Abstract
Constitutive activation of the WNT signaling effector CTNNB1 (β-catenin) in the Sertoli cells of the Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mouse model results in progressive germ cell loss and sterility. In this study, we sought to determine if this phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ and wild-type mice showed that SSC activity is lost in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ testes over time, whereas the mutant testes could not support colonization by wild-type SSCs. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway, which was also found to occur in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ testes. One CTNNB1 target gene encoded the secreted signaling molecule WNT4. We therefore tested the effects of WNT4 on SSC-enriched germ cell cultures, and found that WNT4 induced cell death and reduced SSC activity without affecting cell cycle. Conversely, conditional inactivation of Wnt4 in the Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Furthermore, WNT4 was found to signal via the CTNNB1 pathway in Sertoli cells, suggesting a self-reinforcing positive feedback loop. Collectively, these data indicate for the first time that ectopic activation of a signaling cascade in the stem cell niche depletes SSC activity through a paracrine factor. These findings may provide insight into the pathogenesis of male infertility, as well as embryonic gonadal development.
Highlights
Spermatogonial stem cells (SSCs) are the progenitor population of male germ cells
We discovered that Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ Sertoli cells lose their spermatogonial stem cell (SSC) niche capacity and that this is due to the ectopic expression of WNT4, which acts in a paracrine manner to downregulate SSC activity
Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ Sertoli cells fail to support spermatogonial stem cell activity To determine if germ cell loss in the Ctnnb1tm1Mmt/+; Amhr2tm3(cre)Bhr/+ model could be associated with a loss of SSC activity, reciprocal SSC transplant studies were performed
Summary
Spermatogonial stem cells (SSCs) are the progenitor population of male germ cells. Similar to other stem cell types, they can be directed to one of two cell fate decisions: either self-renewal to maintain the SSC pool, or differentiation into more specialized germ cells that will eventually become spermatozoa. WNT signal transduction can occur via at least three distinct pathways, commonly referred to as the WNT/Ca2+, planar cell polarity and WNT/CTNNB1 or canonical pathway In the latter, a pool of CTNNB1 (b-catenin) protein localizes to the cytoplasm, where it is resides in a large multiprotein complex that notably includes the scaffold proteins APC and AXIN. Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ Sertoli cells exhibited morphological characteristics and gene expression patterns suggestive of incomplete differentiation that appeared in a manner coincident with germ cell loss These data suggested that the WNT/ CTNNB1 pathway disrupts Sertoli cell functions critical to their capacity to support spermatogenesis in the postnatal testis
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