Primary Study On The Roles Of NR4A1/2 And AdipoR/AMPK In The Corpus Luteum Of Pseudopregnant Rats And Pigs

The corpus luteum(CL)is a transient endocrine gland that forms from the mature follicle ovulation.CL formation,development and regression are vital for the breeding ability in domestic animals.Several studies have indicated that NR4A1/2 play important roles in the hormone synthesis,secretion and apoptosis in ovarian cells.Recent studies have found that adiponectin also plays an important role in the mammalian reproduction and cell apoptosis through AMPK,PPAR and p38MAPK pathways.In this study,techniques such as HE,RIA,IHC,real time-PCR and WB were used to study the expression of PGF-induced NR4A1-TV and NR4A2 in the CL of pseudopregnant rats and the expression pattern of NR4A1/2,AMPK and AdipoR during porcine luteal development and regression,which provides a basis for the further study on NR4A1,NR4A2,AMPK and AdipoR involved in the regulation of the luteal function.Major contents are as below:1 PGF induces the expression of NR4A1/2 in the CL of pseudopregnant ratsTo study the roles of NR4A1 and NR4A2 during the prostaglandin F2α analoge(PGF)-induced luteal regression.A sequential PMSG/hCG treatment paradigm was used to induce pseudopregnancy in rats.Rats were then treated with PGF for 0-8 h on day 7 of pseudopregnancy.Rats were killed after treatment with PGF for 0 h,0.5 h,1 h,2 h,4 h and 8 h by cervical dislocation.Ovares were collected to detect the expression of NR4A1/2 by WB and real time-PCR.Results showed that PGF injection did not change the ovarian luteal NR4A1-F expression within 8 h(P>0.05).However,PGF treatment increased the expression of NR4A1-transcript variant(NR4A1-TV)at 0.5 h which peaked at 2 h and returned to low expression at 8 h.The expression pattern of NR4A2 protein was similar to NR4A1-TV protein.The NR4A1-P1 mRNA increased significantly in 0.5 h(P<0.05),sustained at a high level at 1 h,and then decreased.While no changes were found about NR4A1-P2(primer 2)mRNA.All these results suggested that PGF-induced NR4A1-TV and NR4A2 might be involved in the luteal regression of pseudopregnant rats.2 Primary study on the role of NR4A1/2 during porcine luteal development and regressionBased on the natural porcine ovarian corpus luteum,the expression pattern and localization of luteal NR4A1/2 were analyzed to investigate the potential role during the development and regression of the corpus luteum.Porcine ovaries at different stages were collected to isolate the luteal tissues(early phase,middle phase and late phase).RIA was used to determine the progesterone and estrogen concentrations.WB was used to analyze the expression pattern of NR4A1/2 and CYP17A1 and cleaved-Caspase-3.Real time-PCR was used to detect the expression of NR4A1 mRNA.IHC was applied to localize the NR4A1 and cleaved-Caspase-3.RIA results showed that the progesterone level of late luteal tissue was lower than that of the middle corpus luteum,while estrodiol was higher than that of late corpus luteum(corpus albicans)(P<0.05).WB results showed that the CYP17A1 protein expression increased significantly in the late corpus luteum(P<0.05),and no changes were found between early and middle corpus luteum(P>0.05).NR4A1 as well as NR4A2 expression has no significant differences between early(corpus haemorrhagicum)and middle corpus luteum(P>0.05),while both decreased significantly(P<0.05)in the late corpus luteum.Cleaved-Caspase-3 expression level was the lowest in middle corpus luteum,while it was the highest in the late corpus luteum(P<0.05).Real time-PCR results showed that the NR4A1-P3/4 expression pattern was similar and the expression levels in middle CL were much higher than those in the early and the late luteal stages.IHC results showed that the NR4A1 mainly localized in the cytoplasm of luteal steroidogenic cells,while cleaved-Caspase-3 expressed in the cytoplasm of nearly all kinds of luteal cells including steroidogenic cells.All these results suggest that NR4A1 and NR4A2 might be involved in the regulation of progesterone metabolism in porcine corpus luteum,which provides a theoretical basis for the study on NR4A1/2 in the regulation of porcine ovarian function.3 The expression of AdipoR/AMPK during porcine luteal development and regressionBased on the natural ovarian luteal and non-luteal tissues,the expression pattern of AMPK and AdipoR were analyzed to study their potential roles during the development and regression of corpus luteum in pigs.Porcine ovaries at different luteal stages were collected to isolate the luteal tissues and non-luteal tissues,then real time-PCR and WB were used to detect the AMPK and AdipoR expression.The WB results showed that the level of marker protein of luteal steroidogenic cells-StAR protein was the highest in the middle CL,and the lowest in the late CL.No significance was found in the luteal AdipoRl expression at different luteal stages,while the non-luteal AdipoRl expression in the early and middle luteal stage were higher than that in the late luteal stage.The PCR results showed that the luteal AMPK mRNA and AdipoR mRNA levels were the lowest in late CL.The non-luteal AMPKa1 mRNA level was the highest in the late CL stage.AMPKa2 mRNA level was the highest in middle CL.AMPKβ1 mRNA was the highest in middle non-luteal tissues.AMPKγ1 mRNA was the highest in early non-luteal tissues.AMPKγ3 mRNA was the highest in early non-luteal tissues.AdipoRl mRNA was the highest in middle non-luteal tissues.AdipoR2 mRNA was the highest in middle CL(P<0.05).In conclusion,the alternations of AMPK and AdipoR during different stages suggest they might be involved in the luteal development and regression,which provides basic theoretical support for the regulation of porcine luteal function.