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• W2078122977 abstract "When Sertoli and germ cells were co-cultured in vitro in serum-free chemically defined medium, functional anchoring junctions such as cell-cell intermediate filament-based desmosome-like junctions and cell-cell actin-based adherens junctions (e.g. ectoplasmic specialization (ES)) were formed within 1-2 days. This event was marked by the induction of several protein kinases such as phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (PKB; also known as Akt), p21-activated kinase-2 (PAK-2), and their downstream effector (ERK) as well as an increase in PKB intrinsic activity. PI3K, phospho (p)-PKB, and PAK were co-localized to the site of apical ES in the seminiferous epithelium of the rat testis in immunohistochemistry studies. Furthermore, PI3K also co-localized with p-PKB to the same site in the epithelium as determined by fluorescence microscopy, consistent with their localization at the ES. These kinases were shown to associate with ES-associated proteins such as β1-integrin, phosphorylated focal adhesion kinase, and c-Src by co-immunoprecipitation, suggesting that the integrin·laminin protein complex at the apical ES likely utilizes these protein kinases as regulatory proteins to modulate Sertoli-germ cell adherens junction dynamics via the ERK signaling pathway. To validate this hypothesis further, an in vivo model using AF-2364 (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Interestingly, the event of germ cell loss induced by AF-2364 in vivo was also associated with induction of PI3K, p-PKB, PAK-2, and p-ERK as well as a surge in intrinsic PKB activity. Perhaps the most important of all, pretreatment of rats with wortmannin (a PI3K inhibitor) or anti-β1-integrin antibody via intratesticular injection indeed delayed AF-2364-induced spermatid loss from the epithelium. In summary, these results illustrate that Sertoli-germ cell anchoring junction dynamics in the testis are regulated, at least in part, via the β1-integrin/PI3K/PKB/ERK signaling pathway. When Sertoli and germ cells were co-cultured in vitro in serum-free chemically defined medium, functional anchoring junctions such as cell-cell intermediate filament-based desmosome-like junctions and cell-cell actin-based adherens junctions (e.g. ectoplasmic specialization (ES)) were formed within 1-2 days. This event was marked by the induction of several protein kinases such as phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (PKB; also known as Akt), p21-activated kinase-2 (PAK-2), and their downstream effector (ERK) as well as an increase in PKB intrinsic activity. PI3K, phospho (p)-PKB, and PAK were co-localized to the site of apical ES in the seminiferous epithelium of the rat testis in immunohistochemistry studies. Furthermore, PI3K also co-localized with p-PKB to the same site in the epithelium as determined by fluorescence microscopy, consistent with their localization at the ES. These kinases were shown to associate with ES-associated proteins such as β1-integrin, phosphorylated focal adhesion kinase, and c-Src by co-immunoprecipitation, suggesting that the integrin·laminin protein complex at the apical ES likely utilizes these protein kinases as regulatory proteins to modulate Sertoli-germ cell adherens junction dynamics via the ERK signaling pathway. To validate this hypothesis further, an in vivo model using AF-2364 (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) to perturb Sertoli-germ cell anchoring junction function, inducing germ cell loss from the epithelium in adult rats, was used in conjunction with specific inhibitors. Interestingly, the event of germ cell loss induced by AF-2364 in vivo was also associated with induction of PI3K, p-PKB, PAK-2, and p-ERK as well as a surge in intrinsic PKB activity. Perhaps the most important of all, pretreatment of rats with wortmannin (a PI3K inhibitor) or anti-β1-integrin antibody via intratesticular injection indeed delayed AF-2364-induced spermatid loss from the epithelium. In summary, these results illustrate that Sertoli-germ cell anchoring junction dynamics in the testis are regulated, at least in part, via the β1-integrin/PI3K/PKB/ERK signaling pathway. In the seminiferous epithelium of the rat testis, Sertoli-germ cell adhesion is maintained by cell-cell actin-based adherens junctions (AJs) 1The abbreviations used are: AJs, adherens junctions; ES, ectoplasmic specialization(s); BTB, blood-testis barrier; FAK, focal adhesion kinase; PI3K, phosphatidylinositol 3-kinase; SH2, Src homology 2; PPI, polyphosphoinositide; PI-4,5-P2, phosphatidylinositol 4,5-bisphosphate; PI-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PH, pleckstrin homology; PKB, protein kinase B; PDK1, phosphoinositide-dependent kinase-1; PAK, p21-activated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; FA, focal adhesion; DMEM, Dulbecco's modified Eagle's medium; TJ, tight junction; EGF, epidermal growth factor; p-, phospho-; Co-IP, co-immunoprecipitation; GSK-3, glycogen synthase kinase-3; PBS, phosphate-buffered saline; MMP, matrix metalloprotease; MAPK, mitogen-activated protein kinase. and intermediate filament-based desmosome-like junctions (for reviews, see Refs. 1Cheng C.Y. Mruk D.D. Physiol. Rev. 2002; 82: 825-874Crossref PubMed Scopus (475) Google Scholar, 2Vogl A.W. Pfeiffer D.C. Mulholland D. Kimel G. Guttman J. Arch. Histol. Cytol. 2000; 63: 1-15Crossref PubMed Scopus (169) Google Scholar, 3Mruk D.D. Cheng C.Y. Endocr. Rev. 2004; 25: 747-806Crossref PubMed Scopus (662) Google Scholar). The best studied testis-specific AJ type is ectoplasmic specialization (ES). The ES is confined between Sertoli cells (known as the basal ES) at the site of the blood-testis barrier (BTB) as well as between Sertoli cells and spermatids (known as the apical ES) in the adluminal compartment of the epithelium (for reviews, see Refs. 3Mruk D.D. Cheng C.Y. Endocr. Rev. 2004; 25: 747-806Crossref PubMed Scopus (662) Google Scholar and 4Vogl A.W. Int. Rev. Cytol. 1989; 119: 1-56Crossref PubMed Scopus (132) Google Scholar). Most of the studies on cell adhesion function in the testis in the past 2 decades have focused on the apical ES because the biochemical composition of the desmosome-like junction remains largely unexplored in the testis (for reviews, see Refs. 1Cheng C.Y. Mruk D.D. Physiol. Rev. 2002; 82: 825-874Crossref PubMed Scopus (475) Google Scholar, 3Mruk D.D. Cheng C.Y. Endocr. Rev. 2004; 25: 747-806Crossref PubMed Scopus (662) Google Scholar, and 5Toyama Y. Maekawa M. Yuasa S. Anat. Sci. Int. 2003; 78: 1-16Crossref PubMed Scopus (76) Google Scholar). The apical ES is an important anchoring junction device that provides mechanical adhesion of spermatids onto the nourishing Sertoli cells to assist movement of developing spermatids across the epithelium and to ensure proper orientation of spermatids in the epithelium so that fully developed spermatids can be released to the tubule lumen during spermiation. Without this timely event of spermatid movement, spermatogenesis cannot be completed, leading to infertility. Although the morphology of the ES has been characterized for almost 3 decades, its biochemical composition and molecular architecture have not been known until recently. Furthermore, the underlying regulatory mechanism(s) that regulates ES dynamics remains largely unexplored (for reviews, see Refs. 1Cheng C.Y. Mruk D.D. Physiol. Rev. 2002; 82: 825-874Crossref PubMed Scopus (475) Google Scholar, 6de Kretser D.M. Kerr J.B. Knobil E. Neill J. The Physiology of Reproduction. I. Raven Press, New York1988: 837-932Google Scholar, and 7Siu M.K.Y. Cheng C.Y. BioEssays. 2004; 26: 978-992Crossref PubMed Scopus (150) Google Scholar). However, recent studies have shown that ES dynamics are regulated by focal adhesion complex-associated proteins such as β1-integrin, focal adhesion kinase (FAK), and vinculin. Of particular interest is the hypothesis that tyrosine-phosphorylated (activated) FAK is a crucial linker between β1-integrin and other ES components at the apical ES (7Siu M.K.Y. Cheng C.Y. BioEssays. 2004; 26: 978-992Crossref PubMed Scopus (150) Google Scholar, 8Siu M.K.Y. Mruk D.D. Lee W.M. Cheng C.Y. Endocrinology. 2003; 144: 2141-2163Crossref PubMed Scopus (155) Google Scholar). As such, a better understanding of the downstream signaling pathway(s) of integrin and FAK is crucial to the study of ES dynamics. This is also important to developmental biologists and reproductive physiologists because it helps unfold the underlying mechanism that regulates junction restructuring events in the seminiferous epithelium pertinent to spermatogenesis. Autophosphorylation of FAK at Tyr397 can recruit a variety of cytosolic proteins to the plasma membrane (for review, see Ref. 9Parsons J.T. J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1150) Google Scholar). For example, phosphatidylinositol 3-kinase (PI3K) p85α, the adaptor subunit of PI3K, is one of the Src homology 2 (SH2) domain-containing proteins that bind to FAK (10Chen H.C. Appeddu P.A. Isoda H. Guan J.L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 11Cantrell D.A. J. Cell Sci. 2001; 114: 1439-1445Crossref PubMed Google Scholar). Upon this binding, p85α subsequently recruits the PI3K p110 catalytic subunit to the plasma membrane, where it phosphorylates polyphosphoinositides (PPIs; predominantly phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2)) at position 3 of the inositol ring, producing a second messenger, phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5-P3) (for reviews, see Refs. 11Cantrell D.A. J. Cell Sci. 2001; 114: 1439-1445Crossref PubMed Google Scholar, 12Merlot S. Firtel R.A. J. Cell Sci. 2003; 116: 3471-3478Crossref PubMed Scopus (237) Google Scholar, 13Foster F.M. Traer C.J. Abraham S.M. Fry M.J. J. Cell Sci. 2003; 116: 3037-3040Crossref PubMed Scopus (235) Google Scholar). The accumulated PI-3,4,5-P3 acts as membrane anchor that recruits and activates pleckstrin homology (PH) domain-containing proteins such as protein kinase B (PKB; a Ser/Thr kinase also known as Akt), which is a PI3K effector (for reviews, see Refs. 11Cantrell D.A. J. Cell Sci. 2001; 114: 1439-1445Crossref PubMed Google Scholar and 12Merlot S. Firtel R.A. J. Cell Sci. 2003; 116: 3471-3478Crossref PubMed Scopus (237) Google Scholar). The membrane-localized PKB is then activated via phosphorylation at Thr308 and Ser473 by a second phosphoinositide-dependent Ser/Thr kinase, phosphoinositide-dependent kinase-1 (PDK1), and a yet-to-be identified “Ser473 kinase,” respectively. In turn, the activated PKB phosphorylates its downstream effectors at the plasma membrane, in the cytosol, and in the nucleus. Depending on the specific effector that is being activated, diverse biological processes such as glucose metabolism, cell cycle progression, apoptosis, transcription regulation, and cell motility can be modulated by PKB (for reviews, see Refs. 14Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar and 15Scheid M.P. Woodgett J.R. FEBS Lett. 2003; 546: 108-112Crossref PubMed Scopus (346) Google Scholar). The turnover of PI-3,4,5-P3 is mediated by phosphatases, which dephosphorylate the 3′-end of the lipid, leading to a reduction of the pool of lipids capable of PKB binding, thus negatively regulating the PI3K/PKB signaling pathway. PTEN (phosphatase and tensin homolog deleted on chromosome ten) is the major lipid phosphatase that antagonizes the reactions catalyzed by PI3K (16Marino M. Acconcia F. Trentalance A. Mol. Biol. Cell. 2003; 14: 2583-2591Crossref PubMed Scopus (113) Google Scholar). Interestingly, there is accumulating evidence that the PI3K/PKB pathway plays an important role in the formation and stabilization of AJs, linking AJ components to the cytoskeleton in keratinocytes, intestinal epithelial cells, mammary epithelial cells, and Caco-2/15 cells (18Munshi H.G. Ghosh S. Mukhopadhyay S. Wu Y.I. Sen R. Green K.J. Stack M.S. J. Biol. Chem. 2002; 277: 38159-38167Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 19Laprise P. Chailler P. Houde M. Beaulieu J.F. Boucher M.J. Rivard N. J. Biol. Chem. 2002; 277: 8226-8234Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In the testis, PI-4,5-P2 and phosphoinositide-specific phospholipase C have been identified in the ES (20Guttman J.A. Janmey P. Vogl A.W. J. Cell Sci. 2002; 115: 499-505Crossref PubMed Google Scholar), and the PI3K/PKB signaling pathway has recently been shown to be regulated by follicle-stimulating hormone in 20-day-old Sertoli cells (21Meroni S.B. Riera M.F. Pellizzari E.H. Galardo M.N. Cigorraga S.B. J. Endocrinol. 2004; 180: 257-265Crossref PubMed Scopus (49) Google Scholar, 22Khan S.A. Ndjountche L. Pratchard L. Spicer L.J. Davis J.S. Endocrinology. 2002; 143: 2259-2267Crossref PubMed Scopus (71) Google Scholar, 23Meroni S.B. Riera M.F. Pellizzari E.H. Cigorraga S.B. J. Endocrinol. 2002; 174: 195-204Crossref PubMed Scopus (93) Google Scholar). However, little is known regarding the upstream and downstream mediators of the PI3K/PKB signaling pathway in the testis and its contribution to junction dynamics, especially at the apical ES. p21-activated kinases (PAK)-1-3 (Ser/Thr protein kinases) are direct PKB effectors (for reviews, see Refs. 12Merlot S. Firtel R.A. J. Cell Sci. 2003; 116: 3471-3478Crossref PubMed Scopus (237) Google Scholar, 24Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (885) Google Scholar, and 25Kumar R. Vadlamudi R.K. J. Cell. Physiol. 2002; 193: 133-144Crossref PubMed Scopus (99) Google Scholar). The activation of PAKs by direct binding to the active Rac and Cdc42 GTPases has been well established. In addition, PAKs can be activated through a variety of GTPase-independent mechanisms, including the direct phosphorylation by other kinases such as PKB and PDK1 (24Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (885) Google Scholar, 25Kumar R. Vadlamudi R.K. J. Cell. Physiol. 2002; 193: 133-144Crossref PubMed Scopus (99) Google Scholar, 26Zhou G.L. Zhuo Y. King C.C. Fryer B.H. Bokoch G.M. Field J. Mol. Cell. Biol. 2003; 23: 8058-8069Crossref PubMed Scopus (140) Google Scholar). Because of their complicated activation processes and the presence of multiple substrates, PAKs serve as important modulators for a wide range of cellular processes, including cytoskeletal reorganization, cell motility, apoptosis, cell cycle progression, and cell transformation (for review, see Ref. 24Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (885) Google Scholar). One interesting feature of PAKs is their ability to phosphorylate Raf-1 in a PI3K-dependent manner, which, along with integrin, mediates the Ras signaling pathway that leads to activation of the Raf-1/MEK/ERK signaling cascade (for review, see Ref. 25Kumar R. Vadlamudi R.K. J. Cell. Physiol. 2002; 193: 133-144Crossref PubMed Scopus (99) Google Scholar). Besides PAKs, PKB is another crucial downstream mediator of the PI3K/Raf-1 cross-talk in the Ras/Raf-1/MEK/ERK signaling pathway (14Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 27Moelling K. Schad K. Bosse M. Zimmermann S. Schweneker M. J. Biol. Chem. 2002; 277: 31099-31106Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). In the testis, activated ERK has been localized at the apical ES at the time of spermiation, implicating its role in ES disassembly (28Chapin R.E. Wine R.N. Harris M.W. Borchers C.H. Haseman J.K. J. Androl. 2001; 22: 1030-1052Crossref PubMed Scopus (96) Google Scholar). However, the precise mechanism(s) for ERK-mediated ES dynamics remains largely undefined. Furthermore, PI3K, PKB, PAK, and ERK are imperative regulators of cytoskeletal dynamics, conferring cell migration at the focal adhesion (FA) site. Because cytoskeletal rearrangement is an essential process for ES restructuring (for reviews, see Refs. 1Cheng C.Y. Mruk D.D. Physiol. Rev. 2002; 82: 825-874Crossref PubMed Scopus (475) Google Scholar, 2Vogl A.W. Pfeiffer D.C. Mulholland D. Kimel G. Guttman J. Arch. Histol. Cytol. 2000; 63: 1-15Crossref PubMed Scopus (169) Google Scholar, and 7Siu M.K.Y. Cheng C.Y. BioEssays. 2004; 26: 978-992Crossref PubMed Scopus (150) Google Scholar), it is possible that the PI3K/PKB/PAK/ERK pathway is the major downstream signaling cascade of integrin and FAK at the apical ES (7Siu M.K.Y. Cheng C.Y. BioEssays. 2004; 26: 978-992Crossref PubMed Scopus (150) Google Scholar, 8Siu M.K.Y. Mruk D.D. Lee W.M. Cheng C.Y. Endocrinology. 2003; 144: 2141-2163Crossref PubMed Scopus (155) Google Scholar) that regulates ES restructuring. To elucidate the involvement of these kinases in ES dynamics, the expression and activation of these signal mediators during AJ restructuring and their localization in the testis were examined in both in vitro and in vivo models. These findings further support the hypothesis that FA complex-associated proteins are involved in ES dynamics and that the ES is a hybrid cell-cell and cell-matrix actin-based anchoring junction type. Animals—Sprague-Dawley rats were obtained from Charles River Laboratories, Inc. (Kingston, MA). The use of animals reported herein was approved by The Rockefeller University Laboratory Animal Use and Care Committee with Protocol Numbers 00111 and 03017. Primary Sertoli Cell Cultures—Sertoli cells were isolated from the testes of 20-day-old rats (29Grima J. Wong C.C.S. Zhu L.J. Zong S.D. Cheng C.Y. J. Biol. Chem. 1998; 273: 21040-21053Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Cells were plated at high cell density (0.5 × 106 cells/cm2) on 12-well dishes (Corning) coated with Matrigel™ (Collaborative Biochemical Products, Bedford, MA) in 1:1 (v/v) nutrient mixture F-12 and Dulbecco's modified Eagle's medium (DMEM) (3 ml/well) supplemented with growth factors as described (29Grima J. Wong C.C.S. Zhu L.J. Zong S.D. Cheng C.Y. J. Biol. Chem. 1998; 273: 21040-21053Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 30Mruk D.D. Cheng C.Y. J. Biol. Chem. 1999; 274: 27056-27068Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Cultures were incubated in a humidified atmosphere of 95% air and 5% CO2 (v/v) at 35 °C. After 48 h of incubation, cultures were hypotonically treated with 20 mm Tris (pH 7.4) for 2.5 min to lyse residual germ cells (31Galdieri M. Ziparo E. Palombi F. Russo M.A. Stefanini M. J. Androl. 1981; 5: 249-259Crossref Scopus (382) Google Scholar), followed by two successive washes with F-12/DMEM to remove cell debris. The media were replaced every 24 h. The purity of these Sertoli cell cultures was routinely analyzed by electron and light microscopy (32Mruk D.D. Zhu L.J. Silvestrini B. Lee W.M. Cheng C.Y. J. Androl. 1997; 18: 612-622PubMed Google Scholar, 33Lui W.Y. Mruk D.D. Lee W.M. Cheng C.Y. Biol. Reprod. 2003; 68: 1087-1097Crossref PubMed Scopus (156) Google Scholar) as well as by reverse transcription-PCR as described (34Lee N.P.Y. Mruk D.D. Lee W.M. Cheng C.Y. Biol. Reprod. 2003; 68: 489-508Crossref PubMed Scopus (144) Google Scholar). Sertoli cells cultured for 5 days were either lysed in SDS lysis buffer (0.125 m Tris (pH 6.8) at 22 °C containing 1% (w/v) SDS, 2 mm EDTA, 2 mm N-ethylmaleimide, 2 mm phenylmethylsulfonyl fluoride, 1.6% (v/v) 2-mercaptoethanol, 1 mm sodium orthovanadate, and 0.1 μm sodium okadate) for immunoblotting experiments or used for Sertoli-germ cell co-cultures. Germ Cell Isolation—Germ cells were isolated from 90-day-old rat testes by a mechanical procedure (35Aravindan G.R. Pineau C.P. Bardin C.W. Cheng C.Y. J. Cell. Physiol. 1996; 168: 123-133Crossref PubMed Scopus (84) Google Scholar), except that elongating/elongated spermatids were not removed by omitting the glass wool filtration step. Isolated germ cells were incubated with 1 μm gelsolin (Sigma) in F-12/DMEM for 15 min to disrupt residual actin cytoskeleton in the ES structure that might remain associated with elongating/elongated spermatids. This concentration was selected based on a previous study (36Strege P.R. Holm A.N. Rich A. Miller S.M. Ou Y. Sarr M.G. Farrugia G. Am. J. Physiol. Cell Physiol. 2003; 284: C60-C66Crossref PubMed Scopus (63) Google Scholar). The purity of germ cells was >95% when examined microscopically and assessed by other criteria (34Lee N.P.Y. Mruk D.D. Lee W.M. Cheng C.Y. Biol. Reprod. 2003; 68: 489-508Crossref PubMed Scopus (144) Google Scholar). Germ cells were either lysed in SDS lysis buffer for immunoblotting or used for Sertoli-germ cell co-cultures within 1 h after isolation. Sertoli-Germ Cell Co-cultures—Germ cells isolated from adult rat testes were added onto the Sertoli cell epithelium on day 6 after Sertoli cells had been cultured alone for 5 days, forming an intact epithelium (32Mruk D.D. Zhu L.J. Silvestrini B. Lee W.M. Cheng C.Y. J. Androl. 1997; 18: 612-622PubMed Google Scholar), and co-cultured at a Sertoli/germ cell ratio of 1:1 to permit ES assembly. Time 0 represents the time at which germ cells were added onto the Sertoli cell epithelium. Co-cultures were terminated at specific time points by SDS lysis buffer for immunoblotting or by cell lysis buffer (20 mm Tris (pH 7.5) containing 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% (v/v) Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mm sodium orthovanadate, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride) for kinase assay. Using this approach, any changes in target proteins or intrinsic kinase activity could be ascribed to the assembly of Sertoli-germ cell anchoring junctions (viz. apical ES and desmosome-like junctions) because basal ES and tight junctions (TJs) had already been established when Sertoli cells were cultured alone for 5 days. Seminiferous Tubule Cultures—Seminiferous tubules were isolated from the testes of adult rats (∼300 g of body weight) with negligible Leydig cell contamination (37Zwain I.H. Cheng C.Y. Mol. Cell. Endocrinol. 1994; 104: 213-227Crossref PubMed Scopus (28) Google Scholar). Lysates were obtained by homogenizing tubules in immunoprecipitation buffer (0.125 m Tris (pH 6.8) at 22 °C containing 1% (v/v) Nonidet P-40, 2 mm EDTA, 2 mm N-ethylmaleimide, 2 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, and 0.1 μm sodium okadate). Effects of Chelating Agents (e.g. EGTA and EDTA), Vanadate, and the Epidermal Growth Factor (EGF) on the levels of Protein Kinases and Intrinsic PKB Activity—Because several studies have reported that EDTA can interact with vanadate via chelation (39Kustin K. Toppen D.L. J. Am. Chem. Soc. 1973; 95: 3563-3564Google Scholar, 40Przyborowski L. Schwarzenbach G. Zimmermann T. Helv. Chim. Acta. 1965; 48: 1556-1565Crossref Scopus (36) Google Scholar, 41Crans D.C. Comments Inorg. Chem. 1994; 16: 1-33Crossref Scopus (151) Google Scholar, 42Huyer G. Liu S. Kelly J. Moffat J. Payette P. Kennedy B. Tsaprailis G. Gresser M.J. Ramachandran C. J. Biol. 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Soc. 1989; 111: 7597-7607Crossref Scopus (193) Google Scholar) or (ii) by replacing EDTA or EGTA that served as a metalloprotease inhibitor in the buffer with 2 mm 1,10-phenanthroline (a metalloprotease inhibitor) and compared the results with lysis buffers containing EDTA. This applied to different buffers used in this study, including the cell lysis buffer to be used for intrinsic kinase assay (see below). On this note, we anticipated that endogenous protein-tyrosine phosphatase inhibitors could protect samples from unwanted activities of protein-tyrosine phosphatase; however, a complete blockade of protein-tyrosine phosphatase could shift the base line of the intrinsic kinase activity, but plausibly not the trend of activation. Furthermore, EGF (a well known receptor protein-tyrosine kinase), which was present in the Sertoli cell cultures at 2.5 ng/ml as a growth factor in our experiments as described previously (44Cheng C.Y. Mather J.P. Byer A.L. Bardin C.W. Endocrinology. 1986; 118: 480-488Crossref PubMed Scopus (92) Google Scholar, 45Mather J.P. Zhuang L.Z. Perez-Infante V. Phillips D.M. Ann. N. Y. Acad. Sci. 1982; 383: 44-68Crossref PubMed Scopus (91) Google Scholar), has recently been shown to stimulate Sertoli cell ERK activity at 100 ng/ml (46Crepieux P. Marion S. Martinat N. Fafeur V. Vern Y.L. Kerboeuf D. Guillou F. Reiter E. Oncogene. 2001; 20: 4696-4709Crossref PubMed Scopus (171) Google Scholar, 47Crepieux P. Martinat N. Marion S. Guillou F. Reiter E. Arch. Biochem. Biophys. 2002; 399: 245-250Crossref PubMed Scopus (17) Google Scholar). As such, additional controls were included in Sertoli-germ cell co-cultures in which EGF was omitted in the nutrient mixture F-12/DMEM. We expected that the endogenous ERK and phospho (p)-ERK basal levels in co-cultures containing EGF would be higher than in those without EGF; however, its presence plausibly would not interfere with the trend of p-ERK1/2 activation during AJ assembly. Treatment of Rats with AF-2364 to Induce Germ Cell Loss from the Seminiferous Epithelium—AF-2364 (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) was synthesized to a purity of >99.8% as described (48Cheng C.Y. Silvestrini B. Grima J. Mo M.Y. Zhu L.J. Johansson E. Saso L. Leone M.G. Palmery M. Mruk D. Biol. Reprod. 2001; 65: 449-461Crossref PubMed Scopus (135) Google Scholar). This compound has been shown to perturb Sertoli-germ cell adhesion function to induce premature loss of germ cells from the epithelium, causing reversible infertility in rats (48Cheng C.Y. Silvestrini B. Grima J. Mo M.Y. Zhu L.J. Johansson E. Saso L. Leone M.G. Palmery M. Mruk D. Biol. Reprod. 2001; 65: 449-461Crossref PubMed Scopus (135) Google Scholar, 49Grima J. Silvestrini B. Cheng C.Y. Biol. Reprod. 2001; 64: 1500-1508Crossref PubMed Scopus (100) Google Scholar). One of the targets of AF-2364 is the apical ES without affecting the BTB integrity (3Mruk D.D. Cheng C.Y. Endocr. Rev. 2004; 25: 747-806Crossref PubMed Scopus (662) Google Scholar). Adult rats weighing 250-300 g were fed one dose of AF-2364 at 50 mg/kg of body weight. The time when the rats were fed AF-2364 was designated time 0 (control). Thereafter, rats were housed separately for 15 days. Testes were removed at specified time points from a group of three rats for each time point. For immunoblotting, testes were homogenized for lysate preparation either in SDS lysis buffer (for immunoblotting) or in cell lysis buffer (for PKB intrinsic kinase assay). For immunohistochemistry, testes were immediately fixed in 4% paraformaldehyde for paraffin embedding. Electron Microscopy—Electron microscopy was performed to examine the functional ES structures found in Sertoli-germ cell co-cultures, which were terminated 48 h after addition of germ cells to the Sertoli cell epithelium, as described previously (50Lee N.P.Y. Cheng C.Y. Endocrinology. 2003; 144: 3114-3129Crossref PubMed Scopus (105) Google Scholar). Immunoblotting—Protein concentration was estimated by Coomassie Blue dye binding assay using bovine serum albumin as a standard (51Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Proteins (∼100 μg from each sample within an experimental group) were resolved by SDS-PAGE (7.5 or 12.5% T) under reducing conditions (52Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Proteins were electroblotted onto nitrocellulose membranes and immunostained. All primary antibodies were shown to cross-react with the corresponding target proteins in rats as indicated by the manufacturers (Table I). Depending on the origin of the primary antibody, one of the following horseradish peroxide-conjugated secondary antibodies was used: bovine anti-rabbit IgG, bovine anti-goat IgG, or goat anti-mouse IgG. Target proteins in the blots were visualized using an ECL kit (Amersham Biosciences).Table ISources of antibodies and the working dilutions that were used for different experiments in this studyAntibodyVendorCatalog no.UsesaWB, Western blotting or immunoblotting; IP, immunoprecipitation; IH, immunohistochemistry; IF, immunofluorescence microscopy. The working dilutions are indicated in parentheses, except for IP, for which a working dilution of 1:100 was used.Mouse anti-PI3K p85αSanta Cruz Biotechnology Inc. (Santa Cruz, CA)sc-1637WB (1:400), IP, IH (1:50), IF (1:50)Rabbit anti-PI3K p110αCell Signaling Technology, Inc. (Beverly, MA)4254WB (1:1000)Mouse anti-PTENCell Signaling Technology, Inc.9556WB (1:1000), IP, IH (1:50)Rabbit anti-PKBCell Signaling Technology, Inc.9272WB (1:1000), IP, IH (1:50)Rabbit anti-phospho-PKB Thr308Cell Signaling Technology, Inc.4056WB (1:1000)Rabbit anti-phospho-PKB Ser473Cell Signaling Technology, Inc.9271WB (1:1000)Rabbit anti-phospho-PKB Ser473Cell Signaling Technology, Inc." @default.
• W2078122977 created "2016-06-24" @default.
• W2078122977 creator A5048483050 @default.
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• W2078122977 date "2005-07-01" @default.
• W2078122977 modified "2023-10-16" @default.
• W2078122977 title "Sertoli-Germ Cell Anchoring Junction Dynamics in the Testis Are Regulated by an Interplay of Lipid and Protein Kinases" @default.
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• W2078122977 doi "https://doi.org/10.1074/jbc.m501049200" @default.
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