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BOR - Papers in Press, published online ahead of print September 22, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.033530
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BIOLOGY OF REPRODUCTION 72, 164–171 (2005)
DOI: 10.1095/biolreprod.104.033530
© 2005 by the Society for the Study of Reproduction, Inc.

Tyrosine Phosphorylation Generates Multiple Isoforms of the Mitochondrial Capsule Protein, Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx), During Hamster Sperm Capacitation1

Subir K. NagDas2, Virginia P. Winfrey, and Gary E. Olson

Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sperm capacitation is a maturation process, occurring in the female reproductive tract, that produces fertilization-competent spermatozoa. Protein tyrosine phosphorylation represents an important event in capacitation. The present study demonstrates the capacitation-dependent tyrosine-phosphorylation of phospholipid hydroperoxide glutathione peroxidase (PHGPx), the disulfide cross-linked, major structural protein of the sperm mitochondrial capsule. Immunofluorescence microscopy using an antiphosphotyrosine monoclonal antibody (anti-pY20) demonstrated the presence of capacitation-associated tyrosine phosphorylated proteins in the flagellum of hamster spermatozoa. Among the tyrosine-phosphorylated polypeptides (Mr 19 000– 99 000), a 19-kDa polypeptide was the only one that can be solubilized completely by Triton X-100-dithiothreitol (DTT). The 19-kDa polypeptide was purified by anion-exchange chromatography and by immunoaffinity chromatography. Proteomic identification of the 19-kDa polypeptide by nano-electrospray tandem mass spectrometry yielded six peptides that matched the National Center for Biotechnology Information (NCBI) database sequences of bovine PHGPx. Indirect immunofluorescence localized PHGPx to the midpiece of the flagellum and the immunoblot analysis demonstrated its DTT-dependent release from purified flagella. DTT extracts of noncapacitated spermatozoa exhibited a charge train of four major PHGPx isoforms (pIs 7.5– 9.0) by two-dimensional PAGE, whereas capacitated spermatozoa revealed the generation of new acidic PHGPx isoforms with isoelectric points ranging between pH 6.0–7.0 and 4.0–5.0, indicating that it is posttranslationally modified during capacitation. These data suggest that the tyrosine-phosphorylation of PHGPx may represent an important event in the signaling pathway(s) associated with capacitation and could potentially affect mitochondrial function.

PHGPx, signal transduction, sperm, sperm capacitation, sperm mitochondria, tyrosine phosphorylation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After exiting the testis, mammalian spermatozoa undergo morphological and biochemical changes as they travel through the epididymis. As a result of these modifications, spermatozoa acquire progressive motility and fertilizing potential; however, they are still fertilization incompetent. To achieve fertilizing capacity, spermatozoa require residence in the microenvironment of the female tract for a finite period of time [1–5]. This acquisition of functional competence is termed capacitation and was first described independently by Chang [6, 7] and Austin [8, 9]. During capacitation, molecular changes occur in the head that enable spermatozoa to undergo the acrosome reaction in response to zona pellucida or other physiological stimuli, such as progesterone [1, 10, 11], and in the flagellum, where a hyperactivated motility pattern develops (a whiplash-like flagellar movement with large-amplitude, asymmetric bends) [12, 13]. These two physiological processes are considered as the benchmark endpoints of capacitation [1, 14].

In vitro capacitation can be accomplished by incubating cauda epididymal or ejaculated spermatozoa in a defined medium; serum albumin, bicarbonate, and calcium play an important regulatory role in promoting capacitation [3, 15– 17]. Albumin serves as a sink for the removal of plasma membrane cholesterol that leads to the alteration of membrane fluidity observed during capacitation [2, 18–21]. The increase of intracellular pH could be due to the transmembrane movement of bicarbonate anions. The role of calcium during capacitation is controversial. Some investigators have described an increase in intracellular calcium during capacitation, whereas others have reported that no changes in calcium levels occur during this physiological event [1, 22, 23]. Because calcium acts as an effector of enzymes involved in signaling cascade(s), it is assumed that calcium may have a critical role in capacitation [3, 15–17]. Recently, Baker et al. [24] demonstrated that extracellular calcium suppresses tyrosine phosphorylation in both human and mouse spermatozoa by decreasing the availability of intracellular ATP.

Protein tyrosine phosphorylation, a key biochemical event accompanying sperm capacitation, is dependent on the presence of calcium, bicarbonate, and albumin [22, 25– 29]. Sperm protein tyrosine phosphorylation is regulated by a signal transduction pathway involving c-AMP, protein kinase A (PKA), and tyrosine kinases [30, 31]. It has been shown that reactive oxygen species upregulate protein tyrosine phosphorylation of several proteins [32]. Although this signaling cascade generates an array of tyrosine-phosphorylated polypeptides, their molecular characterization is still limited. Only CABYR, a novel calcium-binding fibrous sheath protein [33], VCP, a valosin-containing protein [34], and two members of the A kinase-anchoring protein (AKAP) family [35–37] are ascribed putative functions. The role of the tyrosine-phosphorylated polypeptides in sperm capacitation remains to be elucidated.

We have demonstrated capacitation-dependent tyrosine phosphorylated polypeptides (Mr 19 000–99 000) in the hamster sperm flagellum. The objective of the present study was to identify and to characterize a 19-kDa tyrosine-phosphorylated protein and to elucidate its role in the events associated with capacitation. The results presented here demonstrate that the 19-kDa tyrosine-phosphorylated polypeptide is phospholipid hydroperoxide glutathione peroxidase (PHGPx) and the generation of PHGPx isoforms occurs during in vitro capacitation. The selenoprotein PHGPx is a disulfide-stabilized insoluble structural protein of the sperm mitochondrial capsule [38]. The potential role of PHGPx as related to capacitation is discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Care and use of animals conformed to National Institutes of Health guidelines for humane animal care and use in research, and all protocols were approved by the institutional Animal Care and Use Committee. Mature male golden hamsters were housed in the university animal care facility on a 14L:10D cycle. Animals were killed with CO2, and tissues were collected.

Sperm Preparation and Capacitation

A population of highly motile cauda epididymal spermatozoa was prepared using a swim-up procedure. Fifty microliters of epididymal contents, obtained by puncturing the cauda epididymidis was placed in a 12 x 75-mm tube and overlain with 2 ml of warm (37°C) Tyrode medium (TALP-7) containing 0.3% BSA (fraction V), 0.1 mM hypotaurine, 0.02 mM D-penicilamine and 1 µM epinephrine [39, 40] and incubated for 5 min at 37°C. The upper 0.5 ml, containing the swim-up spermatozoa, was collected, adjusted to 1 x 106 spermatozoa/ml, and then capacitated at 37°C for 3 h in a humidified incubator in a 5% CO2:95% air atmosphere. The sperm motility pattern and acrosomal integrity were evaluated by phase-contrast microscopy. Based on the motility pattern, the spermatozoa were categorized into two groups: motile spermatozoa exhibiting any flagellar movement and hyperactivated spermatozoa exhibiting a whiplash-like flagellar movement with large-amplitude, asymmetric flagellar bends. Approximately, 80~90% of the total spermatozoa population exhibited flagellar movement and ~50% of the motile, capacitated spermatozoa exhibited hyperactivated motility; most of the remaining spermatozoa displayed a linear or circular motility pattern. Equal numbers (107 cells) of capacitated and noncapacitated cauda epididymal spermatozoa were pelleted by centrifugation at 12 000 x g for 5 min and used for SDS-PAGE, Western blotting, or proteomic analysis. Protein concentration of the samples was estimated by the method of Bradford [41].

SDS-PAGE

One-dimensional SDS-PAGE was performed on 12% continuous or 7.5–15% gradient gels [42]. Samples were prepared by solubilizing sperm pellets in equal volumes of SDS-sample buffer at 95°C in the presence of 32 mM dithiothreitol (DTT). To visualize the total polypeptide pattern, gels were stained with Coomassie Brilliant Blue R [43].

Two-dimensional PAGE was performed using a Bio-Rad precast immobilized pH(3–10) gradient gel ready strip for isoelectric focusing (IEF). IEF was done in a Bio-Rad Protean IEF cell following the conditions described in the Bio-Rad manual. DTT-soluble fractions of capacitated and noncapacitated spermatozoa were analyzed by two-dimensional PAGE. Sperm in TNI (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 2 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM sodium fluoride, 1 mm sodium vanadate, and 0.05% sodium azide) were sonicated for four 10-sec intervals with a Branson sonifier at a medium power setting. The suspensions were centrifuged at 1000 x g for 10 min. The pellets were washed three times with TNI and then extracted with 32 mM DTT in TNI for 1 h at 4°C followed by centrifugation at 12 000 x g for 10 min at 4°C. Polypeptides separated either by SDS-PAGE or two-dimensional PAGE were transferred to polyvinylfluoride membranes for immunostaining [44].

Proteomic Analysis

Proteomic analysis of the peptides was performed either at the Harvard Microchemistry Facility using microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry (µLC-MS-MS) on a Finnigan LCQ DECA quadropole ion trap mass spectrometer or at the Vanderbilt Proteomics Core laboratory by MALDI-TOF. Peptide sequences were identified using the National Center for Biotechnology Information (NCBI) Blast programs.

Western Blotting

Immunoblots were incubated for 1 h in blocking buffer composed of 1% BSA, 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 0.1% Tween 20, followed by a 1-h incubation in antiphosphotyrosine (PY20, Transduction Laboratories, Lexington, KY) diluted (1:1000) in blocking buffer. After three washes in wash buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 0.1% Tween 20), the blots were incubated in horseradish peroxidase-conjugated, affinity-purified goat anti-mouse IgG (KPL Laboratories, Gaithersburg, MD) diluted (1:2000) in wash buffer containing 5% nonfat dry milk for 1 h and then washed three times in wash buffer. Immunoreactive protein bands were identified using enhanced chemiluminescence reagents (Pierce Super Signal, Rockford, IL). To validate antibody specificity, antiphosphotyrosine was absorbed with 1 mM O-phospho-DL-tyrosine for 30 min at room temperature before use for immunoblotting.

Immunofluorescence

Both noncapacitated and capacitated spermatozoa were fixed for 60 min on ice with 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, pelleted by centrifugation at 2000 x g for 1 min, resuspended in 150 mM NaCl, 20 mM sodium phosphate, pH 7.4 (PBS), attached to poly-L-lysine-coated coverslips and then permeabilized in –20°C acetone for 10 min. Cells were rinsed in 0.1% Tween-20, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0 (TNT) for 15 min. The cells were then blocked for 1 h at room temperature in TNT containing 5% donkey serum and 2.5% bovine serum albumin (blocking solution) and incubated for 2 h at room temperature either in anti-pY20, diluted 1:500, or anti-PHGPx, diluted 1:1000, in blocking solution. Coverslips were then rinsed three times in TNT containing 1% goat serum (TNT-GS) and incubated either in Cy3-conjugated donkey anti-mouse IgG (1:1000) or Cy3-conjugated donkey anti-rabbit IgG (1:2000) (Jackson Immuno Research Laboratories, West Grove, PA) diluted in blocking solution for 2 h at room temperature. Following several washes in TNT, the slides were examined by epifluorescence and phase-contrast microscopy. As a control, anti-pY20 was preincubated with 1 mM O-phospho-DL-tyrosine for 30 min at room temperature and then used for immunostaining. Alternatively, affinity-purified IgG or nonimmune serum was substituted for primary antibody.

Solubility Properties of Tyrosine-Phosphorylated Polypeptides

To identify tyrosine-phosphorylated proteins associated with particulate sperm structures, a sequential extraction regimen was used. Capacitated spermatozoa (106 cells) were extracted with 0.1% Triton X-100 in TNI for 1 h at 4°C followed by centrifugation at 12 000 x g for 10 min at 4°C. The supernatant was collected and the sperm pellet was re-extracted in TNI containing 0.1% Triton X-100 and 32 mM DTT for 1 h at 4°C followed by centrifugation at 12 000 x g for 10 min at 4°C. The supernatant was collected and the sperm pellet was resuspended in TNI. All fractions were adjusted to the same volume and fractionated by SDS-PAGE followed by immunoblot analyses.

Purification of Tyrosine-Phosphorylated PHGPx

Capacitated spermatozoa were extracted in TNI containing 0.1% Triton X-100, the sperm pellet was then re-extracted in TNI containing 0.1% Triton X-100 and 32 mM DTT for 1 h at 4°C and centrifuged at 12 000 x g for 10 min. The supernatant was dialyzed against 25 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100 (elution buffer), and then applied to a 1.6 x 2.0-cm DEAE-Sephadex column equilibrated with elution buffer. The column was washed extensively with elution buffer and bound polypeptides were eluted with 0.4 M NaCl in elution buffer. An immunoaffinity column (AminoLink Plus, Pierce Chemical Co., Rockford, IL) was prepared by coupling 1 ml resin, at pH 7.2, to phosphotyrosine monoclonal antibody (anti-pY20), following the procedure provided by the manufacturer. The column was washed with TNI containing 0.1% Triton X-100 (TNI-TX). The DEAE column fraction, containing the 19-kDa polypeptide, was dialyzed against TNI-TX and applied to the immunoaffinity column, followed by extensive washes with TNI-TX. The column was eluted with 2 ml of 0.1 M glycine-HCl, pH 2.5, and the eluate was neutralized to pH 7.0, dialyzed against water, freeze dried, analyzed by 12% SDS-PAGE, and stained with Coomassie Brilliant Blue R. The 19-kDa band was excised and the proteomic analysis was done by µLC-MS-MS.

Preparation of PHGPx Antibody

Cauda epididymides were dissected and minced in calcium-free Tyrode solution at 37°C. The sperm suspension was centrifuged at 100 x g for 1 min to sediment tissue fragments, and the supernatant was recentrifuged at 1500 x g for 10 min at 4°C to obtain a sperm pellet. Sperm tails were isolated according to our published procedure [45], extracted with 32 mM DTT in TNI, and centrifuged at 12 000 x g for 10 min at 4°C. The 19-kDa polypeptide in the DTT-soluble flagellar fraction was purified by preparative SDS-PAGE on 12% acrylamide gels, the 19-kDa band was excised, and emulsified in Freund adjuvant, and used for antibody production. A New Zealand White Rabbit was given a primary injection of 19-kDa polypeptide followed by two booster injections at 3-wk intervals. Two weeks after the final booster injection, the rabbit was anesthetized with Nembutal, blood was collected by cardiac puncture, and a serum fraction was prepared. To prepare a PHGPx affinity column, PHGPx was purified from the DTT-soluble flagellar fraction by continuous-elution SDS-PAGE on 12% acrylamide gels using a Model 491 Prep Cell (Bio-Rad Laboratories, Hercules, CA) and coupled to an AminoLink Plus resin, at pH 10.0, following the procedure provided by the manufacturer (Pierce Chemical Co., Rockford, IL). Either whole serum or the monospecific IgG fraction, purified on the PHGPx affinity column, was used for immunocytochemistry, immunoblotting, and immunoprecipitation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pattern of Capacitation-Dependent Tyrosine-Phosphorylated Polypeptides of Cauda Epididymal Spermatozoa

Western blots of capacitated sperm lysates stained with anti-pY20 revealed a spectrum of tyrosine phosphorylated polypeptides of Mr 19 000–99 000 (Fig. 1A, lane 2); no bands were detected in the lysates of noncapacitated spermatozoa (Fig. 1A, lane 1). When the anti-pY20 was preincubated with o-phospho-DL-tyrosine and then used for immunoblotting, no stained bands were observed, suggesting that the polypeptides were specifically phosphorylated on tyrosine residues (data not shown). Blots were subsequently stained with Coomassie Brilliant Blue R (CBB) and an identical pattern of CBB-stained polypeptides was exhibited in the lysates of both noncapacitated (Fig. 1A, lane 3) and capacitated (Fig. 1A, lane 4) spermatozoa. CBB-stained and anti-pY20-stained blots showed that many of the tyrosine-phosphorylated polypeptides are not the major sperm proteins observed by CBB staining.



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  		FIG. 1. Protein tyrosine-phosphorylation pattern of cauda epididymal spermatozoa. A) Western blots of noncapacitated (lanes 1 and 3) and capacitated (lanes 2 and 4) spermatozoa fractionated by reducing SDS-PAGE and stained with antiphosphotyrosine (anti-pY20, lanes 1 and 2) or Coomassie Brilliant Blue (CBB, lanes 3 and 4). No tyrosine phosphorylation was seen in noncapacitated spermatozoa at Time 0 (lane 1). Capacitated spermatozoa (lane 2) exhibit an array of tyrosine-phosphorylated polypeptides at 180 min, including a 19-kDa polypeptide. Equal numbers of spermatozoa were loaded in each lane. B) Time-dependent appearance of tyrosine-phosphorylated sperm proteins during capacitation. Spermatozoa were incubated in capacitation medium for the times indicated at the top of the figure, extracted in SDS-sample buffer, and then subjected to Western blot analysis. C) Densitometric analysis of tyrosine phosphorylation of the 19-kDa polypeptide. D) Immunoblot showing total sperm lysate (lane 1), Triton X-100-soluble sperm proteins (lane 2), Triton X-100-DTT-soluble sperm proteins (lane 3), and Triton X-100-DTT-insoluble proteins (pellet, lane 4) of capacitated spermatozoa stained with anti-pY20

An array of polypeptides (Mr 19 000–99 000) displayed a time-dependent increase in the phosphorylation of tyrosine residues during capacitation (Fig. 1B, lanes 1–8). Among the tyrosine-phosphorylated polypeptides, the tyrosine phosphorylation of the 19-kDa polypeptide initiates after 30 min of capacitation (Fig. 1B, lane 3). Densitometric analysis of the tyrosine phosphorylation of the 19-kDa polypeptide (Fig. 1C) reveals that the tyrosine phosphorylation reaches a plateau after 2–3 h of capacitation; routinely, 3 h of incubation time was used in the subsequent experiments.

Western blots of the total lysates of capacitated spermatozoa (Fig. 1D, lane 1), Triton X-100-soluble fraction (Fig. 1D, lane 2), Triton X-100-DTT-soluble fraction (Fig. 1D, lane 3), and the sperm pellet obtained after Triton X-100-DTT extraction (Fig. 1D, lane 4) stained with anti-pY20 exhibited the complete solubilization of the 19-kDa polypeptide in the presence of both Triton X-100 and DTT. Most other tyrosine-phosphorylated polypeptides remained in the pellet.

Immunofluorescence Localization of Tyrosine-Phosphorylated Polypeptides

The flagellum of capacitated spermatozoa exhibited an intense immunostaining with anti-pY20, while the head was unstained (Fig. 2B). The principal piece segment of the flagellum displayed brighter fluorescence than the midpiece. In contrast, noncapacitated spermatozoa stained with anti-pY20 displayed no fluorescence (Fig. 2D). When anti-pY20 was preabsorbed with o-phospho-DL-tyrosine and used for staining, no fluorescence of the flagellum of the capacitated spermatozoa was observed (data not shown). This experiment demonstrates that capacitation-dependent tyrosine-phosphorylated proteins localize to the flagellum of hamster spermatozoa.



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  		FIG. 2. Immunocytochemical localization of tyrosine-phosphorylated proteins in capacitated (A and B) and noncapacitated (C and D) spermatozoa. Matched phase contrast (A and C) and fluorescence (B and D) photomicrographs of spermatozoa stained with anti-pY20. h, head; mp, midpiece; pp, principal piece. Bar = 10 µm

Purification and Identification of 19-kDa Polypeptide

The 19-kDa polypeptide was released from capacitated spermatozoa by Triton X-100-DTT extraction and then purified by ion-exchange chromatography on DEAE-Sephadex followed by immunoaffinity chromatography on phosphotyrosine antibody-coupled beads. The Triton X-100-DTT-soluble fraction exhibited a complex polypeptide pattern by SDS-PAGE and CBB staining (Fig. 3A, lane 1). In contrast, the purified fraction contained the 19-kDa polypeptide in addition to a few minor bands (Fig. 3A, lane 2). Western blots of the Triton X-100-DTT-soluble fraction (lane 3) and the purified fraction (lane 4) stained with anti-pY20 revealed the presence of the tyrosine-phosphorylated 19-kDa polypeptide in both fractions. Proteomic identification of the 19-kDa polypeptide by µLC-MS-MS analysis yielded six peptides (Fig. 3B) that matched the NCBI protein database sequences of bovine PHGPx.



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  		FIG. 3. A) Coomassie Blue-stained (lanes 1 and 2) and anti-pY20 stained immunoblots (lanes 3 and 4) demonstrating purification of the hamster sperm 19-kDa tyrosine-phosphorylated polypeptide. Lane 1 shows the complex polypeptide pattern present in the Triton X-100-DTT extract of capacitated spermatozoa. Lane 2 demonstrates the purification of the 19-kDa polypeptide by sequential DEAE-Sephadex ion-exchange and anti-pY20 immunoaffinity chromatography. Immunoblots reveal the presence of the 19-kDa tyrosine-phosphorylated polypeptide both in the Triton X-100-DTT-soluble fraction (5 µg protein, lane 3) and in the purified fraction (1 µg protein, lane 4). B) Proteomic identification of the 19-kDa polypeptide by µLC-MS-MS analysis yielded six peptides that matched the NCBI database sequences of PHGPx

Isolation of PHGPx from Sperm Tails for Antibody Preparation

PHGPx is a component of the disulfide-stabilized sperm mitochondrial capsule [38] that can be extracted from the tails with DTT. Tails were isolated from sonicated sperm by centrifugation on discontinuous sucrose density gradients and extracted in TNI containing 32 mM DTT [45]. The DTT-soluble supernatant obtained after centrifugation exhibits a spectrum of polypeptides by reducing SDS-PAGE and CBB staining (Fig. 4A); the major polypeptides are 19 kDa, 26 kDa, and 60 kDa. Proteomic identification of the 19-kDa polypeptide by MALDI-TOF analysis yielded eight peptides (Fig. 4B) that matched the NCBI database sequences of mouse PHGPx. PHGPx was purified from the DTT-soluble fraction of sperm tails by preparative SDS-PAGE and used for the preparation of a specific polyclonal antibody.



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  		FIG. 4. A) Coomassie Blue-stained SDS-PAGE of the DTT-soluble fraction of isolated hamster sperm tails. B) Tryptic peptides of the 19-kDa polypeptide identified by MALDI-TOF

Immunofluorescence Localization of PHGPx

Cauda epididymal spermatozoa immunostained with anti-PHGPx exhibited specific staining of the midpiece of the flagellum; the head and principal piece appear negative (Fig. 5A, panel b). Sperm stained with nonimmune serum exhibited no fluorescence of the flagellum (data not shown).



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  		FIG. 5. A) Paired phase-contrast (panel a) and fluorescence (panel b) images of hamster spermatozoa stained with anti-PHGPx. Bar = 10 µm. B) Immunoblot showing total sperm lysate (lane 1), Triton X-100-soluble sperm proteins (lane 2), Triton X-100-DTT-soluble proteins (lane 3), and Triton X-100-DTT-insoluble proteins (pellet, lane 4) stained with anti-PHGPx. Each lane was loaded with extracts representing 1 x 106 spermatozoa

Western blots of total sperm lysate, Triton X-100-soluble fraction, Triton X-100-DTT-soluble fraction, and Triton X-100-DTT pellet stained with anti-PHGPx exhibited the presence of PHGPx in the sperm lysate (Fig. 5B, lane 1) and in the supernatant fraction obtained after Triton X-100-DTT (Fig. 5B, lane 3) extraction. No immunoreactive PHGPx band was detected in the supernatant fraction of Triton X-100-extracted spermatozoa (Fig. 5B, lane 2) and Triton X-100-DTT pellet (Fig. 5B, lane 4). No band was seen when an identical blot was stained with nonimmune serum (data not shown). The solubility pattern of PHGPx in noncapacitated and capacitated spermatozoa was identical; PHGPx was extracted only in the presence of DTT.

Generation of PHGPx Isoforms During Capacitation of Hamster Spermatozoa

To examine the generation of PHGPx isoforms during capacitation, the DTT-soluble proteins were separated by two-dimensional PAGE and subjected to immunoblot analyses. Blots stained with anti-PHGPx exhibited a charge train of four distinct spots with isoelectric points ranging between pH 7.5 and 9.0 (#1) and one spot of pI 5.2 (#2) in both noncapacitated (Fig. 6A) and capacitated (Fig. 6B) spermatozoa. However, two-dimensional PAGE of capacitated spermatozoa (Fig. 6B) also revealed the generation of PHGPx isoforms with isoelectric points ranging between pH 6.0 and 7.0 (#3) and with pIs ranging between 4.0 and 5.0 (#4). This demonstrates that posttranslational modification of PHGPx occurs during capacitation.



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  		FIG. 6. Western blots, stained with anti-PHGPx, of DTT-soluble polypeptides of noncapacitated (A) and capacitated (B) spermatozoa fractionated by two-dimensional PAGE. Both noncapacitated and capacitated spermatozoa exhibited a charge train of four spots (#1) with isoelectric points ranging between 7.5 and 9.0 and one spot of pI 5.2 (#2); in addition, capacitated spermatozoa revealed the generation of new PHGPx isoforms toward the acidic range with pIs between pH 6.0 and 7.2 (#3) and 4.0 and 5.0 (#4)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Protein tyrosine phosphorylation is one of the important biochemical mechanisms regulating sperm capacitation [3, 15]. In the present study, we found the time-dependent increase in protein tyrosine phosphorylation of hamster spermatozoa in media that supports capacitation. It has been proposed that the tyrosine phosphorylation of an 80-kDa hamster sperm protein, localized to the principal piece of the flagellum, is associated with the acquisition of hyperactivation of sperm motility [46]. Visconti et al. [25] also observed a capacitation-associated increase in protein (Mr 30 000–150 000) tyrosine phosphorylation in hamster spermatozoa. However, their figures also show a capacitation-dependent tyrosine-phosphorylated band less than 24 kDa, which may represent PHGPx. Differences in staining intensity could be due to the different source of phosphotyrosine monoclonal antibody used in this study. The signaling cascade and the tyrosine kinase(s) that regulate the phosphorylation of these substrates have yet to be identified. Kalab et al. [47] demonstrated that the 20-kDa tyrosine-phosphorylated boar sperm polypeptide was the only polypeptide that can be extracted completely in Triton X-100 among the tyrosine phosphorylated polypeptides (Mr 20 000–230 000). However, the localization and the biochemical characterization of the 20-kDa boar sperm polypeptide remain to be elucidated. Our data reveal several new findings, including the localization of capacitation-dependent tyrosine-phosphorylated polypeptides to both the midpiece and the principal piece of hamster spermatozoa and the biochemical characterization of the 19-kDa tyrosine-phosphorylated polypeptide in the Triton X-100 plus DTT-soluble fraction demonstrates that the 19-kDa polypeptide is a disulfide cross-linked, membrane-anchored component.

The molecular mechanism of mammalian sperm capacitation is still poorly understood. Recently, work from several laboratories regarding the identification of tyrosine-phosphorylated polypeptides during capacitation is contributing to our understanding of the molecular basis of sperm capacitation. A calcium-binding polypeptide (CABYR) localized to the fibrous sheath of human sperm flagellum becomes tyrosine phosphorylated during capacitation [33]. Two other protein components of the fibrous sheath (AKAP4 and AKAP3) were previously shown to be phosphorylated during in vitro capacitation [35–37]. Ficarro et al. [34] demonstrated that the tyrosine phosphorylation of a valosin-containing protein (VCP) and the translocation of VCP from the neck to the anterior head occurred during capacitation of human spermatozoa. In the present study, the proteomic identification of the 19-kDa tyrosine-phosphorylated polypeptide purified from the Triton X-100 plus DTT-soluble fraction reveals that the polypeptide is PHGPx, suggesting that the tyrosine phosphorylation of the structural protein of the sperm mitochondrial capsule occurs during capacitation in a time-dependent manner. At present, the molecular characterization of the 19-kDa tyrosine-phosphorylated protein present in the Triton X-100-soluble fraction is not known. Future studies will address this issue.

It is well established that the mammalian testis possesses the highest activity of PHGPx of all mammalian tissues investigated [48, 49] and PHGPx is expressed specifically by spermatids [50, 51]. During differentiation of spermatids into mature spermatozoa, the enzymatically active PHGPx switches to an enzymatically inactive, disulfide cross-linked, insoluble structural protein of the sperm mitochondrial capsule [38, 52]. In the present study, we found that hamster sperm PHGPx is solubilized by 32 mM DTT (Fig. 5B, lane 3). Ursini et al. [38] used a different disulfide-cleavable compound (mercaptoethanol) and guanidine hydrochloride (a denaturing agent) for the solubilization of rat sperm PHGPx. Both studies confirm that the PHGPx of mammalian spermatozoa from the cauda epididymis is a disulfide cross-linked structural protein of the mitochondrial capsule. It is believed that male infertility in selenium-deficient animals, characterized by impaired sperm motility and structural abnormalities of sperm tails, is due to insufficient PHGPx content [53, 54]. Tyrosine 130 of PHGPx is the potential tyrosine-phosphorylation consensus motif as determined by the phosphorylation site prediction program (NetPhos 2.0 Server). At present, the downstream effect(s) of tyrosine phosphorylation of PHGPx is not known. One possibility is that tyrosine phosphorylation can reactivate the catalytic activity of PHGPx. A second potential function is that tyrosine-phosphorylated PHGPx may interact with other signaling protein(s) that could potentially affect mitochondrial function and/or participate in one of the pathways regulating the hyperactivation of sperm motility. Finally, sperm mitochondria degenerate in the egg shortly after fertilization [1, 55] and tyrosine phosphorylation of PHGPx may regulate the stability of the mitochondrial capsule. This is analogous to somatic cells, where phosphorylation of the nuclear lamins regulates the disassembly of the nuclear envelope during mitosis [56]. Additional studies are needed to resolve these issues.

In the present study, we have demonstrated posttranslational modifications of PHGPx during capacitation of hamster spermatozoa. Tyrosine phosphorylation contributes to the generation of a complex charge-variant pattern of PHGPx with multiple isoforms. Whether other posttranslational modifications, such as serine/threonine phosphorylation or tyrosine nitration, also contribute to the charge heterogeneity of PHGPx remains to be investigated. In conclusion, our data suggest that the tyrosine phosphorylation of PHGPx may represent an important event in the signaling cascade(s) associated with capacitation that may impact the regulation of hyperactivation of sperm motility and/or mitochondrial function.


   FOOTNOTES
 
1 Supported by HD044863. Back

2 Correspondence. FAX: 615 343 4539; subir.k.nag.das{at}vanderbilt.edu Back

Received: 23 June 2004.

First decision: 12 July 2004.

Accepted: 3 September 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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