Identification of Extracellular Signal-regulated Kinase 1/2 and p38 MAPK as Regulators of Human Sperm Motility and Acrosome Reaction and as Predictors of Poor Spermatozoan Quality
2008; Elsevier BV; Volume: 283; Issue: 21 Linguagem: Inglês
10.1074/jbc.m710492200
ISSN1083-351X
AutoresTal Almog, Shlomi Lazar, Nachum Reiss, Nir Etkovitz, Eyal Milch, Nir Rahamim, Masha Dobkin-Bekman, Ronit Rotem, M. Kalina, Jacob Ramon, Arieh Raziel, Haim Brietbart, Rony Seger, Zvi Naor,
Tópico(s)Ovarian function and disorders
ResumoMature spermatozoa acquire progressive motility only after ejaculation. Their journey in the female reproductive tract also includes suppression of progressive motility, reactivation, capacitation, and hyperactivation of motility (whiplash), the mechanisms of which are obscure. MAPKs are key regulatory enzymes in cell signaling, participating in diverse cellular functions such as growth, differentiation, stress, and apoptosis. Here we report that ERK1/2 and p38 MAPK are primarily localized to the tail of mature human spermatozoa. Surprisingly, c-Jun N-terminal kinase 1/2, which is thought to be ubiquitously expressed, could not be detected in mature human spermatozoa. ERK1/2 stimulation is downstream to protein kinase C (PKC) activation, which is also present in the human sperm tail (PKCβI and PKCϵ). ERK1/2 stimulates and p38 inhibits forward and hyperactivated motility, respectively. Both ERK1/2 and p38 MAPK are involved in the acrosome reaction. Using a proteomic approach, we identified ARHGAP6, a RhoGAP, as an ERK substrate in PMA-stimulated human spermatozoa. Inverse correlation was obtained between the relative expression level of ERK1 or the relative activation level of p38 and sperm motility, forward progression motility, sperm morphology, and viability. Therefore, increased expression of ERK1 and activated p38 can predict poor human sperm quality. Mature spermatozoa acquire progressive motility only after ejaculation. Their journey in the female reproductive tract also includes suppression of progressive motility, reactivation, capacitation, and hyperactivation of motility (whiplash), the mechanisms of which are obscure. MAPKs are key regulatory enzymes in cell signaling, participating in diverse cellular functions such as growth, differentiation, stress, and apoptosis. Here we report that ERK1/2 and p38 MAPK are primarily localized to the tail of mature human spermatozoa. Surprisingly, c-Jun N-terminal kinase 1/2, which is thought to be ubiquitously expressed, could not be detected in mature human spermatozoa. ERK1/2 stimulation is downstream to protein kinase C (PKC) activation, which is also present in the human sperm tail (PKCβI and PKCϵ). ERK1/2 stimulates and p38 inhibits forward and hyperactivated motility, respectively. Both ERK1/2 and p38 MAPK are involved in the acrosome reaction. Using a proteomic approach, we identified ARHGAP6, a RhoGAP, as an ERK substrate in PMA-stimulated human spermatozoa. Inverse correlation was obtained between the relative expression level of ERK1 or the relative activation level of p38 and sperm motility, forward progression motility, sperm morphology, and viability. Therefore, increased expression of ERK1 and activated p38 can predict poor human sperm quality. Sperm are immotile in the testes and they acquire progressive motility after ejaculation. They cross the uterine cervix, undergo suppression of progressive motility, become capacitated (capable to fertilize), including resumption of another form of motility, hyperactivation, bind the egg, undergo the acrosome reaction, and fertilize it (1Garbers D.L. Nature. 2001; 413: 579-582Crossref PubMed Scopus (18) Google Scholar, 2Eisenbach M. Giojalas L.C. Nat. Rev. Mol. Cell Biol. 2006; 7: 276-285Crossref PubMed Scopus (391) Google Scholar). Relatively little is known about the molecular mechanisms that mediate spermatozoan functions in general and the various forms of motility in particular (1Garbers D.L. Nature. 2001; 413: 579-582Crossref PubMed Scopus (18) Google Scholar, 2Eisenbach M. Giojalas L.C. Nat. Rev. Mol. Cell Biol. 2006; 7: 276-285Crossref PubMed Scopus (391) Google Scholar). MAPK 3The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; OAG, 1-oleoyl-2-acetylglycerol; JNK, c-Jun N-terminal kinase; GPCR, G-protein-coupled receptor; MBP, myelin basic protein; CASA, computer-aided sperm analysis; SM, sperm motility; FPM, forward progression motility; ROC, receiver operating characteristic; AUC, area under the curve; BAPTA/AM, 2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PMA, phorbol myristoyl acetate. cascades play a crucial role in metazoan development, including fate determination, differentiation, proliferation, survival, migration, growth, cell cycle progression, and apoptosis (3Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3218) Google Scholar, 4Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 5Pearson G. Robinson F. Beers Gibson T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3564) Google Scholar, 6Murphy L.O. Blenis J. Trends Biochem. Sci. 2006; 31: 268-275Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 7Yoon S. Seger R. Growth Factors. 2006; 24: 21-44Crossref PubMed Google Scholar). MAPK cascades consist of several tiers of protein kinases that activate each other by sequential phosphorylation. Four major MAPK cascades are known in mammals: ERK1–2, JNK 1–3, p38 α–δ, and ERK5 (big MAPK; BMK) (3Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3218) Google Scholar, 4Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 5Pearson G. Robinson F. Beers Gibson T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3564) Google Scholar, 6Murphy L.O. Blenis J. Trends Biochem. Sci. 2006; 31: 268-275Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 7Yoon S. Seger R. Growth Factors. 2006; 24: 21-44Crossref PubMed Google Scholar). Activation of MAPK is initiated by activation of MAP3Ks by the small G-proteins of the Ras family (e.g. Ras, CDC42, and Rac), followed by sequential activation of MAPKKs and MAPKs. The prototypic ERK cascade is activated by growth factors, mitogens, and G-protein-coupled receptors (GPCR) and consists of Rafs (MAP3K), MEK1/2, ERK1/2, and several MAPK-activating protein kinases (MAPKAPKs). The stress-activated protein kinases are now known as JNK and p38 MAPK. Both are initiated by stress stimuli, GPCRs, inflammatory cytokines, and growth factors. JNK1–3 consists of a sequential activation of Rac1/Cdc42, mixed lineage kinases as MAP3Ks, MAPK kinases 4 and 7, and JNK1–3. The p38 MAPK cascade is composed of sequential activation of MAP3Ks, MKK3/4/6, p38α–δ, and several MAPKAPKs (3Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3218) Google Scholar, 4Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 5Pearson G. Robinson F. Beers Gibson T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3564) Google Scholar, 6Murphy L.O. Blenis J. Trends Biochem. Sci. 2006; 31: 268-275Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 7Yoon S. Seger R. Growth Factors. 2006; 24: 21-44Crossref PubMed Google Scholar). Mature spermatozoa are fully differentiated cells that lack active transcriptional machinery. Hence, it is of great interest to investigate whether ejaculated human spermatozoa express and utilize MAPKs, which specialize in growth, differentiation, and stress. Because spermatozoa lack those functions, the studies might point to novel functions mediated by MAPKs. During spermatogenesis, MAPKs mediate cell division, differentiation, survival, adhesion, and death (8Wong C.H. Cheng C.Y. Dev. Biol. 2005; 286: 1-15Crossref PubMed Scopus (99) Google Scholar). MAPKs were reported to play a role during meiotic progression of mouse spermatocytes (9Sette C. Barchi M. Bianchini A. Conti M. Rossi P. Geremia R. J. Biol. Chem. 1999; 274: 33571-33579Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 10Lu Q. Sun Q.Y. Breitbart H. Chen D.Y. Arch. Androl. 1999; 43: 55-66Crossref PubMed Scopus (47) Google Scholar, 11Rhee K. Wolgemuth D.J. Development (Camb.). 1997; 124: 2167-2177PubMed Google Scholar, 12Di Agostino S. Botti F. Di Carlo A. Sette C. Geremia R. Reproduction. 2004; 128: 25-32Crossref PubMed Scopus (27) Google Scholar) and in adhesion function at the Sertoli-germ cell interface (8Wong C.H. Cheng C.Y. Dev. Biol. 2005; 286: 1-15Crossref PubMed Scopus (99) Google Scholar). On the other hand, the role of MAPK in mature mammalian spermatozoan motility, capacitation, and acrosome reaction is limited and controversial as detailed below (10Lu Q. Sun Q.Y. Breitbart H. Chen D.Y. Arch. Androl. 1999; 43: 55-66Crossref PubMed Scopus (47) Google Scholar, 13Baldi E. Luconi M. Bonaccorsi L. Forti G. Front. Biosci. 1998; 3: D1051-D1059Crossref PubMed Google Scholar, 14de Lamirande E. Gagnon C. Mol. Hum. Reprod. 2002; 8: 124-135Crossref PubMed Scopus (143) Google Scholar, 15du Plessis S.S. Page C. Franken D.R. Andrologia. 2002; 34: 55-59PubMed Google Scholar, 16Weidinger S. Mayerhofer A. Kunz L. Albrecht M. Sbornik M. Wunn E. Hollweck R. Ring J. Kohn F.M. Hum. Reprod. 2005; 20: 456-461Crossref PubMed Scopus (29) Google Scholar, 17Chen W.Y. Ni Y. Pan Y.M. Shi Q.X. Yuan Y.Y. Chen A.J. Mao L.Z. Yu S.Q. Roldan E.R. FEBS Lett. 2005; 579: 4692-4700Crossref PubMed Scopus (28) Google Scholar). In particular the role of JNK and p38 MAPK in ejaculated spermatozoa is not known (8Wong C.H. Cheng C.Y. Dev. Biol. 2005; 286: 1-15Crossref PubMed Scopus (99) Google Scholar). We therefore inquired whether ejaculated mature human spermatozoa express and utilize the various MAPKs (ERK, JNK, and p38) (6Murphy L.O. Blenis J. Trends Biochem. Sci. 2006; 31: 268-275Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 7Yoon S. Seger R. Growth Factors. 2006; 24: 21-44Crossref PubMed Google Scholar), which may orchestrate the fine-tuning of sperm motility. Our results indicate that ERK1/2 and p38 MAPK, but not JNK1/2, are expressed in the tail of ejaculated human spermatozoa. Activation of ERK1/2 is downstream to PKC activation, and two of its isoforms (PKCβI and PKCϵ) are also present in the human sperm tail (18Rotem R. Paz G.F. Homonnai Z.T. Kalina M. Lax J. Breitbart H. Naor Z. Endocrinology. 1992; 131: 2235-2243Crossref PubMed Scopus (0) Google Scholar). Surprisingly, we discovered that ERK stimulates and p38 MAPK inhibits forward and hyperactivated motility, respectively. We also found that both ERK1/2 and p38 MAPK are positively involved in PKC-mediated acrosome reaction. Using a proteomic approach, we identified ARHGAP6, a RhoGAP, as an ERK substrate in PMA-stimulated human spermatozoa. Finally, we found that inverse correlation exists between ERK1 and phospho-p38 and sperm motility, forward progression motility, sperm morphology, and viability. Indeed, increased expression of total ERK1 and activated phospho-p38 MAPK could predict poor human sperm quality. Preparation of Human Spermatozoa—Human semen was obtained from healthy donors with normal sperm density, motility, and morphology according to World Health Organization guidelines (19World Health Organization WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge University Press, Cambridge1999Google Scholar) or from males attending either the Male Infertility Unit, Assaf Harofeh Medical Center, Israel, or the Andrology Laboratory in Assuta Hashalom Medical Center, Tel Aviv, Israel. The human semen was liquefied for 60 min at 36 °C. Sperm was washed twice with Ham's F-10 medium containing bovine serum albumin (BSA, 0.3%) and incubated with the medium for 3 h for capacitation (20Rotem R. Paz G.F. Homonnai Z.T. Kalina M. Naor Z. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7305-7308Crossref PubMed Scopus (89) Google Scholar). PMA or progesterone (Sigma) were added with or without the PKC inhibitor GF109203x (Calbiochem) or the MEK1/2 inhibitors PD98059 and U0126, or the p38 inhibitors SB203580 and PD169316 (Biomol), or the Ca2+ inhibitors EGTA, nifedipine, and BAPTA/AM (Sigma) to the above medium as described in the legends. Immunocytochemistry—Human sperm (1.5 × 106) were collected on glass slides by cytospin (600 rpm). The cells were fixed and permeabilized by cold methanol (10 min), followed by cold acetone (10 min) (20Rotem R. Paz G.F. Homonnai Z.T. Kalina M. Naor Z. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7305-7308Crossref PubMed Scopus (89) Google Scholar). The cells were treated with the respective anti-general MAPK (ERK, JNK, and p38) antibodies (Sigma) or antibodies against mSos, MEK1/2, Raf-1, or tubulin (Santa Cruz Biotechnology) (1:25) for 18 h at 4 °C. Cells were then treated with biotinylated second antibody (1:100) for 30 min. After washing in PBS the cells were further treated for 30 min at room temperature using the avidin-biotin complex method as described previously (20Rotem R. Paz G.F. Homonnai Z.T. Kalina M. Naor Z. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7305-7308Crossref PubMed Scopus (89) Google Scholar). Visualization was performed in solutions containing diaminobenzidine (0.25 mg/ml) and 0.01% H2O2 in PBS. Specificity of the MAPK staining was confirmed by omission of the primary antibody and by the preabsorption of the antibody with an excess of the respective MAPK peptide (Sigma). All the reagents for the immunohistochemistry studies were obtained from Vector Laboratories (Burlingame, CA). Immunoelectron Microscopy—Human sperm were fixed in 2% glutaraldehyde in 0.1 m cacodylate buffer for 60 min at room temperature (21Kalina M. Socher R. Rotem R. Naor Z. J. Histochem. Cytochem. 1995; 43: 439-445Crossref PubMed Scopus (31) Google Scholar). After washing, the tissue samples were dehydrated in acetone and embedded in araldite. The cells were not treated with osmium because of loss of antigenicity in osmium-treated cells. Sections were placed on silver grids, and immunogold labeling was performed as described previously (21Kalina M. Socher R. Rotem R. Naor Z. J. Histochem. Cytochem. 1995; 43: 439-445Crossref PubMed Scopus (31) Google Scholar). Sections were treated with 0.1% Triton X-100 in PBS for 20 min, washed in PBS, and placed in 1% BSA for 1 h. After draining, the sections were incubated with anti-ERK antibody (1:50) (Sigma) for 18 h at 4 °C. After rinsing in 0.05 m Tris-buffered saline (TBS), pH 7.3, the sections were incubated for 1 h with 8- or 15-nm gold-conjugated goat anti-rabbit IgG (Biocell, Cardiff, UK) and diluted 1:10 with TBS, pH 8.4, containing 1% egg albumin. Following the immunostaining, sections were rinsed, and contrast was enhanced with uranyl acetate and lead citrate. Sections were then examined with a JEOL 100B electron microscope. Indirect Immunofluorescence—Sperm were washed and smeared on polylysine slides and then allowed to air-dry. Cells were washed with PBS, permeabilized with Triton X-100, 0.5% buffered in PBS. Nonspecific binding was blocked with 3% BSA buffered in PBS. Cells were probed first with monoclonal anti-p38 (1:100) or anti-ERK1/2 (1:100) polyclonal antibody, washed three times with PBS, and then probed with a secondary anti-rabbit Hilyte Fluor™ 488-labeled antibody or a secondary anti-mouse Hilyte Fluor™ 647-labeled antibody (Anaspec). Slides were viewed with a laser scanning microscope (510, Zeiss). Activation of MAPK Cascades—Capacitated and noncapacitated human spermatozoa were prepared as above and stimulated with PMA, progesterone, or other drugs as indicated (Sigma), and cell extract was used for Western blotting. After stimulation, the cells were diluted immediately with excess Ham's F-10 medium, precipitated by centrifugation at 15,000 × g for 20 s at room temperature, and washed once more, and the resulted pellets were stored at -20 °C until used. The thawed pellets (all these steps were made at 4 °C) were resuspended in a minimal volume of lysis buffer (50 μl per 3 × 107 cells) made of 50 mm Tris-HCl, pH 8.0, 2 mm EGTA, 20 mm NaCl, 1.0 mm sodium orthovanadate, 25 mm β-glycerophosphate, 100 nm okadaic acid, 0.50% Nonidet P-40, 1 mm benzamidine, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 2 mm dithiothreitol. The suspensions tubes were incubated on ice for 10 min and centrifuged (15,000 × g, 15 min, 4 °C). The supernatants, which contained unbound proteins, were collected, and aliquots from each sample (20 μg) were separated on 10% SDS-PAGE followed by Western blotting with mouse monoclonal anti-active (doubly phosphorylated) MAPKs (ERK, JNK, and p38) (Sigma), MPM2 (anti-phospho-Ser/Thr-Pro, Upstate Biotechnology, Inc.), or anti-ARHGAP6 mouse monoclonal antibody (Abnova). Total MAPKs were detected with polyclonal antibodies for the various general MAPKs (Sigma) as a control. The blots were developed with alkaline phosphatase- or horseradish peroxidase-conjugated anti-mouse or anti-rabbit Fab antibodies (Jackson ImmunoResearch). The blots were autoradiographed on Kodak X-100 films, and the phosphorylation was quantified by densitometry (690 densitometer, Bio-Rad). Each band from the anti-phospho-MAPK (ERK, JNK, and p38) was normalized to the corresponding band from the anti-general MAPK antibodies blot for even loading (22Bonfil D. Chuderland D. Kraus S. Shahbazian D. Friedberg I. Seger R. Naor Z. Endocrinology. 2004; 145: 2228-2244Crossref PubMed Scopus (78) Google Scholar). Recombinant Expression of pERK—To obtain doubly phosphorylated ERK, activated human GST-ERK2 was co-expressed with constitutively active MEK1 in BL21 bacteria, as described (23Wilsbacher J.L. Cobb M.H. Methods Enzymol. 2001; 332: 387-400Crossref PubMed Scopus (23) Google Scholar). The bacteria were grown in 2YT medium at 30 °C to an absorbance of 0.6, and then 1 mm isopropyl 1-thio-β-d-galactopyranoside was added for an additional 4 h. Proteins were purified over glutathione-Sepharose 4B (Amersham Biosciences) or nickel-NTA-agarose (Qiagen) according to the manufacturer's instructions. Cell Culture, Transfection, and Immunoprecipitation—Human embryonic kidney 293 cells (HEK293) were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum in 95% air, 5% CO2 at 37 °C. Cells were plated in 100-mm plates 24 h prior to transfection and were transfected with 4 μg of plasmid DNA ARHGAP6-GFP, using the calcium phosphate method, and grown as above. 16 h post-transfection, the medium was replaced, and 48 h later the cells were lysed. Lysis buffer contained 100 mm NaCl, 20 mm Tris-HCl, pH 7.4, 0.5 mm EDTA, 15% glycerol, 0.2% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitors mixture. The lysate was subjected to immunoprecipitation with an anti-green fluorescent protein antibody (Roche Applied Science) conjugated to protein A/G PLUS-agarose (Santa Cruz Biotechnology) by gentle shaking for 3–4 h in 4 °C, and washed three times with lysis buffer. Samples were frozen in -20 °C and were later subjected to in vitro phosphorylation. In Vitro Phosphorylation—Immunoprecipitated ARHGAP6 attached to beads (15 μl) (0.5 μg per reaction) was mixed with immunoprecipitated ERK attached to beads (15 μl). The buffer reaction mix (3×) (75 mm β-glycerophosphate, 1.5 mm dithiothreitol, 3.8 mm EGTA, 0.15 mm orthovanadate, 30 mm MgCl2, 30 μm calmidozolium, 0.3 mm ATP), containing 100 μm [γ-32P]ATP (4000 cpm/pmol), was added to the reaction in a final volume of 30 μl and incubated for 20 min at 30 °C. The reaction was terminated by adding 10 μl of 4× Sample buffer, and the phosphorylated proteins were resolved on SDS-PAGE. Phosphorylation of MBP (8 μg per reaction) by ERK was carried out as a positive control. Assessment of Sperm Motility—Progressive flagellar motility was determined manually (20Rotem R. Paz G.F. Homonnai Z.T. Kalina M. Naor Z. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7305-7308Crossref PubMed Scopus (89) Google Scholar) or by using Computer-aided Sperm Analysis (CASA) (Sperm Analysis System version 12-IVOS, Hamilton Thorne Biosciences, Beverly, MA), which was also used to measure hyperactivation (24Ho H.C. Suarez S.S. Reproduction. 2001; 122: 519-526Crossref PubMed Scopus (265) Google Scholar). Hyperactivation parameters were as follows: curvilinear velocity >100 μm/s, linearity 5 μm. Assessment of Sperm Acrosome Reaction—The percentage of acrosome-reacted sperm was determined microscopically on air-dried sperm smears using FITC-conjugated Pisum sativum agglutinin, which is a fluorescent lectin capable of binding to the acrosomal content. Washed cells (107 cells/ml) were capacitated for 3 h at 37 °C and 0.5% CO2 in F-10 medium supplemented with BSA 3 mg/ml (20Rotem R. Paz G.F. Homonnai Z.T. Kalina M. Naor Z. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7305-7308Crossref PubMed Scopus (89) Google Scholar). The inhibitors were added for the last 10 min of incubation, and then PMA (100 nm) was added for another hour. At the end of incubation an aliquot of the sperm was spread on microscope slides and allowed to air-dry. The sperm were then permeabilized by methanol for 15 min at room temperature, washed once with 25 mm Tris-buffered saline, pH 7.6, for 5 min and twice with H2O at 5-min intervals, air-dried, and then incubated with FITC-P. sativum agglutinin (60 μg/ml) for 1 h, washed twice with H2O at 5-min intervals, and mounted with FluoroGuard Antifade (Bio-Rad). For each experiment, at least 150 cells per slide in duplicates were evaluated. Cells with green staining over the acrosomal cap were considered acrosome-intact; those with equatorial green staining or no staining were considered acrosome-reacted. The analysis of multiple groups was performed by one-way analysis of variance using SPSS software with p < 0.05 considered significant. Correlation between MAPK Levels and Sperm Quality Parameters—Human spermatozoa from 47 males attending the Male Infertility Unit were analyzed for MAPK levels and activity as above and correlated with percent sperm motility (SM), forward progression motility (FPM), morphology, and viability (19World Health Organization WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge University Press, Cambridge1999Google Scholar). The correlations were obtained using Pearson correlation coefficients. Logarithmic transformation (when needed) was used to obtain normal distribution. Aiming to discriminate normal and abnormal motility and FPM samples, single variable analysis was used utilizing Student’s t test. Stepwise logistic regression was used for multivariate analysis. p value <0.05 was considered statistically significant. To define optimal cut points for MAPK levels, receiver operating characteristic (ROC) analysis (25Zweig M.H. Campbell G. Clin. Chem. 1993; 39: 561-577Crossref PubMed Scopus (5341) Google Scholar) was used discriminating normal versus abnormal motility and FPM. We calculated the area under the curve (AUC), which represented the accuracy of the test, and a p value <0.05 was considered predictive. Data Analysis—Results from two or three experiments were expressed as mean ± S.D. Data were subjected to statistical analysis with one-way analysis of variance and Fisher’s protected least significant difference tests, and statistical significance was accepted when p < 0.05. Other statistical tests are detailed in the legends. ERK1/2 (and its cascade members Sos/Raf-1/MEK1) and p38 MAPK, but not JNK, were identified in the tail of mature ejaculated human spermatozoa by immunocytochemistry (Fig. 1A). The ERK cascade (Sos/Raf-1/MEK1/2/ERK1/2) and p38 were localized in the neck and distributed along the mid-, principal, and end pieces of the tail. Tubulin in the axoneme was evenly distributed along the tail. Electron microscopy revealed (Fig. 1B) that ERK1/2 was localized to the outer dense fibers. Confocal microscopy revealed that ERK1/2 is distributed mainly to the entire mid-piece (Fig. 2), whereas p38 is primarily localized to the upper mid-piece (Fig. 2).FIGURE 2Fluorescence microscopy for expression of ERK1/2 and p38 in human spermatozoa. Precapacitated spermatozoa were reacted with anti-ERK (A) and anti-p38 (E) antibodies. Phase-contrast images are shown for ERK1/2 and p38 immunolabeling in B and F, respectively, and negative controls with secondary antibodies are shown in C and G. Phase-contrast images are shown for controls with secondary antibodies in D and H. Like the diaminobenzidine staining of Fig. 1, ERK1/2 was distributed along the tail, whereas p38 seems to localize also in post-acrosomal regions, the mid-piece, and the tail (C). Scale bars indicate 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then identified the presence of active ERK1/2 and p38 in human ejaculated spermatozoa by Western blotting and compared it with the known PMA-stimulated MAPK in the mouse pituitary gonadotrope LβT2 cells (22Bonfil D. Chuderland D. Kraus S. Shahbazian D. Friedberg I. Seger R. Naor Z. Endocrinology. 2004; 145: 2228-2244Crossref PubMed Scopus (78) Google Scholar) (Fig. 3). Human ejaculate spermatozoa express mainly ERK2, which was stimulated by PMA or by the general activator vanadate peroxide (Na3VO4). PMA serves as an analog of diacylglycerol, the physiological activator of PKC, and both stimulants revealed the activation of ERK1. The sperm-ligand progesterone, reported to activate human sperm ERK (26Luconi M. Krausz C. Barni T. Vannelli G.B. Forti G. Baldi E. Mol. Hum. Reprod. 1998; 4: 251-258Crossref PubMed Scopus (80) Google Scholar), had no effect. p38 MAPK in its phosphorylated form was very low in control LβT2 cells but was present in spermatozoa. We could not detect further activation of phospho-p38 by PMA, vanadate, nor progesterone versus a positive effect of PMA in LβT2 cells (Fig. 3). The data suggest that ejaculate sperm p38 MAPK is fully phosphorylated, hence activated. p38 MAPK is encoded by four genes that yield nine isoforms (5Pearson G. Robinson F. Beers Gibson T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3564) Google Scholar), and we could detect mainly p38α and occasionally p38β. JNK1/2 is present in LβT2 cells and is activated by PMA but is undetectable in PMA-, vanadate-, or progesterone-treated spermatozoa. We then incubated spermatozoa for 2.5 h in a capacitation medium (Fig. 4) to reach capacitation in vitro (20Rotem R. Paz G.F. Homonnai Z.T. Kalina M. Naor Z. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7305-7308Crossref PubMed Scopus (89) Google Scholar). Exposure to PMA (15 min) throughout the entire capacitation process further stimulated ERK1/2 levels in comparison with controls (*, p < 0.05; **, p < 0.01). Thus, ERK activation by PMA can be detected in both noncapacitated and capacitated spermatozoa. ERK2 activation by PMA was sensitive to the MEK1/2 inhibitors PD98059 and U0126 (Fig. 5A, left panel), confirming the functioning of a canonical MEK1/2-ERK1/2 cascade (6Murphy L.O. Blenis J. Trends Biochem. Sci. 2006; 31: 268-275Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 7Yoon S. Seger R. Growth Factors. 2006; 24: 21-44Crossref PubMed Google Scholar) in spermatozoa and the use of the two drugs in the studies described below. The selective p38 inhibitor SB203580 had no effect on PMA to ERK signaling, ruling out an inhibitory cross-talk between p38 MAPK and ERK (27Zhang H. Shi X. Hampong M. Blanis L. Pelech S. J. Biol. Chem. 2001; 276: 6905-6908Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The selective pan-PKC inhibitor, GF109203X, completely abolished the PMA-induced activation of ERK1/2, indicating that the PMA effect was PKC-dependent (Fig. 5A, right panel). ERK activation by PMA was insensitive to EGTA (extracellular Ca2+ chelator), to nifedipine (voltage-dependent Ca2+ channel blocker), or to BAPTA/AM (intracellular Ca2+ chelator) (Fig. 5B), unlike LβT2 cells, in which activation of ERK by PMA was markedly reduced by the above Ca2+ inhibitors (Fig. 5B).FIGURE 5A, PMA-induced ERK activation in human spermatozoa is sensitive to MEK1/2 inhibitors and insensitive to p38 inhibitor (left panel) and to the pan-PKC inhibitor GF109203X (right panel). Capacitated human sperm were preincubated with the specific MEK1/2 inhibitors PD98059 (50 μm) and U0126 (50 μm), the p38 inhibitor SB203580 (50 μm) and the PKC inhibitor GF109203X (3 μm) for 15 min and PMA (100 nm) was added for another 15 min. Cell lysates were subsequently prepared and ERK2 activity was determined as in Fig. 3. Bars are mean ± S.D. from three experiments. Means designated by different letters are significantly different (p < 0.05). B, PMA-induced ERK activation is Ca2+-independent. Capacitated human spermatozoa were preincubated with or without various Ca2+ blockers: nifedipine (l-type voltage-dependent Ca2+ channel blocker, 1 μm), EGTA (a general Ca2+ chelator, 5 mm) and BAPTA/AM (intracellular Ca2+ chelator, 50 μm) for 30 min. PMA (100 nm) was then added for 15 min and ERK2 activity was determined as in Fig. 3. A representative blot is shown and bars are mean ± S.D. from three experiments. Means designated by different letters are significantly different (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Incubation of spermatozoa with normal motility (>50% motility) (19World Health Organization WHO Laboratory Ma
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