Retinoic acid regulates sex-specific timing of meiotic initiation in mice

Abstract

In mammals, meiosis is initiated at different time points in males and females, but the mechanism underlying this difference is unknown. Female germ cells begin meiosis during embryogenesis. In males, embryonic germ cells undergo G0/G1 mitotic cell cycle arrest, and meiosis begins after birth. In mice, the Stimulated by Retinoic Acid Gene 8 (Stra8) has been found to be required for the transition into meiosis in both female and male germ cells. Stra8 is expressed in embryonic ovaries just before meiotic initiation, whereas its expression in testes is first detected after birth. Here we examine the mechanism underlying the sex-specific timing of Stra8 expression and meiotic initiation in mice. Our work shows that signaling by retinoic acid (RA), an active derivative of vitamin A, is required for Stra8 expression and thereby meiotic initiation in embryonic ovaries. We also discovered that RA is sufficient to induce Stra8 expression in embryonic testes and in vitamin A-deficient adult testes in vivo. Finally, our results show that cytochrome p450 (CYP)-mediated RA metabolism prevents premature Stra8 expression in embryonic testes. Treatment with an inhibitor specific to RA-metabolizing enzymes indicates that a cytochrome p450 from the 26 family (CYP26) is responsible for delaying Stra8 expression in embryonic testes. Sex-specific regulation of RA signaling thus plays an essential role in meiotic initiation in embryonic ovaries and precludes its occurrence in embryonic testes. Because RA signaling regulates Stra8 expression in both embryonic ovaries and adult testes, this portion of the meiotic initiation pathway may be identical in both sexes.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

RAR signaling is required for Stra8 expression in embryonic ovaries. (A and B) Whole-mount in situ hybridization with Stra8 probe in gonads dissected at E11.5 and cultured for 2 days (A) in control medium or (B) with RAR panantagonist BMS-204493. (C) Whole-mount in situ hybridization with Oct4 probe in gonads dissected at E11.5 and cultured for 2 days with BMS-204493. (D–F) Photomicrographs of hematoxylin/eosin-stained sections of (D) wild-type ovary dissected at E11.5 and cultured for 4 days in control medium or (E and F) Stra8−/− ovaries cultured (E) in control medium or (F) with RA. The long arrow indicates representative cell with meiotic prophase condensation (D). We observed a few pyknotic nuclei in all samples (D–F, short arrows).

Fig. 2.

RA and agonists to all three RARs induce Stra8 expression in testes. Whole-mount in situ hybridization with Stra8 probe in gonads dissected at E12.5 and cultured for 2 days (A) in control medium, (B) with RA, (C) with RA after busulfan treatment, (D) with RAR-α agonist BMS-194753, (E) with RAR-β agonist BMS-213309, or (F) with RAR-γ agonist BMS-270394.

Fig. 3.

Quantitative RT-PCR analysis of Stra8 expression in ROL-injected or control (oil-injected) adult VAD testes compared to preinjection, contralateral testes. (Error bars indicate standard deviation; P < 0.022, paired t test, two sided.)

Fig. 4.

CYP26 activity prevents Stra8 expression in embryonic testes. (A–D and F–I) Whole-mount in situ hybridization with Stra8 probe in gonads dissected at E12.5 and cultured for 2 days in control medium (A and F), with CYP inhibitor ketoconazole (B), with ketoconazole after busulfan treatment (C), with RAR panantagonist BMS-204493 and ketoconazole (D), with CYP26 inhibitor R115866 (G), with R115866 after busulfan treatment (I), or with BMS-204493 and R115866. (E and J) In situ hybridization with Oct4 probe in gonads dissected at E12.5 and cultured for 2 days with BMS-204493 and ketoconazole (E) or with BMS-204493 and R115866 (J).

Fig. 5.

Developmental time course of Cyp26b1 expression in embryonic gonads as revealed by whole-mount in situ hybridization.

Fig. 6.

Stra8 and Cyp26b1 expressed in nonoverlapping domains in E14.5 XYZAL ovotestes. Stra8 is expressed in the polar ovarian regions. Cyp26b1 is expressed in the central testicular region.

Fig. 7.

A proposed model for regulation of meiotic initiation in embryonic gonads.