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Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenasein Spermatozoa

CORRELATION OF ITS LOCALIZATION, TYROSINE PHOSPHORYLATION,AND ACTIVITY DURING SPERMCAPACITATION*
  • Author Footnotes
    ‡ A recipient of a Council of Scientific and Industrial Research fellowship,Government of India.
    Kasturi Mitra
    Footnotes
    ‡ A recipient of a Council of Scientific and Industrial Research fellowship,Government of India.
    Affiliations
    Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007,India
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  • Nandini Rangaraj
    Affiliations
    Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007,India
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  • S. Shivaji
    Correspondence
    To whom correspondence should be addressed. Tel.: 91-40-271-92504; Fax:91-40-271-60591;
    Affiliations
    Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007,India
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.
    ‡ A recipient of a Council of Scientific and Industrial Research fellowship,Government of India.
Open AccessPublished:May 11, 2005DOI:https://doi.org/10.1074/jbc.M500310200
      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(
      • Westhoff D.
      • Kamp G.
      ,
      • Storey B.T.
      • Kayne F.J.
      ,
      • Travis 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.
      ),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”(
      • Austin C.R.
      ,
      • Chang M.C.
      ). 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(
      • Visconti P.E.
      • Galantino-Homer H.
      • Moore G.D.
      • Bailey J.L.
      • Ning X.
      • Fornes M.
      • Kopf G.S.
      ,
      • Jha K.N.
      • Kameshwari D.B.
      • Shivaji S.
      ,
      • de Lamirande E.
      • Leclerc P.
      • Gagnon C.
      ).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(
      • Yanagimachi R.
      ).
      Protein tyrosine phosphorylation is considered to be a hallmark of spermcapacitation(
      • Visconti P.E.
      • Westbrook V.A.
      • Chertihin O.
      • Demarco I.
      • Sleight S.
      • Diekman A.B.
      ,
      • Jha K.N.
      • Shivaji S.
      ,
      • Leclerc P.
      • de Lamirande E.
      • Gagnon C.
      ,
      • Mahony M.C.
      • Gwathmey T.
      ,
      • Si Y.
      • Okuno M.
      ,
      • Urner F.
      • Leppens-Luisier G.
      • Sakkas D.
      ),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(
      • Jha K.N.
      • Shivaji S.
      ,
      • Carrera A.
      • Moos J.
      • Ning X.P.
      • Gerton G.L.
      • Tesarik J.
      • Kopf G.S.
      • Moss S.B.
      ). Recently, tyrosinephosphorylation of AKAP3 has been shown to result in the recruitment ofprotein kinase A to the sperm flagella causing an increase in motility(
      • Luconi M.
      • Carloni V.
      • Marra F.
      • Ferruzzi P.
      • Forti G.
      • Baldi E.
      ). Furthermore, theessentiality of a balance between protein-tyrosine kinase and phosphataseactivities has also been demonstrated as a mandatory requirement for asuccessful acrosome reaction(
      • Tomes C.N.
      • Roggero C.M.
      • De Blas G.
      • Saling P.M.
      • Mayorga L.S.
      ).
      Regulation of metabolic enzymes by phosphorylation has been wellestablished in different cells(
      • Huang K.P.
      • Huang F.L.
      ,
      • El-Maghrabi M.R.
      • Noto F.
      • Wu N.
      • Manes N.
      ,
      • Pilkis S.J.
      • El-Maghrabi M.R.
      • Coven B.
      • Claus T.H.
      • Tager H.S.
      • Steiner D.F.
      • Keim P.S.
      • Heinrikson R.L.
      ).However, the metabolism of spermatozoa is quite distinctly different fromother cells and thus less understood, with spermatozoa having specificisoforms of key metabolic enzymes(
      • Mori C.
      • Welch J.E.
      • Fulcher K.D.
      • O'Brien D.A.
      • Eddy E.M.
      ,
      • Goldberg E.
      ,
      • Dahl H.H.
      • Brown R.M.
      • Hutchison W.M.
      • Maragos C.
      • Brown G.K.
      ).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(
      • Travis A.J.
      • Foster J.A.
      • Rosenbaum N.A.
      • Visconti P.E.
      • Gerton G.L.
      • Kopf G.S.
      • Moss S.B.
      )) and lactatedehydrogenase (localized in the mitochondrial matrix(
      • Blanco A.
      )). 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 (
      • Travis 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.
      ). In ourprevious report we have established the importance of a post pyruvatemetabolic enzyme, dihydrolipoamide dehydrogenase, in hamster spermhyperactivation and acrosome reaction(
      • Mitra K.
      • Shivaji S.
      ). We also identified theenzyme to be a target of the capacitation-dependent protein tyrosinephosphorylation cascade.
      Dihydrolipoamide dehydrogenase, a flavoprotein disulfide oxidoreductase, isthe E3
      The abbreviations used are: E3, dihydrolipoamide dehydrogenase; PDHc,pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase; BSA, bovine serumalbumin; TBS, Tris-buffered saline; PAS, periodic acid-Schiff.
      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(
      • Patel M.S.
      • Vettakkorumakankav N.N.
      • Liu T.C.
      ). It is, canonically, amitochondrial enzyme, the exact localization being in the mitochondrial matrix(
      • Patel M.S.
      • Roche T.E.
      ). 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)(
      • Gazaryan I.G.
      • Krasnikov B.F.
      • Ashby G.A.
      • Thorneley R.N.
      • Kristal B.S.
      • Brown A.M.
      ) as follows.
      Dihydrolipoamide+NAD+reverseforwardlipoamide+NADH+H+REACTION 1DCPIPOX+NADH+H+(dichlorophenolindophenol-oxidized)DCPIPred+NAD+(dichlorophenolindophenol-reduced)REACTION 2NADH+H++O2NAD++H2O2REACTION 3


      Some recent studies show various other functions of this redox activeenzyme, E3 both in vitro(
      • Igamberdiev A.U.
      • Bykova N.V.
      • Ens W.
      • Hill R.D.
      ,
      • Bhushan B.
      • Halasz A.
      • Spain J.C.
      • Hawari J.
      ,
      • Petrat F.
      • Paluch S.
      • Dogruoz E.
      • Dorfler P.
      • Kirsch M.
      • Korth H.G.
      • Sustmann R.
      • de Groot H.
      )and in vivo (
      • Smith A.W.
      • Roche H.
      • Trombe M.C.
      • Briles D.E.
      • Hakansson A.
      ,
      • Bryk R.
      • Lima C.D.
      • Erdjument-Bromage H.
      • Tempst P.
      • Nathan C.
      ). E3 knock-out mice dieearly in development, and the heterozygotes show half of the enzyme activityof that of the normal(
      • Johnson M.T.
      • Yang H.S.
      • Magnuson T.
      • Patel M.S.
      ).
      Pyruvate dehydrogenase complex (PDHc) is a paradigmatic example ofα-ketoacid dehydrogenase complexes hosting E3. Bacterial E3(
      • Wilkinson K.D.
      • Williams Jr., C.H.
      ) and pig heart E3(
      • Matthews R.G.
      • Williams Jr., C.H.
      ) 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(
      • Linn T.C.
      • Pettit F.H.
      • Hucho F.
      • Reed L.J.
      ,
      • Linn T.C.
      • Pettit F.H.
      • Reed L.J.
      ). 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.

      EXPERIMENTAL PROCEDURES

      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)(
      • Bavister B.D.
      ) by swim-up technique(
      • Mitra K.
      • Shivaji S.
      ) 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(
      • Kulanand J.
      • Shivaji S.
      ,
      • Laemmli U.K.
      ). 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(
      • Mitra K.
      • Shivaji S.
      ) 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(
      • O'Farrell P.H.
      ).
      Acrosome Reaction—A minimum of 100 spermatozoa were scoredfor each time point using a phase contrast microscope (Leitz, Germany) with a40× objective (
      • Kulanand J.
      • Shivaji S.
      ). 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(
      • Olson G.E.
      • Winfrey V.P.
      • Davenport G.R.
      ) 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.(
      • Karlsson J.O.
      • Ostwald K.
      • Kabjorn C.
      • Andersson M.
      ).
      Acrosomal matrices were partially dislodged from the sperm head by themethod used for guinea pig spermatozoa(
      • Foster J.A.
      • Friday B.B.
      • Maulit M.T.
      • Blobel C.
      • Winfrey V.P.
      • Olson G.E.
      • Kim K.S.
      • Gerton G.L.
      ). 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(
      • Miething A.
      ). 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.(
      • Patel M.S.
      • Vettakkorumakankav N.N.
      • Liu T.C.
      ). 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.(
      • Gazaryan I.G.
      • Krasnikov B.F.
      • Ashby G.A.
      • Thorneley R.N.
      • Kristal B.S.
      • Brown A.M.
      ) 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 dehydrogenaseControl
      Control represents activity in the presence of NADH as cofactor and withoutheat treatment
      Heat treatment5 mmMICA
      MICA, 5-methoxyindole-2-carboxylic acid
      NADPH
      μmol of NADH/min/μg
      Hamster spermatozoa (non-capacitated)0.16 × 10-30.16 × 10-30
      The value 0 signifies that the activity of the enzyme is totallyinhibited
      0
      The value 0 signifies that the activity of the enzyme is totallyinhibited
      Hamster spermatozoa (capacitated)0.33 × 10-30.33 × 10-30
      The value 0 signifies that the activity of the enzyme is totallyinhibited
      0
      The value 0 signifies that the activity of the enzyme is totallyinhibited
      a Control represents activity in the presence of NADH as cofactor and withoutheat treatment
      b MICA, 5-methoxyindole-2-carboxylic acid
      c The value 0 signifies that the activity of the enzyme is totallyinhibited
      Figure thumbnail gr11
      Fig. 11Stage-specific directional regulation of hamster sperm E3 activityduring capacitation. In vitro enzyme assay of E3 shows that thereverse and forward activities of the enzyme show different regulation duringcapacitation; the reverse activity exhibiting a peak at 4 h and forwardactivity at 5–7 h (A). The activity of the enzyme is expressedas micromoles of NADH/min, normalized with the factor 10-3. Theforward activity and the acrosome reaction appeared to follow the sameprogress during capacitation, the values at each time point being positivelycorrelated to each other (B). The Spearman's correlation coefficientwas found to be 0.874 and was significant at p < 0.01. Theexperiment was performed with three animals.
      Figure thumbnail gr1
      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.
      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.

      RESULTS

      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(
      • Mitra K.
      • Shivaji S.
      ). 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 staining of E3 in hamster spermatozoafrom the cauda and caput region of the epididymides, respectively. Faintstaining was also observed in the principal piece of the flagella in hamsterspermatozoa from the cauda region (Fig.2A). Polyclonal anti-E3 antibody preabsorbed with thepurified protein (100 μg/100 μl) could not detect any distinct stainingin hamster spermatozoa thus proving the authenticity of the localization(Fig. 2C). Monoclonalantibody to the sperm mitochondrial protein Phospholipid hydroperoxideglutathione peroxidase (PHGPx) (kindly donated by Prof. Kühn) was used asa marker for sperm mitochondria, and as anticipated, it localized to the midpiece of spermatozoa (Fig.2D). However the antibody also showed some staining inthe head of hamster spermatozoa, which is in all probability because of thenuclear seleno-protein detected by this antibody as shown previously in ratspermatozoa (
      • Pfeifer H.
      • Conrad M.
      • Roethlein D.
      • Kyriakopoulos A.
      • Brielmeier M.
      • Bornkamm G.W.
      • Behne D.
      ,
      • Roveri A.
      • Casasco A.
      • Maiorino M.
      • Dalan P.
      • Calligaro A.
      • Ursini F.
      ). Furthermore, even whenTriton X-100 was used to expose the mitochondrial proteins(
      • Thompson W.E.
      • Ramalho-Santos J.
      • Sutovsky P.
      ), E3 antibody still failedto convincingly stain the mitochondria in the mid piece, although littlescattered staining was observed in this part(Fig. 2E). It was alsoobserved that detergent treatment obliterated acrosomal staining and thestaining in the principal piece increased. The polyclonal PDHc antibody alsostained the acrosome and the principal piece of the flagella of spermatozoataken from cauda epididymides (Fig.2F), very similar to E3 staining. The preimmune rabbitserum stained only a small part of the acrosome represented as a fine line ofstaining (Fig. 2G), incontrast to the markedly distinct staining of the entire acrosome by the E3antiserum (Fig. 2H).Fig. 2 (I andJ) shows the corresponding bright field images. (The factthat rabbit sera show cross-reactivity in the hamster sperm acrosome was alsoobserved earlier by workers demonstrating localization of p26h(
      • Gaudreault C.
      • El Alfy M.
      • Legare C.
      • Sullivan R.
      )). To investigate the fateof E3 during sperm capacitation, immunofluorescent studies were performed withhamster sperm samples prepared at different time points during capacitation.The intensity of E3 staining in the principal piece of the hamster spermflagellum increased from 0 h (non-capacitated) to the 7th hour ofcapacitation, as shown by a representative spermatozoon from each time point(Fig. 2K). However,the intensity of staining in the acrosome did not seem to change withcapacitation, only around 20% of the spermatozoa appeared to have lost theacrosomal E3 staining at the 7th hour of capacitation.
      Figure thumbnail gr2
      Fig. 2Extra-mitochondrial localization of E3 in hamster spermatozoa.Indirect immunofluorescence studies were performed by using rabbit polyclonalanti-E3 antibody. E3 localizes in the acrosome and principal piece of cauda(A) and caput (B) epidydimal spermatozoa. The preabsorbedanti-E3 antibody fails to give distinct staining (C). Spermmitochondrial marker PHPGx localizes in the sperm mitochondria (mid piece)(D). Triton X-100-treated caudal spermatozoa do not revealmitochondrial localization of E3 (E). PDHc shows similar localizationin the cauda spermatozoa (F). The preimmune rabbit serum stained onlya small part of the acrosome represented as a fine line of staining(G), in contrast to the markedly distinct staining of the entireacrosome by the E3 antiserum (H); the corresponding bright fieldimages are also shown (I and J). There is no change inlocalization of E3 during capacitation as shown by a single representativespermatozoon at each time point of capacitation (K); numbersrepresent the time point of capacitation. The intensity of E3 stainingincreases in the principal piece of the sperm flagella along the time courseof capacitation. The bars in every figure represent 20 μm, whereasin G–I it represents 10 μm.
      To investigate the generality of the extra mitochondrial localization ofE3, immunolocalization of E3 in the caput and cauda epididymal spermatozoa ofanother rodent species (mouse) was carried out, and the results confirmed theextra-mitochondrial acrosomal localization of E3 also in mouse spermatozoa(Fig. 3, A andB). However, attempts to stain human sperm with thepolyclonal E3 antibody raised against pig heart E3 failed to show any positivestaining (data not shown). But when the anti-PDHc antibody, raised againstcorresponding human proteins was used, distinct extra-mitochondrial acrosomalstaining was observed in human spermatozoa(Fig. 3C).
      Figure thumbnail gr3
      Fig. 3Extra mitochondrial localization of E3 in spermatozoa of mouse andhuman. Indirect immunofluorescence studies were performed by using rabbitpolyclonal anti-E3 antibody. E3 shows an acrosomal localization in the mousecaput (A) and cauda spermatozoa (B). PDHc localizes in theacrosome in human spermatozoa (C). The bars represent 10μm.
      Thus, the results taken together bring out clearly that the canonicalmitochondrial protein E3 and also the hosting enzyme complex, PDHc, exhibitextra mitochondrial localization in mammalian spermatozoa; hamster spermatozoashow the whole complex, including E3, to be present both in the acrosome andthe principal piece of the sperm flagella.
      Association of Dihydrolipoamide Dehydrogenase with the Acrosomal Matrixof Hamster Spermatozoa—The calcium ionophore A23187, which is knownto induce complete acrosome reaction(
      • Jaiswal B.S.
      • Eisenbach M.
      • Tur-Kaspa I.
      ), characterized by thetotal release of the acrosomal contents and exposure of the inner acrosomalmembrane (
      • Kopf G.F.
      • Gerton G.L.
      ), was used tocheck whether acrosomal dihydrolipoamide dehydrogenase (E3) is released duringacrosomal exocytosis. A23187 (5 μm for 2 h) could release almostall the E3 from the acrosome as seen in immunofluorescence studies(Fig. 4A). However, inpartial acrosome-reacted spermatozoa, as shown by a representativespermatozoon in phase (Fig.4B, upper panel), E3 was retained in theacrosome (Fig. 4B,lower panel). When immunoblotting was carried out with the sperm samplesimilarly exposed to A23187 for inducing acrosomal exocytosis, theexperimental sample showed less amount of E3 in the spermatozoa than thecontrol (sperm not treated with A23187 at the same time point)(Fig. 4C), indicatingthe release of E3 during acrosomal exocytosis. The same figure also shows therelease of P26h whose homologue, P34H, has been previously shown to bereleased during acrosome reaction in human spermatozoa(
      • Boue F.
      • Blais J.
      • Sullivan R.
      ). Thus the resultsindicate that E3 is released during complete acrosome reaction and notlocalized in the inner acrosomal membrane, which is the part of the acrosomeexposed after acrosome reaction. Furthermore, to investigate whether E3 islocalized in the plasma membrane or the acrosomal matrix, hamster spermatozoawere demembranated by Triton X-100 treatment followed by mechanical shearing,which could partially dislodge the acrosomal matrix of the demembranatedspermatozoa (
      • Foster J.A.
      • Friday B.B.
      • Maulit M.T.
      • Blobel C.
      • Winfrey V.P.
      • Olson G.E.
      • Kim K.S.
      • Gerton G.L.
      ).Immunofluorescence of these hamster spermatozoa with polyclonal anti-E3antibody revealed E3 staining in the Triton X-100-resistant acrosomal matrix.Fig. 4D shows E3staining in Triton X-100-treated hamster spermatozoa in different stages ofdislodgement of the acrosomal matrix. Thus our results indicate that E3 islocalized in the acrosomal matrix of hamster spermatozoa, which is releasedduring complete acrosome reaction.
      Figure thumbnail gr4
      Fig. 4E3 is associated with acrosomal matrix of hamster spermatozoa.Indirect immunofluorescence studies, supported by immunoblotting studies wereperformed by using rabbit polyclonal anti-E3 antibody. Calcium ionophoretreatment (5 μm, 2 h) releases acrosomal E3 from most hamsterspermatozoa (A). A representative partially acrosome reactedspermatozoa has not released E3 (B). E3 in the pellet of calciumionophore-treated (2 h) spermatozoa is less (2ca) than the untreatedcells collected either in the second hour (2c) or before thetreatment (0) (C, right panel). The surface acrosomal markerP26h also shows similar loss upon calcium ionophore treatment (C, leftpanel). Triton X-100-treated spermatozoa do not release E3, which getsdislodged only after mechanical shearing of the treated sperm, as representedby the spermatozoa at different stages of dislodgement of the matrix(D). The bar in A represents 20 μm, inB it represents 5 μm, and in D 10 μm.
      Dihydrolipoamide Dehydrogenase Shows a Mitochondria Type Localizationand Not a Golgi Type in Testicular Spermatocytes of AdultHamster—Acrosome is a Golgi-derived organelle, and thus one expectsdihydrolipoamide dehydrogenase (E3) to localize in the Golgi of thespermatogenic cells. It is known that Golgi exhibits a very characteristicjuxtanuclear localization(
      • Ramalho-Santos J.
      • Schatten G.
      • Moreno R.D.
      ), whereas themitochondrial proteins show perinuclear localization. Thus, animmunohistochemistry approach was used to investigate if dihydrolipoamidedehydrogenase (E3) shows a mitochondria type or a Golgi type staining in theadult testicular cells. Fig.5B shows that the polyclonal E3 antiserum faintly stainedthe whole tubule and the interstitium very strongly (arrows inFig. 5B), whereas thepreimmune serum stained the luminal side of the seminiferous tubules(arrows in Fig.5A). Investigation of the testicular sections at highermagnification revealed E3 staining of the Leydig cells in the interstitium(arrows in Fig.6A, letter “L”). It was alsoobserved that the spermatocytes in the mid-pachytene stage (stage VII)exhibited granulated staining around the nucleus (arrows inFig. 6A), whereas inthe step 1 spermatids (stage I) the stained granules appeared at the Golgipole of the nucleus (arrows inFig. 6C). The parallelPAS-hematoxylin-stained sections show the extent of acrosome formation in thecorresponding stages (Fig. 6, Band D). Comparison of panels C and D inFig. 6 shows that E3accumulates in the Golgi pole (from where acrosome starts forming) in thespermatids. E3 was not detected in the developing acrosome (or the flagella)either in the spermatids or in the matured testicular spermatozoa byimmunohistochemistry, but in spermatozoa released from testis staining couldbe observed in the acrosome by a more sensitive immunofluorescence stainingmethod (Fig. 6E).Thus, E3 shows a mitochondrial type of stain in the spermatocytes and appearsto be in the Golgi pole in the spermatids.
      Figure thumbnail gr5
      Fig. 5Staining of adult hamster testis by rabbit polyclonal E3 antibody.In immunohistochemistry E3 antiserum stains the entire seminiferous tubulesfaintly and stains the interstitium very strongly (arrows inB), whereas the preimmune serum stains only the luminal side of thetubules (arrows in A). Bar represents 100μm.
      Figure thumbnail gr6
      Fig. 6E3 does not show a Golgi type of staining in the testicular cells.Immunohistochemistry of adult hamster testis shows that in stage VII sectionsE3 shows a granulated staining around the pachytene spermatocytes(A). The corresponding PAS-hematoxylin-stained section shows theextended acrosome (arrows in B) in the spermatids. In stageI, E3 shows condensed staining in the Golgi pole of the spermatid nucleus(C). Corresponding PAS-hematoxylin-stained section shows the dot-likeappearance of the acrosome in the early stages of development (arrowsin D). Spermatozoa released from testis shows E3 in the acrosome inimmunofluorescence (E). N, spermatocyte nucleus; n,spermatid nucleus; L, Leydig cells. Bars inA–D represent 10 μm, while in E it represents 20μm.
      Tyrosine Phosphorylation of Dihydrolipoamide Dehydrogenase Detected inthe Principal Piece of Hamster Sperm Flagella— Previously we haddemonstrated that dihydrolipoamide dehydrogenase (E3) showscapacitation-dependent tyrosine phosphorylation in hamster spermatozoa(
      • Mitra K.
      • Shivaji S.
      ). The dual localization ofE3 in hamster spermatozoa as observed in this study prompted us to investigateif E3 is tyrosine-phosphorylated in one or both the locations. Lack ofknowledge of the tyrosine-phosphorylated site of this newly identifiedphosphorylated form of the protein led us to take an indirect approach towardthis question as described below. Fig. 7(A and B) show the staining oftyrosine-phosphorylated proteins using monoclonal anti-phosphotyrosineantibody (4G10), in non-capacitated and capacitated hamster spermatozoa. As inother mammalian spermatozoa(
      • Jha K.N.
      • Shivaji S.
      ,
      • Leclerc P.
      • de Lamirande E.
      • Gagnon C.
      ,
      • Mahony M.C.
      • Gwathmey T.
      ,
      • Si Y.
      • Okuno M.
      ,
      • Urner F.
      • Leppens-Luisier G.
      • Sakkas D.
      ),it was observed that almost all of the capacitation-dependenttyrosine-phosphorylated proteins localize in the flagella of capacitatedhamster spermatozoa. Furthermore, as a modification, the coverslips coatedwith capacitated hamster spermatozoa were preincubated with polyclonal E3antibody to block all the available E3 epitopes (irrespective of the tyrosinephosphorylation status) and were followed by staining of the phosphotyrosineresidues (in any protein). Fig.7C shows that blocking the E3 epitopes markedly reducedthe signal from tyrosine-phosphorylated proteins in the principal piece of thesperm flagella (marked by yellow arrows). The reduction in signal inthe mid piece of the spermatozoa by the same antibody (marked by greenarrows in Fig.7C) could be attributed to preimmune serum, which whenused for preincubation gave a reduced mid piece staining (greenarrows in Fig.7D). Furthermore, preincubation with the polyclonal PDHcantibody was used as another control, which showed very little reduction inthe signal intensity from phosphotyrosine residues(Fig. 7E). Thus, theimmunofluorescence data indicate a substantial contribution of E3 in the poolof the capacitation-dependent tyrosine-phosphorylated proteins in theprincipal piece of hamster sperm flagella.
      Figure thumbnail gr7
      Fig. 7Contribution of tyrosine-phosphorylated E3 to the total tyrosinephosphorylation in the principal piece of hamster sperm flagella.Capacitation-dependent tyrosine phosphorylation in spermatozoa is seen mostlyin the flagella (A, non-capacitated; B, capacitated).C, coverslips blocked with polyclonal E3 antibody reduces thedetection of tyrosine phosphorylation in the principal piece of the flagella(compare yellow arrows in C with B). The preimmuneserum blocking marginally obliterates detection of tyrosine phosphorylation inthe mid piece (green arrows in D and C).E, blocking with polyclonal PDHc antibody does not reduce the signalto a considerable extent compared with that of level reduced by polyclonal E3antibody (compare E with B and C). Barrepresents 10 μm.
      Further dual staining was performed with E3 (fluorescein isothiocyanate)and phosphotyrosine (cy3), in non-capacitated and capacitated hamsterspermatozoa, with the aim of identifying the tyrosine-phosphorylated form ofE3. For this purpose, keeping the result of the previous experiment in mind,the first antibody used for staining was anti-phosphotyrosine antibody (sothat all tyrosine-phosphorylated sites of E3, along with othertyrosine-phosphorylated proteins are bound by the antibody). This was followedby staining with E3 antibody. As a control the reverse incubation (E3 stainingfollowed by anti-phosphotyrosine staining) was also done in a capacitatedsperm sample. Because no tyrosine phosphorylation signal was detected in theacrosome, and E3 was detected only in the principal piece of the spermflagellum, only this part of the cell is being presented in the results of thedual staining experiment. As expected no tyrosine phosphorylation was detectedin the non-capacitated spermatozoa (0 h)(Fig. 8, A–C)for the E3 staining to show colocalization with. In the capacitatedspermatozoa (Fig. 8,D–F) a distinct colocalization was observed thatwas obliterated when sample was first blocked with polyclonal E3 antibody(Fig. 8, G–I).Thus, the results indicated the presence of a tyrosine-phosphorylated form ofE3 in the principal piece of the capacitated hamster sperm flagella.
      Figure thumbnail gr8
      Fig. 8Tyrosine-phosphorylated E3 in the principal piece of the hamster spermflagella. Colocalization was done by confocal microscopy by viewingoptical sections of sperm samples. Colocalization between E3 (green)and phosphotyrosine residues (red) is shown when coverslips wereincubated with anti-phosphotyrosine antibody first. In the non-capacitatedsample absence of tyrosine phosphorylation causes no colocalization(A–C). In the capacitated sample there is a strongcolocalization (D–F), which disappears when E3 antibody is usedfirst to block E3 epitopes (G–I). Bar represents 5μm.
      Time Course of Tyrosine Phosphorylation of DihydrolipoamideDehydrogenase Is Different in Different Locations of HamsterSpermatozoa—To follow the time course of tyrosine phosphorylationof dihydrolipoamide dehydrogenase (E3) in both the locations in hamsterspermatozoa, cell fractionation experiments were done followed by detection ofphosphorylation by immunoblotting with monoclonal anti-phosphotyrosineantibody (4G10). We have previously shown(
      • Jha K.N.
      • Shivaji S.
      ) that the 56-kDa molecularmass protein later identified by us(
      • Mitra K.
      • Shivaji S.
      ) as E3, is the onlytyrosine-phosphorylated protein at this molecular mass range in atwo-dimensional immunoblot with monoclonal anti-phosphotyrosine antibody(4G10). Thus analysis of parallel SDS-PAGE immunoblots of hamster spermlysates, with anti-phosphotyrosine antibody and anti-E3 antibody, suffices forthe analysis of tyrosine phosphorylation of E3. Because the localization of E3is shown to be in the acrosomal matrix and the principal piece of the hamsterspermatozoa (present study), dislodging the acrosomal matrix from the spermcompletely, after harvesting them at different time points of capacitation,would enable the biochemical investigation of the time course of tyrosinephosphorylation of E3 from only the principal piece of the sperm flagella.Fig. 9A shows theprotein loading of acrosome-dislodged spermatozoa at different time points ofcapacitation (by Ponceau S stain), whereas, as evidence supporting acrosomaldislodgement, Fig. 9Bshows the presence of the acrosomal marker P26h only in the intact sperm (Ts)and not in the acrosomal dislodged sperm.Fig. 9C shows that thelevel of E3 in the principal piece of hamster sperm flagella did not varyduring capacitation (taking into consideration the protein loading as inFig. 9A). The presenceof the tyrosine-phosphorylated form of E3 in the principal piece of hamstersperm flagella, as detected by immunofluorescence (Figs.7 and8), is confirmed inFig. 9D. The figurefurther shows the increase in its tyrosine phosphorylation along the timecourse of capacitation till 4th hour of capacitation and decrease again tillthe 7th hour (Fig.9D). Further we followed the time course of tyrosinephosphorylation of E3 in intact hamster spermatozoa (acrosomes not dislodged).With the total protein loaded (Fig.10A) and α tubulin as controls(Fig. 10B) it isclear that the amount of E3 is invariant even in intact hamster spermatozoaduring the time course of capacitation. However, the tyrosine phosphorylationof E3 showed a continuous increase from 0 to 7 h of capacitation in intactspermatozoa (Fig.10D). In the tyrosine phosphoprotein analyses (Figs.9D and10D), the othertyrosine-phosphorylated protein bands (not of interest in this report) arealso shown for the appreciation of the fact that different proteins havedifferent kinetics of phosphorylation during capacitation. The comparison ofthe time course of tyrosine phosphorylation of acrosome intact andacrosome-dislodged sperm clearly indicate the contribution of tyrosinephosphorylation from the acrosomal E3 in the later time points ofcapacitation, being maximum at 7 h (compare Figs.9D and10D). Thus, ourresults indicate differential tyrosine phosphorylation kinetics of E3 in theanalyses of acrosome dislodged sperm (contribution from the principal piece)and that of the acrosome intact sperm (contribution from acrosome andprincipal piece) during hamster capacitation.
      Figure thumbnail gr9
      Fig. 9Time course of capacitation-dependent tyrosine phosphorylation of E3 inacrosome-dislodged hamster spermatozoa. Sperm samples collected atdifferent time points of capacitation (numbers representing hours ofcapacitation) were processed to dislodge acrosomes as shown by the absence ofsignal from an acrosomal marker P26h in the immunoblot analyses (B).P26h signal is shown in the total sperm samples (Ts) where acrosomeswere not dislodged. Immunoblot results indicate lack of any considerablechange in protein concentration of E3 in the acrosomal dislodged spermatozoa(C), taking into consideration the amount of protein loaded in therespective lanes as shown by Ponceau S staining (A). The tyrosinephosphorylation of E3 (bounded by outline), as observed in animmunoblot with monoclonal anti-phosphotyrosine antibody (4G10), increasestill 4 h of capacitation and further decreases till 7 h (D).
      Figure thumbnail gr10
      Fig. 10Time course of capacitation-dependent tyrosine phosphorylation of E3 inintact hamster spermatozoa. The amount of E3 does not vary duringcapacitation (C) (numbers representing hours ofcapacitation) as shown in the immunoblot of samples with equal amount oftubulin (B). Loading of protein is also shown by Ponceau S staining(A). The tyrosine phosphorylation of E3 (bounded by theoutline), as observed in an immunoblot with monoclonalanti-phosphotyrosine antibody (4G10), increases from 0 till 7 h ofcapacitation (D).
      Regulated Directional Activity of Hamster Sperm DihydrolipoamideDehydrogenase during Hamster Sperm Capacitation—To determine theenzymatic status of the dihydrolipoamide dehydrogenase (E3) of hamsterspermatozoa, we first checked for the three important enzymatic properties ofE3, namely, NADH specificity(
      • Massey V.
      • Veeger C.
      ), heat stability (75°C for 5 min) (
      • Lusty C.J.
      ), andinhibition by its specific inhibitor, 5-methoxyindole-2-carboxylic acid(
      • Van Dop C.
      • Hutson S.M.
      • Lardy H.A.
      ). As it is shown inTable I, hamster sperm E3, likeother E3s, was not functional when NADH was replaced by NADPH (zero activity);heat treatment (75 °C for 5 min) did not inactivate hamster sperm E3 (sameactivity to that of the control); and the enzyme was inhibited by5-methoxyindole-2-carboxylic acid, its specific inhibitor (zero activity).Furthermore, the bi-directional dehydrogenase activity of hamster sperm E3 wasassessed at different time points during capacitation in total sperm lysates.The method used for the preparation of the sperm lysate has been shown toextract all the E3 from the spermatozoa into the soluble fraction leavingnothing in the pellet (
      • Mitra K.
      • Shivaji S.
      ).The forward and reverse activities showed different regulation during thecourse of capacitation. The reverse activity of the enzyme (lipoamide wasreduced to dihydrolipoamide accepting electrons from NADH) showed aprogressive increase in activity during capacitation with a peak at 4 h and adecline thereafter (Fig.11A). On the other hand, the forward activity of hamstersperm E3 (dihydrolipoamide was oxidized to lipoamide and NADH was produced asNAD acted as the electron acceptor) increased as capacitation progressed andmaximum activity was obtained at the end of capacitation at 5–7 h(Fig. 11A). Nosignificant correlation between the forward and the reverse activities of theenzyme was found (rs being 0.22, which is not significantat any level). The progress in forward activity was found to positivelycorrelate well with the progress in the number of acrosome-reacted hamsterspermatozoa (Fig.11B). The correlation coefficient(rs) between the forward activity and the number ofacrosome-reacted spermatozoa was found to be 0.874, and the correlation wassignificant at the level of 0.01. Thus, the data suggest a probable controlfor the directionality of hamster sperm E3 activity during capacitation.

      DISCUSSION

      The male gamete, spermatozoon, is a highly specialized cell of the body.During spermiogenesis, in the testis, a thorough reorganization of cellularproteins (and organelles) takes place in the haploid spermatid, reflectedultimately in the unique morphology of the mature spermatozoon. Phospholipidhydroperoxide glutathione peroxidase (PHGPx) is a cytosolic protein in somaticcells, which is found to be associated with the mitochondria in the maturespermatozoa (
      • Roveri A.
      • Casasco A.
      • Maiorino M.
      • Dalan P.
      • Calligaro A.
      • Ursini F.
      ). On the otherhand, Voltage-dependent anion-selective channels (VDAC2 and VDAC3), which aremitochondrial porins in somatic cells, are found to be associated with thecytoskeletal elements (outer dense fibers) in the bovine sperm flagella(
      • Hinsch K.D.
      • De Pinto V.
      • Aires V.A.
      • Schneider X.
      • Messina A.
      • Hinsch E.
      ). It is obvious that suchdramatic changes in localization probably imply fulfillment of specificfunctions. In this report we demonstrate that dihydrolipoamide dehydrogenase,the E3 subunit along with its host pyruvate dehydrogenase complex (PDHc),exhibits a non-canonical localization in mammalian spermatozoa. The enzyme iscanonically localized in mitochondria in the eukaryotic systems investigatedso far (
      • Patel M.S.
      • Roche T.E.
      )(Fig. 1B). However, incyanobacteria a periplasmic form(
      • Engels A.
      • Kahmann U.
      • Ruppel H.G.
      • Pistorius E.K.
      ) and in soya bean anodular form (
      • Moran J.F.
      • Sun Z.
      • Sarath G.
      • Arredondo-Peter R.
      • James E.K.
      • Becana M.
      • Klucas R.V.
      ) have beenfound. In this report we show that dihydrolipoamide dehydrogenase (E3) showsdual localization in the acrosomal matrix and in the principal piece of theflagella of hamster spermatozoa, as we also observed for, the host complex,PDHc (Fig. 2). This result isvery interesting, because it is known that the glycolytic apparatus islocalized in the principal piece of mammalian spermatozoa(
      • Travis 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.
      ) and adds to the idea ofcompartmentalized metabolic pathways in spermatozoa. Mention may be made ofthe fact that the dissolution of the disulfide-rich sperm mitochondrial sheathrequires a reducing agent(
      • Sutovsky P.
      • Moreno R.D.
      • Ramalho-Santos J.
      • Dominko T.
      • Simerly C.
      • Schatten G.
      ). We were able tocompletely solubilize E3 without a reducing agent(
      • Mitra K.
      • Shivaji S.
      ), which further supportsthe extra mitochondrial localization of E3. Reorganization of proteins duringsperm capacitation is a common phenomenon(
      • Cowan A.E.
      • Primakoff P.
      • Myles D.G.
      ,
      • Bronson R.
      • Peresleni T.
      • Golightly M.
      • Preissner K.
      ,
      • Grace K.S.
      • Bronson R.A.
      • Ghebrehiwet B.
      ),but E3 did not show any change in localization during capacitation(Fig. 1K). Theincrease in the intensity of E3 staining in the principal piece of theflagella of a terminally differentiated cell like spermatozoa (also observedfor host complex PDH, data not shown) is probably due to the increase in theaccessibility of the antigen, because immunoblotting showed the presence ofinvariable levels of E3 in the flagella in all time points of capacitation(Fig. 9C). In ourprevious study we have shown that E3 exhibits dual involvement in thephenomena of hyperactivation (enhanced motility) and acrosome reaction duringhamster sperm capacitation(
      • Mitra K.
      • Shivaji S.
      ). In the light of thesestudies the observed dual localization of E3 and PDHc, in the flagella(principal piece) and acrosome of hamster spermatozoa, becomes veryrelevant.
      Acrosome is surrounded by an outer acrosomal membrane (underlying theplasma membrane), and an inner acrosomal membrane (overlying the nucleus)(
      • Kopf G.F.
      • Gerton G.L.
      ). The acrosomalcompartment can be structurally and biochemically divided into a soluble partand a matrix (
      • Olson G.E.
      • Winfrey V.P.
      • Davenport G.R.
      ). Spermacrosome reaction is an exocytotic process during which the sperm plasmamembrane fuses with the underlying (outer) acrosomal membrane andconcomitantly releases the acrosomal proteins thus exposing the proteins ofthe inner acrosomal membrane to aid the sperm in penetrating the oocyte.Acrosin (
      • Baba T.
      • Kashiwabara S.
      • Watanabe K.
      • Itoh H.
      • Michikawa Y.
      • Kimura K.
      • Takada M.
      • Fukamizu A.
      • Arai Y.
      ) and mouse sp56(
      • Kim K.S.
      • Cha M.C.
      • Gerton G.L.
      ) (and its guinea pighomologue AM67 (
      • Foster J.A.
      • Friday B.B.
      • Maulit M.T.
      • Blobel C.
      • Winfrey V.P.
      • Olson G.E.
      • Kim K.S.
      • Gerton G.L.
      )) are twoimportant candidates of the acrosomal matrix. This report adds hamster spermE3 (56 kDa) to the list (Fig.4), which could also be the 56-kDa protein identified in a studyattempting to isolate the acrosomal matrix proteins from hamster spermatozoa(
      • Olson G.E.
      • Winfrey V.P.
      • Davenport G.R.
      ). Acrosome reaction isbelieved to be a gradual and regulated phenomenon. In mouse(
      • Kim K.S.
      • Gerton G.L.
      ) and guinea pig(
      • Kim K.S.
      • Foster J.A.
      • Gerton G.L.
      ) spermatozoa, it has beendemonstrated that the soluble part of the acrosome is released first followedby differential release of matrix components, during acrosome reaction.Therefore, the release of E3 only by complete acrosome-reacted spermatozoa(A23187-treated, Fig.4A) and not by partial acrosome-reacted spermatozoa(Fig. 4B) can beattributed to its localization in the acrosomal matrix.
      In our previous study we could identify the mitochondrial signal sequenceof E3 from hamster testis cDNA(
      • Mitra K.
      • Shivaji S.
      ), thus localization of E3in the extra mitochondrial (acrosomal) locations is quite intriguing.Mitochondrial proteins (
      • Thompson W.E.
      • Ramalho-Santos J.
      • Sutovsky P.
      ,
      • Goldberg E.
      • Sberna D.
      • Wheat T.E.
      • Urbanski G.J.
      • Margoliash E.
      ,
      • Meinhardt A.
      • Parvinen M.
      • Bacher M.
      • Aumuller G.
      • Hakovirta H.
      • Yagi A.
      • Seitz J.
      ) show a typical granularstaining around the nucleus in the spermatocytes in agreement with theobservation that mitochondria are located very close to the outer nuclearmembrane in these cells (
      • Meinhardt A.
      • Wilhelm B.
      • Seitz J.
      ).On the other hand, the Golgi (the organelle from which the acrosomeoriginates) proteins in a spermatocyte appear to have characteristicjuxtanuclear localization, like in any other cell(
      • Ramalho-Santos J.
      • Schatten G.
      • Moreno R.D.
      ). Only recentlyresearchers have begun to realize the connection between Golgi andmitochondria in spermatogenesis. Knock-out mice of Hrb (or Rab)(
      • Kang-Decker N.
      • Mantchev G.T.
      • Juneja S.C.
      • McNiven M.A.
      • van Deursen J.M.
      ) and Golgi-associated PDZ-and coiled-coil motif-containing protein(
      • Yao R.
      • Ito C.
      • Natsume Y.
      • Sugitani Y.
      • Yamanaka H.
      • Kuretake S.
      • Yanagida K.
      • Sato A.
      • Toshimori K.
      • Noda T.
      ), both being Golgiproteins, have been independently shown to have dramatic defects in themitochondrial sheath formation. Our immunohistochemistry results furtherindicate that E3 shows a mitochondrial type staining in the spermatocytes,whereas in the early spermatid it appears to collect near the point from whereacrosome begins to develop from the Golgi(
      • Ramalho-Santos J.
      • Schatten G.
      • Moreno R.D.
      )(Fig. 6); in no other stages inthe testicular sections did E3 appear to be a Golgi protein (data not shown).Thus, our data along with others (as mentioned), hint toward the existence ofa “protein transport machinery” between Golgi and mitochondriaduring the conversion of tetraploid pachytene spermatocytes to haploidspermatozoa.
      During sperm capacitation an array of proteins have been found to betyrosine-phosphorylated (
      • Visconti P.E.
      • Westbrook V.A.
      • Chertihin O.
      • Demarco I.
      • Sleight S.
      • Diekman A.B.
      ).Researchers have proved the involvement of tyrosine phosphorylation events inthe capacitation-associated events hyperactivation(
      • Uma Devi K.
      • Jha K.
      • Patil S.B.
      • Padma P.
      • Shivaji S.
      ,
      • Bajpai M.
      • Asin S.
      • Doncel G.F.
      ) and acrosome reaction(
      • Tomes C.N.
      • Roggero C.M.
      • De Blas G.
      • Saling P.M.
      • Mayorga L.S.
      ), but the presence ofcapacitation-dependent tyrosine-phosphorylated proteins have been mostlydetected in the flagella of spermatozoa and almost none in the acrosome(
      • Urner F.
      • Sakkas D.
      ). We previously reportedE3 as a tyrosine-phosphorylated protein(
      • Mitra K.
      • Shivaji S.
      ), and the duallocalization of E3 necessitated investigation of tyrosine phosphorylation ofE3 in both the locations. We could detect the tyrosine-phosphorylated form ofthis bi-directional enzyme, in the (principal piece of the) flagella ofhamster spermatozoa by immunofluorescence (Figs.7 and8) and further confirmed it byimmunoblot analyses (Fig. 9).Furthermore, our biochemical studies on the time course of E3 tyrosinephosphorylation during capacitation, indirectly suggest the presence of atyrosine-phosphorylated form of E3 also in the acrosome of hamster sperm(compare Figs. 9D and10D); kinetics ofcapacitation-dependent E3 phosphorylation being different in immunoblotanalyses of acrosome intact and acrosome-dislodged spermatozoa. The failure todetect E3 phosphorylation in the acrosome by the immunofluorescence studiescould be due to antigen masking in this tightly packed organelle. It is,however, not clear if the site of the apparent tyrosine phosphorylation inboth the locations are similar, but the presence of nine tyrosines in thesequence of E3 (
      • Mitra K.
      • Shivaji S.
      ) probablyqualifies it to be a protein undergoing hyper-phosphorylation. The differentkinetics of tyrosine phosphorylation of E3 in the acrosome-dislodged sperm(contribution from the principal piece) and that of the acrosome-intact sperm(contribution from acrosome and principal piece) during hamster capacitationthus justifies independent involvement of the bi-directional enzyme E3 inhyperactivation and acrosome reaction, as we have reported before(
      • Mitra K.
      • Shivaji S.
      ).
      Our results, for the first time, show that the forward and the reverseactivities of E3 in hamster spermatozoa are differentially regulated duringcapacitation. The regulation of the reverse activity of the enzyme correlatesto a considerable extent to that of the tyrosine phosphorylation kinetics ofthe E3 in the principal piece (Figs.9D and11A). Furthermore,the strong correlation of the forward activity with the acrosome reaction canalso be extended to the apparent contribution of the tyrosine phosphorylationfrom the acrosomal E3 in the terminal stages of capacitation (5.5–7 h)when acrosome reaction is maximum (Figs.10D and11B). Thus, it ispossible that the tyrosine-phosphorylated E3 in the principal piece of thesperm flagella could be driving more of the reverse reaction, whereas that inthe acrosome could be driving more of its forward reaction. Unlike othersomatic cells, the pyruvate-lactate metabolism and its link to ATP productionin the male gamete is still in the dark for the following reasons:(a) the presence of a mitochondrial lactate dehydrogenase inspermatozoa (
      • Blanco A.
      ); (b)pyruvate metabolism in (bovine) spermatozoa is not linked to oxidativephosphorylation through mitochondrial electron transport chain(
      • Van Dop C.
      • Hutson S.M.
      • Lardy H.A.
      ); and (c)knock-out mice of cytochrome c are fertile, ruling outindispensability of electron transport chain in sperm functioning(
      • Narisawa S.
      • Hecht N.B.
      • Goldberg E.
      • Boatright K.M.
      • Reed J.C.
      • Millan J.L.
      ), whereas knock-out of theglycolytic enzyme GAPDH is defective in motility despite having no deficiencyin oxygen consumption (
      • Miki K.
      • Qu W.
      • Goulding E.H.
      • Willis W.D.
      • Bunch D.O.
      • Strader L.F.
      • Perreault S.D.
      • Eddy E.M.
      • O'Brien D.A.
      ).Thus the role of the pyruvate metabolic enzymes like E3 (in PDHc) and othersin sperm capacitation still remains to be explored.
      This is the first report of the extra mitochondrial localization ofdihydrolipoamide dehydrogenase (E3) along with its host complex, pyruvatedehydrogenase complex (PDHc), in the acrosome and principal piece of the spermflagella. It is important to note here, that most of the earlier studies onmitochondrial activity, including pyruvate metabolism studies, in spermatozoahave used total sperm permeabilized in various ways(
      • Calvin J.
      • Tubbs P.K.
      ,
      • Morton B.E.
      • Lardy H.A.
      ,
      • Morton B.E.
      • Lardy H.A.
      ),and thus the extra mitochondrial contribution might have been overlooked. Themitochondrial type rather than a Golgi type localization of dihydrolipoamidedehydrogenase in the spermatogenic cells suggests a link between the twoorganelles that could deliver the mitochondrial enzyme to the Golgi formingthe acrosome during spermatogenesis. Furthermore, the results presented inthis report bring out clearly the dual involvement of dihydrolipoamidedehydrogenase on the basis of the novel dual localization, dual tyrosinephosphorylation kinetics, and dual regulation of the bi-directional enzymeactivity in sperm capacitation.

      Acknowledgments

      We thank Dr. Jyotsna Dhawan, Dr. Archana B. Siva, and T. Subhash for theirinvaluable help in the project. We sincerely thank Dr. R. A. Harris for a giftof anti PDHc antibody, Dr. R. Sullivan for a gift of anti P26h, and Prof.Kühn for a gift of anti PHGPx antibody used in this report.

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