Estrogen receptors (ERs) are critical for maintaining normal physiology and reproductive functions. In recent years, significant advances have been made in understanding the molecular mechanisms and biological roles of ERα-mediated responses. Despite this progress, the specific mechanisms by which ERα impacts female reproduction and other physiological processes, leading to therapeutic developments for estrogen receptor-associated diseases, remain to be elucidated. In the present study, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively examine the transcriptome dynamics of wild-type (WT) and ERα knockout (KO) mouse ovaries, as well as the differential alterations in signaling pathways. Through our analyses, we identified seven distinct cell populations, including granulosa cells, mesenchyme cells, macrophages, vascular endothelial cells, oocytes, an unknown cell type, ovarian surface epithelium cells (OSE), and neutrophils, which exhibited dynamic changes during the investigation period. These fluctuations likely represent the diverse cell populations' proliferation, differentiation, and cell death processes. While some ovarian somatic cells such as mesenchyme, vascular endothelial cells, and immune cells were included in our analysis, due to limited information regarding other ovarian somatic compartments, we primarily focused on oocytes and granulosa cells. Initially, we examined the transcriptional alterations in oocytes resulting from ERα ablation using high-resolution scRNA-seq. Subsequently, we investigated the granulosa cell fate transition following ERα deletion. Moreover, we discovered that Greb1, an ERα-regulated gene potentially involved in estrogen action, is induced by E2 via ERα binding to ERE in mouse granulosa cells, as previously demonstrated in cancer cells (27). Nevertheless, we observed that ERα ablation impeded GREB1 upregulation and AKT activation by E2 in granulosa cells. It suggested that the ERα-dependent subset of E2 actions may be partially or completely mediated by GREB1, which promotes cell growth and survival in granulosa cell tumors (28). Therefore, our findings provide a novel perspective on the mechanisms and biological functions of ERα in the ovary, expanding our understanding of its roles in reproductive physiology.
The advancement of efficacious treatments for estrogen-associated disorders hinges on comprehending the physiological roles and mechanistic underpinnings of ERα in both human health and disease states. Previously, investigations into the transcriptomic alterations in ovarian cells were primarily conducted using bulk RNA-seq, which generally yields averaged gene expression values across cell populations (29). In the present study, we employed scRNA-seq to elucidate the impact of ERα on oocytes and granulosa cells at the transcriptomic level, thereby refining our understanding of the consequences of ERα ablation on specific cell types. Utilizing this advanced technique, we demonstrated that ERα deletion in the ovary substantially impedes oocyte maturation and ovulation progression due to the disruption of steroid metabolic processes. Moreover, we observed marked alterations in the growth status of both oocytes and granulosa cells following ERα disruption. These findings not only underscore the pivotal role of ERα in female reproductive processes but also lay the groundwork for further in-depth investigations, ultimately contributing to the development of targeted therapeutic strategies for estrogen-related diseases.
To thoroughly investigate the transcriptional regulatory mechanisms of oocytes and granulosa cells in the ERα knockout mouse model, we employed various algorithms and bioinformatic analyses. Initially, we utilized existing data to validate the oocyte developmental trajectory following ERα ablation. Intriguingly, we observed that certain ERα-deleted oocytes were redirected to alternative cell fates, displaying a markedly distinct cell state compared to control oocytes. By scrutinizing distinct gene sets along this trajectory, we pinpointed biological processes related to ovarian steroidogenesis and the ovulatory cycle. Furthermore, we identified several DEGs in oocytes that encode essential components for estradiol synthesis and steroidogenesis, both of which are critical for reproductive processes. For instance, Hsd17b7 predominantly converts estrone to estradiol, particularly during the luteal phase of the rodent ovarian cycle, and is responsible for the final step in estradiol synthesis while being upregulated in ERα KO ovaries (30, 31). ERα ablation also rapidly elevates the expression of the steroidogenic acute regulatory (Star) gene in oocytes, a known luteinization marker gene (32). Abcb1b, which encodes the multidrug-resistant transporter (also referred to as P-glycoprotein), is acutely regulated by CCAAT/Enhancer-Binding Proteins (C/EBP)-α and -β in ovaries and exhibits high expression levels in ERα KO ovaries (33). Although its specific function in luteal cells remains to be elucidated, it may be involved in cholesterol or progesterone transport. Adult female mice lacking Ltbp1 exhibit impaired fertility characterized by ovarian cyst formation and reduced estrogen and progesterone levels (20). Akr1c18 encodes 20α-hydroxysteroid dehydrogenase (20α-HSD), which converts progesterone into the inactive metabolite 20α-hydroxyprogesterone (20α-OHP) (21). Piekorz et al. demonstrated that Akr1c18 deletion in mice results in persistent progesterone production and subsequent parturition failure (34). Additionally, the absence of a functional SCARB1 protein in female mice leads to morphological abnormalities in ovarian follicular structures, similar to the αERKO phenotype (35). Another study reported that Ptgfr plays a role in maintaining the estrous cycle (32). Our findings revealed significantly reduced expression of Ltbp1, Akr1c18, Scarb1, and Ptgfr in ERα-deficient female mice. Consequently, we deduced that ERα KO female mice exhibit a paucity of preantral and small antral follicles, numerous large and hemorrhagic cystic follicles, and an absence of corpora lutea, which may be attributed to the dysregulation of these genes in the ovary.
The most significant changes in composition and cell states were identified in granulosa cells, particularly within follicles based on their stage of development, reflective of their important role in cyclic follicular maturation and hormone production. Early preantral follicle numbers are considered relatively stable during follicle growth and maturation (36), as they are largely unresponsive to gonadotropins (37). In contrast, antral follicles exhibit more variability in numbers and size. Our study revealed that the majority of granulosa cells in preantral follicles originated from control mouse ovaries, while those in antral follicles were primarily derived from ERα KO mouse ovaries. Additionally, the corpus luteum cluster was predominantly identified in control granulosa cells. Genes enriched in this cluster have been previously implicated in the ovulatory process and are regulated by the luteinizing hormone (LH) surge, corroborating that ERα disruption results in ovulation and fertility defects in female mice.
Subsequent bioinformatic analyses of the granulosa cell transcriptome identified numerous differentially expressed genes (DEGs) previously reported as essential for steroid biosynthetic processes and ovulation. For instance, Rhox8 is primarily expressed in mouse ovarian granulosa cells and exhibits peak expression during the periovulatory phase at 8 h post-hCG administration. Rhox8 is specifically stimulated by the progesterone receptor, suggesting its involvement in LH-dependent follicular rupture by inducing secondary progesterone-regulated genes crucial for ovulation (38). In ERα KO mouse granulosa cells, Rhox8 was the most down-regulated gene and may contribute to granulosa cell proliferation, survival, or differentiation. Ovarian follicles lacking FSH or FSH receptors fail to advance to the preovulatory stage, resulting in infertility. A hallmark of preovulatory follicles is the presence of Lhcgr on granulosa cells. The PI3K/AKT pathway activation is required for FSH-induced endogenous Lhcgr mRNA expression in granulosa cells. However, PI3K/AKT pathway disruption and Lhcgr downregulation in ERα-deleted granulosa cells may contribute to ovulation failure and female sterility (39).
Granulosa cells are pivotal in hormone secretion, with Cyp19a1 playing a significant role in E2 synthesis. Cyp19a1 expression is regulated by FSH and LH at the mRNA level. CYP19A1 knockdown modulates hormone secretion and cell proliferation in follicular granulosa cells (40). ERα KO mice exhibit chronically elevated LH, E2, and testosterone due to disrupted negative feedback, with Cyp19a1 significantly decreased in ERα-deleted granulosa cells. Ligand-independent responses are ER-mediated effects observed after activating other pathways, such as insulin-like growth factor 1 (IGF1) receptor-mediated signaling, leading to ER-mediated transcriptional responses independent of estrogenic steroid ligands. Recent studies indicate that IGF1 stimulation can result in ERα recruitment to chromatin, with ChIP-PCR analysis confirming ERα binding to specific ERE sequences of the Igf1 gene (41, 42). Our investigation revealed that Igf1r was significantly upregulated in ERα-deleted granulosa cells, which may regulate granulosa cell growth and proliferation.
In general, the fundamentals of estrogen response can be deduced from the earlier description of ER domains. Pioneer factors such as FOXA1 facilitate accessibility by binding and partially opening chromatin, enabling ER-ERE interactions at appropriate sites within the cell (43). The ER DBD associates with ERE motifs in accessible chromatin regions, while the LBD binds E2, initiating conformational changes in the ER protein. This interaction between E2/ER and transcriptional coactivators, including those with chromatin remodeling activities, is subsequently enabled. In the current study, we aimed to unravel the potential roles and mechanisms of ERα action by conducting cell-based and molecular analyses using mouse granulosa cells. Notably, we demonstrated that ERα-mediated activity could occur through a genomic pathway, stimulating the expression of GREB1 (growth regulation by estrogen in breast cancer 1), a crucial regulator of E2-stimulated epithelial ovarian cancer and granulosa cell tumor cell growth (Fig. 7). Utilizing reporter assays, we showed that ERα could act through an ERE-dependent mechanism in granulosa cells, as previously reported (44). This observation was reinforced by the finding that treatment with the ERα agonist PPT upregulated Greb1 expression, which is necessary for E2-stimulated growth in several hormonally regulated tumors (45). Greb1 upregulation was absent in ERα-deficient granulosa cells, confirming the specificity of ERα activity on Greb1 regulation in these cells. Importantly, our subsequent analyses in granulosa cells revealed that E2-induced Greb1 expression occurs through ERα binding to EREs, as confirmed by ChIP-PCR. Membrane interactions trigger rapid signaling responses (excluding transcriptional components), including activation of intracellular signaling pathways such as AKT and MAPK. This mechanism appears to play a significant role in peripheral E2 cellular responses. Consequently, our study demonstrated that signaling pathways like PI3K-AKT and MAPK are essential for interactions between oocytes and granulosa cells. ERα knockout markedly reduced GREB1 production and AKT activation in E2-treated granulosa cells. We therefore hypothesize that dysregulated genes following ERα knockout in mouse ovaries could offer therapeutic targets to alleviate infertility caused by ERα inactivation.
In conclusion, through the application of scRNA-seq, we have uncovered the transcriptional dynamics of various ovarian cell types, particularly oocytes and granulosa cells, following ERα disruption in female mice. Our findings highlight that ERα deletion may result in impaired ovulatory potential due to the dysregulation of multiple genes, which are potentially essential for steroid biosynthesis and ovulation processes. Furthermore, ERα deletion impacts the proliferation and function of granulosa cells, which play a critical role in oocyte development regulation. These investigations build upon our earlier morphological characterizations of the αERKO ovary and offer additional evidence that the most pronounced ovarian phenotypes in the αERKO arise from substantial transcriptomic alterations. However, given the physiological differences between mice and humans, murine models may not accurately represent human diseases. As such, a more comprehensive understanding of the role and mechanisms of ERα in female reproduction could be achieved by examining samples from individuals with ERα-related disorders. Moving forward, we plan to utilize ovarian tissue samples from patients with ERα inactivation to further elucidate the reproductive mechanisms of ERα, ultimately providing a more in-depth theoretical foundation for the prevention and treatment of reproductive health issues caused by ERα mutations in humans.