Dual Function of the Selenoprotein PHGPx During Sperm Maturation

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Science  27 Aug 1999:
Vol. 285, Issue 5432, pp. 1393-1396
DOI: 10.1126/science.285.5432.1393


The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) changes its physical characteristics and biological functions during sperm maturation. PHGPx exists as a soluble peroxidase in spermatids but persists in mature spermatozoa as an enzymatically inactive, oxidatively cross-linked, insoluble protein. In the midpiece of mature spermatozoa, PHGPx protein represents at least 50 percent of the capsule material that embeds the helix of mitochondria. The role of PHGPx as a structural protein may explain the mechanical instability of the mitochondrial midpiece that is observed in selenium deficiency.

Selenium is essential for male fertility in rodents and has also been implicated in the fertilization capacity of spermatozoa of livestock and humans (1). Selenium deficiency is associated with impaired sperm motility, structural alterations of the midpiece, and loss of flagellum (1). However, three decades after the discovery of selenium as an integral constituent of redox enzymes (2), the molecular basis of the relationship of the essential trace element and male fertility remains obscure. The selenoprotein PHGPx (Enzyme Commission number is abundantly expressed in spermatids and displays high activity in postpubertal testis (3). In mature spermatozoa, however, selenium is largely restricted to the mitochondrial capsule, a keratin-like matrix that embeds the helix of mitochondria in the sperm midpiece (4). A “sperm mitochondria–associated cysteine-rich protein (SMCP)” (5) had been considered to be the selenoprotein accounting for the selenium content of the mitochondrial capsule (4–6). The rat SMCP gene, however, does not contain an in-frame TGA codon (7) that would enable a selenocysteine incorporation (8). In mice, the three in-frame TGA codons of the SMCP gene are upstream of the translation start (5). SMCP can therefore no longer be considered as a selenoprotein. Instead, the “mitochondrial capsule selenoprotein (MCS),” as SMCP was originally referred to (4–7), is here identified as PHGPx.

Routine preparations of rat sperm mitochondrial capsules (9) yielded a fraction that was insoluble in 1% SDS containing 0.2 mM dithiothreitol (DTT) and displayed a vesicular appearance in electron microscopy (Fig. 1A). The vesicles readily disintegrated upon exposure to 0.1 M mercaptoethanol (Fig. 1B) and became fully soluble in 6 M guanidine-HCl. When the solubilized capsule material was subjected to polyacrylamide gel electrophoresis (PAGE), four bands in the 20-kD region were detected (Fig. 1C, left lane). Protein immunoblotting (10) revealed that the most prominent band reacted with PHGPx antibodies (Fig. 1C, right lane). NH2-terminal sequencing (11) of the 21-kD band (46% of total protein content according to stain intensity) revealed that it consisted of at least 95% pure PHGPx. We therefore investigated the composition of the mitochondrial capsules by two-dimensional (2D) electrophoresis (12) (Fig. 2A) followed by microsequencing (13) or matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis (14) for identification (Fig. 2B). The spot migrating with an apparent molecular mass of 21 kD and focusing at a pH near 8 (spot 3) proved to be PHGPx, according to the masses of tryptic peptides detected by MALDI-TOF spectrometry (Fig. 2B). All tryptic fragments yielding MALDI-TOF signals of high intensity could be attributed to PHGPx or trypsin. The predicted NH2-terminal (positions 3 to 12) and COOH-terminal peptides (positions 165 to 170), the fragment corresponding to positions 100 to 105, and those expected from the basic sequence part (residues 119 to 151) were too small to be reliably identified. The fragment corresponding to positions 34 to 48 comprising the active site selenocysteine was not detected either. The more acidic spot 4 of Fig. 2A, the more basic spots 1, 2, and 5, and those exhibiting a smaller apparent molecular mass (spots 6 and 7) also contained PHGPx (15). Spots 1 to 6 were essentially homogeneous. Spot 7 showed a trace of impurity that could not be identified by masses of fragments. Integrated stain intensities of the individual spots indicate that PHGPx constituted about 50% of the capsule material.

Figure 1

Presence of PHGPx in the mitochondrial capsule of rat spermatozoa. (A) Mitochondrial capsule prepared by trypsination and centrifugation (4, 9). (B) The same preparation as shown in (A) but after exposure to 0.1 M 2-mercaptoethanol for 15 min at 4°C. Contamination of the capsule material by mitochondria is evident from the presence of mitochondrial ghosts (arrowhead). Scale bars, 0.3 μm. (C) SDS-PAGE of proteins extracted from capsule material by treatment with 0.1 M 2-mercaptoethanol, 0.1 M tris-HCl (pH 7.5), and 6 M guanidine-HCl. Left lane is stained with colloidal gold; right lane demonstrates presence of PHGPx by protein immunoblotting (10).

Figure 2

Analysis of the composition of the mitochondrial capsule of spermatozoa. (A) Two-dimensional electrophoresis of purified dissolved capsule material. Proteins were focused in a linear pH gradient from 3 to 10 (horizontal direction), then reduced, amidocarboxymethylated, subjected to SDS electrophoresis, and stained with Coomassie blue. MALDI-TOF analysis of the visible spots identified the following proteins (NCBI database): spots 1 to 7, PHGPx (accession number 544434); spots 8 and 9, outer dense fiber protein (accession number P21769); spots 10 and 11, voltage-dependent anion channel-like protein (accession number 540011); spot 12, “stress-activated protein kinase” (accession number 493207); spot 13, glycerol-3-phosphate dehydrogenase (accession number P35571) (22). (B) MALDI-TOF spectrum (overview) of tryptic peptides obtained from PHGPx as found in spot 3. Abscissa, mass/charge ratio (m/z) of the peptide fragments; ordinate, arbitrary units of intensity (a.i.). The insert lists the mass signals 1 to 17 attributed to tryptic fragments of PHGPx with measured m/z values and corresponding residues in the PHGPx sequence. Peaks 8 and 11 correspond to tryptic fragments with oxidized methionine residue; peak 15 corresponds to a fragment with an NH2-terminal pyroglutamyl residue. T, trypsin-derived fragments.

Minor components present in the gel (Fig. 2A, spots 10 to 13) were assigned to mitochondrial proteins or to cytosolic contaminations. Spots 8 and 9 consisted of “outer dense fiber protein,” a cysteine-rich structural sperm protein that is associated with the helix of mitochondria in the midpiece but also extends into the flagellum. SMCP was not detected. This basic protein that becomes superficially associated with the outer mitochondrial membranes in late spermatids and epididymal spermatozoa (5) might have been degraded by trypsination during capsule preparation.

PHGPx was enzymatically inactive in mature spermatozoa prepared from the tail of the epididymis and was not reactivated by the reduced form of glutathione (GSH) in the low millimolar range, as used under conventional test conditions (16). High concentrations of thiols (0.1 M 2-mercaptoethanol or DTT), which in the presence of guanidine fully dissolved the capsule, regenerated a substantial PHGPx activity (17). In fact, the specific activities thereby obtained from mitochondrial capsules (5600 ± 290 mU/mg protein) exceeded, by a factor of 20, the values measured in spermatogenic cells (250 ± 10 mU/mg). Nevertheless, the PHGPx activity regenerated from the capsule material was low relative to its PHGPx protein content. On the basis of the specific activity of pure PHGPx, the reactivated enzyme would be equivalent to less than 3% of the capsule protein, whereas the 2D electrophoresis suggested a PHGPx protein content of at least 50%. The increase of PHGPx activity by the reductive procedure was similarly observed in epididymal spermatozoa (from zero to 3140 ± 200 mU/mg protein) but not in spermatogenic cells from testicular tubules (250 ± 10 to 260 ± 10 mU/mg). The latter observation is consistent with the expression of PHGPx as active peroxidase in round spermatids (3). The switch of PHGPx from a soluble active enzyme to an enzymatically inactive structural protein thus occurs during differentiation of spermatids into spermatozoa (18).

The alternate roles of PHGPx as a glutathione-dependent hydroperoxide reductase or a structural protein are not necessarily unrelated. A feature common to all glutathione peroxidases is a selenocysteine residue, which, together with a tryptophan and a glutamine residue, forms a catalytic triad (19). Therein the selenol group of the selenocysteine residue is oxidized by hydroperoxides with high rate constants. The reaction product, a selenenic acid derivative, R-SeOH, reacts with GSH to form a selenadisulfide bridge between enzyme and substrate, R-Se-S-G, from which the ground-state enzyme is regenerated by a second GSH. In analogy, PHGPx, which is the least specific of the glutathione peroxidases (19), can use protein thiols as alternate substrates to create protein aggregates that are cross-linked by selenadisulfide or disulfide bonds. This likely occurs when cells are exposed to hydroperoxides at low concentrations of GSH, as is documented for late stages of spermatogenesis (20). Proteins derived from epididymal spermatozoa, when exposed to H2O2 in the absence of GSH, yielded a variety of PHGPx-containing aggregates (Fig. 3A). This process depends on the presence of thiol groups in proteins distinct from PHGPx, because under identical conditions only a marginal aggregate formation was observed with pure PHGPx (Fig. 3B).

Figure 3

Formation of PHGPx-containing aggregates by H2O2 in the absence of GSH. (A) Whole rat spermatozoa were solubilized with 0.1 M 2-mercaptoethanol and 6 M guanidine-HCl and freed from low molecular weight compounds as described (17). Aliquots of the protein mixture (0.05 mg of protein) were subjected to SDS-PAGE under reducing (lanes 1 and 2) and nonreducing conditions (lanes 3 and 4) at zero time (lanes 1 and 3) or after 15-min exposure to 75 μM H2O2(lanes 2 and 4). PHGPx-containing bands were detected by protein immunoblotting (10). (B) Lanes 1 to 4 show the same experiments but performed with purified rat testis PHGPx adjusted to the PHGPx content in solubilized spermatozoal proteins. Only traces of dimerized PHGPx and aggregates are seen in the sample exposed to H2O2 for 15 min (lane 4).

Our findings require a fundamental reconsideration of the role of selenium in male fertility. The predominance of the selenoprotein PHGPx in the male reproductive system (3) has been believed to reflect the necessity to shield germ line cells from oxidative damage by hydroperoxides (3, 20). This concept still merits attention with regard to the mutagenic potential of hydroperoxides and probably holds true for the early phases of spermatogenesis. At this stage, phenomena attributed to the enzymatic activity of PHGPx or other glutathione peroxidases—for instance, silencing lipoxygenases, dampening the activation of nuclear factor κB, or inhibiting apoptosis (21)—may also be relevant. Mature spermatozoa, however, depend on PHGPx as a structural protein, because the morphological midpiece alterations that are observed in selenium deficiency likely result from impaired biosynthesis of the selenoprotein. In consequence, it is not the antioxidant capacity of PHGPx but the ability to use hydroperoxides for the formation of a structural element of the spermatozoon that is pivotal for male fertility.

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