Expression of Matrix Metalloproteinases in Rat Uterus During Implantation Phase.

Abstract

In rodents, implantation involves a direct interaction between embryo and uterus that is initiated by a recognition of the hatched, free embryo, followed by membrane apposition. Then, adhesion between the trophectoderm-trophoblast and endometrial luminal epithelium occur. Finally, the embryo passes through the luminal epithelium and epithelial basement membrane and implants in the endometrial stroma. During implantation, dramatic tissue remodeling occurs in the endometrium, and it is thought that matrix metalloproteinases (MMPs) are the key regulators in this process. MMPs are the enzymes that degrade components of the extracellular matrix and are members of the Zinc Metalloproteinase family, having 28 distinct isoforms. There are several studies showed that MMPs were significantly expressed in the rat uterus during the implantation phase. However, the expressional profile of MMPs and its regulation function is not fully elucidated. Additionally, the expression of MMPs during decidualization which is one of the important physiological process in implantation, are still not fully understood. The purpose of the present study is to investigate the expression of MMPs during the implantation phase in the rat uterus. We firstly analyzed the expressional profile of MMPs during the implantation phase. Then the activity of MMPs during decidualization was analyzed by zymography using in vitro decidualization system of the cultured rat endometrial stromal cells (RES). Uteri were obtained from sexually mature rats during the estrous cycle and various stages of early pregnancy (Day 1.5, 3.5, 5.5 and 7.5 of gestation). Total RNA was collected from these uteri. The expression of ß-actin and MMPs (MMP-1, -2, -3, -7, -9 and -11) mRNA were examined by reverse transcription-polymerase chain reaction (RT-PCR). The expression patterns of MMP-2, -3 and 9 mRNA were evaluated at each stage by quantitative real time RT-PCR. Quantitative real time RT-PCR was carried out using a Roche Light Cycler 1.5 system (Roche). The Taqman Master kit (Roche) in combination with the Universal Probe Library for Rat (Roche) was used to assess gene expression. In vitro decidualization of the RES was introduced by the treatment of medroxyprogesterone acetate (MPA). After the treatment for 24h, conditioned medium was collected and the production of MMPs were measured by gelatin zymography. The quantitative measurement of MMPs was performed by densitometric analysis of zymogram using an ImageJ program (Version 1.36). Six MMPs (MMP-1, -2, -3, -7, -9 and 11) mRNA were expressed at estrus cycle and early pregnancy. Expression of MMP-2, -3 and -9 mRNA was not significantly changed during the estrus cycle and early pregnancy. Additionally, there were no significant differences in the expressions of MMP-2 and -9 mRNA during in vitro decidualization. However, the active form of MMP-2 and -9 was significantly increased after the treatment for in vitro decidualization of the RES. These results indicate that the function of MMPs during the implantation phase is not regulated by mRNA expression, but by the activation of MMPs. It is also suggested that RES produces some factor or factors that can activate MMPs during decidualization. This research was supported by a grant from the Ministry of Education, Culture, Sport, Science, and Technology of Japan (Kiban-kenkyu C 18580282).