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Research Article |
Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
ABSTRACT
Sperm mitochondria undergo remodeling during posttesticular maturation that includes extensive disulfide cross-linking of proteins of the outer membrane to form the insoluble mitochondrial capsule. The relationship of these changes to mitochondrial function in mature gametes is unclear. The phospholipid hydroperoxide glutathione peroxidase (GPX4; also termed PHGPx) represents a major disulfide bond-stabilized protein of the mitochondrial capsule, and it is readily released by disulfide-reducing agents. However, in addition to GPX4, we detected a second major protein of 26 kDa (MP26) that was eluted from purified hamster sperm tails by the disulfide-reducing agent dithiothreitol. The objectives of the present study were to identify and characterize MP26 and to explore its potential role in mitochondrial function. Proteomic analysis of MP26 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identified 14 peptides with sequence identity to a member of the short-chain dehydrogenase/reductase superfamily termed P26h, which was implicated previously in hamster sperm-zona binding, and with high sequence similarity to mouse lung carbonyl reductase. Indirect immunofluorescence localized MP26 to the midpiece, and two-dimensional PAGE and immunoblot analysis identified a single MP26 isoform of pI 9.0. Immunoblot analyses of cauda epididymal fluid and of purified sperm plasma membranes and mitochondria revealed the exclusive localization of MP26 to the mitochondrial fraction. These data indicate that MP26 does not function in zona binding; instead, like GPX4, it may be associated with the mitochondrial capsule and play an important role in sperm mitochondrial function.
epididymis, gamete biology, sperm, spermatogenesis
INTRODUCTION
Mammalian spermatozoa undergo a wide variety of changes as they migrate through the epididymis to acquire progressive motility and fertilization potential [1, 2]. One modification is a significant increase in disulfide cross-linking of several flagellar structures, including the mitochondria, in which intermolecular disulfide bonds stabilize the outer mitochondrial membrane, forming a keratin-like mitochondrial capsule [3–8]. The selenoprotein phospholipid hydroperoxide glutathione peroxidase (GPX4; also termed PHGPx) is the major disulfide cross-linked sperm mitochondrial protein, and it plays a significant role in forming the mitochondrial capsule [9]. Other investigators have reported that the sperm mitochondria-associated cysteine-rich protein also is a constituent of the mitochondrial capsule [10, 11], suggesting that this structure may be a complex of several interacting proteins. Although the function of the mitochondrial capsule remains unresolved, ultrastructural studies have demonstrated the association of the regulatory subunit RII of type II cyclic AMP-dependent protein kinase with the outer mitochondrial membrane [12], and two different A kinase-anchoring proteins have been localized to the sperm midpiece [13, 14]. These studies suggest that the mitochondrial capsule might localize a variety of signaling proteins to the midpiece and, thus, could play a critical role in sperm function.
Bedford and Calvin [5] demonstrated that the detergent-dependent solubilization of the outer mitochondrial membrane of mature rat spermatozoa required reduction of disulfide bonds with a disulfide-reducing agent, such as dithiothreitol (DTT), and several studies have shown that the mitochondrial capsule protein GPX4 is released from mitochondria by extraction with disulfide-reducing agents [9, 15]. We detected several polypeptides extracted from purified hamster sperm flagella by DTT, including GPX4 (19 kDa) and a major polypeptide of 26 kDa (MP26), suggesting that it may be associated with the mitochondrial capsule. In the present study, we used both proteomic and immunocytochemical approaches to characterize and to localize MP26 to address its potential role in mitochondrial function. The results presented here demonstrate that MP26 is localized to the mitochondria of the flagellum and is a member of the short-chain dehydrogenase/reductase superfamily.
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 Vanderbilt University's Institutional Animal Care and Use Committee. Mature male golden hamsters were housed in a University facility on a 14L:10D photoperiod and were given free access to food and water. Animals were asphyxiated with CO2, and tissues were collected.
Preparation of Cauda Epididymal Sperm Fractions and Luminal Fluid Samples
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. The sperm pellets were used in the fractionation protocols described below, whereas the supernatant fluid, containing soluble cauda epididymal luminal proteins, was recentrifuged at 100 000 x g for 15 min in a Beckman TL55 rotor. Plasma membrane vesicles from cauda epididymal spermatozoa were isolated according to our previously published procedure [16].
Preparation of Cell Fractions for Western Blot Analysis
To prepare soluble and particulate fractions, cauda epididymal spermatozoa were suspended in TNI (150 mM NaCl, 25 mM Tris-HCl [pH 7.5], 2 mM benzamidine, 1 µg/ml of leupeptin, 1 µg/ml of pepstatin, 1 mM sodium fluoride, 1 mM sodium vanadate, and 0.05% sodium azide), sonicated for four 10-sec intervals with a Branson sonifier at a medium power setting, and centrifuged at 100 000 x g for 30 min in a TL55 rotor (Beckman). To elucidate the solubility of MP26, a sequential extraction regimen was used. Cauda epididymal spermatozoa (106 cells/ml) 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 fractions representing equal sperm numbers were separated by SDS-PAGE for immunoblot analysis.
Isolation of Hamster Sperm Mitochondria
Mitochondria were isolated according to the method described by Hecht and Bradley [17]. Washed pellets of hamster cauda epididymal spermatozoa were suspended in TNI and then sonicated using a blunt probe for three 10-sec intervals with a Branson sonifier at a medium power setting. The sonicated suspension was centrifuged at 1500 x g for 10 min at 4°C, and the sperm pellet was washed two times in TNI by centrifugation at 1500 x g for 10 m. The pellet was resuspended in TNI and then sonicated using a microtip for ten 20-sec intervals at a medium power setting to detach the mitochondria. The homogenized suspension was centrifuged at 1000 x g for 5 min at 4°C to sediment flagellar fragments and heads, and the supernatant was mixed with a one-third volume of a 20% sucrose solution and then centrifuged at 8800 x g for 15 min. The crude mitochondrial pellet was resuspended in a 20% sucrose solution and homogenized with a glass-Teflon homogenizer, and aliquots of the homogenized suspension (3 ml) containing mitochondria were layered on 25 ml of 30%–65% continuous sucrose gradients containing 150 mM NaCl and 25 mM Tris-HCl (pH 7.5). The gradients were centrifuged at 80 000 x g for 2 h in a SW28 rotor (Beckman). The top band, containing mitochondria, was collected, diluted with TNI, and pelleted by centrifugation at 100 000 x g for 1 h in a SW41 rotor (Beckman). Mitochondrial pellets were either used for SDS-PAGE or, to insure sample purity, were prepared for electron microscopic analysis. These samples were fixed in 4% glutaraldehyde in 0.1 M sodium cacodylated buffer, postfixed in 1% OsO4, and embedded in epoxy resin.
Preparation of MP26 Antibody
Sperm tails, isolated according to our previously published procedure [18], were extracted with 32 mM DTT in TNI overnight at 4°C and then centrifuged at 12 000 x g for 10 min at 4°C. The 26-kDa polypeptide in the DTT-soluble fraction was purified by preparative SDS-PAGE on 12% acrylamide gels, and the 26-kDa band was excised, emulsified in Freund adjuvant, and used for antibody production in female guinea pigs. Animals received a total of three antigen injections at 3-wk intervals. Two weeks after the final booster injection, they were anesthetized with Nembutal, after which blood was collected by cardiac puncture and a serum fraction prepared. Monospecific antibody to MP26 (anti-MP26) was then prepared by immunoaffinity chromatography on a MP26 column. The MP26 was purified from flagellar DTT extracts by continuous-elution SDS-PAGE on 12% acrylamide gels using a Model 491 Prep Cell (Bio-Rad Laboratories) and coupled to AminoLink Plus resin (Pierce Chemical Co.) at pH 10.0 according to the manufacturer's instructions. Immune serum was applied to the column, and after washing with TBS (150 mM NaCl, 25 mM Tris-HCl, pH 7.5), bound antibody was eluted with 0.1 M glycine-HCl (pH 2.8). The eluate was neutralized with 1 M Tris and then dialyzed against TBS. The monospecific MP26 was used for immunohistochemistry and immunoblotting.
SDS-PAGE, Western Blot Analysis, and Proteomic Analysis
Samples were solubilized in SDS sample buffer at 95°C in the presence of 32 mM DTT and then analyzed by SDS-PAGE using 12% continuous polyacrylamide gels [19]. To visualize the total polypeptide pattern, gels were stained with Coomassie Brilliant Blue R [20]. For two-dimensional PAGE, Bio-Rad precast immobilized pH-gradient (pH 3–10) strips (7 cm) were used for isoelectric focusing (IEF) in a Bio-Rad Protean IEF cell according to the manufacturer's instructions. For immunoblot analysis, polypeptides were electrophoretically transferred to polyvinylidene fluoride membranes [21]. Protein concentration of the samples was estimated using the method described by Bradford [22].
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 anti-MP26, or, for controls, in nonimmune guinea pig, diluted (1:2000) 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-guinea pig IgG (KPL Laboratories) diluted (1:4000) 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).
Proteomic identification of MP26 was performed at the Vanderbilt University Proteomics Core Laboratory. The MP26 band, excised from SDS-PAGE gels, was subjected to trypsin digestion, and the peptide sequences were determined by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF). Derived peptide sequences were identified using the National Center for Biotechnology Information (NCBI) Blast programs.
Immunocytochemistry
Spermatozoa were fixed for 1 h 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 and 20 mM sodium phosphate (PBS; pH 7.4), attached to poly-L-lysine-coated coverslips, and then permeabilized in acetone at –20°C for 10 min. Cells were rinsed in TNT (0.1% Tween 20, 150 mM NaCl, and 20 mM Tris-HCl [pH 8.0]) 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-MP26 or, for controls, in nonimmune IgG, diluted in blocking solution. Coverslips were then rinsed three times in TNT containing 1% goat serum and incubated for 2 h at room temperature in Cy3-conjugated donkey anti-guinea pig IgG (1:2000; Jackson ImmunoResearch Laboratories) diluted in blocking solution. Following several washes in TNT, the cells were photographed using epifluorescence and phase-contrast microscopy.
RESULTS
Isolation and Identification of Flagellar MP26
Major mitochondrial capsule proteins are readily extracted by solutions containing disulfide-reducing reagents [9, 15]. Flagella, purified from cauda epididymal spermatozoa, were incubated in extraction solutions with or without the disulfide-reducing agent DTT and examined by phase-contrast microscopy. The midpiece of flagella incubated in TNI lacking DTT appeared smooth (Fig. 1a), but the midpiece of flagella extracted with DTT appeared rough and displayed detaching, phase-dense vesicles (Fig. 1b), indicating partial disruption of the mitochondrial sheath.
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The DTT-soluble flagellar fraction exhibited a spectrum of polypeptides by reducing SDS-PAGE and Coomassie blue staining (Fig. 2a). The major polypeptides were 19 kDa (GPX4), 26 kDa (MP26), and 60 kDa. Proteomic analysis of MP26 by MALDI-TOF yielded 14 peptides (Fig. 2b) that matched the NCBI database sequences of a member of the short-chain dehydrogenase/reductase superfamily, which was previously identified as a hamster surface sperm protein, P26h [23], and as mouse lung carbonyl reductase [24]. Our sequence data exhibited 38% coverage of the P26h protein.
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Characterization of Anti-MP26 and Immunolocalization of MP26
Western blot analysis demonstrated the presence of MP26 in testis lysates (Fig. 3a, lane 1), in flagella purified from cauda spermatozoa (Fig. 3a, lane 2), and in the DTT-soluble flagellar fraction (Fig. 3a, lane 3). No MP26 was detected in the particulate flagellar fraction after DTT extraction (Fig. 3a, lane 4). No bands were detected when an identical blot was stained with nonimmune serum (data not shown). Immunoblots of total sperm lysate analyzed by two-dimensional PAGE showed the presence of one isoform of MP26 exhibiting pI 
9.0 (Fig. 3b), which is similar to the reported pI (9.0) of P26h [25]. No spot was seen when an identical blot was stained with nonimmune guinea pig IgG (data not shown).
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Testicular spermatozoa stained with anti-MP26 exhibited specific staining of the midpiece and the cytoplasmic droplet (Fig. 4b), whereas in cauda epididymal spermatozoa, staining was restricted to the midpiece (Fig. 4d). The head and principal piece displayed no detectable staining. Sperm stained with nonimmune guinea pig IgG exhibited no fluorescence (data not shown).
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Biochemical Fractionation of Sperm MP26
The distribution of MP26 in the soluble and particulate fractions of sonicated cauda epididymal spermatozoa was examined by Western blot analysis. The MP26 was present in both the total sperm lysate (Fig. 5, lane 1) and the particulate fraction (Fig. 5, lane 3). The MP26 was not present in the soluble fraction (Fig. 5, lane 2).
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To identify the association of MP26 with specific particulate sperm structures, a sequential extraction strategy was used. Western blots of the total sperm lysate (Fig. 6, lane 1), the Triton X-100-soluble sperm fraction (Fig. 6, lane 2), the Triton X-100-DTT-soluble sperm fraction (Fig. 6, lane 3), and the sperm pellet obtained after Triton X-100-DTT extraction (Fig. 6, lane 4 ) stained with anti-MP26 exhibited the presence of MP26 in the total sperm lysate (Fig. 6, lane 1) and in the supernatant fraction obtained after Triton X-100-DTT (Fig. 6, lane 3) extraction. No band was seen when an identical blot was stained with nonimmune serum (data not shown). These experiments demonstrate that MP26 is a disulfide-stabilized, insoluble flagellar protein.
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The MP26 was present neither in the sperm plasma membrane fraction nor in the cauda epididymal fluid. It has been reported [26] that MP26, previously termed P26h, is present in hamster epididymal fluid and binds the sperm periacrosomal plasma membrane; however, this suggestion was not supported by the above data. Therefore, we performed additional experiments to test if some MP26 also localizes to the sperm plasma membrane and/or epididymal luminal fluid. Previously, we raised a polyclonal antibody to a 23-kDa polypeptide (anti-23kDa) secreted by epididymal epithelium that binds to the plasma membrane of hamster spermatozoa [27]. The anti-23kDa was used as a specific marker of the isolated sperm plasma membrane fraction. Western blots of total sperm lysate (Fig. 7a, lane 1), a purified sperm plasma membrane fraction (Fig. 7a, lane 2), and cauda epididymal fluid (Fig. 7a, lane 3) stained with anti-23kDa, revealed the 23-kDa polypeptide in all fractions. When the blot was reprobed with anti-MP26, MP26 was detected only in the total sperm lysate (Fig. 7b, lane 1). The distribution pattern for GPX4, a 19-kDa mitochondrial capsule protein, was identical to that observed for MP26 (Fig. 7c).
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Biochemical Localization of MP26 in Hamster Sperm Mitochondria
Because MP26 is a member of the dehydrogenase/reductase superfamily and is localized exclusively to the midpiece, we next tested for the presence of MP26 in the mitochondrial fraction isolated from cauda epididymal spermatozoa. The homogeneity of the mitochondrial preparation was demonstrated by electron microscopy (Fig. 8a). Western blots of the DTT-soluble sperm tail fraction (Fig. 8b, lane 1) and of isolated mitochondria (Fig. 8b, lane 2) stained with anti-MP26 each exhibited the presence of MP26 (Fig. 8a). An identical blot stained with antibody to GPX4 showed the presence of a 19-kDa GPX4 band (mitochondrial capsule protein) in both fractions (Fig. 8b, lanes 3 and 4).
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DISCUSSION
We found three major polypeptides (60, 26, and 19 kDa) in the DTT-soluble supernatant of hamster sperm tails. In the present study, the proteomic identification of MP26 was performed using MALDI-TOF. Our data demonstrate that MP26 is a member of the short-chain dehydrogenase/reductase superfamily, with homology to mouse lung carbonyl reductase. Cauda sperm lysates analyzed by two-dimensional PAGE exhibited the presence of a single MP26 isoform (pI 9.0). The biochemical fractionation and immunocytochemical data presented here demonstrate that MP26 is localized to the mitochondrial fraction and is not a secretory product of the epididymis.
Initially, Sullivan and Bleau [28] identified a 26-kDa sperm glycoprotein, termed P26h, that is abundant in the luminal fluid of the proximal hamster epididymis. Epididymal fluid P26h accumulates on the acrosomal cap of hamster spermatozoa occurs as they travel through the epididymis [25, 29–30]. It was proposed that P26h is anchored to the membrane through a phosphaditylinositol linkage and that prostasome-like particles may be involved in the transfer of P26h from the epididymal fluid to the sperm plasma membrane [25]. Later, the same group of investigators cloned a cDNA from a hamster testicular cDNA library and reported that P26h is a member of the short-chain dehydrogenase/reductase superfamily [31]. They also found the predominant expression of P26h transcript in the seminiferous tubules of the testis, not the epididymis, and proposed that the epididymal luminal fluid P26h is secreted by the seminiferous tubules of the testis [31]. They also proposed that P26h may have two different functions, as observed for other moonlighting proteins [32]: first in the testis, as an alcohol dehydrogenase, and then in the epididymis, where it adsorbs on the sperm surface and plays a role during fertilization. The results of the present study are inconsistent with these previous results that MP26 is present in the epididymal fluid and adsorbs onto the periacrosomal plasma membrane of spermatozoa, where it functions in sperm-egg interaction. Furthermore, another group of investigators found that P26h exhibits 87% identity to mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase superfamily, and proposed that P26h mainly exists as a tetrameric dehydrogenase in the mitochondria of testicular cells [24]. Our data, along with the biochemical data of Ishikura et al. [24], however, demonstrate that the hamster sperm 26-kDa dehydrogenase/reductase (MP26) is synthesized in the testis and localizes only to the mitochondria of both testicular and epididymal hamster spermatozoa. This indicates that MP26 is not a moonlighting protein but, instead, exhibits a mitochondria-specific function. Furthermore, our biochemical data show that MP26 has solubility properties similar to those of GPX4, suggesting that it could be associated with the mitochondrial capsule.
The bulk of mitochondrial proteins are synthesized on cytosolic polysomes as precursor proteins carrying amino-terminal presequences that possess import sequence for their targeting to mitochondria [33]. The immunoreactivity observed in the cytoplasmic droplet of testicular spermatozoa (Fig. 4b) may represent a cytosolic form of MP26 that has not yet been imported into the mitochondria. Future studies will address this issue.
Reactive oxygen species and carbonyl compounds are formed in several physiological pathways. They are produced by damaged and immature spermatozoa, are associated with a loss of sperm motility, and have a detrimental effect on sperm-oocyte fusion [34–39]. The primary cellular enzymatic defense systems against damage from reactive oxygen species are the glutathione redox cycle, the thioredoxin cycle, and catalase [40]. Lung carbonyl reductases are involved in the detoxification of carbonyl compounds derived from lipid peroxidation [41, 42]. Ishikura et al. [24] proposed that P26h plays a role in controlling the concentration of 5
-dihydrotestosterone during spermatogenesis. Alternatively, the MP26 localized in sperm mitochondria may be involved in the detoxification of the carbonyl compounds derived from lipid peroxidation, which might provide an enzymatic prevention system in sperm against damage from carbonyl compounds. Further studies will better define the role of MP26 in sperm function.
FOOTNOTES
1 Supported by HD044863 to G.E.O. 
2 Correspondence and current address: Subir K. Nagdas, Department of Natural Sciences, Fayetteville State University, 1200 Murchison Road, Fayetteville, NC 28301. FAX: 910 672 1159; snagdas{at}uncfsu.edu
Received: 1 February 2006.
First decision: 14 February 2006.
Accepted: 8 May 2006.
REFERENCES
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