title: FSH induces the development of circadian clockwork in rat granulosa cells via a gap junction protein Cx43-dependent pathway.
abstract: The present study was designed to assess the relationship between gap junctions and the maturation of a clock system in rat granulosa cells stimulated by follicle-stimulating hormone (FSH). Immature and mature granulosa cells were prepared by puncturing the ovaries of diethylstilbestrol- and equine chorionic gonadotropin (eCG)-treated mouse Period2 (Per2)-dLuc reporter gene transgenic rats, respectively. Mature granulosa cells exposed to dexamethasone (DXM) synchronization displayed several Per2-dLuc oscillations and a rhythmic expression of clock genes. Intriguingly, we observed clear evidence that the FSH stimulation significantly increased the amplitude of Per2 oscillations in the granulosa cells, which was confirmed by the elevation of the Per2 and Rev-erbα (Nr1d1) mRNA levels. FSH also induced a major phase-advance shift of Per2 oscillations. The mature granulosa cells cultured for 2 days with FSH expressed higher mRNA levels of Per2, Rev-erbα, Bmal1 (Arnt1), Lhcgr, and connexin (Cx) 43 (Gja1) compared with the immature granulosa cells. Consistently, our immunofluorescence results revealed abundant Cx43 protein in antral follicles stimulated with eCG and weak or no fluorescence signal of Cx43 in primary and preantral follicles. Similar results were confirmed by Western blotting analysis. Two gap junction blockers, lindane and carbenoxolone (CBX), significantly decreased the amplitude of Per2 oscillations, which further adhered significant decreases in Per2 and Rev-erbα transcript levels. In addition, both lindane and CBX induced a clear phase-delay shift of Per2 oscillations. These findings suggest that FSH induces the development of the clock system by increasing the expression of Cx43.
authors.affiliation: Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: Huatao
authors.initials: H
authors.lastname: Chen
authors.forename: Lijia
authors.initials: L
authors.lastname: Zhao
authors.forename: Guiyan
authors.initials: G
authors.lastname: Chu
authors.forename: Gakushi
authors.initials: G
authors.lastname: Kito
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Yamauchi
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Shigeyoshi
authors.forename: Seiichi
authors.initials: S
authors.lastname: Hashimoto
authors.forename: Masa-aki
authors.initials: MA
authors.lastname: Hattori
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism; D057906:Circadian Clocks / Q000187:drug effects; D056925:Circadian Rhythm Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018031:Connexin 43 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism*; D058787:GABA-A Receptor Antagonists / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D017930:Genes, Reporter / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D049990:Membrane Transport Modulators / Q000494:pharmacology; D051379:Mice; D056950:Period Circadian Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D055647:Rats, Transgenic; D011974:Receptors, LH / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D006063:Chorionic Gonadotropin; D056925:Circadian Rhythm Signaling Peptides and Proteins; D018031:Connexin 43; D058787:GABA-A Receptor Antagonists; C580592:Gja1 protein, rat; D005938:Glucocorticoids; D049990:Membrane Transport Modulators; C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; D011974:Receptors, LH; D003907:Dexamethasone; D005640:Follicle Stimulating Hormone