Expression of ESR2 in GC Lineages is Essential in Ovarian Steroidogenesis and Ovulatory Machinery

Ji-Eun Oh¹, Chanjin Park², Po-Ching Lin^1,2, Mary Bunnell¹, and CheMyong J. Ko^1,2

¹Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, Urbana-Champaign, IL 61802, USA. ²Epivara Inc., Champaign, IL.

Abstract Text:

Estrogen receptors alpha (ESR1) and beta (ESR2) mediate estradiol (E2) actions in estrogen-responsive tissues, including the ovary, uterus, and brain. While both receptors are expressed in these organs, their distribution is highly cell-type specific, with ESR1 predominantly in theca cells and ESR2 in granulosa cells (GCs) in the ovary. Recent evidence challenges the notion of static estrogen receptor expression, suggesting that an ESR1-to-ESR2 switch occurs in some cell lineages. Our previous study showed that transgenic mice with ESR2 ablation in ESR1-expressing lineages (Esr1-iCre/WT; Esr2 flox/flox, hereafter Esr1-Esr2KO) exhibited numerous atretic follicles and anovulation. Further examination confirmed that ESR2 expression was absent in Esr1-Esr2KO GCs, demonstrating that ESR1-to-ESR2 switching occurs in the GC lineage. However, the mechanisms underlying ovarian dysfunction in these mice remain unknown. In this study, we tested the hypothesis that ablation of ESR2 in GC lineages disrupts ovarian steroidogenesis and ovulatory machinery. We compared Esr1-Esr2KO, wild-type (WT), and global Esr2KO mice to assess the impact of ESR2 on ovarian function using hormone measurement, histology, immunohistochemistry (IHC), and RT-PCR. Serum E2 concentrations in both Esr1-Esr2KO and global Esr2KO mice were elevated compared to WT, and there were no changes in testosterone concentration, suggesting increased aromatase (CYP19) activity. RT-PCR was performed using isolated GCs, and showed that Fshr expression was not changed, but Cyp19a1 expression was upregulated in Esr1-Esr2KO GCs, indicating dysregulated steroidogenesis. Since Esr1-Esr2KO mice are anovulatory, we further examined ovulatory gene expression in isolated GCs to identify the mechanism. Key regulators of ovulation, including endothelin-2 (Edn2) and prostaglandin-endoperoxide synthase 2 (Ptgs2), were downregulated in Esr1-Esr2KO GCs, suggesting impaired inflammatory and contractile responses necessary for follicular rupture. Notably, these phenotypes resemble those observed in global Esr2KO mice, reinforcing ESR2’s crucial role in ovulation. As proper GC maturation is essential for steroid hormone production and ovulation, we examined the apoptotic gene, Cas3, in isolated GCs. Cas3 expression was higher in Esr1-Esr2KO GCs than in WT. In support, TUNEL staining indicated greater apoptosis in Esr1-Esr2KO ovaries than in WT ovaries of 30-day-old mice. The observed apoptosis may contribute to the underlying dysfunction in ovarian function associated with the loss of ESR2 in GC lineages. Taken together, this study establishes ESR2 as essential for normal ovarian function in the GCs.