title: ATF6 knockdown decreases apoptosis, arrests the S phase of the cell cycle, and increases steroid hormone production in mouse granulosa cells.
abstract: Activating transcription factor 6 (ATF6), a sensor protein located in the endoplasmic reticulum (ER) membrane, is an important factor in the ER stress signaling pathway. ER stress is known to be involved in folliculogenesis, follicular growth, and ovulation; however, the physiological function of ATF6 in mouse granulosa cells remains largely unknown. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative PCR, Western blotting, flow cytometry, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels in mouse granulosa cells. In the meantime, a recombinant plasmid was used to deplete ATF6 successfully using short hairpin RNA-mediated interference technology, which was verified at both the mRNA and protein levels. Flow cytometry and TUNEL assay analysis indicated that ATF6 depletion decreased apoptosis and arrested the S phase of the cell cycle in mouse granulosa cells. Consistent with these results,p53,caspase-3,B cell lymphoma 2 (Bcl-2)-associated X protein,CCAAT-enhancer-binding protein homologous protein,cyclin A1,cyclin B1, andcyclin D2mRNA expression decreased, whereasBcl-2andglucose-regulated protein 78 kDamRNA expression increased. Interestingly, ATF6 knockdown obviously increased progesterone and estradiol production in mouse granulosa cells.Cytochrome P450 1b1(Cyp1b1) mRNA levels were downregulated, whereasCyp11a1,steroidogenic acute regulatory, andCyp19a1mRNA levels were upregulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increasedinsulin-like growth factor binding protein4(Igfbp4) expression and decreasedhyaluronan synthase 2(Has2),prostaglandin-endoperoxide synthase 2(Ptgs2), andprostaglandin F receptor(Ptgfr) expression in mouse granulosa cells, which are proteins crucial for follicular development. But, after treating with tunicamycin, the levels ofHas2,Ptgs2, andPtgfrincreased relatively, whereasIgfbp4expression decreased. Collectively, these results imply that ATF6, as a key player in ER stress signaling, may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis in mouse granulosa cells, which may indirectly be involved in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells. The present study extends our understanding and provides new insights into the physiological significance of ATF6, a key signal transducer of ER stress, in ovarian granulosa cells.
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China; and.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Yongjie
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China; and.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Huatao
authors.identifier: http://orcid.org/0000-0002-3800-5013
authors.initials: H
authors.lastname: Chen
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China; and.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Lin
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Aihua
authors.initials: A
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China; and.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China; and yapingjin@163.com.|College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Jin
mesh_terms: D051702:Activating Transcription Factor 6 / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002478:Cells, Cultured; D005260:Female; D055785:Gene Knockdown Techniques; D012739:Gonadal Steroid Hormones / Q000378:metabolism*; D006107:Granulosa Cells / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D059807:S Phase Cell Cycle Checkpoints / Q000502:physiology*
chemical_list: D051702:Activating Transcription Factor 6; C497112:Atf6 protein, mouse; D012739:Gonadal Steroid Hormones
keywords: ATF6; depletion; endoplasmic reticulum stress; follicular development; steroidogenesis