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, Fundacioń para la Investigacioń 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 involvement of bicarbonate in germ-cell differentiation through the Methods bicarbonate-sensitive soluble-adenylyl-cyclase pathway. Gene Targeting. The targeting vector for the homologous recombination 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 microscope. 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 structures (micro- cephalia, bicephalia, two tails, or a small tail) versus normal Fig. 1. (A) Targeting strategy for deletion͞inactivation of mouse Ae2. haploid cells, sperm cells were resuspended in PBS͞0.1% poly- Wild-type Ae2 locus, targeting vector, targeted Ae2 allele after homologous vinylpyrrolidone (Fluka), stained with 4Ј,6-diamidino-2- recombination and deleted Ae2 locus by using Cre recombinase, and upstream phenylindole (DAKO), and visualized in a hemocytometer and downstream recombination of Ae2targ allele were assessed by Southern blotting (ES genomic DNA cut with BspHI and probed with Bs-pro) and by PCR under a fluorescent microscope connected to a video camera. (primers are indicated as connected dots in the Ae2targ diagram), respectively. Sperm motility was assessed in PBS (with no polyvinylpyrroli- (B) Initial genotyping to detect targeted mice by Southern blotting of tail done) through the combined flow and image cytometry proce- genomic DNA cut with XbaI and probed with the Xb-pro. (C) PCR genotyping dure (20), which allowed for the identification of haploid cells for routine detection of targeted mice by using primers pr1 and pr2 (repre- with propidium iodine and a further evaluation of their mito- sented in A) on tail genomic DNA. Bs, BspHI site; Xb, XbaI site. chondrial activity by using rhodamine 123 (Molecular Probes).

Analysis of mRNAs. Total RNA was isolated from mouse testes analysis after each fragment subcloning. Finally, vector- according to the guanidinium thiocyanate method by using the backbone-free targeting DNA suitable for electroporation of TRI Reagent (Sigma). Transcriptional expression of several mouse embryonic stem (ES) cells was obtained through diges- genes involved in spermatogenesis was analyzed through a tion of the targeting vector with NotI, electrophoresis in agarose semiquantitative RT-PCR-based procedure (16). For each case, gel, and further electroelution from a gel slide as described (17). bands were evaluated in the linear phase of amplification, i.e., Electroporated cells were screened by PCR, and resultant pos- before reaching the plateau effect with more cycles. The se- itively targeted ES cell clones were confirmed by Southern blot quences and exact positions of the PCR primers are available by using ES genomic DNA digested with BspHI (Bs) and the from the corresponding author on request. Bs-probe (Fig. 1). After a cytogenetic analysis, we chose two clones with a normal karyotype (1C2 and 3B4) and carried out Statistical Analysis. Data are given as mean Ϯ SD. Statistical Cre-catalyzed excisions (18). Coelectroporation with the Cre- analyses were carried out with the statistical program SPSS 11.0. recombinase plasmid pOG231 and the puromycin-expressing Because residuals for each variable had a normal distribution vector pHA262pur (gifts of S. O’Gorman, The Salk Institute for (according to Shapiro–Wilk), we analyzed the data from quan- Biological Studies, San Diego, and H. te Riele, The Netherlands titation of cells in the seminiferous tubules by using parametric Cancer Institute, Amsterdam, respectively) led to enrichment of tests, i.e., with the one-way ANOVA followed by the Student– Cre-expressing ES cells by selection with puromycin, and allowed Newmann–Keuls post hoc test. Motility values (fluorescence for an efficient ganciclovir selection of ES cells with Cre excision mean intensity for rhodamine) and the rate of aberrant structure of the tk-neor box. After PCR screening for positively excised in sperm cells were analyzed with the Student t test.

15848 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2536127100 Medina et al. Downloaded by guest on September 27, 2021 Fig. 2. RT-PCR analysis for the testis expression of Ae2a, Ae2b1 and Ae2b2 mRNA isoforms. Isoform-speciﬁc sense primers (in exons 2, 1b1, and 1b2) and a common antisense primer encompassing the exon3͞exon4 junction were used. GAPDH mRNA was used as the normalizing control. The thick upper band in the 100-bp ladder is 600 bp long.

Results Generation of Ae2-Targeted Mice. A 1.5-kb deletion was produced in mouse Ae2 by gene targeting (Fig. 1). The deleted region is relevant to the three variants Ae2a, Ae2b1, and Ae2b2 normally expressed in mouse testes (Fig. 2); we found that normal mouse testes do not express the intron-5-derived variants Ae2c1 and Ae2c2 (data not shown). Fig. 1 illustrates the strategy used to target one of the alleles of Ae2 in ES cells through homologous recombination. In the first step, the targeted allele incorporated Fig. 3. (A) Macroscopic appearance of testes (with their epididymes) from an engineered Ae2 region flanked by loxP sites that allowed for 20-week-old mice of indicated genotype. (B) Testicular sections from speci- its further excision with Cre recombinase. Removal of the mens shown in A, stained with hematoxylin and eosin to visualize the semi- engineered region deprived the allele of exons 2, 1b1, and 1b2, niferous tubules. (C) TUNEL for in situ detection of apoptotic cells. (D) Nissel’s staining showing apoptotic cells during the process of nuclear fragmentation. which are the initial coding exons for Ae2a, Ae2b1, and Ae2b2 isoforms (16). Targeted ES clones were identified by PCR and Some TUNEL- and Nissel-positive cells are identified by arrows. further assessed by Southern blot analysis (not shown). Two independently targeted ES clones tested for a normal karyotype IV–VII with no spermatozoa and very few elongating (late) contributed to the germ line of chimeric mice and yielded spermatids (Table 1). Also, the number of round (early) sper- progeny heterozygous for the Ae2-targeted allele. After setting Ϫ Ϫ matids is dramatically reduced in Ae2 / mice (Table 1). This up crosses between Ae2 heterozygous mice, PCR genotyping of Ϫ Ϫ reduction is more obvious in older mice (8–9 months old), in F offspring showed the occurrence of Ae2 / offspring (Fig. 2), 2 which spermatid stages become rare (data not shown). The although their number was lower than expected according to Ϫ Ϫ disrupted spermiogenesis in Ae2 / mice is associated with Mendelian distribution (8% instead of 25%). This was at least apoptosis of germ cells, the number of apoptotic bodies within partly caused by an increased perinatal lethality (80% of dead Ϫ Ϫ Ϫ Ϫ the seminiferous tubules being increased in these targeted animals were Ae2 / ). Ae2 / mice that survived the perinatal animals (Table 1 and Fig. 3 C and D). stage exhibited no obvious macroscopic abnormalities. Female Ae2Ϫ/Ϫ mice could become pregnant when crossed ϩ/ϩ ϩ/Ϫ with male Ae2 or Ae2 mice. Male fertility in heterozygous Table 1. Quantitation of germ cell and somatic cell populations mice was similar to that in wild-type mice, as denoted by average in 10 seminiferous tubules of mouse testis specimens litter sizes in the respective cross with female Ae2ϩ/ϩ mice. ϩ/ϩ ϩ/Ϫ Ϫ/Ϫ Ae2 genotype However, female mice (either Ae2 , Ae2 ,orAe2 ) did not P Ϫ/Ϫ become pregnant when crossed with male Ae2 mice, indicat- Cell type Ϫ͞Ϫ (n ϭ 4) ϩ͞Ϫ (n ϭ 4) ϩ͞ϩ (n ϭ 4) (ANOVA) ing male infertility in Ae2Ϫ/Ϫ mice. Spermatogonia 299 Ϯ 90* 353 Ϯ 31 441 Ϯ 41* 0.024 Testis Examinations. The size and weight of the testes in the Ae2Ϫ/Ϫ Spermatocytes 353 Ϯ 47* 439 Ϯ 60 486 Ϯ 74* 0.038 † mice is reduced by 40–60%, whereas no difference is observed Round spermatids 71 Ϯ 25 451 Ϯ 111 365 Ϯ 78 Ͻ0.001 MEDICAL SCIENCES between testes in Ae2ϩ/Ϫ mice and wild-type mice (Fig. 3A). Elongated spermatids 13 Ϯ 11† 359 Ϯ 27 413 Ϯ 122 Ͻ0.001 Histological examination and quantification of cell populations Spermatozoa 0† 632 Ϯ 206 536 Ϯ 111 Ͻ0.001 in the seminiferous tubules showed differences between Ae2Ϫ/Ϫ Sertoli cells 188 Ϯ 27† 299 Ϯ 15 305 Ϯ 26 Ͻ0.001 mice and both heterozygous and wild-type mice (Table 1 and Fig. Apoptotic bodies 80 Ϯ 8‡ 12 Ϯ 5‡ 27 Ϯ 12‡ Ͻ0.001 Ϫ/Ϫ 3B). Thus, Ae2 mice show reductions in most cell populations, Values are presented as mean Ϯ SD. *, significant differences between which may account for the reduction in the testicular size and labeled groups; †, labeled group is significantly different from the two others; Ϫ Ϫ weight. Moreover, Ae2 / mice have a characteristic disruption ‡, labeled groups differ from each other (all P values are Ͻ0.05 according to of spermiogenesis after stage VII, most cells being in stages the Student–Newmann–Keuls post hoc test).

Medina et al. PNAS ͉ December 23, 2003 ͉ vol. 100 ͉ no. 26 ͉ 15849 Downloaded by guest on September 27, 2021 Table 2. Sperm characteristics and motility evaluation Ae2 genotype

Parameter ϩ͞Ϫ (n ϭ 5) ϩ͞ϩ (n ϭ 5)

No. of spermatozoa, ϫ105 80.1 Ϯ 29 77.5 Ϯ 34 Haploid spermatozoa, % 78.2 Ϯ 4 82.4 Ϯ 3.3 Aberrant structure, % 21.8 Ϯ 4 17.6 Ϯ 3.3 Fluorescence mean intensity for 144.7 Ϯ 18 150 Ϯ 12.4 rhodamine 123 as a motility estimation

Values are presented as mean Ϯ SD. No signiﬁcant differences occurred between wild-type and heterozygous mice (all P values are Ͼ0.05 according to the Student t test).

The absolute number of the other populations of germ cells (spermatogonia and spermatocytes) and Sertoli cells are similar in Ae2ϩ/ϩ mice and Ae2ϩ/Ϫ mice (Table 1). Although these cell populations are significantly reduced in Ae2Ϫ/Ϫ mice, their reduction (30–40%) is less dramatic than that observed for round spermatids and further maturing cells (Table 1). On the other hand, the appearance of Sertoli cells and that of Leydig cells is normal in Ae2Ϫ/Ϫ mice. Moreover, these mice show a normal pattern of expression of stem cell factor (Scf or Kit ligand) mRNA in their testis (see below), which suggests a Fig. 4. (A) Sections of the corpus epididymis from specimens shown in Fig. normal functionality of the remaining Sertoli cells (21, 22). Also, Ϫ/Ϫ 3A, stained with hematoxylin and eosin. (B) Two-fold magnification of sec- Leydig cell function appeared to be correct in Ae2 mice, as tions in A; a squamous metaplasia of the epididymal epithelium with a indicated by normal serum levels of testosterone in these animals flattened appearance, and a loss of cilia are observed in Ae2Ϫ/Ϫ mice (Right). (data not shown). (C) TUNEL for in situ detection of apoptotic cells within epididymis. TUNEL- Ϫ Ϫ The testicular alterations observed in Ae2 / mice are virtu- positive cells are identified by arrows. ally absent in heterozygous mice. Thus, the seminiferous tubules in Ae2ϩ/Ϫ mice display a normal spermatogenic wave and cellular composition (Table 1 and Fig. 3B), suggesting that spermiogen- mice. Analysis of the two splice variants of the germ cell sAC, i.e., esis may progress correctly for both Ae2ϩ and Ae2Ϫ haploid germ the full-length sAC (FL-sAC) and the truncated sAC (T-sAC) cells. In fact, the analysis of mature sperm cell characteristics showed minor reductions in Ae2Ϫ/Ϫ testis, mainly the T-sAC such as the rate of aberrant structure and motility estimation variant (Fig. 5). No specific alterations were found in testis ϩ Ϫ showed no differences between Ae2 / mice and wild-type expression of the cAMP-sensitive transcription factor members littermates (Table 2). Moreover, after setting up eight crosses Ϫ/Ϫ ϩ Ϫ ϩ ϩ Creb and Crem, in analyzed Ae2 animals (data not shown). between male Ae2 / mice and female Ae2 / mice, the pro- Finally, the expression of Scf, which in testis is specifically portion of heterozygous and wild-type animals were similar activated in Sertoli cells (22), is normal in targeted mice (Fig. 5). (45% and 55%, respectively).

Epididymis Examinations. The epididymal epithelium in Ae2Ϫ/Ϫ mice shows abnormalities indicative of squamous metaplasia, with a flattened appearance in corpus (Fig. 4A) and cauda, and a cubic distribution in the caput epididymis (not shown). Most of the content in the epididymis of these targeted animals consists of apoptotic bodies together with a few spermatocytes and early spermatids released from the seminiferous tubules and with no mature sperm cells (Fig. 4 B and C). The appearance of the epididymal wall and the frequency of apoptotic cells within the epididymis in heterozygous mice are similar to those in wild-type animals (Fig. 4).

Differential Expression of Testis Genes. Testis steady-state levels of messengers for a few well characterized markers involved in spermatogenesis were analyzed by a semiquantitative RT-PCR procedure (Fig. 5). Expression of the genes for the transition protein 1 (Tp-1) and protamine 1 (Prm1) was each virtually Fig. 5. Expression of indicated mRNAs were analyzed by semiquantitative Ϫ Ϫ absent in Ae2 / testis. By contrast, the levels of transcripts from RT-PCR. All shown bands correspond to amplicons that are in the linear phase other postmeiotic genes, which are normally activated at earlier of amplification at the following number of cycles: 20 cycles for Tp-1 and Prm1; stages of spermiogenesis such as those for acrosin, for a testis- 30 cycles for acrosin, Acp, Atce1, calspermin, and sAC (both splice variants specific form of an actin-capping protein (Acp), and for an FL-sAC and T-sAC); 35 cycles for Scf. RT-PCR for Gapdh mRNA (25 cycles) was ͞ ͞ used as a normalizing control. Each sAC isoform was amplified separately by Atf Creb-like peptide specifically expressed in mid late round using a common sense primer (in exon 9) and isoform-specific antisense Ϫ/Ϫ spermatids (Atce1), were only slightly reduced in Ae2 testis. primers. The sequence of the antisense primer specific for the FL-sAC isoform Similarly, testis levels of the calspermin message, which normally is within exon 11 (the spliced out exon in the T-sAC isoform), whereas that for appear in pachytene primary spermatocytes and continue to the T-sAC isoform encompasses the junction of exons 10 (8 nt) and 12 (12 nt). increase as cells differentiate, were slightly reduced in targeted In the 100-bp ladder, the thick upper band is 600 bp long.

15850 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2536127100 Medina et al. Downloaded by guest on September 27, 2021 Discussion meiotic cell divisions, and they remain until the very end of the As indicated in the introduction, bicarbonate stimulation of differentiation of spermatozoa, when these germ cells are adenylyl cyclase and subsequent increase of intracellular cAMP released to the lumen of the seminiferous tubules. Such bridges levels play an important role for male reproductive processes allow developing germinal haploid cells to share a common such as sperm motility and capacitation (3, 4, 6), the involved cytoplasm with their neighbors and become phenotypically bicarbonate-sensitive adenylyl cyclase being identified as the diploid (24). sAC (7). Recently, a role for bicarbonate and sAC splice variants We investigated the effect of Ae2 deficiency on testis in spermatogenesis was suggested as well (8). For initiating these steady-state mRNA levels for a few well characterized markers processes bicarbonate ions need to enter germ cells by way of an involved in spermatogenesis. Expression of Tp-1 (the gene for anion transporter (4, 9, 10). Ae2 carrier may mediate reversible the transition protein 1, an intermediate nucleoprotein in the Ϫ exchange of ClϪ and HCO across cell membranes and, there- histone-to-protamine exchange), was virtually absent in 3 Ϫ/Ϫ fore, a bicarbonate influx in a gradient-dependent manner. Ae2 testis. Similar results were obtained for protamine 1 Studies in male rats have shown that Ae2 is expressed in (Prm1). Most probably, the undetectable expression of these developing spermatozoa and that Ae2 mRNA levels are in- two haploid germ-cell genes is due to the absence of late creased in the seminiferous tubules from the spermiogenic stage spermatids. On the other hand, expression from other post- VII onward (14), thus suggesting an involvement of the Ae2 gene meiotic genes that are normally activated at early stages of Ϫ/Ϫ in spermatogenesis. spermiogenesis was only slightly reduced in Ae2 testis. For Here we have demonstrated by gene targeting in mice the instance, this was the case of the genes for acrosin (25), Acp critical role for Ae2 in spermiogenesis and male fertility. Male (a testis-specific form of an actin-capping protein) (26, 27), and mice with a 1.5-kb deletion of Ae2 that prevents the expression Atce1 (a Creb-like peptide specifically expressed in mid/late of the three testis Ae2 variants, i.e., Ae2a, Ae2b1, and Ae2b2, are round spermatids) (28). And similar results were obtained for infertile. Histopathological analysis of Ae2Ϫ/Ϫ testes revealed the testis levels of the calspermin message, which normally that spermiogenesis is interrupted after stage VII, with a com- appear in pachytene primary spermatocytes and continue to plete absence of mature spermatozoa and occasional late sper- increase as cells complete meiosis and undergo terminal matids. Interrupted spermiogenesis is associated with an in- differentiation (29). When we analyzed the levels of the two splice variants for the germ cell soluble adenylyl cyclase creased number of apoptotic bodies. Overall, these findings Ϫ Ϫ indicate that expression of Ae2 is crucial for normal progression FL-sAC and T-sAC (8), a minor reduction was found in Ae2 / of spermiogenesis, especially when spermatids start elongation testis for both variants, mainly the T-sAC variant. T-sAC in stage VIII, and suggest that, in the absence of Ae2, germ cells variant seems to be expressed in late spermatogenesis and it reaching middle–late stages (IV–VII) fall in apoptosis. In fact, has been estimated to be responsible for 70% of the adenylyl Ϫ/Ϫ cyclase activity in the adult testis cytosol (8). The expression most of the content in the epididymis of Ae2 animals consists Ϫ Ϫ of apoptotic bodies together with a few spermatocytes and early of sAC in Ae2 / germ cells, albeit at reduced level, suggests spermatids released from the seminiferous tubules. Moreover, that it is already expressed at a stage before the arrest of the epididymal epithelium in these animals shows abnormalities differentiation. Ϫ Ϫ indicative of squamous metaplasia, suggesting that Ae2 may also The spermiogenesis disruption found in the Ae2 / mice Ϫ Ϫ have a relevant homeostatic function in epididymal cells in which resembles that observed in the Crem / mice (30, 31), includ- it is expressed (15). ing the absence of expression of Tp-1 and Prm1. Crem is a In addition to arrested spermiogenesis at middle–late stages, transcriptional factor that is phosphorylated and activated by Ae2Ϫ/Ϫ testes show a reduction in all cell populations within the the cAMP-regulated protein kinase A (32, 33). In germ cells seminiferous tubules (Table 1). Thus, the dramatic reduction in of sexually mature male rodents, cAMP levels are largely the number of round and elongated spermatids (and the absence dependent on the activity of the two splice variants of sAC (8), of spermatozoa) is associated with a moderate but significant which is directly regulated by bicarbonate ions (7, 8). Our decrease (30–40%) in the remaining early germ cell and Sertoli present data suggest that Ae2 is the postulated anion trans- cell populations, and small testis size. Although Sertoli cells seem porter required for the entry of bicarbonate in sperm cells (10). to have a normal phenotype, their reduced number may con- The possibility that the lack of Ae2 prevents the bicarbonate- tribute to the trophic testicular alterations observed in Ae2Ϫ/Ϫ controlled cAMP signaling and renders germ cells like a mice. Crem-deficient state despite their normal Crem expression Male fertility in Ae2ϩ/Ϫ mice is similar to that in wild-type warrants further studies. mice, and heterozygous mice show none of the testicular and epididymal alterations observed in Ae2Ϫ/Ϫ mice. Normal spermatogenic waves and cellular composition in the seminiferous We thank P. Garce´s for excellent technical assistance cutting tissue tubules in Ae2ϩ/Ϫ mice (Table 1) and sperm characteristics slides, F. Arenas for support with illustrations, and Drs. M. Garcıá- (Tables 2) suggest that spermiogenesis may progress correctly Granero and M. Zaratiegui for help with statistics. This work was ϩ Ϫ supported by grants from the Spanish Fondo de Investigaciones Sani- for both Ae2 and Ae2 haploid germ cells. Most probably this tarias (project 01͞0777) and from the Instituto de Salud Carlos III progression is facilitated by the occurrence of intercellular (C03͞02) (to J.F.M.) and from an agreement between Fundacioń para bridges between germinal cells derived from each single la Investigacioń Me´dica Aplicada and the Spanish ‘‘UTE for CIMA spermatogonial cell (2, 23). Intercellular bridges typically project’’ and by a grant from the Netherlands Organization for Scientific result from incomplete cytokinesis at each of the mitotic and Research (program 912-02-073) (to R.P.J.O.E.). MEDICAL SCIENCES

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