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Anion Exchanger 2 Is Essential for Spermiogenesis in Mice

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Anion Exchanger 2 Is Essential for Spermiogenesis in Mice Anion exchanger 2 is essential for spermiogenesis in mice Juan F. Medina*†, Sergio Recalde*, Jesu´ s Prieto*, Jon Lecanda*, Elena Sa´ ez*, Colin D. Funk‡, Paola Vecino§, Marian A. van Roon¶, Roelof Ottenhoffʈ, Piter J. Bosmaʈ, Conny T. Bakkerʈ, and Ronald P. J. Oude Elferinkʈ *Laboratory of Molecular Genetics, Division of Hepatology and Gene Therapy, University Hospital͞School of Medicine, Fundacio´n para la Investigacio´n Medica Aplicada, University of Navarra, E-31008 Pamplona, Spain; ‡Center for Experimental Therapeutics, University of Pennsylvania, Biomedical Research Building II͞III, Philadelphia, PA 19104; §Department of Cell Biology, University of the Basque Country, E-48940 Leioa, Spain; and ¶Facility for Genetically Modified Mice and ʈLaboratory for Experimental Hepatology, AMC Liver Center, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands Edited by Michael J. Welsh, University of Iowa, Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, and approved October 21, 2003 (received for review September 19, 2003) Na؉-independent anion exchangers (AE) mediate electroneutral exchangers (AE), which mediates electroneutral and reversible ؊ ؊ Ϫ Ϫ exchange of Cl for HCO3 ions across cell membranes, being exchange of Cl and HCO3 across cell membranes (12). In involved in intracellular pH and cell volume regulation and in cooperation with other ion carriers, AE proteins are involved in transepithelial hydroionic fluxes. Bicarbonate activation of adeny- intracellular pH and cell volume regulation and in transepithelial lyl cyclase is known to be necessary for sperm motility and sperm hydroionic fluxes and acid͞base transport. Among the four AE capacitation, and a few studies have suggested a possible role of genes identified in mammals thus far (Ae1, Ae2, Ae3, and Ae4) AE carriers in reproduction. Among the four AE genes identified in (12, 13), only Ae2 (Slc4a2) has been shown to be expressed in the mammals thus far, only Ae2 (Slc4a2) has been determined to be male reproductive system, especially in developing spermatozoa expressed in the male reproductive system, especially in develop- (14) and in epididymal epithelium (15). Studies carried out in ing spermatozoa and in epididymal epithelium. Most AE genes normal male rats demonstrated that the levels of Ae2 mRNA in drive alternative transcription, which in mouse Ae2 results in the seminiferous tubules are higher from the spermiogenic stage several Ae2 isoforms. Here, we generated mice carrying a targeted VII onward (14). disruption of Ae2 that prevents the expression of the three AE2 Most AE genes have been found to drive alternative tran- isoforms (Ae2a, Ae2b1, and Ae2b2) normally found in mouse testes. scription. In the mouse Ae2, five N-terminal variants may be .Male Ae2؊/؊ mice (but not female Ae2؊/؊ mice) are infertile transcribed: Ae2a from the upstream promoter, Ae2b1 and Histopathological analysis of Ae2؊/؊ testes shows an interruption Ae2b2 from alternate promoter sequences within intron 2, and of spermiogenesis, with only a few late spermatids and a complete Ae2c1 and Ae2c2 from sequences within intron 5 (16). To study absence of spermatozoa in the seminiferous tubules. The number the specific role of Ae2 in spermatogenesis, we generated mice of apoptotic bodies is increased in the seminiferous tubules and in carrying a targeted disruption of Ae2 that prevents the expres- the epididymis, which also shows squamous metaplasia of the sion of the three AE2 isoforms (Ae2a, Ae2b1, and Ae2b2) epididymal epithelium. Our findings reveal an essential role of Ae2 normally expressed in mouse testes. in mouse spermiogenesis and stress the recently postulated in- volvement of bicarbonate in germ-cell differentiation through the Methods bicarbonate-sensitive soluble-adenylyl-cyclase pathway. Gene Targeting. The targeting vector for the homologous recom- bination was prepared in the Cre͞loxP plasmid pLox-TK-neo (a permatogenesis is the entire process by which a spermato- gift of P. C. Orban, University of British Columbia, Vancouver). Sgonial germ cell is transformed into a uniquely shaped This vector contains the thymidine kinase gene of herpes virus spermatozoon (1). It consists of three sequential phases of cell type2(tk) and the neomycin-phosphotransferase gene (neor), proliferation and differentiation. Successive mitotic divisions of flanked by loxP sites to allow for excision with Cre recombinase. a spermatogonial stem cell maintain the pool of stem cells and Vector restriction sites HindIII–XhoI, BamHI, and XbaI were also give rise to primary spermatocytes. Each primary spermato- used for sequential subcloning of three consecutive DNA frag- cyte undergoes biphasic meiotic divisions, resulting in four ments of the mouse AE2 gene regions named fragments I, II, and haploid round spermatids. Finally, the nuclear and cellular III. These DNA fragments (Fig. 1) were produced by PCR with components of these postmeiotic male germ cells are gradually an Advantage amplification kit with GC-melt (BD Clontech) on remodeled into sperm cells through a process involving complex Ae2 DNA obtained from a genomic library of mouse ES-129͞ structural and biochemical changes referred to as spermiogen- Ola cells (16). Subcloning of fragment I (2-kb long) was followed esis. Consecutive spermatogenic cycles evolve as waves, in which by subcloning of the intermediate fragment II (1.5 kb long) several sequential specific cellular associations of germ cells or within the excisable region (Fig. 1). An XbaI site present in stages can be distinguished. The number of stages varies between fragment II was destroyed before subcloning in the targeting species, and in the mouse each wave can be divided into 12 stages vector to allow for the subsequent subcloning of fragment III (3.2 (2). Mature sperm cells are released from the seminiferous kb long) into the XbaI site of the vector. Fragments I and III are tubules and migrate through the epididymis. The concentration situated outside the vector excisable region (mainly fragment II of bicarbonate has been reported to be important for sperm that includes exons 2, 1b2 and 1b1, each having the ATG start motility (3) and for sperm capacitation (4, 5), which is a codons for AE2a, AE2b2, and AE2b1 isoforms, respectively; see MEDICAL SCIENCES prerequisite for successful fertilization. These effects of bicar- Fig. 1). Correct fragment orientations were assessed by sequence bonate involve activation of a bicarbonate-sensitive adenylyl cyclase (3, 4, 6), recently identified as the soluble adenylyl cyclase (sAC) (7). Recent studies suggested a role for bicarbonate and This paper was submitted directly (Track II) to the PNAS office. sAC splice variants in spermatogenesis as well (8). Abbreviations: AE, anion exchanger; ES, mouse embryonic stem cell; sAC, soluble adenylyl To trigger cAMP-dependent processes, bicarbonate ions enter cyclase; FL-sAC, full length sAC; T-sAC, truncated sAC; TUNEL, terminal deoxynucleotidyl- germ cells by an anion transporter (4, 9, 10) or are produced by transferase-mediated dUTP nick end labeling. † hydration of CO2 through carbonic anhydrase (11). One type of To whom correspondence should be addressed. E-mail: [email protected]. ϩ anion transporters is the family of Na -independent anion © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.2536127100 PNAS ͉ December 23, 2003 ͉ vol. 100 ͉ no. 26 ͉ 15847–15852 Downloaded by guest on September 27, 2021 clones and further cytogenetic analysis, two clones with normal karyotype (1C2-11 and 3B4-4, each deriving from the two original clones), were chosen for expansion and further injection into blastocysts. Ten to 15 targeted 129͞Ola ES cells (from either clone 1C2-11 or 3B4-4) were injected into C57BL͞6 blastocysts isolated at day 3, being further reimplanted into pseudopregnant females as described (19). Male chimeras from both targeted ES clones were mated to FVB females. Offspring were genotyped by PCR and Southern blot analysis, and heterozygotes (Ae2ϩ/Ϫ) were interbred to produce Ae2Ϫ/Ϫ mice. All animals were kept under conventional housing conditions and received humane care according to institutional guidelines. Histopathological Examination of Testis and Epididymis. Tissues were fixed in paraformaldehyde and embedded in paraffin. Serial 5-␮m sections stained with hematoxylin and eosin allowed us to clearly distinguish nuclei of cells and identify their cell type. Cell populations from 10 seminiferous tubules from four different specimens for each genotype were counted under a light micro- scope. Stages were evaluated by carefully examining stage- related spermatid degeneration (2). Apoptosis was determined on 5-␮m sections over poly-L-lysine-coated slides through bright field visualization by using two different procedures: Nissel’s staining (with toluidine blue) and terminal deoxynucleotidyl- transferase-mediated dUTP nick end labeling (TUNEL). For the latter we used an In Situ Cell Death Detection Kit, POD (Roche Molecular Biochemicals), overstained with the peroxi- dase substrate supplied with the kit and counterstained with eosin. Analysis of Spermatozoa. Epididymes from five heterozygous and five wild-type mice were cut in small pieces inside a drop of PBS. After centrifuging at low speed (190 ϫ g), we separated the supernatant with remaining spermatozoa and pelleted these germ cells with a new centrifugation at higher speed (2,000 ϫ g). Spermatozoa were counted in a hemocytometer with a light microscope. To evaluate the rate of aberrant
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