Retinoic acid signaling is dispensable for somatic development and function in the mammalian ovary

Abstract

Retinoic acid (RA) is a potent inducer of cell differentiation and plays an essential role in sex-specific germ cell development in the mammalian gonad. RA is essential for male gametogenesis and hence fertility. However, RA can also disrupt sexual cell fate in somatic cells of the testis, promoting transdifferentiation of male Sertoli cells to female granulosa-like cells when the male sexual regulator Dmrt1 is absent. The feminizing ability of RA in the Dmrt1 mutant somatic testis suggests that RA might normally play a role in somatic cell differentiation or cell fate maintenance in the ovary. To test for this possibility we disrupted RA signaling in somatic cells of the early fetal ovary using three genetic strategies and one pharmaceutical approach. We found that deleting all three RA receptors (RARs) in the XX somatic gonad at the time of sex determination did not significantly affect ovarian differentiation, follicle development, or female fertility. Transcriptome analysis of adult triple mutant ovaries revealed remarkably little effect on gene expression in the absence of somatic RAR function. Likewise, deletion of three RA synthesis enzymes (Aldh1a1-3) at the time of sex determination did not masculinize the ovary. A dominant-negative RAR transgene altered granulosa cell proliferation, likely due to interference with a non-RA signaling pathway, but did not prevent granulosa cell specification and oogenesis or abolish fertility. Finally, culture of fetal XX gonads with an RAR antagonist blocked germ cell meiotic initiation but did not disrupt sex-biased gene expression. We conclude that RA signaling, although crucial in the ovary for meiotic initiation, is not required for granulosa cell specification, differentiation, or reproductive function.

Keywords: DMRT1; Granulosa; Ovary; Retinoic acid; Sertoli.

Figure 1. Efficient conditional deletion of Rar genes

A. Disruption of Sertoli cell gene expression and spermatogenesis in conditional triple-mutant testes. Left panels: section immunofluorescence (IF) of GATA1 in testes from two-month-old Rar triple conditional mutants and controls lacking Sf1-Cre. Right panels: hematoxylin/eosin (H&E) staining of testes from six-month-old triple conditional mutants and controls. Stars indicate GATA1-negative tubule sections, which are much less frequent in mutant testes. Scale bars: 80 microns. B. RNA-seq data from five adult Rar triple conditional mutants (+Cre) and five control ovaries (−Cre). Graph shows number of sequence reads for floxed (indicated with *) and flanking exons in control and conditional mutant ovaries, normalized to total mapped reads for each sample.

Figure 2. Normal morphology and sexual cell fates in Rar triple conditional mutant ovaries

A. Immunofluorescent (IF) staining of FOXL2 and SOX9 in sections of control and triple conditional mutant adult ovaries. Green background is nonspecific antibody staining. Scale bars: 80 microns. B. Principle component analysis (PCA) comparing control and Rar triple conditional mutant ovaries from this study to testes and extensively masculinized DMRT1-expressing ovaries (data from Lindeman et al. (2015). C. Scatter plot showing mRNA expression changes from RNA-seq of adult triple conditional mutant ovaries compared to controls lacking the Sf1-Cre transgene. Red points indicate significant changes (P<0.05, 2-fold or greater expression change), and genes affected are listed in Table 1. D. Scatter plot showing mRNA expression changes from RNA-seq of adult DMRT1-expressing masculinized ovaries compared to transgenic controls lacking Cre. Data for ectopic DMRT1 expression are from Lindeman et al., 2015.

Figure 3. Efficient depletion of Aldh1a1-3 mRNAs at E12.5

Quantitative RT-PCR analysis of Aldh1a1, Aldh1a2 and Aldh1a3 expression in 12.5 days post coitum (12.5 dpc) Aldh1,2,3^flox/flox (red) and CGAG-Cre^tg/+; Aldh1,2,3^flox/flox (blue) ovaries, using Sdha as the normalization control. Bars represent mean +SEM, n = 6 individual ovaries (Cre−) and 18 individual ovaries (Cre+). *** p<0.001.

Figure 4. Efficient depletion of ALDH1A1 and ALDH1A2 in ovaries and mesonephroi of XX conditional mutants

Section IF of XY, XX controls and XX Cag-CreERT2^Tg/+; Aldh1a1-3^fl/fl gonads at E13.5. ALDH1A1 and ALDH1A2 (red), germ cell marker CDH1 (green) and DNA marker DAPI (blue). Scale bars: 50 microns.

Figure 5. Loss of retinaldehyde dehydrogenase genes Aldh1a1-3 in XX gonads and mesonephros does not affect ovarian differentiation

Section IF of XY, XX control and XX Ccag-CreER^Tg/+; Aldh1a1-3^fl/fl gonads at E13.5. Upper panel: SOX9 (red) and germ cell marker TRA98 (green). Lower panel: granulosa cell marker FOXL2 (red) and TRA98 (green). Both panels: DNA marker DAPI (blue). Scale bars: 50 microns.

Figure 6. Expression of dominant-negative RARa in somatic ovaries does not masculinize granulosa cells

Composites images of ovary section IF for FOXL2 (red) and SOX9 (green) with DNA marker DAPI (blue). A. Control ovary containing conditional dnRara transgene but lacking Cre. B. Experimental ovary carrying two copies of dnRAR^fl/fl transgene activated by Sf1-Cre, showing presence of full range of follicle stages as well as large cystic follicles. Scale bars: 100 microns.

Figure 7. BMS treatment blocks meiotic initiation in cultured fetal ovaries

A. Immunofluorescence images of wild type E13.5 ovaries and control and BMS-treated ovaries grown in culture for 72 hours starting at E11.5. GATA4 marks somatic gonadal tissue, TRA98 identifies germ cells, and STRA8 indicates meiotic cells. Scale bars: 20 microns. B. Proportion of meiotic germ cells relative to total number of germ cells in two pairs of fetal ovaries cultured with or without BMS for 72 hours from E11.5. Germ cell counts performed on 5um sections through entire ovary. Dark blue (“Meiotic”) indicates germ cells (TRA98+) strongly expressing STRA8, lighter blue represents germ cells lacking STRA8 (“Non-meiotic”). Light blue (“Ambiguous”) refers to germ cells possibly entering meiosis on the basis of STRA8 expression but lacking strong nuclear STRA8 localization. Number of germ cells scored: animal A, 14 in control ovary and 29 in BMS treated ovary; animal B, 26 in control and 23 in treated.

Figure 8. Fetal sex differentiation is not blocked by RAR antagonist treatment

RT-qPCR of early granulosa and Sertoli markers from gonads dissected at E10.5 and either assayed immediately (t=0) or after 72 hours in culture (t=72). One gonad of each pair was cultured in media with BMS and the other with vehicle. RT-qPCR was performed for the following early markers of somatic ovary differentiation: Foxl2 (A), Irx3 (B), Fst (C), Bmp2 (D), Lypd6 (E); and markers of somatic testis differentiation: Amh (F), Cst9 (G), Gdnf (H), Cyp17a1 (I), Dhh (J). RT-qPCR was performed in triplicate for each condition on RNA isolated from pools of 12–14 gonads. Shown is mean fold-change +/− SD relative to one cultured control testis pool (A–E) and one cultured control ovary pool (F–J). Samples were normalized to Gata4 expression. Primer sequences are listed in Table 2.