Reactive oxygen species (ROS) generation is implicated in stem cell self-renewal in several tissues but is thought to be detrimental for spermatogenesis as well as spermatogonial stem cells (SSCs). Using cultured SSCs, we show that ROS are generated via the AKT and MEK signaling pathways under conditions where the growth factors glial cell line-derived neurotrophic factor and fibroblast growth factor 2 drive SSC self-renewal and, instead, stimulate self-renewal at physiological levels. SSCs depleted of ROS stopped proliferating, but they showed enhanced self-renewal when ROS levels were increased by the addition of hydrogen peroxide, which induced the phosphorylation of stress kinases p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Moreover, ROS depletion in vivo decreased SSC number in the testis, and NADPH oxidase 1 (Nox1)-deficient SSCs exhibited reduced self-renewal division upon serial transplantation. These results suggest that ROS generated by Nox1 play critical roles in SSC self-renewal via the activation of the p38 MAPK and JNK pathways.

Spermatogonial stem cells (SSCs) undergo self-renewal division and support spermatogenesis. Although several cytokines coordinate to drive SSC self-renewal, little is known about the mechanisms underlying this process. We investigated the molecular mechanism by reconstructing SSC self-renewal in vitro without exogenous cytokines. Activation of Ras or overexpression of cyclins D2 and E1, both of which were induced by Ras, enabled long-term self-renewal of cultured spermatogonia. SSCs with activated Ras responded properly to differentiation signals and underwent spermatogenesis, whereas differentiation was abrogated in cyclin transfectants after spermatogonial transplantation. Both Ras- and cyclin-transfected cells produced seminomatous tumors, suggesting that excessive self-renewing stimulus induces oncogenic transformation. In contrast, cells that overexpressed cyclin D1 or D3 failed to make germ cell colonies after transplantation, which indicated that cyclin expression pattern is an important determinant to long-term SSC recolonization. Thus, the Ras-cyclin D2 pathway regulates the balance between tissue maintenance and tumorigenesis in the SSC population.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. In a manner comparable to hematopoietic stem cell transplantation, SSCs colonize the niche of recipient testes and reinitiate spermatogenesis following microinjection into the seminiferous tubules. However, little is known about the homing mechanism of SSCs. Here we examined the role of adhesion molecules in SSC homing. SSCs isolated from mice carrying loxP-tagged beta1-integrin alleles were ablated for beta1-integrin expression by in vitro adenoviral cre transduction. The beta1-integrin mutant SSCs showed significantly reduced ability to recolonize recipient testes in vivo and to attach to laminin molecules in vitro. In contrast, genetic ablation of E-cadherin did not impair homing, and E-cadherin mutant SSCs completed normal spermatogenesis. In addition, the deletion of beta1-integrin on Sertoli cells reduced SSC homing. These results identify beta1-integrin as an essential adhesion receptor for SSC homing and its association with laminin is critical in multiple steps of SSC homing.

SummarySpermatogonial stem cells (SSCs) are required for spermatogenesis. Earlier studies showed that glial cell line-derived neurotrophic factor (GDNF) was indispensable for SSC self-renewal by binding to the GFRA1/RET receptor. Mice with mutations in these molecules showed impaired spermatogenesis, which was attributed to SSC depletion. Here we show that SSCs undergo GDNF-independent self-renewal. A small number of spermatogonia formed colonies when testis fragments from a Ret mutant mouse strain were transplanted into heterologous recipients. Moreover, fibroblast growth factor 2 (FGF2) supplementation enabled in vitro SSC expansion without GDNF. Although GDNF-mediated self-renewal signaling required both AKT and MAP2K1/2, the latter was dispensable in FGF2-mediated self-renewal. FGF2-depleted testes exhibited increased levels of GDNF and were enriched for SSCs, suggesting that the balance between FGF2 and GDNF levels influences SSC self-renewal in vivo. Our results show that SSCs exhibit at least two modes of self-renewal and suggest complexity of SSC regulation in vivo.

Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.

Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, plays an important role in growth factor signaling to the nucleus. However, molecular mechanisms regulating subcellular localization of ERK5 have remained unclear. Here, we show that nucleocytoplasmic shuttling of ERK5 is regulated by a bipartite nuclear localization signal-dependent nuclear import mechanism and a CRM1-dependent nuclear export mechanism. Our results show that the N-terminal half of ERK5 binds to the C-terminal half and that this binding is necessary for nuclear export of ERK5. They further show that the activating phosphorylation of ERK5 by MEK5 results in the dissociation of the binding between the N-and C-terminal halves and thus inhibits nuclear export of ERK5, causing its nuclear import. These results reveal the mechanism by which the activating phosphorylation of ERK5 induces its nuclear import and suggest a novel example of a phosphorylation-dependent control mechanism for nucleocytoplasmic shuttling of proteins.The mitogen-activated protein kinase (MAPK) cascade, one of signaling modules ubiquitous among eukaryotes, transmits extracellular signals from cell surface receptors to specific targets within cells and regulates a wide variety of cellular functions, including cell proliferation, differentiation, and stress responses. The MAPK cascades are composed of three conserved kinases, MAPK, MAPK kinase (MAPKK), and MAPKK kinase. Extracellular stimuli, such as growth factors, induce sequential phosphorylation of the three kinases; stimulus-activated MAPKK kinase phosphorylates MAPKK, which in turn phosphorylates and activates MAPK. Phosphorylated and activated MAPK phosphorylates downstream targets, such as transcription factors, and modulates their function. To date, at least four subfamily members of the MAPK family have been identified: extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun-N-terminal kinases (JNKs), p38, and ERK5. Each molecule is activated by distinct pathways and transmits signals either independently or coordinately. ERK1/2 is activated mainly by mitogenic stimuli, whereas p38 and JNK are activated mainly by stress stimuli or inflammatory cytokines (2,6,8,19,28,31,32,34).ERK5, also known as big MAP kinase 1, is activated by oxidative stress, hyperosmolarity, and several growth factors (11, 13-15, 20, 22, 23, 25, 42). Unlike other MAPK members, ERK5 has a unique large C-terminal region, whose function is not fully elucidated. MEK5 is the upstream MAPKK that specifically phosphorylates and activates ERK5 (23,42). It has been shown that ERK5 directly interacts with, phosphorylates, and activates several transcription factors including c-Myc, Sap1a, c-Fos, Fra-1, and MEF2 family members (11,20,22,35,41). Moreover, ERK5 is shown to regulate transcription through a kinase-independent mechanism that involves its unique C-terminal half (21, 35). ERK5 is important for promoting cell proliferation (12, 23), differentiation (10), and neuronal survival (37). ERK5-null ...

ERK5 plays a crucial role in many biological processes by regulating transcription. ERK5 has a large C-terminal-half that contains a transcriptional activation domain. However, it has remained unclear how its transcriptional activation activity is regulated. Here, we show that the activated kinase activity of ERK5 is required for the C-terminal-half to enhance the AP-1 activity, and that the activated ERK5 undergoes autophosphorylation on its most C-terminal region. Changing these phosphorylatable threonine and serine residues to unphosphorylatable alanines significantly reduces the transcriptional activation activity of ERK5. Moreover, phosphomimetic mutants of the C-terminal-half of ERK5 without an N-terminal kinase domain are shown to be able to enhance the AP-1 activity in fibroblastic cells. These results reveal the role of the stimulus-induced ERK5 autophosphorylation in regulation of gene expression. The mitogen-activated protein kinase (MAPK)2 cascades play an essential role in transducing extracellular signals to cytoplasmic and nuclear effectors, and regulate a variety of cellular functions, including cell proliferation, differentiation, and stress responses. The MAPK cascades are composed of three classes of protein kinases: MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Each MAPK is activated by specific members of MAPKK, which are activated by MAPKKK (1-13). Four subfamily members of the MAPK family have been relatively well studied; extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, and ERK5.ERK5 is activated by oxidative stress, hyperosmolarity, and several growth factors including epidermal growth factor and nerve growth factor (14 -18). MEK5 is the upstream MAPKK that specifically phosphorylates and activates ERK5 (16,19). It has been shown that ERK5 directly phosphorylates and activates several transcription factors including c-Myc, Sap1a, c-Fos, Fra-1, and MEF2 family members (15, 17, 20 -22). ERK5 knock-out mice die in embryonic stages with angiogenic failure and cardiovascular defects, and in the adult stages, ERK5 is required for endothelial cell survival and maintenance of blood vascular integrity (23-26). The targeted deletion of MEK5 also causes early embryonic death with cardiovascular defects (27). Moreover, knockdown studies in Xenopus have shown that ERK5 and MEK5 play an essential role in neural differentiation (28).ERK5 has a large, unique C-terminal-half not found in other MAPK family members (19,29). Because of this unique 400-amino acid extension, ERK5 is also called big MAP kinase 1 (BMK1). The C-terminal-half of ERK5 has two proline-rich domains and a bipartite NLS, and shares no high homology with other proteins (19,30,31). Kasler et al. (32) demonstrated that the C-terminal-half of ERK5 has a potent transcriptional activation domain. It was subsequently shown that the ERK5 C-terminal-half is essential for transactivation of MEF2C (30). Moreover, a recent study has shown that ERK5 has two transactivation domains in its C-termin...

Myc plays critical roles in the self-renewal division of various stem cell types. In spermatogonial stem cells (SSCs), Myc controls SSC fate decisions because Myc overexpression induces enhanced self-renewal division, while depletion of Max, a Myc-binding partner, leads to meiotic induction. However, the mechanism by which Myc acts on SSC fate is unclear. Here we demonstrate a critical link between Myc/Mycn gene activity and glycolysis in SSC selfrenewal. In SSCs, Myc/Mycn are regulated by Foxo1, whose deficiency impairs SSC self-renewal. Myc/Mycn-deficient SSCs not only undergo limited self-renewal division but also display diminished glycolytic activity. While inhibition of glycolysis decreased SSC activity, chemical stimulation of glycolysis or transfection of active Akt1 or Pdpk1 (phosphoinositide-dependent protein kinase 1 ) augmented self-renewal division, and long-term SSC cultures were derived from a nonpermissive strain that showed limited self-renewal division. These results suggested that Myc-mediated glycolysis is an important factor that increases the frequency of SSC self-renewal division.