Constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte exit from quiescence

Abstract

Notch signaling components have long been detected in Sertoli and germ cells in the developing and mature testis. However, the role of this pathway in testis development and spermatogenesis remains unknown. Using reporter mice expressing green fluorescent protein following Notch receptor activation, we found that Notch signaling was active in Sertoli cells at various fetal, neonatal, and adult stages. Since Notch signaling specifies stem cell fate in many developing and mature organ systems, we hypothesized that maintenance and differentiation of gonocytes and/or spermatogonial stem cells would be modulated through this pathway in Sertoli cells. To this end, we generated mutant mice constitutively expressing the active, intracellular domain of NOTCH1 (NICD1) in Sertoli cells. We found that mutant Sertoli cells were morphologically normal before and after birth, but presented a number of functional changes that drastically affected gonocyte numbers and physiology. We observed aberrant exit of gonocytes from mitotic arrest, migration toward cord periphery, and premature differentiation before birth. These events, presumably unsupported by the cellular microenvironment, were followed by gonocyte apoptosis and near complete disappearance of the gonocytes by day 2 after birth. Molecular analysis demonstrated that these effects are correlated with a dysregulation of Sertoli-expressed genes that are required for germ cell maintenance, such as Cyp26b1 and Gdnf. Taken together, our results demonstrate that Notch signaling is active in Sertoli cells throughout development and that proper regulation of Notch signaling in Sertoli cells is required for the maintenance of gonocytes in an undifferentiated state during fetal development.

NOTCH signaling in Sertoli cells regulates gonocyte fate

“…As shown in Figure 2, KIT was indeed markedly upregulated in a sub-population of gonocytes in the mutant. Because KIT expression was upregulated concomitant with a decrease in Pou5f1 and an increase in Sohlh2, Stra8, Sycp3, and Rec8, 53 our data overall establish that the aberrant gonocytes had acquired properties of differentiating spermatogonia.…”

“…To glean additional insight into the molecular signature of the gonocytes in our model and to clarify the role for NOTCH signaling in Sertoli cells, we performed gene expression analysis of whole gonads isolated from E14.5 and E17.5 fetuses. 53 We found that several transcripts were differentially regulated in a manner consistent with NOTCH signaling activation in Sertoli cells and with premature differentiation of germ cells. For instance, RBPJ target genes such as Hey1 and Heyl were upregulated in Sertoli cells, as expected; in germ cells, Nanos2, a gene that suppresses meiosis, 54 was downregulated, and genes that are expressed at the beginning of or during meiosis, such as Stra8 and Rec8, 55,56 were all upregulated (GSE37073).…”

“…Because expression of NOTCH signaling components in a cell does not demonstrate that the pathway is truly activated, our recent study used a transgenic mouse model that expressed green fluorescent protein (GFP) in cells that actively received a NOTCH signal. 53 We observed that the canonical NOTCH signaling pathway is active exclusively in Sertoli cells in the perinatal testis. To circumvent redundancy and compensation in loss-of-function models, we then examined a Sertoli cell-specific NOTCH gainof-function model in which NICD1 was constitutively expressed following Cremediated recombination in Sertoli cells (formerly Amh-cre;Rosa NICD/+ , hereafter referred to as AMH-NICD1).…”

“…53 In addition, γ-secretase inhibitor DAPTinduced downregulation of NOTCH signaling in Sertoli cells in vitro upregulated Gdnf expression levels. These findings therefore strongly suggest that NOTCH signaling is a negative regulator of GDNF production that counteracts the positive effects of FGF2 75 (Fig.…”

“…53 We further examined protein expression of CCND1, as well as CCNB3 and CCND3, to glean additional insight into the stage of differentiation of the aberrant gonocytes. Through immunohistochemistry, we found that CCND1 and CCND3, but not CCNB3, were markedly upregulated in mutant gonocytes at E15.5.…”