Background: In order to study the novel genes related to rat embryonic implantation, a novel implantation-associated gene, Iag-1, was identified and characterized from rat uterus of early pregnancy. Iag-1 was initially derived from suppressive subtracted hybridization of a cDNA library of rat uterus, which was used to analyse differentially expressed genes between the preimplantation and implantation period.
Methods: The full-length cDNA sequence of Iag-1 was cloned from rat uterus on D5.5 of pregnancy by 5'- and 3'-RACE. The expression of Iag-1 in the uterus of early pregnancy, pseudopregnancy, artificial decidualization and activation of delayed implantation was detected by northern blotting, in situ hybridization, western blotting and immunofluorescence. Endometrial stromal cells (ESCs) were isolated from rat uterus. The effect of Iag-1 on ESCs proliferation and apoptosis were determined by MTT assay, TUNEL and Hoechst staining. Apoptosis-related proteins in ESCs were detected by western blotting.
Results: Differential patterns of Iag-1 expression were detected in rat embryo and in the uterus during the peri-implantation period. Iag-1 was specifically localized in glandular epithelium and luminal epithelium. In contrast, the expression of Iag-1 was not significantly altered in uterus of pseudopregnancy and artificial decidualization, but was significantly increased in the uterus after activation of delayed implantation. Stable expression of introduced Iag-1 inhibited the proliferation of in vitro-cultured ESCs. Significant apoptosis was also detected in the ESCs overexpressing Iag-1, along with the enhancement of p53 and Bax protein expression.
Conclusions: Overexpression of Iag-1 can inhibit ESCs proliferation and induce ESCs apoptosis, and p53 and Bax may play an important role in the process of Iag-1-induced apoptosis.