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Testis |
Department of Anatomy and Cell Biology,3 Queen's University, Kingston, Ontario, Canada K7L3N6
Departments of Animal Science and Obstetrics & Gynecology,4 University of Missouri-Columbia, Missouri 65211-5300
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, histone, manchette, nucleus, perinuclear theca, sperm, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Morphological studies indicate that PT assembly occurs in two stages: First, the emergence of the SL occurs in concert with the acrosome development in early spermiogenesis; second, the PAS is assembled during the caudal descent of the manchette in midspermiogenesis [1]. To date, a range of both traditional and nontraditional cytoskeletal proteins have been found to reside within the PT, having a particular subcellular distribution within these distinct regions. Numerous PT proteins typically accumulate in the SL by the onset of spermatid elongation [1, 2], while some initially localize to the membranes of proacrosomic vesicles in round spermatids [2, 3]. With the exception of SubH2Bv, a histone H2B-like variant retained within the SL [3], many PT proteins become associated to the PAS in late elongated spermatids [2]. In the mature spermatozoa, a group of alkali-soluble PT proteins are found throughout both regions of the PT [2], whereas the localization of calicin [7] and cylicin II [8] to the PAS is accompanied by their complete loss of detection in the SL. Other PT proteins, such as cylicin I [9], CPß3 [10], and somatic-type histones [11], are exclusively localized to the PAS in the mature sperm; however, their developmental characterizations during spermiogenesis have yet to be completed.
The transient appearance of the manchette, a microtubule network surrounding the caudal half of spermatid nuclei during midspermiogenesis, is coincident with the period of greatest morphological changes to the sperm head (reviewed in [12]). In midspermiogenesis, the manchette sequentially descends and depolymerizes before spermiation. Mice with abnormal or ectopic manchettes, either by genetic mutation [13] or chemical disruption [14], produce spermatozoa with severe head defects, thereby providing evidence for manchette involvement in sperm-head shaping. Precisely how the manchette might contribute to this function remains unclear. Russell et al. [14] hypothesized that manchette microtubules can wield structural forces onto the developing sperm head, possibly acting as a scaffold to influence shaping. However, recent reports indicate that the manchette contains both nucleocytoplasmic transport elements, such as RanGTPase [15], and microtubule motor proteins, such as kinesins [16, 17] and dyneins [18]. These studies strengthen the notion of manchette propensity for protein and organelle transport, a process termed intramanchette transport (IMT) [19]. While IMT may contribute to multiple features of sperm-head shaping, it directly supports a proposal for the involvement of the manchette in PAS assembly [2]. The latter conjecture was based on two key observations: 1) the appearance of the PAS occurs in the wake of manchette descent and 2) the translational machinery required for the synthesis of PAS components likely resides in the distal cytoplasm. However, no further studies to date have directly addressed the possible relationship between manchette function and PT assembly.
Spermatid nuclear shaping is also driven in part by the extreme changes in nucleoprotein content resulting in a striking remodeling of sperm chromatin (reviewed in [20]). Sequential replacement of somatic and testis-specific histones from chromatin by transition proteins and protamines accelerates condensation of the sperm nucleus during midspermiogenesis. With some exceptions, most notably in humans [21, 22], the majority of somatic histones are removed from the elongating sperm nuclei of most species [20], an event likely facilitated by posttranslational modifications, such as ubiquitination [23, 24] and acetylation [25]. It is widely accepted that histones of nuclear origin are degraded either within the nucleus by chromatin-associated proteases [26, 27] and/or by ubiquitin-mediated proteosomal degradation [28, 29]. However, we have recently identified a population of nonnuclear somatic histones residing within the PAS of the mature bovine sperm [11]. These findings prompted us to assess the localization of somatic histones throughout the course of bovine spermiogenesis, using three immunolocalization techniques, in an attempt to elucidate the developmental origins of PT-derived somatic histones.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testes from mature bulls were collected immediately after slaughter from a local abattoir and processed for immunoperoxidase microscopy and immunogold electron microscopy as previously described by Oko and Maravei [2]. Briefly, for light microscopy, testicular tissue was fixed in Bouin fixative and was paraffin embedded following dehydration in ethanol washes. For electron microscopy, testicular tissue was fixed in 0.5% gluteraldehyde and 4% paraformaldehyde and embedded in LR white (Polysciences Inc., Warrington, PA).
Testicular cells and mature spermatozoa from cauda epididymis were isolated and processed for indirect immunofluorescence microscopy according to methods described elsewhere [30]. Briefly, concentrated testicular cell or epididymal sperm suspensions were isolated in 200 mM phosphate-buffered saline (PBS) from the seminiferous tubules or cauda epididymis, respectively, and allowed to settle onto poly-L-lysine-coated coverslips overlayed with PBS at 37°C. Coverslips were fixed in 2% formaldehyde/PBS for 40 min at room temperature and subsequently stored in PBS at 4°C.
Antibody Preparation and Acquisition
Polyclonal rabbit antihistone serum was affinity purified against recombinant somatic histones H3, H2B, H2A, and H4 (Upstate Biotechnology, Lake Placid, NY), as previously described [11]. Affinity-purified polyclonal anti-SubH2Bv antibodies were also acquired as described elsewhere [3]. The anti-ß-tubulin monoclonal antibody (E7) developed by Dr. M. Klymkowsky was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biological Sciences at the University of Iowa (Iowa City, IA). Monoclonal antibodies against nuclear pore complex protein (NPC) were also commercially acquired (Mab414; CRP Inc., Berkeley, CA).
Immunoperoxidase Staining for Light Microscopy
Processing of paraffin-embedded tissue and subsequent immunoperoxidase staining were conducted as described in [2], with some modifications. An antigen-retrieval technique was used before primary antibody incubation [31]. Slide-mounted deparaffinized testicular sections were placed in a Coplin jar and immersed in a 0.01 M sodium citrate solution, pH 6.0 (total volume 50 ml). Slides were first incubated for 2 min, at high power, followed by a 15-min incubation at 10% power, in a conventional 1100-W microwave (Danby Products Ltd., Guelph, ON, Canada). Slides were allowed to cool to room temperature before equilibration in 300 mM glycine. Immunolabeling was conducted using an avidin-biotin complex (ABC) kit (Vector Labs, Burlingame, CA). Nonspecific sites were sequentially blocked with avidin and biotin blocking serum, followed by 10% normal goat serum (NGS). Primary antibody incubations with all four affinity-purified antihistone antibodies were conducted overnight at 4°C. After washes in 25 mM Tris-buffered saline (TBS) with 0.1% Tween, sections were incubated with biotinylated goat anti-rabbit IgG secondary antibodies (1:200; Vector Labs), followed by incubation with ABC, according to instructions provided by the manufacturers. Peroxidase reactivity was visualized by incubation in 0.05% diaminobenzidine, and sections were counterstained with 0.1% methylene blue. Control sections were incubated in either the absence of primary antibody, with preimmune rabbit serum, or with affinity-purified primary antibodies pretreated with recombinant histone proteins H3, H2B, H2A, and H4 (Upstate Biotechnology, Lake Placid, NY).
Immunogold Labeling for Transmission Electron Microscopy
LR-white-embedded tissue processing and successive procedures for immunogold labeling used in this study were conducted as previously described [2], with some modifications. Following mounting of ultrathin sections on formavar-coated nickel grids, the sections were first blocked with 10% NGS and were then incubated overnight at 4°C, using all four concentrated affinity-purified antihistone antibodies or anti-NPC monoclonal antibodies (1:100). Following washes, sections were incubated with goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to 10-nm gold particles, respectively (1:20; Sigma, Mississauga, ON, Canada). Sections were counterstained with uranyl acetate and lead citrate and examined by transmission electron microscope (EM; Hitachi 7000). Control sections were incubated in either the absence of primary antibodies or with preimmune serum.
Indirect Immunofluorescence of Testicular Cells and Epididymal Sperm
Coverslip-mounted bovine testicular cells and mature spermatozoa from bovine cauda epididymis were processed according to previously described methods [30], with some modifications. Coverslip-mounted cells were permeabilized in 0.1% Triton X-100 made in 0.1 M phosphate-buffered saline (PBS-T) for 40 min at room temperature. Non-specific antibody binding was blocked by incubation in 10% normal goat serum in PBS-T for 25 min. Coverslips were then incubated overnight at 4°C in a mixture of either anti-H3/anti-H2B/anti-H2A/anti-H4 (1:20) and anti-ß-tubulin (1: 200) or anti-SubH2Bv (1:20) and anti-NPC (1:200), made in PBS-T with 1%NGS (PBS-T/N). Following brief washes in PBS-T/N, coverslips were incubated for 40 min in a mixture of either fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse IgG and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat-anti rabbit IgG or FITC-conjugated goat anti-rabbit IgG and TRITC-conjugated goat-anti-mouse IgG (1:80; Zymed Labs Inc., San Francisco, CA). Secondary incubations were diluted in PBS-T/N, and included 4' 6-diamidino-2-phenylindole (DAPI; 5 µg/ ml; Molecular Probes Inc., Eugene, OR). Coverslips were briefly washed in PBS-T/N and then mounted on microscope slides in an antifade-mounting medium (VectaShield; Vector Labs).
Immunofluorescence analysis of bovine testicular and epididymal cells was conducted using a Nikon Eclipse 800 microscope equipped with differential interference contrast (DIC). Images were acquired with a CoolSnap HQ RTE/CCD 1217 digital camera, operated by Metamorph software (Universal Imaging Corp., West Chester, PA). Digital images were edited using Adobe Photoshop CS (Version 8.0) software. Controls for indirect immunofluorescence (IIF) staining by the affinity-purified antihistone primary antibodies was accomplished by either omitting the primary antibody incubation or using preimmune serum in their place.
Staging of Bovine Seminiferous Epithelium
The stages of the cycle of bovine seminiferous epithelium (I–XII) and steps in spermiogenesis (1–14) in testicular sections were identified according to Berndston and Desjardins [32] for light microscopy (LM) and Barth and Oko [33] for EM analysis. The 14 steps of bovine spermiogenesis were defined according to Barth and Oko [33] for EM analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular sections probed with affinity-purified antihistone antibodies yielded identical patterns of immunoperoxidase localization for all histone subtypes, although the general intensity of anti-H3 labeling was remarkably stronger relative to the other three antihistone probes. Thus, the pattern of immunoperoxidase reactivity for the antibodies against each histone subtype will be collectively reported as antihistone labeling.
Antihistone labeling in bovine testicular sections was observed in somatic and spermatogenic cells of the seminiferous epithelium. Specifically, all somatic nuclei, including those of Leydig cells, fibroblasts, and Sertoli cells, were strongly labeled (Figs. 1 and 2). Histone immunodetection was also observed in the nuclei of spermatogonia and spermatocytes in all stages of spermatogenesis and in the metaphase II plate of dividing secondary spermatocytes (Fig. 2B).
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In contrast, histone immunolabeling during spermatid elongation displayed pronounced differences in both intensity and compartmental localization. At the onset of spermatid elongation, diffuse nuclear immunoreactivity was observed in the nuclei of step-7 spermatids (Fig. 1A). Step-8 and step-9 spermatids showed increased intensity of immunoperoxidase reactivity throughout the elongating nucleus, relative to spermatids of previous phases (Fig. 1, A and B). Strong antihistone labeling was apparent in the nuclei of step-10 spermatids (Fig. 1C). In addition, histone immunoreactivity was detected on manchette microtubules, appearing as filamentous material surrounding the caudal nucleus and extending into the distal spermatid cytoplasm. Polarization of antihistone immunoperoxidase reactivity was first apparent in the caudal head region of step-11 spermatids, appearing cylindrical in shape (Fig. 1, D and E). However, it was difficult to discriminate nuclear from perinuclear subcellular localization at this level of resolution. Faint histone immunoperoxidase reactivity in filaments extending beyond the caudal borders of this region was also apparent, indicative of manchette localization. Overall, the detection of immunoreactivity in step-11 spermatids was largely dependent on the cut of the section (Fig. 2A). Nonetheless, reactivity in the apical nuclear regions and in the developing acrosome was undetectable in spermatids of this stage. Immunoperoxidase reactivity remained present in the caudal sperm head and manchette regions of step-12 spermatids (Fig. 2B). Following manchette descent, maturation-phase spermatids (steps 13 and 14) seen in stage-I to stage-VI tubules also showed intense focal labeling to the caudal sperm-head region (Fig. 2, C and D). Although it was difficult to determine subcellular localization of this reactivity within the sperm head at this level of resolution, it was observed that the distal cytoplasm, ensuing residual bodies, and cytoplasmic droplets all remained unlabeled in all elongating spermatids.
Round spermatids from both the Golgi and acrosomic cap phases of spermiogenesis (steps 1–3 and 4–6, respectively) all showed dispersed internuclear labeling (Fig. 2, C and D). No detectable immunoperoxidase reactivity was observed in sections probed with either preimmune serum or secondary antibodies alone (Fig. 2, E and F). Furthermore, somatic and spermatogenic nuclei, manchette microtubules, and caudal sperm-head regions were devoid of any immunoperoxidase reactivity in sections probed with antihistone antibodies preincubated with recombinant histone-blocking proteins (data not shown).
Immunogold Electron Microscopy Analysis of Bovine Spermiogenesis
Somatic Histones Testicular sections probed with the antihistone antibodies by immunogold EM provided further resolution of the subcellular localization previously unidentified by LM. Examination of these sections by EM revealed identical subcellular localization patterns for all four antihistone antibodies. Antihistone gold labeling was apparent throughout the nuclei of all somatic cells of the seminiferous epithelium and interstitium, including Sertoli, Leydig, and fibroblast cells, and of prehaploid spermatogenic cells, including spermatogonia and spermatocytes (data not shown). Antihistone gold particles in early round spermatids from both Golgi and cap phases of spermiogenesis also displayed exclusive nuclear localization, with no detectable labeling of the developing acrosome or periacrosomal compartments (Fig. 3).
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Remarkable changes in the immunogold localization of histones were observed in elongating spermatids from steps 9 to 12, confirming both intranuclear and extranuclear histone localization previously observed by immunoperoxidase light microscopy. Step-9 and step-10 spermatids showed dispersed homogeneous antihistone immunogold labeling throughout the elongating nuclei and in the developing manchette microtubules (Fig. 4, A and B). The nuclei of step-11 spermatids showed a relative decrease in immunogold labeling; however, gold particles continued to be detected on manchette microtubules (Fig. 5, A and B). By late step 11, the near fully condensed nuclei were devoid of gold particle labeling (Fig. 5C). Nuclear gold particles remained undetectable in the condensed and elongated nuclei of step 12 (Fig. 5D) and in the maturation phase nuclei of step-13 and step-14 spermatids (Fig. 6).
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In contrast with the immunoperoxidase study described above, histone immunogold labeling was sparse in the caudal head region of step-12 spermatids (Fig. 5D). In addition to the lack of nuclear immunogold labeling, the developing postacrosomal sheath (PAS) region of the perinuclear theca was also poorly labeled. The assembly of the PAS appeared as discontinuous tracks of electron-dense material outside of the nucleus, formed in the wake of the descending manchette in late step-11 and step-12 spermatids (Fig. 5, C and D). Scant immunogold labeling was detected in the equatorial segment region of the subacrosomal layer (ESL) of step-12 spermatids (Fig. 5D) that was not apparent in step-11 spermatids. However, at the onset of maturation phase following manchette depolymerization, focal gold-particle labeling was apparent in the fully assembled PAS and in the ESL of step-13 spermatids (Fig. 6A). Immunogold labeling persisted in the PAS of step-14 spermatids, but was undetectable in the subacrosomal layer following equatorial segment condensation, at spermiation (Fig. 6B).
Transverse and longitudinal sections of elongating spermatid manchette regions also revealed a transient electron-dense structure, strongly immunogold labeled by the antihistone antibodies. The structure, termed histone-containing body (HCB), was first seen in step-11 spermatids, located between the manchette microtubules, slightly caudal to the base of the nucleus and lateral to the tail axoneme (Fig. 7A). In step-12 spermatids, the HCB was found in the more distal region between the descending manchette microtubules (Fig. 7B). Following manchette descent and depolymerization, the HCB was no longer detectable. Preimmune serum and secondary antibodies alone also did not yield any immunogold labeling to any subcellular structures within the testis sections (data not shown).
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Nuclear Pore Complex To examine whether nuclear pore complexes could still be present and actively transporting histones between spermatid nucleus and cytoplasm, spermatids were immunogold labeled with monoclonal anti-NPC antibodies. NPC proteins showed focal localization to the base of the nucleus at the onset of chromatin condensation, in a region adjacent to the developing redundant nuclear envelope (RNE; Fig. 7C). Though the RNE was unstained by our methods, the immunogold localization of the anti-NPC probe persisted in this region throughout the course of spermiogenesis to spermiation. No immunogold labeling was detected along the lateral borders of the nucleus during the ljspermatid elongation phase.
Indirect Immunofluorescence Analysis of Slide-Mounted Bovine Spermiogenic Cells
Somatic Histones Consistent with the two previously used immunolocalization techniques, antihistone-labeling patterns of isolated testicular cell suspensions visualized by IIF were identical for all four antihistone antibodies. Examination of nuclear and nonnuclear histone immunolocalization patterns in elongating spermatids by IIF were consistent with results obtained by immunoperoxidase LM and immunogold EM. Though undetectable in the nuclei of all elongating spermatids, antihistone labeling was seen along manchette microtubules, identified by its colocalization with anti-ß-tubulin (Fig. 8A). Step-11 and step-12 spermatids also displayed diffuse antihistone labeling in the developing PAS of the PT in the caudal sperm head. Antihistone labeling of the PAS persisted in mature spermatids, and in spermatozoa isolated from the cauda epididymis (Fig. 8B). Spermatogenic cells probed with preimmune serum or fluorescent-conjugated secondary antibodies alone yielded no detectable immunoexpression (data not shown). Furthermore, ejaculated spermatozoa showed immunolocalization patterns identical to cauda epididymal spermatozoa (data not shown).
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Nuclear Pore Complex Double labeling of bovine spermiogenic cells using monoclonal anti-NPC and polyclonal anti-SubH2Bv antibodies revealed rapid relocation of NPC in early spermiogenesis. Premeiotic germ cells displayed even distributions of NPC labeling surrounding the nucleus (Fig. 8C), identified by an absence of SubH2Bv immunofluorescence. However, cap-phase round spermatids (steps 4–7) all showed focal NPC localization to the caudal pole of the nucleus, before nuclear condensation and elongation. These cells were identified by both the subacrosomal marker SubH2Bv, which delineates the forming subacrosomal layer during acrosome development [3], and by a round nuclear shape, as assessed by DAPI staining. NPC labeling persisted at the base of the nuclei of elongating and mature spermatids.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nuclear Histone Transitions During Bovine Spermatid Elongation
With the exception of humans [21, 22], near-complete histone replacement is a hallmark of mammalian spermatid elongation. Consistent for all three immunolocalization techniques, the somatic histone immunolocalization observed in bovine spermatid nuclei was in agreement with both localization and biochemical isolation studies conducted in numerous species [20]. Changes in nuclear immunodetection patterns for all four somatic-core histones were observed in step-8 to step-11 elongating spermatids. During late step-10 and early step-11 spermatids, histone labeling became localized to the caudal pole of the nucleus. This shift in nuclear localization likely represents the removal and/or degradation of somatic histones during spermatid nuclear condensation and temporally correlates with the nuclear occurrence of both acetylated histones [25] and histone acetyltransferases [34] and the expression of ubiquitinated histones [23, 24]. These modifications have been proposed to be the mechanism for histone-chromatin dissociation and eventual histone degradation. The absence of histone immunodetection in spermatid nuclei from late step 11 and beyond also concur with immunolocalization studies performed in several species [35, 36].
Extranuclear Histone Localization During Bovine Spermiogenesis
The combined analysis by three immunolocalization techniques confirmed the localization of somatic histones along manchette microtubules in spermatids from steps 10– 12. Though the conventional technique of immunoperoxidase LM showed varying degrees of histone labeling on the manchette, its detection appeared to be dependent on the slice of the section. Immunogold EM analysis resolved histone localization along step-10 and -11 manchette microtubules, however gold labeling was difficult to detect in step-12 manchettes. Nonetheless, IIF showed conclusive histone colocalization with anti-ß-tubulin decorated manchettes in all spermatids, including those of step 12. Similar to manchette-associated histones, histone localization within the developing perinuclear theca also varied depending on the employed technique. It was evident by both immunoperoxidase LM and IIF analyses that somatic histone immunoexpression remained associated with the caudal sperm head in late step-11 to step-14 spermatids. On the contrary, ultrastructural analysis by EM showed poor immunogold labeling in both the caudal nucleus and the developing PAS of step-11 and -12 elongating spermatids. However, following the descent and depolymerization of manchette microtubules in step-13 spermatids, histone immunogold labeling was exclusively found in the developed PAS and the equatorial segment portion of the subacrosomal layer (ESL), and only in the PAS of step-14 spermatids.
In comparison with results obtained by the other two techniques, the refractory nature of subcellular histone detection in the PAS and manchettes of step-12 spermatids by EM was unexpected. It is possible that histones associated to these structures may be present in relatively lower abundance during this phase of spermatid development, hence, their limited detection by immunogold EM. However, given their detection by IIF, this discrepancy was more likely due to epitope masking by fixation and the sensitivity of the localization technique. Further reinforcing this view were the observations that histone immunoperoxidase expression in the head and manchette of step-12 spermatids by LM was also difficult to detect without the use of an antigen-retrieval technique (data not shown).
A third, novel extranuclear accumulation of somatic histone was found in a previously uncharacterized electron-dense body. We termed this structure the histone-containing body (HCB), which had a transient association with step-11 and -12 manchettes. Though our analysis of the HCB was limited to only ultrastructural examination, its consistent appearance in the distal cytoplasmic region during late acrosome phase of spermiogenesis suggests that it may be a possible site of histone synthesis, degradation, or storage.
Origins of Nonnuclear Somatic Histones in the Perinuclear Theca
We maintain the opinion from our previous study [11] that PT-derived histones are not recycled remnants from nucleoprotein transition during spermatid nuclear elongation and are more likely products of de novo synthesis. Due to evidence of nucleohistone degradation by chromatin-associated proteases [26, 27] and ubiquitin-mediated proteosomal degradation [23, 24, 28, 29], the possibility of nuclear histones recycling for PT incorporation seems unlikely. Though it is plausible that a subset of nuclear histones may evade these proposed degradation pathways, kinetic studies in the mouse testis showed histone synthesis during late spermiogenesis and the persistence of histones synthesized during this period in epididymal spermatozoa [37]. These latter findings are a precedent for de novo histone synthesis during spermiogenesis, possibly including the extranuclear histone pools we describe here. As such, de novo histone synthesis in late spermiogenesis would therefore require delayed translation of mRNA transcripts due to the inactivation of the transcriptional machinery during spermatid elongation. It is clear that the present report is limited in resolving these issues and that further studies in both the kinetics of histone synthesis and nuclear histone turnover during spermiogenesis are required to substantiate this claim. Nonetheless, we have also detected extranuclear histones in rat and mouse spermatids (unpublished observations), thereby supporting the likelihood that this is a common phenomenon among mammalian species.
Contrary to many other PT proteins studied to date [1– 3], we found that somatic histones did not localize to acrosomic membranes, nor did they associate with the SL during spermiogenesis. This finding, coupled with the observation that histones appear late in the PAS of step-11 and step-12 spermatids, strengthens the hypothesis that the distal cytoplasm plays an important role in the synthesis of PAS protein constituents [2]. Due to the absence of apical cytoplasm in the sperm head at this time, it can be assumed that the spermatid translational machinery would be polarized to a region caudal to the nucleus. Studies on PERF 15, a protein residing in perforatorium of murid sperm PT, further support a nonacrosomal phase in mammalian PT development [38, 39]. PERF 15 showed distal cytoplasmic labeling in step-9 to step-12 elongating rat spermatids before the formation of the perforatorium. Although the perforatorium is exclusive to falciform-shaped murid spermatozoa [38], the mechanism for its assembly may be conserved during PAS synthesis in spatulated eutherian sperm.
Interestingly, an accumulation of histones was detected in the ESL of step-12 and -13 spermatids, which was no longer detectable by step-14. Several explanations may be attributed to the lack of ESL histone labeling in step-14 spermatids (Fig. 9). Somatic histones, synthesized in the distal cytoplasm, may rapidly occupy the newly formed PAS region in step 12, extending beyond the apical limits of the PAS proper in step 13. As such, structural changes during equatorial segment condensation may result in masking of histone epitopes in the ESL of step-14 spermatids. It is also conceivable that the combined labeled regions of the PAS and ESL in step-13 spermatids represent the entire PAS proper. Thus, a change in the relative position of the acrosome apically and the PAS proper caudally may also reflect these observations. Another possibility may be the elimination, by degradation or translocation, of histones in ESL before spermiation. Nonetheless, changes in subcellular histone localization patterns appeared to be a common property of several PT proteins during the latter phases of spermiogenesis, most notably actin [40], calicin [7], calmodulin [41], and cylicins [8, 9, 42], whose detection in some cases was reported to vary in a species-specific manner. Clearly, the developmental dynamics of PT morphogenesis during late spermiogenesis warrants further investigation.
Significance of Somatic Histone Localization to the Manchette and Perinuclear Theca
The current model of intramanchette transport (IMT) [19] suggests the manchette serves as a track for the nucleocytoplasmic transport of histones removed from the nucleus during spermatid elongation, which may, in part, be regulated by Ran-GTPase [15]. The model further proposes that ubiquitin-mediated proteolytic degradation of histones may also occur along the manchette, by virtue of manchette localized proteosomal subunits [29] (unpublished observations). Accordingly, high ubiquitin expression has been observed in the cytoplasmic lobe of numerous species [28]. It is important to note that no focal localization of either ubiquitinated histones or ubiquitin has been reported on the manchette to date [23, 28], nor have any ubiquitinated forms of H4 been identified [43]. Taken together, these studies indicate that the manchette-localized histones we report here are likely not ubiquitinated, thereby raising uncertainties in the proposed role of the manchette as a major pathway for proteosomal degradation of nuclear histones.
Our findings also question the strategic positioning of the manchette relative to the elongating nucleus for nucleocytoplasmic transport. Due to their essential absence from the condensed nucleus, it is clear that histones that were not subjected to proteolytic degradation within the nucleus must be exported at the site of NPC aggregation (Fig. 9). In agreement with previous studies in human [44], rat [45], rhesus monkey, and bovine [30] testis, we demonstrated a shift in NPC localization to the base of the bovine spermatid nuclei by EM and IIF. We further conclude that focal NPC localization takes place in round spermatids, before both spermatid elongation and spermatid nucleoprotein transitions. In addition, we failed to detect NPC labeling along the lateral aspect of the nuclear envelope, abutting the parallel apical manchette microtubules in step-9 spermatids. Thus, the export of residual somatic histones during spermatid elongation must take place entirely at the base of the nucleus, a process previously observed with H1t removal in rat spermatids [46]. This would require regulation by nuclear export mechanisms, such as the archetypal exportin/CRM-1-dependent pathway [19], which currently remains uncharacterized in mammalian spermatids. Consequently, if their disposal were truly dependent on the manchette, somatic histones would have to traverse the intramanchette cytoplasmic space from the NPC to the manchette, which would require a specific manchette-targeted protein transport mechanism.
To date, the involvement of the manchette in PT formation has been largely based on morphological studies, demonstrating a relationship between the appearance of the PAS in the wake of manchette descent in late spermiogenesis [2, 5, 47]. The localization of somatic histones to both the manchette and the developing PAS support the proposed mechanism that PT components synthesized in the distal cytoplasm could be transported to the PAS by the manchette [2]. It is possible that the transport of PT-bound proteins, such as somatic histones, may be achieved through previously discussed elements of IMT [19]. Furthermore, other protein translocators, such as the C-terminal kinesin motor proteins KIFC1 and KIFC5A, have also been localized to the manchette [16, 17]. Interestingly, the direction of C-terminal kinesin movement toward the perinuclear ring, the microtubule-organizing center for the manchette, is highly favorable for this proposed manchette function [48]. Further support for the translocation of PT proteins stems from preliminary localization studies of other PAS protein constituents, such as PT 32, which also shares manchette localization before PAS incorporation during bovine spermiogenesis (unpublished observations).
Despite the numerous roles that have been suggested for the PT [1, 49], the function of PT-derived somatic histones remains unelucidated [11]. Due to their lack of association with the acrosome vesicle and the subacrosomal layer of the PT, they likely do not participate in the process of acrosome-nuclear docking, unlike the histone variant SubH2Bv [3]. PT-derived somatic histones may possibly perform structural functions in the maintenance of the PT architecture and, hence, sperm-head shaping. Though this role is somewhat unorthodox for somatic histone proteins, their basic nature confers with the electropositive nature of several other PT proteins, including the cylicins and calicin [1]. However, PT-derived somatic histones were readily extractable by salt or ionic detergent [11], a property that significantly differs from the other extraction-resistant PT protein. As such, their contribution to the structural integrity of the PT is indeed questionable. Although the PT has been implicated in the process of fertilization [6], a role for sperm-borne extranuclear somatic histones, possibly as a readily available histone pool during the initial stages of male pronuclear development, remains to be defined in this process. With regard to assisted reproductive technologies, PT-derived histones could contribute to the ability of mammalian spermatozoa to take up plasmid DNA, a property used in sperm-mediated gene transfer techniques for in vitro fertilization or intracytoplasmic sperm injection [50].
In summary, we have shown that, in addition to the loss of nuclear histone localization in midspermiogenesis, somatic histones associate with manchette microtubules and the developing PAS in elongating spermatids in bovine testis. This unusual nonnuclear localization of somatic core histones provides unique insight into possible roles of the manchette and the synthesis of the PAS during spermatid elongation. Our interpretation of these findings supports the model of IMT for sperm-head shaping, suggesting that the manchette can act as a channel for protein transport. However, whether manchette-localized histones are exclusively involved in PAS assembly remains to be determined. The localization of histones to the developing PAS independent of acrosome formation suggests that PT development is a multifaceted process, where PT substructures are likely derived from different subcellular origins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 613 533 2566; ro3{at}post.queensu.ca
Received: 31 March 2004.
First decision: 20 April 2004.
Accepted: 18 May 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  J. R. Mountjoy, W. Xu, D. McLeod, D. Hyndman, and R. Oko RAB2A: A Major Subacrosomal Protein of Bovine Spermatozoa Implicated in Acrosomal Biogenesis Biol Reprod, August 1, 2008; 79(2): 223 - 232. [Abstract] [Full Text] [PDF]
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 A. T. H. Wu, P. Sutovsky, G. Manandhar, W. Xu, M. Katayama, B. N. Day, K.-W. Park, Y.-J. Yi, Y. W. Xi, R. S. Prather, et al. PAWP, a Sperm-specific WW Domain-binding Protein, Promotes Meiotic Resumption and Pronuclear Development during Fertilization J. Biol. Chem., April 20, 2007; 282(16): 12164 - 12175. [Abstract] [Full Text] [PDF]
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C. Fitzgerald, C. Sikora, V. Lawson, K. Dong, M. Cheng, R. Oko, and F. A. van der Hoorn Mammalian Transcription in Support of Hybrid mRNA and Protein Synthesis in Testis and Lung J. Biol. Chem., December 15, 2006; 281(50): 38172 - 38180. [Abstract] [Full Text] [PDF]
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W.-X. Yang, H. Jefferson, and A. O. Sperry The Molecular Motor KIFC1 Associates with a Complex Containing Nucleoporin NUP62 That Is Regulated During Development and by the Small GTPase RAN Biol Reprod, April 1, 2006; 74(4): 684 - 690. [Abstract] [Full Text] [PDF]
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