|Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
The Sertoli cell line, TTE3,and Leydig cell line, TTE1, were established. Their characters are already reported. In TTE1 and TTE3 cells, cell growth was suppressive at nonpermissive temperature. This indicated that the cells were not transformed.
We investigated their morphological examination for cell-cell adhesion. In TTE3 cells, they formed more tight-junction in nonpermissive temperature. In TTE1 cells they did not form tight-junction.
In TTE3, the cells expressed mRNAs encoding steel factor, inhibin-α, transferrin, follicle stimulating hormone receptor and sulfated glycoprotein-2. Moreover, vimentin and zonula occludens-1 were observed in the cytoplasm and on the boundaries of the cells, respectively. Interestingly expression levels of transferrin and zonula occludens-1(tight junction component) were significantly elevated nonpermissive temperature.
In TTE1, alkaline phosphatase and 3β-hydroxysteroid dehydrogenase(HSD) activities or expression of cytokeratin and vimentin were observe
d. Reverse transcription-polymerase chain reaction(RT-PCR) analysis indicated that TTE1 cells expressed mRNAs encoding 17β-HSD typesl and 3, and inhibin-α.
In future, we want to make a model for addressing important questions regarding the nature of Leydig cells, Sertoli cells, and germ cell interactions.
To identify differentially expressed genes in the process of Sertoli cell and Leydig cell differentiation, we carried out microarray analysis of TTE3 cells and TTE1 cells cultured at permissive and nonpermissive temperatures. In TTE3, of the 1080 genes(IntelliGene microarray) analyzed, the level of 29 genes were changed, with 20 genes showing increased levels of expression and the remaining 9 genes showing decreased levels. In TTE1, of the 1081 genes(Atlass microarray), the levels of 31 genes were changed, with 24 genes showing increased levels of expression and the remaining 7 genes showing decreased levels. Tie2 was the most changed transcript, with a 13.5-fold upregulation under the differentiated condition.
In the next step, we want to study these genes related to cell-cell adhesion(or tight junction) whether their expression will change or not. Less