Isoforms of the Mitochondrial Capsule Protein: RESULTS
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.
An array of polypeptides (M 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.
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-Sephad-ex followed by immunoaffinity chromatography on phos-photyrosine 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 ^LC-MS-MS analysis yielded six peptides (Fig. 3B) that matched the NCBI protein database sequences of bovine PHGPx.
Isolation of PHGPx from Sperm Tails for Antibody Preparation
PHGPx is a component of the disulfide-stabilized sperm mitochondrial capsule 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. 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.
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).
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.
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 antiphospho-tyrosine (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-phos-phorylated polypeptides at 180 min, including a 19-kDa polypeptide. Equal numbers of spermatozoa were loaded in each lane. B) Time-dependent appearance of ty-rosine-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.
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 ^m.
FIG. 3. A) Coomassie Blue-stained (lanes 1 and 2) and anti-pY20stained 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 immunoaffnity chromatography. Immunoblots reveal the presence of the 19-kDa tyrosine-phosphorylated polypeptide both in the Triton X-100-DTT-soluble fraction (5 ^g protein, lane 3) and in the purified fraction (1 ^g protein, lane 4). B) Proteomic identification of the 19-kDa polypeptide by ^LC-MS-MS analysis yielded six peptides that matched the NCBI database sequences of PHGPx.
FIG. 5. A) Paired phase-contrast (panel a) and fluorescence (panel b) images of hamster spermatozoa stained with anti-PHGPx. Bar = 10 ^m. 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.
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).