title: 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.
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Shaanxi 712100, China.
authors.forename: Fenglei
authors.initials: F
authors.lastname: Chen
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: Diqi
authors.initials: D
authors.lastname: Yang
authors.forename: Xin
authors.initials: X
authors.lastname: Wen
authors.forename: Tagwa Norain
authors.initials: TN
authors.lastname: Mahmoud
authors.forename: Dong
authors.initials: D
authors.lastname: Zhou
authors.forename: Keqiong
authors.initials: K
authors.lastname: Tang
authors.forename: Pengfei
authors.initials: P
authors.lastname: Lin
authors.forename: Aihua
authors.initials: A
authors.lastname: Wang
authors.forename: Yaping
authors.initials: Y
authors.lastname: Jin
mesh_terms: D000818:Animals; D017209:Apoptosis; D001141:Aromatase / Q000378:metabolism; D002453:Cell Cycle; D059447:Cell Cycle Checkpoints; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D006107:Granulosa Cells / Q000166:cytology* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D006080:Ovarian Follicle / Q000378:metabolism; D010060:Ovulation; D034741:RNA, Small Interfering / Q000378:metabolism; D016196:S Phase; D013256:Steroids / Q000378:metabolism
chemical_list: C415829:Herpud1 protein, mouse; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D013256:Steroids; D004958:Estradiol; D001141:Aromatase