Reply to Chen and Liu: Role of GDNF from peritubular myoid cells in the testis stem cell niche.

Chen and Liu (1) agree with our main conclusion that glial cell line-derived neurotrophic factor (GDNF) produced by peritubular myoid (PM) cells signals to undifferentiated spermatogonia and regulates their maintenance (2), but argue that a PM cell-specific gene knockout is impossible because the myosin heavy polypeptide 11 smooth muscle-Cre (Myh11-Cre) is not expressed specifically in PM cells. Chen and Liu (1) note that in previous studies the Myh11-Cre transgene was expressed in vascular smoothmuscle cells in a variety of tissues (3). However, what is important is whether the transgene and the target gene are coexpressed in the same cell type in the testis. The Myh11-Cre transgene was used previously to produce a PM cell conditional androgen receptor (Ar) gene knockout (cKO) (4). We showed MYH11 and Myh11-Cre are expressed in PM cells and in blood vessels in the testis, but that GDNF is detected in PM cells and not in blood vessels in the testis (2, 5). Thus, even if the Gdnf gene is disrupted in vascular smooth muscle cells in the testis, it is unlikely to influence GDNF levels in the testis stem cell niche. The other major concern of Chen and Liu (1) is that Sertoli cell function might be affected by the PM cell Gdnf cKO, as occurred in the PM cell Ar cKO (4). However, the AR probably regulates multiple PM cell genes that influence Sertoli cells, whereas GDNF acts directly on undifferentiated spermatogonia. Chen and Liu (1) also suggest the effect of interactions between Sertoli cells and PM cells on Gdnf expression in vivo should be determined, observation of PM cell-specific markers and generation of PM cell-specific Cre transgenes would be helpful, and Sertoli cell-specific knockout ofGdnf is urgently required. We agree there is a need to understand the effects of Gdnf expression on Sertoli and PM cell interactions, and stated previously that “coregulatory networks might exist between PM and Sertoli cells that influence the levels of GDNF and other factors in the testis niche that deserve further study” (5), and “generation of mice with a cKO of genes for GDNF or other factors in Sertoli cells or in other cells in the testis will be needed to determine their roles in regulating SSC [spermatogonial stem cell] development in vivo” (2). We also agree that identifying PM cell-specific markers and generating PM cell-specific Cre transgenes, as well as developing new approaches for dissecting the functions of PM cells in vivo, are needed to understand how the microenvironment of the testis stem cell niche is regulated. We thank Chen and Liu (1) for their interest in our studies and their thoughtful comments.