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• W2035168650 abstract "Spermatozoa are cells distinctly different from other somatic cells of thebody, capacitation being one of the unique phenomena manifested by thisgamete. We have shown earlier that dihydrolipoamide dehydrogenase, apost-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosinephosphorylation, and the functioning of the enzyme is required forhyperactivation (enhanced motility) and acrosome reaction of hamsterspermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70,887–899). In this report we have investigated the localization of thismitochondrial enzyme in spermatozoa revealing non-canonicalextra-mitochondrial localization of the enzyme in mammalian spermatozoa. Inhamster spermatozoa, dihydrolipoamide dehydrogenase along with its hostcomplex, the pyruvate dehydrogenase complex, are localized in the acrosome andin the principal piece of the sperm flagella. The localization ofdihydrolipoamide dehydrogenase, however, appears to be in the mitochondria inthe spermatocytes, but in spermatids it appears to show a juxtanuclearlocalization (like Golgi). The capacitation-dependent time course of tyrosinephosphorylation of dihydrolipoamide dehydrogenase appears to be different inthe principal piece of the flagella and the acrosome in hamster spermatozoa.Activity assays of this bi-directional enzyme suggest a strong correlationbetween the tyrosine phosphorylation and the bi-directional enzyme activity.This is the first report of a direct correlation of the localization, tyrosinephosphorylation, and activity of the important metabolic enzyme,dihydrolipoamide dehydrogenase, implicating dual involvement and regulation ofthe enzyme during sperm capacitation. Spermatozoa are cells distinctly different from other somatic cells of thebody, capacitation being one of the unique phenomena manifested by thisgamete. We have shown earlier that dihydrolipoamide dehydrogenase, apost-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosinephosphorylation, and the functioning of the enzyme is required forhyperactivation (enhanced motility) and acrosome reaction of hamsterspermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70,887–899). In this report we have investigated the localization of thismitochondrial enzyme in spermatozoa revealing non-canonicalextra-mitochondrial localization of the enzyme in mammalian spermatozoa. Inhamster spermatozoa, dihydrolipoamide dehydrogenase along with its hostcomplex, the pyruvate dehydrogenase complex, are localized in the acrosome andin the principal piece of the sperm flagella. The localization ofdihydrolipoamide dehydrogenase, however, appears to be in the mitochondria inthe spermatocytes, but in spermatids it appears to show a juxtanuclearlocalization (like Golgi). The capacitation-dependent time course of tyrosinephosphorylation of dihydrolipoamide dehydrogenase appears to be different inthe principal piece of the flagella and the acrosome in hamster spermatozoa.Activity assays of this bi-directional enzyme suggest a strong correlationbetween the tyrosine phosphorylation and the bi-directional enzyme activity.This is the first report of a direct correlation of the localization, tyrosinephosphorylation, and activity of the important metabolic enzyme,dihydrolipoamide dehydrogenase, implicating dual involvement and regulation ofthe enzyme during sperm capacitation. Spermatozoa, the haploid cells, are unique compared with other cells withrespect to their morphology and functionality; needless to say the underlyingsignaling mechanisms in a functional spermatozoon are also unique. Themetabolic pathways in a mature spermatozoon are compartmentalized(1Westhoff D. Kamp G. J. CellSci. 1997; 110: 1821-1829Google Scholar, 2Storey B.T. Kayne F.J. Fertil. Steril. 1975; 26: 1257-1265Abstract Full Text PDF PubMed Google Scholar, 3Travis A.J. Jorgez C.J. Merdiushev T. Jones B.H. Dess D.M. Diaz-Cueto L. Storey B.T. Kopf G.S. Moss S.B. J. Biol. Chem. 2001; 276: 7630-7636Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar),which probably enables them to survive in two different kinds of milieu, themale and the female reproductive tract. The residence time in the femalereproductive tract is an obligatory event in the life cycle of a spermatozoonand has been termed “capacitation”(4Austin C.R. Nature. 1952; 170: 326Crossref PubMed Scopus (509) Google Scholar,5Chang M.C. Nature. 1951; 168: 697-698Crossref PubMed Scopus (973) Google Scholar). During capacitationspermatozoa undergo multifaceted changes in aspects like metabolism,intracellular ion concentrations, plasma membrane fluidity, and thus membranereorganization, intracellular pH, intracellular cAMP concentration, andgeneration of reactive oxygen species(6Visconti P.E. Galantino-Homer H. Moore G.D. Bailey J.L. Ning X. Fornes M. Kopf G.S. J.Androl. 1998; 19: 242-248Crossref PubMed Google Scholar, 7Jha K.N. Kameshwari D.B. Shivaji S. Cell. Mol. Biol. (Noisyle-grand). 2003; 49: 329-340PubMed Google Scholar, 8de Lamirande E. Leclerc P. Gagnon C. Mol. Hum. Reprod. 1997; 3: 175-194Crossref PubMed Scopus (391) Google Scholar).These cellular alterations during capacitation bring about three physiologicalchanges: (a) hyperactivation (enhanced motility), (b)tyrosine phosphorylation in an array of proteins, and (c) acrosomereaction (release of the acrosomal contents); the first two events show atemporal correlation with capacitation, whereas acrosome reaction is taken tobe the end point of capacitation(9Yanagimachi R. Knobil E. Neill J.D. MammalianFertilization: The Physiology of Reproduction. Raven Press Ltd., NewYork1994: 189-317Google Scholar). Protein tyrosine phosphorylation is considered to be a hallmark of spermcapacitation(10Visconti P.E. Westbrook V.A. Chertihin O. Demarco I. Sleight S. Diekman A.B. J. Reprod.Immunol. 2002; 53: 133-150Crossref PubMed Scopus (290) Google Scholar, 11Jha K.N. Shivaji S. Mol.Reprod. Dev. 2002; 61: 258-270Crossref PubMed Scopus (46) Google Scholar, 12Leclerc P. de Lamirande E. Gagnon C. Free Radic. Biol. Med. 1997; 22: 643-656Crossref PubMed Scopus (255) Google Scholar, 13Mahony M.C. Gwathmey T. Biol. Reprod. 1999; 60: 1239-1243Crossref PubMed Scopus (83) Google Scholar, 14Si Y. Okuno M. Biol.Reprod. 1999; 61: 240-246Crossref PubMed Scopus (157) Google Scholar, 15Urner F. Leppens-Luisier G. Sakkas D. Biol. Reprod. 2001; 64: 1350-1357Crossref PubMed Scopus (104) Google Scholar),but only a few of these proteins have been identified, and the functionalsignificance of the respective tyrosine phosphorylation has been ascertainedin only a few cases. The protein kinase A-anchoring protein(s), AKAP(s),localized in the principal piece of the sperm flagella, have been explored ina greater detail in this regard(11Jha K.N. Shivaji S. Mol.Reprod. Dev. 2002; 61: 258-270Crossref PubMed Scopus (46) Google Scholar,16Carrera A. Moos J. Ning X.P. Gerton G.L. Tesarik J. Kopf G.S. Moss S.B. Dev.Biol. 1996; 180: 284-296Crossref PubMed Scopus (284) Google Scholar). Recently, tyrosinephosphorylation of AKAP3 has been shown to result in the recruitment ofprotein kinase A to the sperm flagella causing an increase in motility(17Luconi M. Carloni V. Marra F. Ferruzzi P. Forti G. Baldi E. J. Cell Sci. 2004; 117: 1235-1246Crossref PubMed Scopus (94) Google Scholar). Furthermore, theessentiality of a balance between protein-tyrosine kinase and phosphataseactivities has also been demonstrated as a mandatory requirement for asuccessful acrosome reaction(18Tomes C.N. Roggero C.M. De Blas G. Saling P.M. Mayorga L.S. Dev. Biol. 2004; 265: 399-415Crossref PubMed Scopus (55) Google Scholar). Regulation of metabolic enzymes by phosphorylation has been wellestablished in different cells(19Huang K.P. Huang F.L. J.Biol. Chem. 1980; 255: 3141-3147Abstract Full Text PDF PubMed Google Scholar, 20El-Maghrabi M.R. Noto F. Wu N. Manes N. Curr. Opin. Clin. Nutr. Metab.Care. 2001; 4: 411-418Crossref PubMed Scopus (30) Google Scholar, 21Pilkis S.J. El-Maghrabi M.R. Coven B. Claus T.H. Tager H.S. Steiner D.F. Keim P.S. Heinrikson R.L. J. Biol. Chem. 1980; 255: 2770-2775Abstract Full Text PDF PubMed Google Scholar).However, the metabolism of spermatozoa is quite distinctly different fromother cells and thus less understood, with spermatozoa having specificisoforms of key metabolic enzymes(22Mori C. Welch J.E. Fulcher K.D. O'Brien D.A. Eddy E.M. Biol. Reprod. 1993; 49: 191-203Crossref PubMed Scopus (80) Google Scholar, 23Goldberg E. MethodsEnzymol. 1975; 41: 318-323Google Scholar, 24Dahl H.H. Brown R.M. Hutchison W.M. Maragos C. Brown G.K. Genomics. 1990; 8: 225-232Crossref PubMed Scopus (174) Google Scholar).Because there is little or no cytoplasm in spermatozoa, most of thecytoplasmic enzymes exhibit non-canonical localization in two domains of thesperm, namely, the head and the flagellum. The head hosts the nucleus and theacrosome; the mid-piece of the flagellum hosts all the mitochondria and thetail piece, which is further divided into a principal piece and an end piece,and harbors cytoskeletal elements. The two ideal examples of non-canonicallocalization of metabolic enzymes in spermatozoa are the sperm-specificisoforms of hexokinase (localized in the mid piece and principal piece of theflagella and in the sperm head(25Travis A.J. Foster J.A. Rosenbaum N.A. Visconti P.E. Gerton G.L. Kopf G.S. Moss S.B. Mol. Biol. Cell. 1998; 9: 263-276Crossref PubMed Scopus (115) Google Scholar)) and lactatedehydrogenase (localized in the mitochondrial matrix(26Blanco A. Johns Hopkins Med.J. 1980; 146: 231-235PubMed Google Scholar)). The non-canonicallocalizations of metabolic enzymes indicate there is an extra mitochondrialenergy production center; the ATP generated after glycolysis has been shown tobe the source of tyrosine phosphorylation during capacitation of mousespermatozoa (3Travis A.J. Jorgez C.J. Merdiushev T. Jones B.H. Dess D.M. Diaz-Cueto L. Storey B.T. Kopf G.S. Moss S.B. J. Biol. Chem. 2001; 276: 7630-7636Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In ourprevious report we have established the importance of a post pyruvatemetabolic enzyme, dihydrolipoamide dehydrogenase, in hamster spermhyperactivation and acrosome reaction(27Mitra K. Shivaji S. Biol.Reprod. 2004; 70: 887-899Crossref PubMed Scopus (44) Google Scholar). We also identified theenzyme to be a target of the capacitation-dependent protein tyrosinephosphorylation cascade. Dihydrolipoamide dehydrogenase, a flavoprotein disulfide oxidoreductase, isthe E3 1The abbreviations used are: E3, dihydrolipoamide dehydrogenase; PDHc,pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase; BSA, bovine serumalbumin; TBS, Tris-buffered saline; PAS, periodic acid-Schiff. component ofα-ketoacid dehydrogenase multienzyme complexes(28Patel M.S. Vettakkorumakankav N.N. Liu T.C. Methods Enzymol. 1995; 252: 186-195Crossref PubMed Scopus (29) Google Scholar). It is, canonically, amitochondrial enzyme, the exact localization being in the mitochondrial matrix(29Patel M.S. Roche T.E. FASEBJ. 1990; 4: 3224-3233Crossref PubMed Scopus (502) Google Scholar). Dihydrolipoamideacetyltransferase, another component of the same multienzyme complexes, is thephysiological substrate of E3. The enzyme, E3, is active as a dimer, and themain catalytic actions of E3 are dehydrogenase (bidirectional,Reaction 1), diaphorase(Reaction 2), and oxidase(Reaction 3)(30Gazaryan I.G. Krasnikov B.F. Ashby G.A. Thorneley R.N. Kristal B.S. Brown A.M. J. Biol.Chem. 2002; 277: 10064-10072Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) as follows.Dihydrolipoamide+NAD+ reverse↑↓forwardlipoamide+NADH+H+REACTION 1DCPIPOX+NADH+H+→(dichlorophenolindophenol-oxidized)DCPIPred+NAD+(dichlorophenolindophenol-reduced)REACTION 2NADH+H++O2→NAD++H2O2REACTION 3 Some recent studies show various other functions of this redox activeenzyme, E3 both in vitro(31Igamberdiev A.U. Bykova N.V. Ens W. Hill R.D. FEBS Lett. 2004; 568: 146-150Crossref PubMed Scopus (42) Google Scholar, 32Bhushan B. Halasz A. Spain J.C. Hawari J. Biochem. Biophys. Res. Commun. 2002; 296: 779-784Crossref PubMed Scopus (51) Google Scholar, 33Petrat F. Paluch S. Dogruoz E. Dorfler P. Kirsch M. Korth H.G. Sustmann R. de Groot H. J.Biol. Chem. 2003; 278: 46403-46413Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar)and in vivo (34Smith A.W. Roche H. Trombe M.C. Briles D.E. Hakansson A. Mol. Microbiol. 2002; 44: 431-448Crossref PubMed Scopus (57) Google Scholar,35Bryk R. Lima C.D. Erdjument-Bromage H. Tempst P. Nathan C. Science. 2002; 295: 1073-1077Crossref PubMed Scopus (328) Google Scholar). E3 knock-out mice dieearly in development, and the heterozygotes show half of the enzyme activityof that of the normal(36Johnson M.T. Yang H.S. Magnuson T. Patel M.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14512-14517Crossref PubMed Scopus (68) Google Scholar). Pyruvate dehydrogenase complex (PDHc) is a paradigmatic example ofα-ketoacid dehydrogenase complexes hosting E3. Bacterial E3(37Wilkinson K.D. Williams Jr., C.H. J. Biol. Chem. 1981; 256: 2307-2314Abstract Full Text PDF PubMed Google Scholar) and pig heart E3(38Matthews R.G. Williams Jr., C.H. J. Biol. Chem. 1976; 251: 3956-3964Abstract Full Text PDF PubMed Google Scholar) have been shown to beregulated by alteration of the NAD:NADH ratio. However, aphosphorylation-dephosphorylation cycle of the α subunit of the E1component of PDHc seems to be a stronger regulator in eukaryotes(39Linn T.C. Pettit F.H. Hucho F. Reed L.J. Proc. Natl. Acad. Sci. U. S. A. 1969; 64: 227-234Crossref PubMed Scopus (259) Google Scholar,40Linn T.C. Pettit F.H. Reed L.J. Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 234-241Crossref PubMed Scopus (520) Google Scholar). No direct regulation ofE3 has been observed as far as pyruvate metabolism is concerned. This paper reveals non-canonical extra mitochondrial localization ofdihydrolipoamide dehydrogenase (E3) in mammalian spermatozoa; in hamsterspermatozoa the enzyme shows dual localization in the acrosome and in theprincipal piece of sperm flagella. The data further demonstrate a strongpositive correlation between the tyrosine phosphorylation status of the enzymeand its bi-directional enzymatic activity in the two locations in the hamsterspermatozoa during capacitation, indicating a dual control of the metabolicenzyme during the cellular event. Spermatozoa Collection and in Vitro Capacitation—Male Goldenhamsters (Mesocricetus auratus) aged 6 months were used asexperimental animals. All animal experiments were performed in accordance withthe guidelines of the Institutional Animal Ethics Committee of the Centre forCellular and Molecular Biology. Spermatozoa were collected from the caudalepididymides into TALP (modified Tyrode's medium, a medium known to supportcapacitation of hamster spermatozoa)(41Bavister B.D. GameteRes. 1989; 23: 139-158Crossref PubMed Scopus (240) Google Scholar) by swim-up technique(27Mitra K. Shivaji S. Biol.Reprod. 2004; 70: 887-899Crossref PubMed Scopus (44) Google Scholar) and thereafter counted ina Makler chamber using a computer-assisted semen analyzer (HTM-CEROS, HamiltonThorne, Maryland, MD). Aliquots from the swim-up were used for differentstudies. Hamster spermatozoa maintained in TALP medium in 5% CO2 at37 °C attained capacitation within 3–5 h(42Kulanand J. Shivaji S. Andrologia. 2001; 33: 95-104Crossref PubMed Scopus (58) Google Scholar, 43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). For the time-courseexperiments spermatozoa maintained in TALP medium were harvested at the timepoints mentioned in the study. Detection of Phosphorylation and Protein Levels—5 ×106 spermatozoa were used to study tyrosine phosphorylation bySDS-PAGE immunoblot analysis(27Mitra K. Shivaji S. Biol.Reprod. 2004; 70: 887-899Crossref PubMed Scopus (44) Google Scholar) with monoclonalanti-phosphotyrosine (αPY) antibody (Upstate) as follows: (a)blocking with 5% nonfat milk, (b) incubation with 1:10,000 dilutionof primary antibody (αPY) in 1% BSA in TBS-T (TBS containing 0.1% Tween20), and (c) incubation with 1:10,000 dilution of secondary antibodyconjugated with horseradish peroxidase in 1% BSA in TBS-T. These steps wereinterspersed with washes in TBS-T. The blots were then developed using theEnhanced Chemiluminescence kit (Amersham Biosciences). Immunoblotting of thetwo-dimensional-PAGE was also performed as described above while thetwo-dimensional-PAGE was run according to the method of O'Farrell(44O'Farrell P.H. J. Biol.Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar). Acrosome Reaction—A minimum of 100 spermatozoa were scoredfor each time point using a phase contrast microscope (Leitz, Germany) with a40× objective (42Kulanand J. Shivaji S. Andrologia. 2001; 33: 95-104Crossref PubMed Scopus (58) Google Scholar). Thesamples were stained with eosin Y (0.25% in TALP medium) and scored forspontaneous acrosome-reacted spermatozoa. The spermatozoa undergoing or havingundergone acrosome reaction were counted as positive. The results wereexpressed as a percentage of acrosome-reacted spermatozoa. Generation of Antibody—Antibody against E3 was raised inrabbit by injecting 200 μg of pig heart E3 as the antigen (Sigma). Allinjections were given subcutaneously, the first three being in Freund'scomplete adjuvant and the later ones (one or two) in Freund's incompleteadjuvant. Indirect Immunofluorescence and Confocal Studies—Spermatozoamaintained in TALP medium were collected while carefully ignoring the pelletof dead cells at the bottom of the microcentrifuge tubes. Spermatozoa werewashed (1000 rpm for 5 min at room temperature) and fixed with 2% formaldehyde(10 min) prepared freshly in TBS. The fixed sperm suspension was then coatedproperly on clean glass coverslips and air dried (37 °C). Cells on thecoverslips were freshly permeabilized by dipping in ice-cold (-20 °C)methanol (20 s) and were blocked (5% BSA in TBS) followed by incubations withprimary and appropriate secondary antibody (made in 1% BSA in TBS). All theincubation steps were interspersed with 3–4 washes in TBS. MitoTrackerCmxRos (Molecular Probes, Eugene, OR) staining of MCF-7 cells grown oncoverslips was performed using 200 nm dye for 45 min in serum-freemedium. Coverslips were washed with serum-free medium after which the cellswere fixed with 3.5% formaldehyde (in the same medium) for 10 min. Afterimmunostaining coverslips were processed for antibody staining as before andmounted on clean glass slides using Antifade (Vector) as the mounting mediumand viewed in an Axioplan 2 epifluorescence microscope (Carl Zeiss Inc.).Colocalization studies were done using a laser scanning confocal microscope,LSM510 Meta (Carl Zeiss Inc.). The dyes used were fluorescein isothiocyanateand Cy3 (or MitoTracker dye) for the dual staining, which were excited at 488nm and 543 nm laser lines, respectively. Optical sections (0.2 μm each) ofthe sperm samples were obtained during the scanning, and for each sample twoto five innermost sections were projected. Dissolution of Acrosomal Matrix—The acrosomal matrices weredislodged from the spermatozoa following an established protocol(45Olson G.E. Winfrey V.P. Davenport G.R. Biol. Reprod. 1988; 39: 1145-1158Crossref PubMed Scopus (39) Google Scholar) with littlemodifications. The spermatozoa were harvested at different stages ofcapacitation and placed on ice. The sperm pellets were then washed twice withcold TBS (500 × g for 5 min), resuspended in HEPES buffer (10mm HEPES and 140 mm NaCl, pH 7.2) containing 0.1% TritonX-100 and 0.25 mm phenylmethylsulfonyl fluoride and once again kepton ice for 1 h. After vortexing the spermatozoa were then subjected tohomogenization in a Dounce homogenizer. They were further kept on ice for 30min after which spermatozoa were centrifuged (350 × g for 5min) and washed again in TBS. SDS-PAGE extracts were prepared as mentionedbefore. Protein estimation was done with the extracts according to the methodof Karlsson et al.(46Karlsson J.O. Ostwald K. Kabjorn C. Andersson M. Anal. Biochem. 1994; 219: 144-146Crossref PubMed Scopus (59) Google Scholar). Acrosomal matrices were partially dislodged from the sperm head by themethod used for guinea pig spermatozoa(47Foster J.A. Friday B.B. Maulit M.T. Blobel C. Winfrey V.P. Olson G.E. Kim K.S. Gerton G.L. J. Biol. Chem. 1997; 272: 12714-12722Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Briefly, hamsterspermatozoa were collected from the cauda epididymides in a buffer having 20mm sodium acetate (pH 5.2) and 0.15 m NaCl (along with0.2 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, and0.5 μg/ml aprotinin) and were washed at 500 × g (5 min) at 4°C. The sperm pellet was then suspended in the same buffer with 0.625%Triton X-100, and the suspension was passed through a 26-gauge needle 20times. Although this procedure did not dislodge all the acrosomal matricescompletely, it loosened them and spermatozoa could be identified at differentstages of acrosomal matrix loss. Immunohistochemistry—Testes, dissected out from the animalsand rinsed in TBS, were fixed overnight in Bouin's fixative (70%water-saturated picric acid, 20% formaldehyde, and 5% acetic acid) after whichthe tissue was washed in 70% alcohol and dehydrated in a graded series ofalcohol. Molds were prepared with paraffin wax, and 5-μm-thick sectionswere collected on slides coated with 0.5% gelatin. Sections weredeparaffinized in xylene (20 min), hydrated through graded alcoholincubations, and finally transferred to distilled water. Deparaffinizedhydrated sections were stained with PAS-hematoxylin, and staging of theseminiferous tubules was done according to Miething(48Miething A. Adv. Anat. Embryol.Cell Biol. 1998; 140: 1-92Crossref PubMed Google Scholar). Parallel sections wereprocessed for immunohistochemistry; they were blocked with 5% BSA (in TBS)followed by primary and appropriate secondary antibody (made in 1% BSA in TBS)incubations. Each incubation was interspersed with gentle washes in TBS.Finally, the color was developed by incubating the sections with nitro bluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate in the presence of 0.1mm levamisole to inhibit any endogenous alkaline phosphataseactivity. Sections were rinsed in distilled water and mounted in 30% glyceroland viewed under the bright field of Axioplan 2 microscope (Carl ZeissInc.). Enzyme Assay—The detergent-soluble sperm lysates wereprepared, with sperm pellets harvested at different time points duringcapacitation, according to the method of Patel et al.(28Patel M.S. Vettakkorumakankav N.N. Liu T.C. Methods Enzymol. 1995; 252: 186-195Crossref PubMed Scopus (29) Google Scholar). In brief, a pellet of100 million spermatozoa was suspended in 200 μl of hypotonic phosphatebuffer with Triton X-100 (1%), protease inhibitors (0.2 mmphenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, and 0.5 μg/mlaprotinin), and sodium orthovanadate (1 mm) and kept on ice for 1h. The suspension of cells was then subjected to three to four freeze-thawcycles and again kept on ice for 1 h and centrifuged at 14,000 rpm for 15 min.5 μl of the supernatant was used for the enzyme assay (equivalent to 2.5× 106 spermatozoa). The enzyme assay was performed in a200-μl volume according to Gazaryan et al.(30Gazaryan I.G. Krasnikov B.F. Ashby G.A. Thorneley R.N. Kristal B.S. Brown A.M. J. Biol.Chem. 2002; 277: 10064-10072Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) with some modifications.The substrates for the assay were dihydrolipoamide (8 mm) andlipoamide (4 mm) for the forward and reverse reactions,respectively. NAD (0.32 mm) and NADH (0.16 mm) were usedas cofactors for forward and reverse reactions, respectively. The activity ofthe enzyme was measured as a change in optical density(A340) in a UV-visible spectrophotometer (Shimadzu); theunit of activity was expressed as (micromoles of NADH/min)/μg of totalprotein in Table I. However, inthe experiment where E3 activity has been compared at different time points ofcapacitation, the enzyme activity was expressed as micromoles of NADH/min andfurther normalized by a factor of 10-3 as inFig. 11. This is because ofthe fact that a lot of acrosomal proteins are lost during capacitation, whichwould overestimate the activity at the time points when the percentage ofacrosome reacted spermatozoa is high.Table IReverse activity of hamster spermatozoal E3 under differentconditions The “reverse activity” is the activity of E3 toreduce lipoamide to dihydrolipoamide accepting electrons from NADH. Activityof E3 is expressed in units representing change in micromoles ofNADH/min/μg of sperm lysate. Similar results were obtained for forwardactivity (data not shown). The values represent E3 activity of approximately2.5 × 106 spermatozoa.Dihydrolipoamide dehydrogenaseControlaControl represents activity in the presence of NADH as cofactor and withoutheat treatmentHeat treatment5 mmMICAbMICA, 5-methoxyindole-2-carboxylic acidNADPHμmol of NADH/min/μgHamster spermatozoa (non-capacitated)0.16 × 10-30.16 × 10-30cThe value 0 signifies that the activity of the enzyme is totallyinhibited0cThe value 0 signifies that the activity of the enzyme is totallyinhibitedHamster spermatozoa (capacitated)0.33 × 10-30.33 × 10-30cThe value 0 signifies that the activity of the enzyme is totallyinhibited0cThe value 0 signifies that the activity of the enzyme is totallyinhibiteda Control represents activity in the presence of NADH as cofactor and withoutheat treatmentb MICA, 5-methoxyindole-2-carboxylic acidc The value 0 signifies that the activity of the enzyme is totallyinhibited Open table in a new tab Fig. 1Validation of the polyclonal anti-E3 antibody. Two-dimensional-PAGEperformed with this polyclonal antibody detected only a single expected spot(A, left panel); increasing molecular weight (MW) andisoelectric point (PI) are directed by arrows. The othercomponents of the PDHc were detected in SDS-PAGE immunoblot using polyclonalanti-PDHc antibody; the identity of the bands are indicated on theright (A, right panel). Colocalization of PDHc and E3 withthe MitoTracker dye in MCF-7 cell line (B). MCF-7 cells were doublystained with MitoTracker (panel A) and polyclonal anti-PDHc antibody(panel B, PDHc)/anti-E3 antibody (panel B, E3)/anti-E3antibody preabsorbed with purified E3 (panel B, Preabs)/rabbitpreimmune serum (panel B, Preimm). Corresponding colocalized imagesare shown in panel C. The bar represents 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Statistical Analysis—The correlation between acrosomereaction and forward activity of hamster sperm E3 was done using Spearman'scorrelation coefficient (rs) using SPSS, version 11.0.1;significance of correlation between the parameters was also checked using thesame software. For the sake of comparison the values of forward activity of E3were normalized with the percentage viability of spermatozoa at the respectivetime point, because during capacitation a reduction in viability wasobserved. Validation of the Rabbit Polyclonal Antibody Raised against PurifiedPig Heart Dihydrolipoamide Dehydrogenase—The polyclonal antibodyraised against purified pig heart dihydrolipoamide dehydrogenase (E3) wasvalidated by two-dimensional-PAGE immunoblots and immunofluorescence methods.The two-dimensional-PAGE immunoblot of hamster sperm lysate with thepolyclonal anti-E3 antibody detected a single spot(Fig. 1A, leftpanel), which was previously identified as E3 by N-terminal sequencing(27Mitra K. Shivaji S. Biol.Reprod. 2004; 70: 887-899Crossref PubMed Scopus (44) Google Scholar). Because E3 is a part ofthe pyruvate dehydrogenase complex (PDHc), a polyclonal antibody raisedagainst the whole PDHc except E3 (kindly donated by Dr. R. A. Harris) was usedto detect the other components of the complex in immunoblot of sperm extracts,as shown in the right panel ofFig. 1A. E3 along with its host complex, PDHc, are canonically mitochondrialproteins. Therefore, it is expected that both E3 and PDHc would colocalizewith the MitoTracker used as a mitochondrial marker. Thus, dual staining wasperformed in a mammalian cell line, MCF-7, using the polyclonalanti-E3/anti-PDHc antibody and the MitoTracker dye. The upper twopanels of Fig. 1Bshow staining of the polyclonal PDHc antibody (PDHc) and that of thepolyclonal anti-E3 antibody (E3), respectively. The correspondingimages in Fig. 1A showthe MitoTracker staining, which is very similar to the corresponding antibodystaining. The corresponding panels of panel C show the merged imageof the MitoTracker and the PDHc/E3 staining, demonstrating clearcolocalization of the two as expected. Furthermore, preabsorption of thepolyclonal E3 antibody with purified E3 protein (100 μg/100 μl) failedto detect any antigen in the cells (panel B, Preabs), and thepreimmune serum also showed no staining (panel B, Preimm). Thecorresponding panels in panels A and C show the MitoTrackerstaining and the merged images, respectively. Our polyclonal anti-E3 antibodyalso could detect the E3 in immunoblots of extracts of MCF-7 cell lines (datanot shown). Thus the results taken together validate the specificity of thepolyclonal anti-E3 antibody, which is thus used for further experiments todetect dihydrolipoamide dehydrogenase (E3) both in immunoblots andimmunofluorescence methods. Extra Mitochondrial Localization of Dihydrolipoamide Dehydrogenase inMammalian Epididymal Spermatozoa—The localization ofdihydrolipoamide dehydrogenase (E3) has been investigated in spermatozoa fromthe hamster caput and cauda (non-capacitated) epididymides by indirectimmunofluorescence using polyclonal E3 antibody.Fig. 2 (A andB) shows acrosomal stain" @default.
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• W2035168650 date "2005-07-01" @default.
• W2035168650 modified "2023-09-30" @default.
• W2035168650 title "Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenasein Spermatozoa" @default.
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