Effects Of Prostaglandin PGF_2? And Clock Gene Bmal1 On Prostaglandin F Receptor Gene Expression In Chicken Uterine Epithelial Cells

Circadian rhythm affects many physiological processes in animals,including cardiovascular system,energy metabolism,immunity,hormone secretion,and cell regeneration.The mechanism of hormone ligand-receptor and circadian clock regulating the expression of prostaglandin F receptor gene?PTGFR?in chicken uterus was studied from the perspective of biological cycle change.The effects of clock genes and hormone on the expression of hormone receptor gene were analyzed,and the potential regulatory relationship between clock genes and PTGFR gene was unraveled.In our study,Lohmann layers during the growth and peak laying period were selected as experimental materials,and also explore the morphological changes of uterus under different physiological conditions.The dynamic changes of prostaglandins?PGs?,estradiol?E₂?,follicle stimulating hormone?FSH?,and luteinizing hormone?LH?in blood were detected.The relationships among the change of hormone concentration,expression of hormone receptor genes,and clock genes expression abundances in the two critical periods were studied.Uterine epithelial cells were cultured in vitro,primary cells were identified by immunohistochemistry,and cell growth curve was determined.The cells were treated with different concentrations of hormones,CCK8 kits was used to detect the effect of hormones on proliferation of uterine epithelial cells in vitro.At the cellular level,siRNA interfered with the core clock gene Bmal1.Real-time fluorescence quantitative detection of the clock genes Bmal1 and Per2,and time-controlled gene Rev-erb and hormone receptor gene PTGFR expression levels in the experiments,while exploring the effects of PGF2?hormone on the clock genes expression.The results are as follows:?1?Layers laid eggs from 8:00 to 10:00 a.m.Plasma reproductive hormones of hens change regularly in different periods of the day.The content of reproductive hormones in the laying period is generally higher than that in the growing period.The change of hormone receptor gene expression is consistent with that of corresponding hormones.Except for LH hormone,other three hormones do not fluctuate greatly in the growing period.The content of PGs in the laying period was significantly higher than that at the growing period,and the expression of PTGFR was significantly increased before and after laying eggs?P<0.05?,compared with other time points;FSH and E₂ were significantly increased after laying eggs?18:00??P<0.01?.?2?HE staining of tissue sections showed that the morphological changes of uterus during laying period mainly as follows:the thickness of endometrium increased at ZT8?14:00?and the muscular layer decreased at ZT4?10:00?,the thickness of uterine wall increased at ZT16-ZT20?22:00⁰2:00?,but the thickness of serosa did not change much.?3?The uterus epithelial cells of laying hens were successfully cultured,and the effects of PGF₂?and E₂ hormones on cell proliferation were determined.Among them,50 nM of PGF₂?and 10 nM of E₂ could significantly?P<0.05?promote the proliferation of uterine epithelial cells in vitro.However,the effect of combined treatment of the two hormones was not obvious?P>0.05?.When treated with 100 nM PGF₂?,compared with the control group,the expression of Bmal1 in uterine epithelial cells decreased significantly at 0²4 h?P<0.05?,Per2 mRNA was the opposite,and the amplitude of Per2 increased significantly.The expression of PTGFR decreased significantly?P<0.05?or remained unchanged?P>0.05?,while the expression of Bmal1 and Per2 did not change significantly at 24⁴8 h?P>0.05?.However,the expression of PTGFR increased significantly?P<0.05?or remained unchanged?P>0.05?.After treatment with PGF₂?for 0²4 h,the expression of PTGFR protein was increased and expressed rhythmically.?4?The rhythm model of uterine epithelial cells in vitro was successfully established by the treatment of serum shock,and the effect of clock gene on the expression of PTGFR gene was explored.Serum shock caused regular expression of PTGFR,and the expression of PTGFR significantly decreased?P<0.05?after interference with the clock gene Bmal1,suggesting that the clock gene can regulate the expression of PTGFR.In summary,the clock genes can regulate PTGFR expression;PGF₂?can regulate PTGFR and clock gene expression.The change of PTGFR expression is the result of the regulation of both.