Granulosa cell lineage undergo a transition from ERα- to ERβ-expressing cells during follicular development

Estrogen receptors alpha (ERα, ESR1) and beta (ERβ, ESR2) are co-expressed in most estrogen-responsive tissues, but their expression tends to be mutually exclusive at the cellular level. In the adult ovary, ESR2 is expressed in granulosa cells (GCs) but not in theca cells or ovarian surface epithelium (OSE), whereas ESR1 is expressed in theca and OSE cells. Interestingly, recent studies showed that subpopulation of GCs is of OSE origin, suggesting that GC lineage cells lose ESR1 expression and gain ESR2 expression during the OSE cell-to-GC transition. In this study, we report that GC lineage cells initially express ESR1 but later lose their expression and gain ESR2 expression and that endothelial cell-derived TGFβ1 promotes such ESR1-to-ESR2 change in the developing ovary. To investigate the ovarian differential gene expression in individual cells, WT ovaries were collected from ED 16.5, PND 7, and PND 14. For single-cell RNA-seq analyses, ovaries were dissociated. Ovaries were removed, cut into small pieces, transferred to 2 ml of collagenase digestion solution containing 3.5 U of collagenase type I, 1000 U of deoxyribonuclease I, and 40 mg of BSA in H-199 media, then placed in a water bath at 37 °C for 30 min. After incubation, tissues were further dissociated by repeatedly passing them through an 18-G needle attached to a 3-ml syringe. Then, the digested tissue suspension was filtered through a 40-μm filter, centrifuged at 300 × g for 5 min, then resuspended in 1 ml of PBS containing 1% BSA. Single-cell suspensions with a viability of >90% by Trypan Blue staining were converted into individually barcoded cDNA libraries with the Single-Cell 3’ Chromium kit version 2 from 10X Genomics following the manufacturer’s protocols.