Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M500310200 on May 11, 2005

J. Biol. Chem., Vol. 280, Issue 27, 25743-25753, July 8, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/27/25743    most recent
M500310200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mitra, K.
Right arrow Articles by Shivaji, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mitra, K.
Right arrow Articles by Shivaji, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenase in Spermatozoa

CORRELATION OF ITS LOCALIZATION, TYROSINE PHOSPHORYLATION, AND ACTIVITY DURING SPERM CAPACITATION*

Kasturi Mitra{ddagger}, Nandini Rangaraj, and S. Shivaji§

From the Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

Received for publication, January 10, 2005 , and in revised form, April 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatozoa are cells distinctly different from other somatic cells of the body, capacitation being one of the unique phenomena manifested by this gamete. We have shown earlier that dihydrolipoamide dehydrogenase, a post-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosine phosphorylation, and the functioning of the enzyme is required for hyperactivation (enhanced motility) and acrosome reaction of hamster spermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70, 887–899). In this report we have investigated the localization of this mitochondrial enzyme in spermatozoa revealing non-canonical extra-mitochondrial localization of the enzyme in mammalian spermatozoa. In hamster spermatozoa, dihydrolipoamide dehydrogenase along with its host complex, the pyruvate dehydrogenase complex, are localized in the acrosome and in the principal piece of the sperm flagella. The localization of dihydrolipoamide dehydrogenase, however, appears to be in the mitochondria in the spermatocytes, but in spermatids it appears to show a juxtanuclear localization (like Golgi). The capacitation-dependent time course of tyrosine phosphorylation of dihydrolipoamide dehydrogenase appears to be different in the principal piece of the flagella and the acrosome in hamster spermatozoa. Activity assays of this bi-directional enzyme suggest a strong correlation between the tyrosine phosphorylation and the bi-directional enzyme activity. This is the first report of a direct correlation of the localization, tyrosine phosphorylation, and activity of the important metabolic enzyme, dihydrolipoamide dehydrogenase, implicating dual involvement and regulation of the enzyme during sperm capacitation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatozoa, the haploid cells, are unique compared with other cells with respect to their morphology and functionality; needless to say the underlying signaling mechanisms in a functional spermatozoon are also unique. The metabolic pathways in a mature spermatozoon are compartmentalized (1–3), which probably enables them to survive in two different kinds of milieu, the male and the female reproductive tract. The residence time in the female reproductive tract is an obligatory event in the life cycle of a spermatozoon and has been termed "capacitation" (4, 5). During capacitation spermatozoa undergo multifaceted changes in aspects like metabolism, intracellular ion concentrations, plasma membrane fluidity, and thus membrane reorganization, intracellular pH, intracellular cAMP concentration, and generation of reactive oxygen species (6–8). These cellular alterations during capacitation bring about three physiological changes: (a) hyperactivation (enhanced motility), (b) tyrosine phosphorylation in an array of proteins, and (c) acrosome reaction (release of the acrosomal contents); the first two events show a temporal correlation with capacitation, whereas acrosome reaction is taken to be the end point of capacitation (9).

Protein tyrosine phosphorylation is considered to be a hallmark of sperm capacitation (10–15), but only a few of these proteins have been identified, and the functional significance of the respective tyrosine phosphorylation has been ascertained in 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 in a greater detail in this regard (11, 16). Recently, tyrosine phosphorylation of AKAP3 has been shown to result in the recruitment of protein kinase A to the sperm flagella causing an increase in motility (17). Furthermore, the essentiality of a balance between protein-tyrosine kinase and phosphatase activities has also been demonstrated as a mandatory requirement for a successful acrosome reaction (18).

Regulation of metabolic enzymes by phosphorylation has been well established in different cells (19–21). However, the metabolism of spermatozoa is quite distinctly different from other cells and thus less understood, with spermatozoa having specific isoforms of key metabolic enzymes (22–24). Because there is little or no cytoplasm in spermatozoa, most of the cytoplasmic enzymes exhibit non-canonical localization in two domains of the sperm, namely, the head and the flagellum. The head hosts the nucleus and the acrosome; the mid-piece of the flagellum hosts all the mitochondria and the tail piece, which is further divided into a principal piece and an end piece, and harbors cytoskeletal elements. The two ideal examples of non-canonical localization of metabolic enzymes in spermatozoa are the sperm-specific isoforms of hexokinase (localized in the mid piece and principal piece of the flagella and in the sperm head (25)) and lactate dehydrogenase (localized in the mitochondrial matrix (26)). The non-canonical localizations of metabolic enzymes indicate there is an extra mitochondrial energy production center; the ATP generated after glycolysis has been shown to be the source of tyrosine phosphorylation during capacitation of mouse spermatozoa (3). In our previous report we have established the importance of a post pyruvate metabolic enzyme, dihydrolipoamide dehydrogenase, in hamster sperm hyperactivation and acrosome reaction (27). We also identified the enzyme to be a target of the capacitation-dependent protein tyrosine phosphorylation cascade.

Dihydrolipoamide dehydrogenase, a flavoprotein disulfide oxidoreductase, is the E31 component of {alpha}-ketoacid dehydrogenase multienzyme complexes (28). It is, canonically, a mitochondrial enzyme, the exact localization being in the mitochondrial matrix (29). Dihydrolipoamide acetyltransferase, another component of the same multienzyme complexes, is the physiological substrate of E3. The enzyme, E3, is active as a dimer, and the main catalytic actions of E3 are dehydrogenase (bidirectional, Reaction 1), diaphorase (Reaction 2), and oxidase (Reaction 3) (30) as follows.

(REACTION 1)

(REACTION 2)

(REACTION 3)

Some recent studies show various other functions of this redox active enzyme, E3 both in vitro (31–33) and in vivo (34, 35). E3 knock-out mice die early in development, and the heterozygotes show half of the enzyme activity of that of the normal (36).

Pyruvate dehydrogenase complex (PDHc) is a paradigmatic example of {alpha}-ketoacid dehydrogenase complexes hosting E3. Bacterial E3 (37) and pig heart E3 (38) have been shown to be regulated by alteration of the NAD:NADH ratio. However, a phosphorylation-dephosphorylation cycle of the {alpha} subunit of the E1 component of PDHc seems to be a stronger regulator in eukaryotes (39, 40). No direct regulation of E3 has been observed as far as pyruvate metabolism is concerned.

This paper reveals non-canonical extra mitochondrial localization of dihydrolipoamide dehydrogenase (E3) in mammalian spermatozoa; in hamster spermatozoa the enzyme shows dual localization in the acrosome and in the principal piece of sperm flagella. The data further demonstrate a strong positive correlation between the tyrosine phosphorylation status of the enzyme and its bi-directional enzymatic activity in the two locations in the hamster spermatozoa during capacitation, indicating a dual control of the metabolic enzyme during the cellular event.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Spermatozoa Collection and in Vitro Capacitation—Male Golden hamsters (Mesocricetus auratus) aged 6 months were used as experimental animals. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Ethics Committee of the Centre for Cellular and Molecular Biology. Spermatozoa were collected from the caudal epididymides into TALP (modified Tyrode's medium, a medium known to support capacitation of hamster spermatozoa) (41) by swim-up technique (27) and thereafter counted in a Makler chamber using a computer-assisted semen analyzer (HTM-CEROS, Hamilton Thorne, Maryland, MD). Aliquots from the swim-up were used for different studies. Hamster spermatozoa maintained in TALP medium in 5% CO2 at 37 °C attained capacitation within 3–5 h (42). For the time-course experiments spermatozoa maintained in TALP medium were harvested at the time points mentioned in the study.

Detection of Phosphorylation and Protein Levels—5 x 106 spermatozoa were used to study tyrosine phosphorylation by SDS-PAGE immunoblot analysis (27) with monoclonal anti-phosphotyrosine ({alpha}PY) antibody (Upstate) as follows: (a) blocking with 5% nonfat milk, (b) incubation with 1:10,000 dilution of primary antibody ({alpha}PY) in 1% BSA in TBS-T (TBS containing 0.1% Tween 20), and (c) incubation with 1:10,000 dilution of secondary antibody conjugated with horseradish peroxidase in 1% BSA in TBS-T. These steps were interspersed with washes in TBS-T. The blots were then developed using the Enhanced Chemiluminescence kit (Amersham Biosciences). Immunoblotting of the two-dimensional-PAGE was also performed as described above while the two-dimensional-PAGE was run according to the method of O'Farrell (44).

Acrosome Reaction—A minimum of 100 spermatozoa were scored for each time point using a phase contrast microscope (Leitz, Germany) with a 40x objective (42). The samples were stained with eosin Y (0.25% in TALP medium) and scored for spontaneous acrosome-reacted spermatozoa. The spermatozoa undergoing or having undergone acrosome reaction were counted as positive. The results were expressed as a percentage of acrosome-reacted spermatozoa.

Generation of Antibody—Antibody against E3 was raised in rabbit by injecting 200 µg of pig heart E3 as the antigen (Sigma). All injections were given subcutaneously, the first three being in Freund's complete adjuvant and the later ones (one or two) in Freund's incomplete adjuvant.

Indirect Immunofluorescence and Confocal Studies—Spermatozoa maintained in TALP medium were collected while carefully ignoring the pellet of dead cells at the bottom of the microcentrifuge tubes. Spermatozoa were washed (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 coated properly on clean glass coverslips and air dried (37 °C). Cells on the coverslips were freshly permeabilized by dipping in ice-cold (-20 °C) methanol (20 s) and were blocked (5% BSA in TBS) followed by incubations with primary and appropriate secondary antibody (made in 1% BSA in TBS). All the incubation steps were interspersed with 3–4 washes in TBS. MitoTracker CmxRos (Molecular Probes, Eugene, OR) staining of MCF-7 cells grown on coverslips was performed using 200 nM dye for 45 min in serum-free medium. Coverslips were washed with serum-free medium after which the cells were fixed with 3.5% formaldehyde (in the same medium) for 10 min. After immunostaining coverslips were processed for antibody staining as before and mounted on clean glass slides using Antifade (Vector) as the mounting medium and 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 isothiocyanate and Cy3 (or MitoTracker dye) for the dual staining, which were excited at 488 nm and 543 nm laser lines, respectively. Optical sections (0.2 µm each) of the sperm samples were obtained during the scanning, and for each sample two to five innermost sections were projected.

Dissolution of Acrosomal Matrix—The acrosomal matrices were dislodged from the spermatozoa following an established protocol (45) with little modifications. The spermatozoa were harvested at different stages of capacitation and placed on ice. The sperm pellets were then washed twice with cold TBS (500 x g for 5 min), resuspended in HEPES buffer (10 mM HEPES and 140 mM NaCl, pH 7.2) containing 0.1% Triton X-100 and 0.25 mM phenylmethylsulfonyl fluoride and once again kept on ice for 1 h. After vortexing the spermatozoa were then subjected to homogenization in a Dounce homogenizer. They were further kept on ice for 30 min after which spermatozoa were centrifuged (350 x g for 5 min) and washed again in TBS. SDS-PAGE extracts were prepared as mentioned before. Protein estimation was done with the extracts according to the method of Karlsson et al. (46).

Acrosomal matrices were partially dislodged from the sperm head by the method used for guinea pig spermatozoa (47). Briefly, hamster spermatozoa were collected from the cauda epididymides in a buffer having 20 mM sodium acetate (pH 5.2) and 0.15 M NaCl (along with 0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin) and were washed at 500 x 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 20 times. Although this procedure did not dislodge all the acrosomal matrices completely, it loosened them and spermatozoa could be identified at different stages of acrosomal matrix loss.

Immunohistochemistry—Testes, dissected out from the animals and rinsed in TBS, were fixed overnight in Bouin's fixative (70% water-saturated picric acid, 20% formaldehyde, and 5% acetic acid) after which the tissue was washed in 70% alcohol and dehydrated in a graded series of alcohol. Molds were prepared with paraffin wax, and 5-µm-thick sections were collected on slides coated with 0.5% gelatin. Sections were deparaffinized in xylene (20 min), hydrated through graded alcohol incubations, and finally transferred to distilled water. Deparaffinized hydrated sections were stained with PAS-hematoxylin, and staging of the seminiferous tubules was done according to Miething (48). Parallel sections were processed 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 blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate in the presence of 0.1 mM levamisole to inhibit any endogenous alkaline phosphatase activity. Sections were rinsed in distilled water and mounted in 30% glycerol and viewed under the bright field of Axioplan 2 microscope (Carl Zeiss Inc.).

Enzyme Assay—The detergent-soluble sperm lysates were prepared, with sperm pellets harvested at different time points during capacitation, according to the method of Patel et al. (28). In brief, a pellet of 100 million spermatozoa was suspended in 200 µl of hypotonic phosphate buffer with Triton X-100 (1%), protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin), and sodium orthovanadate (1 mM) and kept on ice for 1 h. The suspension of cells was then subjected to three to four freeze-thaw cycles 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 x 106 spermatozoa). The enzyme assay was performed in a 200-µl volume according to Gazaryan et al. (30) with some modifications. The substrates for the assay were dihydrolipoamide (8 mM) and lipoamide (4 mM) for the forward and reverse reactions, respectively. NAD (0.32 mM) and NADH (0.16 mM) were used as cofactors for forward and reverse reactions, respectively. The activity of the enzyme was measured as a change in optical density (A340) in a UV-visible spectrophotometer (Shimadzu); the unit of activity was expressed as (micromoles of NADH/min)/µg of total protein in Table I. However, in the experiment where E3 activity has been compared at different time points of capacitation, the enzyme activity was expressed as micromoles of NADH/min and further normalized by a factor of 10-3 as in Fig. 11. This is because of the fact that a lot of acrosomal proteins are lost during capacitation, which would overestimate the activity at the time points when the percentage of acrosome reacted spermatozoa is high.


View this table:
[in this window]
[in a new window]
  		TABLE I
Reverse activity of hamster spermatozoal E3 under different conditions The "reverse activity" is the activity of E3 to reduce lipoamide to dihydrolipoamide accepting electrons from NADH. Activity of E3 is expressed in units representing change in micromoles of NADH/min/µg of sperm lysate. Similar results were obtained for forward activity (data not shown). The values represent E3 activity of approximately 2.5 x 106 spermatozoa.

 
Statistical Analysis—The correlation between acrosome reaction and forward activity of hamster sperm E3 was done using Spearman's correlation coefficient (rs) using SPSS, version 11.0.1; significance of correlation between the parameters was also checked using the same software. For the sake of comparison the values of forward activity of E3 were normalized with the percentage viability of spermatozoa at the respective time point, because during capacitation a reduction in viability was observed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Validation of the Rabbit Polyclonal Antibody Raised against Purified Pig Heart Dihydrolipoamide Dehydrogenase—The polyclonal antibody raised against purified pig heart dihydrolipoamide dehydrogenase (E3) was validated by two-dimensional-PAGE immunoblots and immunofluorescence methods. The two-dimensional-PAGE immunoblot of hamster sperm lysate with the polyclonal anti-E3 antibody detected a single spot (Fig. 1A, left panel), which was previously identified as E3 by N-terminal sequencing (27). Because E3 is a part of the pyruvate dehydrogenase complex (PDHc), a polyclonal antibody raised against the whole PDHc except E3 (kindly donated by Dr. R. A. Harris) was used to detect the other components of the complex in immunoblot of sperm extracts, as shown in the right panel of Fig. 1A.

E3 along with its host complex, PDHc, are canonically mitochondrial proteins. Therefore, it is expected that both E3 and PDHc would colocalize with the MitoTracker used as a mitochondrial marker. Thus, dual staining was performed in a mammalian cell line, MCF-7, using the polyclonal anti-E3/anti-PDHc antibody and the MitoTracker dye. The upper two panels of Fig. 1B show staining of the polyclonal PDHc antibody (PDHc) and that of the polyclonal anti-E3 antibody (E3), respectively. The corresponding images in Fig. 1A show the MitoTracker staining, which is very similar to the corresponding antibody staining. The corresponding panels of panel C show the merged image of the MitoTracker and the PDHc/E3 staining, demonstrating clear colocalization of the two as expected. Furthermore, preabsorption of the polyclonal E3 antibody with purified E3 protein (100 µg/100 µl) failed to detect any antigen in the cells (panel B, Preabs), and the preimmune serum also showed no staining (panel B, Preimm). The corresponding panels in panels A and C show the MitoTracker staining and the merged images, respectively. Our polyclonal anti-E3 antibody also could detect the E3 in immunoblots of extracts of MCF-7 cell lines (data not shown). Thus the results taken together validate the specificity of the polyclonal anti-E3 antibody, which is thus used for further experiments to detect dihydrolipoamide dehydrogenase (E3) both in immunoblots and immunofluorescence methods.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 1.
Validation of the polyclonal anti-E3 antibody. Two-dimensional-PAGE performed with this polyclonal antibody detected only a single expected spot (A, left panel); increasing molecular weight (MW) and isoelectric point (PI) are directed by arrows. The other components of the PDHc were detected in SDS-PAGE immunoblot using polyclonal anti-PDHc antibody; the identity of the bands are indicated on the right (A, right panel). Colocalization of PDHc and E3 with the MitoTracker dye in MCF-7 cell line (B). MCF-7 cells were doubly stained with MitoTracker (panel A) and polyclonal anti-PDHc antibody (panel B, PDHc)/anti-E3 antibody (panel B, E3)/anti-E3 antibody preabsorbed with purified E3 (panel B, Preabs)/rabbit preimmune serum (panel B, Preimm). Corresponding colocalized images are shown in panel C. The bar represents 10 µm.

 
Extra Mitochondrial Localization of Dihydrolipoamide Dehydrogenase in Mammalian Epididymal Spermatozoa—The localization of dihydrolipoamide dehydrogenase (E3) has been investigated in spermatozoa from the hamster caput and cauda (non-capacitated) epididymides by indirect immunofluorescence using polyclonal E3 antibody. Fig. 2 (A and B) shows acrosomal staining of E3 in hamster spermatozoa from the cauda and caput region of the epididymides, respectively. Faint staining was also observed in the principal piece of the flagella in hamster spermatozoa from the cauda region (Fig. 2A). Polyclonal anti-E3 antibody preabsorbed with the purified protein (100 µg/100 µl) could not detect any distinct staining in hamster spermatozoa thus proving the authenticity of the localization (Fig. 2C). Monoclonal antibody to the sperm mitochondrial protein Phospholipid hydroperoxide glutathione peroxidase (PHGPx) (kindly donated by Prof. Kühn) was used as a marker for sperm mitochondria, and as anticipated, it localized to the mid piece of spermatozoa (Fig. 2D). However the antibody also showed some staining in the head of hamster spermatozoa, which is in all probability because of the nuclear seleno-protein detected by this antibody as shown previously in rat spermatozoa (49, 50). Furthermore, even when Triton X-100 was used to expose the mitochondrial proteins (51), E3 antibody still failed to convincingly stain the mitochondria in the mid piece, although little scattered staining was observed in this part (Fig. 2E). It was also observed that detergent treatment obliterated acrosomal staining and the staining in the principal piece increased. The polyclonal PDHc antibody also stained the acrosome and the principal piece of the flagella of spermatozoa taken from cauda epididymides (Fig. 2F), very similar to E3 staining. The preimmune rabbit serum stained only a small part of the acrosome represented as a fine line of staining (Fig. 2G), in contrast to the markedly distinct staining of the entire acrosome by the E3 antiserum (Fig. 2H). Fig. 2 (I and J) shows the corresponding bright field images. (The fact that rabbit sera show cross-reactivity in the hamster sperm acrosome was also observed earlier by workers demonstrating localization of p26h (52)). To investigate the fate of E3 during sperm capacitation, immunofluorescent studies were performed with hamster sperm samples prepared at different time points during capacitation. The intensity of E3 staining in the principal piece of the hamster sperm flagellum increased from 0 h (non-capacitated) to the 7th hour of capacitation, 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 with capacitation, only around 20% of the spermatozoa appeared to have lost the acrosomal E3 staining at the 7th hour of capacitation.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 2.
Extra-mitochondrial localization of E3 in hamster spermatozoa. Indirect immunofluorescence studies were performed by using rabbit polyclonal anti-E3 antibody. E3 localizes in the acrosome and principal piece of cauda (A) and caput (B) epidydimal spermatozoa. The preabsorbed anti-E3 antibody fails to give distinct staining (C). Sperm mitochondrial marker PHPGx localizes in the sperm mitochondria (mid piece) (D). Triton X-100-treated caudal spermatozoa do not reveal mitochondrial localization of E3 (E). PDHc shows similar localization in the cauda spermatozoa (F). The preimmune rabbit serum stained only a small part of the acrosome represented as a fine line of staining (G), in contrast to the markedly distinct staining of the entire acrosome by the E3 antiserum (H); the corresponding bright field images are also shown (I and J). There is no change in localization of E3 during capacitation as shown by a single representative spermatozoon at each time point of capacitation (K); numbers represent the time point of capacitation. The intensity of E3 staining increases in the principal piece of the sperm flagella along the time course of capacitation. The bars in every figure represent 20 µm, whereas in G–I it represents 10 µm.

 
To investigate the generality of the extra mitochondrial localization of E3, immunolocalization of E3 in the caput and cauda epididymal spermatozoa of another rodent species (mouse) was carried out, and the results confirmed the extra-mitochondrial acrosomal localization of E3 also in mouse spermatozoa (Fig. 3, A and B). However, attempts to stain human sperm with the polyclonal E3 antibody raised against pig heart E3 failed to show any positive staining (data not shown). But when the anti-PDHc antibody, raised against corresponding human proteins was used, distinct extra-mitochondrial acrosomal staining was observed in human spermatozoa (Fig. 3C).

Thus, the results taken together bring out clearly that the canonical mitochondrial protein E3 and also the hosting enzyme complex, PDHc, exhibit extra mitochondrial localization in mammalian spermatozoa; hamster spermatozoa show the whole complex, including E3, to be present both in the acrosome and the principal piece of the sperm flagella.

Association of Dihydrolipoamide Dehydrogenase with the Acrosomal Matrix of Hamster Spermatozoa—The calcium ionophore A23187 [GenBank] , which is known to induce complete acrosome reaction (53), characterized by the total release of the acrosomal contents and exposure of the inner acrosomal membrane (54), was used to check whether acrosomal dihydrolipoamide dehydrogenase (E3) is released during acrosomal exocytosis. A23187 [GenBank] (5 µM for 2 h) could release almost all the E3 from the acrosome as seen in immunofluorescence studies (Fig. 4A). However, in partial acrosome-reacted spermatozoa, as shown by a representative spermatozoon in phase (Fig. 4B, upper panel), E3 was retained in the acrosome (Fig. 4B, lower panel). When immunoblotting was carried out with the sperm sample similarly exposed to A23187 [GenBank] for inducing acrosomal exocytosis, the experimental sample showed less amount of E3 in the spermatozoa than the control (sperm not treated with A23187 [GenBank] at the same time point) (Fig. 4C), indicating the release of E3 during acrosomal exocytosis. The same figure also shows the release of P26h whose homologue, P34H, has been previously shown to be released during acrosome reaction in human spermatozoa (55). Thus the results indicate that E3 is released during complete acrosome reaction and not localized in the inner acrosomal membrane, which is the part of the acrosome exposed after acrosome reaction. Furthermore, to investigate whether E3 is localized in the plasma membrane or the acrosomal matrix, hamster spermatozoa were demembranated by Triton X-100 treatment followed by mechanical shearing, which could partially dislodge the acrosomal matrix of the demembranated spermatozoa (47). Immunofluorescence of these hamster spermatozoa with polyclonal anti-E3 antibody revealed E3 staining in the Triton X-100-resistant acrosomal matrix. Fig. 4D shows E3 staining in Triton X-100-treated hamster spermatozoa in different stages of dislodgement of the acrosomal matrix. Thus our results indicate that E3 is localized in the acrosomal matrix of hamster spermatozoa, which is released during complete acrosome reaction.



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 3.
Extra mitochondrial localization of E3 in spermatozoa of mouse and human. Indirect immunofluorescence studies were performed by using rabbit polyclonal anti-E3 antibody. E3 shows an acrosomal localization in the mouse caput (A) and cauda spermatozoa (B). PDHc localizes in the acrosome in human spermatozoa (C). The bars represent 10 µm.

 



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4.
E3 is associated with acrosomal matrix of hamster spermatozoa. Indirect immunofluorescence studies, supported by immunoblotting studies were performed by using rabbit polyclonal anti-E3 antibody. Calcium ionophore treatment (5 µM, 2 h) releases acrosomal E3 from most hamster spermatozoa (A). A representative partially acrosome reacted spermatozoa has not released E3 (B). E3 in the pellet of calcium ionophore-treated (2 h) spermatozoa is less (2ca) than the untreated cells collected either in the second hour (2c) or before the treatment (0) (C, right panel). The surface acrosomal marker P26h also shows similar loss upon calcium ionophore treatment (C, left panel). Triton X-100-treated spermatozoa do not release E3, which gets dislodged only after mechanical shearing of the treated sperm, as represented by the spermatozoa at different stages of dislodgement of the matrix (D). The bar in A represents 20 µm, in B it represents 5 µm, and in D 10 µm.

 



View larger version (78K):
[in this window]
[in a new window]
  		FIG. 5.
Staining of adult hamster testis by rabbit polyclonal E3 antibody. In immunohistochemistry E3 antiserum stains the entire seminiferous tubules faintly and stains the interstitium very strongly (arrows in B), whereas the preimmune serum stains only the luminal side of the tubules (arrows in A). Bar represents 100 µm.

 
Dihydrolipoamide Dehydrogenase Shows a Mitochondria Type Localization and Not a Golgi Type in Testicular Spermatocytes of Adult Hamster—Acrosome is a Golgi-derived organelle, and thus one expects dihydrolipoamide dehydrogenase (E3) to localize in the Golgi of the spermatogenic cells. It is known that Golgi exhibits a very characteristic juxtanuclear localization (56), whereas the mitochondrial proteins show perinuclear localization. Thus, an immunohistochemistry approach was used to investigate if dihydrolipoamide dehydrogenase (E3) shows a mitochondria type or a Golgi type staining in the adult testicular cells. Fig. 5B shows that the polyclonal E3 antiserum faintly stained the whole tubule and the interstitium very strongly (arrows in Fig. 5B), whereas the preimmune serum stained the luminal side of the seminiferous tubules (arrows in Fig. 5A). Investigation of the testicular sections at higher magnification revealed E3 staining of the Leydig cells in the interstitium (arrows in Fig. 6A, letter "L"). It was also observed that the spermatocytes in the mid-pachytene stage (stage VII) exhibited granulated staining around the nucleus (arrows in Fig. 6A), whereas in the step 1 spermatids (stage I) the stained granules appeared at the Golgi pole of the nucleus (arrows in Fig. 6C). The parallel PAS-hematoxylin-stained sections show the extent of acrosome formation in the corresponding stages (Fig. 6, B and D). Comparison of panels C and D in Fig. 6 shows that E3 accumulates in the Golgi pole (from where acrosome starts forming) in the spermatids. E3 was not detected in the developing acrosome (or the flagella) either in the spermatids or in the matured testicular spermatozoa by immunohistochemistry, but in spermatozoa released from testis staining could be observed in the acrosome by a more sensitive immunofluorescence staining method (Fig. 6E). Thus, E3 shows a mitochondrial type of stain in the spermatocytes and appears to be in the Golgi pole in the spermatids.



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 6.
E3 does not show a Golgi type of staining in the testicular cells. Immunohistochemistry of adult hamster testis shows that in stage VII sections E3 shows a granulated staining around the pachytene spermatocytes (A). The corresponding PAS-hematoxylin-stained section shows the extended acrosome (arrows in B) in the spermatids. In stage I, E3 shows condensed staining in the Golgi pole of the spermatid nucleus (C). Corresponding PAS-hematoxylin-stained section shows the dot-like appearance of the acrosome in the early stages of development (arrows in D). Spermatozoa released from testis shows E3 in the acrosome in immunofluorescence (E). N, spermatocyte nucleus; n, spermatid nucleus; L, Leydig cells. Bars in A–D represent 10 µm, while in E it represents 20 µm.

 
Tyrosine Phosphorylation of Dihydrolipoamide Dehydrogenase Detected in the Principal Piece of Hamster Sperm Flagella— Previously we had demonstrated that dihydrolipoamide dehydrogenase (E3) shows capacitation-dependent tyrosine phosphorylation in hamster spermatozoa (27). The dual localization of E3 in hamster spermatozoa as observed in this study prompted us to investigate if E3 is tyrosine-phosphorylated in one or both the locations. Lack of knowledge of the tyrosine-phosphorylated site of this newly identified phosphorylated form of the protein led us to take an indirect approach toward this question as described below. Fig. 7 (A and B) show the staining of tyrosine-phosphorylated proteins using monoclonal anti-phosphotyrosine antibody (4G10), in non-capacitated and capacitated hamster spermatozoa. As in other mammalian spermatozoa (11–15), it was observed that almost all of the capacitation-dependent tyrosine-phosphorylated proteins localize in the flagella of capacitated hamster spermatozoa. Furthermore, as a modification, the coverslips coated with capacitated hamster spermatozoa were preincubated with polyclonal E3 antibody to block all the available E3 epitopes (irrespective of the tyrosine phosphorylation status) and were followed by staining of the phosphotyrosine residues (in any protein). Fig. 7C shows that blocking the E3 epitopes markedly reduced the signal from tyrosine-phosphorylated proteins in the principal piece of the sperm flagella (marked by yellow arrows). The reduction in signal in the mid piece of the spermatozoa by the same antibody (marked by green arrows in Fig. 7C) could be attributed to preimmune serum, which when used for preincubation gave a reduced mid piece staining (green arrows in Fig. 7D). Furthermore, preincubation with the polyclonal PDHc antibody was used as another control, which showed very little reduction in the signal intensity from phosphotyrosine residues (Fig. 7E). Thus, the immunofluorescence data indicate a substantial contribution of E3 in the pool of the capacitation-dependent tyrosine-phosphorylated proteins in the principal piece of hamster sperm flagella.

Further dual staining was performed with E3 (fluorescein isothiocyanate) and phosphotyrosine (cy3), in non-capacitated and capacitated hamster spermatozoa, with the aim of identifying the tyrosine-phosphorylated form of E3. For this purpose, keeping the result of the previous experiment in mind, the first antibody used for staining was anti-phosphotyrosine antibody (so that all tyrosine-phosphorylated sites of E3, along with other tyrosine-phosphorylated proteins are bound by the antibody). This was followed by staining with E3 antibody. As a control the reverse incubation (E3 staining followed by anti-phosphotyrosine staining) was also done in a capacitated sperm sample. Because no tyrosine phosphorylation signal was detected in the acrosome, and E3 was detected only in the principal piece of the sperm flagellum, only this part of the cell is being presented in the results of the dual staining experiment. As expected no tyrosine phosphorylation was detected in the non-capacitated spermatozoa (0 h) (Fig. 8, A–C) for the E3 staining to show colocalization with. In the capacitated spermatozoa (Fig. 8, D–F) a distinct colocalization was observed that was 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 of E3 in the principal piece of the capacitated hamster sperm flagella.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 7.
Contribution of tyrosine-phosphorylated E3 to the total tyrosine phosphorylation in the principal piece of hamster sperm flagella. Capacitation-dependent tyrosine phosphorylation in spermatozoa is seen mostly in the flagella (A, non-capacitated; B, capacitated). C, coverslips blocked with polyclonal E3 antibody reduces the detection of tyrosine phosphorylation in the principal piece of the flagella (compare yellow arrows in C with B). The preimmune serum blocking marginally obliterates detection of tyrosine phosphorylation in the mid piece (green arrows in D and C). E, blocking with polyclonal PDHc antibody does not reduce the signal to a considerable extent compared with that of level reduced by polyclonal E3 antibody (compare E with B and C). Bar represents 10 µm.

 



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 8.
Tyrosine-phosphorylated E3 in the principal piece of the hamster sperm flagella. Colocalization was done by confocal microscopy by viewing optical sections of sperm samples. Colocalization between E3 (green) and phosphotyrosine residues (red) is shown when coverslips were incubated with anti-phosphotyrosine antibody first. In the non-capacitated sample absence of tyrosine phosphorylation causes no colocalization (A–C). In the capacitated sample there is a strong colocalization (D–F), which disappears when E3 antibody is used first to block E3 epitopes (G–I). Bar represents 5 µm.

 
Time Course of Tyrosine Phosphorylation of Dihydrolipoamide Dehydrogenase Is Different in Different Locations of Hamster Spermatozoa—To follow the time course of tyrosine phosphorylation of dihydrolipoamide dehydrogenase (E3) in both the locations in hamster spermatozoa, cell fractionation experiments were done followed by detection of phosphorylation by immunoblotting with monoclonal anti-phosphotyrosine antibody (4G10). We have previously shown (11) that the 56-kDa molecular mass protein later identified by us (27) as E3, is the only tyrosine-phosphorylated protein at this molecular mass range in a two-dimensional immunoblot with monoclonal anti-phosphotyrosine antibody (4G10). Thus analysis of parallel SDS-PAGE immunoblots of hamster sperm lysates, with anti-phosphotyrosine antibody and anti-E3 antibody, suffices for the analysis of tyrosine phosphorylation of E3. Because the localization of E3 is shown to be in the acrosomal matrix and the principal piece of the hamster spermatozoa (present study), dislodging the acrosomal matrix from the sperm completely, after harvesting them at different time points of capacitation, would enable the biochemical investigation of the time course of tyrosine phosphorylation of E3 from only the principal piece of the sperm flagella. Fig. 9A shows the protein loading of acrosome-dislodged spermatozoa at different time points of capacitation (by Ponceau S stain), whereas, as evidence supporting acrosomal dislodgement, Fig. 9B shows the presence of the acrosomal marker P26h only in the intact sperm (Ts) and not in the acrosomal dislodged sperm. Fig. 9C shows that the level of E3 in the principal piece of hamster sperm flagella did not vary during capacitation (taking into consideration the protein loading as in Fig. 9A). The presence of the tyrosine-phosphorylated form of E3 in the principal piece of hamster sperm flagella, as detected by immunofluorescence (Figs. 7 and 8), is confirmed in Fig. 9D. The figure further shows the increase in its tyrosine phosphorylation along the time course of capacitation till 4th hour of capacitation and decrease again till the 7th hour (Fig. 9D). Further we followed the time course of tyrosine phosphorylation of E3 in intact hamster spermatozoa (acrosomes not dislodged). With the total protein loaded (Fig. 10A) and {alpha} tubulin as controls (Fig. 10B) it is clear that the amount of E3 is invariant even in intact hamster spermatozoa during the time course of capacitation. However, the tyrosine phosphorylation of E3 showed a continuous increase from 0 to 7 h of capacitation in intact spermatozoa (Fig. 10D). In the tyrosine phosphoprotein analyses (Figs. 9D and 10D), the other tyrosine-phosphorylated protein bands (not of interest in this report) are also shown for the appreciation of the fact that different proteins have different kinetics of phosphorylation during capacitation. The comparison of the time course of tyrosine phosphorylation of acrosome intact and acrosome-dislodged sperm clearly indicate the contribution of tyrosine phosphorylation from the acrosomal E3 in the later time points of capacitation, being maximum at 7 h (compare Figs. 9D and 10D). Thus, our results indicate differential tyrosine phosphorylation kinetics of E3 in the analyses of acrosome dislodged sperm (contribution from the principal piece) and that of the acrosome intact sperm (contribution from acrosome and principal piece) during hamster capacitation.



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 9.
Time course of capacitation-dependent tyrosine phosphorylation of E3 in acrosome-dislodged hamster spermatozoa. Sperm samples collected at different time points of capacitation (numbers representing hours of capacitation) were processed to dislodge acrosomes as shown by the absence of signal from an acrosomal marker P26h in the immunoblot analyses (B). P26h signal is shown in the total sperm samples (Ts) where acrosomes were not dislodged. Immunoblot results indicate lack of any considerable change in protein concentration of E3 in the acrosomal dislodged spermatozoa (C), taking into consideration the amount of protein loaded in the respective lanes as shown by Ponceau S staining (A). The tyrosine phosphorylation of E3 (bounded by outline), as observed in an immunoblot with monoclonal anti-phosphotyrosine antibody (4G10), increases till 4 h of capacitation and further decreases till 7 h (D).

 



View larger version (76K):
[in this window]
[in a new window]
  		FIG. 10.
Time course of capacitation-dependent tyrosine phosphorylation of E3 in intact hamster spermatozoa. The amount of E3 does not vary during capacitation (C) (numbers representing hours of capacitation) as shown in the immunoblot of samples with equal amount of tubulin (B). Loading of protein is also shown by Ponceau S staining (A). The tyrosine phosphorylation of E3 (bounded by the outline), as observed in an immunoblot with monoclonal anti-phosphotyrosine antibody (4G10), increases from 0 till 7 h of capacitation (D).

 
Regulated Directional Activity of Hamster Sperm Dihydrolipoamide Dehydrogenase during Hamster Sperm Capacitation—To determine the enzymatic status of the dihydrolipoamide dehydrogenase (E3) of hamster spermatozoa, we first checked for the three important enzymatic properties of E3, namely, NADH specificity (57), heat stability (75 °C for 5 min) (58), and inhibition by its specific inhibitor, 5-methoxyindole-2-carboxylic acid (59). As it is shown in Table I, hamster sperm E3, like other 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 (same activity to that of the control); and the enzyme was inhibited by 5-methoxyindole-2-carboxylic acid, its specific inhibitor (zero activity). Furthermore, the bi-directional dehydrogenase activity of hamster sperm E3 was assessed at different time points during capacitation in total sperm lysates. The method used for the preparation of the sperm lysate has been shown to extract all the E3 from the spermatozoa into the soluble fraction leaving nothing in the pellet (27). The forward and reverse activities showed different regulation during the course of capacitation. The reverse activity of the enzyme (lipoamide was reduced to dihydrolipoamide accepting electrons from NADH) showed a progressive increase in activity during capacitation with a peak at 4 h and a decline thereafter (Fig. 11A). On the other hand, the forward activity of hamster sperm E3 (dihydrolipoamide was oxidized to lipoamide and NADH was produced as NAD acted as the electron acceptor) increased as capacitation progressed and maximum activity was obtained at the end of capacitation at 5–7 h (Fig. 11A). No significant correlation between the forward and the reverse activities of the enzyme was found (rs being 0.22, which is not significant at any level). The progress in forward activity was found to positively correlate well with the progress in the number of acrosome-reacted hamster spermatozoa (Fig. 11B). The correlation coefficient (rs) between the forward activity and the number of acrosome-reacted spermatozoa was found to be 0.874, and the correlation was significant at the level of 0.01. Thus, the data suggest a probable control for the directionality of hamster sperm E3 activity during capacitation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The male gamete, spermatozoon, is a highly specialized cell of the body. During spermiogenesis, in the testis, a thorough reorganization of cellular proteins (and organelles) takes place in the haploid spermatid, reflected ultimately in the unique morphology of the mature spermatozoon. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a cytosolic protein in somatic cells, which is found to be associated with the mitochondria in the mature spermatozoa (50). On the other hand, Voltage-dependent anion-selective channels (VDAC2 and VDAC3), which are mitochondrial porins in somatic cells, are found to be associated with the cytoskeletal elements (outer dense fibers) in the bovine sperm flagella (60). It is obvious that such dramatic changes in localization probably imply fulfillment of specific functions. 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 is canonically localized in mitochondria in the eukaryotic systems investigated so far (29) (Fig. 1B). However, in cyanobacteria a periplasmic form (61) and in soya bean a nodular form (62) have been found. In this report we show that dihydrolipoamide dehydrogenase (E3) shows dual localization in the acrosomal matrix and in the principal piece of the flagella of hamster spermatozoa, as we also observed for, the host complex, PDHc (Fig. 2). This result is very interesting, because it is known that the glycolytic apparatus is localized in the principal piece of mammalian spermatozoa (3) and adds to the idea of compartmentalized metabolic pathways in spermatozoa. Mention may be made of the fact that the dissolution of the disulfide-rich sperm mitochondrial sheath requires a reducing agent (63). We were able to completely solubilize E3 without a reducing agent (27), which further supports the extra mitochondrial localization of E3. Reorganization of proteins during sperm capacitation is a common phenomenon (64–66), but E3 did not show any change in localization during capacitation (Fig. 1K). The increase in the intensity of E3 staining in the principal piece of the flagella of a terminally differentiated cell like spermatozoa (also observed for host complex PDH, data not shown) is probably due to the increase in the accessibility of the antigen, because immunoblotting showed the presence of invariable levels of E3 in the flagella in all time points of capacitation (Fig. 9C). In our previous study we have shown that E3 exhibits dual involvement in the phenomena of hyperactivation (enhanced motility) and acrosome reaction during hamster sperm capacitation (27). In the light of these studies the observed dual localization of E3 and PDHc, in the flagella (principal piece) and acrosome of hamster spermatozoa, becomes very relevant.

Acrosome is surrounded by an outer acrosomal membrane (underlying the plasma membrane), and an inner acrosomal membrane (overlying the nucleus) (54). The acrosomal compartment can be structurally and biochemically divided into a soluble part and a matrix (45). Sperm acrosome reaction is an exocytotic process during which the sperm plasma membrane fuses with the underlying (outer) acrosomal membrane and concomitantly releases the acrosomal proteins thus exposing the proteins of the inner acrosomal membrane to aid the sperm in penetrating the oocyte. Acrosin (67) and mouse sp56 (68) (and its guinea pig homologue AM67 (47)) are two important candidates of the acrosomal matrix. This report adds hamster sperm E3 (56 kDa) to the list (Fig. 4), which could also be the 56-kDa protein identified in a study attempting to isolate the acrosomal matrix proteins from hamster spermatozoa (45). Acrosome reaction is believed to be a gradual and regulated phenomenon. In mouse (69) and guinea pig (70) spermatozoa, it has been demonstrated that the soluble part of the acrosome is released first followed by differential release of matrix components, during acrosome reaction. Therefore, the release of E3 only by complete acrosome-reacted spermatozoa (A23187 [GenBank] -treated, Fig. 4A) and not by partial acrosome-reacted spermatozoa (Fig. 4B) can be attributed to its localization in the acrosomal matrix.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 11.
Stage-specific directional regulation of hamster sperm E3 activity during capacitation. In vitro enzyme assay of E3 shows that the reverse and forward activities of the enzyme show different regulation during capacitation; the reverse activity exhibiting a peak at 4 h and forward activity at 5–7 h (A). The activity of the enzyme is expressed as micromoles of NADH/min, normalized with the factor 10-3. The forward activity and the acrosome reaction appeared to follow the same progress during capacitation, the values at each time point being positively correlated to each other (B). The Spearman's correlation coefficient was found to be 0.874 and was significant at p < 0.01. The experiment was performed with three animals.

 
In our previous study we could identify the mitochondrial signal sequence of E3 from hamster testis cDNA (27), thus localization of E3 in the extra mitochondrial (acrosomal) locations is quite intriguing. Mitochondrial proteins (51, 71, 72) show a typical granular staining around the nucleus in the spermatocytes in agreement with the observation that mitochondria are located very close to the outer nuclear membrane in these cells (73). On the other hand, the Golgi (the organelle from which the acrosome originates) proteins in a spermatocyte appear to have characteristic juxtanuclear localization, like in any other cell (56). Only recently researchers have begun to realize the connection between Golgi and mitochondria in spermatogenesis. Knock-out mice of Hrb (or Rab) (74) and Golgi-associated PDZ- and coiled-coil motif-containing protein (75), both being Golgi proteins, have been independently shown to have dramatic defects in the mitochondrial sheath formation. Our immunohistochemistry results further indicate that E3 shows a mitochondrial type staining in the spermatocytes, whereas in the early spermatid it appears to collect near the point from where acrosome begins to develop from the Golgi (56) (Fig. 6); in no other stages in the 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 of a "protein transport machinery" between Golgi and mitochondria during the conversion of tetraploid pachytene spermatocytes to haploid spermatozoa.

During sperm capacitation an array of proteins have been found to be tyrosine-phosphorylated (10). Researchers have proved the involvement of tyrosine phosphorylation events in the capacitation-associated events hyperactivation (76, 77) and acrosome reaction (18), but the presence of capacitation-dependent tyrosine-phosphorylated proteins have been mostly detected in the flagella of spermatozoa and almost none in the acrosome (78). We previously reported E3 as a tyrosine-phosphorylated protein (27), and the dual localization of E3 necessitated investigation of tyrosine phosphorylation of E3 in both the locations. We could detect the tyrosine-phosphorylated form of this bi-directional enzyme, in the (principal piece of the) flagella of hamster spermatozoa by immunofluorescence (Figs. 7 and 8) and further confirmed it by immunoblot analyses (Fig. 9). Furthermore, our biochemical studies on the time course of E3 tyrosine phosphorylation during capacitation, indirectly suggest the presence of a tyrosine-phosphorylated form of E3 also in the acrosome of hamster sperm (compare Figs. 9D and 10D); kinetics of capacitation-dependent E3 phosphorylation being different in immunoblot analyses of acrosome intact and acrosome-dislodged spermatozoa. The failure to detect E3 phosphorylation in the acrosome by the immunofluorescence studies could be due to antigen masking in this tightly packed organelle. It is, however, not clear if the site of the apparent tyrosine phosphorylation in both the locations are similar, but the presence of nine tyrosines in the sequence of E3 (27) probably qualifies it to be a protein undergoing hyper-phosphorylation. The different kinetics 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 capacitation thus justifies independent involvement of the bi-directional enzyme E3 in hyperactivation and acrosome reaction, as we have reported before (27).

Our results, for the first time, show that the forward and the reverse activities of E3 in hamster spermatozoa are differentially regulated during capacitation. The regulation of the reverse activity of the enzyme correlates to a considerable extent to that of the tyrosine phosphorylation kinetics of the E3 in the principal piece (Figs. 9D and 11A). Furthermore, the strong correlation of the forward activity with the acrosome reaction can also be extended to the apparent contribution of the tyrosine phosphorylation from the acrosomal E3 in the terminal stages of capacitation (5.5–7 h) when acrosome reaction is maximum (Figs. 10D and 11B). Thus, it is possible that the tyrosine-phosphorylated E3 in the principal piece of the sperm flagella could be driving more of the reverse reaction, whereas that in the acrosome could be driving more of its forward reaction. Unlike other somatic cells, the pyruvate-lactate metabolism and its link to ATP production in the male gamete is still in the dark for the following reasons: (a) the presence of a mitochondrial lactate dehydrogenase in spermatozoa (26); (b) pyruvate metabolism in (bovine) spermatozoa is not linked to oxidative phosphorylation through mitochondrial electron transport chain (59); and (c) knock-out mice of cytochrome c are fertile, ruling out indispensability of electron transport chain in sperm functioning (79), whereas knock-out of the glycolytic enzyme GAPDH is defective in motility despite having no deficiency in oxygen consumption (80). Thus the role of the pyruvate metabolic enzymes like E3 (in PDHc) and others in sperm capacitation still remains to be explored.

This is the first report of the extra mitochondrial localization of dihydrolipoamide dehydrogenase (E3) along with its host complex, pyruvate dehydrogenase complex (PDHc), in the acrosome and principal piece of the sperm flagella. It is important to note here, that most of the earlier studies on mitochondrial activity, including pyruvate metabolism studies, in spermatozoa have used total sperm permeabilized in various ways (81–83), and thus the extra mitochondrial contribution might have been overlooked. The mitochondrial type rather than a Golgi type localization of dihydrolipoamide dehydrogenase in the spermatogenic cells suggests a link between the two organelles that could deliver the mitochondrial enzyme to the Golgi forming the acrosome during spermatogenesis. Furthermore, the results presented in this report bring out clearly the dual involvement of dihydrolipoamide dehydrogenase on the basis of the novel dual localization, dual tyrosine phosphorylation kinetics, and dual regulation of the bi-directional enzyme activity in sperm capacitation.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} A recipient of a Council of Scientific and Industrial Research fellowship, Government of India. Back

§ To whom correspondence should be addressed. Tel.: 91-40-271-92504; Fax: 91-40-271-60591; E-mail: shivas{at}ccmb.res.in.

1 The abbreviations used are: E3, dihydrolipoamide dehydrogenase; PDHc, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase; BSA, bovine serum albumin; TBS, Tris-buffered saline; PAS, periodic acid-Schiff. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jyotsna Dhawan, Dr. Archana B. Siva, and T. Subhash for their invaluable help in the project. We sincerely thank Dr. R. A. Harris for a gift of 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.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Westhoff, D., and Kamp, G. (1997) J. Cell Sci. 110,1821 -1829[Abstract]
  2. Storey, B. T., and Kayne, F. J. (1975) Fertil. Steril. 26,1257 -1265[Medline] [Order article via Infotrieve]
  3. Travis, A. J., Jorgez, C. J., Merdiushev, T., Jones, B. H., Dess, D. M., Diaz-Cueto, L., Storey, B. T., Kopf, G. S., and Moss, S. B. (2001) J. Biol. Chem. 276,7630 -7636[Abstract/Free Full Text]
  4. Austin, C. R. (1952) Nature 170, 326[CrossRef][Medline] [Order article via Infotrieve]
  5. Chang, M. C. (1951) Nature 168,697 -698[Medline] [Order article via Infotrieve]
  6. Visconti, P. E., Galantino-Homer, H., Moore, G. D., Bailey, J. L., Ning, X., Fornes, M., and Kopf, G. S. (1998) J. Androl. 19,242 -248[Free Full Text]
  7. Jha, K. N., Kameshwari, D. B., and Shivaji, S. (2003) Cell. Mol. Biol. (Noisyle-grand) 49, 329-340[Medline] [Order article via Infotrieve]
  8. de Lamirande, E., Leclerc, P., and Gagnon, C. (1997) Mol. Hum. Reprod. 3,175 -194[Abstract/Free Full Text]
  9. Yanagimachi, R. (1994) in Mammalian Fertilization: The Physiology of Reproduction (Knobil, E., and Neill, J. D., eds) pp. 189-317, Raven Press Ltd., New York
  10. Visconti, P. E., Westbrook, V. A., Chertihin, O., Demarco, I., Sleight, S., and Diekman, A. B. (2002) J. Reprod. Immunol. 53,133 -150[CrossRef][Medline] [Order article via Infotrieve]
  11. Jha, K. N., and Shivaji, S. (2002) Mol. Reprod. Dev. 61,258 -270[CrossRef][Medline] [Order article via Infotrieve]
  12. Leclerc, P., de Lamirande, E., and Gagnon, C. (1997) Free Radic. Biol. Med. 22, 643-656[CrossRef][Medline] [Order article via Infotrieve]
  13. Mahony, M. C., and Gwathmey, T. (1999) Biol. Reprod. 60,1239 -1243[Abstract/Free Full Text]
  14. Si, Y., and Okuno, M. (1999) Biol. Reprod. 61,240 -246[Abstract/Free Full Text]
  15. Urner, F., Leppens-Luisier, G., and Sakkas, D. (2001) Biol. Reprod. 64,1350 -1357[Abstract/Free Full Text]
  16. Carrera, A., Moos, J., Ning, X. P., Gerton, G. L., Tesarik, J., Kopf, G. S., and Moss, S. B. (1996) Dev. Biol. 180,284 -296[CrossRef][Medline] [Order article via Infotrieve]
  17. Luconi, M., Carloni, V., Marra, F., Ferruzzi, P., Forti, G., and Baldi, E. (2004) J. Cell Sci. 117,1235 -1246[Abstract/Free Full Text]
  18. Tomes, C. N., Roggero, C. M., De Blas, G., Saling, P. M., and Mayorga, L. S. (2004) Dev. Biol. 265,399 -415[CrossRef][Medline] [Order article via Infotrieve]
  19. Huang, K. P., and Huang, F. L. (1980) J. Biol. Chem. 255,3141 -3147[Free Full Text]
  20. El-Maghrabi, M. R., Noto, F., Wu, N., and Manes, N. (2001) Curr. Opin. Clin. Nutr. Metab. Care 4,411 -418[CrossRef][Medline] [Order article via Infotrieve]
  21. Pilkis, S. J., El-Maghrabi, M. R., Coven, B., Claus, T. H., Tager, H. S., Steiner, D. F., Keim, P. S., and Heinrikson, R. L. (1980) J. Biol. Chem. 255,2770 -2775[Abstract/Free Full Text]
  22. Mori, C., Welch, J. E., Fulcher, K. D., O'Brien, D. A., and Eddy, E. M. (1993) Biol. Reprod. 49, 191-203[Abstract]
  23. Goldberg, E. (1975) Methods Enzymol. 41,318 -323[Medline] [Order article via Infotrieve]
  24. Dahl, H. H., Brown, R. M., Hutchison, W. M., Maragos, C., and Brown, G. K. (1990) Genomics 8, 225-232[CrossRef][Medline] [Order article via Infotrieve]
  25. Travis, A. J., Foster, J. A., Rosenbaum, N. A., Visconti, P. E., Gerton, G. L., Kopf, G. S., and Moss, S. B. (1998) Mol. Biol. Cell 9,263 -276[Abstract/Free Full Text]
  26. Blanco, A. (1980) Johns Hopkins Med. J. 146,231 -235[Medline] [Order article via Infotrieve]
  27. Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70,887 -899[Abstract/Free Full Text]
  28. Patel, M. S., Vettakkorumakankav, N. N., and Liu, T. C. (1995) Methods Enzymol. 252,186 -195[Medline] [Order article via Infotrieve]
  29. Patel, M. S., and Roche, T. E. (1990) FASEB J. 4,3224 -3233[Abstract]
  30. Gazaryan, I. G., Krasnikov, B. F., Ashby, G. A., Thorneley, R. N., Kristal, B. S., and Brown, A. M. (2002) J. Biol. Chem. 277,10064 -10072[Abstract/Free Full Text]
  31. Igamberdiev, A. U., Bykova, N. V., Ens, W., and Hill, R. D. (2004) FEBS Lett. 568,146 -150[CrossRef][Medline] [Order article via Infotrieve]
  32. Bhushan, B., Halasz, A., Spain, J. C., and Hawari, J. (2002) Biochem. Biophys. Res. Commun. 296,779 -784[CrossRef][Medline] [Order article via Infotrieve]
  33. Petrat, F., Paluch, S., Dogruoz, E., Dorfler, P., Kirsch, M., Korth, H. G., Sustmann, R., and de Groot, H. (2003) J. Biol. Chem. 278,46403 -46413[Abstract/Free Full Text]
  34. Smith, A. W., Roche, H., Trombe, M. C., Briles, D. E., and Hakansson, A. (2002) Mol. Microbiol. 44, 431-448[CrossRef][Medline] [Order article via Infotrieve]
  35. Bryk, R., Lima, C. D., Erdjument-Bromage, H., Tempst, P., and Nathan, C. (2002) Science 295,1073 -1077[Abstract/Free Full Text]
  36. Johnson, M. T., Yang, H. S., Magnuson, T., and Patel, M. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,14512 -14517[Abstract/Free Full Text]
  37. Wilkinson, K. D., and Williams, C. H., Jr. (1981) J. Biol. Chem. 256,2307 -2314[Abstract/Free Full Text]
  38. Matthews, R. G., and Williams, C. H., Jr. (1976) J. Biol. Chem. 251,3956 -3964[Abstract/Free Full Text]
  39. Linn, T. C., Pettit, F. H., Hucho, F., and Reed, L. J. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 227-234[Abstract/Free Full Text]
  40. Linn, T. C., Pettit, F. H., and Reed, L. J. (1969) Proc. Natl. Acad. Sci. U. S. A. 62, 234-241[Abstract/Free Full Text]
  41. Bavister, B. D. (1989) Gamete Res. 23,139 -158[CrossRef][Medline] [Order article via Infotrieve]
  42. Kulanand, J., and Shivaji, S. (2001) Andrologia 33,95 -104[CrossRef][Medline] [Order article via Infotrieve]
  43. Laemmli, U. K. (1970) Nature 227,680 -685[CrossRef][Medline] [Order article via Infotrieve]
  44. O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007 -4021[Abstract/Free Full Text]
  45. Olson, G. E., Winfrey, V. P., and Davenport, G. R. (1988) Biol. Reprod. 39,1145 -1158[Abstract]
  46. Karlsson, J. O., Ostwald, K., Kabjorn, C., and Andersson, M. (1994) Anal. Biochem. 219,144 -146[CrossRef][Medline] [Order article via Infotrieve]
  47. Foster, J. A., Friday, B. B., Maulit, M. T., Blobel, C., Winfrey, V. P., Olson, G. E., Kim, K. S., and Gerton, G. L. (1997) J. Biol. Chem. 272,12714 -12722[Abstract/Free Full Text]
  48. Miething, A. (1998) Adv. Anat. Embryol. Cell Biol. 140,1 -92[Medline] [Order article via Infotrieve]
  49. Pfeifer, H., Conrad, M., Roethlein, D., Kyriakopoulos, A., Brielmeier, M., Bornkamm, G. W., and Behne, D. (2001) FASEB J. 15,1236 -1238[Free Full Text]
  50. Roveri, A., Casasco, A., Maiorino, M., Dalan, P., Calligaro, A., and Ursini, F. (1992) J. Biol. Chem. 267,6142 -6146[Abstract/Free Full Text]
  51. Thompson, W. E., Ramalho-Santos, J., and Sutovsky, P. (2003) Biol. Reprod. 69, 254-260[Abstract/Free Full Text]
  52. Gaudreault, C., El Alfy, M., Legare, C., and Sullivan, R. (2001) Biol. Reprod. 65, 79-86[Abstract/Free Full Text]
  53. Jaiswal, B. S., Eisenbach, M., and Tur-Kaspa, I. (1999) Mol. Hum. Reprod. 5, 214-219[Abstract/Free Full Text]
  54. Kopf, G. F., and Gerton, G. L. (1991) in Mammalian Sperm Acrosome and the Acrosome Reaction (Wassarman, P. M., ed) pp. 153-203, CRC Press, Boca Raton, FL
  55. Boue, F., Blais, J., and Sullivan, R. (1996) Biol. Reprod. 54,1009 -1017[Abstract]
  56. Ramalho-Santos, J., Schatten, G., and Moreno, R. D. (2002) Biol. Reprod. 67,1043 -1051[Abstract/Free Full Text]
  57. Massey, V., and Veeger, C. (1961) Biochim. Biophys. Acta 48,33 -47[Medline] [Order article via Infotrieve]
  58. Lusty, C. J. (1963) J. Biol. Chem. 238,3443 -3452[Free Full Text]
  59. Van Dop, C., Hutson, S. M., and Lardy, H. A. (1977) J. Biol. Chem. 252,1303 -1308[Abstract/Free Full Text]
  60. Hinsch, K. D., De Pinto, V., Aires, V. A., Schneider, X., Messina, A., and Hinsch, E. (2004) J. Biol. Chem. 279,15281 -15288[Abstract/Free Full Text]
  61. Engels, A., Kahmann, U., Ruppel, H. G., and Pistorius, E. K. (1997) Biochim. Biophys. Acta 1340, 33-44[CrossRef][Medline] [Order article via Infotrieve]
  62. Moran, J. F., Sun, Z., Sarath, G., Arredondo-Peter, R., James, E. K., Becana, M., and Klucas, R. V. (2002) Plant Physiol 128,300 -313[Abstract/Free Full Text]
  63. Sutovsky, P., Moreno, R. D., Ramalho-Santos, J., Dominko, T., Simerly, C., and Schatten, G. (2000) Biol Reprod 63,582 -590[Abstract/Free Full Text]
  64. Cowan, A. E., Primakoff, P., and Myles, D. G. (1986) J. Cell Biol. 103,1289 -1297[Abstract/Free Full Text]
  65. Bronson, R., Peresleni, T., Golightly, M., and Preissner, K. (2000) Mol. Hum. Reprod. 6, 977-982[Abstract/Free Full Text]
  66. Grace, K. S., Bronson, R. A., and Ghebrehiwet, B. (2002) Biol. Reprod. 66, 823-829[Abstract/Free Full Text]
  67. Baba, T., Kashiwabara, S., Watanabe, K., Itoh, H., Michikawa, Y., Kimura, K., Takada, M., Fukamizu, A., and Arai, Y. (1989) J. Biol. Chem. 264,11920 -11927[Abstract/Free Full Text]
  68. Kim, K. S., Cha, M. C., and Gerton, G. L. (2001) Biol. Reprod. 64,36 -43[Abstract/Free Full Text]
  69. Kim, K. S., and Gerton, G. L. (2003) Dev. Biol. 264,141 -152[CrossRef][Medline] [Order article via Infotrieve]
  70. Kim, K. S., Foster, J. A., and Gerton, G. L. (2001) Biol. Reprod. 64,148 -156[Abstract/Free Full Text]
  71. Goldberg, E., Sberna, D., Wheat, T. E., Urbanski, G. J., and Margoliash, E. (1977) Science 196,1010 -1012[Abstract/Free Full Text]
  72. Meinhardt, A., Parvinen, M., Bacher, M., Aumuller, G., Hakovirta, H., Yagi, A., and Seitz, J. (1995) Biol. Reprod. 52,798 -807[Abstract]
  73. Meinhardt, A., Wilhelm, B., and Seitz, J. (1999) Hum. Reprod. Update 5,108 -119[Abstract/Free Full Text]
  74. Kang-Decker, N., Mantchev, G. T., Juneja, S. C., McNiven, M. A., and van Deursen, J. M. (2001) Science 294,1531 -1533[Abstract/Free Full Text]
  75. Yao, R., Ito, C., Natsume, Y., Sugitani, Y., Yamanaka, H., Kuretake, S., Yanagida, K., Sato, A., Toshimori, K., and Noda, T. (2002) Proc. Natl. Acad. Sci. U. S. A. 99,11211 -11216[Abstract/Free Full Text]
  76. Uma Devi, K., Jha, K., Patil, S. B., Padma, P., and Shivaji, S. (2000) Andrologia 32, 95-106[CrossRef][Medline] [Order article via Infotrieve]
  77. Bajpai, M., Asin, S., and Doncel, G. F. (2003) Arch. Androl. 49,229 -246[Medline] [Order article via Infotrieve]
  78. Urner, F., and Sakkas, D. (2003) Reproduction 125,17 -26[Abstract]
  79. Narisawa, S., Hecht, N. B., Goldberg, E., Boatright, K. M., Reed, J. C., and Millan, J. L. (2002) Mol. Cell. Biol. 22,5554 -5562[Abstract/Free Full Text]
  80. Miki, K., Qu, W., Goulding, E. H., Willis, W. D., Bunch, D. O., Strader, L. F., Perreault, S. D., Eddy, E. M., and O'Brien D, A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101,16501 -16506[Abstract/Free Full Text]
  81. Calvin, J., and Tubbs, P. K. (1978) Eur. J. Biochem. 89,315 -320[Medline] [Order article via Infotrieve]
  82. Morton, B. E., and Lardy, H. A. (1967) Biochemistry 6,50 -56[CrossRef][Medline] [Order article via Infotrieve]
  83. Morton, B. E., and Lardy, H. A. (1967) Biochemistry 6,43 -49[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 ReproductionHome page
S. A Khan, A. R Suryawanshi, S. A Ranpura, S. V Jadhav, and V. V Khole
Identification of novel immunodominant epididymal sperm proteins using combinatorial approach
Reproduction, July 1, 2009; 138(1): 81 - 93.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
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.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
V. Kumar, N. Rangaraj, and S. Shivaji
Activity of Pyruvate Dehydrogenase A (PDHA) in Hamster Spermatozoa Correlates Positively with Hyperactivation and Is Associated with Sperm Capacitation
Biol Reprod, November 1, 2006; 75(5): 767 - 777.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/27/25743    most recent
M500310200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mitra, K.
Right arrow Articles by Shivaji, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mitra, K.
Right arrow Articles by Shivaji, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement