GATA4 deficiency impairs ovarian function in adult mice

Abstract

Transcription factor GATA4 is expressed in granulosa cells and, to a lesser extent, in other ovarian cell types. Studies of mutant mice have shown that interactions between GATA4 and its cofactor, ZFPM2 (also termed FOG2), are required for proper development of the fetal ovary. The role of GATA4 in postnatal ovarian function, however, has remained unclear, in part because of prenatal lethality of homozygous mutations in the Gata4 gene in mice. To circumvent this limitation, we studied ovarian function in two genetically engineered mouse lines: C57BL/6 (B6) female mice heterozygous for a Gata4-null allele, and 129;B6 female mice in which Gata4 is deleted specifically in proliferating granulosa cells using the Cre-loxP recombination system and Amhr2-cre. Female B6 Gata4(+/-) mice had delayed puberty but normal estrous cycle lengths and litter size. Compared to wild-type mice, the ovaries of gonadotropin-stimulated B6 Gata4(+/-) mice were significantly smaller, released fewer oocytes, produced less estrogen, and expressed less mRNA for the putative GATA4 target genes Star, Cyp11a1, and Cyp19. Gata4 conditional knockout (cKO) mice had a more severe phenotype, including impaired fertility and cystic ovarian changes. Like Gata4(+/-) mice, the ovaries of gonadotropin-stimulated cKO mice released fewer oocytes and expressed less Cyp19 than those of control mice. Our findings, coupled with those of other investigators, support the premise that GATA4 is a key transcriptional regulator of ovarian somatic cell function in both fetal and adult mice.

FIG. 1.

Delayed puberty in B6 Gata^+/− female mice. A) Representative estrous cycles from WT and Gata^+/− mice. B) Average ages for first vaginal cornification and the onset of cyclicity. Note the significant delay in the onset of cyclicity in Gata^+/− mice. C) Proportion of time spent in each estrous stage (n = 10 for each genotype). Error bars represent SD. D, diestrus; E, estrus; M, metestrus; P, proestrus.

FIG. 2.

Ostensibly normal follicular development in the ovaries of randomly cycling adult Gata^+/− mice. A–C) Ovaries from 3-mo-old WT (A and B) or Gata4^+/− (C) mice were subjected to immunoperoxidase staining to assess ovarian morphology and to visualize the distribution of GATA4 within this tissue. Note that GATA4 is expressed in granulosa and theca cells associated with primordial, preantral, and antral follicles of both WT and Gata4^+/− mice. Bar = 75 μm. D and E) Histomorphometric analysis of follicle development in WT (n = 10) and Gata4^+/− (n = 6) mice. Serial ovarian sections from 7-wk-old WT and Gata4^+/− mice were processed, and the numbers of healthy (nonatretic; D) or atretic (E) follicles were estimated. Error bars represent SD. AnF, antral follicles; CL, corpora lutea; PF, primordial and primary follicles; PrF, preantral follicles.

FIG. 3.

Impaired ovarian response to gonadotropins in Gata^+/− mice. A) Weanling WT (solid line) or Gata^+/− (dashed line) mice were treated with one of the following regimens: 1) no stimulation; 2) eCG for 48 h; 3) eCG for 48 h, followed by hCG for 18 h; or 4) eCG for 48 h, followed by hCG for 5 days. Ovaries were harvested and weighed (n = 8 for each genotype). *P < 0.05. Error bars represent SD. B and C) Tissue sections of WT (B) and Gata^+/− (C) ovaries 48 h after eCG stimulation. The Gata^+/− ovary is smaller and contains fewer antral follicles. Bar = 0.3 mm. D) Oocyte yields after gonadotropin-induced ovulation. WT and Gata^+/− mice were treated with eCG, followed 48 h later by hCG. Oocytes were harvested from oviducts and counted 16 h after hCG treatment. Each data point represents the oocyte yield of an individual mouse. Horizontal lines represent mean values. E and F) Sections of WT (E) and Gata^+/− (F) uteri 48 h after eCG stimulation. Note the hypoestrogenic appearance of the heterozygous uterus. Glandular elements of the endometrial layer are less complex, and only scattered glands are detected in the stroma. No difference was found in mean body weight between the WT and Gata^+/− mice (13.8 ± 1.6 and 14.4 ± 1.6 g, respectively). Bar = 0.3 mm. G) Serum E₂ levels in WT (white bar, n = 7) and Gata^+/− (black bar, n = 7) uteri 48 h after eCG stimulation. *P < 0.05. Error bars represent SD.

FIG. 4.

Reduced expression of mRNA for Gata4 and steroidogenic factors in the ovaries of gonadotropin-stimulated Gata^+/− mice. Weanling WT (solid line, n = 5) or Gata^+/− (dashed line, n = 5) mice were treated with one of the following regimens: 1) no stimulation; 2) eCG for 48 h; 3) eCG for 48 h, followed by hCG for 18 h; or 4) eCG for 48 h, followed by hCG for 5 days. Ovarian RNA was subjected to qRT-PCR for Gata4 (A), Star (B), Cyp11a1 (C), and Cyp19 (D). Error bars represent SD. *P < 0.05, **P < 0.01.

FIG. 5.

Gata4 mRNA is not expressed in the müllerian duct or adult mouse uterus. Cryosections of Embryonic Day 18.5 mouse embryo (A and B) and Embryonic Day 9.5 gravid uterus (C and D) were subjected to in situ hybridization for GATA4 using radiolabeled riboprobe. Shown are corresponding bright-field (A and C) and dark-field (B and D) photomicrographs. Note that Gata4 mRNA is expressed in fetal ovary (ov) and in endodermal cells of the small intestine (si) and yolk sac (ys), but not in the müllerian duct (md), embryonic ectoderm (em), decidua (de), or uterus (ut). Bar = 100 μm.

FIG. 6.

Amhr2-cre-mediated deletion of Gata4^Flox in the mouse ovary. A) Amhr2^cre/+ mice were crossed with homozygous R26R mice to generate R26R^+/−;Amhr2^cre/+ mice. Ovaries were cryosectioned and stained with X-gal. Note the variegated pattern of Cre activity in a secondary follicle; both lacZ-positive (arrow) and lacZ-negative (arrowhead) granulosa cells are evident. B) Ovarian RNA was isolated from pairs of 2-mo-old control (Gata4^Flox/Flox;Amhr2^+/+) or cKO mice and subjected to RT-PCR analysis with primers that distinguish the intact floxed allele from the recombined allele lacking exons 3–5 (Gata4^Δex3–5). Note that a transcript derived from recombined allele is seen in the cKO mice but not the control mice. The identity the third band in the cKO mice is unclear. C and D) Ovaries from 7-wk-old Gata4^Flox/+;Amhr2^cre/+ (C) or cKO (D) mice were sectioned and subjected to immunoperoxidase staining to visualize the distribution of GATA4 within this tissue. GATA4 immunoreactivity was seen in granulosa and theca cells of both the Gata4^Flox/+;Amhr2^cre/+ and cKO mice. Bar = 75 μm. AAnF, atretic antral follicles; AnF, antral follicles; PF, primordial and primary follicles; PrF, preantral follicles.

FIG. 7.

Morphological and functional abnormalities in the ovaries of cKO mice. A and B) Hematoxylin-and-eosin-stained sections of ovaries from 7-wk-old Gata4^Flox/+;Amhr2^cre/+ and cKO mice, respectively. Bar = 200 μm. C and D) Histomorphometric analysis of follicle development in 7-wk-old Gata4^Flox/+;Amhr2^cre/+ (n = 10) and cKO (n = 6) mice ovaries. Serial ovarian sections were processed, and the numbers of healthy (nonatretic; D) or atretic (E) follicles were estimated. E–H) Ovaries from 6-mo-old Gata4^Flox/+;Amhr2^cre/+ (E and G) and cKO (F and H) mice. The arrowheads and asterisk highlight large cysts in the cKO ovary. Bar = 200 μm. I) Serum AMH levels in Gata4^Flox/+;Amhr2^cre/+ (n = 3–7) and cKO (n = 5–7) mice of varying ages. Error bars represent SD. The bars denoted by a differ significantly (P < 0.01) from that denoted by b but not from one another.

FIG. 8.

Aberrant response to superovulation in cKO mice. Immature mice were treated with eCG, followed 48 h later by hCG. Tissue samples were harvested for analysis 18 h after hCG treatment. A and B) Ovaries from eCG/hCG-treated (A) Gata4^Flox/+;Amhr2^cre/+ and (B) cKO mice. Note the presence of hemorrhagic follicles (arrowheads) in the cKO ovary. C) Oocyte yields after superovulation. Each data point represents the oocyte yield of an individual mouse. The horizontal lines represent the mean values. The sample sizes were Gata4^Flox/+;Amhr2^cre/+ (n = 11) and cKO (n = 13).

FIG. 9.

Reduced expression of mRNA for Gata4 and steroidogenic factors in the ovaries of gonadotropin-stimulated cKO mice. Weanling Gata4^Flox/+;Amhr2^cre/+ (solid line, n = 5) or cKO (dashed line, n = 5) mice were treated with one of the following regimens: 1) no stimulation; 2) eCG for 48 h; 3) eCG for 48 h, followed by hCG for 18 h; or 4) eCG for 48 h, followed by hCG for 5 days. Ovarian RNA was subjected to qRT-PCR for Gata4 (A), Star (B), Cyp11a1 (C), and Cyp19 (D). Error bars represent SD. *P < 0.05, **P < 0.01.