Expression of transcriptional factor EB (TFEB) in differentiating spermatogonia potentially promotes cell migration...

Spermatogenesis is a complex process involving the self-renewal and differentiation of spermatogonia into mature spermatids in the seminiferous tubules. During spermatogenesis, germ cells migrate from the basement membrane to cross the blood-testis barrier (BTB) and finally reach the luminal side of the seminiferous epithelium. However, the mechanism for regulating the migration of germ cells remains unclear. In this study, we focused on the expression and function of transcriptional factor EB (TFEB), a master regulator of lysosomal biogenesis, autophagy and endocytosis, in spermatogenesis.

The expression pattern of the TFEB in mouse testes were investigated by Western blotting and immunohistochemistry analyses. Either undifferentiated spermatogonia or differentiating spermatogonia were isolated from testes using magnetic-activated cell sorting based on specific cell surface markers. Differentiation of spermatogonia was induced with 100 nM retinoic acid (RA). shRNA was used to knock down TFEB in cells. TFEB expression was detected by immunofluorescence, qRT-PCR, and Western blotting. Cell migration was determined by both transwell migration assay and wound healing assay applied to a cell line of immortalized spermatogonia, GC-1 cells.

During testicular development, TFEB expression was rapidly increased in the testes at the period of 7 days post-partum (dpp) to 14 dpp, whereas in adult testis, it was predominantly localized in the nucleus of spermatogonia at stages VI to VIII of the seminiferous epithelial cycle. Accordingly, TFEB was observed to be mainly expressed in differentiating spermatogonia and was activated for nuclear translocation by RA treatment. Moreover, knockdown of TFEB expression by RNAi did not affect spermatogonial differentiation, but significantly reduced cell migration in GC-1 cells.

These findings imply that regionally distinct expression and activation of TFEB was strongly associated with RA signaling, and therefore may promote cell migration across the BTB and transport along the seminiferous epithelium.