Meiosis occurs normally in the fetal ovary of mice lacking all retinoic acid receptors

Abstract

Gametes are generated through a specialized cell differentiation process, meiosis, which, in ovaries of most mammals, is initiated during fetal life. All-trans retinoic acid (ATRA) is considered as the molecular signal triggering meiosis initiation. In the present study, we analyzed female fetuses ubiquitously lacking all ATRA nuclear receptors (RAR), obtained through a tamoxifen-inducible cre recombinase-mediated gene targeting approach. Unexpectedly, mutant oocytes robustly expressed meiotic genes, including the meiotic gatekeeper STRA8. In addition, ovaries from mutant fetuses grafted into adult recipient females yielded offspring bearing null alleles for all Rar genes. Thus, our results show that RAR are fully dispensable for meiotic initiation, as well as for the production of functional oocytes. Assuming that the effects of ATRA all rely on RAR, our study goes against the current model according to which meiosis is triggered by endogenous ATRA in the developing ovary. It therefore revives the search for the meiosis-inducing substance.

Fig. 1. Expression of RARs in the female gonad and in germ cells during embryonic development.

(A and B) Detection of RARA and RARG (red signals) on frontal histological sections of an E11.5 wild-type female embryo: Expression of RARG is confined to the precartilaginous anlage of a vertebra, while that of RARA is more widespread and includes notably the undifferentiated gonad. (B′) Same section as (B) stained with H&E. (C to E) Enlargement of the box in (A): RARA (in red) is detected in the nuclei of some somatic cells of the gonad (arrowheads) and of the coelomic epithelium; in contrast, the large, rounded nuclei characteristic of germ cells (arrows) do not exhibit anti-RARA immunostaining. Nuclei are counterstained with DAPI (blue signal). Ao, aorta; CE, coelomic epithelium; Go, gonad; Me, mesonephros; SC, spinal cord; V, vertebra. Scale bar (in E), 160 μm (A to B′) and 30 μm (C to E). (F) Expression of Rara, Rarb, and Rarg determined by RNA-seq of 14,750 single germ cells isolated from gonads between E10.5 and E16.5. Smoothed expression curves of Rara, Rarb, and Rarg in male (blue lines) and female (pink lines) germ cells ordered by computed pseudotime. The red-shaded boxes indicate the time of meiosis initiation in the fetal ovary. (G and H) RT-qPCR analysis comparing the expression levels and distributions of mRNAs in single germ cells from control and mutant ovaries at E13.5 and E14.5. The violin plot width and length represent, respectively, the number of cells and the range of expression (Log2Ex). The box-and-whisker plots illustrate medians, ranges, and variabilities of the collected data. The histograms show the percentages of expressing cells in each group. Photo credits: Norbert B. Ghyselinck and Manuel Mark, IGBMC.

Fig. 2. Excision of RAR after administration of TAM at E10.5.

(A to H) Detection of RARG (red signal) on frontal histological sections at similar levels of control and mutant embryos at E11.5, namely, 24 hours after TAM administration. (A to D) RARG is strongly expressed in precartilaginous vertebrae, in periocular and corneal mesenchyme, in conotruncal ridges, and in epidermis of the control embryo. (E to H) Expression of RARG is nearly abolished in the tissues of the mutant embryo. White arrowheads indicate few nuclei that are still expressing RARG in the mutant tissues. Nuclei are counterstained with DAPI (blue signal). (I to N) ISH showing expression of Rara mRNA (I to L) and of Ppib mRNA (M and N) detected by red color staining in control and mutant embryos at E11.5. Only a few red dots indicative of Rara mRNA are observed in the mutant. In contrast, red dots indicative of the ubiquitously expressed Ppib mRNA were as numerous in mutant and control embryos. (O) Western blot analysis of protein extracts from TAM-treated E11.5 whole control and mutant embryos, using anti-RARA, anti-RARG, or anti-GAPDH antibodies. (P) PCR analysis of genomic DNA extracted from TAM-treated E11.5 control and mutant embryos used for protein extraction. This experiment proves efficient excision of the Rara and Rarg alleles in the whole embryos, as assessed by the absence of L2 alleles in the mutants. The asterisk points to unspecific DNA fragments amplified in all samples. Ao, dorsal aorta; C, cornea; CT, conotruncal ridges; D, dermis; E, epidermis; Go, gonadal ridge; L, lens; M, dorsal mesentery of the hindgut; Po, periocular mesenchyme; R, retina; SV, subcardinal vein. The dotted lines mark off the periphery of the heart outflow tract. White arrowheads indicate nuclei that are still expressing RARG in the mutant tissues. Scale bar (in H), 160 μm (A and E), 80 μm (B, C, F, G, I, J, M, and N), and 40 μm (D, H, K, and L). Photo credits: Norbert B. Ghyselinck and Manuel Mark, IGBMC.

Fig. 3. Markers of meiotic prophase I are robustly expressed at E14.5 in ovaries of mutants lacking RARs.

(A) Detection of meiotic cells expressing SYCP3 (green nuclear signal) and REC8 or STRA8 (red nuclear signals) on consecutive, 5-μm-thick, transverse histological sections at four different levels of the ovaries from control and mutant fetuses, as indicated. DDX4 (red cytoplasmic signal) is present in all germ cells. The positions of histological sections along the anteroposterior axis are indicated in terms of distance from the anterior pole of the ovary (i.e., 50, 200, 350, and 500 μm). + 5 and + 10 μm: The indicated histological sections “REC8” and “STRA8” on each line are, respectively, 5 and 10 μm apart from the indicated section “DDX4+SYCP3” that was used to establish the total number of germ cells. Nuclei are counterstained with DAPI (blue signal). Scale bar, 60 μm. (B) Average of the total number of germ cells present at the four different levels of the ovary illustrated in (A) in four control (white bars) and four mutant (gray bars) fetuses at E14.5. (C) Mean percentages of germ cells expressing SYCP3, REC8, and STRA8 in four control (white bars) and four mutant (gray bars) fetuses at E14.5. The asterisk (C) indicates a significant difference (P < 0.05). Photo credits: Norbert B. Ghyselinck and Manuel Mark, IGBMC.

Fig. 4. Evidence that gene excision has actually occurred in meiotic cells 4 days after administration of TAM.

(A to F) Detection of the meiotic markers SYCP3, REC8, or STRA8 (red nuclear signals) and of mGFP (green membranous signal) on consecutive, 5-μm-thick, transverse histological sections at two different levels of the ovary of a mutant fetus at E14.5. Efficient excision of the reporter transgene by cre/ERT² is assessed by mGFP expression in virtually all meiotic germ cells. Possible exceptions (i.e., red nuclei without a green contour) are indicated by white arrowheads. The position of histological sections along the anteroposterior axis is indicated on the left side in terms of distance from the anterior pole of the ovary (i.e., 200 and 350 μm). + 5 and + 10 μm: The indicated histological sections “REC8+mGFP” and “STRA8+mGFP” on each line are, respectively, 5 and 10 μm apart from the indicated section “SYCP3+mGFP.” Nuclei are counterstained with DAPI (blue signal). Scale bar (F), 60 μm. (G and H) PCR analysis of genomic DNA extracted from ovaries of control and mutant fetuses at E13.5, as indicated. (I) RT-qPCR analysis comparing the levels and distributions of mRNAs in single germ cells from control and mutant ovaries at E13.5 and E14.5. The violin plot width and length represent, respectively, the number of cells and the range of expression (Log2Ex). The box-and-whisker plots illustrate medians, ranges, and variabilities of the collected data. The histograms show the percentages of expressing cells in each group. NS, not significantly different; ***P < 0.001. Photo credits: Norbert B. Ghyselinck and Manuel Mark, IGBMC.

Fig. 5. Mutant fetuses generated upon TAM injection at E9.5 display a spectrum of congenital defects typically observed at E14.5 in compound RAR-knockout mutants.

Frontal histological sections at similar levels of E14.5 control and mutant female fetuses. (A and B) In mutants, the ventral portion of the retina (vR) is reduced in size in comparison to the dorsal retina (dR), the lens is rotated ventrally, and the eyelid folds (Ey) are fused together. (C and D) Mutants display mesenchymal condensations indicating the position of the salivary glands [asterisks in (D)], but their epithelial portion [SG in (C)] is absent. (E and F) In mutants, the thickness of the compact layer of the myocardium (green arrowheads) is markedly reduced in both right and left ventricles (rV and lV, respectively). (G and H) Mutants display hypoplasia of the right and left lungs (rL and lL, respectively). (I and J) Mutants have hypoplastic kidneys (K). Note that at this level of the pelvic cavity, the section does not pass through the mutant ovary, which is of normal size and located anteriorly. (K and L) Mutants lack the Müllerian duct [MD in (K)]. Sections were stained with H&E. H, heart; Oe, esophagus; Ov, ovary; PP, palatal process; SG, salivary glands; T, tongue; WD, Wolffian duct. Scale bar (in L), 160 μm (A and B), 320 μm (C to F, I, and J), 640 μm (G and H), and 60 μm (K and L). Photo credits: Norbert B. Ghyselinck and Manuel Mark, IGBMC.

Fig. 6. All germ cells have entered meiosis at E15.5 in ovaries of mutants lacking RARs.

(A and B) Detection of germ cells (DDX4 positive, red cytoplasmic signal) expressing SYCP3 (green nuclear signal): In both control (A) and mutant (B) ovaries, almost all germ cells express SYCP3 at high levels; exceptions (i.e., cells expressing SYCP3 at low to undetectable levels) are indicated by white arrowheads. (C and D) High-power magnification views of germ cells immunostained for detection of SYCP1 (red signal) and SYCP3 (green signal): Thread-like structures of SYCP1 and SYCP3 indicate the zygotene stage (Z). (E and F) Detection of SYCP1 or SYCP3 (red nuclear signals) and mGFP (green membranous signal) in the ovary of a mutant fetus also bearing the mT/mG reporter transgene. All germ cells that have experienced cre-mediated recombination (mGFP positive) contain thread-like structures of SYCP1 and SYCP3. Nuclei are counterstained with DAPI (blue signal). (G and H) Upper panels: Mice grafted with control (G) or mutant (H) E17.5 ovaries and mated with a CD1 male yielded black/agouti progenies, which were numbered #1 to #8. M1 and M2 are the recipient mothers. Lower panels: PCR analysis of genomic DNA extracted from white and black/agouti progenies as indicated. Scale bar (in B), 60 μm (A and B) and 10 μm (C to F). Photo credits: Norbert B. Ghyselinck and Manuel Mark, IGBMC.