Preliminary Analysis Of Uterine Phenotype In Rtcb^flox/flox;Pgr^cre/wt Mice

Uterus is an important organ for the gestation of offspring,and maintaining its normal physiological function is of great importance for the fertility and health of females.The structure of the uterus is mainly composed of the epithelium(luminal epithelium and glandular epithelium),stroma and muscular layer(circular muscle and longitudinal muscle).Structural integrity is pre-requisite for ensuring normal uterine physiologic function.RTCB is an RNA ligase found in post-animals.It is also involved in early embryonic development and antibody secretion.However,there has been no report about the role of Rtcb in the uterus.Previous studies in our laboratory have shown that Rtcb is highly expressed in the uterine epithelium during the pre-implantation period of the embryo and is up-regulated by estrogen.Based on the current facts,this study used Rtcb^flox/flox;Pgr^cre/wt gene knockout mice at 10 and 15 days,8 and 25 weeks of age after birth to study the effect of Rtcb on uterine development,to understand the role of RTCB in the uterus,and to identify human innate Candidate genes for sexual uterine dysplasia provide ideas and have certain guiding significance for the prevention and treatment of related diseases.In this study,Rtcb^flox/flox mice were crossed with Pgr^cre/cre tool mice to obtain female Rtcbflox/flox;Pgrcre / wt knockout mice.The phenotype of the knockout mouse uterus was preliminarily analyzed through immunohistochemistry,Western Blotting,Real-time PCR and other experimental techniques,and the effect of Rtcb on uterine development and function was explored.The main experimental results are as follows:1.25-week-old adult Rtcb^flox/flox;Pgr^cre/wt knockout mice are infertile and have abnormal uterine structure.(1)Sexually mature Rtcb^flox/flox;Pgr^cre/wt female mice and wild-type male mice are caged for 6 months,the total litter size is 0,and they do not have a normal reproductive ability.The knockout efficiency of the Rtcb gene in the uterus of knockout mice was detected from three levels of DNA,RNA,and protein.Our findings indicate that Rtcb was knocked out.(2)The tissue morphology of the uterus of the knockout mouse was analyzed by HE staining and immunohistochemistry.The results showed that compared with the control group,the knockout mice had abnormal uterine structures,with only stroma and longitudinal muscles,but no circular muscles and epithelial tissues.Further analysis of marker proteins expressed specifically in the uterine epithelium,gland,stroma but myometrium showed that compared to wild-type mice,KRT8(epithelium)and FOXA2(gland)were not detected in the uterus of knockout mice),While the levels of expression of Vimentin(stroma)and SMA(muscle layer)decreased.(3)The detection results of cell proliferation and apoptosis in 25-week-old mice's uterus showed no significant difference in the number of cell proliferation and apoptosis in the knockout mice's uterus compared to the wild type,but the apoptosis in the knockout mice's uterus had a significant difference.2.8 weeks old sexual maturity Rtcb^flox/flox;Pgr^cre/wt knockout mice uterine structure abnormalities.(1)HE staining and immunohistochemistry results showed that compared with wild-type mice,the uterine structure of 8-week-old knockout mice was abnormal,similar to the phenotype of 25-week-old knockout mice.Circular muscle and epithelial tissue.(2)The protein expression of KRT8,FOXA2,Vimentin,and SMA in the uterus was identified using western Blotting.KRT8 and FOXA2 were not expressed in the 8-week-old knockout mice,and the expression of Vimentin and SMA decreased.(3)The detection results of cell proliferation and apoptosis in the 8-week-old mice's uterus showed that the number of cell proliferation in the knockout mice's uterus was very small,and in apoptosis,there was a significant difference.3.10 and 15 days after birth Rtcb^flox/flox;Pgr^cre/wt knockout mouse uterus structure is normal.0-15 days after birth is an important period for the differentiating the various types of cells in the uterine structure.To further determine the time at which the abnormality of the uterus caused by the absence of RTCB was examined for expression of RTCB,PGR,KRT8,FOXA2,Vimentin,and SMA by immunohistochemistry and Western Blotting,this study selected day 0 after birth(PND0),PND3,PND5,Uterine tissues of PND7,PND10 and PND15 mice.The results showed that:(1)in the uterus of PND0-15 wild-type mice,RTCB is predominantly expressed in the epithelium,and a small amount is expressed in the stroma and myometrium;the expression patterns of other proteins are consistent with the literature studies.(2)The uterus of PND10 and PND15 knockout mice had normal morphology and complete structure.The expressions of RTCB,PGR,KRT8,FOXA2,Vimentin,and SMA in the uterus of the knockout mice were the same in wild-type mice.(3)The findings of cell proliferation and apoptosis showed that there was no significant difference in the number of cell proliferation in the uterus of PND10 and PND15 knockout mice and wild-type mice,and there was no obvious apoptosis in the uterus of both groups.In summary,this study shows that Rtcb plays a significant role in the structure and physiological function of the uterine mouse.Rtcb knockout results in abnormal development of between 15 days and 8 weeks after birth,but the specific time is yet to be determined by further studies.