Ovarian granulosa cells are essential to gonadotrophin-regulated estrogen production, female cycle maintenance and fertility. The epithelial Na+ channel (ENaC) is associated with female fertility; however, whether and how it plays a role in ovarian cell function(s) remained unexplored. Here, we report patch-clamp and Na+ imaging detection of ENaC expression and channel activity in both human and mouse ovarian granulosa cells, which are promoted by pituitary gonadotrophins, follicle stimulating hormone (FSH) or luteinizing hormone (LH). Cre-recombinase- and CRISPR-Cas9-based granulosa-specific knockout of ENaC α subunit (Scnn1a) in mice resulted in failed estrogen elevation at early estrus, reduced number of corpus luteum, abnormally extended estrus phase, reduced litter size and subfertility in adult female mice. Further analysis using technologies including RNA sequencing and Ca2+ imaging revealed that pharmacological inhibition, shRNA-based knockdown or the knockout of ENaC diminished spontaneous or stimulated Ca2+ oscillations, lowered the capacity of intracellular Ca2+ stores and impaired FSH/LH-stimulated transcriptome changes for estrogen production in mouse and/or human granulosa cells. Together, these results have revealed a previously undefined role of ENaC in modulating gonadotrophin signaling in granulosa cells for estrogen homeostasis and thus female fertility.

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

Abstract Disclosure: Y. Que: None. X. Ma: None. Y. Wu: None. J. Chen: None. J. Guo: None. Y. Ruan: None. In response to glucose elevation or other physiological stimuli, pancreatic islet β cells are excited to mobilize intracellular Ca2+ leading to insulin secretion, which is a complex cellular event with underlying mechanisms not fully elucidated. The present study explored possible involvement of the epithelial Na+ channel (ENaC) in β cell excitability and insulin secretion. Analyzing human databases, primary rat/mouse pancreatic tissues as well as RINm5F, a rat β-cell line, we confirmed the expression of Scnn1a, Scnn1b and Scnn1g genes (encoding ENaC subunits, α, β and γ, respectively) in human and rodent β cells. To our surprise, inhibiting this Na+ channel by selective blockers, amiloride (1-10 µM) or benzamil (1 µM), did not retard insulin secretion, but instead triggered a slow membrane depolarization with electrical bursts (41.5 ± 5.9 mV, measured by patch-clamp, n = 5-6), elicited substantial Ca2+ oscillations (135.2 ± 2.5% of baseline, by Fura-2 imaging, n = 256-380) and promoted insulin secretion (158.2 ± 25.6% of control, by ELISA, t-test, p < 0.05, n = 6) in RINm5F or isolated mouse β cells. siRNA-based knockdown of ENaCα, the rate-limiting subunit of ENaC, in RINm5F cells confirmed that deficiency of ENaC induced a significant increase in insulin secretion (230.2 ± 20.2% of control, t-test, p < 0.001, n = 6). Proteomic analysis of RINm5F cells (n = 5) through mass spectrometry showed that signaling pathways key to glucose metabolism and insulin secretion were significantly activated in RINm5F cells with ENaC knockdown in comparison with control cells, consistently suggesting a role of ENaC deficiency in exciting β cells to release insulin. We next built a mouse model with β cell-specific knockout of ENaCα (Scnn1afl/fl, Ins1-Cre+), which exhibited disturbed responses in glucose tolerance test in comparison with the loxp-negative Cre control mice (Scnn1awt/wt, Ins1-Cre+). Taken together, these results have suggested an important role of ENaC in regulating the excitability of β cells and insulin secretion, which may contribute to the understanding of Na+ environment in relation to insulin homeostasis. This work was supported in part by National Natural Science Foundation of China (82071599), Areas of Excellence Scheme of Hong Kong (AoE/M-402/20) and Bai Cheng Bai Yuan Start-up Fund (I2022A008). Presentation: Saturday, June 17, 2023

Cystic fibrosis transmembrane conductance regulator (CFTR), known as an epithelial Cl− channel, is increasingly noted to be expressed in the nervous system, although whether and how it plays a role in neuronal excitability is unclear. Given the association of CFTR with fertility, we tested here possible involvement of CFTR in regulating hypothalamic neuron excitability. Patch-clamp and Ca2+ imaging showed that pharmacological inhibition of CFTR evoked electrical pulses and Ca2+ spikes in primary rat hypothalamic neurons, which was dependent on extracellular Cl−. Hypothalamic neurons in brain-slice preparations from adult female mice with CFTR mutation (DF508) exhibited significantly reduced electrical pulses as compared to the wild-type controls. Removal of extracellular Cl− eliminated hypothalamic electrical pulses in the wild-type brain slices, which was reversible by subsequent addition of Cl−. In adult female mice, Ca2+ indicator (GCaMP6s)-based fiber-photometry showed that hypothalamic Ca2+ activities in vivo were enhanced at the proestrus/estrus phase as compared to the diestrus phase of the female cycle. Such estrus-associated hypothalamic activities were largely diminished in DF508 female mice, together with delayed puberty and disturbed female cycles. Therefore, these findings suggest a critical role of CFTR in modulating hypothalamic neuron excitability, which may account for the disturbed female cycles and reduced female fertility associated with CFTR mutations.

The regulatory interaction between two typical epithelial ion channels, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC), for epithelial homeostasis has been noted, although the underlying mechanisms remain unclear. Here, we report that in a human endometrial epithelial cell line (ISK), shRNA-based stable knockdown of ENaC produced a biphasic effect: a low (∼23%) degree of ENaC knockdown resulted in significant increases in CFTR mRNA and protein levels, CFTR-mediated Cl− transport activity as well as intracellular cAMP concentration, while a higher degree (∼50%) of ENaC knockdown did not further increase but restored CFTR expression and cAMP levels. The basal intracellular Ca2+ level of ISK cells was lowered by ENaC knockdown or inhibition in a degree-dependent manner. BAPTA-AM, an intracellular Ca2+ chelator that lowers free Ca2+ concentration, elevated cAMP level and CFTR mRNA expression at a low (5 µM) but not a high (50 µM) dose, mimicking the biphasic effect of ENaC knockdown. Moreover, KH-7, a selective inhibitor of soluble adenylyl cyclase (sAC), abolished the CFTR upregulation induced by low-degree ENaC knockdown or Ca2+ chelation, suggesting the involvement of sAC-driven cAMP production in the positive regulation. A luciferase reporter to indicate CFTR transcription revealed that all tested degrees of ENaC knockdown/inhibition stimulated CFTR transcription in ISK cells, suggesting that the negative regulation on CFTR expression by the high-degree ENaC deficiency might occur at post-transcription stages. Additionally, similar biphasic effect of ENaC knockdown on CFTR expression was observed in a human bronchial epithelial cell line. Taken together, these results have revealed a previously unidentified biphasic regulatory role of ENaC in tuning CFTR expression involving Ca2+-modulated cAMP production, which may provide an efficient mechanism for dynamics and plasticity of the epithelial tissues in various physiological or pathological contexts.

Mitochondria are critical organelles in eukaryotes that efficiently generate adenosine 5'-triphosphate (ATP) for various biological activities, and any defect in the process of ATP synthesis may lead to mitochondrial dysfunction and directly link to a variety of medical disorders. Monitoring the ATP variations in cells is key for innovative early diagnosis of mitochondrial diseases. Herein, multifunctional single-layered graphene quantum dots (s-GQDs) with bright green emission were constructed, which exhibit strong binding affinity for ATP and good mitochondria targeting ability. Using the proposed s-GQDs, we successfully discriminated the primary smooth muscle cells isolated from the transgenic mouse (heterozygote sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) 2 C674S knock-in mouse) with mitochondrial disorders or their littermate controls, indicating s-GQDs as promising probes for the study of cell metabolism and mitochondrial malfunction-related diseases, and targeting endoplasmic reticulum stress is an effective way to modulate metabolic pathways relevant to SERCA 2 inactivity mitochondrial dysfunction.