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Divergent Roles of CYP26B1 and Endogenous Retinoic Acid in Mouse Fetal Gonads - PubMed

  • ️Tue Jan 01 2019

Divergent Roles of CYP26B1 and Endogenous Retinoic Acid in Mouse Fetal Gonads

Laura Bellutti et al. Biomolecules. 2019.

Abstract

In female mammals, germ cells enter meiosis in the fetal ovaries, while in males, meiosis is prevented until postnatal development. Retinoic acid (RA) is considered the main inducer of meiotic entry, as it stimulates Stra8 which is required for the mitotic/meiotic switch. In fetal testes, the RA-degrading enzyme CYP26B1 prevents meiosis initiation. However, the role of endogenous RA in female meiosis entry has never been demonstrated in vivo. In this study, we demonstrate that some effects of RA in mouse fetal gonads are not recapitulated by the invalidation or up-regulation of CYP26B1. In organ culture of fetal testes, RA stimulates testosterone production and inhibits Sertoli cell proliferation. In the ovaries, short-term inhibition of RA-signaling does not decrease Stra8 expression. We develop a gain-of-function model to express CYP26A1 or CYP26B1. Only CYP26B1 fully prevents STRA8 induction in female germ cells, confirming its role as part of the meiotic prevention machinery. CYP26A1, a very potent RA degrading enzyme, does not impair the formation of STRA8-positive cells, but decreases Stra8 transcription. Collectively, our data reveal that CYP26B1 has other activities apart from metabolizing RA in fetal gonads and suggest a role of endogenous RA in amplifying Stra8, rather than being the initial inducer of Stra8. These findings should reactivate the quest to identify meiotic preventing or inducing substances.

Keywords: CYP26-enzymes; electroporation; fetal gonad; meiotic entry; retinoic acid.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyzes, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1

Rar are expressed in germ and somatic cells of fetal gonads. Analysis of mRNA expression of the three Rar (Rara, Rarb, and Rarg) by RT-qPCR in purified populations of 13.5 dpc Oct4-GFP fetal gonads. Testicular (Te) cell populations: germ, interstitial and Sertoli cells; ovarian (Ov) cell populations: germ and pregranulosa cells. Values are normalized to the housekeeping gene β-Actin. mRNA expression levels are expressed as percentage of maximum (i.e., mean values for highest conditions are defined as 100%). Different letters indicate significantly different data (multiple comparisons ANOVA). Mean ± SEM, n = 3.

Figure 2
Figure 2

Retinoic acid (RA) increases Leydig cell testosterone production. Testosterone secreted (pg/h/testis) by 12.5 dpc WT (n = 7) and Rara −/− (KO Rara, n = 5) testes cultured for 1, 2, and 3 days (d1, d2, and d3, respectively) in the presence or not of 10-6M RA (CT and RA respectively); d3 testes are stimulated by LH during the last 3 h of culture (d3 + LH). * p < 0.05; ** p < 0.01 (parametric t-test) Mean ± SEM.

Figure 3
Figure 3

RA inhibits Sertoli cell proliferation. (A,B) 11.5 and 13.5 dpc WT and Rara −/− mutant (KO Rara) testes were cultured for 36 h and 48 h, respectively, in the presence or not of 10-6M RA (CT and RA). Percentage of BrdU-positive/AMH-positive Sertoli cells was measured. (C) 13.5 dpc WT and Rara −/− mutant (KO Rara) testes were cultured for 48 h in the presence or not of 1 µM of Talarazol (CT and tal). (D) Percentage of BrdU-positive/AMH-positive Sertoli cells in testes of Cyp26b1 +/+ and Cyp26b1 −/− 13.5 dpc embryos. Mean ± SEM, n = 3. ** p < 0.01 (two-way ANOVA). (E) Immunohistochemical detection of Ki67 and AMH in sections of 13.5 dpc fetal testes. Red arrows indicate Ki67-negative/AMH-positive cells; black arrows indicate Ki67-positive/AMH-positive cells. Scale bar: 10 µm.

Figure 4
Figure 4

Short-term inhibition of RA signaling does not prevent Stra8 expression. Fetal ovaries were collected at 12.2 dpc (t0) and cultured for 6 h (t6) in culture medium (CT) or in medium with 10–7 M RA or 10–6 M BMS 493. mRNA expression levels of RA-target genes (Stra8, Cyp26a1, and Rarb) were analyzed. Stra8 values were normalized to the GC specific marker Ddx4; Cyp26a1 and Rarb values were normalized to the housekeeping gene β-Actin. RNA levels are expressed as percentage of controls (i.e., t6 CT values are defined as 1) Mean ± SEM, n = 3–5. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001 unpaired t-test.

Figure 5
Figure 5

Ectopic expression of CYP26A1 does not reduce the number of STRA8-positive germ cells. (A) Schematic representation of the experimental protocol used to electroporate plasmids into 12.2 dpc fetal gonads: Intracardiac injection of plasmids diluted in PBS and trypan blue; after 30 min one gonad was electroporated (E). The contralateral gonad is used as a non-electroporated control (NE). E and NE gonads were cultured for 48 h on inserts (organotypic culture). (B) Red Fluorescence Protein (RFP) detection in ovaries (Ov) NE or E with RFP plasmid. White dotted lines encircle gonads (G). Adjacent mesonephros (M) is indicated. Scale bar: 200 µm. (C) 12.2 dpc fetal ovaries were non-electroporated (NE) or electroporated with GFP (E GFP, green), CYP26B1 (E b1, blue) or CYP26A1 (E a1, orange). After 48 h of culturing, the percentage of STRA8-positive/DDX4-positive cells is represented. GFP: n = 5; CYP26B1: n = 7; CYP26A1: n = 7. Different letters indicate significantly different data (multiple comparisons ANOVA). (D) Immunohistochemical detection of STRA8 and DDX4 in sections of fetal E or NE ovaries. Red stars indicate STRA8-negative/DDX4-positive cells; red arrows indicate STRA8-positive/DDX4-positive cells. Scale bar: 50 µm (E) mRNA expression level of Stra8. Values are normalized to Ddx4 (germ cell specific marker). Ovaries were non-electroporated (NE) or electroporated with CYP26B1 (E b1) or CYP26A1 (E a1). The RA treatment of CYP26A1-electroporated ovaries prevented the decrease of Stra8 expression. * p = 0.041; ** p = 0.0071 (paired t-test between E and contralateral NE ovaries; unpaired t-test between ovaries from different embryos) n = 5.

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References

    1. Feng C.-W., Bowles J., Koopman P. Control of mammalian germ cell entry into meiosis. Mol. Cell. Endocrinol. 2014;382:488–497. doi: 10.1016/j.mce.2013.09.026. - DOI - PubMed
    1. Guerquin M.-J., Duquenne C., Lahaye J.-B., Tourpin S., Habert R., Livera G. New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice. Dev. Boil. 2010;346:320–330. doi: 10.1016/j.ydbio.2010.08.002. - DOI - PubMed
    1. McLaren A. Primordial germ cells in the mouse. Dev. Biol. 2003;262:1–15. - PubMed
    1. Koubova J., Menke D.B., Zhou Q., Capel B., Griswold M.D., Page D.C. Retinoic acid regulates sex-specific timing of meiotic initiation in mice. PNAS. 2006;103:2474–2479. doi: 10.1073/pnas.0510813103. - DOI - PMC - PubMed
    1. E Baltus A., Menke D.B., Hu Y.-C., Goodheart M.L., E Carpenter A., De Rooij D.G., Page D.C. In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication. Nat. Genet. 2006;38:1430–1434. doi: 10.1038/ng1919. - DOI - PubMed

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