Gonocytes (or prospermatogonia) are the precursors to spermatogonial stem cells (SSCs), which provide the foundation for spermatogenesis through their ability to both self-renew and generate daughter cells. Despite their relative importance, th regulatory mechanisms that govern gonocyte maintenance in the fetus and transition to SSCs after birth are poorly understood. Using transgenic mice, we established that constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte loss-the first suggestion of the potential role of this signaling pathway in the testis. We then inhibited NOTCH activation in mouse Sertoli cells and observed an increase in germ cell numbers and testicular size. Therefore dysregulation of NOTCH signaling induces either sterility (NOTCH overactivation) or hyperplasia that could enhance predisposition to testicular cancer (NOTCH downregulation). This proposal will test the hypotheses that NOTCH activity, through its target effectors HEY1 and HEYL, downregulates two crucial molecules that maintain the undifferentiated states of germ cells: GDNF and CYP26B1. We will use NOTCH overactivation, NOTCH lack of function and wild type mouse models to test whether the transcriptional repressors HEY1 and/or HEYL directly influence the expression of GDNF and CYP26B1 when NOTCH is activated.
In Aim1, we will investigate the temporal expression of Hey1 and HeyL transcription factors by qPCR, and use ChIP-PCR to demonstrate direct binding of these repressors to the Gdnf promoter. Further, we will test whether failure of maintaining gonocyte quiescence in our NOTCH lack-of-function model leads to a carcinoma-in-situ-like (CIS-like) phenotype.
In Aim 2, we will investigate the role of NOTCH signaling on the expression of CYP26B1, an enzyme that blocks germ cell differentiation. Using ChIP-PCR analysis, we will demonstrate that HEY1/HEYL transcription factors directly bind to the Cyp26b1 promoter to downregulate its expression. Finally, we will test whether overexpression of NOTCH signaling truly leads to a Sertoli cell-only syndrome through inhibition of CYP26B1.
In Aim 3, using germ cell-Sertoli cells co-cultures, we will test the hypothesis that germ cells regulate NOTCH activity in Sertoli cells and therefore can regulate their own numbers. Altogether, this proposal will demonstrate for the first time that NOTCH signaling modulates the expression of two molecules essential for germ cell proliferation and maintenance of the undifferentiated state, and is a component of normal germ cell homeostasis. Dysregulation of this pathway will induce sterility or germ cell hyperplasia.
Male sterility due to germ cell maturation arrest is not a rare event, and increases in incidence of testicular cancer have been reported in many countries around the world over the last 40 years. Many studies have now demonstrated that these pathologies originate during the fetal stage, and that dysregulations of the germ cell environment provided by Sertoli cells, which are the nursing cells of the testis, play an important role. This project intends to elucidate how the NOTCH signaling pathway in normal Sertoli cells maintains proper germ cell development in the perinatal testis, and how its overactivation or deletion induces male sterility or an environment permissive to germ cell hyperplasia and possibly testicular cancer.