Distribution of GFRA1-expressing spermatogonia in adult mouse testis

Abstract

In mice and other mammals, spermatogenesis is maintained by spermatogonial stem cells (SSCs), a cell population belonging to undifferentiated type A spermatogonia. In the accepted model of SSC self-renewal, Asingle (As) spermatogonia are the stem cells, whereas paired (Apaired (Apr)) and chained (Aaligned (Aal)) undifferentiated spermatogonia are committed to differentiation. This model has been recently challenged by evidence that As and chained (Apr and Aal), undifferentiated spermatogonia are heterogeneous in terms of gene expression and function. The expression profile of several markers, such as GFRA1 (the GDNF co-receptor), is heterogeneous among As, Apr and Aal spermatogonia. In this study, we have analysed and quantified the distribution of GFRA1-expressing cells within the different stages of the seminiferous epithelial cycle. We show that in all stages, GFRA1C chained spermatogonia (Apr to Aal) are more numerous than GFRA1C As spermatogonia. Numbers of chained GFRA1C spermatogonia are sharply reduced in stages VII-VIII when Aal differentiate into A1 spermatogonia. GFRA1 expression is regulated by GDNF and in cultures of isolated seminiferous tubules, we found that GDNF expression and secretion by Sertoli cells is stage-dependent, being maximal in stages II-VI and decreasing thereafter. Using qRT-PCR analysis, we found that GDNF regulates the expression of genes such as Tex14, Sohlh1 and Kit (c-Kit) known to be involved in spermatogonial differentiation. Expression of Kit was upregulated by GDNF in a stage-specific manner. Our data indicate that GDNF, besides its crucial role in the self-renewal of stem cells also functions in the differentiation of chained undifferentiated spermatogonia.

Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling

“…1). Although PLZF (Costoya et al 2004), SALL4 (Gassei & Orwig 2013), and CDH1 (Tokuda et al 2007) are expressed in all undifferentiated A-spermatogonia at all stages, GFRA1 (Grasso et al 2012), LIN28 (Zheng et al 2009), NANOS2 (Suzuki et al 2009), and NGN3 (Yoshida et al 2004) are primarily expressed in specific types of undifferentiated A-spermatogonia. In addition, ID4 and PAX7 are specific to type A single spermatogonia (Oatley et al 2011, Aloisio et al 2014.…”

Identification of 5-Bromo-2’-Deoxyuridine-Labeled Cells during Mouse Spermatogenesis by Heat-Induced Antigen Retrieval in Lectin Staining and Immunohistochemistry

“…Normal mouse IgG (10 μg/ml; Vector Laboratories) was used instead of the anti-BrdU antibody as a negative control. In IHC with fluorescence detection, the sections were treated with 1% bovine serum albumin alone and then incubated overnight at 4C with the mouse monoclonal anti-BrdU antibody (1:150), either alone for single immunofluorescence or in combination with a goat polyclonal anti-rat GFRA1 antibody (1:200; Neuromics, Edina, MN) (Naughton et al 2006;Grasso et al 2012), rabbit polyclonal anti-mouse PLZF antibody (1: 200, Sigma-Aldrich) (Meistrich and Hess 2013), rabbit polyclonal anti-mouse CADM1 antibody developed in our laboratory (0.5 μg/ml) (Wakayama et al 2003(Wakayama et al , 2007Nakata et al 2012), or goat anti-mouse Gata4 antibody (1: 400; Santa Cruz Biotechnology, Dallas, TX) (Imai et al 2004) for double immunofluorescence microscopy. After washing, the immunoreactive sites were visualized by incubating the sections for 1 hr at room temperature with the anti-mouse IgG antibody conjugated with Alexa Fluor 594 (1:400; Molecular Probes, Eugene, OR) alone for single immunofluorescence or in combination with the anti-rabbit or anti-goat IgG antibodies conjugated with Alexa Fluor 488 (1:400; Molecular Probes) for double immunofluorescence.…”

“…Undifferentiated spermatogonia, i.e., AsAal, randomly proliferate during stages X-II, stop proliferation thereafter, and Aal spermatogonia finally differentiate without dividing into A1 spermatogonia in stages VII-VIII (de Rooij 2001). Differentiated spermatogonia, i.e., A1-B, divide in a highly synchronized manner in particular spermatogenic stages; for example, A1 spermatogonia in stages VIII-IX and B spermatogonia in stages V-VI (Grasso et al 2012).…”

Yangjing Capsule extract promotes proliferation of GC-1 spg cells <i>via</i> up-regulated POU3F1 pathway

“…The GFRα1 is expressed in all stages of type A spermatogonia (35), and it is a necessary component of the GFRα1/RET complex. The RET alone is unable to bind with GDNF unless it is co-expressed with the GFRα1 receptor.…”