FRI389 Granulosa Cell-specific Knockout Of Scnn1a Disturbs Estrus Cycle And Impairs Estrogen Production In Mice

Abstract

Abstract Disclosure: X. Ma: None. R. Xu: None. Y. Que: None. J. Chen: None. Y. Ruan: None. Estrogens are produced primarily by ovarian granulosa cells in females, the homeostasis of which is not only a prerequisite for female reproduction but also essential to the overall health. Studies suggested Na+ environment/intake in relation to ovarian functions, although the underlying mechanism remains unclear. In the present study, we explored possible expression and function of the epithelial Na+ channel (ENaC) in granulosa cells. We analyzed an available single-cell RNA sequencing database of human ovarian cells, mouse ovarian tissues as well as a human granulosa cell line (KGN), which showed ENaC expression and its channel activities (measured by patch-clamp) in granulosa cells. Since ENaCα (Scnn1a) is the rate-limiting subunit for ENaC to function, we established a granulosa cell-specific Scnn1a knockout mouse model (Scnn1afl/fl, Cyp19a1-Cre). In such a conditional knockout (cKO) model, the estrus cycle of the female mice at reproductive ages of 8 to 10-week-old was found to be disturbed with the ratio of proestrus/estrus versus metestrus/diestrus significantly higher (t-test, p < 0.001) in cKO (1.8 ± 0.1, n = 8) compared to that of the Cre-negative control (Scnn1afl/fl, 1.0 ± 0.1, n = 8) mice. Histological analysis of the ovarian tissues showed a fewer (t-test, p < 0.05) number of corpus luteum in cKO mice (1.9 ± 0.8 per ovary, n = 8) than that of the control mice (6.2 ± 1.5 per ovary, n = 6). We next isolated the granulosa cells from the mouse model and tested their responses to gonadotropins in vitro. In granulosa cells from the control mice, the estradiol level in the culture medium (measured by ELISA) was significantly increased by the treatment of FSH (100 ng/ml, 48 hours) and subsequent LH (100 ng/ml, 18 hours) as compared to that of cells without FSH/LH treatment (33.0 ± 12.3 vs. 5.1 ± 0.9 ng/ml, t-test, p < 0.05, n = 6). Whereas, in cKO granulosa cells, no such elevation of estradiol level in response to FSH/LH was detected (7.6 ± 1.8 vs. 6.5 ± 1.3 ng/ml, t-test, p > 0.05, n = 6), suggesting impaired estrogen production with ENaCα knockout. Consistently, quantitative PCR results showed significant downregulation of Cyp19a1 (-96.1 ± 0.9%), Lhcgr (-99.5 ± 0.1%) and Fshr (- 67.9 ± 7.2%), three key steroidogenic genes, in FSH/LH-treated cKO cells, as compared to that of FSH/LH-treated control cells (n = 6, t-test, p < 0.05). Taken together, these results have suggested a previously undefined role of ENaC in granulosa cells for estrogen production in response to gonadotrophins maintaining female cycle homeostasis. This work was supported by National Natural Science Foundation of China (82071599). Presentation: Friday, June 16, 2023