Ovol2, a zinc finger transcription factor, is dispensable for spermatogenesis in mice.

Ovol2, a mouse homolog of Drosophila ovo, was identified as a zinc finger transcription factor predominantly expressed in testis. However, the function of Ovol2 in postnatal male germ cell development remains enigmatic. Here, we firstly examined the mRNA and protein levels of Ovol2 in developing mouse testes by RT-qPCR and western blot and found that both mRNA and protein of Ovol2 are continually expressed in postnatal developing testes from postnatal day 0 (P0) testes to adult testes (P56) and exhibits its higher level at adult testis. Further testicular immuno-staining revealed that OVOL2 is highly expressed in the spermatogonia, spermatocytes and round spermatids. Interestingly, our conditional ovol2 knockout mouse model show that loss of ovol2 in embryonic germ cells does not affect fecundity in mice. Our data also show that Ovol1 may have compensated for the loss of Ovol2 functions in germ cells. Overall, our data indicate that ovol2 is dispensable for germ cell development and spermatogenesis.

Main text

Ovol2, a mouse homolog of Drosophila ovo, was identified as a zinc finger transcription factor predominantly expressed in testis [1]. Previous studies revealed that Ovol2 exhibits its functions in keratinocyte transient proliferation and differentiation [2], mouse embryonic stem cells differentiation [3] and primordial germ cell development [4]. However, the function of Ovol2 in postnatal male germ cell development remains enigmatic. Thus, we firstly examined the mRNA and protein levels of Ovol2 in multiple adult mouse tissues by RT-qPCR and western blot. We found that both the mRNA and protein of Ovol2 are highly expressed in testis and lung (Fig. 1a-c). We then examined the expression levels of Ovol2 in postnatal developing testes and found that both mRNA and protein of Ovol2 are continually expressed in postnatal developing testes from postnatal day 0 (P0) testes to adult testes (P56) and exhibits its highest level at adult testis (Fig. 1d-f). Further testicular immuno-staining revealed that OVOL2 is highly expressed in the germ cells (spermatogonia, spermatocytes and round spermatids) (Fig. 1g and Additional file 1: Figure S1A-D). Thus we hypothesized that Ovol2 could play an important role in postnatal germ cell development and spermatogenesis.

Fig. 1

Ovol2 is expressed in spermatogenic cells in mice. a RT-qPCR analyses of Ovol2 mRNA levels in nine organs of adult mice. Data are presented as mean ± SEM, n = 5. b Western blot analyses of OVOL2 protein levels in nine organs of adult mice. GAPDH served as a loading control. c Quantification analyses of OVOL2 protein levels in nine organs. Data are presented as mean ± SEM, n = 3. d RT-qPCR analyses of Ovol2 mRNA levels in developing testes. Testes at postnatal Day 0 (P0), P7, P14, P21, P28, P35, and P56 were analyzed. Data are presented as mean ± SEM, n = 5. e Western blot analyses of OVOL2 protein levels in developing testes. Testes at postnatal Day 0 (P0), P7, P14, P21, P28, P35, P42 and P56 were analyzed. GAPDH served as a loading control. f Quantification analyses of OVOL2 protein levels in developing testes. Data are presented as mean ± SEM, n = 3. g Immunofluorescence showing the localization of OVOL2 in P21 and adult testis sections. Note: Spg, spermatogonia; Spc, spermatocytes; Rds, round spermatids; eLg, elongated spermatids; Ser, sertoli cells; Ley, leydig cells. Scale bar = 50 μm

Due to Ovol2 conventional mutant mice displayed an embryonic mortality [4], we tried to specific knockout of Ovol2 in mouse germ cells to determine the physiological roles of Ovol2 in germ cell development. We then generated Ovol2 conditional knockout mouse model by crossing Ovol2-floxed mice with Vasa-cre (Cre was specifically activated at embryonic day 15.5) to inactive Ovol2 gene in testicular germ cells (Fig. 2a). The genotype of Ovol2 germ cell-specific knockout mice (Vasa-Cre; Ovol2, hereafter called Vasa-cKO) was confirmed by PCR-based genotyping analyses (Fig. 2b). In addition to genotyping analyses, both mRNA and proteins of Ovol2 were appeared to be significantly reduced in Vasa-cKO testes compared with that of WT controls by RT-qPCR, Western blot and Immunofluorescence analyses (Fig. 2c-e and Additional file 1: Figure S1E). Therefore, these data suggest that Ovol2 was specifically inactivated in testes efficiently.

Fig. 2

Ovol2 is dispensable for spermatogenesis in mice. a Schematic illustration of the targeting strategy for generating a complete inactivation of Ovol2 in mouse testes. Mice containing the floxed allele were crossed with Vasa-Cre mouse lines to generate male germ cell-specific deletion of Ovol2 (Vasa-Cre was activated at embryonic day 15.5, E15.5). P1/2 indicate the position of primers used for detection of floxed and WT alleles. P3/4 indicate the position of primers used for detection of delete allele after Cre recombination. b Representative PCR genotyping results showing that the floxed (lox) and the WT (+) alleles can be detected as larger (223 bp) and a shorter (125 bp) bands, respectively. The last two right lanes are the Vasa-Cre transgene detection. M, marker; NC, negative control. c RT-qPCR analyses of Ovol2 mRNA levels in WT and cKO (Vasa-cre; Ovol2) adult testes. Wt1 gene was used as RNA quality control. Data are presented as mean ± SEM, n = 6. **P < 0.01 by student t-test. d Representative Western blot analysis of OVOL2 protein levels in WT and cKO (Vasa-cre; Ovol2) adult testes. GAPDH served as a loading control. e Quantification analysis of OVOL2 protein levels in WT and cKO adult testes. Data are presented as mean ± SEM, n = 6. *P < 0.01 by student t-test. f Gross morphology of the testis and the epididymis in WT and cKO adult mice. g Histogram showing the testis weights in WT and cKO adult mice. Data are presented as mean ± SEM, n = 6. h Periodic acid-Schiff (PAS) staining showing the histology of testes from adult WT and cKO mice. Scale bar = 50 μm. i RT-qPCR assays showing elevated Ovol1 mRNA levels in cKO adult testes. Data are presented as mean ± SEM, n = 6. *P < 0.05 by student t-test. j Representative Western blot analysis of OVOL1 protein levels in WT and cKO adult testes. GAPDH served as a loading control. k Quantification analysis of OVOL1 protein levels in WT and cKO adult testes. Data are presented as mean ± SEM, n = 6. *P < 0.05 by student t-test

To investigate the fertility of Vasa-cKO mice, we bred the Vasa-cKO males with fertility-proven WT females for at least 5 months. Unexpectedly, Vasa-cKO breeding pairs can produce comparable litter size to WT breeding pairs (data not shown), which indicated that Vasa-cKO male mice are completely fertile. Consistent with this fertile phenotype, testis gross morphology and weights are comparable between Vasa-cKO and WT control mice (Fig. 2f-g). Histological analyses further revealed that Vasa-cKO testes display normal spermatogenesis (Fig. 2h). To further confirm the Vasa-Cre recombined deletion efficiency in DNA level, we detected the delete allele of Ovol2 in the offspring derived from Vasa-cKO male breeding pairs by PCR-based DNA analyses. As we expected, all of pups are contained Ovol2 delete allele (Additional file 1: Figure S2). Together, these data indicate that Ovol2 is not essential for spermatogenesis and male germ cell development in mice despite its high expression in testis.

Ovol1, another Drosophila ovo mouse homologue, was confirmed to express in overlapping tissues with Ovol2, such as skin, kidney and testis. Ablation of Ovol1 in mice led to abnormal male germ cell development and male infertility, and Ovol1 is essential for spermatogenesis [5]. Thus, we analyzed the expression levels of Ovol1 in adult WT and Vasa-cKO testes by RT-qPCR. Interestingly, both Ovol1 mRNA and protein levels were significantly increased in Vasa-cKO testes compared with those of WT testes (Fig. 2i-k). Therefore, these data suggest that Ovol1 may have compensated for the loss of Ovol2 functions in germ cells, which leads to normal phenotype in Vasa-Cre;Ovol2 mice. However, it is worthwhile pointing out that other transcription factors need to be elucidated in future, which may contribute to the compensation of OVOL2 loss-of -function in male germ cells. Overall, in this study, we report that Ovol2 is dispensable for testicular germ cell development and spermatogenesis in mice, and provide a molecular therapeutic clue for human male infertility caused by genetic mutation.

Conclusion

Both mRNA and protein of Ovol2 are continually expressed in postnatal developing testes from postnatal day 0 (P0) testes to adult testes (P56) and exhibits its highest level at adult testis. Ovol2 is dispensable for testicular germ cell development and spermatogenesis in mice. Ovol1 may have compensated for the loss of Ovol2 functions in germ cells, which leads to normal phenotype in Ovol2 conditional mutation mice.

Additional file 1: Figure S1: The localization of OVOL2 in mouse testicular sections was revealed by immunofluorescence. (A) Co-­-immunofluorescent staining for OVOL2 and WT1 (a Sertoli cell marker) antibodies on P21 WT testicular section showing OVOL2 expressed in Sertoli cells. Nuclei were stained with DAPI. (B) Co-­-immunofluorescent staining for OVOL2 and γ-­-H2A.X antibodies on P21 testicular section showing OVOL2 expressed in pachytene spermatocytes. Nuclei were stained with DAPI. (C) Co-­- immunofluorescent staining for OVOL2 and γ-­-H2A.X antibodies on P56 testicular section showing OVOL2 expressed in round spermatids. Nuclei were stained with DAPI. (D) Co-­-immunofluorescent staining for OVOL2 and γ-­-H2A.X antibodies on P56 testicular section showing OVOL2 expressed in elongating spermatids. Nuclei were stained with DAPI. (E) Co-­-immunofluorescent staining for OVOL2 and γ-­-H2A.X antibodies on WT and Vasa-­-cKO (Vasa-­-cre; Ovol2lox/lox) testis sections at adulthood. Nuclei were stained with DAPI. Arrowheads indicate Sertoli cell; Arrows indicate Leydig cells. Scale bar bar = 50 μm. Figure S2. PCR based genotyping of the offspring derived from the Vasa-­-cKO (Vasa-­- cre; Ovol2lox/del) male breeding pairs. All pups are contains delete allele.