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Dipartimento di Biologia,3 Università di Milano, 20133 Milano, Italy
Dipartimento di Biotecnologie e Bioscienze,4 Università di Milano-Bicocca, 20126 Milano, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sperm, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We [10] recently identified, by molecular screening of a mouse embryo cDNA library, a novel deubiquitinating enzyme, which we named mUBPy, that was found to be the mouse UBP homolog to the human hUBPy, identified in a human myeloblast cell line [11]. The mUBPy is expressed in adult mice predominantly in the nervous system, in agreement with its discovery as a protein able to interact with the mouse brain-specific Ras-guanine nucleotide exchange factor CDC25 Mm and, unexpectedly, in the testis [10]. Indeed, the bulk of the experimental findings about the role of the ubiquitin system in the mammalian testis has been recently acquired, starting from the pioneer work of Roest and coworkers [12] on the HR6B-KO mice; although HR6B is widely expressed in mouse tissues, HR6B-KO mice present only a defect, i.e., male infertility. Two other mouse models generated by gene targeting of components of the ubiquitin system, i.e., mHR23B-KO [13] and Siah1a-KO [14], are characterized by a male sterile phenotype due to two specific and temporally regulated blocks of spermatogenesis. So the ubiquitin system plays a key role in the progression of correct spermatogenesis. The majority of the members of the ubiquitin system so far identified in the testis belong, however, to the ubiquitination pathway [12– 15], while very little is known about the components of the opposite pathway, i.e., deubiquitination. The only deubiquitinating enzyme so far characterized in the testis is the rat testis-specific UBP-t, which is present in two isoforms, UBP-t1 and UBP-t2 [9]. There are no other data about testis deubiquitinases, apart from some information derived from studies on genetic alterations. It has been, in fact, suggested that deletions in the human USP9Y gene, the human homologue of the Drosophila fat facets gene [16], which codifies a putative ubiquitin C-terminal hydrolase, are associated with cases of azoospermia [17]. Moreover, an intragenic deletion in the gene codifying PGP 9.5/Uch-l1, a deubiquitinating enzyme of the UCH group [18], is the responsible for the gad mutation, a pathological mouse disorder characterized by progressive neurodegeneration due to accumulation of ubiquitinated protein conjugates in nerve terminals [19]. Kwon and coworkers [20] have recently reported that gad mutation affects also spermatogenesis.
In this work, we report the biochemical and cellular characterization of the deubiquitinating enzyme mUBPy in differentiating and mature mouse male germ cells. Our preliminary results [21] suggested a possible interaction between MSJ-1, the mouse DnaJ chaperone protein evolutionarily conserved for spermiogenesis in vertebrates [22, 23], and mUBPy in spermatogenic cells. Here we show that endogenous sperm mUBPy colocalizes and is effectively able to interact with MSJ-1. Moreover, male germ cell mUBPy is enzymatically active toward endogenous substrates, while the proteasomes, the multisubunit protein complexes, which catalyze the degradation of multiubiquitin-conjugated proteins, exhibit an intracellular distribution that overlaps that of mUBPy in both spermatogenetic cells and mature spermatozoa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant GST-mUBPy (542–660) and anti-mUBPy antibodies were obtained as described [10]. GST-MSJ-1 fusion protein, control GST, and anti-MSJ-1 antibodies were obtained as described [24]. The rabbit polyclonal antibodies to 20S proteasome core (PW 8155) and to ubiquitin conjugate (UG 9510) were purchased from Affiniti. GST antibodies (sc-459) were from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated anti-rabbit antibodies used for chemiluminescence and ECL were from Amersham-Pharmacia; HRP-conjugated affinity purified antirabbit antibodies used in immunohistochemistry were from Chemicon International, and the diaminobenzidine (DAB) substrate (1 718 096) was purchased from Roche. Alexa 488 antirabbit, Alexa 568 antirabbit, and Alexa 488 antimouse were from Molecular Probes.
Animals, Tissues, and Cell Collections
Animal care and handling were in accordance with policies on the care and use of animals promulgated by the ethical committee of the University of Milano following the guidelines of the Italian Minister of Health, DL 27 gennaio 1992, N. 116. CD-1 male mice, obtained by Charles River (Italy) and housed under conventional, controlled standard conditions were used. Tissue homogenates were obtained immediately after dissection as described [10]. For testis developmental studies, the gonads were collected from animals at Days 8, 16, 21, 23, and 35 of postnatal life. Spermatogenic cell suspensions and epididymal spermatozoa were obtained essentially as reported previously [25], with the exception of the addition of 100 µg/ml soybean trypsin inhibitor in the sedimentation/washing steps after the trypsin digestion.
Western Blotting
Proteins from spermatogenic cell suspensions and epididymal spermatozoa were obtained after cell lysis in extraction buffers. To solubilize the protein of interest, the detergent final concentration in the extraction buffer was increased more and more, i.e., from the mildest to the strongest concentration; Triton X-100 was 0.2%, 0.5%, 1%, and 2% in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM EDTA, 5 mM benzamidine, 100 µg/ ml leupeptin, 10 mM NaF, plus a complete protease inhibitor cocktail (P8340; Sigma). Cell lysis was additionally carried out also in RIPA buffer, i.e., 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulphate, 10 mM NaF plus the protease inhibitors as in the Triton buffers. After sonication (five 5-sec bursts; a Bransonic sonifier set at 50 W), detergent was added and the samples were incubated for 30 min at 4°C with gentle shaking. The insoluble material was pelleted by centrifugation (14 000 x g, 15 min, 4°C) and resuspended in 2x SDS-PAGE sample buffer; the supernatant was the cell lysate. Proteins were separated on SDS-PAGE and transferred to nitrocellulose to be immunoprobed with the specific antibodies, followed by horseradish peroxidase-conjugated secondary antibodies and chemiluminescence. When necessary, membranes were stripped [26] to be reprobed with other antibodies. To neutralize antibodies, standard procedures were used, i.e., antibody at the appropriate concentration was combined with a fivefold (by weight) excess of the protein/peptide immunogen in a small volume (500 µl) of PBS and incubated overnight at 4°C with gentle rotation. The antibody/peptide mixture, clarified by centrifugation, was then diluted into the appropriate buffer according to the successive experimental applications. Protein concentration was assessed by comparison with bovine serum albumin using the Bio-Rad DC protein assay.
Enzymatic Assays
Protein deubiquitination assay was essentially as previously described [10]. Briefly, proteins from spermatogenic cells were extracted in 2% Triton extraction buffer containing 20 mM N-ethylmaleimide (NEM). The cleared extract was dialyzed at 4°C against 50 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 2 mM dithiothreitol and then centrifuged and washed several times in a Centricon-3 concentrator (Amicon) to remove residual NEM. Concomitantly, mUBPy was immunoprecipitated from a 2% Triton spermatogenic cell extract by incubation with affinity-purified mUBPy polyclonal antibodies, followed by addition of Protein A-Sepharose beads. The precipitated immunocomplex was washed three times in lysis buffer and once in lysis buffer without detergent. Successively, it was incubated with the spermatogenic cell extract in the presence of 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin for 20 min at 34°C. To the control sample, before the enzymatic assay, NEM was added to the final concentration of 20 mM. Reactions were stopped by addition of 2x SDS sample buffer and boiling for 10 min; then the samples were subjected to SDS-PAGE, transfer blot, and immunodecoration with antiubiquitin antibodies.
MSJ-1/mUBPy Protein Interaction Assays
Recombinant full-length MSJ-1, obtained and purified as described [24], was used as a bait protein for both far Western and GST pull-down assays. In far Western assays, RIPA buffer-extracted sperm proteins were electrophoresed and blotted onto nitrocellulose membrane. The protein denaturation/renaturation process was essentially as already reported [26]. After incubation in the blocking solution, 5% notfat milk in TBST (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.2% Tween 20), membranes were incubated for 2 h at room temperature with GST/MSJ-1 or GST alone (5 µg/ml) in TBST (containing 3 µM reduced glutathione and 0.5% notfat milk). Direct interactions between MSJ-1 and blotted proteins were detected by rabbit affinity-purified GST antibodies. For GST pull-down assays, freshly prepared 2% Triton X-100 extracted sperm proteins were incubated with 5 µg of purified GST/MSJ-1 or GST alone for 2 h at 4°C, followed by 1 h of incubation with glutathione-Sepharose beads. The complexed beads, recovered by centrifugation, were washed four times in lysis buffer and solubilized in SDS-PAGE sample buffer to be first resolved by SDS-PAGE and then analyzed by Western blotting.
Immunohistochemistry and Immunocytochemistry
Mouse testes were fixed at 4°C in buffered paraformaldehyde and embedded in paraffin. Deparaffinized sections (7 µm) were processed for immunohistochemistry. Endogenous peroxidase activity was blocked by using 0.3% H2O2 in PBS for 15 min at room temperature. After washings and blocking of nonspecific binding sites in 3% bovine serum albumin in PBS for 45 min at room temperature, sections were incubated with the primary antibody, diluted 1:200–1:400 in 1% BSA in PBS, for 1 h in a humidity chamber. Control samples were incubated with the neutralized antibody. After incubation in horseradish peroxidase-conjugated secondary antibody, samples were incubated in the DAB substrate working solution until the desired staining was achieved (5–10 min). Nuclei counterstaining was carried out with hematoxylin.
Freshly prepared spermatogenic cell suspensions and epididymal spermatozoa, smeared on slides treated with 3-aminopropyltriethoxy-silane, were methanol fixed, processed for blocking of nonspecific sites, and subjected to double fluorescent staining. First, cells were immunostained with the indicated primary antibody (1:200–1:400 dilution), followed by Alexa 488- or Alexa 568-conjugated IgG as the secondary antibody. In control samples, primary antibody was omitted or replaced with the neutralized primary antibody. Nuclei counterstaining was carried out with DAPI (1 µg/ml). Cells were examined as described [24].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To verify the presence of mUBPy in male germ cells, we carried out immunoblot analyses with the polyclonal antibodies raised against recombinant mUBPy we generated as previously described [10]. Spermatogenic cells isolated from adult mouse testis gave a strong immunopositive signal (Fig. 1A, lane 1), detected also in testis homogenate (Fig. 1A, lane 2), corresponding to a protein doublet of 125 kDa, a molecular weight in agreement with the expected size of the mUBPy open reading frame (122,579 Da); mature epididymal spermatozoa also were mUBPy immunopositive, although at a considerably lower level (Fig. 1A, lane 3). The mUBPy is expressed throughout spermatogenesis (Fig. 1B). Protein homogenates obtained from testes of male mice at different postnatal ages were mUBPy immunopositive at 8 days (spermatogonia) as well as at 16 (pachytene spermatocytes), 23 (differentiating spermatids), and 35 (sexually mature) days. As to its extraction, the deubiquitinating enzyme is not easily solubilizable. By using an extraction buffer containing 0.5% Triton X-100, most protein remains in the detergent-insoluble fraction, whereas the opposite recovery could be obtained increasing the detergent final concentration to 2% Triton X-100. The mUBPy is completely solubilized by sonication and extraction in RIPA buffer for 40 min at 4°C; this means that, if not all, at least a great bulk of mUBPy is not cytosolic but might be associated with membranes and/or cytoskeletal elements. The protein is in vitro susceptible to rapid degradation. More proteolytic products (Fig. 1C, lane 1) were in evidence when immunoblot analyses were carried out with, not fresh, but frozen and thawed protein samples; these products are mUBPy specific because they were not recognized by the neutralized mUBPy antibodies (Fig. 1C, lane 2). When assayed for its enzymatic activity, the endogenous mUBPy was shown to be active. Immunoprecipitated mUBPy, in fact, greatly reduced the amount of ubiquitinated spermatogenic proteins recognized by antiubiquitin antibodies (Fig. 2, compare lane 2 with lane 1). Conversely when NEM, an inhibitor of cysteine proteases used to inhibit the UBPs [11], was added to the enzymatic assay mixture, the immunoprecipitated mUBPy failed to generate deubiquitination of spermatogenic proteins as shown in Figure 2, lane 3.
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Immunocytochemical Analysis of mUBPy Expression
Our immunoblotting data do not indicate a developmentally regulated expression of mUBPy, suggesting that the protein is expressed throughout spermatogenesis. We tested the presence of mUBPy also by immunohistochemistry. Analyzing adult mouse testis sections (Fig. 3A), meiotic spermatocytes and round spermatids are the cells more intensively immunostained; both elongating spermatids and testicular spermatozoa are mUBPy immunopositive whereas spermatogonia resting directly on the basal lamina are mUBPy immunonegative (Fig. 3, A and B). Significantly, the testicular interstitial tissue is not stained by the mUBPy immunolabeling, thus revealing mUBPy as a gene product restricted to the seminiferous epithelium only (Fig. 3A). The germ cell specificity of mUBPy expression is evident also in 21-day-old testis, the postnatal age at which the first wave of meiotic divisions has just taken place; whereas meiotic cells and the early round spermatids are strongly mUBPy immunopositive, all intertubular cells are mUBPy immunonegative (Fig. 3C). A control picture is shown in Figure 3D.
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We are particularly interested in spermiogenesis, so to better specify the intracellular localization of mUBPy in the different stages of spermiogenesis, we analyzed freshly prepared suspensions of postmeiotic cells by immunofluorescence. All samples were counterstained with DAPI to follow concomitantly the nuclear shape modifications. In round spermatids, mUBPy shows a diffuse, scattered distribution with a remarkable perinuclear localization (Fig. 4A). Figure 4, B through D, illustrates successive stages of the cap phase when the acrosomal vesicle is developing and the nucleus is acquiring a bent shape: mUBPy immunostains step by step the profiles of the acrosomal vesicle. Figure 4E shows a more differentiated stage, when the acrosomal vesicle is well developed and mUBPy marks its contour by a punctate dot staining. The enlargement of the merge image of Figure 4F allows appreciation of how, in late differentiating spermatids, the mUBPy labeling delineates the outlines of the acrosomal vesicle, which, at this stage, covers most of the nuclear surface. So our preliminary observation was restricted to elongating spermatids only [21] that mUBPy labeling is very similar to that of MSJ-1, the mouse spermatogenic cell-specific DnaJ protein [24], could be extended in the present study to the various subtypes of differentiating spermatids and with a particular regard to the process of acrosomogenesis. We analyzed also epididymal spermatozoa, which were mUBPy immunopositive in immunoblotting analysis and immunohistochemistry. As shown in Figure 5A, mUBPy maintains its periacrosomal localization in mature spermatozoa, staining also the centrosomal region and, less intensively, the tail principal piece. Figure 5B provides a magnification of mUBPy labeling in the mouse sperm head; it is impressive in its similarity with that displayed by MSJ-1 (Fig. 5C). The finding that, in mouse spermatozoa, a deubiquitinating enzyme colocalizes with a molecular chaperone led us to further experiments addressed to verify whether a) sperm mUBPy is able to interact with the chaperone MSJ-1; and b) proteasomes, the giant multimeric protease complexes to which ubiquitinated proteins are transferred to be degraded, localize in sites mUBPy and/or are MSJ-1 immunopositive.
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MSJ-1/mUBPy Protein Interaction
A preliminary far Western experiment using recombinant GST-mUBPy (542–660) suggested that mUBPy could interact with several proteins from spermatogenic cells, including MSJ-1 [21]. To give further evidence of this in vitro MSJ-1/mUBPy interaction, we here performed two additional protein interaction assays probing, this time, recombinant MSJ-1 as the bait protein and endogenous sperm proteins as the prey. Figure 6A shows the far Western results. Several proteins, solubilized from mature spermatozoa with RIPA buffer, interacted in the protein overlay with GST/MSJ-1 (lane 2), but did not with the control GST alone (lane 1); an asterisk (lane 2) marks, in particular, a reactive protein of about 125 kDa. When the filter was stripped to be immunoprobed with mUBPy antibody, the 125-kDa protein was specifically recognized as mUBPy (lane 3). A more direct piece of evidence for protein interaction was given by the pull-down experiment reported in Figure 6B. Freshly Triton-solubilized sperm proteins were directly incubated in suspension with GST/MSJ-1 or control GST for protein-affinity precipitation; the precipitated bound complexes were electrophoresed to then be immunoblotted with mUBPy antibody. The antibody recognized specifically a protein of 125 kDa in the precipitated GST/ MSJ-1 protein complex (Fig. 6B, lane 2), but not in the GST complex (Fig. 6B, lane 1). Altogether, these results strongly suggest that the deubiquitinase and the chaperone could effectively interact in vivo in sperm cells.
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Proteasome in Mouse Male Germ Cells
The presence of the proteasome in male germ cells has already been reported, albeit with some conflicting results. These discrepancies, regarding essentially the putative sites of proteasome localization, could be due to the fact that spermatozoa from different animal species have been studied, from the sea urchin [27] to the ascidian Halocynthia roretzi [28], salmon [29], and, for the mammals, human [30] and rat [31]. Moreover, being that the proteasome is a giant multimeric protease complex formed by more subunits, the different panel of antibodies used in the few studies with an immunocytochemical approach might be responsible for the nonconcordant results, as it was also for the same sperm species [30, 32]. For our study, we used commercially available antibodies, which, under our experimental conditions, gave highly reproducible results, i.e., the rabbit polyclonal antibody to 20S proteasome 
/ß subunits from Affiniti. First, we tested the antibody specificity by Western immunoblot analysis on protein extracts from mouse male germ cells. As shown in Figure 7A, the antibody recognized bands attributable to the core subunits at 25–30 kDa, in agreement with what was expected, plus some minor bands at 45–50 kDa of a slightly different intensity according to the protein extraction conditions. Then, we used this antibody for immunolocalization analysis. Figure 7B shows proteasome distribution in spermatogenic cells, i.e., more specifically, a spermatocyte (1), a round spermatid (2), a late round spermatid (3), an elongating spermatid (4), and a testicular spermatozoa (5). In spermatocytes (1), proteasomes are widely distributed, with a preferential cytoplasmic localization along the perinuclear region, as occurs also for mUBPy (unpublished results). In spermatids at the cap-phase stage (2 and 3), proteasomes, although still diffusely distributed, concentrate and mark like an immunofluorescent luminous halo the outlines of the nascent and developing acrosomal vesicle. The periacrosomal staining by proteasome is well evident also in elongating spermatids (4) and testicular spermatozoa (5). In mature epididymal spermatozoa, proteasomes surround the acrosome with an intense immunolabeling and mark more weakly the centrosomal area and a portion of the tail principal piece (Fig. 7C), as they do already in testicular sperm. So, intriguingly, it turns out that mUBPy, MSJ-1, and proteasomes reposition during haploid cytodifferentiation, moving from their foregoing intracellular sites toward the developing acrosome and sperm centrosome. Significantly, mUBPy, MSJ-1, and proteasomes maintain these localizations in mature spermatozoa.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spermatogenesis is a complex process by which spermatogonial stem cells divide and differentiate to produce sperm. During spermiogenesis, the cytomorphogenetic phase, a massive cell remodeling occurs and protein ubiquitination is heavily involved [15]. Little is known, however, about the opposite process, i.e., protein deubiquitination. Here we show that the mouse ubiquitin-specific protease mUBPy is expressed throughout spermatogenesis and its expression within the testis is restricted only to the germ cell component. The mUBPy is present as a protein doublet of 125 kDa; at the moment, we do not know the meaning of this doublet; Naviglio and coworkers [11], however, also reported a protein doublet of 130 kDa for hUBPy, the human homologue of mUBPy, and suggested that hUBPy could be a phosphoprotein. Spermatogenic mUBPy is enzymatically active, being able, when immunoprecipitated, to deubiquitinate endogenous protein substrates; consequently, mUBPy could exert in vivo an active role in the male germ cell ubiquitin/proteasome system. Immunohistochemistry revealed the highest mUBPy protein staining in spermatocytes and round spermatids followed by differentiating spermatids and spermatozoa, while resting spermatogonia are mUBPy immunonegative. This suggests that the functional requirement of mUBPy could vary according to the different phases of spermatogenesis. At the present, there is no experimental evidence about a role of mUBPy in spermatocytes. The hUBPy, the human homologue, is thought to regulate the overall function of the ubiquitin-proteasome pathway in cultured cells; more specifically, hUBPy activity has been related to the different phases of the mitotic cell cycle [11]. Moreover, recently, VCIP135, a new rat deubiquitinating enzyme-like protein, has been suggested to play a role in the mitotic cycle by mediating reassembly of the fragmented Golgi cisternae after mitosis [35]. It might be that, in mouse spermatocytes, mUBPy exerts a role for the progression of the meiotic cycle.
Particularly interesting results were provided by immunocytochemistry. During spermiogenesis, mUBPy undergoes a fine repositioning, moving from the scattered spots concentrated mainly along the perinuclear zona, where both the Golgi and centrosome are located, toward the cytosolic surface of the forming acrosome, a golgian derivative. The mUBPy labeling follows, step by step, the acrosome development and, in mature epididymal spermatozoa, mUBPy still stains the cytosolic surface of the acrosome and the centrosomal region. Because this pattern of intracellular localization was exhibited also by MSJ-1, a chaperone able to interact with the testis-specific heat shock protein Hsp70-2 in agreement with its nature of DnaJ protein [24], it might emerge, in line with several recent findings about a strict interplay between chaperones and members of the ubiquitin/proteasome system [2–4], a cooperation between mUBPy and MSJ-1, as our preliminary results already suggested [21]. The capacity of mUBPy to interact with MSJ-1 has been confirmed by two different assays just in the cells, mature spermatozoa, where the two proteins colocalize. Further support of a functional association between mUBPy and MSJ-1 has been provided by our study on the proteasomes. During mouse spermiogenesis, these multienzymatic protease complexes move exactly as mUBPy, i.e., from the predominant perinuclear localization toward the developing acrosome; moreover proteasomes, just as mUBPy and MSJ-1, surround as a halo the acrosome of mature epididymal spermatozoa and mark the centrosome. This proteasome repositioning in mouse male germ cells could not be casual, but is indicative for a concerted action with mUBPy and MSJ-1 because it is well known that, when equipped with the appropriate signaling molecules, proteasomes, generally diffuse in the cell but with a preferential localization in the centrosome [36], could be deployed to different cellular locations, wherever their action is needed [5].
As to the possible critical functions of mUBPy, MSJ-1, and proteasomes, a distinction between differentiating spermatids and mature spermatozoa is necessary. During the massive spermatid remodeling, new proteins are synthesized for providing novel structures and organelles, such as the acrosome and the flagellum, while other unnecessary proteins and organelles, are eliminated and/or degraded. Consequently, the cell machineries of protein folding and protein degradation have to operate intensively and correctly. Malfunctions of the cellular protein folding/degradation systems are hallmarks of a growing number of neurodegenerative [19, 37] and nonneurodegenerative [4] disorders, in the human as well as in the mouse, and some of these disorders are characterized also by male infertility or reduced fertility [20, 37, 38]. The mUBPy, MSJ-1, and proteasomes could play a role in the balance between protein folding and degradation during sperm morphogenesis and, in particular, acrosomogenesis. Indeed, MSJ-1 has already been shown to be related to acrosomogenesis [23]. Wobbler mice, a still-unidentified mutation characterized by a progressive motoneuron degeneration due to protein accumulation, present another defect, i.e., male infertility due to spermatozoa lacking a real acrosome and with reduced motility [39]. MSJ-1 is significantly underexpressed as both a transcript and a protein in the testis [23] and nervous system [40] of wobbler mice.
As for mature spermatozoa, cells known to be devoid of rubbish proteins, mUBPy, MSJ-1, and proteasomes could be involved in the mouse sperm acrosome reaction, as suggested for sea urchin sperm [27] and human sperm [41] proteasome, and/or in sperm penetration through the egg coatings, as recently proposed for the ascidian sperm proteasome [42], now that the mammalian protease acrosin has been shown not to be the sperm zona-lysin [43]. Moreover, mUBPy, MSJ-1, and proteasomes colocalize also in the sperm centrosomal region; consequently, they might be recruited in some of the numerous functions coordinated by the centrosome [36, 44, 45]. In this regard, it is worth mentioning what has recently been discovered by Lee and coworkers [46]. CeUBP130, the 130-kDa deubiquitinating enzyme of Caenorhabditis elegans, localizes in the sperm centrosome. CeUBP130-defective sperm fail to form asters necessary for mitotic spindle formation in the fertilized oocyte, while wild-type sperm could rescue the embryonic lethality due to CeUBP130 deficiency [46]. So, a novel, unexpected function for a deubiquitinating enzyme of the UBP group has been discovered. It might be that mUBPy, alone or in association with MSJ-1 and/or proteasome, could be involved in the formation of a functional microtubule-organizing center in the mammalian zygote.
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FOOTNOTES |
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2 Correspondence: Giovanna Berruti, Department of Biology, University of Milan, Via Celoria 26, 20133 Milano, Italy. FAX: 39 025 031 4802; giovanna.berruti{at}unimi.it
Received: 9 April 2004.
First decision: 6 May 2004.
Accepted: 22 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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protein: characterization of its cellular and developmental pattern of expression in the male germ line. Mol Reprod Dev 1997 47:370-379[CrossRef][Medline]This article has been cited by other articles:
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  V. Y. Rawe, E. S. Diaz, R. Abdelmassih, C. Wojcik, P. Morales, P. Sutovsky, and H. E. Chemes The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans Hum. Reprod., March 1, 2008; 23(3): 573 - 580. [Abstract] [Full Text] [PDF]
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 G Manandhar, D Feng, Y-J Yi, L Lai, J Letko, J Laurincik, M Sutovsky, J L Salisbury, R S Prather, H Schatten, et al. Centrosomal protein centrin is not detectable during early pre-implantation development but reappears during late blastocyst stage in porcine embryos. Reproduction, September 1, 2006; 132(3): 423 - 434. [Abstract] [Full Text] [PDF]
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