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• W2000519963 abstract "In vitro addition of stem cell factor (SCF) to c-kit-expressing A1–A4spermatogonia from prepuberal mice stimulates their progression into the mitotic cell cycle and significantly reduces apoptosis in these cells. SCF addition results in a transient activation of extracellular signal-regulated kinases (Erk)1/2 as well as of phosphatidylinositol 3-kinase (PI3K)-dependent Akt kinase. These events are followed by a rapid re-distribution of cyclin D3, which becomes predominantly nuclear, whereas its total cellular amount does not change. Nuclear accumulation of cyclin D3 is coupled to transient activation of the associated kinase activity, assayed using the retinoblastoma protein (Rb) as a substrate. These events were followed by a transient accumulation of cyclin E, stimulation of the associated histone H1-kinase activity, a delayed accumulation of cyclin A2, and Rb hyper-phosphorylation. All the events associated with SCF-induced cell cycle progression are inhibited by the addition of either a PI3K inhibitor or a mitogen-activated protein-kinase kinase (MEK) inhibitor, indicating that both MEK and PI3K are essential for c-kit-mediated proliferative response. On the contrary, the anti-apoptotic effect of SCF is not influenced by the separate addition of either MEK or PI3K inhibitors. Thus, SCF effects on mitogenesis and survival in c-kit expressing spermatogonia rely on different signal transduction pathways. In vitro addition of stem cell factor (SCF) to c-kit-expressing A1–A4spermatogonia from prepuberal mice stimulates their progression into the mitotic cell cycle and significantly reduces apoptosis in these cells. SCF addition results in a transient activation of extracellular signal-regulated kinases (Erk)1/2 as well as of phosphatidylinositol 3-kinase (PI3K)-dependent Akt kinase. These events are followed by a rapid re-distribution of cyclin D3, which becomes predominantly nuclear, whereas its total cellular amount does not change. Nuclear accumulation of cyclin D3 is coupled to transient activation of the associated kinase activity, assayed using the retinoblastoma protein (Rb) as a substrate. These events were followed by a transient accumulation of cyclin E, stimulation of the associated histone H1-kinase activity, a delayed accumulation of cyclin A2, and Rb hyper-phosphorylation. All the events associated with SCF-induced cell cycle progression are inhibited by the addition of either a PI3K inhibitor or a mitogen-activated protein-kinase kinase (MEK) inhibitor, indicating that both MEK and PI3K are essential for c-kit-mediated proliferative response. On the contrary, the anti-apoptotic effect of SCF is not influenced by the separate addition of either MEK or PI3K inhibitors. Thus, SCF effects on mitogenesis and survival in c-kit expressing spermatogonia rely on different signal transduction pathways. stem cell factor amino acids extracellular signal-regulated kinase phosphatidylinositol 3-kinase retinoblastoma protein mitogen-activated protein-kinase kinase phosphate-buffered saline glutathione S-transferase terminal dUTP nick-end labeling cyclin-dependent kinase The tyrosine kinase receptor encoded by the c-kit gene and its ligand stem cell factor (SCF)1 play a fundamental role in gametogenesis (1Sette C. Dolci S. Geremia R. Rossi P. Int. J. Dev. Biol. 2000; 44: 599-608PubMed Google Scholar). Most mutations of either c-kit or SCF genes (W and Steel mutations, respectively) result in the loss of primordial germ cells in the embryonal gonad, whereas some Steel mutations affect gametogenesis after birth (2Dolci S. Williams D.E. Ernst M.K. Resnick J.L. Brannan C.I. Lock L.F. Lyman S.D. Boswell H.S. Donovan P.J. Nature. 1991; 352: 809-811Crossref PubMed Scopus (421) Google Scholar, 3Brannan C.I. Bedell M.A. Resnick J.L. Eppig J.J. Handel M.A. Williams D.E. Lyman S.D. Donovan P.J. Jenkins N.A. Copeland N.G. Genes Dev. 1992; 6: 1832-1842Crossref PubMed Scopus (118) Google Scholar). c-kit expression is high in primordial germ cells and is down-regulated in germ cells of the fetal gonad at around 13.5 days postcoitum (4Manova K. Bachvarova R.F. Dev. Biol. 1991; 146: 312-324Crossref PubMed Scopus (197) Google Scholar). It is resumed in perinatal oocytes at the end of meiotic prophase and in proliferating spermatogonia at around 6 days postpartum (5Manova K. Nocka K. Besmer P. Bachvarova R.F. Development. 1990; 110: 1057-1069Crossref PubMed Google Scholar, 6Sorrentino V. Giorgi M. Geremia R. Besmer P. Rossi P. Oncogene. 1991; 6: 149-151PubMed Google Scholar, 7Yoshinaga K. Nishikawa S. Ogawa M. Hayashi S. Kunisada T. Fujimoto T. Nishikawa S.-I. Development. 1991; 113: 689-699PubMed Google Scholar). In the adult testis, c-kit expression is absent in undifferentiated spermatogonia (8Schrans-Stassen B.H. van de Kant H.J. de Rooij D.G. van Pelt A.M. Endocrinology. 1999; 140: 5894-5900Crossref PubMed Google Scholar), high in differentiating spermatogonia from type A1 to B (5Manova K. Nocka K. Besmer P. Bachvarova R.F. Development. 1990; 110: 1057-1069Crossref PubMed Google Scholar, 6Sorrentino V. Giorgi M. Geremia R. Besmer P. Rossi P. Oncogene. 1991; 6: 149-151PubMed Google Scholar, 7Yoshinaga K. Nishikawa S. Ogawa M. Hayashi S. Kunisada T. Fujimoto T. Nishikawa S.-I. Development. 1991; 113: 689-699PubMed Google Scholar, 8Schrans-Stassen B.H. van de Kant H.J. de Rooij D.G. van Pelt A.M. Endocrinology. 1999; 140: 5894-5900Crossref PubMed Google Scholar), and turned off in meiotic and postmeiotic cells (6Sorrentino V. Giorgi M. Geremia R. Besmer P. Rossi P. Oncogene. 1991; 6: 149-151PubMed Google Scholar, 7Yoshinaga K. Nishikawa S. Ogawa M. Hayashi S. Kunisada T. Fujimoto T. Nishikawa S.-I. Development. 1991; 113: 689-699PubMed Google Scholar). A truncated form of the c-kit kinase, possibly playing a role during sperm-induced egg activation at fertilization, is expressed during spermiogenesis (9Rossi P. Marziali G. Albanesi C. Charlesworth A. Geremia R. Sorrentino V. Dev. Biol. 1992; 152: 203-207Crossref PubMed Scopus (94) Google Scholar, 10Albanesi C. Geremia R. Giorgio M. Dolci S. Sette C. Rossi P. Development. 1996; 122: 1291-1302Crossref PubMed Google Scholar, 11Sette C. Bevilacqua A. Bianchini A. Mangia F. Geremia R. Rossi P. Development. 1997; 124: 2267-2274Crossref PubMed Google Scholar, 12Sette C. Bevilacqua A. Geremia R. Rossi P. J. Cell Biol. 1998; 142: 1063-1074Crossref PubMed Scopus (104) Google Scholar). c-kit expression in differentiating spermatogonia has led to the hypothesis that the SCF/c-kit interaction is required for the proliferation and/or survival of these cells. Several lines of evidence support this hypothesis. In vivo injection of antibodies directed against the extracellular region of c-kit selectively blocks proliferation and induces apoptosis of c-kit expressing type A spermatogonia but not of c-kit negative undifferentiated spermatogonia (7Yoshinaga K. Nishikawa S. Ogawa M. Hayashi S. Kunisada T. Fujimoto T. Nishikawa S.-I. Development. 1991; 113: 689-699PubMed Google Scholar, 13Packer A.I. Besmer P. Bachvarova R.F. Mol. Reprod. Dev. 1995; 42: 303-310Crossref PubMed Scopus (142) Google Scholar). Furthermore, a mutation in the c-kit docking site for the p85 subunit of phosphatidylinositol 3-kinase (PI3K), introduced by a knock-in strategy, causes a dramatic reduction of the spermatogonial population in the prepuberal testis (14Blume-Jensen P. Jiang G. Hyman R. Lee K.F. O'Gorman S. Hunter T. Nat. Genet. 2000; 24: 157-162Crossref PubMed Scopus (275) Google Scholar, 15Kissel H. Timokhina I. Hardy M.P. Rothschild G. Tajima Y. Soares V. Angeles M. Whitlow S.R. Manova K. Besmer P. EMBO J. 2000; 19: 1312-1326Crossref PubMed Scopus (308) Google Scholar). A loss of spermatogonia during postnatal development is also observed in a peculiarSteel mutation, Sl 17H (3Brannan C.I. Bedell M.A. Resnick J.L. Eppig J.J. Handel M.A. Williams D.E. Lyman S.D. Donovan P.J. Jenkins N.A. Copeland N.G. Genes Dev. 1992; 6: 1832-1842Crossref PubMed Scopus (118) Google Scholar). Finally,in vitro addition of SCF, which is expressed by Sertoli cells (16Rossi P. Albanesi C. Grimaldi P. Geremia R. Biochem. Biophys. Res. Commun. 1991; 176: 910-914Crossref PubMed Scopus (105) Google Scholar, 17Rossi P. Dolci S. Albanesi C. Grimaldi P. Ricca R. Geremia R. Dev. Biol. 1993; 155: 68-74Crossref PubMed Scopus (195) Google Scholar) under FSH control (17Rossi P. Dolci S. Albanesi C. Grimaldi P. Ricca R. Geremia R. Dev. Biol. 1993; 155: 68-74Crossref PubMed Scopus (195) Google Scholar, 18Yan W. Linderborg J. Suominen J. Toppari J. Endocrinology. 1999; 140: 1499-1504Crossref PubMed Scopus (63) Google Scholar), selectively stimulates DNA synthesis in type A but not in type B spermatogonia (17Rossi P. Dolci S. Albanesi C. Grimaldi P. Ricca R. Geremia R. Dev. Biol. 1993; 155: 68-74Crossref PubMed Scopus (195) Google Scholar, 19Hakovirta H. Yan W. Kaleva M. Zhang F. Vanttinen K. Morris P.L. Sode M. Parvinen M. Toppari J. Endocrinology. 1999; 140: 1492-1498Crossref PubMed Google Scholar). The series of molecular events leading to G1 progression, G1/S transition, and mitosis have been established in several somatic cell types synchronized in G0 through serum starvation (20Lundberg A.S. Weinberg R.A. Eur. J. Cancer. 1999; 35: 531-539Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 21Pestell R.G. Albanese C. Reutens A.T. Segall J.E. Lee R.J. Arnold A. Endocr. Rev. 1999; 20: 501-534Crossref PubMed Scopus (321) Google Scholar, 22Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5159) Google Scholar, 23Johnson D.G. Walker C.L. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 295-312Crossref PubMed Scopus (570) Google Scholar). Synthesis of D-type cyclins and the assembly and nuclear translocation of cyclin D/cyclin-dependent kinase 4/6 (cdk4/6) complexes is required for commitment to G1entry, whereas the consequent cyclin E accumulation and activation of the associated cyclin-dependent kinase 2 (cdk2) allows progression through G1 (20Lundberg A.S. Weinberg R.A. Eur. J. Cancer. 1999; 35: 531-539Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 21Pestell R.G. Albanese C. Reutens A.T. Segall J.E. Lee R.J. Arnold A. Endocr. Rev. 1999; 20: 501-534Crossref PubMed Scopus (321) Google Scholar, 22Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5159) Google Scholar, 23Johnson D.G. Walker C.L. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 295-312Crossref PubMed Scopus (570) Google Scholar). Cyclin D·cdk4/6 complexes trigger initial phosphorylation of the retinoblastoma protein (Rb) and titrate cdk2 inhibitors (cip1/kip1family), thus de-repressing cyclin E/cdk2 activity. Hyperphosphorylation of Rb by cyclin E/cdk2 is followed by release of the Rb-associated transcription factor E2F, which activates cyclin E transcription in a positive feedback loop, allowing the burst of cyclin E accumulation and activity in a narrow window coincident with the G1/S transition. E2F transcriptional activity is required to elicit timely induction of genes required for S phase progression, such as cyclin A2. Progression through the S phase coincident with the appearance of cyclin A2/cdk2 activity is followed by rapid down-regulation of cyclin E levels (20Lundberg A.S. Weinberg R.A. Eur. J. Cancer. 1999; 35: 531-539Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 21Pestell R.G. Albanese C. Reutens A.T. Segall J.E. Lee R.J. Arnold A. Endocr. Rev. 1999; 20: 501-534Crossref PubMed Scopus (321) Google Scholar, 22Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5159) Google Scholar, 23Johnson D.G. Walker C.L. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 295-312Crossref PubMed Scopus (570) Google Scholar). We report evidence that SCF acts as a mitogenic factor in cultured c-kit-expressing spermatogonia and that both mitogen-activated protein kinase kinase (MEK)- and PI3K-dependent pathways are required for the proliferative response. The mitogenic effect is not accompanied by an increase in total cellular amount of cyclin D3 (24Feng L.X. Ravindranath N. Dym M. J. Biol. Chem. 2000; 275: 25572-25576Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), but it is associated with a rapid change in its subcellular localization. We also show that SCF is an anti-apoptotic factor for spermatogonia, but the MEK- or the PI3K-dependent pathways are not sufficient on their own to promote the survival response. Spermatogonia were obtained from either 5/6- or 8-day-old Swiss CD-1 mice, as reported previously (17Rossi P. Dolci S. Albanesi C. Grimaldi P. Ricca R. Geremia R. Dev. Biol. 1993; 155: 68-74Crossref PubMed Scopus (195) Google Scholar). Spermatogonial stem cells and proliferating but undifferentiated spermatogonia are the prevalent germ cell types at 5–6 days of age, whereas differentiating (type A1–A4, intermediate, and type B) spermatogonia predominate at 8 days of age (25Bellve A.R. Cavicchia J.C. Millette C.F. O'Brien D.A. Bhatnagar Y.M. Dym M. J. Cell Biol. 1977; 74: 68-85Crossref PubMed Scopus (1131) Google Scholar, 26Kong Sung W. Komatsu M. Jagiello G.M. Gamete Res. 1986; 14: 245-254Crossref Scopus (9) Google Scholar). Briefly, germ cell suspensions were obtained by sequential collagenase-hyaluronidase-trypsin digestions of freshly withdrawn testes from 20 animals. To release cells completely, after the trypsin treatment, the pellet was resuspended in 1 ml of culture medium and pipetted at least 30 times and then brought to 20 ml with culture medium adding 2 mg/ml DNase and 10% fetal calf serum. Cell suspension was plated in Petri dishes (5 ml/dish) for 3 h in a humidified incubator at 32 °C to promote adhesion of somatic cells. At the end of this pre-plating treatment, enriched germ cell suspensions were washed from fetal calf serum, and spermatogonia were then cultured in Eagle's minimal essential medium supplemented with 1 mmdl-lactic acid, 2 mm sodium pyruvate, non-essential amino acids (Life Technologies, Inc.). For time course experiments, spermatogonia were either left untreated or stimulated with SCF (100 ng/ml, Genzyme) at different time points and then they were processed as described below. Where indicated cells were also incubated 1 h before SCF addition with 10 µm U0126 (catalog number V1121, Promega), with 10 µm LY294002 (catalog number 270-038-M005, Alexis), or with 1 µm tyrphostin AG490 (catalog number 658401, Calbiochem), all dissolved in Me2SO. In these experiments, an equal volume of the Me2SO solvent was also added in control and SCF-treated cultures. Nuclear morphology of spermatogonia after the pre-plating time and after 24 h of culture in the absence or constant presence of SCF and/or the signaling inhibitors was assessed after hypotonic shock of 105 cells (75 mm KCl) followed by fixation in methanol:acetic acid solution (3:1). Cells were then dropped onto glass slides to allow spreading of the nuclei and stained with Giemsa solution. Spermatogonia nuclei were judged as in interphase, metaphase, or apoptotic and counted from quadruplicate experiments. Somatic nuclei were excluded from the counts, and purity of spermatogonia was assessed as about 85% after the pre-plating treatment and almost 100% after 24 h of culture. DNA synthesis was studied by [3H]thymidine incorporation followed by autoradiography as previously described (17Rossi P. Dolci S. Albanesi C. Grimaldi P. Ricca R. Geremia R. Dev. Biol. 1993; 155: 68-74Crossref PubMed Scopus (195) Google Scholar). In these experiments, incubation with [3H]thymidine was performed during the last 4 h of the 24 h culture period. Cells were harvested, washed in cold PBS, and homogenized at 4 °C in lysis buffer containing 10 mm Hepes, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.1 mm EGTA, 0.5 mm dithiothreitol, 10 mm β-glycerophosphate, 0.1 mm sodium vanadate, and 1/100th (v/v) of a pre-formed protease inhibitors mixture (P8340, Sigma). Total cellular proteins were transferred to polyvinylidene difluoride membranes after SDS-PAGE. Membranes were blocked with PBS buffer containing 5% fat-free milk and 0.1% Tween 20 for 1 h at room temperature and then hybridized with primary antibodies. After hybridization with secondary antibodies conjugated to horseradish peroxidase, the immunocomplexes were detected with Supersignal West Pico detection reagent (Pierce). Primary antibodies used are as follows: anti-phospho-Akt and anti-Akt rabbit polyclonal (PhosphoPlus Akt (Ser-473) antibody kit, catalog number 9270, New England Biolabs Inc.); anti-phospho Erk1/2 mouse monoclonal antibody (sc-7383, Santa Cruz Biotechnology, Inc.); anti-Erk2 rabbit polyclonal (sc-154, Santa Cruz Biotechnology, Inc.); anti-cyclin D3 mouse monoclonal antibody (sc-6283, Santa Cruz Biotechnology, Inc.); anti-cyclin E rabbit polyclonal antibody (sc-481, Santa Cruz Biotechnology, Inc.); anti-cyclin A2 rabbit polyclonal antibody (sc-751, Santa Cruz Biotechnology, Inc.); anti-Rb (aa 332–344) mouse monoclonal antibody (14001A, PharMingen); anti-Rb (carboxyl terminus) rabbit polyclonal antibody (sc-50, Santa Cruz Biotechnology Inc.); anti-p21Cip1/Waf1 mouse monoclonal antibody (catalog number sc-6246, Santa Cruz Biotechnology, Inc.); anti-c-Myc rabbit polyclonal antibody (sc-788, Santa Cruz Biotechnology, Inc.); anti-cdk4 goat polyclonal antibody (sc-260-G, Santa Cruz Biotechnology, Inc.). 2 × 106 viable cells were harvested and homogenized in 40 µl of a modified lysis buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10% glycerol, 1 mm dithiothreitol, 0.1% Tween 20, 10 mm β-glycerophosphate, 1 mM NaF, 0.1 mmsodium vanadate, 0.1 mm phenylmethylsulfonyl fluoride) containing 0.4 µl of a pre-formed protease inhibitors mixture (P8340, Sigma). One hundred µg of proteins from clarified supernatants of whole cell lysates were incubated with 20 µg/ml anti-cyclin D3 monoclonal antibody or anti-cyclin E polyclonal antibody for 2 h at 4 °C on a rotating shaker. The immunocomplexes were recovered with protein G-Sepharose or protein A-Sepharose (Sigma), respectively, for 1 h at 4 °C, washed three times at 4 °C with PBS, 0.5% bovine serum albumin and once with the specific kinase reaction buffer (50 mm Hepes, pH 7.5, 10 mm MgCl2, and 1 mm dithiothreitol). Kinase assays were performed at 30 °C for 30 min in a 20-µl volume of kinase reaction buffer containing 10 mm β-glycerophosphate, 0.1 mmsodium vanadate, 0.2 µl of a pre-formed protease inhibitors mixture (P8340, Sigma), 2.5 mm EGTA, 50 µm ATP, 0.1 mm protein kinase A inhibitor, 3 µCi of [γ-32P]ATP/reaction, and the following specific substrates: 0.5 µg/reaction histone H1 (type III-S, Sigma) for cyclin E/Cdk2 and 0.5 µg/reaction GST-Rb (sc-4112, Santa Cruz Biotechnology Inc.) for cyclin D3/Cdk4/6. Reactions were terminated by addition of 4× Laemmli buffer. Samples were boiled, and proteins were separated by SDS-PAGE. Phosphorylated substrates were visualized by autoradiography. Control and 1-h SCF-treated spermatogonia, preincubated or not with U0126 or LY294002, were spotted onto poly-l-lysine glass slides and fixed for 10 min at room temperature in 2% paraformaldehyde. Cells were washed in PBS, permeabilized 10 min with PBS, 0.1% Triton X-100 and incubated for 30 min at room temperature with PBS, 0.5% bovine serum albumin. Cells were incubated overnight at 4 °C in a humidified chamber with mouse monoclonal anti-cyclin D3 antibody at a final concentration of 2 µg/ml and then 1 h at room temperature with cyanin 3-conjugated anti-mouse IgG (Calbiochem). Slides were washed and mounted in 50% glycerol in PBS and immediately examined by fluorescence microscopy. Nuclei were counterstained with 1 µg/ml Hoechst (catalog number 33342, Sigma). Control experiments were performed using mouse non-immune IgGs instead of the specific antibody. For in situ detection of apoptotic cell death, control and SCF-treated spermatogonia, preincubated or not with U0126, LY294002, or AG490, after a 24-h period of culture were spotted onto poly-l-lysine glass slides, fixed for 10 min at room temperature in 2% paraformaldehyde, and subjected to TUNEL assay with an in situ cell death detection kit (catalog number 1684817,Roche Molecular Biochemicals) by following the manufacturer's instructions. Nuclei were counterstained with 1 µg/ml Hoechst (catalog number 33342, Sigma). Slides where then examined by fluorescence microscopy. Cultures of germ cells obtained from 8-day-old mice are particularly enriched in differentiating spermatogonia (25Bellve A.R. Cavicchia J.C. Millette C.F. O'Brien D.A. Bhatnagar Y.M. Dym M. J. Cell Biol. 1977; 74: 68-85Crossref PubMed Scopus (1131) Google Scholar, 26Kong Sung W. Komatsu M. Jagiello G.M. Gamete Res. 1986; 14: 245-254Crossref Scopus (9) Google Scholar), which express high levels of c-kit (5Manova K. Nocka K. Besmer P. Bachvarova R.F. Development. 1990; 110: 1057-1069Crossref PubMed Google Scholar, 6Sorrentino V. Giorgi M. Geremia R. Besmer P. Rossi P. Oncogene. 1991; 6: 149-151PubMed Google Scholar, 7Yoshinaga K. Nishikawa S. Ogawa M. Hayashi S. Kunisada T. Fujimoto T. Nishikawa S.-I. Development. 1991; 113: 689-699PubMed Google Scholar, 8Schrans-Stassen B.H. van de Kant H.J. de Rooij D.G. van Pelt A.M. Endocrinology. 1999; 140: 5894-5900Crossref PubMed Google Scholar, 14Blume-Jensen P. Jiang G. Hyman R. Lee K.F. O'Gorman S. Hunter T. Nat. Genet. 2000; 24: 157-162Crossref PubMed Scopus (275) Google Scholar). Fig. 1shows that, after 24 h of culture, several nuclei with characteristic features of apoptosis, such as reduced size and intense chromatin staining, can be observed in untreated cells. In SCF-treated cultures, the frequency of such cells is clearly reduced (see the last paragraph of this section), and a clear increase in the number of mitotic figures (nuclei showing condensed metaphase chromosomes) can be appreciated. These data confirm that SCF is required to maintain the proliferative state of differentiating spermatogonia cultured in vitro (17Rossi P. Dolci S. Albanesi C. Grimaldi P. Ricca R. Geremia R. Dev. Biol. 1993; 155: 68-74Crossref PubMed Scopus (195) Google Scholar,24Feng L.X. Ravindranath N. Dym M. J. Biol. Chem. 2000; 275: 25572-25576Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). We studied DNA synthesis and cell cycle progression in these cultures by using [3H]thymidine incorporation and metaphase counting. SCF induces a 2-fold increase in the number of [3H]thymidine incorporating cells and a 3-fold increase of metaphase counts with respect to the control after 24 h of culture (Table I). We also analyzed the effects of SCF addition in germ cell populations from 5- to 6-day-old mice, when undifferentiated spermatogonia are the predominant cell types (25Bellve A.R. Cavicchia J.C. Millette C.F. O'Brien D.A. Bhatnagar Y.M. Dym M. J. Cell Biol. 1977; 74: 68-85Crossref PubMed Scopus (1131) Google Scholar, 26Kong Sung W. Komatsu M. Jagiello G.M. Gamete Res. 1986; 14: 245-254Crossref Scopus (9) Google Scholar), and c-kit expression is not detectable (5Manova K. Nocka K. Besmer P. Bachvarova R.F. Development. 1990; 110: 1057-1069Crossref PubMed Google Scholar, 7Yoshinaga K. Nishikawa S. Ogawa M. Hayashi S. Kunisada T. Fujimoto T. Nishikawa S.-I. Development. 1991; 113: 689-699PubMed Google Scholar, 8Schrans-Stassen B.H. van de Kant H.J. de Rooij D.G. van Pelt A.M. Endocrinology. 1999; 140: 5894-5900Crossref PubMed Google Scholar, 27Tajima Y. Sawada K. Morimoto T. Nishimune Y. J. Reprod. Fertil. 1994; 102: 117-122Crossref PubMed Scopus (45) Google Scholar,28Ohta H. Yomogida K. Dohmae K. Nishimune Y. Development. 2000; 127: 2125-2131PubMed Google Scholar). No stimulation of cell cycle progression was observed in these cells (% of 3H-labeled cells in control cultures, 9.20 ± 0.05; in SCF-treated cultures, 9.40 ± 1.85; % cells in mitosis in control cultures, 1.25 ± 0.35; in SCF-treated cultures, 0.70 ± 0.30).Table IMEK- and PI3K-dependent stimulation of DNA synthesis and cell cycle progression induced by SCF in spermatogonia from 8-day-old mice cultured for 24 hControlSCFU0126SCF + U0126LY-294002SCF + LY-294002AG490SCF + AG490% 3H-labeled cells1-aPercentage ± S.D. of [3H]thymidine labeled nuclei observed out of 500 cells in autoradiographic slides. See Ref. 17 for further experimental details. (n = 8)19.58 ± 3.8734.90 ± 2.011-bStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.0001.19.52 ± 3.131-cStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.18.92 ± 1.631-cStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.15.02 ± 5.441-cStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.13.92 ± 4.151-cStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.23.57 ± 2.981-cStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.43.92 ± 0.821-bStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.0001.% cells in metaphase1-dPercentage ± S.D. of cells with nuclei showing condensed metaphase chromosomes out of 500 cells stained with Giemsa (see Fig. 1). (n = 5)1.08 ± 0.312.94 ± 0.501-eStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p value versuscontrol, p < 0.01.0.62 ± 0.241-cStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.0.62 ± 0.211-cStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.1.12 ± 0.321-cStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.1.31 ± 0.301-cStatistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.ND1-fND, not determined.ND1-fND, not determined.1-a Percentage ± S.D. of [3H]thymidine labeled nuclei observed out of 500 cells in autoradiographic slides. See Ref. 17Rossi P. Dolci S. Albanesi C. Grimaldi P. Ricca R. Geremia R. Dev. Biol. 1993; 155: 68-74Crossref PubMed Scopus (195) Google Scholar for further experimental details.1-b Statistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.0001.1-c Statistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p values versuscontrol, p < 0.5.1-d Percentage ± S.D. of cells with nuclei showing condensed metaphase chromosomes out of 500 cells stained with Giemsa (see Fig. 1).1-e Statistical analysis (analysis of variance test) was performed using a program PSI-plot 3.0 from Polysoftware International, p value versuscontrol, p < 0.01.1-f ND, not determined. Open table in a new tab c-kit signaling pathways activated in cell cycle progression have been shown to involve PI3K, MEK, and Janus-activated kinase 2 (JAK2) in different cell types (14Blume-Jensen P. Jiang G. Hyman R. Lee K.F. O'Gorman S. Hunter T. Nat. Genet. 2000; 24: 157-162Crossref PubMed Scopus (275) Google Scholar, 15Kissel H. Timokhina I. Hardy M.P. Rothschild G. Tajima Y. Soares V. Angeles M. Whitlow S.R. Manova K. Besmer P. EMBO J. 2000; 19: 1312-1326Crossref PubMed Scopus (308) Google Scholar, 29Wu M. Hemesath T.J. Takemoto C.M. Horstmann M.A. Wells A.G. Price E.R. Fisher D.Z. Fisher D.E. Genes Dev. 2000; 14: 301-312PubMed Google Scholar, 30Weiler S.R. Mou S. DeBerry C.S. Keller J.R. Ruscetti F.W. Ferris D.K. Longo D.L. Linnekin D. Blood. 1996; 87: 3688-3693Crossref PubMed Google Scholar). In mouse spermatogonia, PI3K activation has been shown to be involved in SCF-dependent proliferation (14Blume-Jensen P. Jiang G. Hyman R. Lee K.F. O'Gorman S. Hunter T. Nat. Genet. 2000; 24: 157-162Crossref PubMed Scopus (275) Google Scholar, 15Kissel H. Timokhina I. Hardy M.P. Rothschild G. Tajima Y. Soares V. Angeles M. Whitlow S.R. Manova K. Besmer P. EMBO J. 2000; 19: 1312-1326Crossref PubMed Scopus (308) Google Scholar, 24Feng L.X. Ravindranath N. Dym M. J. Biol. Chem. 2000; 275: 25572-25576Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar); however, the possible involvement of MEK- and JAK2-dependent pathways has not been studied. To investigate whether these c-kit-activated signaling pathways mediate the mitogenic activation of spermatogonia observed in vitro, the proliferation assays were performed in the presence of inhibitors selective for each of the three different pathways: the MEK inhibitor U0126, the JAK2 inhibitor tyrphostin AG490, and the PI3K inhibitor LY294002 (Table I). The inhibition of the MEK pathway abolished the SCF-induced increase in both [3H]thymidine incorporation and metaphase counts, demonstrating that the integrity of this pathway is required for SCF induction of mitogenesis. Inhibition of PI3K pathway also abolished SCF mitogenic effect, indicating that both MEK and PI3K pathways are required. On the contrary, inhibition of JAK2 signaling had no effect on SCF-stimulated [3H]thymidine incorporation. Since the MEK and PI3K inhibitors were effective in the inhibition of SCF-induced proliferation" @default.
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• W2000519963 title "Signaling through Extracellular Signal-regulated Kinase Is Required for Spermatogonial Proliferative Response to Stem Cell Factor" @default.
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