The Center for Animal Resources and Development (CARD) at Kumamoto University was established in 1998 as a mouse bank for the collection, archiving and supply of genetically modified mice. Previously, we reported on the viability of genetically modified mouse embryos cryopreserved for 10 years and confirmed that live pups were obtained from the frozen embryos. In this study, we evaluated the effect of storing cryopreserved mouse embryos for 20 years on their viability and developmental ability,

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... as well as the percentage of pups with transgenes. We compared a short-term storage group, a 10-year storage group, and a 20-year storage group in 10 strains of genetically engineered mice. The percentage of morphologically normal embryos after thawing, the percentage of live pups, and the percentage of live pups with transgenes in the 20-year storage zone ranged from 77.6% to 92.5%, 18.0% to 66.7%, and 28.6% to 77.8% respectively. These results indicate that it is possible to produce live pups carrying transgenes from two-cell embryos taken from genetically modified mice and cryopreserved for 20 years. The process of implantation consists of apposition of the blastocyst, attachment to the uterine epithelium and decidualization of the uterine stroma. Leptin and its receptor play a role in female reproduction, and previous reports have shown that leptin signaling enhances blastocyst attachment. However, its functional role in decidualization remains unclear. In this study, we investigated in detail the expression of LepR in the uterus during implantation of mouse embryos. C57BL/6 female mice were mated with fertile or vasectomized males to induce pregnancy or pseudopregnancy (D1, vaginal plug). The uterine tissue was collected on the D4-8 of pregnancy. Artificial decidualization was induced by intraluminally infusing of sesame oil into one uterine horn on D4 of pseudopregnancy. After 48 hrs, the uteri were collected, and used to analyze the expression of LepR by RT-PCR, western blot, and immunohistochemistry (IHC). LepR mRNA levels were maximal on D4 of pregnancy, then gradually declined during decidualization. In contrast, protein levels increased from D4 to D8 of pregnancy. Consistent with these results, LepR mRNA levels were low, while protein levels high in artificially induced decidua. Furthermore, LepR protein signals were detected in the decidual tissue by IHC.These results suggest that leptin signaling also play an important role in peri-implantation period and contribute to decidualization.