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• W2610471268 abstract "Mammalian sperm feature a specialized secretory organelle on the anterior part of the sperm nucleus, the acrosome, which is essential for male fertility. It is formed by a fusion of Golgi-derived vesicles. We show here that the predominantly Golgi-resident Na+/H+ exchanger NHE8 localizes to the developing acrosome of spermatids. Similar to wild-type mice, Nhe8−/− mice generated Golgi-derived vesicles positive for acrosomal markers and attached to nuclei, but these vesicles failed to form large acrosomal granules and the acrosomal cap. Spermatozoa from Nhe8−/− mice completely lacked acrosomes, were round-headed, exhibited abnormal mitochondrial distribution, and displayed decreased motility, resulting in selective male infertility. Of note, similar features are also found in globozoospermia, one of the causes of male infertility in humans. Germ cell-specific, but not Sertoli cell-specific Nhe8 disruption recapitulated the globozoospermia phenotype, demonstrating that NHE8's role in spermiogenesis is germ cell-intrinsic. Our work has uncovered a crucial role of NHE8 in acrosome biogenesis and suggests that some forms of human globozoospermia might be caused by a loss of function of this Na+/H+ exchanger. It points to NHE8 as a candidate gene for human globozoospermia and a possible drug target for male contraception. Mammalian sperm feature a specialized secretory organelle on the anterior part of the sperm nucleus, the acrosome, which is essential for male fertility. It is formed by a fusion of Golgi-derived vesicles. We show here that the predominantly Golgi-resident Na+/H+ exchanger NHE8 localizes to the developing acrosome of spermatids. Similar to wild-type mice, Nhe8−/− mice generated Golgi-derived vesicles positive for acrosomal markers and attached to nuclei, but these vesicles failed to form large acrosomal granules and the acrosomal cap. Spermatozoa from Nhe8−/− mice completely lacked acrosomes, were round-headed, exhibited abnormal mitochondrial distribution, and displayed decreased motility, resulting in selective male infertility. Of note, similar features are also found in globozoospermia, one of the causes of male infertility in humans. Germ cell-specific, but not Sertoli cell-specific Nhe8 disruption recapitulated the globozoospermia phenotype, demonstrating that NHE8's role in spermiogenesis is germ cell-intrinsic. Our work has uncovered a crucial role of NHE8 in acrosome biogenesis and suggests that some forms of human globozoospermia might be caused by a loss of function of this Na+/H+ exchanger. It points to NHE8 as a candidate gene for human globozoospermia and a possible drug target for male contraception. Spermatogenesis is the process in which spermatozoa, the male gametes, are produced. After cycles of mitosis and meiosis, the haploid spermatids undergo drastic morphological changes to transform from a spherical cell into a polarized, elongated spermatozoon in a process called spermiogenesis (1Cooke H.J. Saunders P.T.K. Mouse models of male infertility.Nat. Rev. Genet. 2002; 3: 790-801Crossref PubMed Scopus (244) Google Scholar). In this process, a large, mainly Golgi-derived membrane-enclosed structure, the acrosome, is formed on the anterior part of the sperm head. The acrosome plays a crucial role during fertilization. Mice and men in which all spermatozoa lack acrosomes are infertile (2Dam A.H.D.M. Feenstra I. Westphal J.R. Ramos L. van Golde R.J.T. Kremer J.A.M. Globozoospermia revisited.Hum. Reprod. Update. 2007; 13: 63-75Crossref PubMed Scopus (216) Google Scholar, 3Okabe M. The cell biology of mammalian fertilization.Development. 2013; 140: 4471-4479Crossref PubMed Scopus (131) Google Scholar). The Na+/H+ exchanger (NHE) 2The abbreviations used are: NHENa+/H+ exchangerGDVGolgi-derived vesicleWTwild-typePNApeanut agglutininTEMtransmission electron microscopePFAparaformaldehyde NHE8 is an integral membrane protein with 12 transmembrane domains as suggested by structures of bacterial homologues (4Hunte C. Screpanti E. Venturi M. Rimon A. Padan E. Michel H. Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH.Nature. 2005; 435: 1197-1202Crossref PubMed Scopus (522) Google Scholar). NHE8 (SLC9A8) belongs to the SLC9A gene family that comprises nine members in mammals (5Orlowski J. Grinstein S. Na+/H+ exchangers.Compr. Physiol. 2011; 1: 2083-2100Crossref PubMed Scopus (79) Google Scholar). These can be divided into two subfamilies: a plasma membrane subfamily including NHE1–5 and an organellar subfamily comprising NHE6–9. Plasma membrane NHEs are involved in the absorption of salt and water in epithelia and in regulating cytoplasmic pH, osmolarity, and cell volume. Knock-out mouse models have implicated these transporters in disorders like epilepsy and seizures, diarrhea, or metabolic acidosis (6Schultheis P.J. Clarke L.L. Meneton P. Miller M.L. Soleimani M. Gawenis L.R. Riddle T.M. Duffy J.J. Doetschman T. Wang T. Giebisch G. Aronson P.S. Lorenz J.N. Shull G.E. Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.Nat. Genet. 1998; 19: 282-285Crossref PubMed Scopus (703) Google Scholar, 7Bell S.M. Schreiner C.M. Schultheis P.J. Miller M.L. Evans R.L. Vorhees C.V. Shull G.E. Scott W.J. Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures.Am. J. Physiol. Cell Physiol. 1999; 276: C788-C795Crossref PubMed Google Scholar, 8Cox G.A. Lutz C.M. Yang C.L. Biemesderfer D. Bronson R.T. Fu A. Aronson P.S. Noebels J.L. Frankel W.N. Sodium/hydrogen exchanger gene defect in slow-wave epilepsy mutant mice.Cell. 1997; 91: 139-148Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 9Bourgeois S. Meer L.V. Wootla B. Bloch-Faure M. Chambrey R. Shull G.E. Gawenis L.R. Houillier P. NHE4 is critical for the renal handling of ammonia in rodents.J. Clin. Invest. 2010; 120: 1895-1904Crossref PubMed Scopus (56) Google Scholar). Organellar NHEs may regulate luminal ion concentrations, in particular the pH, of organelles in the secretory and endocytic pathways. These organelles display distinct, mostly acidic, luminal pH, which is crucial for organellar function (10Rivinoja A. Hassinen A. Kokkonen N. Kauppila A. Kellokumpu S. Elevated Golgi pH impairs terminal N-glycosylation by inducing mislocalization of Golgi glycosyltransferases.J. Cell. Physiol. 2009; 220: 144-154Crossref PubMed Scopus (97) Google Scholar, 11Huang C. Chang A. pH-dependent cargo sorting from the Golgi.J. Biol. Chem. 2011; 286: 10058-10065Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Orlowski J. Grinstein S. Emerging roles of alkali cation/proton exchangers in organellar homeostasis.Curr. Opin. Cell Biol. 2007; 19: 483-492Crossref PubMed Scopus (90) Google Scholar). The specific pH of each organelle is determined by a balance of V-ATPase-mediated proton pumping and a “proton leak” pathway (13Forgac M. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology.Nat. Rev. Mol. Cell Biol. 2007; 8: 917-929Crossref PubMed Scopus (1081) Google Scholar, 14Ouyang Q. Lizarraga S.B. Schmidt M. Yang U. Gong J. Ellisor D. Kauer J.A. Morrow E.M. Christianson syndrome protein NHE6 modulates TrkB endosomal signaling required for neuronal circuit development.Neuron. 2013; 80: 97-112Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 15Kondapalli K.C. Hack A. Schushan M. Landau M. Ben-Tal N. Rao R. Functional evaluation of autism-associated mutations in NHE9.Nat. Commun. 2013; 4: 2510Crossref PubMed Scopus (73) Google Scholar). Several studies in yeast (16Brett C.L. Tukaye D.N. Mukherjee S. Rao R. The yeast endosomal Na+(K+)/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking.Mol. Biol. Cell. 2005; 16: 1396-1405Crossref PubMed Scopus (238) Google Scholar) and mammalian cells (14Ouyang Q. Lizarraga S.B. Schmidt M. Yang U. Gong J. Ellisor D. Kauer J.A. Morrow E.M. Christianson syndrome protein NHE6 modulates TrkB endosomal signaling required for neuronal circuit development.Neuron. 2013; 80: 97-112Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 15Kondapalli K.C. Hack A. Schushan M. Landau M. Ben-Tal N. Rao R. Functional evaluation of autism-associated mutations in NHE9.Nat. Commun. 2013; 4: 2510Crossref PubMed Scopus (73) Google Scholar, 17Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation.J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 18Roxrud I. Raiborg C. Gilfillan G.D. Strømme P. Stenmark H. Dual degradation mechanisms ensure disposal of NHE6 mutant protein associated with neurological disease.Exp. Cell Res. 2009; 315: 3014-3027Crossref PubMed Scopus (45) Google Scholar, 19Xinhan L. Matsushita M. Numaza M. Taguchi A. Mitsui K. Kanazawa H. Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin.Am. J. Physiol. Cell Physiol. 2011; 301: C1431-C1444Crossref PubMed Scopus (46) Google Scholar) demonstrated that overexpression or knockdown of organellar NHEs leads to alkalinization or acidification of the organellar lumen, respectively, suggesting that these transporters provide a proton leak. The importance of organellar NHEs is underscored by mutations in NHE6 and NHE9 that lead to neurological disorders such as autism and epilepsy in humans (20Gilfillan G.D. Selmer K.K. Roxrud I. Smith R. Kyllerman M. Eiklid K. Kroken M. Mattingsdal M. Egeland T. Stenmark H. Sjøholm H. Server A. Samuelsson L. Christianson A. Tarpey P. et al.SLC9A6 mutations cause X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome.Am. J. Hum. Genet. 2008; 82: 1003-1010Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 21Morrow E.M. Yoo S.-Y. Flavell S.W. Kim T.-K. Lin Y. Hill R.S. Mukaddes N.M. Balkhy S. Gascon G. Hashmi A. Al-Saad S. Ware J. Joseph R.M. Greenblatt R. Gleason D. Ertelt J.A. Apse K.A. Bodell A. Partlow J.N. Barry B. Yao H. et al.Identifying autism loci and genes by tracing recent shared ancestry.Science. 2008; 321: 218-223Crossref PubMed Scopus (559) Google Scholar). Na+/H+ exchanger Golgi-derived vesicle wild-type peanut agglutinin transmission electron microscope paraformaldehyde NHE8 (SLC9A8) is widely expressed in murine tissue, with high mRNA levels found in kidney, liver, skeletal muscle, and testes (17Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation.J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 22Goyal S. Vanden Heuvel G. Aronson P.S. Renal expression of novel Na+/H+ exchanger isoform NHE8.Am. J. Physiol. Renal Physiol. 2003; 284: F467-F473Crossref PubMed Scopus (178) Google Scholar). NHE8 localizes predominantly to mid- to trans-Golgi stacks, where it may alkalinize the luminal pH (17Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation.J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). NHE8 was also reported to be present on multivesicular bodies (23Lawrence S.P. Bright N.A. Luzio J.P. Bowers K. The sodium/proton exchanger NHE8 regulates late endosomal morphology and function.Mol. Biol. Cell. 2010; 21: 3540-3551Crossref PubMed Scopus (42) Google Scholar) and somewhat surprisingly at the apical brush-border membrane of epithelial cells of the intestine and renal proximal tubules (22Goyal S. Vanden Heuvel G. Aronson P.S. Renal expression of novel Na+/H+ exchanger isoform NHE8.Am. J. Physiol. Renal Physiol. 2003; 284: F467-F473Crossref PubMed Scopus (178) Google Scholar, 24Xu H. Chen R. Ghishan F.K. Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8.Am. J. Physiol. Gastrointest. Liver Physiol. 2005; 289: G36-G41Crossref PubMed Scopus (81) Google Scholar, 25Goyal S. Mentone S. Aronson P.S. Immunolocalization of NHE8 in rat kidney.Am. J. Physiol. Renal Physiol. 2005; 288: F530-F538Crossref PubMed Scopus (93) Google Scholar). Nhe8−/− mice show impaired colonic mucin synthesis and bicarbonate secretion (26Xu H. Zhang B. Li J. Wang C. Chen H. Ghishan F.K. Impaired mucin synthesis and bicarbonate secretion in the colon of NHE8 knockout mice.Am. J. Physiol. Gastrointest. Liver Physiol. 2012; 303: G335-G343Crossref PubMed Scopus (41) Google Scholar, 27Liu C. Xu H. Zhang B. Johansson M.E.V. Li J. Hansson G.C. Ghishan F.K. NHE8 plays an important role in mucosal protection via its effect on bacterial adhesion.Am. J. Physiol. Cell Physiol. 2013; 305: C121-C128Crossref PubMed Scopus (34) Google Scholar), a dysfunction of the retinal pigment epithelium (28Jadeja S. Barnard A.R. McKie L. Cross S.H. White J.K. on behalf of the Sanger Mouse Genetics Project Robertson M. Budd P.S. MacLaren R.E. Jackson I.J. Mouse Slc9a8 mutants exhibit retinal defects due to retinal pigmented epithelium dysfunction.Invest. Ophthalmol. Vis. Sci. 2015; 56: 3015-3026Crossref PubMed Scopus (10) Google Scholar, 29Xia C.-H. Liu H. Cheung D. Tang F. Chang B. Li M. Gong X. NHE8 is essential for RPE cell polarity and photoreceptor survival.Sci. Rep. 2015; 5: 9358Crossref PubMed Scopus (10) Google Scholar), and male infertility (28Jadeja S. Barnard A.R. McKie L. Cross S.H. White J.K. on behalf of the Sanger Mouse Genetics Project Robertson M. Budd P.S. MacLaren R.E. Jackson I.J. Mouse Slc9a8 mutants exhibit retinal defects due to retinal pigmented epithelium dysfunction.Invest. Ophthalmol. Vis. Sci. 2015; 56: 3015-3026Crossref PubMed Scopus (10) Google Scholar, 30Xu H. Chen H. Li J. Zhao Y. Ghishan F.K. Disruption of NHE8 expression impairs Leydig cell function in the testes.Am. J. Physiol. Cell Physiol. 2015; 308: C330-C338Crossref PubMed Scopus (20) Google Scholar). In addition to NHE8, two other NHEs are known to affect male fertility in mice. Loss of plasma membrane NHE3 (SLC9A3) leads to increased fluid volume in the epididymis, thereby diminishing sperm concentration (31Zhou Q. Clarke L. Nie R. Carnes K. Lai L.W. Lien Y.H. Verkman A. Lubahn D. Fisher J.S. Katzenellenbogen B.S. Hess R.A. Estrogen action and male fertility: roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 14132-14137Crossref PubMed Scopus (170) Google Scholar). Loss of the so-called sperm NHE (sNHE, a member (SLC9C1) of the SLC9C gene family) drastically reduces sperm motility and causes male infertility (32Wang D. King S.M. Quill T.A. Doolittle L.K. Garbers D.L. A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility.Nat. Cell Biol. 2003; 5: 1117-1122Crossref PubMed Scopus (196) Google Scholar). sNHE, which contains additional voltage-sensor and nucleotide binding domains, localizes to the flagellum of mouse spermatozoa, where it may participate in a signaling complex at the plasma membrane (33Wang D. Hu J. Bobulescu I.A. Quill T.A. McLeroy P. Moe O.W. Garbers D.L. A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC).Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9325-9330Crossref PubMed Scopus (127) Google Scholar). Here we analyzed the role of NHE8 in male reproductive function. Mice lacking NHE8 globally or specifically only in germ cells produced round-headed spermatozoa lacking the acrosomal cap. This resembles human globozoospermia which likewise is associated with infertility. Instead of acrosomes, Nhe8−/− spermatids displayed isolated, sometimes nucleus-attached Golgi-derived vesicles (GDVs). Hence NHE8 may play a crucial role in the fusion of GDVs to form acrosomes. Our work suggests NHE8 as a candidate gene for human globozoospermia and as a possible drug target for male contraception. We established Nhe8−/− mice (Fig. 1A) to elucidate physiological roles of NHE8. Wild-type (WT), Nhe8+/−, and Nhe8−/− mice were born at Mendelian ratio (Fig. 1H), grew normally, and all littermates were superficially indistinguishable. Complete loss of NHE8 protein in Nhe8−/− mice was confirmed in several organs by immunoblot analysis which in the WT showed NHE8 signals of ∼60 and 85 kDa as reported (22Goyal S. Vanden Heuvel G. Aronson P.S. Renal expression of novel Na+/H+ exchanger isoform NHE8.Am. J. Physiol. Renal Physiol. 2003; 284: F467-F473Crossref PubMed Scopus (178) Google Scholar) (Fig. 1B). NHE8 expression was prominent in kidney, testes, and epididymis, but very low in the female reproductive system (Fig. 1B). Although we frequently obtained offspring from Nhe8−/− females, male Nhe8−/− mice never produced progeny, as reported previously (26Xu H. Zhang B. Li J. Wang C. Chen H. Ghishan F.K. Impaired mucin synthesis and bicarbonate secretion in the colon of NHE8 knockout mice.Am. J. Physiol. Gastrointest. Liver Physiol. 2012; 303: G335-G343Crossref PubMed Scopus (41) Google Scholar, 30Xu H. Chen H. Li J. Zhao Y. Ghishan F.K. Disruption of NHE8 expression impairs Leydig cell function in the testes.Am. J. Physiol. Cell Physiol. 2015; 308: C330-C338Crossref PubMed Scopus (20) Google Scholar). Vaginal plugs observed in breedings with male Nhe8−/− mice indicated normal mating behavior, libido, copulation, and ejaculation. Body (WT, 27.5 ± 1.5 g; Nhe8−/−, 26.4 ± 1.0 g; n = 12) and testes weight (WT, 0.57 ± 0.03% of body weight; Nhe8−/−, 0.59 ± 0.04% of body weight; n = 12) was unchanged in Nhe8−/− mice. Their male reproductive systems appeared macroscopically normal (Fig. 1C), as did their seminiferous tubules (Fig. 1E), the diameters of which were unchanged (Fig. 1I). Seminiferous tubules of both WT and Nhe8−/− mice contained spermatozoa (Fig. 1F) and the epididymal sperm concentration was not significantly different (Fig. 1J). Contrasting with the typical elongated heads of WT spermatozoa (Fig. 1G, top), Nhe8−/− spermatozoa had round heads, within seminiferous tubules (Fig. 1G, bottom) or the cauda epididymis (Fig. 1D). Their motility was drastically reduced (Fig. 1K). Nhe8−/− spermatozoa lacked acrosomes and showed an abnormal distribution of mitochondria which were found in sperm heads instead of forming a sheath around the initial part of the flagellum. This was revealed by crosses with “green sperm” mice (34Hasuwa H. Muro Y. Ikawa M. Kato N. Tsujimoto Y. Okabe M. Transgenic mouse sperm that have green acrosome and red mitochondria allow visualization of sperm and their acrosome reaction in vivo.Exp. Anim. 2010; 59: 105-107Crossref PubMed Scopus (94) Google Scholar) that express dsRed in mitochondria and GFP in acrosomes (Fig. 2A) and by labeling acrosomes with peanut agglutinin (PNA) (35Mortimer D. Curtis E.F. Miller R.G. Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon.J. Reprod. Fertil. 1987; 81: 127-135Crossref PubMed Scopus (152) Google Scholar) and mitochondria with MitoTracker® (Fig. 2B) and ascertained by transmission electron microscopic (TEM) images (Fig. 3, A–D). Because the mitochondrial sheath provides ATP for flagellar movement (36Ramalho-Santos J. Varum S. Amaral S. Mota P.C. Sousa A.P. Amaral A. Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.Hum. Reprod. Update. 2009; 15: 553-572Crossref PubMed Scopus (321) Google Scholar), this finding might explain the reduced motility of Nhe8−/− spermatozoa.Figure 3Defective formation of the acrosome and mitochondrial sheath in Nhe8−/− mice is shown. A and B, TEM images of epididymal spermatozoa from WT (A) and Nhe8−/− (B) mice. x: axoneme, m: mitochondria. Red boxes indicate areas of A and B that are shown in higher magnification in C and D, respectively. E–G, TEM images from WT mice showing different phases of spermiogenesis. E, Golgi phase. F, cap phase. G, maturation phase. H–J, equivalent images from Nhe8−/− mice. H, electron-dense vesicles attached to the nuclear envelop can be found. Acrosomal granules or acrosomal caps were not detected. I, single dense vesicles were found in nuclear notches. J, acrosome-less spermatozoa with irregularly shaped nuclei. g: Golgi apparatus; a: acrosome or acrosomal granule; m: mitochondrial sheath; x: cilia crossections. Nuclei highlighted in red. K–M, magnification of the areas delineated by red lines in E, H, and I, respectively, reveal the acroplaxome (red arrows).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The lack of Golgi-derived acrosomes upon ablation of the Golgi-resident NHE8 protein (17Nakamura N. Tanaka S. Teko Y. Mitsui K. Kanazawa H. Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation.J. Biol. Chem. 2005; 280: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) points to a germ cell-intrinsic effect, but also defects in germ-cell nurturing Sertoli cells or hormone-producing Leydig cells may cause infertility (37Zhang F.P. Poutanen M. Wilbertz J. Huhtaniemi I. Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice.Mol. Endocrinol. 2001; 15: 172-183Crossref PubMed Scopus (346) Google Scholar). X-Gal staining of testes from mice carrying the lacZ reporter gene in the Nhe8 locus (Fig. 1A) revealed Nhe8 transcription in seminiferous tubules, but not in intertubular Leydig cells (Fig. 2C). Immunofluorescence detected NHE8 protein in WT (but not Nhe8−/−) germ cells, where it localized to an acrosome-like structure in close proximity to the nucleus (Fig. 2D). NHE8 perfectly co-localized with the acrosomal marker PNA in all four phases of spermiogenesis (Fig. 2E). Absence of immunoreactivity in Nhe8−/− testes (Fig. 2D) confirmed the specificity of labeling of preacrosomal granules. However, it cannot strictly guarantee that NHE8 is expressed on mature acrosomes because these structures are not present in Nhe8−/− testes. Comparison of spermiogenesis between the genotypes showed that only the Golgi phase appeared normal in Nhe8−/− mice (Fig. 2F). Instead of a single acrosomal granule that grows and flattens in WT mice, many small PNA-positive vesicles were detected on the nuclear surface in Nhe8−/− spermatids (Fig. 2F, cap phase). In the maturation phase, the resulting acrosome-less Nhe8−/− spermatids failed to obtain the typical elongated shape, although nuclei were condensed (Fig. 2F, maturation phase). TEM of WT testis showed the generation of acrosomal granules by fusion of GDVs, their attachment to the nucleus (Fig. 3E), growth, and flattening (Fig. 3F), as well as nuclear elongation (Fig. 3G). By contrast, in Nhe8−/− mice GDVs accumulated in the region between the Golgi and the nucleus (Fig. 3, H and I) but failed to generate larger granules or acrosomal caps. GDVs were more electron-dense in Nhe8−/− cells. Although spermatozoa in the seminiferous tubules of Nhe8−/− mice had condensed chromatin, their nuclei frequently displayed irregular shapes (Fig. 3J). The preacrosomal granule is attached to the nucleus by a cytoskeletal scaffold, the acroplaxome (38Kierszenbaum A.L. Rivkin E. Tres L.L. Acroplaxome, an F-actin-keratin-containing plate, anchors the acrosome to the nucleus during shaping of the spermatid head.Mol. Biol. Cell. 2003; 14: 4628-4640Crossref PubMed Scopus (165) Google Scholar). The acroplaxome was visible in TEM as a dense plate in the subacrosomal space in WT spermatids (Fig. 3K) as well as in the space between the nucleus and the GDVs in Nhe8−/− spermatids (Fig. 3, L and M). This indicates that the defects in acrosome biogenesis are probably caused by impaired fusion of GDVs, whereas tethering of these vesicles to the nuclear envelope seems to be unaffected. Our data suggest that NHE8 is directly involved in acrosome biogenesis and that disruption of this process causes the infertility of Nhe8−/− mice. To test this hypothesis, Nhe8 was specifically deleted in Sertoli or germ cells by crossing Nhe8lox/lox mice with AMH-Cre (39Lécureuil C. Fontaine I. Crepieux P. Guillou F. Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice.Genesis. 2002; 33: 114-118Crossref PubMed Scopus (174) Google Scholar) and Stra8-iCre (40Sadate-Ngatchou P.I. Payne C.J. Dearth A.T. Braun R.E. Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice.Genesis. 2008; 46: 738-742Crossref PubMed Scopus (197) Google Scholar) mice, respectively. Specificity of Cre expression was ascertained by crosses with R26R reporter mice (41Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat. Genet. 1999; 21: 70-71Crossref PubMed Scopus (4153) Google Scholar) (Figs. 4A and 5A). In the following, we refer to the resulting Sertoli cell- and germ cell-specific knock-out mice as SC-ΔNHE8 and GC-ΔNHE8 mice, respectively. NHE8 protein levels of SC-ΔNHE8 testes were indistinguishable from WT mice, indicating that NHE8 is not, or is only at very low levels, expressed in Sertoli cells (Fig. 4B). As expected, male SC-ΔNHE8 mice were fertile and always produced offspring when mated over a 2-month period (n = 7 SC-ΔNHE8 males). Neither morphology and diameter of seminiferous tubules (Fig. 4, C and E), nor sperm morphology was affected (Fig. 4, E and F). Spermatozoa from SC-ΔNHE8 mice displayed normal acrosomal caps and mitochondrial sheaths (Fig. 4G). Hence NHE8 is dispensable in Sertoli cells for normal sperm development and fertility. In contrast, testicular NHE8 protein levels were drastically reduced in GC-ΔNHE8 mice (Fig. 4B), indicating that in mouse testes NHE8 is predominantly expressed in germ cells. The testicular phenotype of GC-ΔNHE8 mice appeared identical to that of Nhe8−/−, except for the finding that ∼5% of spermatozoa from GC-ΔNHE8 mice appeared normal, which can be attributed to the known ∼95% deletion efficiency of Stra8-iCre mice (40Sadate-Ngatchou P.I. Payne C.J. Dearth A.T. Braun R.E. Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice.Genesis. 2008; 46: 738-742Crossref PubMed Scopus (197) Google Scholar). The identical phenotype included male infertility (n = 15 GC-ΔNHE8 males), normal morphology and diameter of the seminiferous tubules (Figs. 4C and 5C), and the presence of round-headed sperm (Fig. 5, C and D) lacking both acrosomes and mitochondrial sheaths (Fig. 5E). Acrosome biogenesis was similarly affected in global and germ cell-specific Nhe8−/− mice (Fig. 5F and Fig. 6, A–F). The only ∼95% deletion efficiency of Stra8-iCre mice allowed us to directly compare WT and NHE8-deficient cells side by side in sections from GC-ΔNHE8 mice. NHE8-positive cells displayed acrosomal caps and elongated nuclei (Fig. 6, G and H, yellow arrows), whereas NHE8-deficient cells in the same tubule lacked acrosomes and had round nuclei. We conclude that NHE8 is required in germ cells for intact acrosome biogenesis, sperm development, and male fertility.Figure 5Germ cell-specific disruption of Nhe8 leads to round-headed acrosome-less spermatozoa. A, X-gal staining on frozen tissue sections from Rosa26R+/−, Stra8-Cre+ mice, and control mice (Rosa26R+/−; Stra-Cre−). B and C, H&E staining on testes sections from (B) Nhe8lox/lox and (C) GC-ΔNHE8 mice. Right panels show a magnification of the areas delineated by broken lines. Sperm heads indicated by yellow arrows. D, H&E staining of isolated sperm from WT and GC-ΔNHE8 mice. E, fluorescent labeling of the acrosomal cap (PNA, red), the crescent-shaped nucleus (DAPI, blue), and the mitochondrial sheath (MitoTracker®, green) on isolated spermatozoa of WT and GC-ΔNHE8 mice. F, PNA labeling (green) illustrating the normal development of the nucleus-apposed acrosome in Nhe8lox/lox mice in contrast to the impaired biogenesis in GC-ΔNHE8 mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Defective acrosome biogenesis in GC-ΔNHE8 mice is shown. A–C, TEM images from Nhe8lox/lox mice representing the (A) Golgi, (B) cap, or (C) maturation phase of spermiogenesis. D–F, TEM images from GC-ΔNHE8 mice. D and E, electron-dense vesicles accumulate between the Golgi and the nuclear envelope, but acrosomal granules or acrosomal caps were not found. F, in mature spermatozoa, DNA is condensed, but sperm heads are not elongated and lack acrosomal caps. g, Golgi apparatus; a, acrosome. Nuclei are in red. G, remaining NHE8 signal in GC-ΔNHE8 mice co-localizes with the acrosome in the cap phase of spermiogenesis (indicated by yellow arrows). Spermatids lacking NHE8 lack acrosomes, having instead PNA-positive vesicles attached to their nucleus. H, besides the round-headed, acrosome-less spermatozoa, spermatozoa with intact acrosomes and elongated nuclei can be found in some tubules in GC-ΔNHE8 mice. These spermatozoa express NHE8 (indicated by yellow arrows).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Spermatogenesis is the long process that leads from diploid spermatogonia to spermatozoa, highly differentiated haploid motile gametes. The complexity of this differentiation process implies that muta" @default.
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• W2610471268 date "2017-06-01" @default.
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• W2610471268 title "Loss of the Na+/H+ exchanger NHE8 causes male infertility in mice by disrupting acrosome formation" @default.
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