Male germ cells and photoreceptors, both dependent on close cell-cell interactions, degenerate upon ClC-2 Cl(-) channel disruption

Abstract

The functions of some CLC Cl(-) channels are evident from human diseases that result from their mutations, but the role of the broadly expressed ClC-2 Cl(-) channel is less clear. Several important functions have been attributed to ClC-2, but contrary to these expectations ClC-2-deficient mice lacked overt abnormalities except for a severe degeneration of the retina and the testes, which led to selective male infertility. Seminiferous tubules did not develop lumina and germ cells failed to complete meiosis. Beginning around puberty there was a massive death of primary spermatocytes and later also of spermatogonia. Tubules were filled with abnormal Sertoli cells, which normally express ClC-2 in patches adjacent to germ cells. In the retina, photoreceptors lacked normal outer segments and degenerated between days P10 and P30. The current across the retinal pigment epithelium was severely reduced at P36. Thus, ClC-2 disruption entails the death of two cell types which depend on supporting cells that form the blood-testes and blood-retina barriers. We propose that ClC-2 is crucial for controlling the ionic environment of these cells.

Fig. 1. Generation of ClC-2 knock-out mice. (A) Targeting construct. The sequence between the BstEII and SwaI restriction sites of a genomic clone (top) was replaced by a neomycin resistance cassette (bottom). This deletes exons 3–7 totally or partially (exons are shown in black). A diphtheria toxin α gene (dta) was used to select against random integration. (B) Southern analysis of genomic DNA from parental ES cells (left) and a correctly targeted ES cell clone (Clcn2^+/–) (right). The DNA was restricted with NheI, and the blot was probed with the NotI–HindIII fragment shown in (A). Correctly targeted cells have an additional shorter NheI fragment. (C) Northern analysis of testes mRNA from +/+, +/– and –/– mice using a 5′ (left) and a 3′ (right) ClC-2 probe. (D) Immunoblot of testes protein lysate from +/+ and –/– mice using an antibody directed against the ClC-2 C-terminus. It detects a protein of the correct size (∼100 kDa) in +/+, but not in –/– mice. An antibody against actin was used as a loading control. (E) Macroscopic male reproductive tract phenotype. Testes of adult Clcn2^–/– mice (right) are smaller than those of +/+ males (left). Epididymis and seminal vesicles are of the same size. Testes from +/– animals were not different from those of +/+ mice (not shown).

Fig. 2. Histological analysis of post-natal testes development. Semi-thin sections of testes from WT animals (A) and ClC-2^–/– mice (B) obtained 1 (a), 2 (b), 3 (c), 4 (d) and 10 weeks (e) after birth. At 2 weeks, the germinal epithelium is already disorganized in –/– mice (Bb). The normal development leading to a fully developed spermatogenesis in +/+ animals at 10 weeks (Ae) is thwarted in –/– animals. Clusters of dying spermatocytes are present at 3 and 4 weeks (Bc and Bd, arrows) and gradually disappear until the tubule is filled exclusively with abnormal Sertoli cells with long cytoplasmic extrusions. (Ca and Da) Testes sections of Clcn2 +/+ (Ca) and –/– (Da) mice (12 weeks of age) were stained for cytochrome P450, which is indicative for Leydig cells. This reveals a relative Leydig cell hyperplasia in adult knock-out mice. (Cb–Ce and Db–De) Germ cells stained by the GCNA1 antibody (Enders and May, 1994) in Clcn2^+/– (C) and Clcn2^–/– (D) animals at 2 (b), 3 (c), 4 (d) and 6 weeks (e) of age. Germ cells are gradually lost after 3 weeks of age, with only occasional GCNA1-positive cells being detected at 6 weeks (De). After 3 months, no GCNA1-positive cells could be detected (not shown).

Fig. 3. Immunolocalization of ClC-2 in the testes. (A) Histochemical staining for ClC-2 in WT mice and (B) ClC-2^–/– mice at 2 weeks after birth. (C and D) ClC-2 staining in 10-week-old WT animals; (D) was obtained at a higher magnification. ClC-2 is expressed in Sertoli cell membranes mainly in the basal third of the seminiferous tubule, also adjacent to spermatogonia that are at the cis side of the blood–testis barrier (D). Clcn2^–/– testes lack immunoreactivity.

Fig. 4. Inwardly rectifying Cl^– currents in Leydig and Sertoli cells are mediated by ClC-2. Whole-cell patch–clamp measurements were performed on purified Sertoli (A and B) or Leydig (C and D) cells from +/+ (A and C) or –/– (B and D) animals. Membrane voltages were clamped from +40 to –140 mV in steps of 20 mV. The hyper polarization-activated Cl^– current typical for ClC-2 is missing in cells from –/– animals. Similar currents were obtained from >15 cells of each cell type. The kinetics of ClC-2 currents is variable, and the difference between currents in (A) and (C) is not significant.

Fig. 5. Expression of germ cell markers in Clcn2^+/– and Clcn2^–/– mice during development. Northern analysis of testes RNA (∼10 µg total RNA per lane) using probes for RNAs encoding proacrosin binding protein (Pbp), a germ-cell-specific cyclin (Ccna1) and protamine 1 (Prm1). Total testes RNA from Clcn2^+/– (left) and Clcn2^–/– mice (right) at weeks 2, 3, 4 and 6 after birth was analyzed. The loading control GAPDH indicates that less RNA has been loaded for 4- and 6-week-old +/– animals, in part explaining the lower signals in these lanes. None of these markers is detected in –/– animals.

Fig. 6. Retinal degeneration in Clcn2^–/– mice. Retinal sections of Clcn2^–/– mice at lower magnification show the temporal pattern of the rapid loss of photoreceptor cells (A–E) between post-natal day P10 (A), P14 (B), P20 (C), P31 (D) and P65 (E). At P10, the thickness and cell number in the ONL did not differ from heterozygous animals or WT controls (not shown). A detailed view at higher magnification demonstrates the development of the inner (IS) and outer segment (OS) between P10 (F) and P18 (H) in heterozygous animals compared with Clcn2^–/– mutant mice (G and I). In Clcn2^–/– mice the OS is less developed at P10 (G) and completely absent at P18 (I), when the IS is also reduced in length. Arrows in (G) hint at dislocated photoreceptor cells beneath the RPE in Clcn2^–/– mice. Electron micrographs of this retinal zone at P10 (J and K). In Clcn2^–/– mice, the IS and OS are disorganized, the outer segments are compacted, extremely shortened and whirled (K), whereas in heterozygous controls (J) they protrude and elongate towards the RPE in regularly stacked discs. INL, inner nuclear layer. Scale bars: (A–E), 20 µm; (F–I), 10 µm; (J and K), 5 µm.

Fig. 7. Localization and function of ClC-2 in the retina and the RPE. (A–C) Expression of ClC-2 in the retina by in situ hybridization (B) and western blotting (C). Membranes from mouse retina (mRet) and mouse RPE (mRPE) were analyzed. (A) A Giemsa-stained retinal section. In (B), the ClC-2 transcript was detected in the RPE, the inner nuclear layer (INL) and the ganglia cell layer (GCL). The staining of the ONL, which comprises the nuclei of photoreceptors, was somewhat weaker. (D) Results from Ussing chamber experiments of P36 RPE preparations. The transepithelial voltage, V_te, and the equivalent short circuit current I′_sc are significantly reduced in Clcn2^–/– animals (n = 5) compared with +/– animals (n = 10); error bars indicate SEM.