Fetal Estrogens are not Involved in Sex Determination But Critical for Early Ovarian Differentiation in Rabbits

Abstract

AROMATASE is encoded by the CYP19A1 gene and is the cytochrome enzyme responsible for estrogen synthesis in vertebrates. In most mammals, a peak of CYP19A1 gene expression occurs in the fetal XX gonad when sexual differentiation is initiated. To elucidate the role of this peak, we produced 3 lines of TALEN genetically edited CYP19A1 knockout (KO) rabbits that were devoid of any estradiol production. All the KO XX rabbits developed as females with aberrantly small ovaries in adulthood, an almost empty reserve of primordial follicles, and very few large antrum follicles. Ovulation never occurred. Our histological, immunohistological, and transcriptomic analyses showed that the estradiol surge in the XX fetal rabbit gonad is not essential to its determination as an ovary, or for meiosis. However, it is mandatory for the high proliferation and differentiation of both somatic and germ cells, and consequently for establishment of the ovarian reserve.

Keywords: AROMATASE; CYP19A1; TALEN; ovary; rabbit; sex determination.

Figure 1.

TALEN induced indel mutations in the rabbit CYP19A1 gene. (A) Structure of the rabbit aromatase gene, with already described tissue specific promoters (69), ie, the I.1 placental promoter at around 80 kb upstream of the transcription start point (tsp, marked by squared arrows) and the 2 proximal ovarian promoters I.r and II. The enlargement shows the sequence of exon II with the initiation site of translation (ATG in green letters). The 2 subunits (left and right arms) of the TALEN are shown as green symbols and the targeted sequence is written in red. The horizontal black arrows indicate the primers used for the PCR detection of mutants in founders (F0/R0 set) and for routine qPCR genotyping (F1/R1 set). (B) Sequences of mutant alleles: Δ339, Δ498, and Δ829. Capital letters refer to the exon sequence and lowercase letters to the intron sequence. The deletion spans 339 nucleotides (nt) in the Δ339 mutant allele (149 nt at the 3′ end of exon II and 190 nt of intron 2) and 498 nt in the Δ498 mutant allele (148 nt at the 3′ end of exon II and 350 nt of intron 2), both with the insertion of a few nucleotides at the repair position. In the Δ829 allele, the mutation consisted in the elimination of 829 nt encompassing 250 nt upstream of the start of exon II (transcription start site of the gene as regards the ovarian promoter), the totality of the second exon (263 nt) and 316 nt in the second intron. All mutations suppressed the ATG codon and the splice donor site at the 3′ extremity of exon II.

Figure 2.

Estradiol and testosterone in serum from female rabbits and in gonads from fetuses. Serum samples were collected from 2 to 14 months after birth. Fetal gonads were collected 28 days after mating. Pairs of gonads were immediately frozen at –80°C. Steroid concentrations were measured by GC/MS in wild-type XX rabbits (black points), ARO^+/– rabbits (green points) and ARO^–/– rabbits (red points). The number of serum samples analyzed from ARO^–/– females is shown in brackets in red. Note the difference of scale between male and female serum levels. Gonad contents were assayed in the pool of both gonads from 1 animal. The same samples were used to assay testosterone and estradiol.

Figure 3.

Ovaries from adult ARO^–/– rabbits were small with few follicles and filled with collagen-rich connective tissue. Ovaries from wild-type female rabbits (ARO^+/+, A and B) and from the mutant ARO^–/– line Δ339 (C and D) were fixed in PAF then HES-stained. The females were 5.5 months old. Two distinct sections of the same ovary are shown for the ARO^–/– mutant in order to reveal follicles that were otherwise not visible. Enlarged zones highlight the different types of follicles. Black arrows = Call–Exner bodies. Black stars = accumulation of fibrous tissue. Red stars = remnants of degenerated follicles. “p” = primordial follicle; I = primary follicle; II = secondary follicle; IC = interstitial cells. The phenotype of the ARO^–/– ovary was similar in the other lines, Δ498 and Δ829 (Supplementary Figure 3 (29)).

Figure 4.

RSPO2, FOXL2, and AMH were detected in wild-type (WT)and ARO^–/– adult ovaries. In situ hybridization to localize the mRNA of RSPO2 and FOXL2 genes and for the immunodetection of AROMATASE and AMH in adult ovaries. Ovaries were collected from WT or ARO^–/– Δ329 females aged 5-6 months. The expression of RSPO2 (A) and FOXL2 (B) genes was detected using ISH probes. AROMATASE (C) and AMH (D) were detected by immunohistochemistry on 2 adjacent sections. The RSPO2 and FOXL2 probes respectively labeled the cytoplasm of oocytes (brown dots) and of granulosa cells (red dots) from all follicles, from the primordial to the antrum stages in both WT and ARO^–/– rabbits. Some thecal cells were also positive for FOXL2 labeling in both genotypes. Mural granulosa cells from pre-ovulatory follicles were positively labeled by the anti-AROMATASE antibody in WT ovaries (brown colored); no cells were positive in ARO^–/– ovaries. The anti-AMH antibody labeled most granulosa cells of growing follicles in WT ovaries (brown colored), except those positive for AROMATASE. In ARO^–/– ovaries, all follicles with antrum were positively labeled. Similar staining was observed in females from the other 2 strains (Supplementary Figure 4 (29)). The graph represents AMH serum levels in WT (black points), ARO^+/– (green points) and ARO^–/– (red points) females.

Figure 5.

DMRT1 and FOXL2 immunodetection in wild-type (WT) and ARO^–/– ovaries. Ovaries were collected from WT or ARO^–/– Δ339, Δ498, and Δ829 females aged 5-6 months. Immunolabeling was performed simultaneously on adult testis as a positive control in order to detect DMRT1- and SOX9-positive cells in seminiferous tubes. Granulosa cells from a small number of large antrum follicles were positively labeled by the anti-DMRT1 antibody in ARO^–/– ovaries in lines Δ498 and Δ829 but not in Δ339, simply because of the lack of large antrum follicles; few cells were DMRT1-positive in WT ovaries.

Figure 6.

CYP19A1, FOXL2, RSPO1, WNT4, and estrogen receptor genes in wild-type 20 dpc fetal ovaries. The cellular localization of gene expression was determined using dual ISH with blue (CYP19A1, ESR1, ESR2) or red (FOXL2) labeling. Each image represents the epithelial surface of the ovary and the tissue beneath with an enlargement. Some cells were probably labeled by 2 probes. The diagrams represent mRNA fold changes relative to the lowest point of each graph. Each point represents 1 RNA sample extracted from both gonads of 1 animal at the developmental stage indicated. The same RNA samples are analyzed in the 4 diagrams. The vertical red arrow indicates birth, which occurs 31 days after conception in the rabbit. The CYP19A1, FOXL2, RSPO1 and WNT4 graphs were obtained from data published by Daniel-Carlier et al., 2013 and re analyzed to show fold changes and individual values.

Figure 7.

Morphology and detection of OCT4 and KI67 in ovaries from ARO^+/– and ARO^–/– 18 dpc old fetuses. Gonads were collected at 18 dpc from ARO^+/– and ARO^–/– fetuses, fixed in Bouin’s then HES stained (A) or fixed in PAF and then treated for antibody labeling (B and C). (A) HES staining. The surface epithelium appeared as a continuous layer of epithelial cells. Red stars indicate dense nuclei that might correspond to pyknotic nuclei. Arrows indicate figures of mitoses. (B) Detection of OCT4-positive cells. Immune-labeling was visualized using a fluorescent secondary antibody. The nuclei of positive cells (large, round, green-labeled) were dispersed throughout the gonad in both ARO^+/– and ARO^–/– ovaries. (C) Detection of KI67-positive cells. Immune labeling was visualized using a peroxidase coupled secondary antibody. Positive cells with brown colored nuclei were found at the surface epithelium, and in rows of cells delimiting cell clusters. The red double arrow indicates the thickness of the surface epithelium enriched with KI67-positive cells. Green stars point to brown light-colored large nuclei, possibly corresponding to germ cells.

Figure 8.

Gradual modifications to ARO^–/– ovaries at 20 and 22 dpc. Gonads were collected from ARO^+/– and ARO^–/– fetuses, fixed in Bouin’s then HES stained (A1, B1). Ovigerous nests and cords are highlighted by a dotted white line in A1-3, B1-3. The red double arrow indicates the thickness of the nascent cortex. Blue stars point to connective tissue. In situ hybridization was performed to visualize simultaneously 2 RNA targets to show ESR1 (blue points) and FOXL2 (fast red points) RNAs (A2, B2), and ESR1 (blue) and OCT4 (fast red) RNAs (A3, B3). The lower panel is the fluorescence observation of the fast red OCT4 ISH labeling, showing the overall density of OCT4-positive cells (A4, B4). The ESR1/FOXL2 ISH image in the control ovary is the same as that presented in Fig. 6.

Figure 9.

Mitotic activity and DNA fragmentation at 22 dpc. The marker of mitotic activity KI67 (A), and that of double-strand breaks γH2AX (B) were localized by immunohistochemistry in sections from PAF-fixed ovaries from ARO^+/– and ARO^–/– fetuses at 22 dpc. The KI67 antibody labeled the nuclei of most cells in the coelomic epithelium, and numerous cells inside the ovary in both genotypes. Cells with flat nuclei that were KI67-positive (red arrowheads) intermittently surrounded the ovigerous cords. Cells with large round nuclei that were positive for KI67 labeling (probably germ cells) were mainly found inside the cords (green stars). The red double arrow indicates the thickness of the surface epithelium enriched with KI67-positive cells. Expression of the KI67 gene did not differ in all the ovaries. The γH2AX antibody labeled large round shaped nuclei, probably corresponding to germ cells. Note the disconnection between the coelomic epithelium and the newly forming ovigerous cords in ARO^–/– ovaries. The density of γH2AX-positive cells did not differ in wild-type and ARO^–/– Ovaries.

Figure 10.

Gene expression in 20 and 22 dpc ovaries from wild-type (WT) and ARO^–/– fetuses. Ovaries were collected from WT and mutant fetuses. The diagrams represent mRNA fold changes relative to the lowest point of each graph. Each point represents 1 RNA sample extracted from both gonads of 1 animal at the developmental stage indicated. The same RNA samples are analyzed in the 6 diagrams. Red triangles refer to mutants from the Δ339 line, and red points from the Δ829 line. No samples from the Δ498 line were analyzed. The blue point in the SOX9 diagram shows the SOX9 mRNA level measured in the 2 testes of 1 20 dpc WT fetus. Horizontal bars represent the medians. The density of cells positive for OCT4 is shown, together with the diagram showing expression of the OCT4 gene.

Figure 11.

Germ cells were committed to meiosis in wild-type (WT) and ARO^–/– ovaries from 28 dpc old fetuses. Ovaries were fixed in PAF, then HES stained or analyzed by ISH. (A) Morphology of the ovaries. The images show the difference in overall size of the gonads and the different thicknesses of the cortex. The OCT4 (B) and DDX4 (C) ISH probes labeled pluripotent germ cells and differentiating germ cells, respectively. The STRA8 ISH probe (D) labeled germ cells committed to meiosis. The FOXL2 ISH probe (E) labeled differentiating somatic cells. Note that the latter were mainly distributed around and within the ovigerous cords in WT ovaries, but in contrast were dispersed throughout the gonad in the ARO^–/– rabbit. As in all ISH images, positive labeling appears as colored dots.

Figure 12.

Meiosis in wild-type (WT) and ARO^–/– ovaries. Ovaries collected at 34 dpc (3-4 days after birth) were fixed in Bouin’s then HES stained. The stages of meiosis were identified as previously published (70). (A) In WT ovaries, large ovigerous cords were filled with numerous germ cells in zygotene (a1, red arrows). Few pachytene stages were observed in the inner part of the cords (a1, blue arrows). (B) In ARO^–/– ovaries, the rare ovigerous cords were filled with a small number of germ cells in zygotene (b1). As in WT ovaries, some germ cells in pachytene were found in the inner part of the cords (b2). In both ovaries, aberrant pictures of cell nuclei were characterized by punctiform or condensed and dark colored chromatin (red stars). Blue stars indicate the connective tissue already observed, mostly in ARO^–/– ovaries.

Figure 13.

Expression of marker genes at meiosis. Germ cell marker genes (DDX4, STRA8, SPO11) and somatic cell marker genes (FOXL2, RSPO1, WNT4) were analyzed using ISH-specific probes and quantitative PCR. In wild-type ovaries, numerous germ cells positive for DDX4, STRA8 and SPO11 filled the ovigerous cords; FOXL2-positive pregranulosa cells were found around and within the cords. In ARO^–/– ovaries, germ cells positive for these same ISH probes were also found but were much less numerous; the FOXL2 labeling was very faint. The diagrams represent mRNA fold changes relative to the lowest point of each graph. Total RNA was extracted from the 2 gonads of each animal from wild-type (WT), ARO^+/– (+/-) and ARO^–/– (-/-) rabbits; the same RNA samples were analyzed in the diagrams. Horizontal bars represent the medians. As regard to the germ cell marker genes STRA8 and SPO11, ratio to DDX4 values are reported in the graphs on the right. Statistical significance (P < .01).

Figure 14.

Early follicle differentiation. Ovaries were collected at 16 or 19 dpp then treated with Bouin’s or PAF fixative and processed for HES staining or ISH labeling, respectively. (A) HES staining of 19 dpp sections. Blue stars indicate the abnormal persistence of connective tissue, devoid of differentiating oocytes or follicles, at the outer part of the cortex and in the medulla of ARO^–/– ovaries. The green arrows highlight some of the primordial follicles in formation with a discontinuous wall of granulosa cells. Early formed primordial follicles surrounded by a wall of regular shape granulosa cells were found in wild-type (WT) and ARO^–/– ovaries (white arrows). (B,C) ISH labeling using FOXL2 and RSPO2 gene specific probes. The FOXL2 and RSPO2 probes labeled granulosa cells and oocytes, respectively, in both WT and ARO^–/– ovaries. Note the huge reduction in FOXL2 positive cells in ARO^–/– ovaries when compared with WT.