PARENT SESSION
Sex Determination

MALE GONADAL STEM LIKE CELLS ORIGINATED FROM MIGRATED MESONEPHRIC CELLS.

Nishino, Koichiro^1,3, Sato, Naoko¹, Tanaka, Satoshi¹, Tojo, Hideaki², Shiota, Kunio¹, ^{1 3 2}

ABSTRACT- In mouse embryo, morphological differences between males and females in the fetal gonads are the first visible as a formation of testicular cords at 12.5 days post coitum (dpc). The migration of mesonephric cells from adjacent mesonephros plays an important role for the formation of testicular cords during testis development. In our previous study we found the three types of morphology among the migrated mesonephric cells in vitro: these are a fibroblastic cell type, a round cell type, and a cluster-forming small cell type. Of these, the cluster-forming small cells are probably male gonadal stem like cells. In the present study, we tried to establish the gonadal stem cells from the migrated mesonephric cells. The migrated mesonephric cells was observed as Enhanced Green Fluorescent Protein (EGFP)-positive cells in the EGFP-negative XY gonads through the use of our organ coculture system by using EGFP-transgenic mice. The living migrated cells including the three cell types were FACS sorted from the XY gonads by taking advantage of EGFP expression. Expression of desmin only in the fibroblastic cells suggested that they are precursor cells for peritubular myoid cells. The round cells may be precursor cells for Leydig cells because some of the migrated cells were 3-HSD-positive. The cluster-forming small cells proliferated well unlike fibroblastic cells and round cells, and some of the cells differentiated to round cells, indicating that the cluster-forming cells show the character of gonadal stem cells. The migrated mesonephric cells did not express Sry gene, indicating that the cluster-forming cells are not able to differentiate into Sertoli cells. The immortalized cells from the cluster-forming cells were established in the culture condition with LIF and L-glutamine enriched medium, and embryonic fibroblast cell feeder. For further analysis of the potential characters as the male gonadal stem like cells, we cloned several lines of the cells. These cells are powerful tool for understanding molecular mechanism of testis development.

KEY WORDS: gonad, mesonephros, cell migration, stem cell