Identification of Spermatogonial Stem Cell Subsets by Morphological Analysis and Prospective Isolation

Abstract

Spermatogenesis is maintained by a pool of spermatogonial stem cells (SSCs). Analyses of the molecular profile of SSCs have revealed the existence of subsets, indicating that the stem cell population is more heterogeneous than previously believed. However, SSC subsets are poorly characterized. In rodents, the first steps in spermatogenesis have been extensively investigated, both under physiological conditions and during the regenerative phase that follows germ cell damage. In the widely accepted model, the SSCs are type Asingle (As) spermatogonia. Here, we tested the hypothesis that As spermatogonia are phenotypically heterogeneous by analyzing glial cell line-derived neurotrophic factor (GDNF) family receptor a1 (GFRA1) expression in whole-mounted seminiferous tubules, via cytofluorimetric analysis and in vivo colonogenic assays. GFRA1 is a coreceptor for GDNF, a Sertoli cell-derived factor essential for SSC self-renewal and proliferation.Morphometric analysis demonstrated that 10% of As spermatogonia did not express GFRA1 but were colonogenic, as shown by germ cell transplantation assay. In contrast, cells selected for GFRA1 expression were not colonogenic in vivo. In human testes, GFRA1 was also heterogeneously expressed in Adark and in Apale spermatogonia, the earliest spermatogonia. In vivo 5-bromo-2 0 -deoxyuridine administration showed that both GFRA1 1 and GFRA1 2As spermatogonia were engaged in the cell cycle, a finding supported by the lack of long-term label-retaining As spermatogonia. GFRA1 expression was asymmetric in 5% of paired cells, suggesting that As subsets may be generated by asymmetric cell division. Our data support the hypothesis of the existence of SSC subsets and reveal a previously unrecognized heterogeneity in the expression profile of As spermatogonia in vivo.

Distribution of GFRA1-expressing spermatogonia in adult mouse testis

“…The analysis of clone distribution revealed that while the numbers of clones of 1 and 2 GFRA1C spermatogonia remain constant in all stages, clones of 4 and 8 GFRA1C spermatogonia are differentially distributed with a significant decrease of GFRA1C Aal4 in stages VII-VIII and relatively large numbers of GFRA1C Aal4 and Aal8 clones in stages IX-XI. This further supports the concept of heterogeneity of gene expression among undifferentiated spermatogonia during the epithelial cycle (Grisanti et al 2009, Nakagawa et al 2010.…”

“…The total number of ZBTB16C spermatogonia, including type As, Apr and Aal spermatogonia, their corresponding GFRA1 staining status and Sertoli cell nuclei were counted within the grid on randomly selected tubules. A ZBTB16C cell was described as type As if surrounding cells within 25 mm lacked ZBTB16 staining and exhibited a dissimilar nuclear morphology; furthermore, doublets of cells were designated type Apr when the internuclear distance was below 25 mm (Tegelenbosch & de Rooij 1993, Grisanti et al 2009 …”

“…The expression profile of CDH1, LIN28, GFRA1, NANOS2, NANOS3 and NEUROG3 (NGN3) is heterogeneous among As, Apr and Aal spermatogonia (Grisanti et al 2009, Suzuki et al 2009, Zheng et al 2009, Nakagawa et al 2010; moreover, different subsets of As cells may be generated through an asymmetric division (Grisanti et al 2009, Suzuki et al 2009). Data from transgenic animal models were interpreted to mean that two functionally distinct stem cell compartments exist: the actual and the potential stem cell compartments, which may play different roles in different conditions.…”

Renoprotective Effects of Cotransplanted Allogeneic Testicular Sertoli Cells in a Renal Acute Rejection Model in Rats

“…[4][5][6][7][8][9][10][11][12][13] In cotransplanted with allogeneic cells, Sertoli cells not only are immune privileged, but also assist fibromyoblasts and islet cells in escaping immune rejection, which allows the long-term survival of the grafts. [14][15][16][17][18][19] Such privilege is thought to be associated with the FasL expressed on the surface of Sertoli cells.…”

Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis

“…In undifferentiated spermatogonia, transplantable stem cell activity was detected both in Gfra1 pos and in Gfra1 neg (likely to be Neurog3 pos ) fractions (Hofmann et al 2005a, Nakagawa et al 2007, Grisanti et al 2009). It is noteworthy that in vitro culture that retains the stem cell activity, another big breakthrough in this field, has been developed taking advantage of this transplantationbased stem cell assay (Kanatsu- Shinohara et al 2003, Kubota et al 2004.…”