Sertoli cells are the somatic cells in the testis that are essential for testis formation and spermatogenesis [1, 32–34]. Proliferation of ISCs is important for male fertility because each Sertoli cell is able to support a limited number of germ cells [35, 36]. Hormones such as FSH [37–39], estrogen  and thyroid hormones  as well as various paracrine growth factors including insulin-like growth factors I and II (IGF-I and IGF-II) , fibroblast growth factor (FGF) and somatomedin-C , activin , transforming growth factor-α (TGF-α) , and interleukin-1(IL-1)  are important in regulating the proliferation of ISCs. GDNF, a protein remotely related to the TGF-β super family members, and its receptor components GFRα1, RET and NCAM have been reported to be expressed in several types of testicular cells, implying a role of GDNF signaling in spermatogenesis. The role of GDNF signaling in spermatogonial proliferation and differentiation has been unequivocally demonstrated by in vivo studies using Gdnf
mice and mice with specific over-expression of Gdnf in testis  and by in vitro studies in which GDNF was indentified as an essential factor for spermatogonial stem cell culture . However, the role of GDNF in Sertoli cells is controversial. No Sertoli cell abnormality was reported in Gdnf
mice and mice with Gdnf testis-specific over-expression while its stimulatory effect on the proliferation of Sertoli cells was observed in cultured neonatal rat seminiferous tubules and in Sertoli cells from fetal mouse testis [2, 3, 16]. In the present study, we used cultured ISCs from neonatal mice to show that GDNF stimulates the proliferation of ISCs derived from 4-5-day-old mice.
GFRα1 has been used as a specific surface marker for the identification and purification of mouse SSCs in some studies [29, 47, 48]. However, in the present study, we detected GFRα1 expression in cultured ISCs at both the mRNA and protein levels. As indicated by the results of He et al. [29, 47, 48], the protein level of GFRα1 in ISCs seemed to be much lower than in SSCs and was probably regarded as background signal in immunohistochemistry experiments by those authors. GDNF is known to signal through a multi-component receptor system consisting of GFRα1 and one of the two co-receptor subunits RET [7, 8] or NCAM [14, 49–51]. GFRa1 is the main ligand binding subunit and therefore is indispensible for the function of the receptor complex. Because the expression of RET was not detected in ISCs both at the mRNA and the protein levels, it is almost certain that the signal transduction is mediated by NCAM. Therefore, we focused on studying the role of NCAM in mediating GDNF signaling in ISCs although we could not fully exclude that additional or alternative receptors and pathways might also be involved in mediating the action of GDNF in ISCs. The essential role of NCAM in ISCs was supported by results from both NCAM antibody blocking and siRNA knockdown experiments. The observation that mRNA and protein of NCAM but not RET were expressed in cultured ISCs was consistent with one previous report .
It has been reported that GDNF signaling through the phosphorylation of ERK1/2 and AKT mediated the self-renewal division and proliferation of SSCs . We observed in this study that GDNF stimulation also led to a rapid increase of ERK1/2 phosphorylation levels at Thr202/Tyr204 in cultured ISCs, but AKT phosphorylation levels did not change during this process. Significantly, the increase in ERK1/2 phosphorylation and ISC proliferation could be completely blocked by PD98059, with the phosphorylation level of AKT not changing during this process, suggesting that GDNF signaled through the ERK1/2 pathway in ISCs to execute its pro-proliferation function.
Proliferation of TM4 cells was not stimulated by GDNF although they possessed the same set of GDNF receptor subunits as ISCs. It is well known that TM4, an immortalized Sertoli cell line from mouse testis, has lost some characteristics of primary Sertoli cells. For example, FSH responsiveness was reduced in TM4 cells compared with primary Sertoli cell cultures . We also noticed that CLU, a Sertoli cell marker, was not expressed by TM4 cells. Therefore, it is possible that downstream components of the GDNF signaling pathways have been changed in TM4 cells during its conversion from a primary culture to an immortalized cell line. Based on these characteristics of TM4, we used it as a negative control for studying ISC proliferation stimulated by GDNF in the present study. It will be instructive to elucidate the difference in GDNF signaling between ISCs and TM4 cells in future studies as the results would explain why TM4 cells and ISCs are different in response to GDNF treatment.