Fig 1.
Inactivation of Wdr62 causes infertility of both male and female mice.
(A) Wdr62−/− mice were viable and no obvious developmental abnormities were noted at adult stage. (B) The size of ovary from Wdr62−/− mice was dramatically reduced compared with that of control mice. (C and D) Both female and male Wdr62−/− mice were completely infertile. The ovarian follicles at different developmental stages were observed in (E, asterisks) control ovaries at 2 month of age, but not in ovaries from (F) Wdr62−/− mice. Data are presented as the mean ± SEM. ns, p > 0.05, *p < 0.05; and **p < 0.01.
Fig 2.
The expression of meiosis-related genes was dramatically reduced in germ cells from Wdr62−/− ovaries.
The expression of meiosis-related genes in germ cells from control and Wdr62-deficient ovaries was examined by immunofluorescence and real-time PCR analysis. In control ovaries, STRA8 protein (red) was detected in a few germ cells (green, white arrows) at (A) E12.5 and in most of germ cells at (B) E13.5. No STRA8 protein was expressed in germ cells (green) from Wdr62−/− ovaries at (E) E12.5 and (F) E13.5. (C, red) SYCP3 and (D, red) γH2AX proteins were detected in most of germ cells (green, white arrows) in control ovaries at E13.5. No (G) SYCP3 or (H) γH2AX proteins were detected in germ cells (green, white arrowheads) from Wdr62−/− ovaries at E13.5. (I) Quantitative analyses of meiotic germ cells in control and Wdr62−/− ovaries at E13.5. (J) The expression of meiotic genes was analyzed by RT-PCR using purified germ cells. The mRNA levels of meiotic genes were also significantly reduced in purified Wdr62−/− germ cells at E13.5, whereas the expression of Dazl and Mvh was not changed. The expression of the pluripotent genes Oct4, Nanog, Sox2 and Stella was dramatically increased in purified Wdr62-deficient germ cells. Data are presented as the mean ± SEM. ns, p > 0.05, *p < 0.05; and **p < 0.01.
Fig 3.
Stra8 and Sycp3 expression was not induced by RA treatment in germ cells from Wdr62−/− testes during the embryonic stage.
The testes from E13.5 control and Wdr62−/− embryos were cultured in vitro for 72 hours and treated with 1 μM RA. The expression of meiotic genes was examined by immunofluorescence and real-time PCR analysis. Both (A, red) STRA8 and (B, red) SYCP3 proteins were detected in germ cells (green, white arrows) from control testes. No (C) STRA8 or (D) SYCP3 proteins were detected in germ cells (green, white arrowheads) from Wdr62−/− testes. (E) Quantitative analyses of STRA8- and SYCP3-positive germ cells in RA-treated control and Wdr62−/− testes in vitro at E13.5. (F) The mRNA levels of meiotic genes were significantly increased in the control testes with RA treatment, whereas it was not induced by RA treatment in the Wdr62−/− testes. Data are presented as the mean ± SEM. ns, p > 0.05, *p < 0.05; and **p < 0.01.
Fig 4.
The Stra8 promoter was activated by Wdr62 and JNK signaling.
(A) Stra8 expression was significantly induced by Wdr62 when retinoic acid (RA) was present. (B) Stra8 promoter activity could not be activated by either the WDR62 protein WD40 or JNK binding domain combined with RA treatment. (C) The AP-1 promoter was synergistically activated by Wdr62 transfection and RA treatment. (D) Stra8 mRNA levels were significantly increased in F9 cells when JNK1 and/or JNK2 was overexpressed. (E) Wdr62 was required for RA-induced JNK signaling activation. (F-G) Relative protein levels of WDR62 and P-JNK. Data are presented as the mean ± SEM (n = 3). ns, p > 0.05, *p < 0.05; and **p < 0.01.
Fig 5.
RA-induced meiosis-associated gene expression was attenuated by JNK inhibitor treatment.
Control ovaries and testes at E13.5 and P2 were cultured in vitro and treated with 1 μM RA and/or 1 μM JNK inhibitor SP600125. The expression of meiotic genes was examined by immunofluorescence and real-time PCR. The number of (E) STRA8-positive germ cells (red, white arrowheads) in the ovaries was dramatically reduced when SP600125 was present in the culture medium compared with the (A) control group (red, white arrows). (F) The expression of SYCP3 in female germ cells was also dramatically reduced with SP600125 treatment, and meiosis was blocked during early leptotene stage (white arrowheads) compared with the (B) control group (red, white arrows). (C and D) STRA8- and SYCP3-positive germ cells (white arrows) were detected in the testes with RA treatment. (G and H) No STRA8- or SYCP3-positive germ cells (white arrowheads) were noted in the testes with combined RA and SP600125 treatment. (I and J) Quantitative analyses of STRA8- and SYCP3-positive germ cells in control and RA/SP600125-treated ovaries and testes. (K) The mRNA levels of meiotic genes were significantly decreased in SP600125-treated ovaries compared with control ovaries. (L) RA-induced meiotic gene expression in male germ cells was completely blocked by the JNK inhibitor SP600125 treatment. The number of STRA8- and SYCP3-positive germ cells in (O and P) the SP600125-treated testes was dramatically reduced compared with (M and N) the control group. (Q) Quantitative analyses of STRA8- and SYCP3-positive germ cells in control and SP600125-treated testes. (R) The mRNA level of meiotic genes was significantly decreased in SP600125-treated testes compared with control testes. Data are presented as the mean ± SEM. ns, p > 0.05, *p < 0.05; and **p < 0.01.
Fig 6.
The defects of germ cell development in Wdr62−/− females were partially rescued by JNK1 overexpression.
Numerous MVH-positive germ cells and growing follicles were observed in (A) control (arrows) and (C) rescued ovaries (arrows) at P7, but not in (B) Wdr62−/− ovaries. (D) Quantification of germ cell numbers in control, rescued and Wdr62−/− ovaries at P7. Many corpora lutea (CL) were observed in (E) 8-week control (asterisks) and (G) rescued ovaries (asterisks) primed with PMSG and hCG, but not in (F) ovaries from Wdr62−/− mice. Live pups were obtained from (H) control and (I) rescued Wdr62−/− females via superovulation and embryo transplantation. Data are presented as the mean ± SEM. ns, p > 0.05, *p < 0.05; and **p < 0.01.
Fig 7.
Mutations of WDR62 gene were detected in patients with premature ovarian insufficiency (POI).
(A) Genomic structure of WDR62 and chromatograms of two mutations identified in POI patients by Sanger sequencing. Alignment of the coding strand of WDR62 in nine eutherian mammals from Ensembl database. (B) The characteristics of WDR62 novel mutations detected in the two patients. (C)The mutations of WDR62 gene observed in POI patients play a dominant negative role in regulating Stra8 expression. The Stra8 promoter activity induced by wild-type WDR62 (WT) and WDR62 carrying the mutations detected in POI patients (M1 and M2) was analyzed by luciferase assay. The Stra8 promoter was activated by WT when RA was present, but not by mutants. The WDR62 induced Stra8 promoter activity was attenuated by co-transfection of WT and mutant expression vectors. Data are presented as the mean ± SEM. ns, p > 0.05, *p < 0.05; **p < 0.01.