Uterine development and fertility are dependent on gene dosage of the nuclear receptor coregulator REA

Abstract

Although the effectiveness of nuclear hormone-receptor complexes is known to depend on coregulator partner proteins, relatively little is known about the roles of coregulators in uterine development and early stages of pregnancy and implantation. Because conventional genetic deletion of the coregulator, repressor of estrogen receptor activity (REA), was embryonic lethal, we here study REA conditional knockout mice generated by cre-loxP recombination, in which REA function was abrogated only in progesterone receptor-expressing tissues, to define the roles of REA in postembryonic stages and in a tissue-specific manner. We find that REA has gene dose-dependent activity impacting uterine development and fertility. Conditional homozygous mutant (REA(d/d)) mice developed to adulthood and showed normal ovarian function, but females were infertile with severely compromised uterine development and function characterized by cell cycle arrest, apoptosis, and altered adenogenesis (endometrial gland morphogenesis), resulting in failure of implantation and decidualization. By contrast, mice heterozygous for REA (REA(f/d)) had a very different phenotype, with estradiol treatment resulting in hyperstimulated, large uteri showing increased proliferation of luminal epithelial cells, and enhanced fluid imbibition associated with altered regulation of aquaporins. These REA(f/d) female mice showed a subfertility phenotype with reduced numbers and sizes of litters. These findings highlight that uterine development and regulation of estrogen receptor activities show a bimodal dependence on the gene dosage of REA. Optimal uterine development and functional activities require the normal gene dosage of REA, with partial or complete deletion resulting in hyperresponsiveness or underresponsiveness to hormone and subfertility or infertility, respectively.

Fig. 1.

Uterine growth and cell proliferation in response to E2 are enhanced in REA^f/d (heterozygous) mice and reduced in REA^d/d (homozygous) mice and are accompanied by changes in the expression of AQP genes. A, Uteri from vehicle or E2-treated REA^f/f, REA^f/d, and REA^d/d mice. Mice, 21 d of age, were injected once daily with control vehicle or E2 for 4 d, and uteri were harvested 24 h after the last injection. B, Uterine weight gain in response to E2 was monitored and normalized to each animal's body weight. C, BrdU immunohistochemistry in uteri from REA^f/f, REA^f/d, and REA^d/d mice treated with vehicle (Veh) or E2 for 4 d and injected ip with BrdU at 2 h before harvesting of uteri (magnification, ×20). Scale bar, 200 μm. D, Percentage of BrdU-positive epithelial cells/total cells monitored in at least six ×40 fields. E, mRNA levels of AQP known to be expressed in the mouse uterus were monitored by qRT-PCR in mice at age 21 d treated with control vehicle or E2 for 4 d. Uteri were harvested and RNA isolated at 24 h after the last injection of E2 or vehicle. The data are mean ± sd (n = 10 per group), and mRNA levels are illustrated as relative expression normalized to 36B4 by vehicle-treated wild type. *, P < 0.05; **, P < 0.01.

Fig. 2.

Early pregnancy events in REA^f/f and REA^d/d mice: superovulation and implantation activities. A, Female mice for each genotype at 24 d of age were subjected to a superovulatory dose of the gonadotropins PMSG and hCG. Oocytes were then collected from their oviducts and counted. B, Follicular development of the ovary was assessed by histologic examination after treatment with PMSG only for 48 h. C, Formation of corpora lutea (CL) was assessed by histologic examination after complete superovulation treatment. Note the presence of numerous mature follicles and CL (indicated as arrow) in wild-type and mutant ovaries. D, Representative uteri from REA^f/f and REA^d/d mice at 5.5 dpc. Arrows indicate sites of implantation. E, Implantation sites were visually counted by the localized retention of Chicago Blue dye. PMSG, Pregnant mare's serum goandotropin; hCG, human chorionic goandotropin.

Fig. 3.

REA^d/d mice show impaired uterine decidual response in vivo and in vitro. A, Representative gross anatomy of uteri from REA^f/f and REA^d/d mice after decidual stimulation. The left horn was stimulated, and the right horn was not stimulated (see Materials and Methods). Note the dramatic increase in size of the left horn of REA^f/f uteri after stimulation. Lower panels show REA in the stimulated REA^f/f left horn by immunohistochemistry (IHC). Note high expression of REA in the decidual zone. B, Expression of molecular markers for decidualization measured by qRT-PCR at 0 h and 48 h after stimulation. C, In vitro decidual response of uterine stromal cells. Mouse stromal cells from d-4 pregnant uteri were infected by mock or control adenovirus or different multiplicity of infection (MOI) of AdCre, and then exposed to 10 nm E2 and 1 μm P4 for times up to 96 h. REA mRNA (C) and protein levels (D) were then analyzed by qRT-PCR (24–96 h) and immunoblotting (96 h), respectively. E, Molecular markers specific to stromal cells were monitored at 96 h by qRT-PCR. Data are mean ± sd. *, P < 0.05; **, P < 0.01. Wnt4, Wingless-related MMTV integration site 4; PRP, prolactin-related protein; VDR, vitamin D receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 4.

REA^d/d mice show altered uterine growth and maturation. A, Uterine morphology was examined in hematoxylin- and eosin-stained sections from uteri of REA^f/f and REA^d/d mice at PND5, PND10, PND14, and PND21. B, REA gene is excised in the uteri of REA^d/d mice from as early as age 5 d (PND5). REA deletion was confirmed by genotyping. C, REA mRNA level was monitored by qRT-PCR in uteri from PND5, PND10, PND14, and PND21. Values are mean ± sd (n = 10 per group), and mRNA levels are illustrated as relative expression normalized to 36B4 by wild-type PND5. *, P < 0.05; **, P < 0.01. Immunohistochemical detection of REA (D) and PR from PND5, PND10, and PND14 uteri of REA^f/f and REA^d/d mice (E). Magnification is the same for D and E. Scale bar, 200 μm.

Fig. 5.

Uterine developmental gene expression is altered in REA^d/d mutant mice. A, mRNA levels of molecular markers (BMP2, Hoxa10, EGFR, and HGF) implicated in uterine development as monitored by qRT-PCR. B, The cell cycle inhibitor p21 mRNA expression levels are elevated in the uterus of REA^d/d mice, as measured by qRT-PCR. C, p21 protein levels are elevated in the REA^d/d uterus as observed by immunohistochemistry (IHC). D, Reduction of REA expression is correlated with increased p21 expression as measured by IHC (PND10 uterus). Scale bar, 200 μm. Values are mean ± sd (n = 10 per group), and mRNA levels are illustrated as relative expression normalized to 36B4 by wild-type PND5. *, P < 0.05; **, P < 0.01.

Fig. 6.

REA^d/d mice have decreased neonatal uterine cell proliferation and increased apoptosis. A, Representative histologic sections of BrdU immunohistochemistry in uteri of REA^f/f and REA^d/d d-14 mice. Note decreased BrdU incorporation in the REA^d/d uterus. Scale bar, 200 μm. B, Representative fluorescence images of TUNEL staining in uterine sections from REA^f/f and REA^d/d d-14 mice. C, Immunohistochemical detection of active-caspase-3 in uteri of REA^f/f and REA^d/d d-14 mice. Scale bar, 100 μm.

Fig. 7.

Model for the relationship between REA gene dosage and ER activity and optimal uterine function and fertility. Estrogen activity in the uterus was enhanced with reduction of REA level in REA^f/d mice, thereby allowing reduced repression of E2-driven ER activity, resulting in an increased uterine weight gain and enhanced cell proliferation and gene expression with E2 treatment. However, further depletion of REA (REA^d/d mice) does not elicit a further enhancement of E2 stimulation (long dashes), because this is associated with loss of an essential cell survival function of REA that results in cell cycle arrest and apoptosis, leading to the impairment of REA-dependent activities essential for ER action, and uterine development and function. Thus, the normal gene dosage of REA is required for optimal fertility, with hyperresponsiveness to E2 in REA^f/d mice and underresponsiveness to hormone in REA^d/d mice resulting in subfertility or infertility, respectively.