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Sex-specific timing of meiotic initiation is regulated by Cyp26b1 independent of retinoic acid signalling - PubMed

  • ️Sat Jan 01 2011

Sex-specific timing of meiotic initiation is regulated by Cyp26b1 independent of retinoic acid signalling

Sandeep Kumar et al. Nat Commun. 2011.

Abstract

Sex-specific initiation of meiosis in the fetal ovary has been suggested to require retinoic acid (RA) for induction of Stra8, with expression of the RA-degrading enzyme Cyp26b1 in fetal testis delaying meiosis until postnatal development. In this study, we investigate Raldh2(-/-) mice lacking RA synthesis and signalling in mesonephros and adjacent gonad and reveal that Stra8 expression in the fetal ovary does not require RA signalling. In contrast to previous observations, we find that Stra8 is expressed in the absence of physiologically detectable levels of RA. Ketoconazole inhibition of Cyp26b1 in Raldh2(-/-) testis allows RA-independent induction of Stra8, but only when the mesonephros remains attached, pointing to a non-RA signal from the mesonephros that induces Stra8 in the adjacent gonad. These findings demonstrate that Cyp26b1 prevents the onset of meiosis by metabolizing a substrate other than RA that controls Stra8 expression, thus changing the paradigm for how studies on Cyp26 function are conducted.

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Conflict of interest statement

Competing financial interests: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Genetic loss of RA synthesis does not affect Stra8 expression in fetal ovary

Stra8 mRNA was detected in gonads by whole-mount in situ hybridization. (a–f) E13.5 ovary/mesonephros; (a) wild-type whole-mount, (b) wild-type vibratome section, (c) Raldh2−/− whole-mount, (d) Raldh2−/− vibratome section, (e) Raldh2−/−;Raldh3−/− whole-mount (f) Raldh2−/−;Raldh3−/− vibratome section. (g, h) E12.0 ovary/mesonephros. (i, j) E12.5 ovary/mesonephros. (k, l) E14.5 ovary/mesonephros. (m–o) E13.5 testis/mesonephros. Meso, mesonephros; ov, ovary; tes, testis. n = 3 for all wild-type (WT) and Raldh2−/− (R2 −/−) specimens; n = 2 for all Raldh2−/−;Raldh3−/− (R2 −/−;R3 −/−) specimens; scale bar, 200 μm.

Figure 2
Figure 2. Germ cells in Raldh2 mutant ovary undergo meiosis

(a, b) Expression of Scp3 in E13.5 wild-type and Raldh2−/− ovaries examined by whole-mount in situ hybridization; scale bar, 200 μm. (c–e) Immunostaining for γ-H2AX in E13.5 ovary (c, d) and testis (e); arrows indicate female germ cells positive for γ-H2AX detection; scale bar, 10 μm. (f, g). Confocal images of nuclei stained with DAPI (4′,6-diamidino-2-phenylindole) in E14.5 wild-type and Raldh2−/− ovaries; arrows indicate representative germs cells in meiotic prophase; scale bar, 10 μm. Meso, mesonephros; ov, ovary. n = 3 for each specimen.

Figure 3
Figure 3. Mesonephros and gonads of Raldh mutants exhibit no RA signalling activity

Detection of RA activity in embryonic ovary/mesonephros. (a, b) RARE-lacZ expression in E12.0 mesonephros/ovary of wild-type (WT) and Raldh2−/− (R2 −/−) embryos; n = 2; scale bar, 200 μm. (c, d) RARE-lacZ expression in E13.5 mesonephros/ovary; staining in kidney from the same embryo verifies that the embryo carries RARE-lacZ; n = 3; scale bar, 200 μm. (e) Detection of RA activity by culturing E13.5 ovary and mesonephros tissues from WT, Raldh2−/− and Raldh2−/−;Raldh3−/− embryos on a monolayer of F9 RA-reporter cells followed by staining for β-galactosidase activity; this experiment was performed using multiple tissue fragments in duplicate experiments with similar results as shown here; scale bar, 50 μm. k, kidney; meso, mesonephros; ov, ovary.

Figure 4
Figure 4. Validation of RARE-lacZ transgene sensitivity to detect gonadal RA activity

RA activity detected by RARE-lacZ expression in wild-type E13.5 gonads cultured in the absence (control) or presence of a physiological dose of RA (25 or 100 nM). (a–c) Ovary/mesonephros. (d–f) Testis/mesonephros. Meso, mesonephros; ov, ovary; tes, testis. n = 4 for each condition; scale bar, 200 μm.

Figure 5
Figure 5. RA-independent induction of Stra8 in Raldh2 mutant testis

(a) Detection of RA activity by analysis of RARE-lacZ expression in testis/mesonephros of E13.5 wild-type (WT) and Raldh2−/− (R2 −/−) embryos; n = 3. (b) Intact testis/mesonephros complexes or separated testis from E13.5 wild-type (WT) or Raldh2−/− (R2 −/−) embryos were cultured in the absence (untreated) or presence of 0.7 μM ketoconazole to inhibit Cyp26b1 activity, then analysed for Stra8 mRNA; similar results were obtained in duplicate experiments. (c) RARE-lacZ staining of wild-type E13.5 testis/mesonephros cultured in the absence (untreated) or presence of 0.7 μM ketoconazole; n = 2. Meso, mesonephros; tes, testis; scale bar, 200 μm.

Figure 6
Figure 6. Comparison of RA receptor binding to the Stra8 and RARb promoters in ovary

(a) The promoter region of mouse Stra8 is displayed showing the location of a putative RARE as well as binding sites for PCR primers used for chromatin immunoprecipitation (ChIP) analysis. (b) ChIP results for Stra8 using RAR antibodies compared to input DNA (diluted 100-fold) detected with primers flanking the Stra8 RARE. (c) ChIP results for RARb detected with primers flanking its RARE. (d) Quantitation of RAR binding (% input) from the above ChIP results was performed by determining the amount of RAR-specific signal compared with input DNA for either Stra8 (open bar) or RARb (solid bar); ChIP results are from two independent experiments using different chromatin preparations (range is indicated above bars). IgG, antibody negative control; IP, immunoprecipitate; M, markers for DNA size in kilobase pairs (kb); NS, non-specific primers for region located several kb from either Stra8 or RARb RAREs; S, specific primers for RARE.

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