PGF-induced Expression And Localization Of MAPK And Immediate Early Genes(IEGs;ATF3,NR4A1 And NR4A2) In Pseudopregnant Rat Corpus Luteum

The CL plays a central role in the regulation of ovarian cyclicity and maintenance of pregnancy through biosynthesis and secretion of P4.If fertilization does not occur,the CL undergoes regression to prepare for the next ovulation.In rodents,luteal demise is characterized by marked decrease in P4 production(functional regression)followed by apoptotic death(structural regression).In this study,techniques such as HE,ELISA,IHC,RT-PCR,real-time PCR and Western blot were used to analyze prostaglandin F2α analogue(PGF)-induced MAPK and immediate early genes(IEGs;ATF3,NR4A1 and NR4A2)during luteal regression in rats.The main contents are as follows:1.PGF induces expression of activating transcription factor 3(ATF3)and activates MAPK signaling in the rat corpus luteum.The current study was conducted to evaluate the expression of ATF3,in association with the activation of mitogen-activated protein kinases(MAPK)during PGF-induced luteal regression in rats.A sequential PMSG/hCG treatment paradigm was used to obtain a single,well-defined generation of corpora lutea(CL)in rats.Rats were treated with PGF for 0-4 h on day 7 of pseudopregnancy.Results showed that serum progesterone(P4)concentrations declined in a time dependent manner.Western blot results revealed that ATF3 increased within 2 h post-PGF injection(P<0.01).Phosphorylated ERK1/2(p-ERK)and JNK(p-JNK)increased within 30 min(P<0.01)and then were gradually reduced in response to PGF.In contrast,the levels of phosphorylated p38 MAPK(p-p38)were not significantly altered.The immunostaining density for p-ERK decreased from the periphery to the center of the corpus luteum following treatment with PGF,while ATF3 was expressed uniformly in the nuclei of luteal steroidogenic cells.These results indicate that treatment with PGF could induce increases in MAPK phosphorylation,especially in p-ERK,which might be correlated with the increases in ATF3 expression and the decline in P4 concentrations.2.Expression and localization of NR4A1 and NR4A2 in PGF-induced degradation of corpus luteum in rats.To investigate the expression and localization pattern of nuclear receptor NR4A1 and NR4A2 in PGF-induced degradation of corpus luteum in rats,we set a model of pseudopregnancy rat.Rats were treated with PGF for 0-4 h(0 h,30 min,1 h,2 h and 4 h)on day 7 of pseudopregnancy.RT-PCR and real-time PCR analysis were used to detect the expression of ovarian NR4A1 and NR4A2 mRNA.IHC analysis was used to localize NR4A1 and NR4A2.The results of IHC analysis revealed that NR4A1 protein mainly expressed in the cytoplasm of luteal cells,the oocytes and theca cells.After PGF treatment,NR4A1 mRNA levels increased first and then decreased,and the peaked expression were in about 30 min-1 h which were significantly different(P<0.01),compared with the saline group.Meanwhile we found that NR4A2 was unevenly distributed in CL,and mainly expressed in the cytoplasm of luteal cells.However,the levels of NR4A2 mRNA about 30 min-1 h after PGF treatment were consistent with NR4A1 mRNA,which were significantly different(P<0.01),compared with the saline group.Moreover,the expression of NR4A1 was more susceptible than NR4A2.These results indicate that the nuclear receptor NR4A1 and NR4A2 are closely related to PGF-induced degradation of corpus luteum in rats.