The Role Of ADAMTS18 In The Development Of Female Reproductive System In Mice

ADAMTS(A disintegrin and metalloproteinase with thrombospondin motifs)family is a kind of secretory type,multi-domain,Zinc-ion dependent,solution of integrin metal protease family.The main role of these family members is to cut the extracellular matrix(ECM)and thus plays a regulatory role in the physiological processes of tissue remodeling,development,reproduction,inflammation,central nervous system,tumor,angiogenesis and other diseases.Previous studies have shown that ADAMTS family played an important role in the regulation of reproductive phylogeny.The development of reproductive system is a complex process,including embryonic and postnatal development.Mice have two sets of reproductive tubes in early embryonic stage,including the Wolffian duct and the Mullerian duct,in which the Wolffian duct develops into the male reproductive system,including the testes,and the Mullerian duct develops into the female ovaries,fallopian tubes,uterus,and upper vagina.Female C57BL/6 mice’s vagina opens at the 5th week,and it is closed before the 5th week.Vaginal opening plays an important role in the excretion of uterine and cervical secretions and mating of adult mice.ADAMTS18 is an "orphan metalloproteinase" closely related to embryonic development and early organ morphogenesis.Our research group constructed Adamts18 knock out(KO)mice in the previous study.In the breeding process,it was found that fertility of KO female mice decreased significantly,and some KO mice had the symptoms of vaginal atresia.KO mice with symptoms of vaginal atresia were accompanied by uterine effusion.Fertility plays an important role in population reproduction and expansion,so it is of great importance to study the causes of vaginal atresia in mice caused by ADAMTS18 deficiency.Methods: 1)Hematoxylin and eosin(H&E)staining methods were used to compare the morphological differences between WT(wide type)mice and KO mice.2)Quantitative Real-Time-PCR(qRT-PCR)and in situ hybridization(ISH)techniques were used to study the expression of Adamts18 mRNA in the female genital tract of WT mice from 1 week to 6 weeks old.3)Vaginal length of WT mice and KO mice was measured at 1,2 and 5 weeks old.4)MRNA levels of molecules related to vaginal atresia in 1 week old WT mice and KO mice were detected by qRT-PCR.5)E15.5(embryonic day 15.5)WT mice and KO mice embryos paraffin sections were cut to compare whether there were differences on fusion of Mullerian duct.6)TUNEL method was used to detect the apoptosis at the vaginal end of WT mice and KO mice at 5 weeks old to explore the mechanism of vaginal atresia in mice caused by ADAMTS18 deficiency.Results: 1)QRT-PCR results showed that Adamts18 mRNA was highly expressed in the vagina of mice from 1 week to 5 weeks,expressed in the ovaries and fallopian tubes of mice at 1 week,and rarely detected in the ovaries and fallopian tubes of mice from 2 weeks to 6 weeks and in the uterus of mice from 1 week to 6 weeks.2)In in situ hybridization,Adamts18 mRNA showed the highest level of expression in the epithelial and muscular layer of the vagina of mice at 1 week,and sustained expression in the vagina of mice at 1 week to 5 weeks,suggesting that it played an important role in vaginal development.3)There was no significant difference in vaginal length between 1 week mice and 2 weeks mice,and vagina of 5 weeks vaginal atremia mice were significantly shorter than those of 5 weeks WT mice 4)At 2 and 5 weeks old,the appearance of vagina of KO mice was deformed,and there was obvious swelling in 2/3 of the vagina.5)Compared with WT mice,expression level of Bcl-2 mRNA in vagina of KO mice at 1 week increased significantly,suggesting that apoptosis of vaginal cells in KO mice at 1 week decreased.6)Analysis of paraffin sections of E15.5 mouse embryos showed that some KO mice with vaginal atresia may have problems in the fusion of Mullerian ducts in the embryonic development stage,and the two Mullerian ducts were not fused together.7)Apoptosis detection showed that apoptosis of vaginal terminal cells in KO mice was reduced at 5 weeks compared with WT mice.In summary,ADAMTS18 deletion has a two-stage effect on the development of female mouse reproductive system: 1)At embryonic stage,some KO mice have an obstruction to Mullerian duct fusion,which may result in formation of a vaginal barrier in postembryonic mice;2)After birth,ADAMTS18 plays an important role in vaginal elongation of mice,which may influence the apoptosis of vaginal terminal cells at 5 weeks,thus forming a phenotype of vaginal atresia in KO mice.This study expands the function of ADAMTS18,reveals important role of ADAMTS18 in development of female reproductive system in mice,and provides an idea for clinical treatment of female vaginal atresia and other diseases.