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. 2017 Jul 1;158(7):2330-2343.
doi: 10.1210/en.2016-1916.

Defining the Role of Estrogen Receptor β in the Regulation of Female Fertility

M A Karim Rumi  1   2 , Prabhakar Singh  1   2 , Katherine F Roby  1   3 , Xiao Zhao  1   2 , Khursheed Iqbal  1   2 , Anamika Ratri  1   2 , Tianhua Lei  1   2 , Wei Cui  1   2   3 , Shaon Borosha  1   4 , Pramod Dhakal  1   2 , Kaiyu Kubota  1   2 , Damayanti Chakraborty  1   2 , Jay L Vivian  1   2 , Michael W Wolfe  1   4 , Michael J Soares  1   2   5
Affiliations

Defining the Role of Estrogen Receptor β in the Regulation of Female Fertility

M A Karim Rumi et al. Endocrinology. .

Abstract

Estrogens are essential hormones for the regulation of fertility. Cellular responses to estrogens are mediated by estrogen receptor α (ESR1) and estrogen receptor β (ESR2). In mouse and rat models, disruption of Esr1 causes infertility in both males and females. However, the role of ESR2 in reproductive function remains undecided because of a wide variation in phenotypic observations among Esr2-mutant mouse strains. Regulatory pathways independent of ESR2 binding to its cognate DNA response element have also been implicated in ESR2 signaling. To clarify the regulatory roles of ESR2, we generated two mutant rat models: one with a null mutation (exon 3 deletion, Esr2ΔE3) and the other with an inframe deletion selectively disrupting the DNA binding domain (exon 4 deletion, Esr2ΔE4). In both models, we observed that ESR2-mutant males were fertile. ESR2-mutant females exhibited regular estrous cycles and could be inseminated by wild-type (WT) males but did not become pregnant or pseudopregnant. Esr2-mutant ovaries were small and differed from WT ovaries by their absence of corpora lutea, despite the presence of follicles at various stages of development. Esr2ΔE3- and Esr2ΔE4-mutant females exhibited attenuated preovulatory gonadotropin surges and did not ovulate in response to a gonadotropin regimen effective in WT rats. Similarities of reproductive deficits in Esr2ΔE3 and Esr2ΔE4 mutants suggest that DNA binding-dependent transcriptional function of ESR2 is critical for preovulatory follicle maturation and ovulation. Overall, the findings indicate that neuroendocrine and ovarian deficits are linked to infertility observed in Esr2-mutant rats.

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Figures

Figure 1.
Figure 1.
Translation of WT and mutant ESR2 proteins. (a) Schematic presentation of the rat Esr2 gene (NC_005105.4) followed by predicted translation products of Esr2 mRNA (XM_006240221.3) indicate that the full-length rat ESR2 protein possesses 567 amino acids (DBD is underlined). (b) Deletion of exon 3 leads to a frame shift and two premature stop codons after amino acid 140, whereas (c) deletion of exon 4 causes a loss of 39 amino acids from the DBD (truncated DBD is underlined), resulting in a truncated protein of 528 amino acids. Full-length rat Esr2 cDNA was amplified by RT-PCR, cloned into pCMV-SC vector, and exon 3 or exon 4 deletion was performed by site-directed mutagenesis. (d) Transfection of ΔE3 constructs into 293FT cells failed to express detectable ESR2 protein; (e) however, ΔE4 constructs expressed ESR2 proteins of lower molecular weight. (f) ERE-dependent luciferase (Luc) activity was assessed by cotransfection of WT or ΔE4 ESR2 expression vectors and ERE-reporter vector into HeLa cells and stimulated with E2. Partial loss of the DBD caused failure of ΔE4 ESR2 to induce the ERE-reporter activity. (g) AP1-reporter activity was assessed by cotransfection of WT or ΔE4 ESR2 expression vector and a TRE-reporter construct into HeLa cells and stimulated with tamoxifen. No significant differences in transactivation activity were observed between WT and ΔE4 ESR2 expression vectors. Results are expressed as mean ± standard error of the mean and the induction of Luc activity was expressed as fold induction with E2 stimulation. Asterisk indicates significant differences between Esr2-mutant and WT means (P < 0.05). ns, not significant.
Figure 2.
Figure 2.
Targeted disruption of the rat Esr2 gene. (a) Schematic presentation of the rat Esr2 gene (NC_005105.4) and ZFN target sites within exon 3 and exon 4. (b) RT-PCR was performed on RNA samples from WT and Esr2ΔE3 mutant ovaries. PCR primers were designed for exon 2 and exon 5. Sequencing of the RT-PCR products showed the presence of two different ΔE3 mutant transcripts arising from alternative splicing between (c) exons 2 and 4 or (d) exons 2 and 5. (c) Splicing between exons 2 and 4 was predominant generating a frameshift and premature stop codons after amino acid 140. (d) Exon 2 to 5 splicing also generated a frameshift, 17 aberrant codons after amino acid 140 followed by a premature stop codon. (e) RT-PCR was performed on RNA samples from WT and Esr2ΔE4-mutant ovaries to detect ΔE4-mutant transcripts. Sequencing of the RT-PCR products indicated that (f) ZFN-edited ΔE4-mutant transcript is indistinguishable from (g) the naturally occurring endogenous ΔE4 isoform. Western blot analyses of granulosa cell proteins demonstrated undetectable ESR2 protein in (h) ΔE3-mutant ovaries and (i) a truncated ESR2 protein in ΔE4-mutant ovaries. (j, k) Full-length WT and mutant ESR2 coding sequences were amplified by RT-PCR, cloned into a mammalian expression vector with a C-terminal FLAG-tag, and expressed in 293FT cells. Western blot analysis with anti-FLAG antibody did not detect expression of (j) recombinant ΔE3 ESR2 protein but detected (k) a truncated ΔE4 ESR2 protein.
Figure 3.
Figure 3.
Assessment of postnatal development and fertility of Esr2 mutant rats. (a, b) Body weight of ΔE3- and ΔE4-mutant male and female rats were compared with WT rats and no significant difference was observed. (c) Onset of puberty in Esr2-mutant males (preputial separation) and mutant females (vaginal opening) was similar to that of their WT littermates. (d) Both Esr2ΔE3- and Esr2ΔE4-mutant males were fertile and their fertility was comparable to that of WT males. However, Esr2ΔE3- and Esr2ΔE4-mutant females did not become pregnant (n ≥ 12 in each group). (e–g) Vaginal cytology of adult (8–12 weeks old) Esr2ΔE3- and Esr2ΔE4-mutant females showed cyclic changes similar to WT female rats. When mutant females cohabitated with WT males, they mated but did not become pregnant or pseudopregnant. (h–j) Changes in serum hormone levels during specific stages of the estrous cycle were examined in WT vs Esr2ΔE3- and Esr2ΔE4-mutant females. Samples were collected at 0800 hours on the morning of proestrus (Pm), 2000 hours on the evening of proestrus (Pe), and 0800 hours on the morning of estrus (Em), and the first day of diestrus (Dm; n = 6 on collection day). Results are presented as mean ± standard error of the mean. Asterisks indicate significant differences between Esr2 mutant and WT means (P < 0.05). Serum E2, LH, and follicle-stimulating hormone (FSH) concentrations were measured. Serum E2 concentrations were significantly lower during proestrus in Esr2-mutant rats vs WT control rats. In addition, LH and FSH levels during the evening of proestrus were significantly lower in Esr2ΔE3- and Esr2ΔE4-mutant female rats vs WT control rats. D, diestrus; E, estrus; P, proestrus.
Figure 4.
Figure 4.
Effects of ESR2 disruption on the male reproductive tract. The reproductive tracts of adult WT and Esr2ΔE3- and Esr2ΔE4-mutant males (12 to 14 weeks of age) were examined, including gross appearance and weights for (a–c) testes, (d–f) epididymides, and (g–i) seminal vesicles. Sample sizes for the organ weight measurements were ≥10 per genotype. Representative hematoxylin and eosin–stained tissue sections of (j–l) testis, (m–o) caput epididymis, and (p–r) cauda epididymis from WT and Esr2ΔE3- and Esr2ΔE4-mutant male rats are presented. No significant differences in reproductive tracts were observed between the WT and Esr2-mutant male rats. Scale bars, 500 μM. Epid, epididymis; SV, seminal vesicle; wt., weight.
Figure 5.
Figure 5.
Female reproductive tract in Esr2-mutant rats. Ovaries of (a, c) Esr2ΔE3- and (b, c) Esr2ΔE4-mutant rats were smaller than those of WT rats (n = 6). Histological examination of hematoxylin and eosin (H&E)–stained ovary sections shows the presence of (d, g) multiple corpora lutea in WT ovaries, whereas (e, h) Esr2ΔE3- or (f, i) Esr2ΔE4-mutant ovaries were characterized by the absence of corpora lutea despite presence of numerous follicles in various stages of development. [Boxed areas of (d–f) are magnified in (g–i).] Uteri of (j, l) Esr2ΔE3- and (k, l) Esr2ΔE4-mutant rats were smaller than those of WT rats (n = 6). H&E-stained uterine sections show that (n) Esr2ΔE3- and (o) Esr2ΔE4-mutant uteri possessed all three definitive uterine compartments: myometrium, endometrial stroma, and epithelium; however, all were relatively smaller than that found in (m) the WT uterine sections. Scale bars, 1000 μM (d–f and m–o) and 100 μM (g–i). Results are presented as mean ± standard error of the mean. Asterisks indicate significant differences between Esr2 mutant and WT means (P < 0.05). a, antrum; cl, corpus luteum; gr, granulosa layer; o, oocyte; wt, weight.
Figure 6.
Figure 6.
Ovarian responses to gonadotropin stimulation in Esr2-mutant rats. (a) Four-week-old WT and Esr2-mutant female rats were treated with equine chorionic gonadotropin (eCG) and hCG, as indicated. (b) Gonadotropin treatment resulted in an increase in ovarian weight (wt.) in all three genotypes but was less effective with the ΔE3- and ΔE4-mutant ovaries compared with WT. (c) Esr2ΔE3- and Esr2ΔE4-mutant rats did not ovulate after eCG and hCG treatment (n = 10 per group). Histological examination of hematoxylin and eosin–stained ovary sections revealed that (d, g) WT ovaries contained follicles at various stages of maturation and corpora lutea, whereas (e, h) Esr2ΔE3- or (f, i) Esr2ΔE4- mutant ovaries exhibited numerous follicles and the absence of corpora lutea. *P < 0.05. Scale bars, 1000 μM (d–f) and 100 μM (g–i). Sac, sacrifice.
Figure 7.
Figure 7.
Gonadotropin-induced ovarian transcript responses in Esr2-mutant rats. RNA was extracted from gonadotropin-stimulated ovaries and expression of potential ESR2 target genes was examined by quantitative RT-PCR. (a) Four-week old WT and Esr2-mutant female rats were treated with eCG and hCG as indicated. Animals were euthanized and ovaries harvested from untreated animals (basal) 48 hours after eCG administration (eCG 48h) and 4 hours after hCG administration (hCG 4h). (b–j) WT ovaries responded with an upregulation of (b) Pgr, (c) Arnt2, (d) Runx2, (e) Ptgs2, (f) Adamts1, (g) Hpgd, (h) Srxn1, (i) Fam110c, and (j) Akap12. (k, l) However, ΔE3- and ΔE4-mutant ovaries did not respond to the gonadotropin treatment. In contrast, expression of (k) Trim61 and (l) Pou5f1 was uniquely upregulated in gonadotropin-treated Esr2-mutant ovaries. (m) Ahr expression remained unchanged after gonadotropin treatment. Results are expressed as mean ± standard error of the mean of gonadotropin-induced fold changes relative to basal levels (n = 4 per group). Asterisks indicate significant differences between Esr2-mutant and WT means (P < 0.05). eCG, equine chorionic gonadotropin; Rel exp, relative expression.
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