22 Jan

Isoforms of the Mitochondrial Capsule Protein: MATERIALS AND METHODS

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 75mm 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 |xM epinephrine 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.

SDS-PAGE

One-dimensional SDS-PAGE was performed on 12% continuous or 7.5-15% gradient gels. 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.

Two-dimensional PAGE was performed using a Bio-Rad precast immobilized pH 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 benza-midine, 1 |xg/ml leupeptin, 1 |xg/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.

Proteomic Analysis

Proteomic analysis of the peptides was performed either at the Harvard Microchemistry Facility using microcapillary reverse-phase HPLC nanoelectrospray tandem mass spectrometry (|xLC-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, an-tiphosphotyrosine 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 immu-noaffinity 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 ^LC-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, 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 immunocytochem-istry, immunoblotting, and immunoprecipitation.

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