Loss of basigin expression in the uterus reduces fertility in female mice

basigin;implantation;decidualizaiton;infertility;mouse model

Li, Kailiang

关键词: basigin;    implantation;    decidualizaiton;    infertility;    mouse model;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/107985/LI-DISSERTATION-2020.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Basigin (BSG), a member of the immunoglobulin superfamily, is a transmembrane glycoprotein expressed in many cell types.It is involved in neurological processes, lymphocyte migration, angiogenesis, tissue remodeling and lactate transport. Our data show that Bsg is expressed in the reproductive tract and is important for fertility in both males and females. I hypothesized that loss of Bsg expression in the uterus reduces fertility in female mice by altering implantation, decidualization, lactate transporters and angiogenesis. In mice, global ablation of Bsg is embryonic lethal, which complicates the studies on the role of BSG in reproduction. To overcome the embryonic lethality, we generated a progesterone receptor (PR)-Cre Bsg conditional knockout (cKO) mouse model and used this animal model to test this hypothesis. The aims of the present study were 1) to validate the Bsg cKO animal model and investigate the fertility phenotype associated with this animal model; 2) to investigate whether BSG is required for luminal epithelial cell and basement membrane integrity at the time of implantation; 3) to examine whether BSG regulates decidualization process in vivo and in vitro; and 4) to investigate whether loss of BSG affects angiogenesis and lactate transport.Successful deletion of uterine Bsg was confirmed by quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC) and immunofluorescence (IF) staining. qRT-PCR indicated much lower Bsg mRNA levels in the cKO uteri compared to the controls. BSG was expressed in the luminal epithelial and stromal cells in the control mouse uteri. In the cKO mouse uteri, Bsg expression was lost in these PR positive cells, and only presented in PR negative cells such as immune cells and endothelial cells. BSG expression in the kidney was not affected, indicating it was PR-positive cell specific. Breeding study results showed that the cKO females had significantly reduced fertility about 40% compared to the controls. They had much smaller litter size and litter frequency. The fertility of the cKO mice decreased more severely as they aged compared to the controls. Ovulation, progesterone production, fertilization and early embryo development were not affected in the cKO mice, suggesting that dysfunction occurred primarily in the uterus. Day 5 pregnant uteri were collected to analyze implantation. The results showed that about 70% cKO mice did not show signs of implantation. In these animals the luminal epithelial cells still showed E-cadherin and cytokeratin at the site of implantation, indicating an intact luminal epithelial cell layer. In contrast, E-cadherin and cytokeratin in the control uteri were lost at the site of implantation, and only presented at the regions of luminal epithelium far from the embryo. The luminal epithelium basement membrane in the cKO mice was intact, not disrupted as in the controls. Many embryos in the cKO mice were restrained in a continuous layer of luminal epithelium, not able to break down the epithelium and penetrate the basement membrane. To investigate whether BSG regulates decidualization, an artificially induced decidualization experiment was performed. I found that the cKO mice had reduced response to the artificial stimulus compared to a robust response in the controls. The decidua size was also significantly smaller in the cKO mice. qRT-PCR results showed that decidual genes Cebpβ and Bmp2 were significantly downregulated in the cKO decidua. IHC results showed that CEBPβ and HAND2 protein expression were lower in the induced decidua and day 5 pregnant uteri in the cKO mice. Mouse endometrial stromal cell (MESC) culture experiments showed there was no difference in cell proliferation, but qRT-PCR results showed that Prl8a2, Cebpβ, Bmp2 and Hand2 were significantly downregulated in the cKO MESCs. These results suggest that BSG is required for proper decidualization in vivo and in vitro.BSG is involved in angiogenesis. To test whether loss of BSG leads to abnormal angiogenesis, day 6 pregnant uteri were stained with the angiogenic marker CD31. There was significantly lower abundance of CD31 in the cKO mouse uteri compared to the controls. BSG is a chaperone protein for the lactate transporter MCT1/4. The abundance and localization of MCT1 were altered in the cKO mouse uteri in vivo. A lactate assay of medium from MESCs collected from both genotypes showed that the cKO MESCs secreted less lactate than the controls. These data indicate that loss of BSG leads to reduction in angiogenesis and lactate transport. Collectively, data in this dissertation suggest that uterine expression of BSG is required for proper implantation, decidualization, angiogenesis and lactate transport and loss of BSG results in severe subfertility in female mice.