Spatiotemporal Expression And Function Of Metastasis Inhibitory Factor CD82/KAI1 Gene In Uteri Of Pseudopregnant And Implantation Delayed Mice

Objective: Embryo implantation is an important and complicated physiological process in viviparous mammalian and human reproduction. Trophoblastic cells invading into endometrium and forming the placenta are key steps in embryo implantation. Comparative research shows that there are striking similarities in behavioral patterns between invasive trophoblastic cells and cancer cells. CD82/KAI1 gene, one member of tumor metastasis suppressor factors, plays an important role in inhibiting invasion and metastasis of cancer cells. To better understand CD82/KAI1 function in embryo peri-implantation, we investigated its expression in uteri of pseudopregnant and implantation-delayed mice, and pre-implantation embryos by RT-PCR, immunohistochemistry. Moreover, in vitro culture of embryos with and in vivo uterine horn injection of CD82/KAI1antibody were conducted.Methods: 1. pseudopregnant mice. Adult Kunming female mice (6-8 weeks old) were mated with vasectomized males of the same strain. The day vaginal plug is positive in the morning is designated as D1. From D1 to D8, mouse whole uteri were collected for RNA extraction and frozen sectioning. 2. implantation- delayed mice. Pregnant mice were produced by mating with fertile males. To induce and maintain delayed implantation, mice were ovariectomized at 08:00–09:00 on the morning of D4 and received daily injection of P4 (1 mg/mouse) from D5 to D7. To terminate delayed implantation and induce blastocyst activation, those P4 primed, delayed implanting mice were given an injection of E2 (100 ng/mouse) on the third day of the delay (D7). Mice were killed 24 h later after treatment with the respective steroid hormones. Pregnancy was confirmed by recovering embryos from one uterine horn. Active implantation was identified by visually detectable implantation site after Trypan blue dye injection. 3. adult female mice were superovulated by an intra-peritoneal injection of PMSG followed by a single injection of hCG 48 h later, and then mated with fertile males of the same strain. Embryos (2-cell, 4-cell, 8-cell, morula and blastula) were collected from reproductive tracts for mRNA extraction and immunofluorescence test. 4. 8-cell embryos were in vitro cultured with several concentrations of CD82/KAI1 antibody, percentage of embryo cleavage, blastocyst hatching and total cell number of blastocyst were observed. 5. one side uterine horn of D4 mouse was injected with CD82/KAI1 antibody while the other side was injected with serum as control. Number of implantation sites were examined on D8. Results: 1.CD82/KAI1 mRNA was expressed in pseudopregnant mouse uterus during D1-8, and it was dramatically abundant on D4 and D5. CD82/KAI1 protein was expressed mainly in stromal cells and glandular epithelial cells during D1-8. No expression was detected in luminal epithelial cells during D1-4 while a gradually increased expression was observed in luminal epithelial cells from D5 to D8. 2.Expression of CD82/KAI1 mRNA was markedly decreased from D4 to D8 in delayed-implantation, but it increased after estradiol injection. CD82/KAI1 protein was weakly positive on D5, but negative on D6-D8, but strong positive in stroma-cells (decidual cells) after estradiol injection. 3.CD82/KAI1 mRNA was expressed in all stages of pre-implantation embryos and significantly intensive at morula and blastula stages. Consistently, its protein was detected in cell membrane and cytoplasm of all stage embryos. Interestingly, it was strongly expressed in nuclei of blastomeres at morula stage. 4.CD82/KAI1 antibody significantly inhibits 8-cell embryos developing into blastocysts (1: 400, P < 0.05), blastocysts hatching from zona pellucida (1: 800, P < 0.05), and decreases total cell number of each blastocyst in a dose-dependent manner. 5.More implanted embryos were observed on D8 in the injected horn with CD82 antibody compared with the control (8.35±0.17 vs 4.52±0.24, P<0.05).Conclusions: 1.Expression of CD82/KAI1 in uterus was embryo-independent in pregnant mice. 2.Expression of CD82/KAI1 in mouse uterus might be regulated by estrogen. 3.CD82/KAI1 could promote embryo development and inhibit embryo implantation in mice. Overall, CD82/KAI1 might partcipate in regulating in embryo implantation in mice.