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• W2805997767 abstract "Spermatogenesis is a highly complex developmental process that occurs primarily in seminiferous tubules of the testes and requires additional maturation steps in the epididymis and beyond. Mutations in many different genes can lead to defective spermatozoa and hence to male infertility. Some of these genes encode for ion channels and transporters that play roles in various processes such as cellular ion homeostasis, signal transduction, sperm motility, and the acrosome reaction. Here we show that germ cell–specific, but not Sertoli cell–specific, disruption of Lrrc8a leads to abnormal sperm and male infertility in mice. LRRC8A (leucine-rich repeat containing 8A) is the only obligatory subunit of heteromeric volume-regulated anion channels (VRACs). Its ablation severely compromises cell volume regulation by completely abolishing the transport of anions and osmolytes through VRACs. Consistent with impaired volume regulation, the cytoplasm of late spermatids appeared swollen. These cells failed to properly reduce their cytoplasm during further development into spermatozoa and later displayed severely disorganized mitochondrial sheaths in the midpiece region, as well as angulated or coiled flagella. These changes, which progressed in severity on the way to the epididymis, resulted in dramatically reduced sperm motility. Our work shows that VRAC, probably through its role in cell volume regulation, is required in a cell-autonomous manner for proper sperm development and explains the male infertility of Lrrc8a−/− mice and the spontaneous mouse mutant ébouriffé. Spermatogenesis is a highly complex developmental process that occurs primarily in seminiferous tubules of the testes and requires additional maturation steps in the epididymis and beyond. Mutations in many different genes can lead to defective spermatozoa and hence to male infertility. Some of these genes encode for ion channels and transporters that play roles in various processes such as cellular ion homeostasis, signal transduction, sperm motility, and the acrosome reaction. Here we show that germ cell–specific, but not Sertoli cell–specific, disruption of Lrrc8a leads to abnormal sperm and male infertility in mice. LRRC8A (leucine-rich repeat containing 8A) is the only obligatory subunit of heteromeric volume-regulated anion channels (VRACs). Its ablation severely compromises cell volume regulation by completely abolishing the transport of anions and osmolytes through VRACs. Consistent with impaired volume regulation, the cytoplasm of late spermatids appeared swollen. These cells failed to properly reduce their cytoplasm during further development into spermatozoa and later displayed severely disorganized mitochondrial sheaths in the midpiece region, as well as angulated or coiled flagella. These changes, which progressed in severity on the way to the epididymis, resulted in dramatically reduced sperm motility. Our work shows that VRAC, probably through its role in cell volume regulation, is required in a cell-autonomous manner for proper sperm development and explains the male infertility of Lrrc8a−/− mice and the spontaneous mouse mutant ébouriffé. Spermatogenesis, the production of male gametes, takes place in seminiferous tubules that harbor Sertoli cells and germ cells at various developmental stages. Sertoli cells envelop and provide a specialized environment for the developing spermatozoa. Following cycles of mitosis and meiosis, spherical, haploid spermatids transform into elongated and polarized spermatozoa (1Cooke H.J. Saunders P.T. Mouse models of male infertility.Nat. Rev. Genet. 2002; 3 (12360237): 790-80110.1038/nrg911Crossref PubMed Scopus (244) Google Scholar). This transformation, known as spermiogenesis, includes formation of the acrosome, chromatin condensation, and reshaping of the nucleus that results in the typical sperm head shape and the assembly of a mitochondrial sheath around the axoneme of the flagellum. It culminates in spermiation, in which residual cytoplasm of the elongated spermatids is shed off and spermatozoa are released into the lumen of the seminiferous tubule (2O'Donnell L. Nicholls P.K. O'Bryan M.K. McLachlan R.I. Stanton P.G. Spermiation: the process of sperm release.Spermatogenesis. 2011; 1 (21866274): 14-3510.4161/spmg.1.1.14525Crossref PubMed Google Scholar, 3O'Donnell L. Mechanisms of spermiogenesis and spermiation and how they are disturbed.Spermatogenesis. 2014; 4 (26413397): e97962310.4161/21565562.2014.979623Crossref PubMed Google Scholar). Seminiferous tubules merge into the rete testis and finally into the efferent ducts (4Haschek W.M. Rousseaux C.G. Wallig M.A. Male Reproductive System. Elsevier Science Publishing Co., Inc., New York2010: 553-597Google Scholar), which guide spermatozoa to the epididymis. During epididymal transit, spermatozoa undergo further maturation, thereby acquiring abilities required for fertilization of the oocyte (5Cooper T.G. Yeung C.-H. Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet.Microsc. Res. Tech. 2003; 61 (12672120): 28-3810.1002/jemt.10314Crossref PubMed Scopus (151) Google Scholar, 6Cooper T.G. The epididymis, cytoplasmic droplets and male fertility.Asian J. Androl. 2010; 1397: 130-138Google Scholar, 7Yeung C.H. Barfield J.P. Cooper T.G. Physiological volume regulation by spermatozoa.Mol. Cell. Endocrinol. 2006; 250 (16446027): 98-10510.1016/j.mce.2005.12.030Crossref PubMed Scopus (81) Google Scholar). Mature spermatozoa are then stored in the cauda epididymis until ejaculation. During their development and maturation, male germ cells are exposed to changes in extracellular osmolality. From roughly isoosmotic conditions in seminiferous tubules, the osmolality increases to up to ∼410 mOsm in the epididymis (5Cooper T.G. Yeung C.-H. Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet.Microsc. Res. Tech. 2003; 61 (12672120): 28-3810.1002/jemt.10314Crossref PubMed Scopus (151) Google Scholar, 7Yeung C.H. Barfield J.P. Cooper T.G. Physiological volume regulation by spermatozoa.Mol. Cell. Endocrinol. 2006; 250 (16446027): 98-10510.1016/j.mce.2005.12.030Crossref PubMed Scopus (81) Google Scholar, 8Joseph A. Shur B.D. Ko C. Chambon P. Hess R.A. Epididymal hypo-osmolality induces abnormal sperm morphology and function in the estrogen receptor α knockout mouse.Biol. Reprod. 2010; 82 (20130266): 958-96710.1095/biolreprod.109.080366Crossref PubMed Scopus (64) Google Scholar). Although the maintenance of a near-constant cell volume in face of hyper- or hypotonic challenges is crucial for cells in general, this is believed to be of particular importance for male germ cells (7Yeung C.H. Barfield J.P. Cooper T.G. Physiological volume regulation by spermatozoa.Mol. Cell. Endocrinol. 2006; 250 (16446027): 98-10510.1016/j.mce.2005.12.030Crossref PubMed Scopus (81) Google Scholar). To counteract shrinkage or swelling under hyper- and hypotonic conditions, cells have developed two mechanisms, namely regulatory volume increase and regulatory volume decrease (RVD), 2The abbreviations used are: RVDregulatory volume decreaseVRACvolume-regulated anion channelKIknock-inKOknock-outHAhemagglutininEGFPenhanced green fluorescent proteinTEMtransmission EMPNApeanut agglutininDAPI4′,6′-diamidino-2-phenylindoleH&Ehematoxylin and eosin. respectively (9Lang F. Busch G.L. Ritter M. Völkl H. Waldegger S. Gulbins E. Häussinger D. Functional significance of cell volume regulatory mechanisms.Physiol. Rev. 1998; 78 (9457175): 247-30610.1152/physrev.1998.78.1.247Crossref PubMed Scopus (1583) Google Scholar, 10Hoffmann E.K. Lambert I.H. Pedersen S.F. Physiology of cell volume regulation in vertebrates.Physiol. Rev. 2009; 89 (19126758): 193-27710.1152/physrev.00037.2007Crossref PubMed Scopus (1087) Google Scholar, 11Jentsch T.J. VRACs and other ion channels and transporters in the regulation of cell volume and beyond.Nat. Rev. Mol. Cell Biol. 2016; 17 (27033257): 293-30710.1038/nrm.2016.2910.1038/nrn.2016.28Crossref PubMed Scopus (182) Google Scholar). Previous studies demonstrated that the high osmolality in the epididymis is important for sperm maturation (8Joseph A. Shur B.D. Ko C. Chambon P. Hess R.A. Epididymal hypo-osmolality induces abnormal sperm morphology and function in the estrogen receptor α knockout mouse.Biol. Reprod. 2010; 82 (20130266): 958-96710.1095/biolreprod.109.080366Crossref PubMed Scopus (64) Google Scholar) and that the ability of spermatozoa to regulate their volume during epididymal transit (5Cooper T.G. Yeung C.-H. Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet.Microsc. Res. Tech. 2003; 61 (12672120): 28-3810.1002/jemt.10314Crossref PubMed Scopus (151) Google Scholar, 12Yeung C.H. Anapolski M. Setiawan I. Lang F. Cooper T.G. Effects of putative epididymal osmolytes on sperm volume regulation of fertile and infertile c-ros transgenic mice.J. Androl. 2004; 25 (14760007): 216-22310.1002/j.1939-4640.2004.tb02781.xCrossref PubMed Scopus (58) Google Scholar, 13Cooper T.G. Yeung C.-H. Wagenfeld A. Nieschlag E. Poutanen M. Huhtaniemi I. Sipilä P. Mouse models of infertility due to swollen spermatozoa.Mol. Cell. Endocrinol. 2004; 216 (15109745): 55-6310.1016/j.mce.2003.10.076Crossref PubMed Scopus (54) Google Scholar) and in the female reproductive tract (7Yeung C.H. Barfield J.P. Cooper T.G. Physiological volume regulation by spermatozoa.Mol. Cell. Endocrinol. 2006; 250 (16446027): 98-10510.1016/j.mce.2005.12.030Crossref PubMed Scopus (81) Google Scholar, 14Milenkovic A. Brandl C. Milenkovic V.M. Jendryke T. Sirianant L. Wanitchakool P. Zimmermann S. Reiff C.M. Horling F. Schrewe H. Schreiber R. Kunzelmann K. Wetzel C.H. Weber B.H. Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (25941382): E2630-E263910.1073/pnas.1418840112Crossref PubMed Scopus (86) Google Scholar) can have an effect on their motility. Upon excessive swelling, e.g. caused by impaired RVD, spermatozoa change the shape of their flagella to reduce membrane tension (7Yeung C.H. Barfield J.P. Cooper T.G. Physiological volume regulation by spermatozoa.Mol. Cell. Endocrinol. 2006; 250 (16446027): 98-10510.1016/j.mce.2005.12.030Crossref PubMed Scopus (81) Google Scholar). This usually results in a coiling or angulation of flagella that impairs their forward motility and thus the ability to pass the female reproductive tract and fertilize the egg (7Yeung C.H. Barfield J.P. Cooper T.G. Physiological volume regulation by spermatozoa.Mol. Cell. Endocrinol. 2006; 250 (16446027): 98-10510.1016/j.mce.2005.12.030Crossref PubMed Scopus (81) Google Scholar). Abnormalities of sperm flagella, referred to as teratozoospermia, are a common cause of infertility in mice and men (15Wambergue C. Zouari R. Fourati Ben Mustapha S. Martinez G. Devillard F. Hennebicq S. Satre V. Brouillet S. Halouani L. Marrakchi O. Makni M. Latrous H. Kharouf M. Amblard F. Arnoult C. et al.Patients with multiple morphological abnormalities of the sperm flagella due to DNAH1 mutations have a good prognosis following intracytoplasmic sperm injection.Hum. Reprod. 2016; 31 (27094479): 1164-117210.1093/humrep/dew083Crossref PubMed Scopus (63) Google Scholar, 16De Kretser D.M. Baker H.W. Infertility in men: recent advances and continuing controversies.J. Clin. Endocrinol. Metab. 1999; 84 (10522977): 3443-345010.1210/jcem.84.10.610110.1210/jc.84.10.3443Crossref PubMed Scopus (229) Google Scholar, 17Huynh T. Mollard R. Trounson A. Selected genetic factors associated with male infertility.Hum. Reprod. Update. 2002; 8 (12099633): 183-19810.1093/humupd/8.2.183Crossref PubMed Scopus (123) Google Scholar, 18Escalier D. Knockout mouse models of sperm flagellum anomalies.Hum. Reprod. Update. 2006; 12 (16565154): 449-46110.1093/humupd/dml013Crossref PubMed Scopus (80) Google Scholar). regulatory volume decrease volume-regulated anion channel knock-in knock-out hemagglutinin enhanced green fluorescent protein transmission EM peanut agglutinin 4′,6′-diamidino-2-phenylindole hematoxylin and eosin. A key player in RVD is the volume-regulated anion channel (VRAC; Ref. 11Jentsch T.J. VRACs and other ion channels and transporters in the regulation of cell volume and beyond.Nat. Rev. Mol. Cell Biol. 2016; 17 (27033257): 293-30710.1038/nrm.2016.2910.1038/nrn.2016.28Crossref PubMed Scopus (182) Google Scholar) (also known as volume-sensitive outwardly rectifying anion channel, or VSOR (19Okada Y. Volume expansion-sensing outward-rectifier Cl− channel: fresh start to the molecular identity and volume sensor.Am. J. Physiol. 1997; 273 (9316396): C755-C78910.1152/ajpcell.1997.273.3.C755Crossref PubMed Google Scholar)). These plasma membrane channels, which are ubiquitously expressed in vertebrate cells, are normally closed under resting conditions and open upon cell swelling. VRAC-mediated efflux of organic osmolytes and Cl−, the latter paralleled by K+ efflux through independent K+ channels, decreases intracellular osmolality and thereby reduces cell volume by driving water out of the cell (11Jentsch T.J. VRACs and other ion channels and transporters in the regulation of cell volume and beyond.Nat. Rev. Mol. Cell Biol. 2016; 17 (27033257): 293-30710.1038/nrm.2016.2910.1038/nrn.2016.28Crossref PubMed Scopus (182) Google Scholar, 20Pedersen S.F. Okada Y. Nilius B. Biophysics and physiology of the volume-regulated anion channel (VRAC)/volume-sensitive outwardly rectifying anion channel (VSOR).Pflügers Arch. 2016; 468 (26739710): 371-383Crossref PubMed Scopus (105) Google Scholar). Only recently, VRAC was discovered to be constituted by LRRC8 heteromers (21Voss F.K. Ullrich F. Münch J. Lazarow K. Lutter D. Mah N. Andrade-Navarro M.A. von Kries J.P. Stauber T. Jentsch T.J. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC.Science. 2014; 344 (24790029): 634-63810.1126/science.1252826Crossref PubMed Scopus (397) Google Scholar) that are formed by the obligatory subunit LRRC8A (21Voss F.K. Ullrich F. Münch J. Lazarow K. Lutter D. Mah N. Andrade-Navarro M.A. von Kries J.P. Stauber T. Jentsch T.J. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC.Science. 2014; 344 (24790029): 634-63810.1126/science.1252826Crossref PubMed Scopus (397) Google Scholar, 22Qiu Z. Dubin A.E. Mathur J. Tu B. Reddy K. Miraglia L.J. Reinhardt J. Orth A.P. Patapoutian A. SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel.Cell. 2014; 157 (24725410): 447-45810.1016/j.cell.2014.03.024Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar) and at least one other member of the LRRC8 protein family (LRRC8B–E) (21Voss F.K. Ullrich F. Münch J. Lazarow K. Lutter D. Mah N. Andrade-Navarro M.A. von Kries J.P. Stauber T. Jentsch T.J. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC.Science. 2014; 344 (24790029): 634-63810.1126/science.1252826Crossref PubMed Scopus (397) Google Scholar). LRRC8 proteins have four transmembrane helices followed by a long cytoplasmic tail that contains many leucine-rich repeats. In part based on their similarity to pannexins and connexins, LRRC8 proteins were believed to assemble to hexameric channels (21Voss F.K. Ullrich F. Münch J. Lazarow K. Lutter D. Mah N. Andrade-Navarro M.A. von Kries J.P. Stauber T. Jentsch T.J. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC.Science. 2014; 344 (24790029): 634-63810.1126/science.1252826Crossref PubMed Scopus (397) Google Scholar, 23Abascal F. Zardoya R. LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication.Bioessays. 2012; 34 (22532330): 551-56010.1002/bies.201100173Crossref PubMed Scopus (119) Google Scholar, 24Syeda R. Qiu Z. Dubin A.E. Murthy S.E. Florendo M.N. Mason D.E. Mathur J. Cahalan S.M. Peters E.C. Montal M. Patapoutian A. LRRC8 proteins form volume-regulated anion channels that sense ionic strength.Cell. 2016; 164 (26824658): 499-51110.1016/j.cell.2015.12.031Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), as recently confirmed by cryo-EM structures (25Deneka D. Sawicka M. Lam A.K.M. Paulino C. Dutzler R. Structure of a volume-regulated anion channel of the LRRC8 family.Nature. 2018; (29769723 in press)10.1038/s41586-018-0134-yCrossref PubMed Scopus (112) Google Scholar). Depending on the LRRC8 subunit composition, VRACs can also conduct a wide range of organic compounds (26Lutter D. Ullrich F. Lueck J.C. Kempa S. Jentsch T.J. Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels.J. Cell Sci. 2017; 130 (28193731): 1122-1133Crossref PubMed Scopus (94) Google Scholar, 27Planells-Cases R. Lutter D. Guyader C. Gerhards N.M. Ullrich F. Elger D.A. Kucukosmanoglu A. Xu G. Voss F.K. Reincke S.M. Stauber T. Blomen V.A. Vis D.J. Wessels L.F. Brummelkamp T.R. et al.Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs.EMBO J. 2015; 34 (26530471): 2993-300810.15252/embj.201592409Crossref PubMed Scopus (174) Google Scholar). The general importance of LRRC8 channels became evident from the severe phenotypes of Lrrc8a−/− mice, which were reported shortly after the identification of LRRC8A as an obligatory VRAC constituent (28Kumar L. Chou J. Yee C.S. Borzutzky A. Vollmann E.H. von Andrian U.H. Park S.Y. Hollander G. Manis J.P. Poliani P.L. Geha R.S. Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function.J. Exp. Med. 2014; 211 (24752297): 929-94210.1084/jem.20131379Crossref PubMed Scopus (75) Google Scholar). These mice display high pre- and postnatal mortality, growth retardation, curly hair, and abnormalities in several tissues. Importantly, females and males lacking LRRC8A are sterile (28Kumar L. Chou J. Yee C.S. Borzutzky A. Vollmann E.H. von Andrian U.H. Park S.Y. Hollander G. Manis J.P. Poliani P.L. Geha R.S. Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function.J. Exp. Med. 2014; 211 (24752297): 929-94210.1084/jem.20131379Crossref PubMed Scopus (75) Google Scholar) for so-far unknown reasons. It was only recently discovered that the spontaneous mouse mutant ébouriffé (29Lalouette A. Lablack A. Guenet J.L. Montagutelli X. Segretain D. Male sterility caused by sperm cell-specific structural abnormalities in ebouriffé, a new mutation of the house mouse.Biol. Reprod. 1996; 55 (8828840): 355-36310.1095/biolreprod55.2.355Crossref PubMed Scopus (14) Google Scholar) carries a mutation that truncates the cytoplasmic carboxyl terminus of LRRC8A (30Platt C.D. Chou J. Houlihan P. Badran Y.R. Kumar L. Bainter W. Poliani P.L. Perez C.J. Dent S.Y.R. Clapham D.E. Benavides F. Geha R.S. Leucine-rich repeat containing 8A (LRRC8A)-dependent volume-regulated anion channel activity is dispensable for T-cell development and function.J. Allergy Clin. Immunol. 2017; 140 (28192143): 1651-165910.1016/j.jaci.2016.12.974Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). This mouse mutant shares several pathological features (29Lalouette A. Lablack A. Guenet J.L. Montagutelli X. Segretain D. Male sterility caused by sperm cell-specific structural abnormalities in ebouriffé, a new mutation of the house mouse.Biol. Reprod. 1996; 55 (8828840): 355-36310.1095/biolreprod55.2.355Crossref PubMed Scopus (14) Google Scholar) with Lrrc8a−/− mice (28Kumar L. Chou J. Yee C.S. Borzutzky A. Vollmann E.H. von Andrian U.H. Park S.Y. Hollander G. Manis J.P. Poliani P.L. Geha R.S. Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function.J. Exp. Med. 2014; 211 (24752297): 929-94210.1084/jem.20131379Crossref PubMed Scopus (75) Google Scholar). The more benign phenotype of ébouriffé mice may be explained by the observation that their VRAC currents are strongly reduced but not abolished (30Platt C.D. Chou J. Houlihan P. Badran Y.R. Kumar L. Bainter W. Poliani P.L. Perez C.J. Dent S.Y.R. Clapham D.E. Benavides F. Geha R.S. Leucine-rich repeat containing 8A (LRRC8A)-dependent volume-regulated anion channel activity is dispensable for T-cell development and function.J. Allergy Clin. Immunol. 2017; 140 (28192143): 1651-165910.1016/j.jaci.2016.12.974Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The first characterization of ébouriffé mice focused on their male sterility, which was attributed to structural defects of sperm cells (29Lalouette A. Lablack A. Guenet J.L. Montagutelli X. Segretain D. Male sterility caused by sperm cell-specific structural abnormalities in ebouriffé, a new mutation of the house mouse.Biol. Reprod. 1996; 55 (8828840): 355-36310.1095/biolreprod55.2.355Crossref PubMed Scopus (14) Google Scholar). It remains, however, unclear whether a complete loss of LRRC8A would have similar consequences and whether these pathologies are cell-autonomous outcomes of a reduction of VRAC currents in germ cells or in Sertoli cells. In this study, we investigated the role of LRRC8A in spermatogenesis using several mouse models. Whereas mice lacking LRRC8A specifically in Sertoli cells were completely fertile, LRRC8A was indispensable in germ cells for the normal development of mature spermatozoa and for male fertility. In the absence of LRRC8A, late spermatids displayed severe disorganization of the mitochondrial sheath in the midpiece region and a drastically swollen cytosolic compartment. Spermatozoa showed flagellar coiling or angulation, features that were previously described with abnormal cell swelling upon RVD failure (7Yeung C.H. Barfield J.P. Cooper T.G. Physiological volume regulation by spermatozoa.Mol. Cell. Endocrinol. 2006; 250 (16446027): 98-10510.1016/j.mce.2005.12.030Crossref PubMed Scopus (81) Google Scholar). As the basis for exploring the role of VRAC in male fertility, we first determined the expression of all LRRC8 subunits in testis and epididymis. It is generally believed that VRAC is ubiquitously expressed in all vertebrate tissues and cells (11Jentsch T.J. VRACs and other ion channels and transporters in the regulation of cell volume and beyond.Nat. Rev. Mol. Cell Biol. 2016; 17 (27033257): 293-30710.1038/nrm.2016.2910.1038/nrn.2016.28Crossref PubMed Scopus (182) Google Scholar, 20Pedersen S.F. Okada Y. Nilius B. Biophysics and physiology of the volume-regulated anion channel (VRAC)/volume-sensitive outwardly rectifying anion channel (VSOR).Pflügers Arch. 2016; 468 (26739710): 371-383Crossref PubMed Scopus (105) Google Scholar, 31Nilius B. Eggermont J. Voets T. Buyse G. Manolopoulos V. Droogmans G. Properties of volume-regulated anion channels in mammalian cells.Prog. Biophys. Mol. Biol. 1997; 68 (9481145): 69-11910.1016/S0079-6107(97)00021-7Crossref PubMed Scopus (320) Google Scholar), which is consistent with the wide expression pattern of all LRRC8 genes gleaned from EST database analysis (21Voss F.K. Ullrich F. Münch J. Lazarow K. Lutter D. Mah N. Andrade-Navarro M.A. von Kries J.P. Stauber T. Jentsch T.J. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC.Science. 2014; 344 (24790029): 634-63810.1126/science.1252826Crossref PubMed Scopus (397) Google Scholar). Indeed, Western blotting analysis identified the obligatory VRAC subunit LRRC8A in testis and epididymis and in all other tissues examined (Fig. 1A). With the exception of LRRC8E, the other LRRC8 isoforms were also significantly expressed in those tissues (Fig. 1A). The glutamate transport-enhancing subunit LRRC8E (26Lutter D. Ullrich F. Lueck J.C. Kempa S. Jentsch T.J. Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels.J. Cell Sci. 2017; 130 (28193731): 1122-1133Crossref PubMed Scopus (94) Google Scholar), known to be almost absent from brain and blood cells (21Voss F.K. Ullrich F. Münch J. Lazarow K. Lutter D. Mah N. Andrade-Navarro M.A. von Kries J.P. Stauber T. Jentsch T.J. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC.Science. 2014; 344 (24790029): 634-63810.1126/science.1252826Crossref PubMed Scopus (397) Google Scholar), was prominently expressed in the epididymis but was only barely detectable in testes (Fig. 1A). The testicular expression pattern of Lrrc8a was investigated using knock-in (KI) mice expressing β-gal under the control of the endogenous Lrrc8a promoter (32Stuhlmann T. Planells-Cases R. Jentsch T.J. LRRC8/VRAC anion channels enhance β-cell glucose sensing and insulin secretion.Nat. Commun. 2018; 9 (29773801): 197410.1038/s41467-018-04353-yCrossref PubMed Scopus (55) Google Scholar). Blue LacZ staining was scattered over the whole width of seminiferous tubules (Fig. 1B), suggesting that Lrrc8a is expressed in Sertoli cells and in germ cells of all developmental stages. Because the antibodies we have generated against the essential VRAC subunit LRRC8A (21Voss F.K. Ullrich F. Münch J. Lazarow K. Lutter D. Mah N. Andrade-Navarro M.A. von Kries J.P. Stauber T. Jentsch T.J. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC.Science. 2014; 344 (24790029): 634-63810.1126/science.1252826Crossref PubMed Scopus (397) Google Scholar, 27Planells-Cases R. Lutter D. Guyader C. Gerhards N.M. Ullrich F. Elger D.A. Kucukosmanoglu A. Xu G. Voss F.K. Reincke S.M. Stauber T. Blomen V.A. Vis D.J. Wessels L.F. Brummelkamp T.R. et al.Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs.EMBO J. 2015; 34 (26530471): 2993-300810.15252/embj.201592409Crossref PubMed Scopus (174) Google Scholar) work only in Western blots (Fig. 1A) and not in immunohistochemistry, we generated KI mice in which we fused three hemagglutinin (HA) peptide tags to the carboxyl terminus of LRRC8A. These tags were inserted by CRISPR-Cas9–mediated recombination in fertilized mouse oocytes obtained from crosses of WT and Lrrc8alox/lox (32Stuhlmann T. Planells-Cases R. Jentsch T.J. LRRC8/VRAC anion channels enhance β-cell glucose sensing and insulin secretion.Nat. Commun. 2018; 9 (29773801): 197410.1038/s41467-018-04353-yCrossref PubMed Scopus (55) Google Scholar) mice. The resulting Lrrc8aHA/HA and Lrrc8alox-HA/lox-HA mice allowed the detection of LRRC8A by Western blotting and immunohistochemistry using commercial antibodies against the HA tag, with WT mice serving as negative controls. Lrrc8alox-HA/lox-HA mice additionally permit to ascertain Cre-mediated, cell type–specific disruption of Lrrc8a. Consistent with the lacZ staining (Fig. 1B), immunofluorescent labeling of testis sections of Lrrc8aHA/HA mice revealed broad expression of LRRC8A all over the seminiferous tubules (Fig. 1C). We observed a remarkably strong radial staining that extended from the outer circumference to the lumen of the seminiferous tubules, a pattern that is suggestive of Sertoli cells. It is possible that the remaining scattered and weaker labeling represents germ cells, but the low signal intensity precluded a definite conclusion. Considering the complexity of spermatogenesis, including the important interplay of germ cells with Sertoli cells, the male infertility of Lrrc8a−/− mice (28Kumar L. Chou J. Yee C.S. Borzutzky A. Vollmann E.H. von Andrian U.H. Park S.Y. Hollander G. Manis J.P. Poliani P.L. Geha R.S. Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function.J. Exp. Med. 2014; 211 (24752297): 929-94210.1084/jem.20131379Crossref PubMed Scopus (75) Google Scholar) might be due to primary defects in different testicular cell types. For instance, the male infertility of mice lacking the ClC-2 Cl− channel has been tentatively attributed to a defect in Sertoli rather than germ cells (33Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhopadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20 (11250895): 1289-129910.1093/emboj/20.6.1289Crossref PubMed Scopus (259) Google Scholar). To identify the cell type in which absence of LRRC8A causes male infertility, we generated different conditional LRRC8A knock-out (KO) mouse models. We first crossed Lrrc8alox/lox (32Stuhlmann T. Planells-Cases R. Jentsch T.J. LRRC8/VRAC anion channels enhance β-cell glucose sensing and insulin secretion.Nat. Commun. 2018; 9 (29773801): 197410.1038/s41467-018-04353-yCrossref PubMed Scopus (55) Google Scholar) or Lrrc8alox-HA/lox-HA mice with AMH-Cre mice (34Lécureuil C. Fontaine I. Crepieux P. Guillou F. Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice.Genesis. 2002; 33 (12124943): 114-11810.1002/gene.10100Crossref PubMed Scopus (174) Google Scholar), which express the Cre-recombinase specifically in Sertoli cells. In the following, we refer to the resulting Sertoli cell-specific LRRC8A KO as SC-Δ8A and SC-Δ8A-HA, respectively. Immunofluorescent analysis of testes from SC-Δ8A-HA mice (Fig. 2A) showed that LRRC8A could no longer be detected in seminiferous tubules. However, Western blotting analysis showed only a moderate reduction of LRRC8A protein levels in testes compared with Lrrc8alox/lox controls (Fig. 2B), suggesting that LRRC8A is also expressed in testicular cell types other than Sertoli cells. Despite the prominent expression of LRRC8A in Sertoli cells, we failed to detect any morphological changes of SC-Δ8A testes compared with Lrrc8alox/lox con" @default.
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• W2805997767 title "LRRC8/VRAC anion channels are required for late stages of spermatid development in mice" @default.
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