GILZ-Dependent Modulation of Mtorc1 Regulates Spermatogonial Maintenance Hue M
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© 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev165324. doi:10.1242/dev.165324 STEM CELLS AND REGENERATION RESEARCH ARTICLE GILZ-dependent modulation of mTORC1 regulates spermatogonial maintenance Hue M. La1,2,*, Ai-Leen Chan1,2,*, Julien M. D. Legrand1,2, Fernando J. Rossello1,2, Christina G. Gangemi1,2, Antonella Papa3, Qiang Cheng4, Eric F. Morand4 and Robin M. Hobbs1,2,‡ ABSTRACT prospermatogonia) upon migration to the basement membrane of Male fertility is dependent on spermatogonial stem cells (SSCs) that the seminiferous cords. The undifferentiated population contains self-renew and produce differentiating germ cells. Growth factors isolated spermatogonia (A-single or As) plus chains of cells produced within the testis are essential for SSC maintenance interconnected by cytoplasmic bridges. Two-cell chains are but intrinsic factors that dictate the SSC response to these stimuli known as A-paired (Apr) whereas chains of four or more cells are poorly characterised. Here, we have studied the role of GILZ, are known as A-aligned (Aal). Lineage-tracing studies demonstrate α a TSC22D family protein and spermatogenesis regulator, in that SSCs are marked by GFR 1 and typically As and Apr,whereas spermatogonial function and signalling. Although broadly expressed most undifferentiated cells, particularly Aal, are committed in the germline, GILZ was prominent in undifferentiated spermatogonia progenitors and marked by NGN3 (Hara et al., 2014; Nakagawa + and Gilz deletion in adults resulted in exhaustion of the GFRα1+ et al., 2010). NGN3 Aal may revert to SSCs through chain SSC-containing population and germline degeneration. GILZ loss fragmentation, particularly upon tissue damage (Nakagawa et al., γ was associated with mTORC1 activation, suggesting enhanced 2010). Immunofluorescence demonstrates that RAR and SOX3 α − growth factor signalling. Expression of deubiquitylase USP9X, an preferentially mark GFR 1 progenitors, although they are detectable mTORC1 modulator required for spermatogenesis, was disrupted in at lower levels in SSC-containing fractions (Ikami et al., 2015; Lord Gilz mutants. Treatment with an mTOR inhibitor rescued GFRα1+ et al., 2018; Suzuki et al., 2012). Although transplantation indicates α − spermatogonial failure, indicating that GILZ-dependent mTORC1 that SSCs are also found in the GFR 1 undifferentiated population inhibition is crucial for SSC maintenance. Analysis of cultured (Garbuzov et al., 2018), progenitors of homeostatic testis can exhibit undifferentiated spermatogonia lacking GILZ confirmed aberrant transplantation capacity (Carrieri et al., 2017; Nakagawa et al., activation of ERK MAPK upstream mTORC1 plus USP9X 2007). ID4 marks cells with potent transplantation capacity and is α + downregulation and interaction of GILZ with TSC22D proteins. Our primarily restricted to the GFR 1 population (Chan et al., 2014; α data indicate an essential role for GILZ-TSC22D complexes in Helsel et al., 2017), supporting the theory that GFR 1 marks an ensuring the appropriate response of undifferentiated spermatogonia SSC-enriched fraction. to growth factors via distinct inputs to mTORC1. Spermatogonial differentiation is marked by c-KIT induction and formation of A1 spermatogonia that undergo mitotic divisions and KEY WORDS: Spermatogonial stem cells, GILZ, mTORC1, TSC22D via A2,A3 and A4, Intermediate (In) and B-type spermatogonia family, Spermatogenesis generate meiotic spermatocytes (de Rooij, 1998). Undifferentiated and differentiating spermatogonia are both present within the INTRODUCTION seminiferous epithelium basal layer. Spermatogenesis is a cyclic Sustained production of spermatozoa is dependent on mitotic germ process and the seminiferous epithelium can be divided into 12 cells with self-renewal potential known as spermatogonial stem stages in the mouse (I-XII). Tubules at a given stage contain cells (SSCs) (de Rooij, 1998; Kanatsu-Shinohara and Shinohara, spermatogonia at a specific differentiation step (de Rooij, 1998). 2013). Murine spermatogonia are divided into undifferentiated Undifferentiated cells are present at all cycle stages but mitotic plus differentiating fractions; SSCs represent a subset of the activity and the relative proportions of SSCs and progenitors vary. undifferentiated pool whereas remaining undifferentiated cells GDNF is a growth factor produced by Sertoli and peritubular act as committed progenitors. Undifferentiated spermatogonia myoid cells within the testis that promotes SSC self-renewal via are generated postnatally from foetal germ cells (gonocytes or the GFRα1/RET receptor (Chen et al., 2016; Kanatsu-Shinohara and Shinohara, 2013). Basic fibroblast growth factor (bFGF) is produced by germ and somatic cells within the testis, and 1Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria 3800, Australia. 2Development and Stem Cells Program, Monash independently promotes SSC self-renewal (Ishii et al., 2012; Biomedicine Discovery Institute and Department of Anatomy and Developmental Takashima et al., 2015). Undifferentiated spermatogonia can be Biology, Monash University, Melbourne, Victoria 3800, Australia. 3Cancer Program, cultured in vitro in the presence of GDNF and bFGF (Kanatsu- Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia. 4Centre Shinohara and Shinohara, 2013). Signalling pathways mediating the for Inflammatory Diseases, School of Clinical Sciences at Monash Health, Monash effects of these growth factors have been elucidated, e.g. the University, Melbourne, Victoria 3800, Australia. extracellular-regulated kinase (ERK) mitogen-activated protein *These authors contributed equally to this work kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT ‡Author for correspondence ([email protected]) (Hasegawa et al., 2013; Ishii et al., 2012; Lee et al., 2007; Oatley et al., 2007; Takashima et al., 2015). RAS activation drives SSC H.M.L., 0000-0003-4549-7017; Q.C., 0000-0002-5066-5583; R.M.H., 0000- 0002-3853-2614 self-renewal via induction of cyclins D2 plus E1, and is upstream ERK MAPK and PI3K/AKT (Lee et al., 2009). Aberrant activation Received 16 March 2018; Accepted 6 August 2018 of growth factor-dependent signalling can, however, be detrimental. DEVELOPMENT 1 STEM CELLS AND REGENERATION Development (2018) 145, dev165324. doi:10.1242/dev.165324 Ablation of phosphatase and tensin homolog (PTEN), a negative potential role for GILZ in establishing the SSC pool may underlie regulator of PI3K/AKT, drives SSC exhaustion (Goertz et al., 2011). germline failure (Ngo et al., 2013b; Romero et al., 2012). That growth factor signalling is tightly regulated in SSCs is To define GILZ function in mature SSCs and characterise indicated by the key role of transcription factor FOXO1 in SSC effectors, we developed an inducible knockout model allowing function (Goertz et al., 2011); phosphorylation of FOXO1 by AKT GILZ ablation in adults. We identified an essential role for GILZ inhibits nuclear localisation and activity (Calnan and Brunet, 2008). in SSC maintenance through mTORC1 inhibition. GILZ also The transcription factor promyelocytic leukaemia zinc finger promoted expression of spermatogenic regulators independently (PLZF) is expressed by undifferentiated spermatogonia plus early of mTORC1 control. Analysis of GILZ-associated proteins differentiating cells and promotes SSC self-renewal (Buaas et al., indicated crosstalk between GILZ and other TSC22D proteins in 2004; Costoya et al., 2004; Hobbs et al., 2010). SALL4 is expressed undifferentiated spermatogonia. in undifferentiated and differentiating spermatogonia, and is crucial for SSC activity plus spermatogonial differentiation (Chan et al., RESULTS 2017; Hobbs et al., 2012). PLZF and SALL4 modulate responses of Rapid SSC depletion upon acute Gilz deletion in adults undifferentiated spermatogonia to niche factors by regulating Gilz is essential for spermatogenesis but its role in SSCs poorly pathway components (Chan et al., 2017; Hobbs et al., 2010). PLZF understood (Ngo et al., 2013b; Romero et al., 2012; Suarez et al., enhances sensitivity of undifferentiated spermatogonia to GDNF 2012). Whole-mount immunofluorescence of seminiferous tubules through mammalian target of rapamycin complex 1 (mTORC1) from adult wild-type mice demonstrated that GILZ was present in inhibition (Hobbs et al., 2010). mTORC1 promotes cell growth and the cytosol of undifferentiated and differentiating spermatogonia + + plays crucial roles in stem cell regulation (Laplante and Sabatini, (Fig. 1A,B). GILZ was detected in GFRα1 As and Apr plus SOX3 2012). mTORC1 activity is higher in progenitors than SSCs and Aal, indicating expression in SSCs and progenitors (Chan et al., aberrant mTORC1 activation promotes SSC exhaustion, indicating a 2017; Hara et al., 2014; Suzuki et al., 2012). From Id4IRES-GFP adult role in differentiation (Hobbs et al., 2015; Wang et al., 2016). testis, over 85% of GFRα1+ but less than 10% of SOX3+ cells were Deleting the mTOR kinase blocks spermatogonial proliferation and ID4+ (Fig. S1A,B), supporting use of GFRα1 and SOX3 as stem differentiation, consistent with roles of mTORC1 (Serra et al., 2017). and progenitor markers, respectively (Best et al., 2014; Helsel et al., mTORC1 promotes translation of factors required for differentiation, 2017). Id4 expression overlapped poorly with RARγ, an alternative including SOHLH1 and SOHLH2 (Busada et al., 2015). marker