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Testis |
Biology Laboratory,2
Department of Biochemistry,3
Department of Obstetrics and Gynecology,4 University of Yamanashi, Faculty of Medicine, Tamaho-cho, Yamanashi, 409-3898, Japan
Department of Histology and Embryology,5 Institute of Biology, State University of Campinas, Campinas, Brazil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reactive oxygen species are generated continually as by-products of aerobic metabolism in cells and are damaging to all cellular constituents, including protein, DNA, and lipids [3–5]. It has been proposed that oxidative damage is a possible cause of male infertility involving disruptions of spermatogenesis [6]. Thus, the action of antioxidant enzymes in spermatogenic cells would be essential for the maturation to achieve normal sperm function. Spermatogenic cells possess a range of enzymatic defense systems to counter oxidative stress, including superoxide dismutase, catalase, and a selenoenzyme, glutathione peroxidase [7, 8]. Nutritional studies indicate that selenium is essential for male fertility [9].
Among the four types of glutathione peroxidases, phospholipid hydroperoxide glutathione peroxidase (PHGPx; E.C. 1.11.1.12) is reported to be the most abundant in the rat testis [10]. Also, evidence has accumulated that PHGPx is a multifunctional protein (i.e., a structural protein as well as an antioxidant of mature sperm) [11]. Recently, such multifunctional proteins were termed moonlighting proteins [12]. In the rat testis, PHGPx is present in mitochondria and, to a lesser extent, in nuclei [13, 14]. In sperm mitochondria, disulfide bonds are formed between PHGPx during the final stages of spermiogenesis [11]. Consequently, the PHGPx in the mature sperm mitochondria, losing its enzymatic activity, functions as a structural protein of the mitochondrial capsule [11]. Also, study of the PHGPx levels in human sperm indicated that some cases of male infertility might be related to the reduced expression of PHGPx [15, 16]. In the sperm nucleus, PHGPx forms disulfide bonds between the nuclear protein protamines to condense the DNA during the final stages of spermiogenesis [1]. However, the clinical significance of PHGPx in the nucleus of human sperm is still not clear.
It is necessary to study the spatiotemporal changes in the expression of sperm PHGPx to reveal the biological significance of PHGPx during spermatogenesis and spermiogenesis, because a multifunctional PHGPx is thought to be essential for the spermatogenesis and the function of the mature sperm. Recently, Tramer et al. [17] demonstrated, using biochemical techniques, that the activity of PHGPx was highest in the testis of 3-mo-old rats and in the epididymal sperm of 6-mo-old rats. They also demonstrated, using electron microscopy, that the subcellular distributions of PHGPx in the spermatogenic cells and epididymal sperm were unchanged with aging. However, to our knowledge, the changes in the PHGPx levels of subcellular compartments in spermatogenic cells during the spermatogenesis and spermiogenesis have not been clarified.
To elucidate this issue, we adopted immunoelectron microscopic methods, which make possible the quantitative localization of antigen at each step of the cell's development; it is not easy to do this using biochemical methods because of the difficulty in isolating spermatogenic cells at the distinct step. Our goal was to reveal the roles of PHGPx in human fertility, so we generated an antibody against the carboxyl terminal region of human PHGPx. Because the primary structure of PHGPx is well conserved among vertebrates, our antibody against human PHGPx can be used for the study of rodent PHGPx. Here, we showed the spatiotemporal changes to PHGPx levels in subcellular compartments during spermatogenesis and spermiogenesis using adult rat testis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Japanese white rabbits (weight, 3–4 kg; age, 10 wk) and male Wistar rats (weight, 230–240 g; age, 9 wk) were fed appropriate standard diets and water ad libitum until used. The animal experiments were performed in accordance with the Guidance for Animal Experiments, University of Yamanashi.
Preparation of Antibody
Rabbit antibody to the 15-amino acid sequence at the C-terminus of human PHGPx (MEEPLVIEKDLPHYF) was prepared as described for 15-lipoxygenase [18]. The peptide-specific antibody was purified by affinity-column chromatography using a peptide-conjugated CH-Sepharose 4B column (Amersham Biosciences, Tokyo, Japan). Alexa 549-conjugated goat anti-rabbit (immunoglobulin) IgG was purchased from Molecular Probes (Eugene, OR).
It was confirmed that antibodies produced in two rabbits reacted with the peptide conjugated with BSA (Fraction V; Sigma-Aldrich, St. Louis, MO) at all concentrations ranging from 10 to 1000 ng of peptide but not with BSA (data not shown). When the PHGPx activity of the mitochondrial extracts from rat testis was assayed spectrophotometrically by the method of Maiorino et al. [19] using phosphatidyl choline hydroperoxide as a substrate, it was confirmed that the PHGPx activity in the extract decreased as the membrane pieces absorbing separately both antibodies increased if a fixed amount of the extract was incubated with the membrane pieces (data not shown).
Western Blot Analysis of Antibody
Heavy mitochondrial fractions were prepared from homogenates of rat testis by differential centrifugation. Briefly, the homogenate in 0.25 M sucrose-10 mM Hepes-KOH buffer (pH 7.2) was centrifuged at 800 g for 2 min. The resulting supernatant was centrifuged at 10,000 g for 1 min and pellet was suspended in a 0.1 M phosphate buffer containing 0.1% Triton X-100 and protease inhibitors (all from Sigma-Aldrich), 10 µM PMSF, 4 µM leupeptin, 4 µM chymostatin, 4 µM pepstatin, and 4 µM bestatin [20]. Nuclear fractions were collected from rat testis as described previously [21] and suspended in 0.25 M sucrose and 1 mM MgCl2. Protein concentrations were determined by the bicinchoninic acid method (Pierce Chemical, Rockford, IL) with BSA as a standard. The protein concentrations of the extracts were adjusted to 2 mg/ml, mixed with one volume of sample buffer for SDS-PAGE, and heated in boiling water for 2 min. Five micrograms of each sample were analyzed by Western blotting.
Immunofluorescence Microscopy
Rat testes were dissected out and cut into small tissue blocks in ice-cold fixative and then fixed further with gentle stirring for 2 h. The fixative consisted of 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.1 M Hepes-KOH buffer (pH 7.4). Rat epididymal sperm were collected from the caudal epididymis and fixed in the same way. Fixed tissues and cells were embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan) and frozen at -20°C. Frozen sections (thickness, 10 µm) were cut with a Coldtome (Sakura Finetechnical) and mounted on clean glass slides. Sections were then treated with 0.05% SDS in 0.1 M Tris-HCl buffer (pH 7.2) for 30 min at room temperature, followed by 2-h incubation with anti-PHGPx antibody. After being washed in PBS, sections were incubated with Alexa 549-conjugated goat anti-rabbit IgG for 1 h at room temperature. For immunohistochemical control, preimmune serum was used instead of the primary specific antibody, followed by Alexa 549 secondary antibody.
Rat epididymal sperm were smeared on clean glass slides, dried in air, and stored at -70°C. Smear preparations were treated with 6 M guanidine-HCl, 10 mM glutathione, 10 mM dithiothreitol (DTT), or 10 mM 2-mercaptoethanol (2-ME) for 30 min at room temperature and fixed in 4% paraformaldehyde for 30 min. The preparations were immunostained and mounted as described above. Sections and smear preparations were examined using a Leica TCS 4D confocal laser-scanning microscope (Leica Microsystems, Mannheim, Germany).
Postembedding Immunoelectron Microscopy
Rat testes and epididymal sperm were treated with fixative containing 4% paraformaldehyde and 0.25% glutaraldehyde as described above. Small tissue blocks and sperm were dehydrated with a graded series of ethanol concentrations and embedded in LR White (London Resin, Reading, England) or Lowicryl K4M (Chemische Werke Lowi, Waldkraiburg, Germany) at -20°C. Thin sections were cut with a diamond knife equipped with a Reichert Ultracut R (Leica, Vienna, Austria), mounted on nickel grids, and incubated overnight with affinity-purified anti-PHGPx antibody (5 µg/ml) at 4°C, followed by protein A-gold probes with 15- or 8.5-nm diameter gold particles. Sections were stained with 2% uranyl acetate for 8 min and lead citrate for 30 sec and then examined with a Hitachi H7500 electron microscope (Hitachi, Tokyo, Japan) at an acceleration voltage of 80 kV.
Quantitative Analysis of the Gold Labeling for PHGPx in Subcellular Compartments
After the immunogold staining for PHGPx with a 15-nm protein A-gold probe, 10 electron micrographs of spermatogonia, primary spermatocytes, and spermatids in the various developing steps (steps 1–19) were taken at 10 000x magnification and enlarged fourfold. The steps were determined according to the morphological criteria of Russel et al. [22]. For analysis of gold labeling density, the areas of mitochondria, nuclei, and cytoplasmic matrix of spermatogenic cells were estimated using a digitizer tablet and SigmaScan software (Jandel Scientific, San Rafael, CA) attached to a computer, and gold particles located in those areas were counted. The labeling density was expressed as gold particles/µm2 for each compartment. For analysis of relative gold labeling in a unit area of sections, the total number of gold particles located in each compartment contained in a 100-µm2 area of a section was counted.
Histogram Analysis of the Association of PHGPx with Mitochondrial Membranes in the Middle Piece of Epididymal Sperm
Sections of LR White-embedded rat testis were immunostained for PHGPx with an 8.5-nm protein A-gold probe, and spermatids in steps 18–19 were analyzed. Twenty digital electron micrographs were taken at 40 000x magnification, and threefold magnifications of them were printed. The width (L) of the intermembrane space between the outer and inner mitochondrial membranes and the shortest distance (l) from the center of the gold particles to the middle of L were measured under a stereoscopic microscope using an ocular micrometer. Approximately 500 gold particles were counted, and the frequency of the particles (%) was plotted against l to make a histogram.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We analyzed mitochondrial and nuclear fractions isolated from rat testis and isolated heads of rat epididymal sperm. Strong signal was noted in the lanes for mitochondrial fractions and rat sperm (Fig. 1, lanes 1 and 3). The molecular mass of the signal was calculated to be 19 kDa. The nuclear fraction isolated from testis and sperm heads also showed a weak 19-kDA band (Fig. 1, lanes 2 and 4). Minor signals with larger molecular masses developed in all lanes. Among these signals, that corresponding to 34 kDa was observed only in the sample containing nuclei (Fig. 1, lanes 2–4).
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Confocal Laser Microscopic Observation of Rat Testis and Epididymal Sperm
Staining patterns in rat testis obtained using the two antibodies were essentially similar. Our antibodies stained mitochondria of spermatid (Fig. 2a) and the middle pieces of developing spermatozoa, which were seen as small rings in cross-sections or as rods in longitudinal sections (Fig. 2b). Weak fluorescence was also observed in small cytoplasmic granules and nuclei of spermatocytes (Fig. 2a). In addition, the antibodies stained crescent structures that seemed to be early stage acrosomes (Fig. 2a).
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In epididymal sperm, positive but weak staining was localized in the middle piece, whereas the head was very weakly stained. The principal piece and end piece of the tail were constantly negative (data not shown). It has been reported that treatments of epididymal sperm with strong chaotropic agents and with DTT, glutathione, or 2-ME make the antibody more accessible to the protein in the sperm head [17]. Thus, we tried to treat smear preparations of rat epididymal sperm with 6 M guanidine-HCl, 10 mM glutathione, 10 mM DTT, or 10 mM 2-ME before immunostaining. Treatments with all four agents enhanced the reactivity of sperm with antibody (Fig. 2, c and d), although their effects varied. In the treatment with 6 M guanidine-HCl (Fig. 2c), mitochondria were strongly stained, whereas nuclei strongly reacted after the treatment with 10 mM glutathione (Fig. 2d). The effects of 10 mM DTT or 10 mM 2-ME on mitochondria were the same as those of 6M guanidine-HCl (data not shown).
Postembedding Immunoelectron Microscopy
In spermatogenic cells, signals for PHGPx were noted in mitochondria, cytosol, acrosomes, chromatoid bodies, mitochondria-associated material, mitochondrial aggregates, and granulated bodies in residual bodies. We describe the precise localization of PHGPx in individual subcellular structures. The differentiation of spermatogenic cells was broken down into steps according to ultrastructural criteria [22]. The positive staining was abolished in control sections incubated with preimmune serum or peptide-absorbed antibody, followed by protein A-gold probe (data not shown).
Cytoplasmic matrix Cytoplasmic matrix was homogeneously stained. The staining intensity was essentially low compared with that in other compartments but was significant (Figs. 3–8).
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Mitochondria Mitochondria were stained throughout the differentiation (Figs. 3– 8). In spermatogonia, PHGPx signals were present in the matrix of mitochondria, and some were associated with the mitochondrial periphery (Fig. 3). Mitochondria in zygotene spermatid were stained weakly. In pachytene spermatids, gold particles were observed in the matrix of rod-like mitochondria, some of which elongated and aggregated (Fig. 4). Intermitochondrial dense material (intermitochondrial cement) [23] between the aggregated mitochondria was also labeled with gold particles (Fig. 4, insert). In step 1–5 spermatids, mitochondria moved to beneath the cell membrane, and the intracristal space was expanded as described by De Martino et al. [24]. The PHGPx signals were present in the matrix of these mitochondria (Fig. 5). Labeling intensity increased gradually as the process progressed. In step 9–10 spermatids, mitochondria increased in size and began to move to the flagellum. Gold labeling for PHGPx was heavier than before, and most of the labeling was present in the matrix (Fig. 6). At steps 15–17, mitochondria attached to flagellar axoneme but did not yet surround it spirally (Fig. 7). A space was observed between adjacent mitochondria lining up along the axoneme. In cross-sections of spermatids, it was found that mitochondria attached to each other in small areas (Fig. 7, insert). Gold particles for PHGPx were noted in the matrix, and some were associated with the intermembrane space. At steps 18–19, mitochondria became smaller and exhibited no further expansion of the intracristal space (Fig. 8, a and b). They closely attached to each other and almost compactly surrounded the flagellar axoneme to form a spiral structure as described previously [24]. Heavy staining with gold particles was observed in these mitochondria. Many gold particles were located in the intermembrane space. In epididymal sperm, the spiral arrangement of mitochondria in the middle piece was completed and mitochondria compactly attached to each other. Gold labeling for PHGPx was closely associated with the intermembrane space, and few signals were present in the matrix (Fig. 9b).
Nuclei In the nuclei of spermatogonia, gold staining for PHGPx was diffuse in the euchromatin area (Fig. 3). In the nuclei of pachytene spermatocytes characterized by the synaptonemal complex, gold particles tended to associate with heterochromatin and, especially, with electron-dense aggregates, which showed a very similar profile to that of early chromatoid bodies (Fig. 10a). The labeling intensity in the nuclei was low and did not change until step 12. As chromatin condensation started, the nuclear staining intensity and localization of PHGPx changed dramatically. In step 16–18 spermatids and epididymal sperm, PHGPx signals were observed along the nuclear envelope and in electron-lucent spots in condensed nucleoplasm (Figs. 7, 8a, and 9a). In the caudal part of the nucleus in spermatids of steps 16–18, an electron-lucent area was observed to be surrounded by a redundant nuclear envelope in which heavy staining was noted on fibrous materials (Fig. 12).
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Chromatoid bodies Chromatoid bodies appeared in pachytene spermatocytes and developed until steps 7–8. In early pachytene spermatid, chromatoid bodies were frequently present just adjacent to the nuclear envelope, and a very similar structure was observed in the nucleoplasm. These structures were heavily stained for PHGPx (Fig. 10a). In step 3–8 spermatids, the chromatoid bodies enlarged, and their electron density increased. Gold particles were associated with the periphery or electron-lucent spots of the dense material of these chromatoid bodies (Fig. 10, b and c). After step 9, the structure and profile of the chromatoid bodies changed greatly, and a typical morphology was no longer observed. Therefore, the continuity was not clear. In step 18 spermatids, cytoplasmic dense structures resembling chromatoid bodies were labeled for PHGPx (Fig. 10d).
Acrosomes The formation of acrosomes started from step 2 or 3. The PHGPx signals appeared in acrosomal granules of proacrosomal vesicles at a very early stage of the formation (Fig. 11a). Gold labeling in the acrosomal granules increased gradually as the acrosomes developed (Fig. 11, b–d). In epididymal sperm, PHGPx was localized to the apical and ventral parts of the acrosome (Fig. 11e).
Others We detected PHGPx in three subcellular structures, the origins of which were not very clear. The mitochondria-associated structure described by Clermont et al. [25] was heavily stained (Fig. 13). This structure appeared in step 16 spermatid and disappeared soon after. The other two structures were present only in residual bodies. Mitochondrial aggregates, which are reported to be positive for cytochrome sc [26], were strongly labeled for PHGPx (Fig. 14, arrows). Granulated bodies [25], which are frequently located near mitochondrial aggregates, were moderately stained for PHGPx (Fig. 14, arrowheads).
Quantitative Immunoelectron Microscopy
To clarify the changes in the amount of PHGPx during spermatogenesis, we analyzed the labeling density (gold particles/unit area) at each step in spermatogenic cells. We and others have reported that the labeling density (gold particles/unit area) is proportional to the concentration of antigen [27], which might correspond with the amount of PHGPx assayed biochemically. We then used this method to analyze the relative amount of PHGPx in each organelle during rat spermatogenesis.
To see the changes in the amount of PHGPx in the whole cell, we analyzed the number of gold particles in a unit area of cells (100 µm2), including organelles and the cytoplasmic matrix. As shown in Figure 15A, the gold labeling started to increase from spermatids at steps 5–8 and was twofold the level at step 1. Afterward, the labeling sharply increased, reaching approximately 1500 particles in step 18–19 spermatids, which was approximately sevenfold the level at steps 5–8.
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Next, we analyzed the changes in the amount of PHGPx in the mitochondria, nuclei, and cytoplasm at each step in the same manner. The mitochondrial labeling density (gold particles/µm2) of zygotene spermatocytes decreased by approximately one-third compared to that of spermatogonial mitochondria. The mitochondrial labeling density increased slowly until step 1, which was followed by a rapid increase until steps 12–14. After step 15, the labeling density did not change greatly and was similar to the level in epididymal sperm (Fig. 15C). The mitochondrial labeling density of epididymal sperm was approximately threefold that of step 1 spermatids.
In nuclei, the amount of PHGPx did not change, as was the case in mitochondria (Fig. 15D). The nuclear labeling density remained low and did not change from spermatogonia to step 8 spermatid, but in step 12–14 spermatids, it suddenly increased and became approximately 18-fold that in step 8 spermatids. Afterward, the labeling density was kept constantly high.
Analysis of the labeling density in cytoplasm that did not contain mitochondria and nuclei showed cytoplasmic-specific changes. The cytoplasmic labeling density was essentially low, compared with mitochondria, and did not change (Fig. 15B).
The labeling density in chromatoid bodies gradually decreased from pachytene spermatocytes to step 18 spermatids, although chromatoid bodies lost their typical shape after step 11 (Fig. 15E).
Distribution of PHGPx in Mitochondria of Epididymal Sperm
As described above, during the development of spermatogenic cells, mitochondrial PHGPx signals were observed in the matrix and the periphery of mitochondria (Figs. 6 and 7). However, at the end of spermatogenesis, the signals completely moved from the matrix to the outermost membrane region (Fig. 9b). To confirm the distribution of the signals of PHGPx in the mitochondrial periphery in more detail, we analyzed precisely the location of gold particles in this region. The results are shown in Figure 16. Gold particles were located in the intermembrane space, and the distribution peak of PHGPx was located near the outer membrane in the intermembrane space.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In seminal tubules, positive staining for PHGPx was noted in developing acrosomes and in cross-sectioned and longitudinally sectioned middle pieces of developing sperm. In addition, weak staining was observed in nuclei of spermatogenic cells and spermatids late in development. These results are mostly consistent with the published biochemical data [30].
Immunofluorescence staining of epididymal sperm for PHGPx was not as strong as expected from immunoelectron microscopic staining if preparations were not treated with chaotropic or reducing agents. The fact that pretreatment with agents enhanced the staining of sperm suggests that the access by antibody to PHGPx in mature sperm is unable to be improved. Although the effects of the agents varied, mitochondria were heavily stained but nuclei weakly stained after treatment with 6 M guanidine-HCl, 10 mM DTT, or 10 mM 2-ME. Conversely, mitochondria were weakly stained but nuclei heavily stained after treatment with 10 mM glutathione. The difference of staining patterns with agents would imply differences in the associative strength of PHGPx with mitochondrial capsules or nuclei. On the other hand, in the thin sections used for immunoelectron microscopy, epitope seems to be exposed on the surface. In contrast to the nuclear staining, acrosomes were stained at immunofluorescence as well as at immunoelectron microscopic examination.
Sites of localization of PHGPx in rat testis are similar to those described by Tramer et al. [17]. Major sites for PHGPx in rat testis were mitochondria and nuclei [13]. Mitochondrial labeling decreased when spermatogonia differentiated into zygotene spermatocytes; afterward, the labeling increased gradually. During these steps, mitochondria dynamically moved from aggregates, in which they were attached to each other via intermitochondrial material (pachytene), to the cell periphery (step 1 spermatid) and then moved to the flagella (step 13 spermatid) as described previously [24]. In the present study, we showed, to our knowledge for the first time, that PHGPx was present in intermitochondrial material, although to a lesser extent than in mitochondria. We speculate that PHGPx in intermitochondrial material might function as a glue protein to paste mitochondria together. The morphology of mitochondria also changed during these steps. In spermatogonia, mitochondria exhibited a typical profile without expansion of the intracristal space [24]. In pachytene spermatocytes, they elongated, and the expansion of intracristal space began. Afterward, mitochondria became rounded, and the intracristal space gradually enlarged as mitochondria increased in size until step 15. In the process of development from the early steps to step 18, the gold particles were present in the matrix.
In step 18 spermatids and in epididymal sperm, mitochondria surrounded spirally and compactly flagellar axonemes, and the expansion of intracristal space was no longer observed. The gold particles moved from the matrix to the peripheral region after the step 19 and were only present in the outermost region. Quantitative analysis indicates that the distribution peak of PHGPx was located near the outer membrane in the intermembrane space. Given the fact that the antibody used here is for the C-terminal region of PHGPx and that PHGPx has one putative transmembrane domain in its N-terminal region, part of PHGPx (N-terminal) might be exposed outside of the outer membrane. It was reported that PHGPx changed from a soluble antioxidant enzyme to an enzymatically inactive structural protein [11] during spermatogenesis. The present study suggests that this functional change is synchronized with the relocalization of PHGPx from the matrix to the intermembrane space, although the mechanism of this translocation remains to be elucidated.
In the present study, we showed quantitatively that PHGPx accumulated suddenly in nuclei after step 12. At this point, chromatin condensation was considerably progressed. In the nuclei of spermatids at step 16, heavy gold labeling for PHGPx was present in electron-lucent spots within the nucleoplasm. In the nuclei of spermatids at steps 15–17, two regions were identified: One was the nucleoplasm where chromatin condensation was ending and surrounded by a nuclear membrane without nuclear pores, and the other was characterized by an electron-lucent matrix with various fibrous materials and enveloped by a so-called redundant nuclear membrane with pores [31]. The present study showed that heavy gold labeling for PHGPx was associated with the fibrous materials of the latter region. It is unclear whether PHGPx serves as an antioxidant and/or a structural protein in this region. It has been reported that at the late steps of spermiogenesis, sperm nuclear protein histones are replaced by transition proteins and, finally, by cysteine-rich protamines [1] and that the sulfhydryl residues of the protamines are successively oxidized to form disulfide bonds among them [1, 32, 33]. During the process, sperm DNA becomes highly compacted to protect it from various external stresses before fertilization [1, 33]. Recently, Pfeifer et al. [14] demonstrated that the nuclear PHGPx acts as a protamine thiol peroxidase responsible for the formation of cross-linked protamine disulfide. Therefore, the PHGPx around the electron-lucent spots is considered to function as a protamine thiol peroxidase. In the nuclei of spermatids at step 16, increased signals for PHGPx were also observed along the nuclear envelope, but to our knowledge, the biological function of the PHGPx located in these regions remains to be elucidated. Interestingly, an increased association of PHGPx with the membrane was also observed in the mitochondria during the late steps of spermiogenesis. Thus, it is tempting to speculate that the PHGPx associated with the nuclear envelope may have a role as a structural protein similar to the PHGPx associated with the mitochondrial outer membrane.
The localization of PHGPx in acrosomes has been reported [17]. The present study showed that PHGPx was localized in acrosomes from the early to late steps, including in mature sperm, in which the major site for PHGPx was acrosomal granules. It would be likely that acrosomal PHGPx is stored as antioxidant to operate during fertilization, including acrosomal reactions.
We first demonstrated here the localization of PHGPx in chromatoid bodies and clearly showed a gradual decrease in the amount of PHGPx in the organelle with progress through the developmental steps of spermatids by a quantitative analysis. In the present study, PHGPx signals were noted in the nuclear material having a very similar electron density and structure to those of chromatoid bodies, suggesting that the material associated with PHGPx in the nucleus would form chromatoid bodies. Also, the present study showed that PHGPx was localized to the intermitochondrial material, suggesting that it is one of the components of chromatoid bodies. It was proposed that chromatoid bodies would be derived from the nucleus [34, 35] and the cytoplasm of spermatids, because they contain DNA, RNA, subunits of small nuclear ribonucleoprotein particles, and several proteins [38, 46–51]. It was also speculated that the chromatoid body originates from the intermitochondrial cement that appears in pachytene spermatids [23]. Our observations support these proposals. However, the function of PHGPx in chromatoid bodies is unclear.
Other cytoplasmic components stained with anti-PHGPx antibody were "mitochondria-associated material" [25], which was most prominent in step 17–18 spermatids, and "mitochondrial aggregates" and "granulated bodies" in residual bodies [25]. The origin and fate of "mitochondrial-associated material" and "granulated bodies" are not known. Mitochondrial aggregates seem to be formed by an accumulation of surplus or defective mitochondria that still have cytochrome [26].
As shown here, PHGPx markedly changes in amount and distribution in subcellular compartments during spermatogenesis and spermiogenesis. So far, three kinds of PHGPx have been reported, and their mRNAs are differentially transcribed from one gene through alternative splicing [14] or alternative usage of the transcription/translation start site [41]. The origin of PHGPx in cytoplasmic components other than the nucleus and mitochondria is unclear. However, PHGPx should be produced precisely at each step using the differential transcription mechanism and transferred to each site. Ursini et al. [11] clearly demonstrated, using an in vitro system, that PHGPx forms aggregates with proteins having thiol groups when exposed to H2O2 in the absence of glutathione. The PHGPx might be involved in important functions through orchestration with other proteins. We supposed that proteins interacting with PHGPx might be present in various compartments of spermatogenic cells. This could explain the localization diversity of PHGPx in these cells. Thus, PHGPx is considered to be a typical moonlighting protein [12]. So, a deficiency of PHGPx affected various functions at several steps, and the mechanisms of infertility caused by deficient PHGPx [15, 16] would not be simple. Elucidation of the functions of a moonlighting protein PHGPx specific for different subcellular compartments in each step would be essential for understanding the roles of PHGPx in spermatogenesis.
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FOOTNOTES |
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Received: 15 November 2002.
First decision: 12 December 2002.
Accepted: 7 April 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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