoform and splice variant (reviewed in Strehler and Zacharias (16Strehler E.E. Zacharias D.A. Physiol. Rev. 2001; 81: 21-50Crossref PubMed Scopus (484) Google Scholar)). The identification of physical and functional interaction partners of the Ca2+ pump has given insights into the putative functions of PMCAs as regulators of Ca2+-dependent signal transduction processes (17Kim E. DeMarco S.J. Marfatia S.M. Chishti A.H. Sheng M. Strehler E.E. J. Biol. Chem. 1998; 273: 1591-1595Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 18DeMarco S.J. Strehler E.E. J. Biol. Chem. 2001; 276: 21594-21600Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 19DeMarco S.J. Chicka M.C. Strehler E.E. J. Biol. Chem. 2002; 277: 10506-10511Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Schuh K. Uldrijan S. Telkamp M. Rothlein N. Neyses L. J. Cell Biol. 2001; 155: 201-205Crossref PubMed Scopus (134) Google Scholar, 21Schuh K. Uldrijan S. Gambaryan S. Roethlein N. Neyses L. J. Biol. Chem. 2003; 278: 9778-9783Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Interaction of PMCA2 and -4 “b” splice variants was shown to be mediated by the PDZ-(PSD-95/Dlg/ZO-1) domain of the corresponding interaction partner and the C termini of the PMCA isoform (which harbors a typical PDZ domain binding motif (17Kim E. DeMarco S.J. Marfatia S.M. Chishti A.H. Sheng M. Strehler E.E. J. Biol. Chem. 1998; 273: 1591-1595Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar)). Both modes of interaction with PDZ domain-containing proteins, specific and promiscuous binding to different PDZ domains, have been demonstrated (18DeMarco S.J. Strehler E.E. J. Biol. Chem. 2001; 276: 21594-21600Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 19DeMarco S.J. Chicka M.C. Strehler E.E. J. Biol. Chem. 2002; 277: 10506-10511Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In addition to the overlapping expression pattern of the four PMCA isoforms and the diversity generated by alternative splicing, the specificity of interaction with other proteins adds a further level of complexity in determining the physiological functions of each isoform. Gene ablation in mice using homologous recombination in embryonic stem cells represents one possibility to evaluate the function of proteins in vivo and to address the isoform-specific functions of a certain protein. This strategy has been successfully used to generate PMCA2-deficient mice that suffer from deafness and balance deficits (22Kozel P.J. Friedman R.A. Erway L.C. Yamoah E.N. Liu L.H. Riddle T. Duffy J.J. Doetschman T. Miller M.L. Cardell E.L. Shull G.E. J. Biol. Chem. 1998; 273: 18693-18696Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), supported by analyses of “deafwaddler” and “wriggle mouse Sagami” mouse strains, both showing a phenotype comparable with the PMCA2-deficient mice and also harboring spontaneous mutations in the PMCA2 gene (23Street V.A. McKee-Johnson J.W. Fonseca R.C. Tempel B.L. Noben-Trauth K. Nat. Genet. 1998; 19: 390-394Crossref PubMed Scopus (231) Google Scholar, 24Takahashi K. Kitamura K. Biochem. Biophys. Res. Commun. 1999; 261: 773-778Crossref PubMed Scopus (90) Google Scholar). To clarify the in vivo function of PMCA4, we generated PMCA4-deficient mice and studied the physiological effects of this gene deficiency. PMCA4 deficiency does not impair development to adulthood but leads to male infertility due to impaired sperm motility. Cloning of Mouse PMCA4 Isoforms—Mouse PMCA4 isoforms were cloned by rapid amplification of cDNA ends from a mouse testis Marathon-Ready™ double-stranded cDNA library (catalog number 7455-1, Clontech) using the Advantage® 2 PCR system (Clontech) and the following gene-specific primers: mouse PMCA4 forward, 5′-GTC TGA TCA TGT CTG TCC TCA CAG TTG-3′, and mouse PMCA4 reverse, 5′-GCA GCC CCT CTG GCA CAG CCA CT-3′. Polymerase chain reaction was performed as suggested by the manufacturer, and the resulting PCR fragments were cloned into pCR®-XL-TOPO vector (Invitrogen) and subsequently sequenced with the standard T7 and M13 reverse primers. The resulting sequences were analyzed and aligned with DNAMAN 4.0 software (Lynnon BioSoft), and the transmembrane helices were predicted with HMMTOP Version 2.0 at www.enzim.hu/hummtop (25Tusnady G.E. Simon I. Bioinformatics (Oxf.). 2001; 17: 849-850Crossref PubMed Scopus (1565) Google Scholar). Mouse PMCA4b and -4a sequences have been deposited in GenBank™, and the accession numbers are AY560895 and AY560896, respectively. Northern Blotting and Immunofluorescence Stainings—To determine the expression pattern of PMCA4 in mouse testis, we hybridized the MessageMap™ Northern blot (each lane containing 2 μg of poly(A)+ RNA, Stratagene, catalog number 776900) with the 32P-dATP-labeled full-length PMCA4b cDNA according to the manufacturer's protocol and reprobed the subsequently stripped membrane with a 32P-dATP-labeled β-actin probe. The subcellular distribution of PMCA4b was determined by immunofluorescence stainings. Isolated sperm were allowed to swim out from cauda epididymidis in sperm preparation buffer (MediCult, catalog number 10680060) for 15 min, and the suspension was streaked out on poly-lysine-coated slides, air-dried for 10 min, and fixed and stained as described previously (21Schuh K. Uldrijan S. Gambaryan S. Roethlein N. Neyses L. J. Biol. Chem. 2003; 278: 9778-9783Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The PMCA4-specific polyclonal antiserum was also described previously (26Stauffer T.P. Guerini D. Carafoli E. J. Biol. Chem. 1995; 270: 12184-12190Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Generation of PMCA4-deficient Mice—A 12-kb genomic DNA fragment containing exons 2–4 of the mouse PMCA4 gene was isolated from a 129Sv mouse genomic library in the λFix™II vector (Stratagene). The targeting vector was generated by inserting the homologous BglII/KpnI and BspMII/NotI fragments into the pPNT vector (27Tybulewicz V.L. Crawford C.E. Jackson P.K. Bronson R.T. Mulligan R.C. Cell. 1991; 65: 1153-1163Abstract Full Text PDF PubMed Scopus (1159) Google Scholar) (Fig. 3A). 20 μg of linearized targeting vector was electroporated into E14.1 embryonic stem cells. Neomycin- and ganciclovir-resistant clones (500 μg/ml G418 and 2 μm ganciclovir) were screened for homologous recombination by Southern blot using the external BamHI/BglII fragment of the mouse PMCA4 gene as a probe (Fig. 3A). This probe hybridizes to a 7.0-kb BamHI fragment (endogenous pmca4 allele) and a 3.3-kb fragment (disrupted allele). Embryonic stem cells that had undergone homologous recombination were injected into C57Bl/6 blastocysts, and the manipulated blastocysts were transferred into pseudo-pregnant foster mice to generate chimeras. Chimeric males were mated to C57Bl/6 females to test for germ line transmission of the targeted PMCA4 allele. PMCA4-deficient mice were obtained by appropriate inbreeding of heterozygous offspring and subsequent genotyping. RT-PCR and Western Blotting—The absence of PMCA4 mRNA in PMCA4-deficient mice was tested by RT-PCR using the OneStep RT-PCR kit (Qiagen) and the following PMCA4-specific (not species-specific) primers: PMCA4 forward, 5′-CTG AGG AAG CTC ATGGAG C-3′, and PMCA4 reverse, 5′-CGG AAA/G TGC TTC TCT TTG C-3′. To test for the absence of PMCA4 at the protein level in sperm, isolated sperm from one cauda epididymidis were collected by centrifugation and boiled for 5 min in 200 μl of Laemmli sample buffer (Bio-Rad). 20 μl of each lysate was separated on a 10% SDS-PAGE, blotted onto a nitrocellulose membrane (Schleicher & Schuell), and the membranes were cut at the level of the 75-kDa protein marker band. The upper half was tested for PMCA4 expression using the PMCA4-specific monoclonal antibody JA9 (28Caride A.J. Filoteo A.G. Enyedi A. Verma A.K. Penniston J.T. Biochem. J. 1996; 316: 353-359Crossref PubMed Scopus (128) Google Scholar) (NeoMarkers, 1:500 dilution), and the lower half was probed for actin (polyclonal goat anti-actin, Santa Cruz Biotechnology, catalog number sc-1616, 1:500 dilution) to check for equal loading of the samples. Blocking and antibody incubations were done with 5% milk in phosphate-buffered saline/0.05% Tween 20. Signals were detected with specific horseradish peroxidase-labeled secondary antibodies and the ECL detection system (Amersham Biosciences). Tissue Sections of Testis and Histological Stainings—Tissue sections were frozen and prepared as described previously (21Schuh K. Uldrijan S. Gambaryan S. Roethlein N. Neyses L. J. Biol. Chem. 2003; 278: 9778-9783Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and stained with hematoxylin and eosin using a standard protocol. Cytological staining of mouse sperm was carried out using SpermacStain™ (Stain Enterprises Inc.) according to the manufacturer's instructions. In Vitro Fertilization—Sperm were collected from cauda epididymidis in sperm preparation buffer (MediCult) and capacitated in vitro for 2 h at 37 °C. Oocytes were prepared from C57Bl/6 females that had been synchronized with 10 units of pregnant mare serum gonadotropin (Sigma) and 10 units of human chorionic gonadotropin (Sigma) 48 and 14 h prior to oocyte collection. Eggs were flushed from oviducts in M2 medium (Sigma) and cultivated in M16 medium (Sigma) in 5% CO2 at 37 °C. In vitro fertilization capacity was tested as described previously (29Perez G.I. Robles R. Knudson C.M. Flaws J.A. Korsmeyer S.J. Tilly J.L. Nat. Genet. 1999; 21: 200-203Crossref PubMed Scopus (311) Google Scholar). In brief, eggs were incubated with ∼105 wild-type or 105 PMCA4-deficient sperm for 24 h at 37 °C, and eggs that had divided to the two-cell stage were counted as indicative of successful fertilization. Estimation of Sperm Motility and Measurement of Intracellular Calcium—Sperm were collected from cauda epididymidis as described The containing sperm was into a and the cells were at 37 for min or with the PMCA inhibitor diacetate succinimidyl ester sperm motility was using the system for mouse and upper were and respectively. were and and and have been In addition to the of sperm and were with of motility To intracellular Ca2+ in capacitated sperm, were prepared in sperm preparation buffer (MediCult) as described capacitated for min, and with 10 μm for min at 37 °C. The cells were subsequently counted and at a of in sperm preparation buffer with 2 in were with of and of with a using the protocol of the Intracellular Ca2+ the capacitation were on a given of For of intracellular calcium was with μm calcium and was in the of 2 in sperm preparation cDNA of mouse PMCA4b splice variant and the C of mouse variant were cloned from a testis cDNA library by rapid amplification of cDNA ends and rapid amplification of cDNA of predicted protein sequences with human E.E. P. R. R. T. Filoteo A.G. Penniston J.T. Carafoli E. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and T.P. Shull G.E. Biochem. J. 1995; PubMed Scopus Google Scholar) PMCA4b and -4a a of (Fig. Mouse PMCA4b 10 predicted transmembrane domains, a in the plasma which is a typical of The C of mouse PMCA4b harbors a typical PDZ domain binding motif most specificity of binding to certain PDZ domains, as shown previously for other human PMCA splice variants (17Kim E. DeMarco S.J. Marfatia S.M. Chishti A.H. Sheng M. Strehler E.E. J. Biol. Chem. 1998; 273: 1591-1595Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 18DeMarco S.J. Strehler E.E. J. Biol. Chem. 2001; 276: 21594-21600Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 19DeMarco S.J. Chicka M.C. Strehler E.E. J. Biol. Chem. 2002; 277: 10506-10511Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Schuh K. Uldrijan S. Telkamp M. Rothlein N. Neyses L. J. Cell Biol. 2001; 155: 201-205Crossref PubMed Scopus (134) Google Scholar, 21Schuh K. Uldrijan S. Gambaryan S. Roethlein N. Neyses L. J. Biol. Chem. 2003; 278: 9778-9783Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, DeMarco S.J. Strehler E.E. N. Acad. Sci. 2003; PubMed Scopus Google Scholar). The C of mouse has a level of to splice variants of other (Fig. human mouse the function of this motif Although previously shown to be in (14Brandt P. Neve R.L. Kammesheidt A. Rhoads R.E. Vanaman T.C. J. Biol. Chem. 1992; 267: 4376-4385Abstract Full Text PDF PubMed Google Scholar), multiple Northern blot expression of the PMCA4 in mouse testis (Fig. In the actin mRNA of various are most the PMCA4 signal in expression of PMCA4 in mouse polyclonal antibody the of PMCA4 and with mouse PMCA4 (26Stauffer T.P. Guerini D. Carafoli E. J. Biol. Chem. 1995; 270: 12184-12190Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) was used to determine subcellular of PMCA4 protein in wild-type mouse The protein was in the of the sperm tail, the the forward, and to a to the mouse sperm (Fig. the described calcium channel was also in the of the tail, and gene deletion leads to severely reduced sperm motility and male thus the role of Ca2+ signaling in sperm motility (5Ren D. Navarro B. Perez G. Jackson A.C. Hsu S. Shi Q. Tilly J.L. Clapham D.E. Nature. 2001; 413: 603-609Crossref PubMed Scopus (712) Google Scholar). To the physiological function of PMCA4 in we the PMCA4 gene in embryonic stem cells by homologous recombination (Fig. 3A). The and of the were by the homologous embryonic stem cells were injected into blastocysts and into pseudo-pregnant foster germ line transmission of the PMCA4-deficient mice were obtained by heterozygous of the PMCA4 gene was by Southern (Fig. and the absence of the mRNA and of the protein was shown by RT-PCR and Western blot (Fig. C and of mated heterozygous males and females were in the that PMCA4 deficiency not embryonic mice were from their wild-type with to and mated with wild-type or heterozygous not show in a homozygous PMCA4-deficient line not be both homozygous males and females were demonstrated female and male 10 males a of to in or were homozygous males with PMCA4-deficient the had a of sperm of and sperm in and in sperm of mice and their wild-type (Fig. and In vitro fertilization were performed to test the of PMCA4-deficient sperm to of of eggs incubated with capacitated wild-type sperm and of of eggs incubated with PMCA4-deficient sperm the 24 h in PMCA4-deficient sperm were also to to zona pellucida, that these sperm undergo the reaction in To a into the regulation of intracellular calcium of PMCA4-deficient sperm we have the intracellular calcium preparation and capacitation of sperm from from PMCA4-deficient mice in with sperm of their wild-type a of for the intracellular calcium of wild-type sperm was to be and the intracellular calcium in sperm was of one given in in This the previously suggested pivotal role of the PMCA in the regulation of calcium and calcium in sperm G. B. J. Physiol. 2003; PubMed Scopus Google Scholar), but analyses of the regulation of intracellular calcium in different of sperm have to be to the functions of the PMCA4 in the fertilization a of the in the motility of and wild-type sperm was Sperm were by standard for sperm motility with a sperm a number of PMCA4-deficient sperm were immotile motility or as with sperm of wild-type of in and of motility that and were severely impaired in PMCA4-deficient mice (Fig. deletion of PMCA4 leads to strongly impaired sperm be to this by using the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester 10 μm severely reduced the motility of wild-type sperm of in of motility in for the importance of PMCA in the regulation of sperm motility. in the of μm has been shown previously to be specific inhibitor of the plasma membrane calcium pump D.A. J. Physiol. 1999; Scopus Google Scholar, C. J. Physiol. PubMed Google Scholar, C. W. 1995; PubMed Scopus Google Scholar). In we a highly specific of male infertility in with a deletion of the PMCA4 The is crucial to sperm motility but not to in vitro fertilization The PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester the of gene deletion on sperm motility. as for the development of and has a number of that be used for for to in the testis or and is the of 5-(and-6)-carboxyeosin diacetate succinimidyl but the eosin and are used as a in and for and a of for further the to a of Furthermore, PMCA4 a gene for the of and of male Nat. Cell Biol. 2002; Google Scholar, 2003; PubMed Scopus Google Scholar). are to for and for to for of embryonic stem and to and Roethlein for