| Author: | Ma, Xiyang |
| Title: | Role of the epithelial sodium channel (ENaC) in ovarian granulosa cell function |
| Advisors: | Ruan, Y. C. Sharon (BME) |
| Degree: | Ph.D. |
| Year: | 2022 |
| Subject: | Ovaries -- Diseases -- Animal models Infertility, Female Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Biomedical Engineering |
| Pages: | xv, 125 pages : color illustrations |
| Language: | English |
| Abstract: | Ovarian dysfunction accounts for over 40% of female infertility. However, the molecular mechanisms underlying ovarian function and diseases remain unclear. The epithelial sodium channel (ENaC) is best known for conducting Na+ and thus fluid absorption across epithelial tissues such as those in the airway and kidney. Interestingly, the expression of ENaC was detected in human granulosa cells, although the possible roles of ENaC in ovarian function remain unexplored. Previous studies have suggested that ENaC in uterine endometrial epithelial cells plays a role in inflammatory signal transduction involving proteases, Ca2+ mobilization and prostaglandins. Given the known involvement of proteases, Ca2+ mobilization and prostaglandins in ovarian functions such as folliculogenesis and/or ovulation, we hypothesized that ENaC in the granulosa cells might play a role in the regulation of ovarian function. The present study demonstrates that ENaC is functionally expressed in human and mouse ovarian granulosa cells, as shown by RT-PCR, western blot, immunofluorescence, patch-clamp, Na+ imaging and membrane potential measurement results obtained from primary mouse granulosa cells, a human granulosa cell line (KGN) and primary human granulosa cells from women undergo in vitro fertilization treatment. Such ENaC expression in granulosa cells is subject to the regulation by gonadotropins (i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH)). Pharmacological inhibition of ENaC, siRNA-based transient knockdown or shRNA-based stable knockdown of ENaCα, the rate-limiting subunit of ENaC, attenuates spontaneous Ca2+ oscillation and FSH-promoted Ca2+ mobilization in granulosa cell in vitro models. Proteomic analysis by mass spectrometry was performed on primary cultured mouse granulosa cells treated with siRNAs targeting ENaCα in comparison with control cells treated with non-silencing siRNAs, which reveals proteins associated with signaling pathways including estrogen-dependent gene expression, innate immune response, prostaglandin production and collagen degradation to be downregulated in cells with ENaC knockdown. Consistent proteomic analysis results were obtained from cell models with shRNA-based stable knockdown of ENaC. Analysis using western blot, qPCR or ELISA confirms that ENaC knockdown causes genes associated with Ca2+ transport to be downregulated. Ca2+ regulated downstream events such as cyclooxygenase-2 (COX-2) activation to produce prostaglandin E2 (PGE2), and aromatase (cyp19a1) activation to produce estrogen are affected by ENaC deficiency. The next part of the present study was performed to test the effect of ENaC deficiency on granulosa cells and ovarian function in vivo. First, microinjection of ENaC inhibitor, amiloride, or ENaCα-targeting siRNAs into peri-ovary region, significantly inhibited ovulation in mice. Second, a conditional knockout (cKO) mouse model with the granulosa-cell-specific knockout of the ENaC (ENaCαgc-/-) was established using CRISPR-Cas9 and Cre/LoxP technologies to cross breed ENaCα (SCNN1A)-flox and cyp19a1-cre mice. ENaCαgc-/- mice exhibited a dysregulated estrus cycle with the estrus phase significantly prolonged than the control mice. Consistently, estrogen production was observed to be disturbed in ENaCαgc-/- mice. Likewise, the fertility of ENaCαgc-/- mice was affected as shown by significant reduction in litter size. The isolated granulosa cells from the cKO model consistently showed impaired Ca2+ mobilization, as well as reduction in a transcription factor CREB key to the transcriptional activation of Ptgs2 and cyp19a1. Taken together, the present study has demonstrated a previously undefined role of ENaC in modulating Ca2+ mobilization, prostaglandin and estrogen production that is key to the function of granulosa cells to realize or promote folliculogenesis and ovulation in the ovary. Abnormality in ENaC may underlie ovarian disorder and female infertility/subfertility cases with currently unclear etiology. The demonstrated effect of ENaC deficiency on ovarian endocrine function to produce ovary hormone may also have further implications in other system diseases beyond the ovary or female reproduction. |
| Rights: | All rights reserved |
| Access: | open access |
Copyright Undertaking
As a bona fide Library user, I declare that:
- I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
- I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
- I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.
Please use this identifier to cite or link to this item:
https://theses.lib.polyu.edu.hk/handle/200/13429