Abnormal spermatogenesis and male infertility in testicular zinc finger protein Zfp318-knockout mice

Abstract

Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.

Keywords: male infertility; sperm count; spermatogenesis; testis; zinc finger.

Figure 1

Localization of Zfp318 mRNA in testis. (A, B) In situ hybridization analysis of Zfp318 mRNA localization in the testes of adult mice for a short transcript (A) and a long transcript (B). The hybridization signals (blue) are confined to the luminal compartments. (C) Higher magnification reveals that the long transcript is localized in the nuclei of pachytene spermatocytes through round spermatids. Roman numerals indicate the stage of the cycle of the mouse seminiferous epithelium. Bars: 50 μm.

Figure 2

Immunohistochemistry of ZFP318. (A) This antibody recognizes both forms of ZFP318 in extract from the normal adult testis. The long‐form ZFP318 is the major splice variant. There is no signals in extract from the Zfp318 ^−/− testis. Anti‐GAPDH‐HRP detection and CBBR (Coomassie Brilliant Blue R‐250) staining of the same membrane show as a loading control. (B) Normal adult testis immunostained with the antibody and with hemotoxylin. Roman numerals indicate the stage of the cycle of the mouse seminiferous epithelium. Bar: 50 μm.

Figure 3

Sections of the normal adult testis stained with HE (A, C and E) and the neighboring sections immunostained using an antibody against ZFP318 (B, D and F). Roman numerals reveal stage of the cycle of the mouse seminiferous epithelium. Positive signals are observed in the nuclei of spermatogonia (B and F, arrowheads), of pachytene spermatocytes (B and D, arrows) and of the secondary spermatocytes (F, arrows). Nuclei of round spermatids (B, white arrowheads) and of leptotene spermatocytes (D, arrowheads) also show weak reactions. Bar: 100 μm.

Figure 4

Zfp318 ^−/− male mice (129SvJ) display reduced body, testes, and epididymides weights and less mature spermatozoa. (A) Zfp318 ^−/− mice (open circle, n = 10) display approximately 10% reduction in body weight compared to Zfp318 ^+/+ mice (closed circle, n = 10). (B) Zfp318 ^−/− (10 weeks old) testes display ~22.3% reduction in weight (n = 10). (C) Zfp318 ^−/− (10 weeks old) epididymides display approximately 30.8% reduction in weight (n = 6). A t‐test was used for the evaluation of statistical significance. *P < 0.05, **P < 0.001. (D, E) Flow cytometric analysis of the DNA content of the whole testis (D) and epididymis (E). (D) 1C, haploid spermatids; 2C, diploid cells at the G1 phase of the cell cycle (mainly Sertoli cells and spermatogonia); 4C, cells at the G2 phase of the cell cycle (mainly primary spermatocytes). Each panel is representative of four or six animals, with the data averages stated in Results. (E) 1C (mature), mature haploid spermatids with condensed nuclear DNA; 1C, haploid spermatids; 2C, diploid cells at the G1 phase of the cell cycle (mainly epithelial cells of the epididymis and spermatogonia). Each panel is representative of three or four animals, with the data averages stated in Results.

Figure 5

Histological analysis of testes and epididymides of the Zfp318 ^−/− mouse. PAS‐hematoxylin‐stained sections of testes (A‐D) and caput epididymides (E and F). Depicted areas in A–D are enlarged in a–d, respectively. Roman numerals in A and C show the stage of the cycle of the mouse seminiferous epithelium. Seminiferous tubules in B and D were regarded as the corresponding stage of A and C, respectively. The wild‐type male shows normal testicular morphology (A and C), whereas the mutant testis shows affected spermatogenesis including vacuole formation (B, asterisk) and sparsely located elongated spermatids (D). Note the cells with the large nuclei (b and d, arrowheads) among the normal round spermatids (arrows), comparable to those of pachytene spermatocytes (b and d, white arrowheads). Accumulation of the sperm is seen in the lumen of the wild‐type epididymal duct (E). Sloughed germ cells with few sperm are observed in the mutant (F). Bars: 50 μm.