A combination of small molecular inhibitors YAC increases expression levels of DMRT1 in the culture of sertoli cells from the transitional zone of mouse testis

There is a cell population capable of active proliferation in culture in the compartment of adult mouse testis consisting of transitional zone of seminiferous tubules and rete testis. These cells were termed Sertoli cells of the transitional zone because they express many specific Sertoli cell genes in culture. A feature of these cells is a low level of Dmrt1 expression, a transcription factor important for Sertoli cell differentiation and male sex determination. In the current research, the culture conditions have been optimized to increase Dmrt1 expression in Sertoli cells of the transitional zone and then functional properties of these cells have been tested. It was shown that a combination of small molecular inhibitors YAC (Y-27632, A-83-01 and CHIR99021) substantially increases the number of Sertoli cells of the transitional zone expressing Dmrt1. It was demonstrated that Sertoli cells of the transitional zone maintained in medium with YAC remain the high level of Dmrt1 expression even after their transfer to 3D culture where they form seminiferous tubule-like structures with neonatal testicular cells. According to immunofluorescence and real-time PCR data Sertoli cells treated with YAC supported germ cell differentiation to the zygotene stage of meiotic prophase I in 3D culture. These results suggest that Sertoli cells of the transitional zone maintained in medium with YAC closely resemble Sertoli cells from seminiferous tubules and can function as Sertoli cells in 3D culture.