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BOR - Papers in Press, published online ahead of print July 19, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.053587
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BIOLOGY OF REPRODUCTION 75, 767–777 (2006)
DOI: 10.1095/biolreprod.106.053587
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Activity of Pyruvate Dehydrogenase A (PDHA) in Hamster Spermatozoa Correlates Positively with Hyperactivation and Is Associated with Sperm Capacitation1

Vivek Kumar , Nandini Rangaraj , and Sisinthy Shivaji 2

Centre for Cellular and Molecular Biology, Hyderabad 500 007, India

ABSTRACT

Unravelling the molecular basis of capacitation is crucial to our understanding the basis of acquisition of fertilization competence by spermatozoa. In two recent studies, we have demonstrated that dihydrolipoamide dehydrogenase, which is a post-pyruvate metabolic enzyme and one of the components of pyruvate dehydrogenase complex, undergoes capacitation-dependent tyrosine phosphorylation, and that the activity of the enzyme correlates with capacitation events in the hamster spermatozoa. However, it is not clear as to whether other components of the pyruvate dehydrogenase complex are also crucial for sperm capacitation. In this report, we have identified pyruvate dehydrogenase A2 (PDHA2), a constituent of pyruvate dehydrogenase A (PDHA), which is a component of pyruvate dehydrogenase complex that exhibits tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing that hamster sperm PDHA2 is a testis-specific phosphotyrosine that is associated with the fibrous sheath of hamster spermatozoa. The localization of PDHA2 in spermatozoa was investigated using antibodies to PDHA, which is the active tetrameric protein that consists of a homodimer of PDHA2 and PDHB. Both immunofluorescence and confocal studies indicated a unique non-canonical, extramitochondrial localization for PDHA in the principal piece of hamster spermatozoa. It was also observed that PDHA colocalized with AKAP4 in the fibrous sheath of the spermatozoon. The enzymatic activity of PDHA was positively correlated with hyperactivation but not with the acrosome reaction. Given the localization of PDHA and the evidence that its activity correlates positively with hyperactivation and that its PDHA2 subunit exhibits capacitation-associated protein tyrosine phosphorylation, it appears that PDHA2 is associated with the process of capacitation.

acrosome reaction, confocal microscopy, hamster sperm, hyperactivation, immunofluorescence, PDHA, phosphorylation, protein tyrosine, sperm, sperm capacitation

INTRODUCTION

Although freshly ejaculated mammalian spermatozoa are not sufficiently competent to fertilize the oocyte, they achieve competence after a finite period of residence in the female reproductive tract. This time-dependent process, during which mammalian spermatozoa become fertilization competent, is termed capacitation [1–4]. During capacitation, spermatozoa undergo two distinct physiological changes: hyperactivation, during which spermatozoa gain the momentum to proceed towards the oocyte, and the acrosome reaction, which facilitates penetration of the oocyte. Concomitant with these changes, activation of a signal transduction cascade that leads to increased tyrosine phosphorylation of proteins is observed in mammalian spermatozoa [5–8]. Some of these capacitation-induced tyrosine phosphorylated proteins have been identified, and they include the 95-kDa fibrous sheath protein (AKAP3) [9], the calcium-binding tyrosine phosphorylation regulator protein (CABYR) [10], the E1ß subunit of pyruvate dehydrogenase (PDHB) [11], and the A Kinase Anchoring Proteins (AKAPs) in human spermatozoa [12], as well as AKAP4 [13], dihydrolipoamide dehydrogenase (DLD) [14, 15], and phospholipid hydroperoxide glutathione peroxidase (GPX4) [16] in hamster spermatozoa, and the 90-kDa heat shock protein (HSP90AA1) [17] in mouse spermatozoa. Despite the identification of these tyrosine phosphorylated proteins and attempts to understand their roles in capacitation, the molecular basis for capacitation is still poorly understood [7].

A simple straightforward method to unravel the molecular basis of capacitation is to identify components that are essential for capacitation in vitro, and then work around these components so as to understand the process. It is in this context that it is important to note that the carbon sources glucose, pyruvate, and lactate are essential for in vitro capacitation of mammalian spermatozoa [14, 18]. Of these carbon sources, glucose has been shown to be of primary importance for capacitation because of its ability to generate energy through the glycolytic pathway [19, 20], and due to its requirement for capacitation-associated protein tyrosine phosphorylation [20–22]. In hamster spermatozoa, it has been shown that pyruvate-lactate supports the acrosome reaction (more so than glucose) [18]. Recently, we have provided in vitro evidence for the essential roles of glucose in the early stages and of pyruvate-lactate in the later stages of capacitation in hamster spermatozoa [14]. Indeed, the DLD component of the pyruvate dehydrogenase complex, which metabolizes pyruvate directly, was identified as the post-pyruvate-lactate metabolic enzyme involved in hamster sperm capacitation [14]. In a more recent study, we have established a direct correlation between localization, tyrosine phosphorylation, and the activity of DLD during hamster sperm capacitation [15]. However, since DLD is not the only protein that undergoes capacitation-associated protein tyrosine phosphorylation in hamster spermatozoa [13, 14], identification of the other proteins and characterization of their functions would further unravel the molecular basis for sperm capacitation. It is also logical to assume that, in addition to DLD, other components that constitute the pyruvate dehydrogenase complex (PDH) may also be involved in sperm capacitation. Pyruvate dehydrogenase complex (PDH), which converts pyruvate to acetyl CoA during aerobic oxidation of glucose, consists of seven subunits: PDHA, PDHB, dihydrolipoyl acetyltransferase (DLAT), DLD, PDHX (E3-binding protein), a pyruvate dehydrogenase-specific kinase, and a pyruvate dehydrogenase-specific phosphatase [23–25].

In the present study, we demonstrate, to the best of our knowledge for the first time, that pyruvate dehydrogenase A2 (PDHA2), a testis-specific isozyme, undergoes time-dependent tyrosine phosphorylation during capacitation in hamster spermatozoa. Mammalian pyruvate dehydrogenase is present both as a somatic form (PDHA1) and as a testis-specific form (PDHA2) [26]. The gene for PDHA1 (Pdha1) is located on p22.1 of the human X chromosome, contains 10 introns, and spans approximately 17 kb [26]. The gene for PDHA2 is located on chromosome 4 (Pdha2), completely lacks introns, and possesses the characteristics of a functional processed gene [27]. PDHA2 is a homodimer and makes a functionally active heterotetramer PDHA together with another homodimer, PDHB, which is reported to be tyrosine phosphorylated in human spermatozoa during capacitation (11). PDHA (2{alpha} + 2ß) has two active sites and requires thiamine pyrophosphate (TPP) and Mg++ as cofactors for its enzymatic activity. PDHA catalyzes the decarboxylation of pyruvate, thereby forming a tightly bound enzyme intermediate and liberating carbon dioxide. Thus, although much is known about PDHA1, few studies have been conducted on the function and role of the testis-specific PDHA2. PDHA2 is considered to be one of the most important subunits of PDH complex, since perturbations in the activity of this subunit lead to reduced or total loss of function of the entire complex, resulting in a loss of pyruvate metabolism and ATP production. PDHA2 perturbations are known to cause neurological abnormalities and lactic acidosis [23].

In this study, PDHA2 was identified as a tyrosine-phosphorylated protein from the proteome of capacitated spermatozoa of the Golden Hamster (Mesocricetus auratus). N-terminal sequencing of the protein and the translated cDNA sequence indicated that the protein was the mammalian testis-specific PDHA2. This paper reveals non-canonical extramitochondrial localization of PDHA in the principal piece of hamster spermatozoa. Furthermore, the kinetics of phosphorylation of PDHA2 and the activity of PDHA correlate with the capacitation status of hamster spermatozoa.

MATERIALS AND METHODS

Preparation of Spermatozoal Suspension

Cauda epididymal spermatozoa were collected from the distal epididymis of 6-month-old Golden Hamsters (Mesocricetus auratus), following the procedure described earlier [14, 15]. Spermatozoa from the cauda epididymides were collected directly into TALP, a medium that supports the capacitation of hamster spermatozoa [28]. The motile swim-up spermatozoa were then collected and counted in a Makler chamber using HTM-CEROS (Hamilton Thorne, Beverly, MA) Computer Assisted Semen Analyzer (CASA), as described earlier [14]. Aliquots of the sperm suspension were in vitro capacitated by incubating the suspension in TALP medium in a CO2 incubator set at 37°C and flushed with 5% CO2 in air. TALP, which is modified Tyrode medium [28], was prepared in MilliQ water that contained 114 mM NaCl, 3.16 mM KCl, 2 mM CaCl2.2H2O, 0.35 mM MgCl2.6H2O, 25 mM NaHCO3, 12.5 mM sodium lactate, and 5 mM glucose. The pH of the medium was adjusted to 7.6 and the osmolality to 280–300 mmol/kg. Prior to use, the medium was supplemented with PHE, which is a cocktail of motility stimulators (2 mM penicillamine, 10 mM hypotaurine, 100 µm epinephrine), 0.18 mM pyruvate, and 3 mg/ml BSA. This cocktail was developed by Dow and Bavister (1989) and is demonstrated to sustain the motility of hamster spermatozoa during capacitation and in vitro fertilization. Spermatozoa collected at 0 h were designated as non-capacitated, and those that were collected at 5 h were designated as capacitated spermatozoa. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Ethics Committee of the Centre for Cellular and Molecular Biology, Hyderabad, India.

Extraction of Sperm Proteins for One- and Two-Dimensional Gel Electrophoresis

Spermatozoal suspensions were collected and centrifuged twice at 9500 rpm for 5 min at 4°C in 1 ml TBS (20 mM Tris [pH 7.4], 137 mM NaCl), with 1 mM sodium orthovanadate as a tyrosine phosphatase inhibitor. For one-dimensional SDS-PAGE analysis, 5 x 106 spermatozoa were boiled at 100°C for 10 min in Laemmli's buffer [29]. Solubilized samples were then centrifuged at 14000 rpm for 15 minutes and then resolved on a 10% gel. For two-dimensional PAGE (2D-PAGE) analysis [30] 50 x 106 spermatozoa were lysed by incubating the sperm suspension in urea lysis buffer (9.5 M urea, 2% NP-40, 1.6% ampholytes [pH 5–8], 0.4% ampholytes [pH 3–10], and 5% ß-mercapto-ethanol) with 1 mM sodium orthovanadate at 4°C for 1 h. Subsequently, the suspension was centrifuged at 14000 rpm for 15 min, and the proteins were resolved in the first phase by isoelectric focusing using gel strips that contained 9.5 M urea, 4% acrylamide, 2% NP-40, 1.6% ampholyte (pH 5–8), 0.4% ampholyte (pH 3–10) and 5% ß-mercapto-ethanol. The strips were run for 3 h at 400 V, using 0.025 M phosphoric acid as the anode buffer and 0.05 M NaOH as the cathode buffer. The gel strips were then removed, and equilibrated in equilibration buffer (60 mM Tris-Cl, [pH 6.8], 2.3% SDS, 5% ß-mercapto-ethanol, 10% glycerol) prior to second dimension electrophoresis in a 10% polyacrylamide gel. The proteins were stained with Coomassie brilliant blue R250.

Immunoblotting

For immunoblotting, sperm proteins that were separated by SDS-PAGE or 2D-PAGE were electrotransferred onto a nitrocellulose membrane at 0.8 mA/cm2 for 1 h. Subsequently, the membrane was stained with 0.1% Ponceau S, to check for equal loading of the proteins. The membranes were then blocked with 5% (w/v) non-fat milk in TBST for 1 h at room temperature, washed, and incubated with the primary antibody prepared in TBST (0.01% Tween [v/v], 1% BSA [w/v)]). The antibody dilutions used were 1:10 000 for the monoclonal antiphosphotyrosine antibody (clone 4G10; Promega, Madison, WI), 1:10 000 for the monoclonal antiphosphoserine antibody (Sigma Chemical Co., St. Louis, MO), and 1:1000 for the monoclonal anti-PDHA2 antibody (clone 9H9AF5; Molecular Probes, Eugene, OR). After the incubation period, the membranes were washed (four times, 5 min each with TBST), and incubated in 1% BSA-TBST that contained the appropriate secondary antibody for 1 h at room temperature. The secondary antibody used was either conjugated to horseradish peroxidase (Sigma) or to alkaline phosphatase (Sigma) and was used at a concentration of 1:10 000. The blots were developed using the ECL kit (Amersham Biosciences, Piscataway, NJ). The immunoblots shown are representative blots from three independent experiments using materials from different animals.

N-Terminal Sequencing

Hamster spermatozoal proteins were resolved by 2-D PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (wet with methanol), stained with 0.1% Ponceau S, and visualized. A protein of 41 kDa and pI 8.4 was excised, washed in 100 % methanol for 10 min, and subsequently washed in 50% methanol for 1 h to remove glycine. The membrane piece was then air-dried and subjected to N-terminal sequencing using an automated protein sequencer (Applied Biosystems).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from hamster testis, brain, liver, spleen, kidney, heart, muscle, ovary, and oviduct using the Tri-reagent according to the manufacturers protocol (Sigma). Reverse transcription was performed with 50 U of Expand reverse transcriptase (Roche, Basel, Switzerland) using oligo(dT) as the primer. PCR was performed with the following Pdha2 primers: for FPE1, forward 5'-CGTTTCTCCATGAGGAAAATGCTG-3' and reverse, RPL 5'-TCATACTGTGTCCATGATAAC-3'. These primers were designed based on conserved regions in the Pdha2 of mouse (P35487), rat (AAH78757), and human (P29803). Thermal cycling was carried out in a thermal cycler (PTC-200 DNA apparatus; MJ Research) using the following PCR conditions: one cycle at 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. RT-PCR of glyceraldehyde phosphate dehydrogenase (Gapdh) was carried out as a positive control for cDNA amplification from all tissues. The primers used for this purpose were as follows: GF forward, 5'-TGAAGGTCGGTGTGAACGGATTTG-3' and GR reverse, 5'-TGATGGCATGGACTGTGGTCATGA-3' [31]. The PCR conditions comprised one cycle at 94°C for 5 min, followed by 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min. The Gapdh primers yielded a product of 534 bp.

Sequencing of Hamster Testis ?>Pdha2?> cDNA

Hamster testis cDNA was used as a template to amplify the hamster testis Pdha2. The primers FPE1 forward (5'-CGTTTCTCCATGAGGAAAATGCTG-3') and RPL reverse (5'TCATACTGTGTCCATGATAAC-3') amplified an 850-bp fragment, whereas primers RP4 forward (5'-CGGCTAACCAAGGGCAGGTATT-3') and RP2 reverse (5'-TTGGTGGTAGAGGTAATTGGCTA-3') amplified a 500-bp overlapping fragment of the hamster testis Pdha2. The PCR products were separated on 1% agarose gels, and the bands were eluted from the gel with the Qiagen Gel Elution Kit (Qiagen, Hilden, Germany) and sequenced in an automated sequencer (Applied Biosystems) using the above four primers and two other nested primers (NE3, 5'-GTTCTCCAACGACGCCACCTGTGA-3' and FP1, 5'-GTTATCATGGACACAGTATGA-3').

Solubilization of PDHA2 from Hamster Spermatozoa

Attempts were made to solubilize the PDHA2 protein of hamster spermatozoa. In this protocol, spermatozoa (10 x 106 cells) were extracted with 5 mM DTT and 0.05% Triton X-100 in TNI (150 mM NaCl, 25 mM Tris-HCl [pH 7.5] 2.5 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate) for 1 h at 4°C, to solubilize the fibrous sheath proteins of the hamster spermatozoal [32]. Subsequently, the sperm suspension was centrifuged at 14 000 rpm for 10 min at 4°C and the detergent-soluble supernatant and the detergent-insoluble pellet were collected, boiled at 100°C for 10 min in Laemmli buffer, and subjected to immunoblot analysis using the monoclonal anti-PDHA2 antibody (clone 9H9AF5).

Localization of PDHA in Hamster Spermatozoa by Indirect Immunofluorescence

These studies were carried out essentially as we described previously [15]. In brief, spermatozoa were allowed to undergo capacitation in TALP medium, as described above, and at the required time-points they were collected, centrifuged at 1000 rpm for 5 minutes at room temperature, and fixed for 10 min in 2% formaldehyde that was prepared freshly in TBS. The fixed sperm suspensions were then coated onto clean glass coverslips and air-dried (37°C). The cells on the coverslips were then permeabilized by dipping in ice-cold (–20°C) methanol for 20 s, and were subsequently blocked with 5% BSA in TBS, followed by incubations with the primary and appropriate secondary antibody in TBS that contained 1% BSA. All of the incubations were interspersed with 3–4 washes with TBS. After immunostaining, the coverslips were mounted on clean glass slides using antifade (Vector Laboratories, Burlingame, CA) as the mounting medium, and viewed under an Axioplan2 epifluorescence microscope (Carl Zeiss Inc., Jena, Germany).

Colocalization of PDHA in Hamster Spermatozoa by Confocal Microscopy

Colocalization of PDHA and AKAP4 in hamster spermatozoa was performed with specific antibodies to PDHA and AKAP4. Anti-AKAP4 was generated in mice, as described previously [13]. Polyclonal anti-PDHA antibody was a gift from Prof J.G. Lindsay, University of Glasgow, UK. The antibody was raised in rabbit against bovine PDHA. For the colocalization studies, sperm processed as described above were initially treated with anti-PDHA antibody for 2 h, washed with TBS for 3 to 5 min, and then treated with Cy3-labeled anti-rabbit IgG for 1 h. The coverslip was washed, and then exposed to anti-AKAP4 antibody for 2 h, washed, and treated with FITC-conjugated anti-mouse IgG. Subsequently, the coverslip was transferred to a slide and visualized as described above. The procedure followed for the colocalization of PDHA and phosphotyrosine was similar to the steps described above, except that antiphosphotyrosine monoclonal antibody was used in place of the anti-AKAP4 antibody.

Colocalization studies of PDHA with AKAP4 and with phosphotyrosine in hamster spermatozoa were carried out using the LSM510 Meta laser scanning confocal microscope (Carl Zeiss). The dyes used for dual staining were FITC and Cy3, which were excited at 488 nm and 543 nm, respectively. Optical sections (0.2-µm thickness) of the sperm samples were obtained during the scanning and two to five of the innermost sections were projected for each sample [15].

Preparation of the Hamster Fibrous Sheath and Outer Dense Fiber

The hamster fibrous sheaths (FS) and outer dense fibers (ODF) of hamster spermatozoa were prepared essentially according to the procedure described previously [33, 34]. In this procedure, demembranated hamster spermatozoa were used as the starting material for the preparation of FS and ODF. Hamster spermatozoa were demembranated by treating with 1% Triton X-100 for 1–2 minutes at room temperature. Subsequently, the suspension was washed twice with TBS and centrifuged at 2000 x g for 5 min, and the pellet containing the demembranated hamster spermatozoa was recovered. This pellet was then treated with 0.1% Triton X-100 and 2 mM dithiothreitol (DTT) in 50 mM TBS (pH 9) for 3–5 min, to remove the mitochondrial sheaths (MS). The MS-free spermatozoa were then treated with 6 M urea in 50 mM TBS and 2 mM DTT at pH 7.6 for 3 h, to separate the FS from the demembranated sperm pellet. Subsequently, the extract was mixed with equal volume of 50 mM TBS buffer containing 1.8 M sucrose and 1 % Triton X-100 and centrifuged at 9000 x g for 15 min. FS, which appeared as an opalescent band, was recovered [33], washed, and centrifuged at 10 000 x g for 15 min at 4°C. The MS-free spermatozoal pellet was again treated with 0.03% SDS for 3 h, to dissolve selectively the FS and axoneme (32) and to collect the ODF pellet by centrifugation at 5000 x g for 10 min. Protease inhibitors (1 mM PMSF and 10 µg/ml leupeptin) were included in all the steps for the preparation of FS and ODF. Appropriate membrane markers, which included anti-AKAP4 antibody for FS and anti-ODF2 antibody for ODF2, were used to confirm the purity of each preparation.

Assessment of Hyperactivation

Hyperactivation of hamster spermatozoa was assessed using the CASA according to the criteria described previously [8, 13, 14, 35]. Non-hyperactivated spermatozoa exhibited planar motility, while the hyperactivated spermatozoa exhibited a circular or helical type of motility. The results are expressed as the percentage of spermatozoa that were hyperactivated. Three or four fields were recorded and analyzed for each time-point, and the number of spermatozoa analyzed varied from a minimum of 100 spermatozoa to a maximum of about 150 spermatozoa. The percentages of motile spermatozoa were also ascertained using the CASA system.

Assessment of Acrosome Reaction

Spermatozoa were stained with eosin Y (0.25% in medium), and only live spermatozoa were scored for spontaneous acrosome-reacted spermatozoa using a phase-contrast microscope (Leitz Messetechnik, Wetzlar, Germany) with a 40x objective, as described previously [14, 15]. A minimum of 100 spermatozoa was scored for each time-point, and the results are expressed as the percentages of acrosome-reacted spermatozoa.

PDHA Assay

The activity of PDHA in hamster spermatozoa capacitated in TALP was assayed using the methods described previously [36, 37]. [1-14C] Pyruvate was used as the substrate and the formation of the 14CO2, end-product of the reaction was quantified. In this experiment, aliquots of spermatozoa at specific time-points of capacitation (0–7 h) were removed, rapidly cooled to 4°C, and pelleted at 4000 rpm for 5 min. The spermatozoal pellet was then lysed at 4°C in TBS buffer that contained 0.5% Triton X-100, 1 mM PMSF, and 1 mg/ml aprotinin. Finally, the spermatozoal lysate was suspended in TBS that contained 1 mM thiamine pyrophosphate, 2 mM sodium arsenite, 2 mM MgCl2, 0.1 mM dichlorophenolindophenol (DCPIP), and 0.05 µCi [1-14C] pyruvate, transferred to an airtight conical flask and incubated for 2 h at 37°C in a water bath. The evolved 14CO2 was then trapped onto a filter paper soaked in 2 N NaOH, which was also suspended in an airtight flask. The filter papers with the entrapped 14CO2 at the end of the reaction were dried and counted in a scintillation counter. Reactions carried out in the absence of the spermatozoal lysate served as the blank.

Statistical Methods

The Student t-test was used to determine significant differences in enzymatic activity during capacitation. Spearmans correlation coefficient (rS) (SPSS, version 11.0.1) was used to determine the significance of the correlation between PDHA activity and hyperactivation and acrosome reaction. The level of significance for the differences in PDHA activity was determined by the Student t-test (P < 0.05).

RESULTS

Identification of a Basic 41-kDa Tyrosine-Phosphorylated Protein in Capacitated Hamster Spermatozoa

The present study confirms our earlier observations [13, 14] that hamster spermatozoa that are incubated in a medium that is conducive for capacitation for 5 h at 37°C in a CO2 incubator exhibit time-dependent and capacitation-associated increases in the tyrosine phosphorylation of proteins in the range of 35–95 kDa and pI 4–9 (Fig. 1, A–D), as judged by immunoblot analysis with monoclonal antiphosphotyrosine antibody (4G10). However, when the antibody to phosphotyrosine was pre-incubated with 10 mM O-phospho-L-tyrosine before use, the antibody did not cross-react with any of the proteins on the immunoblot (data not shown), thus confirming the specificity of the antibody for phosphotyrosine. In non-capacitated spermatozoa, phosphorylation of proteins at tyrosine residues was not detected (Fig. 1C). The prominent tyrosine-phosphorylated proteins identified to date in capacitated hamster spermatozoa include hamster pro-AKAP4 (97 kDa), AKAP4 (previously known as AKAP83; 83 kDa), the DLD component of pyruvate dehydrogenase (56 kDa) [13, 14, 15], and GPX4 [16]. In the present study, we identified a basic tyrosine-phosphorylated protein of molecular mass 41 kDa and pI8.4 that was present only in capacitated hamster spermatozoa (Fig. 1D). BLAST analysis [38] of the 15 N-terminal residues of this protein (41 kDa and pI 8.4) (Fig. 1E) showed that the candidate protein was PDHA2 (EC 1.2.4.1), which is a constituent of PDHA (Fig. 1E). The first residue of the candidate protein aligned with the 31st residue of PDHA2 (EC 1.2.4.1), which indicates that the 41-kDa tyrosine-phosphorylated protein is PDHA2, the mature testis-specific form of pyruvate dehydrogenase in hamster spermatozoa [39].


Figure 1
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  		FIG. 1. A–D). 2D-PAGE and immunoblot analyses of the total proteins of non-capacitated (A and C) and capacitated (B and D) hamster spermatozoa. In A and B, the gels were stained with Coomassie brilliant blue R250, whereas in C and D, the gels were probed with monoclonal antiphosphotyrosine antibody. The molecular mass standards and pI values are indicated. Note that PDHA2, which has a molecular mass of 41 kDa, separates into three different protein spots with pI values of 8.2, 8.4, and 8.6. E) Comparison of the N-terminal sequence of the 41-kDa and pI-8.4 protein of capacitated hamster spermatozoa with the PDHA2 protein of Rattus norvegicus. F) Immunoblot analysis of the total proteins of capacitated hamster spermatozoa with monoclonal the anti-PDHA2 antibody. G) Immunoblot analysis of PDHA2 of hamster spermatozoa at different time-points (0–7 h) during capacitation using the anti-PDHA2 antibody. The anti-{alpha} tubulin antibody was used as a control for equal loading of protein. H) Solubilization experiments indicate that PDHA2 of hamster spermatozoa is associated with the detergent-soluble supernatant (S) fraction and not the detergent insoluble pellet (P) fraction, following immunoblot analysis with the anti-PDHA2 antibody. Solubilization of hamster spermatozoa by 0.05% Triton-X 100, followed by immunoblot analysis with the anti-PDHA antibody indicates that both PDHA2 (41 kDa) and PDHB (36 kDa) are associated with the pellet fraction (TP) and not with the soluble fraction (TS)

Furthermore, using the monoclonal anti-PDHA2 antibody (clone 9H9AF5; Molecular Probes) it was further confirmed that the candidate protein is indeed PDHA2 (Fig. 1F). In addition to this protein, two other proteins of identical molecular masses but with pIs of 8.2 and 8.6 were also found to cross-react with the monoclonal anti-PDHA2 antibody (Fig. 1F). Thus, it appears that PDHA2 exists as three different spots in the blots of hamster spermatozoa (Fig. 1F). The levels of these three proteins were unchanged during the process of capacitation (Fig. 1G). The same blot was stripped and reprobed with the anti-{alpha} tubulin antibody, to confirm equal loading. Densitometric evaluation of the protein levels of PDHA2 did not show significant differences during the process of capacitation (data not shown). The reason why we studied the levels of PDHA2 during capacitation was to demonstrate that the levels of the protein were unaltered during capacitation but that the phosphorylation levels changed (compare Fig. 1, C, D, and G).

Immunoblot analysis of the detergent-soluble (supernatant) and detergent-insoluble (pellet) fractions of hamster spermatozoa using monoclonal anti-PDHA2 antibody clearly indicated a single protein band of 41 kDa, which was present only in the soluble fraction of hamster spermatozoa treated with 5 mM DTT and 0.05% Triton X-100. This indicates that PDHA2 is associated with the detergent soluble fraction of sperm (Fig. 1H). PDHA2 was not detected in the detergent-insoluble fraction (Fig. 1H). Treatment with Triton X-100 alone failed to solubilize PDHA2 (Fig. 1H), and this protein was detected only in the pellet fraction.

The activity of PDH complex in eukaryotic cells is known to be regulated by the phosphorylation and dephosphorylation of serine residues on PDHA2 [40]. 2D-PAGE immunoblot analysis using monoclonal antiphosphoserine antibody (clone PTR8) revealed that PDHA2 was phosphorylated at the serine residue both in the non-capacitated and capacitated spermatozoa, and densitometric evaluation indicated that the intensity of the phosphorylation of PDHA2 remained unchanged during capacitation (data not shown).

Testis-Specific Expression of the Hamster ?>Pdha2?> Gene

The primer set FPE1 and RPL specifically amplified a fragment of 850 bp when cDNA from hamster testis was used as the template. No amplification was observed when cDNAs from somatic tissues, such as the liver, brain, kidney, spleen, heart, muscle, ovary, and oviduct (Fig. 2A), were used. After sequencing and BLAST analysis, the amplified product was found to be 76%, 84% and 85% similar to the testis-specific forms of Pdha2 in human, rat, and mouse, respectively. Gapdh primers were used as a positive control to amplify the cDNA of Gapdh from all the above tissues (Fig. 2B).


Figure 2
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  		FIG. 2. Tissue-specific expression of Pdha2.A) Tissue-specific expression of Pdha2 analyzed by RT-PCR using total RNA from the testis (T), liver (L), brain (B), kidney (K), heart (H), muscle (M), spleen (S), ovary (OV), and oviduct (OI) of hamster using the testis-specific primers FPE1 and RPL. B) Amplification of Gapdh as a positive control for the amplification of cDNA from all the above tissues. In both A and B, the 100-bp DNA ladder is used as a size marker in the first lane

Sequencing of Testis-Specific Isozyme from Hamster Sperm ?>Pdha2?> cDNA

The primer sets FPE1-RPL and RP4-RP2 amplified two overlapping fragments of Pdha2 cDNA of 850 bp and 500 bp, respectively, when hamster testis cDNA was used as the template. These fragments were sequenced using the above four primers and two other nested primers (NE3 and FP1) and the sequences were aligned, to yield a cDNA sequence of 1.12 kb for Pdha2 (EMBL accession no. AM279660). In silico translation of the cDNA sequence yielded the sequence of the hamster PDHA2 protein, which matched with the testis-specific form of rodent PDHA2 and showed >80% similarity with the rat (AAH 78757.1; 82%) and mouse (P35487; 81%) testis-specific forms of PDHA2, respectively. The obtained sequence did not cover the 16 amino acid residues at the C-terminus [41]. The in silico-translated sequence included functionally important domains, such as the thiamine pyrophosphate (TPP)-binding domain (GDGAAQGQVAEAYNLSALWKLPCVF), the catalytic domain (TYRYHGHSMSDPGISYRTREEVQSMRS), and interestingly, the mitochondrial signal sequence (MRKMLASLVSHVFSGVNQKPAMRGLLASLH). The TPP-binding domain and the catalytic domain have been shown to be conserved in the PDHA2 proteins of humans, yeasts, and bacteria [41]. The testis-specific form of the hamster PDHA2 differed from the somatic form of PDHA2 in the rat (CAA78146.1) and mouse (AAH07142.1) to the same extent (23%), although for both forms of the TPP-binding domain, the catalytic domain and signal sequence were conserved. The testis and somatic isoforms of PDHA2 in the mouse also differed by 11% [27].

Validation of Rabbit Polyclonal Antibody against Bovine Pyruvate Dehydrogenase (PDHA)

The commercially procured monoclonal anti-PDHA2 antibody, which successfully recognized PDHA2 as a 41-kDa protein in capacitated hamster spermatozoa by 2D-PAGE immunoblot analysis (Fig. 1G), was not suitable for localizing PDHA2 in hamster spermatozoa in immunofluorescence studies. Various dilutions of the antibody (up to 1:1) did not yield any positive signals, which implies that this monoclonal antibody is unable to recognize the epitope associated with both intact and demembranated spermatozoa (data not shown). It is known that a homodimer of PDHA2 and a homodimer of PDHB together form the functionally active PDHA. Therefore, by localizing PDHA, one could indirectly infer the localization of both PDHA2 and PDHB, provided that both these subunits are present. In fact, when the polyclonal anti-PDHA antibody was used, it was observed that the anti-PDHA antibody cross-reacted with two proteins of molecular mass 36 kDa and 41 kDa (Fig. 3A). The 41-kDa protein appeared as three distinct spots and was earlier identified as PDHA2 by N-terminal sequencing (Fig. 1G). The 36-kDa protein was identified as PDHB based on its molecular mass (36 kDa) and pI (4.5–5.5), and also by MALDI analysis, as determined previously [42] (Fig. 3A). The designation of the 41-kDa and 36-kDa proteins as PDHA2 and PDHB, respectively, was also confirmed using the antipyruvate dehydrogenase complex antibody (a generous gift from Dr. R.A. Harris, Indiana University School of Medicine) which was raised against whole pyruvate dehydrogenase complex (PDHc) without the DLD component, and which is known to cross-react with DLAT, PDHA2, and PDHB (Fig. 3B). Thus, these results validate the specificity of the polyclonal anti-PDHA antibody for PDHA2 and PDHB. This polyclonal anti-PDHA antibody was used in all the immunoblotting and immunofluorescence experiments to detect PDHA2 and PDHB.


Figure 3
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  		FIG. 3. Immunoblot analysis of hamster sperm proteins using the polyclonal anti-PDHA antibody and antipyruvate dehydrogenase complex (PDHc) antibody. Hamster sperm proteins were subjected to 2D-PAGE and Western blot analysis with the polyclonal anti-PDHA antibody. All three protein spots for PDHA2 (41 kDa) and PDHB (36 kDa) are apparent (A). The anti-PDHc antibody detects DLAT (72 kDa), PDHA2, and PDHB (B). The molecular masses and isoelectric points (pI) are indicated

Extramitochondrial Localization of PDHA in Mammalian Spermatozoa

Localization of PDHA in hamster caudal spermatozoa was studied using the anti-PDHA antibody. Intense staining was observed exclusively in the principal piece (PP; Cy3: red) (Fig. 4A). No staining was observed in the head (H), midpiece (MP) or other regions of the tail (Fig. 4, A and B). PDHA is a mitochondrial enzyme, and therefore, it was anticipated that PDHA would be present in the midpiece (mitochondrial compartment) rather than in the principal piece. The absence of staining in the midpiece could be due to the inability of the antibody to penetrate the mitochondria. To rule out this possibility, immunofluorescence staining was performed following permeabilization of spermatozoa with Triton X-100, which is known to expose mitochondrial proteins. In these permeabilized spermatozoa, staining was observed only in the principal piece (Fig. 4, B and C). Furthermore, when monoclonal antibody to sperm mitochondrial protein phospholipid glutathione peroxidase (GPX4) (donated by Prof. Kuhn, Humboldt University Medical School Charite, Germany) was used as a marker for sperm mitochondria (midpiece), as anticipated, localization was observed only in the midpiece (MP) of the spermatozoa (FITC: green) (Fig. 4D). Immunofluorescence studies using the secondary antibody alone did not yield any staining of the spermatozoa (Fig. 4E).


Figure 4
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  		FIG. 4. A–F). Immunofluorescent localization of PDHA and the sperm mitochondrial marker GPX4 in hamster spermatozoa using the anti-PDHA antibody and anti-GPX4 antibody, respectively. PDHA is present only in the principal piece (PP) of the spermatozoa of both non-capacitated (A and C) and capacitated (F) spermatozoa. The intensity of staining remains unaltered during the process of capacitation (F). Staining is not observed in the endpiece (EP), midpiece (MP) or the head (H) region (blue due to DAPI staining). Triton-X 100-treated sperm also show PDHA in the principal piece (C). GPX4 localizes to the midpiece of the spermatozoa (D). B) Phase-contrast micrograph of A. Intact acrosomes are visible in three of the spermatozoa. Spermatozoa treated with only the secondary antibody do not show any localization to the spermatozoa (E). G–I) Immunofluorescent localization of PDHA in cat and human spermatozoa using the anti-PDHA antibody. In the cat spermatozoa, PDHA localizes to the posterior region of the head (H) and the principal piece (PP) of the tail (G), whereas in the human spermatozoa, PDHA is present only in the posterior region of the head (H) (H). Bar = 20 µm. Immunoblot analysis was also performed on cat and human spermatozoal proteins using the anti-PDHA antibody, which detects PDHA2 (41 kDa) and PDHB (36 kDa) (I)

In addition, to investigate the fate of PDHA during hamster sperm capacitation, immunofluorescence studies were performed with hamster spermatozoa samples prepared at different time-points during capacitation. Staining was observed only in the principal piece, and the intensity of staining remained unaltered in this region of the spermatozoa both in the non-capacitated (0–1.5 h) and capacitated spermatozoa (3–7 h) (Fig. 4F).

The generality of the non-canonical extramitochondrial localization of PDHA observed in hamster spermatozoa was also investigated in ejaculated human and cat spermatozoa using the anti-PDHA antibody. In both cat and human spermatozoa, staining was not observed in the midpiece. The staining pattern of cat spermatozoa differed from that of hamster spermatozoa, in that PDHA appeared to be present in the principal piece and in the posterior region of the head (Fig. 4G). In human spermatozoa, staining was seen only in the posterior region of the head (Fig. 4H). Immunoblot analysis of cat and human spermatozoal proteins confirmed that the PDHA antibody cross-reacted with both PDHA2 (41 kDa) and PDHB (36 kDa) (Fig. 4F).

Localization of PDHA in Hamster Spermatozoa by Confocal Microscopy

The localization of PDHA in the fibrous sheaths (FS) of the flagella of hamster spermatozoa was performed by confocal microscopy using the polyclonal anti-PDHA antibody and the anti-AKAP4 antibody of hamster, which we have previously demonstrated to be localized in the FS of hamster spermatozoa. Hamster spermatozoa showed localization of both AKAP4 and PDHA in the principal piece, and merging of the images indicated that AKAP4 and PDHA were colocalized (Fig. 5A), which implies that PDHA is present in the FS of hamster spermatozoa. Reverse staining using the anti-AKAP4 antibody first and then the anti-PDHA antibody also revealed strong colocalization of PDHA and AKAP4 in the FS of hamster spermatozoa (data not shown). In the confocal studies, optical sections (0.2-µm thickness) of the spermatozoa were obtained and the innermost sections in which the localization was observed were merged to ascertain colocalization [15].


Figure 5
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  		FIG. 5. A). Localization of PDHA in the fibrous sheath of hamster spermatozoa by confocal microscopy. Confocal microscopy shows colocalization of PDHA (Cy3: red) with the FS marker AKAP4 (FITC: green) in the principal piece of hamster caudal spermatozoa. B) Immunoblot analyses using the anti-AKAP4 antibody (panel 1), anti-ODF2 antibody (panel 2), and anti-PDHA antibody (panel 3) confirm that PDHA2 and PDHB are present in the FS but not in the ODF. ODF markers are not detected in the FS but are present in the ODF (panel 2). The FS marker AKAP4 is detected only in the FS fraction (panel 1). C) Colocalization of PDHA and phosphotyrosine proteins in the principal piece of capacitated hamster spermatozoa by confocal microscopy. The spermatozoon was first labeled with the antiphosphotyrosine antibody (green) followed by the anti-PDHA antibody (red)

Subcellular Localization of PDHA in Hamster Spermatozoa

Hamster spermatozoa were fractionated into heads, tails, FS, and ODF and subjected to immunoblot analysis using the anti-PDHA antibody. The head did not yield any immunoreactive band in Western blots (data not shown), whereas in the tail, two bands with molecular masses of 41 kDa and 36 kDa, which corresponded to PDHA2 and PDHB, respectively, were associated with the FS and not the ODF. Using the anti-AKAP4 antibody as a marker for FS and the anti-outer dense fiber 2 (anti-ODF2) antibody as a marker for ODF (gift from Dr Sigrid Hoyer-Fender, Georg August Universitat, Gottingen, Germany), it was demonstrated that the FS and ODF cross-reacted with the respective antibodies (Fig. 5B). The anti-AKAP4 antibody cross-reacted with AKAP4 (83-kDa protein in FS) [13] and the anti-ODF2 antibody cross-reacted with two proteins with molecular masses of 45 kDa and 70–80 kDa, which are characteristic of the ODF2 protein [43] (Fig. 5B). These results confirm the association of PDHA with the FS of hamster spermatozoa.

Tyrosine Phosphorylation of PDHA in the Principal Piece of Hamster Spermatozoa

Colocalization of PDHA and tyrosine phosphorylation in capacitated hamster spermatozoa was monitored by confocal microscopy using the anti-PDHA antibody and antiphosphotyrosine antibody (Fig. 5C). The results indicate co-localization of PDHA and tyrosine phosphorylation in the sperm tail (Fig. 5C) but not in the head region (data not shown).

Pyruvate Dehydrogenase Activity during Hamster Spermatozoal Capacitation

The activity of PDHA in hamster spermatozoa capacitated in TALP was assayed using an established method [36, 37] (for details see Materials and Methods). PDHA was enzymatically active both in non-capacitated and capacitated spermatozoa (Fig. 6A). This activity increased progressively with capacitation and attained a peak at between 3 and 5 h, and declined marginally thereafter. The activity at 3 h was significantly different to the activities at 0 h and 7 h (P < 0.05). Furthermore, the activity peak at 3 h coincided with maximum number of spermatozoa hyperactivated at this time-point. Correlation studies indicated a positive correlation between enzyme activity and the percentage of hyperactivated spermatozoa (Fig. 6A), with a correlation coefficient (rS) of 0.863 and significance at the level of 0.01. However, when the activity of PDHA was compared with the number of spermatozoa undergoing the acrosome reaction, the correlation coefficient (rs) was 0.274 and this was not significant at the level of 0.01 (Fig. 6A). The increase in PDHA activity also matched the temporal sequence of increased tyrosine phosphorylation of PDHA2 during capacitation (compare Fig. 6, A and B). The motility of the spermatozoa during the course of the experiment was around 80%.


Figure 6
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  		FIG. 6. A). Correlation of PDHA activity with hamster sperm hyperactivation and acrosome reaction. The PDHA activity (black square) and the percentage hyperactivated spermatozoa (white square) are positively correlated (Spearmans correlation coefficient = 0.863) and are significant at P < 0.01. A correlation coefficient of 0.274 exists for the relationship between PDHA activity and percentage acrosome reaction (white circle). Asterisks indicate significant differences (P < 0.05, as determined by the Student t-test) in PDHA activity at 3 h compared to the PDHA activities at 0 h and 7 h. The percentage motility during the period of incubation was around 80%. The experiments were performed three times with three different animals. B) Capacitation-dependent increase in tyrosine phosphorylation of PDHA2 in hamster spermatozoa. The histogram represents the intensity of PDHA2 tyrosine phosphorylation determined with a densitometer

DISCUSSION

Mammalian sperm capacitation, which is a process that confers fertilization competence upon spermatozoa, was discovered about half a century ago [4, 5, 7]. However, the molecular basis of capacitation is still not completely understood [8, 13, 44–46]. In this study, we demonstrate that PDHA2, a testis-specific form that metabolizes pyruvate directly, is tyrosine-phosphorylated in capacitated hamster spermatozoa. The molecular weight of hamster sperm PDHA2 is 41 kDa and it exists as three different tyrosine-phosphorylated proteins with identical molecular masses but with pI values of 8 to 8.6. These protein spots cross-reacted with the PDHA2 monoclonal antibody. PDHA2 in maize also exists as multiple protein spots [47]. PDHA2 is not associated with the detergent-soluble fraction of hamster spermatozoa. The cDNA-derived amino acid sequence of hamster sperm PDHA2 exhibits >80% similarity with the testis-specific PDHA2 proteins of rat and mouse. Furthermore, the TPP-binding motif and the catalytic site are conserved as in all the PDHA2 sequences. The tissue specificity was confirmed using a set of primers specific for the testis form (Fig. 2). In all, 18 tyrosine residues are present in PDHA2. Fourteen of the tyrosine residues are conserved in mouse, rat, hamster, and human, and some of these conserved residues are located at sites that are important for the activity of the enzyme, such as the residue at position 195 adjacent to the TPP-binding domain and three residues within the proposed catalytic domain at positions 292, 294, and 306. Furthermore, the tyrosine residues at positions 157, 247, and 277 are predicted to be potential sites for tyrosine phosphorylation (NetPhos 2.0 Server) [48]. Two of these residues, at positions 157 and 247, are conserved in mouse, rat, hamster, and human. Fujinoki et al. [42] have observed that PDHB is phosphorylated at the serine residue and is associated with the regulation of motility activation in hamster spermatozoa. In human spermatozoa, PDHB has been identified as being tyrosine-phosphorylated during capacitation using the MALDI approach [11]. The present study is the first report on tyrosine phosphorylation of PDHA2 in a spermatozoon.

Studies in hamster spermatozoa have revealed the extramitochondrial localization of enzymes, such as the ATP synthase F1ß subunit (ATP5B) in the principal piece [49], DLD in the principal piece and head [15], and PDHB in the flagella, of hamster spermatozoa [42]. Similarly, the voltage-dependent anion channels VDAC2 and VDAC3, which are mitochondrial porins in somatic cells, have been found to be associated with the ODF in bovine sperm flagella [50], and phospholipid glutathione peroxidase (GPX4), which is another cytosolic protein in somatic cells, has also been found to be associated with mitochondria in mature rat spermatozoa [51]. In this study, we demonstrate that PDHA exhibits extramitochondrial non-canonical localization in the principal piece of hamster spermatozoa (Fig. 4A), in the principal piece and in the posterior region of the head of cat spermatozoa (Fig. 4G), and in the posterior region of the head of human spermatozoa (Fig. 4H), in contrast to its canonical, mitochondrial localization in somatic cells. Re-organization of proteins during sperm capacitation is an important phenomenon [52], although PDHA does not show any change in localization during the time course of capacitation (Fig. 4F). It is noteworthy that since the homology between the testis and somatic isoforms of PDHA is high, the polyclonal anti-PDHA antibody used in this study is likely to recognize both isoforms.

Spermatozoal fibrous sheaths are known to act as a scaffold for protein kinase A (PKA)- anchoring proteins and glycolytic enzymes [53–56]. In hamster spermatozoa, PDHA2 is associated with FS (Fig. 5B) and can be solubilized using 0.05% Triton-X 100 and DTT (Fig. 1H). Confocal studies also confirmed that PDHA in hamster spermatozoa is associated with FS (Fig. 5A), based on colocalization with AKAP4, a marker of FS and PDHA (Fig. 5A). This result is interesting, since it is already known that the glycolytic apparatus is localized in the principal piece of mammalian spermatozoa, and lends support to the idea of compartmentalized metabolic pathways in spermatozoa [53]. The association of PDHA with FS highlights the importance of PDHA in providing byproducts, such as NADH [57, 58], which has been implicated in the facilitation of pyruvate metabolism in the mitochondria of pancreatic islets [59]. The presence of the mitochondrial localizing signal sequence in PDHA is intriguing (data not shown), considering that the enzyme is localized at an extramitochondrial site, the principal piece of the spermatozoon.

A capacitation-associated increase in protein tyrosine phosphorylation has been observed in spermatozoa of a number of mammalian species [60]. As yet, it is not clear whether phosphorylation is a cause or effect of capacitation. In eukaryotes, the phosphorylation-dephosphorylation cycle of PDHA2 is a strong regulator of PDH activity [61, 62], and the entire pyruvate dehydrogenase complex is enzymatically inactive when phosphorylated [39, 63]. In contrast, the hamster spermatozoal PDHA2 does not exhibit any capacitation-dependent variation in serine phosphorylation (data not shown), although it is possible that the particular serine residue that is phosphorylated is not the same in non-capacitated and capacitated spermatozoa. In hamster spermatozoa, the activity of PDHA (Fig. 6A) follows the temporal sequence of increase in protein tyrosine phosphorylation of PDHA2 during capacitation, which peaks around the third to the fifth hour of capacitation and persists until the seventh hour [13] (Fig. 6B). It is also interesting to note that the PDHA activity profile and the temporal increase in the percentage of hyperactivated spermatozoa are positively correlated with a correlation coefficient (rs) of 0.863, which implies that PDHA activity and hyperactivation are positively correlated. If this contention is correct, it should be possible to demonstrate that inhibition of PDHA inhibits hyperactivation. We also observed that PDHA activity is not correlated with the acrosome reaction.

Our data on the phosphorylation, localization, and enzymatic activity of PDHA in mammalian spermatozoa clearly indicate the involvement of PDHA in sperm capacitation. A question arises as to the role of PDHA in sperm capacitation. There is definitive evidence that the pyruvate dehydrogenase complex is crucial for the generation of reactive oxygen species (ROS) (unpublished results and [64]), which have been implicated in capacitation [65, 66]. Furthermore, an increase in the activity of pyruvate dehydrogenase complex may lead to increased utilization of pyruvate-lactate, which in turn leads to the generation of more NADH. The NADH thus generated can be used by NADH oxidase to generate the ROS required for capacitation. NADH oxidase activity and ROS generation have also been linked to the motility of human spermatozoa [67]. Studies are in progress to identify the role of extramitochondrially localized PDHA in sperm capacitation and to understand the contribution of pyruvate to the overall energy metabolism in mammalian spermatozoa.

ACKNOWLEDGMENTS

We thank Dr. Archana B. Siva, Dr. Kasturi Mitra, Dr. Paneer Doss, Dr. G Umapathy, and Dr. Sadanand Sontakke for their valuable suggestions and technical help. We thank Dr. G. Lindsay for the anti-PDHA antibody, Dr. R. Harris for the anti-PDHc antibody, Dr. Kuhn for the anti-GPX4 antibody, and Dr. Sigrid Hoyer Fender for the anti-ODF2 antibodies.

FOOTNOTES

2 Correspondence: Sisinthy Shivaji, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. FAX: 00 91 40 27160591; shivas{at}ccmb.res.in Back

1 Supported by a UGC fellowship from the Government of India (to V.K.). Back

Received: 4 May 2006.

First decision: 30 May 2006.

Accepted: 17 July 2006.

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V. Kumar, V. Kota, and S. Shivaji
Hamster Sperm Capacitation: Role of Pyruvate Dehydrogenase A and Dihydrolipoamide Dehydrogenase
Biol Reprod, August 1, 2008; 79(2): 190 - 199.
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