Primary Study On The Expression Of RGMb And Its Role In The Hu Ewe Uteri

RGMb is one of members in RGM family.As the co-receptor of BMP2/4,RGMb enhances the signal transduction of BMP which is involved in the regulation of the uterine function.Hu ewes,the local sheep in Jiangsu and Zhejiang area of our country,are famous for the high fecudity.In this research,the tissue expression of RGMb in Hu ewe were firstly detected by qPCR and IHC.Secondly,the effect of food restriction on the expression of RGMb and members of BMP system were detected by qPCR and WB.Finally,the effect of RGMb on the viability of endometrial cells and its mechanism were examined by techniques such as cell transfection.The main contents are as following:1.The tissue expression of RGMb and localization of RGMb in the uteri of Hu ewesTo investigate RGMb the sequence of CDS in Hu ewe,RNA from the uterus of Hu ewe on day 110 of pregnancy were collected to analyze the sequence of CDS in Hu ewes by RT-PCR combined with sequencing.To investigate the tissue expression of RGMb in Hu ewes,a total of 15 tissues such as kidney,pituitary,ovary and uterus were collected from 3-month-old female Hu ewes(n=3)to analyze the expression of RGMb by qPCR.To investigate the localization of RGMb protein in the uterus of Hu ewes,the uteri of 3-month-old,7-month-old and pregnant Hu ewes(D110)were collected to detect the localization of RGMb protein by IHC.The sequences were compared with those of mice,rats,pigs and cattle,respectively,and results showed that the homology were more than 80%.RGMb mRNA was detected in 14 tissues of Hu ewes,the relative expression of RGMb was greater than 0.5 in uteri when the relative expression of RGMb in hypothalamus was set as 1.IHC staining showed that RGMb was strongly expressed in the endometrial luminal and glandular epithelial cells,and weakly expressed in the circular muscle.In conclusion,RGMb can be expressed and localized in the uteri of Hu ewes,which provides a theoretical basis for further study on the RGMb in the regulation of uterine funtion in Hu ewes.2.Impact of food restriction on the expression of RGMb and BMP system members in the uteri of Hu ewesThe effects of food restriction on the expression of RGMb and BMP system members were explored in Hu ewes.Hu ewes on day 35 of pregnancy(D 35)were divided into two groups(n=8):ewes in the control(C)group were fed with food and water ad libitum and ewes in the restriction(R)group were one-half of the total complete diet of the control group.At D 110,uteri(n=8)were collected to evaluate the expression of BMP2,BMP4,BMPR1A,BMPR1B,BMPR2 and RGMb by qPCR and WB.Results showed that the uterine RGMb in the R group was significantly lower(P<0.05)than that in the C group,while there was no significant differences in RGMb mRNA(P>0.05).The expression of BMP4 and BMP2 mRNAs in the R group were both higher(P<0.05)than those in the C group.However,the expression of BMP receptors(BMPRIA,BMPR1B and BMPR2)were significant decreased in the R group(P<0.05).In conclusion,food restriction affects the expression of BMP system members,especially the RGMb protein level.3.Effect of RGMb down-regulation on the endometrial glandular epithelial cells and its primary mechanismTo investigate the effect of RGMb on the endometrial glandular epithelial cells and its mechanism,Ishikawa cells were transfected with siRGMb.The cell viability was detected by CCK-8,and members of MAPK signaling were dectected by WB.The results showed that Ishikawa cell viability of RGMb knockdown was significantly lower than the control cells(P<0.05).The level of p-ERK1/2 protein was significant down-regulated in the RGMb konckdown Ishikawa cells(P<0.05),while there was no significant changes in the level of p-P38(P>0.05).In conclusion,RGMb affects the cell viability of Ishikawa and p-ERKl/2 level,which provides a theoretical basis for further study on the RGMb in the regulation of the uterine funtion of Hu ewes.