| Blastocyst implantation is a key step for successful establishment and maintenance of pregnancy during mammalian reproduction. Successful implantation is dependent upon the interaction between the blastocyst and the uterus. The most fundamental feature of this process is the synchronized development of the embryo to the blastocyst stage and differentiation of the uterus to the receptive state. Angiogenesis are crucial to successful implantation, because administration of AGM-1470 (a nonspecific angiogenesis inhibitor) to pregnant mice resulted in implantation failure due to impaired decidualization and vascular development in the placenta.Angiopoietin (Ang) is another discovered endothelial cell-specific pro-angiogenic factor family discovered after VEGF. This family includes four members withTie-2 as their co-receptor. According to our previous data of implantation and decidualization gene chips, the expression of Ang-2 and Ang-3 was significantly up-regulated compared with control group. The aim of this study was to investigate the expression of Ang-2 and Ang-3 expression in mouse uterus during early pregnancy and pseudopregnancy, delayed implantation and artificial decidualization, and its regulation by steroid hormones using in situ hybridization and RT-PCR. The expression of Ang-2 and Ang-3 in the oocyte and embryos at the zygote, 2-cell, 4-cell, 8-cell, morula and blastocyst stages was also determined by RT-PCR. Furthermore, we examined Ang-2 and Ang-3 expression in mouse ovary during sexual maturation, follicular development induced by eCG, ovulation induced by hCG and corpus luteum formation and regression.There was no detectable Ang-2 mRNA signal in mouse uterus on days 1-5 of pregnancy. On day 6 of pregnancy, a low level of Ang-2 mRNA signal was found in the decidual cells surrounding the embryo. On day 7 of pregnancy, Ang-2 mRNA was distinctly expressed in the primary deciduas of mesometrial side. The pattern of Ang-2 expression on day 8 was similar to that observed on day 7 but at much higher level. To verify Ang-2 expression, RT-PCR was also performed. A significantly high level of Ang-2 expression was detected on day 8 of pregnancy, although Ang-2 expression was seen throughout days 1–8. No detectable Ang-2 mRNA signal was found in the uterus from days 1 to 5 of pseudopregnancy, delayed uterus and activated implantation sites by in situ hybridization. However, Ang-2 expression was detected throughout days 1–5 of pseudopregnancy by RT-PCR and highly expressed on day 3. Compared with the delayed uterus, Ang-2 mRNA was highly expressed in activated implantation sites. Ang-2 mRNA was strongly detected in the decidualized cells under artificial decidualization, while Ang-2 mRNA signal was not detected in the uninjected control uterus. Furthermore, the RT-PCR result was consistent with the above results. The levels of Ang-2 mRNA showed a gradual increase after estrogen injection and with peak levels at 12h. Progesterone injection resulted in a decline in uterine Ang-2 mRNA levels that reaching a nadir by 12h. These results indicated that estrogen induced the expression of Ang-2, while progesterone inhibited it. This was further confirmed by the injection of estrogen plus progesterone subsequently. To determine the effect of estrogen and progesterone on Ang-2 expression was mediated via their nuclear receptors, the ovariectomized mice were injected with estrogen plus antiestrogen (ICI 182,780) and progesterone plus antiprogestin (RU486), and the control group was only injected with estrogen and progesterone respectively. The results showed that Ang-2 expression significantly decreased after 12h of injecting estrogen plus ICI 182,780, while the Ang-2 expression was significantly increased after 12h of injecting progesterone plus RU486. While the in situ hybridization results showed that Ang-2 expression could only be detected in the uterine luminal epithelium and glandular epithelial cells after 12h of injecting estrogens. Ang-2 mRNA was not detected in the oocytes and preimplantation mouse embryos. These results suggested that Ang-2 participated in the process of mouse embryo implantation and decidualization, and was regulated by the activated embryo and itself. Estrogen could induce Ang-2 expression through its receptor, while progesterone could inhibit its expression through its receptor.From days 1 to 5, there was no visible Ang-3 mRNA signal in the uterus. On day 6 of pregnancy, a low level of Ang-3 mRNA signal was also found in the primary deciduas of mesometrial.side. Ang-3 mRNA expression gradually increased along with the development of decidua, and the expression scope was also expanded. On day 8 of pregnancy, Ang-3 mRNA was strongly detected in the primary and secondary decidualized zone of mesometrial side. The results of RT-PCR indicated that Ang-3 mRNA was slightly expressed in mouse uterus on days 1-4 of pregnancy, and the expression was increased on days 5 of pregnancy (time for embryo implantation), continuously and gradually increased and reached the highest level on day 8 of pregnancy with the process of decidualization. Moreover, the expression of Ang-3 mRNA on the implantation site on day 5 was significantly higher than that of the non-implantation site. The results of in situ hybridization showed that Ang-3 mRNA was not expressed in the uterus from days 1 to 5 of pseudopregnancy, as well as delayed implantation uterus and activated implantation sites. While the result of RT-PCR showed that Ang-3 was expressed with a low level on 1-5 days of pseudopregnant mouse uterus, and with the highest level on day 3 of pseudopregnancy. Ang-3 mRNA was highly expressed in activated implantation sites compared with the delayed uterus. Ang-3 mRNA was strongly detected in the decidualized cells under artificial decidualization, while no detectable signals in the uninjected control uterus. Furthermore, the RT-PCR result was consistent with the above results. After injection of estrogen, progesterone and estrogen plus progesterone, the expression of Ang-3 was gradually increased in ovariectomized mice uterus and reached the highest level at 12 hours, indicating that estrogen and progesterone induced the expression of Ang-3.To determine whether the effects of estrogen and progesterone on Ang-3 expression was mediated via their nuclear receptors, the ovariectomized mice were injected with estrogen plus ICI 182,780 and progesterone plus RU486, and the control group was only injected with estrogen and progesterone respectively. The results indicated that Ang-3 expression was significantly decreased after 12h of injecting ICI 182,780, while no significant changes was occurred after 12h of injecting RU486. While in situ hybridization results showed that the expression of Ang-3 was not detected in the mouse uterus after injection of estrogen, progesterone, estrogen plus progesterone. Ang-3 mRNA was also not detected in the oocytes and preimplantation mouse embryos. Taken together, Ang-3 is involed in the process of mouse embryo implantation and decidualization, and was regulated by the activated embryo and itself. Estrogen could induce Ang-3 expression through its receptor, while whether progesterone could induce Ang-3 expression through its receptor still needs to be further addressed.Ang-2 mRNA was expressed in granular cells and theca cells of developing follicle. In mature follicles, Ang-2 mRNA was only expressed in theca cells at low level. However, Ang-2 was highly expressed in granular cells of atretic follicles. This suggested that Ang-2 may play some roles in follicular development, atresia and ovulation process. Simultaneously, Ang-2 was also expressed in the corpus luteum after ovulation, indicating that Ang-2 may be involved in corpus luteum formation and regression. Ang-3 was not detected in the mouse ovary during sexual maturation, follicular development, ovulation and corpus luteum formation and regression.In summary, Ang-2 and Ang-3 are closely related to mouse embryo implantation and decidualization. Estrogen can induce the expression of Ang-2 and Ang-3 while progesterone inhibits the expression of Ang-2 in the ovariectomized mouse uterus. Ang-2 may play a role during mouse follicular development, ovulation and corpus luteum formation and regression.
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