Simultaneous gene deletion of gata4 and gata6 leads to early disruption of follicular development and germ cell loss in the murine ovary

Abstract

Granulosa cell formation and subsequent follicular assembly are important for ovarian development and function. Two members of the GATA family of transcription factors, GATA4 and GATA6, are expressed in ovarian somatic cells early in development, and their importance in adult ovarian function has been recently highlighted. In this study, we demonstrated that the embryonic loss of Gata4 and Gata6 expression within the ovary results in a strong down-regulation of genes involved in the ovarian developmental pathway (Fst and Irx3) as well as diminished expression of the pregranulosa and granulosa cell markers SPRR2 and FOXL2, respectively. Postnatal ovaries deficient in both Gata genes show impaired somatic cell proliferation and arrested follicular development at the primordial stage, where oocytes are either enclosed by one layer of squamous granulosa cells or remain in germ cell nests/clusters. Furthermore, germ cell nests and primordial follicles are predominantly localized to the central region of the Sf1Cre; Gata4(flox/flox) Gata6(flox/flox) ovaries, where the boundary between the medulla and cortex is almost nonexistent. Lastly, most of the oocytes are lost early in development in conditional double mutant ovaries, which confirms the importance of normally differentiated granulosa cells as supporting cells for oocyte survival. Thus, both GATA4 and GATA6 proteins are fundamental regulators of granulosa cell differentiation and proliferation, and consequently of proper follicular assembly during normal ovarian development and function.

Keywords: differentiation; granulosa cells; ovarian development.

FIG. 1

Gene expression analysis in E13.5 control and Sf1Cre; Gata4^flox/flox Gata6^flox/flox samples. A–J) Representative sections of wild-type control (A–E) and Sf1Cre; Gata4^flox/flox Gata6^flox/flox (F–J) ovaries at Embryonic Day 13.5 (E13.5). Ovarian sections were stained with antibodies against GATA4 (green) and GATA6 (red) (A and F); the small proline-rich protein 2 (SPRR2; red) and the pluripotent germ cell marker OCT3/4 (green) (C and H); and the cell proliferation marker bromodeoxyuridine (BrdU; green) and the nuclear receptor SF1 (red) (E and J). B, G, D, and I are higher magnifications of A, F, C, and H, respectively. B and G are visualized for GATA4 staining. Note a decrease in SPRR2 expression in the mutant ovary (H, I). Bars = 100 μm (A, C, E, F, H, J), 50 μm (D, I), and 20 μm (B, G). K–M) Gonad-mesonephros complexes from an XX wild-type control (K), XX Axin2^LacZ; Sf1Cre; Gata4^flox/flox Gata6^flox/flox (L), and XY Axin2^LacZ; Sf1Cre; Gata4^flox/+ Gata6^flox/flox (M) were stained with X-gal for LacZ expression. Note the comparable staining of the ovaries (K, L) and the lack of LacZ expression in the male gonad (M), as expected. Gonad (g); mesonephros (m); posterior side of the gonad (p). N) Gene expression analysis by qPCR of wild-type controls and Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovaries at E13.5. Genes examined included Irx3, Fst, Foxl2, Sprr2d, Mvh, Oct4, Gata4, and Gata6, and the results are shown as the means ± SEM of fold change relative to wild-type controls from at least three biological replicates with significance considered at *P < 0.05, **P < 0.01, and ***P < 0.001.

FIG. 2

Gene expression analysis in E15.5 and E18.5 control and Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovarian samples. A–N) Representative images of wild-type control (A–D, I–K) and Sf1Cre; Gata4^flox/flox Gata6^flox/flox (E–H, L–N) ovarian sections at E15.5 (A–H) and E18.5 (I–N). E15.5 sections were stained for GATA4 (green) and GATA6 (red) (A and E), for the granulosa cell marker, FOXL2 (green), and the phosphorylated histone family protein H2A (Gamma-H2AX; red) (B and F), or for the universal germ cell marker mouse vasa homolog (MVH; red) (D, H). C and G are higher magnifications of B and F, respectively, visualized for FOXL2 staining. Bars = 50 μm (A, B, E, F) and 20 μm (C, D, G, H). Ovarian sections at E18.5 were stained for GATA4 (green) and GATA6 (red) (I and L), for FOXL2 (green) and MVH (red) (J and M), or for bromodeoxyuridine to measure cell proliferation (BrdU; green) (K and N). Nuclei are stained by DAPI (blue). Note in J that germ cells are mainly localized to the cortical region in control ovaries (shown by arrows) but not in the Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovaries (arrowheads in M). Bars = 50 μm (I–N). O, P) Quantitative RT-PCR analysis of changes in gene expression in Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovaries at E15.5 (O) and E18.5 (P). Transcripts examined were Irx3, Fst, Foxl2, Mvh, Wnt4, Bmp2, and Gata4. The results are shown as the means ± SEM of fold change relative to wild-type controls from at least three biological replicates. Data were analyzed by Student t-test (two-tailed) with significance considered at *P < 0.05, **P < 0.01, and ***P < 0.001.

FIG. 3

The loss of Gata4 and Gata6 expression causes an early block in follicular development and somatic cell proliferation. A–L) Sections of wild-type control (A–F) and Sf1Cre; Gata4^flox/flox Gata6^flox/flox (G–L) ovaries at Postnatal Day 4 (PND 4). Sections were stained for GATA4 (green) and GATA6 (red) (A and G), for anti-Müllerian hormone (AMH; green) and Wilm's Tumor 1 (WT1; red) (B and H), or for bromodeoxyuridine (BrdU; green) and mouse vasa homologue (MVH; red) (C and I). Nuclei are stained by DAPI (blue). D–F and J–L are higher magnifications of A–C and G–I, respectively. P, primary follicle; PA, preantral follicle; Pr, primordial follicle. Bars = 100 μm (A–C, G–I), 50 μm (E, F, L), and 20 μm (D, J, K). M) Quantitative changes in gene expression of Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovaries. Genes examined were Amh, Figla, Fst, Foxl2, Mvh, and Gata4; the results are shown as the means ± SEM of fold change relative to wild-type controls (n = 4). N) Changes in somatic cell proliferation in Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovaries. Results are shown as the percentage of BrdU-positive cells relative to the number of DAPI-positive cells from three different females. Data were analyzed by Student t-test (two-tailed) with significance considered at *P < 0.05, **P < 0.01, and ***P < 0.001.

FIG. 4

Structural organization of control and Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovaries at PND 4. Representative sections of wild-type control (A–D) and Sf1Cre; Gata4^flox/flox Gata6^flox/flox (E–H) ovaries at Postnatal Day 4 (PND 4) stained either for spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 protein (SOHLH1; green) (A and E) or Laminin (green) (C and G). C, D, G, and H are higher magnifications of A, B, E, and F, respectively, visualized for both marker and nuclear staining (DAPI, blue). Bars = 100 μm (A, B, E, F), 50 μm (C, D, G), and 20 μm (H). Note primordial follicles in the cortical region of wild-type ovaries (arrows in A, C, and D) and clusters of germ cells in the center of Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovaries (arrowheads in G and H). Pr, primordial follicle; P, primary follicle; PA, preantral follicle; Co, cortex; Me, medulla.

FIG. 5

Increased cell death at PND 6 in conditional double mutant ovaries. Sections of wild-type control (A–F) and Sf1Cre; Gata4^flox/flox Gata6^flox/flox (G–L) ovaries at Postnatal Day 6 (PND 6). Sections were stained with hematoxylin and eosin (H&E) (A and G), for GATA4 (green) and GATA6 (red) (B and H), or for the cell proliferation marker, histone 3 (yellow) (E and K). Detection of apoptotic cells was performed by TUNEL analysis using TMR Red-conjugated dUTP to identify apoptotic nuclei (yellow) and MVH (green) to identify germ cells (C and I). D, J, F, and L are higher magnifications of A, G, C, and I, respectively. Bars = 100 μm (A, B, E, G), 50 μm (C, D, H–K), and 20 μm (F, L). Nuclei are stained by DAPI (blue).

FIG. 6

Loss of oocytes by PND 9 in the Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovaries. Sections of wild-type control (A–F) and Sf1Cre; Gata4^flox/flox Gata6^flox/flox (G–L) ovaries at Postnatal Day 9 (PND 9). Sections were stained with hematoxylin and eosin (H&E) (A and G), antibodies against GATA4 (green) and GATA6 (red) (C and I), mouse vasa homolog (MVH; red) (D and J), or FOXL2 (green) and Wilm's tumor 1 (WT1; red) (E and K). B, H, F, and L are higher magnifications of A, G, E, and K, respectively. In B and D, arrows show primordial follicles in the ovarian cortex, and in H and J, arrowheads point to the few primordial follicles remaining in the central region of the Sf1Cre; Gata4^flox/flox Gata6^flox/flox ovaries. Bars = 100 μm (A, C–E, G, J, K), 50 μm (B, I, H), and 20 μm (F, L). Pr, primordial follicle; P, primary follicle; PA, preantral follicle.