Overactivation of Notch Signaling in Fetal Sertoli Cells Results in an Immediate Loss of Germ Cells Due to Apoptosis.

Abstract

Notch signaling pathway components have long been demonstrated in Sertoli cells and all germ cell subtypes in developing and mature testes, however the role of this pathway in spermatogenesis remains entirely unknown. Because published studies seem to disagree on the pattern of expression of these components, we re-examined the postnatal expression of the activated form of NOTCH1 (NOTCH1 intracellular domain; NICD1) through immunohistochemistry. Our data indicated that NICD1 is ubiquitously present in the neonatal and adult mouse testis with punctate nuclear staining in Sertoli cells, spermatogonia and spermatocytes, but not round or elongated spermatids. Since the Sertoli cell plays an essential role in the maintenance and fate determination of germ cells, we hypothesized that perinatal alteration of Notch signaling in these cells would result in an alteration of gonocyte or spermatogonial stem cell behavior. To this end, mutant mice lacking NOTCH1—or overexpressing NICD1—in Sertoli cells were generated using the Cre-lox system and Amh-cre. Mice lacking NOTCH1 in Sertoli cells were normal and fertile. Upon analysis of mutants overexpressing NICD1 in Sertoli cells, we found that adult male mice were infertile, had significantly reduced testis weights (~90% reduced), and a complete absence of all germ cells, including SSCs (Sertoli-only phenotype). Analysis of fetal and newborn testes revealed a significant reduction in germ cells starting at E18.5, indicating that Notch signaling in Sertoli cells might modulate prespermatogonia/gonocyte maintenance. TUNEL assay demonstrated the emergence of apoptotic gonocytes beginning at E15.5. Gene expression analysis of mutant vs. normal Sertoli cells in culture revealed differential expression of a number of markers, including a significant decrease (~80%) in Gdnf. These results so far indicate that in these mutants, the loss of gonocytes and subsequent germ cells is due to the amplification of death signals from Sertoli cells and/or suboptimal concentrations of growth factors. In conclusion, Notch signaling in Sertoli cells might control gonocyte number and proliferation through apoptosis and/or dosage of growth factors, in particular GDNF. Current studies are aimed at understanding the mechanisms underlying these processes. Supported by NICHD R01 HD044543 and NIEHS T32-ES007326 (poster)