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Oestrogen in the Chick Embryo Can Induce Chromosomally Male ZZ Left Gonad Epithelial Cells to Form an Ovarian Cortex That Can Support Oogenesis

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Oestrogen in the Chick Embryo Can Induce Chromosomally Male ZZ Left Gonad Epithelial Cells to Form an Ovarian Cortex That Can Support Oogenesis

Silvana Guioli et al. Development.

Abstract

In chickens, the embryonic ovary differentiates into two distinct domains before meiosis: a steroidogenic core (the female medulla), overlain by the germ cell niche (the cortex). The differentiation of the medulla is a cell-autonomous process based on chromosomal sex identity (CASI). In order to address the extent to which cortex differentiation depends on intrinsic or extrinsic factors, we generated models of gonadal intersex by mixing ZW (female) and ZZ (male) cells in gonadal chimeras, or by altering oestrogen levels of ZW and ZZ embryos. We found that CASI does not apply to the embryonic cortex. Both ZW and ZZ cells can form the cortex and this can happen independently of the phenotypic sex of the medulla as long as oestrogen is provided. We also show that the cortex-promoting activity of oestrogen signalling is mediated via estrogen receptor alpha within the left gonad epithelium. However, the presence of a medulla with an 'intersex' or male phenotype may compromise germ cell progression into meiosis, causing cortical germ cells to remain in an immature state in the embryo.

Keywords: Chicken embryo; Gonadal chimera; Oestrogen; Ovary differentiation; Sex determination.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Perturbing oestrogen levels at embryonic D7-7.5 (HH31) affects cortex formation in ZW and ZZ embryos. (A-H) Sections from left gonads at D10 (HH36) (A-D) or D17 (HH43) (E-H) double-stained for the Sertoli marker SOX9 (red) and a germ cell marker (VASA or P63; green) in ZW controls (A,E), ZZ controls (B,F), ZW gonads treated with fadrozole (ZW-Fa) (C,G) and ZZ gonads treated with β-oestradiol (ZZ-E2) (D,H). Decreasing oestrogen in ZW embryos after sex determination compromises the differentiation of the ovarian cortex; adding β-oestradiol in ZZ embryos after sex determination induces the formation of a cortex on top of a male medulla. White dotted lines highlight the cortex-medulla border.
Fig. 2.
Fig. 2.
ZW and ZZ cells can both contribute to the somatic component of the cortex domain. (Aa) Schematic illustrating the transplant procedure. D18 (HH44) gonadal chimeras were generated by transplanting posterior lateral plate/intermediate mesoderm from the left side of donor GFP embryos (green) into the left side of recipient wild-type embryos at D1.5-2 (HH10). (Ab-Ad) Whole-mount images of the resulting gonads dissected at D18 (HH44) showing the chimeric left gonad enriched in donor GFP (green) cells in ZW donor into ZW host (ZW/ZW) (Ab), ZZ donor into ZW host (ZZ/ZW) (Ac) and ZW donor into ZZ host (ZW/ZZ) (Ad). White dashed and dotted lines highlight the chimeric left gonad (ovarian and testis domain, respectively). (Ba-Bo) Immunofluorescence images of sections from the left chimeric gonads showing the germ cells using the marker VASA (blue), the female-specific steroidogenic cells using the marker P450 aromatase (AROM; red) and the GFP-positive donor cells (green) in ZW/ZW control chimera (Ba-Be), ZZ/ZW chimera (Bf-Bj) and ZW/ZZ chimera (Bk-Bo). The schematics summarise the immunostaining results (see also Fig. S3), showing the ovarian domain (pink dotted line border), the cortical germ cells nests (superficial areas delimited by black dashed lines), the testis domain (blue dotted line) and the GFP-enriched area (green). ZW/ZW (Ba-Be) and ZZ/ZW (Bf-Bj) chimeras are ovaries, and the ZW/ZZ (Bk-Bo) is an ovotestis. White arrowheads indicate a small cortical germ cell nest overlaying a patch of AROM donor cells (yellow arrowheads) in the transition area between ovary and testis. (Ca-Cf) High-resolution images of cortical areas from ZW/ZW, ZZ/ZW and ZW/ZZ chimera sections immunostained for the extracellular matrix protein Fibronectin 1 (FN; blue) and the germ cell marker P63 (grey); nuclei are counterstained with DAPI (red). GFP (green) can be found in the cortex epithelium (orange arrows) and in somatic cells close to the germ cells nests, either positive (yellow arrows) or negative (white arrows) for FN. The ZZ donor cells in the ZZ/ZW chimera (Cc,Cd) and the ZW donor cells in the ZW/ZZ chimera (Ce,Cf) can both contribute the somatic components of the cortical domain. Epithelial donor cells (orange arrows) and host cells (arrowheads) may intermingle. Dotted lines delineate cortex borders.
Fig. 3.
Fig. 3.
Induction of meiosis is compromised in the cortical germ cells of D17 embryos subject to oestrogen level alterations. (A) D17 (HH43) left gonad sections immunostained for γH2AX (green) and P63 (red); nuclei are counterstained with DAPI (blue). (B) Sections from the D17 (HH43) left gonad shown in A immunostained for γH2AX (green) and SYCP3 (red). ZW-WT, ZW wild type; ZW-Fa, ZW treated with fadrozole from D7-7.5 (HH31); ZW-E2, ZW treated with β-oestradiol at D7-7.5; ZZ-E2, ZZ treated with β-oestradiol at D7-7.5; ZW-Fa(sr), ZW, partially sex-reversed gonad, treated with fadrozole at D4; ZZ-E2(sr), ZZ, partially sex-reversed gonad, treated with β-oestradiol at D4. All gonadal models have a cortical domain containing germ cells. In ZW-Fa ovary and ZW-E2 ovary, most cortical germ cells express SYCP3 like the ZW-WT control. In ZW-Fa(sr) ovotestis, very few germ cells express γH2AX and SYCP3 (orange dotted circled areas). In ZZ-E2(sr) ovotestis and ZZ-E2 testis overlain by a cortex, some cortical germ cells express γH2AX but none expresses SYCP3. White dotted line highlights the cortical domain borders. See Fig. S4 for the medullary structure of the ZZ-E2, ZW-Fa(sr) and ZZ-E2(sr) models.
Fig. 4.
Fig. 4.
The expression pattern of mitotic-meiotic switch markers is affected in the cortical germ cells of embryos subject to oestrogen levels alteration. (A) RNA in situ analysis of STRA8 expression on sections from the left gonad of D14 (HH40) and D17 (HH43) embryos. ZW, female wild-type control; ZZ-E2, ZZ treated with β-oestradiol at D7-7.5 (HH31); ZW-E2, ZW treated with β-oestradiol at D7-7.5 (HH31); ZW-Fa, ZW treated with fadrozole at D7-7.5 (HH31). STRA8 expression is severely compromised in the gonadal cortical germ cells from ZZ embryos exposed to β-oestradiol. (Ba-Bl) Immunofluorescence detection of DMRT1 (red), LHX9 (green) and Fibronectin 1 (FN; blue) on gonadal cryostat sections from the following D17 (HH43) embryos: ZW, female wild-type control (Ba-Bc); ZZ-E2, ZZ treated with β-oestradiol at D7-7.5 (HH31) (Bd-Bf); ZZ-E2(sr), ZZ treated with β-oestradiol at D4 (HH23) (partially sex reversed) (Bg-Bi); ZW-Fa(sr), ZW treated with fadrozole at D4 (HH23) (partially sex reversed) (Bj-Bl). Right-hand panels show high-magnification images of the boxed areas on the left; (Bb,Be,Bh,Bk) LHX9, FN and DMRT1; (Bc,Bf,Bi,Bl) DMRT1 only. All samples were cut sagittally (left gonad shown) apart from ZZ-E2, which was cut transversally (left and right gonads shown). White dotted lines mark the cortex-medulla border. In the left wild-type ovary, LHX9 marks cortical somatic cells, FN highlights the cortex-medulla border cells (Guioli and Lovell-Badge, 2007) and DMRT1 marks cortical germ cells, not somatic cortical cells (LHX9 positive). In the control ZW female, only the cortical germ cells at the gonad poles are DMRT1 positive (white arrows). In ZZ-E2, ZZ-E2(sr) and ZW-Fa(sr), DMRT1 is expressed in many germ cells across the cortex.
Fig. 5.
Fig. 5.
In mixed-sex gonadal chimeras ZZ somatic cells provide an adequate niche for progression of cortical germ cell into meiosis. (A-D) Sections from chimeric left gonads at D18 (HH44) immunostained for SYCP3 (red); donor cells are GFP positive (green); nuclei are counterstained with DAPI (blue). (A) ZW donor into ZW host (ZW/ZW) control chimeric ovary; (B) ZZ donor into ZW host (ZZ/ZW); (C) ZW donor into ZZ host (ZW/ZZ); (D) high-magnification images of cortical areas (orange dashed boxes) from A-C (top to bottom, respectively). In ZW/ZW and in ZZ/ZW chimeric left ovaries, most cortical germ cells express SYCP3. In the ZW/ZZ chimeric left ovotestis, SYCP3 is expressed only in some cortical germ cells (see Fig. S3 for visualisation of the distribution of all the germ cells). The ZZ donor cortical somatic cells in the ZZ/ZW chimera provide an adequate niche for progression of germ cells into meiosis. Both ZW and ZZ cortical germ cells can progress into meiosis. However, in the ZW/ZZ ovotestis meiosis is compromised. White dotted line shows the cortex-medulla border.
Fig. 6.
Fig. 6.
In ovo suppression of epithelial ERα activity disrupts cortex differentiation. (A) Electroporation of a dominant-negative isoform of ERα (dnERα) to the gonadal epithelium. (Aa) Schematic of the inducible TET-ON plasmid system used to express dnERα (see Materials and Methods for details), LBD, ligand binding domain. (Ab,Ac) Whole-mount images of two ZW left gonads electroporated at D2.5 (HH15-17) and screened at D10 (HH36); targeted cells are highlighted by expression of an EGFP reporter (green). (Ad-Ai) Fluorescence images of D10 (HH36) gonad sections immunostained for P450 aromatase (AROM; red), or the germ cell marker VASA (red) and GFP (green); (see Fig. S7 for the EGFP pattern alone) from ZW control (Ad,Ag) and ZW electroporated (Ae,Af,Ah,Al) gonads. (Aj-Al) PITX2 RNA in situ expression pattern in ZW control (Aj) and ZW electroporated (Ak,Al) gonads. White dotted lines show the cortex/medulla border. (B) Electroporation of RCAS retroviral DNA expressing ERα shRNA (RCAS-shER1) to the gonadal epithelium. (Ba) Schematic of the construct: RCAS-shERα expressing ERα-specific short hairpin RNA molecules in tandem with an EGFP reporter. (Bb) Whole-mount image of a ZW left gonad electroporated with RC-shER1 at D2.5 (HH15-17) and screened at embryonic D9 (HH35-36) based on the EGFP reporter expression. (Bc-Bf) Fluorescence images of D9 (HH35-36) gonad sections immunostained for P450 aromatase (AROM; red) or the germ cell marker VASA (red) (see Fig. S7 for the EGFP pattern alone) from ZW control (Bc,Be) and ZW-RCAS-shER1 (Bd,Bf) gonads. The cortex is compromised by downregulation of ERα activity in both model systems compared with controls. Four out of eight samples from the two experiments showed the severe cortical phenotype in well-electroporated regions. See Fig. S8 for immunofluorescence results on sections from electroporation of various control vectors expressing an EGFP reporter (minimum of three replicates per control vector analysed).

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