Herp depletion arrests the S phase of the cell cycle and increases estradiol synthesis in mouse granulosa cells

Abstract

The endoplasmic reticulum (ER) stress response has been implicated in the development, atresia and luteinization of ovarian follicles. However, there have been few reports concerning the role of Herp, an ER stress-induced protein, in follicular development. The present study aims to detect the distribution and cyclic variations of Herp during the estrous cycle and to reveal the roles of Herp in regulating the cell cycle, apoptosis and steroid hormone biosynthesis in mouse granulosa cells. In this study, immunohistochemistry staining showed that Herp expression was primarily in the granulosa cells and oocytes. Furthermore, we constructed recombinant lentiviral vectors for Herp short hairpin interfering RNA (shRNA) expression; immunofluorescence staining, real-time quantitative PCR (RT-qPCR) and western blot analysis revealed that Herp was successfully knocked down. Flow cytometry showed that knockdown of Herp arrested granulosa cells at the S phase of the cell cycle. More importantly, ELISA analysis revealed that Herp knockdown significantly upregulated the concentration of estradiol (E2) in the culture supernatants. RT-qPCR was performed to determine the regulatory mechanism of Herp knockdown in the cell cycle, and in steroid synthesis, RT-qPCR analysis revealed that Herp knockdown upregulated the mRNA expression of steroidogenic enzymes (Cyp19a1) and downregulated metabolic enzymes (Cyp1b1) and cell cycle factors (cyclin A1, cyclin B1 and cyclin D2). These results suggest that Herp may regulate the cell cycle and hormone secretions in mouse granulosa cells. The present study helps to elucidate the physiological functions of Herp as they relate to reproduction.

IRS2 depletion inhibits cell proliferation and decreases hormone secretion in mouse granulosa cells

“…Expression of the lentiviral vector (carrying green fluorescence protein (GFP)) was used to evaluate virus-mediated transfection efficiency, under a fluorescent microscope. Transfection of mouse GCs with shRNA-IRS2 lentiviral vector was carried out, as our previous report [ 22 ]. The mouse GCs were seeded into 6-well plates, which were cultured to ~60−70% confluence and infected by addition of 1 × 10 8 TU/ml lentivirus (10 μl), 5 μg/ml polybrene and complete medium.…”

An immortalized steroidogenic goat granulosa cell line as a model system to study the effect of the endoplasmic reticulum (ER)-stress response on steroidogenesis

“…In female mammals, only 1% of follicles reach ovulation, causing the remaining follicles to undergo atresia [ 1 ]. Apoptosis of granulosa cells and degeneration of theca cells are considered to be crucial reasons for follicular atresia [ 2 ]. Granulosa cells also play a vital role in the secretion of sex steroids, such as estrogen and progesterone, in the follicle and are indispensable for oocyte maturation [ 2 , 3 , 4 ].…”

“…Apoptosis of granulosa cells and degeneration of theca cells are considered to be crucial reasons for follicular atresia [ 2 ]. Granulosa cells also play a vital role in the secretion of sex steroids, such as estrogen and progesterone, in the follicle and are indispensable for oocyte maturation [ 2 , 3 , 4 ]. Previous in vitro studies using primary granulosa cells have elucidated changes in the molecular mechanisms of steroid synthesis, and the regulation of granulosa cell function [ 5 ].…”

Selenium Attenuates Chronic Heat Stress-Induced Apoptosis via the Inhibition of Endoplasmic Reticulum Stress in Mouse Granulosa Cells

“…Additionally, a major factor that can affect serum estradiol levels and/or ovary is the number of granulosa cells. In the normal physiological process in the ovary, a certain degree of cell death can be observed in the granulosa cells, which plays an important role in accurately modulating the discarded decrepit cells, thereby maintaining the self-renewal of the granulosa cell population and normal estradiol levels [46,47]. However, excessive or severe granulosa cell death induced by damage to ovary usually results in reduced estradiol levels, ultimately leading to female reproductive dysfunction [48].…”