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Over the entire reproductive lifespan in mammals, a fixed number of primordial follicles serve as the source of mature oocytes. Uncontrolled and excessive activation of primordial follicles can lead to depletion of the ovarian reserve. We observed that disruption of estrogen receptor β (ESR2) signaling results in increased activation of primordial follicles in Esr2-null (Esr2-/-) rats. However, follicle assembly was unaffected, and the total number of follicles remained comparable between neonatal wild-type and Esr2-/- ovaries. While the activated follicle counts were increased in Esr2-/- ovary, the number of primordial follicles were markedly decreased. Excessive recruitment of primordial follicles led to premature ovarian senescence in Esr2-/- rats and was associated with reduced levels of serum AMH and estradiol. Disruption of ESR2 signaling through administration of a selective antagonist (PHTPP) increased the number of activated follicles in wildtype rats, whereas a selective agonist (DPN) decreased follicle activation. In contrast, primordial follicle activation was not increased in the absence of ESR1, indicating that the regulation of primordial follicle activation is ESR2 specific. Follicle activation was also increased in Esr2 mutants lacking the DNA binding domain, suggesting a role for the canonical transcriptional activation function. Both primordial and activated follicles express ESR2, suggesting a direct regulatory role for ESR2 within these follicles. We also detected that loss of ESR2 augmented the activation of AKT, ERK, and mTOR pathways. Our results indicate that the lack of ESR2 upregulated both granulosa and oocyte factors, which can facilitate AKT and mTOR activation in Esr2-/- ovaries leading to increased activation of primordial follicles., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hypothalamic expression of Kiss1 plays an essential role in the onset of puberty, gonadal development, and ovulation. Estrogens regulate the expression of Kiss1 in the hypothalamus through estrogen receptor-α. Kiss1 is also expressed in the ovary, where its expression correlates with the onset of puberty and progression of the estrous cycle. To date, estrogen regulation of Kiss1 expression in the ovary has not been investigated. We recently observed that gonadotropin-induced Kiss1 expression was absent in Esr2-null rat ovaries even though Esr1 was present. Wild-type granulosa cells abundantly expressed Kiss1 and oocytes expressed the Kiss1 receptor. We characterized estrogen receptor-β (ESR2) regulation of Kiss1 expression in granulosa cells by identifying granulosa cell-specific transcript variants and potential regulatory regions. The Kiss1 promoter, an upstream enhancer, and a downstream enhancer all possessed conserved estrogen response elements (EREs) and showed active histone marks in gonadotropin-stimulated granulosa cells. The transcriptionally active Kiss1 promoter, as well as the enhancers, also revealed enrichment for ESR2 binding. Furthermore, activity of a Kiss1 promoter construct was induced after overexpression of ESR2 and was blocked upon mutation of an ERE within the promoter. Finally, pregnant mare serum gonadotropin and human chorionic gonadotropin administration induced phosphorylation of ESR2 and upregulated the AP-1 proteins FOSL2 and JUNB in granulosa cells. Activated MAPK ERK2 was associated with the ESR2 phosphorylation in granulosa cells, and AP-1 factors could synergistically activate the Kiss1 promoter activity. These gonadotropin-induced changes paralleled Kiss1 expression in granulosa cells. We conclude that gonadotropin-stimulated Kiss1 expression in granulosa cells is dependent on both the activation of ESR2 and the upregulation of AP-1.

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., (Copyright © 2017 Endocrine Society.)

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17β-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action.

The hormone adiponectin has been shown to be important in maintaining insulin sensitivity throughout the body, whereas potential effects on the placenta have not been assessed. Pregnancy constitutes a unique physiological environment in which metabolism has a profound effect on the health of both the mother and the developing fetus. It is imperative that a delicate balance in glucose delivery be maintained between maternal tissues and the fetal/placental unit. Adiponectin's role in regulating peripheral insulin responsiveness suggests it may be a factor in maintaining this balance during gestation as well. Examination of human cytotrophoblast cells revealed that mRNA for both adiponectin receptors, adipoR1 and adipoR2, are abundantly expressed at term. We were, however, unable to reliably detect mRNA for adiponectin in primary cytotrophoblasts. Expression of both receptors was maintained after induction of syncytium formation by exogenous epidermal growth factor treatment. Treatment of cytotrophoblasts with adiponectin resulted in a significant drop, as assessed by quantitative RT-PCR, in expression for a number of genes involved in the endocrine function of the placenta, including the chorionic gonadotropin subunits, placental lactogen, and some steroidogenic enzymes. Immunofluorescent staining for connexin 43 and desmoplakin in primary trophoblasts revealed that adiponectin does not inhibit syncytialization of trophoblast cells in culture. Taken together, these data describe a novel role for maternal adiponectin in regulating the placental environment. Determination of the effects of such adipokines on the maternal-fetal interface is increasingly important, because the incidence of pregnancies complicated by gestational diabetes remains a significant health problem in developed countries.

PRL-like protein C variant (PLP-Cv) is a newly identified member of the PRL family. PLP-Cv is specifically expressed in the chorioallantoic placenta by two distinct cell populations: trophoblast giant cells and spongiotrophoblast cells. To gain some insight regarding the control of PLP-Cv gene expression and the regulatory factors controlling trophoblast giant cell and spongiotrophoblast cell lineages, we have initiated a structural and functional analysis of the PLP-Cv promoter. The activities of a series of PLP-Cv promoter constructs, ranging in size from 4.5 kb to 50 bp, ligated to a luciferase reporter have been assessed in the Rcho-1 trophoblast cell line (restricted to trophoblast giant cell differentiation) and in a primary spongiotrophoblast cell culture system after transient transfection. PLP-Cv promoter constructs containing 4.5 kb to 149 bp of 5'-flanking DNA possessed full activity in the trophoblast giant cell model. A region located between -149 and -124 bp upstream of the PLP-Cv transcription start site was found to be essential for activation of the PLP-Cv promoter. Spongiotrophoblast cells required additional PLP-Cv 5'-flanking DNA for full activity. A region located between -2518 and -2242 bp upstream of the PLP-Cv transcription start site significantly enhanced PLP-Cv promoter in spongiotrophoblast cells. In conclusion, mechanisms underlying the activation of the PLP-Cv promoter are different in trophoblast giant cells vs. spongiotrophoblast cells.