Abstract
Cytokine-dependent renewal of stem cells is a fundamental requisite for tissue homeostasis and regeneration. Spermatogonial progenitor cells (SPCs) including stem cells support life-long spermatogenesis and male fertility, but pivotal phosphorylation events that regulate fate decisions in SPCs remain unresolved. Here, we described a quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of SPCs following sustained stimulation with glial cell-derived neurotrophic factor (GDNF), an extrinsic factor supporting SPC proliferation. Stimulated SPCs contained 3382 identified phosphorylated proteins and 12141 phosphorylation sites. Of them, 325 differentially phosphorylated proteins and 570 phosphorylation sites triggered by GDNF were highly enriched for ERK1/2, GSK3, CDK1, and CDK5 phosphorylating motifs. We validated that inhibition of GDNF/ERK1/2-signaling impaired SPC proliferation and increased G2/M cell cycle arrest. Significantly, we found that proliferation of SPCs requires phosphorylation of the mTORC1 component Raptor at Ser863 Tissue-specific deletion of Raptor in mouse germline cells results in impaired spermatogenesis and progressive loss of spermatogonia, but in vitro increased phosphorylation of Raptor by raptor over-expression in SPCs induced a more rapidly growth of SPCs in culture. These findings implicate previously undescribed signaling networks in governing fate decision of SPCs, which is essential for the understanding of spermatogenesis and of potential consequences of pathogenic insult for male infertility.
Highlights
Mitotic self-renewal of stem cells is essential for tissue homeostasis and regeneration and usually relies on extrinsic stimuli from cytokines that are released by supporting cells within the stem cell niche
After digestion of extracted proteins with trypsin, purified Tandem Mass Tag (TMT)-labeled peptides from untreated (GDNF starved) and treated (GDNF replenished) replicates were pooled, and phosphopeptides were enriched with IMAC followed by titanium dioxide (TiO2) beads [21] (Fig. 1A)
Consistent with a distinct temporal regulation of different intracellular phosphorylation events in response to a stimulus, our study did not detect differential PI3K/AKT kinase activity after sustained stimulation of spermatogonial progenitor cells (SPCs) with glial cell line-derived neurotrophic factor (GDNF). Activation of this pathway, which has been previously shown to be involved in SPC self-renewal, is an immediate change likely within 30 min in response to GDNF stimulation that would not be detected in our experimental set-up [16, 17]
Summary
Mitotic self-renewal of stem cells is essential for tissue homeostasis and regeneration and usually relies on extrinsic stimuli from cytokines that are released by supporting cells within the stem cell niche. Mitotic division and initial differentiation of SSCs produces Apaired (Apr) and Aaligned (Aal) type germ cells, which remain connected through intercellular bridges [1]. These cells are the spermatogonial progenitor cells (SPCs) of the male testis that give rise to all cells of the spermatogenic lineage and support life-long spermatogenesis [2]. We validated that the kinase ERK1/2-associated signaling network is essential for proliferation of SPCs. Our data indicate that Ser863 phosphorylation of the mTORC1 complex partner raptor may represent a crucial step for SPC self-renewal and mitotic proliferation. These data provide new insight into the mechanisms that govern fate decision of SPCs and male reproduction
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