Anion exchanger 2 is essential for spermiogenesis in mice

Abstract

Na+-independent anion exchangers (AE) mediate electroneutral exchange of Cl- for HCO3- ions across cell membranes, being involved in intracellular pH and cell volume regulation and in transepithelial hydroionic fluxes. Bicarbonate activation of adenylyl cyclase is known to be necessary for sperm motility and sperm capacitation, and a few studies have suggested a possible role of AE carriers in reproduction. Among the four AE genes identified in mammals thus far, only Ae2 (Slc4a2) has been determined to be expressed in the male reproductive system, especially in developing spermatozoa and in epididymal epithelium. Most AE genes drive alternative transcription, which in mouse Ae2 results in several Ae2 isoforms. Here, we generated mice carrying a targeted disruption of Ae2 that prevents the expression of the three AE2 isoforms (Ae2a, Ae2b1, and Ae2b2) normally found in mouse testes. Male Ae2-/- mice (but not female Ae2-/- mice) are infertile. Histopathological analysis of Ae2-/- testes shows an interruption of spermiogenesis, with only a few late spermatids and a complete absence of spermatozoa in the seminiferous tubules. The number of apoptotic bodies is increased in the seminiferous tubules and in the epididymis, which also shows squamous metaplasia of the epididymal epithelium. Our findings reveal an essential role of Ae2 in mouse spermiogenesis and stress the recently postulated involvement of bicarbonate in germ-cell differentiation through the bicarbonate-sensitive soluble-adenylyl-cyclase pathway.

Fig. 1.

(A) Targeting strategy for deletion/inactivation of mouse Ae2. Wild-type Ae2 locus, targeting vector, targeted Ae2 allele after homologous recombination and deleted Ae2 locus by using Cre recombinase, and upstream and downstream recombination of Ae2^targ allele were assessed by Southern blotting (ES genomic DNA cut with BspHI and probed with Bs-pro) and by PCR (primers are indicated as connected dots in the Ae2^targ diagram), respectively. (B) Initial genotyping to detect targeted mice by Southern blotting of tail genomic DNA cut with XbaI and probed with the Xb-pro. (C) PCR genotyping for routine detection of targeted mice by using primers pr1 and pr2 (represented in A) on tail genomic DNA. Bs, BspHI site; Xb, XbaI site.

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 exon3/exon4 junction were used. GAPDH mRNA was used as the normalizing control. The thick upper band in the 100-bp ladder is 600 bp long.

Fig. 3.

(A) Macroscopic appearance of testes (with their epididymes) from 20-week-old mice of indicated genotype. (B) Testicular sections from specimens shown in A, stained with hematoxylin and eosin to visualize the seminiferous tubules. (C) TUNEL for in situ detection of apoptotic cells. (D) Nissel's staining showing apoptotic cells during the process of nuclear fragmentation. Some TUNEL- and Nissel-positive cells are identified by arrows.

Fig. 4.

(A) Sections of the corpus epididymis from specimens shown in Fig. 3A, stained with hematoxylin and eosin. (B) Two-fold magnification of sections in A; a squamous metaplasia of the epididymal epithelium with a flattened appearance, and a loss of cilia are observed in Ae2^-/- mice (Right). (C) TUNEL for in situ detection of apoptotic cells within epididymis. TUNEL-positive cells are identified by arrows.

Fig. 5.

Expression of indicated mRNAs were analyzed by semiquantitative RT-PCR. All shown bands correspond to amplicons that are in the linear phase of amplification at the following number of cycles: 20 cycles for Tp-1 and Prm1; 30 cycles for acrosin, Acp, Atce1, calspermin, and sAC (both splice variants 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 using a common sense primer (in exon 9) and isoform-specific antisense primers. The sequence of the antisense primer specific for the FL-sAC isoform is within exon 11 (the spliced out exon in the T-sAC isoform), whereas that for the T-sAC isoform encompasses the junction of exons 10 (8 nt) and 12 (12 nt). In the 100-bp ladder, the thick upper band is 600 bp long.