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BOR - Papers in Press, published online ahead of print January 16, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.062901
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BIOLOGY OF REPRODUCTION 78, 577–585 (2008)
DOI: 10.1095/biolreprod.107.062901
© 2008 by the Society for the Study of Reproduction, Inc.

Gamete Biology; 

Immunocytochemical and Ultrastructural Analyses of the Function of the Ubiquitin-Proteasome System During Spermiogenesis with the Use of the Inhibitors of Proteasome Proteolytic Activity in the Alga, Chara vulgaris1

Agnieszka Wojtczak  2 and Maria Kwiatkowska 

Department of Cytophysiology, University of Lódz, 90-231 Lódz, Poland

ABSTRACT

Spermiogenesis in Chara vulgaris and in animals share many common features, including exchange of nucleohistones into nucleoprotamines, remodeling and extreme condensation of chromatin, formation of flagellae and of microtubule manchette, and decrease in cytoplasm volume. In C. vulgaris, spermiogenesis is not preceded by meiosis since this alga is a haplobiont. In the present work we showed that in early spermiogenesis characterized by a significant metabolic activity of spermatids, the inhibitors of proteasomes did not visibly change their ultrastructure but significantly prolonged this process. At late stages of spermiogenesis, MG-132 and epoxomicin dramatically changed the structure of nuclei: regular fibrillar and lamellar structure of chromatin was disturbed and clusters of grains corresponding to aggresomes appeared, but the nucleus shape and cytoplasm structure were the same as in the controls. Immunocytochemical studies revealed that these inhibitors blocked disappearance of histones from nuclei while the structures corresponding to aggresomes were clusters of undegraded ubiquitinated histones, since they gave positive immunosignals indicating the presence of ubiquitin and histones.

aggresomes, Chara vulgaris, epoxomicin, gamete biology, MG-132, proteasomes, spermiogenesis, sperm maturation, ubiquitin

INTRODUCTION

Transformation of somatic chromatin into unique, extremely condensed chromatin of male generative cells during spermiogenesis is one of the most dramatic processes of remodeling. Chromatin of mammalian spermatozoa is over sixfold more condensed than that of mitotic chromosomes [1, 2]. In spite of its crucial biological role, the mechanisms controlling specific reorganization of a haploid genome during spermiogenesis are poorly known. There are only suggestions concerning the putative role of histone variants as well as the role of posttranslational modifications of histones before and during the incorporation of basic proteins characteristic of sperm [3]. Ubiquitination of histones is one such modification.

Ubiquitin is a 76-amino acid protein common both in Eukaryota [4] and Prokaryota [5]. It can be covalently bound to acceptor proteins via a multistep enzymatic pathway (ubiquitin-activating, -conjugating, and -ligating enzymes - E1, E2, E3). Tandem ligation of ubiquitin to a single substrate molecule results in polyubiquitination [4, 6]. This generally marks protein for ATP-dependent, selective degradation by the proteasome—a large, highly conserved multicatalitic complex 26S—present in all eukaryotes to date. This complex consists of the 20S catalytic core and PA700 (19S) regulatory complex [4, 6–8]. Structural analysis of the proteasome by cryoelectron tomography demonstrated the central chamber and two antechambers of the core complex, and 18 constitutive subunits of the 19S regulatory complex [9].

Ubiquitination of histones during spermiogenesis was first described by Agell et al. [10] in roosters during the exchange of nucleohistones for nucleoprotamines. These authors suggested that ubiquitin binding with histones causes chromatin relaxation, making binding of DNA with protamines and remodeling of chromatin possible. This hypothesis was later confirmed by these authors when they investigated spermiogenesis in chickens [11], mice [12, 13], and rats [14, 15]. This hypothesis was further confirmed by experiments in mutated mice involving ubiquitin ligase HR6B, the enzyme conjugating with ubiquitin. Ubiquitin ligase HR6B inactivation causes male infertility [16], if this inactivation is very strong [17]. High activity of the ubiquitin-conjugating enzyme E2, which binds ubiquitin with the histone H2A in rat testes [18] as well as intensifies expression of all genes of ubiquitin—UnI, UbII, Ub-t52, and Ub-t80—in mature chicken testes [19], also supports the key role of this protein during spermiogenesis. Moreover, it has been observed that Ubl4b, the ubiquitin-related retrogene appearing during late spermiogenesis in mice [20], as well as Usp42–deubiquitinating enzyme [21] and Cullin3—which together with KLHL10 forms CUL3-based ubiquitin E3 ligase [22]— are present during late spermiogenesis, which may also indicate their significant role in sperm differentiation.

On the other hand, use of the immunogold technique and anti-proteasome antibodies for ultrastructural analyses has revealed proteasomes along salmon sperm flagellae [23], in acrosomal and postacrosomal regions of human sperm [24], and in the sperm head in humans and rats [25]. The latter authors (Haraguchi et al. [25]) have also presented a precise quantitative analysis of ubiquitin and proteasomes during consecutive stages of spermatogenesis in rats.

It is now believed that during late stages of spermiogenesis in mammals, ubiquitination and deubiquitination systems as well as proteasomes play an important role in chromatin remodeling during which histones are replaced with nucleus-specific transition proteins and then with protamines [22, 25, 26, 27].

Plant spermatozoids, which can actively move in water due to flagellae and in which exchange of histones into protamine-type proteins takes place during spermiogenesis, are found in Marchantia polymorpha [28], Chara vulgaris [29–32], and Chara tomentosa [32, 33]. However, to the best of our knowledge the ubiquitin-proteasome system has been investigated only in C. vulgaris [34–36].

Immunocytochemical studies with anti-ubiquitin and anti-proteasome antibodies has revealed antigenic signals in spermatids during all ten stages of spermiogenesis in Chara vulgaris [36]. In early and mid spermatids (stages II-VI) the strongest antigen signals indicating the presence of proteasomes and ubiquitin have been observed in cytoplasm. During stages VII and VIII when cytochemical, immunocytochemical and electrophoretic analyses reveal the exchange of histones for protamine-type proteins [32, 37, 38], stronger antigen signals have been observed in nuclei than in cytoplasm. In late spermiogenesis both these types of signals are very weak. On this basis a hypothesis has been put forward that the ubiquitin-proteasome system plays a crucial role throughout spermiogenesis.

To verify this hypothesis, we used multiple inhibitors of proteasome proteolytic activity in the present research: epoxomicin, lactacystin, clasto-lactacystin β-lactone, MG-115, and MG-132. These substances primarily irreversibly inhibit chymotrypsine-like activity of proteasomes [39–41]. Application of the inhibitors of proteasome proteolytic activity is one of the main methods for examining ubiquitin-proteasome system functions [6, 41–43]. This method has not been used previously to analyze spermiogenesis. The present results fully confirm the earlier suggestions: the activity of the ubiquitin-proteasome system is indispensable for the proper course of early spermiogenesis and for nuclear protein replacement and changes of the nucleus structure at later stages as well as for removal of superfluous histones.

MATERIALS AND METHODS

Apical parts of Chara vulgaris thalli were obtained from plants grown in an artificial pond located in Rogów Arboretum (Poland). Antheridia were taken from III-V node pleuridia counting from the apical buds. Before the onset of the experiment the plants were cultivated for a few days in tanks containing water from the natural environment and with a natural photoperiod, i.e., 14L:10D. Antheridia of C. vulgaris from all ten stages of spermiogenesis (I-X) were examined.

Treatment with Proteasomal Inhibitors

The thallus fragments carrying antheridia were divided into two groups and were incubated for 24 and 48 h in proteasome inhibitors: 10-µM lactacystin (Sigma), 10-µM clasto-lactacystin β-lactone (Sigma), 10-µM epoxomicin (Sigma), 100-µM MG-115 (carbobenzoxyl-leucinyl-leucinyl-norvalinal; Calbiochem), 100-µM MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinal; Calbiochem).

The solutions of all proteasome inhibitors were prepared with water from the natural environment. The solutions of epoxomicin, MG-115, and MG-132 were prepared with DMSO (Sigma) in the ratio 20 mg/ml.

Plants incubated in the inhibitors and rinsed in pond water, and the control material—untreated or treated only with DMSO, without the inhibitor—were fixed in Carnoy solution (ethanol:acetic acid, 3:1 v/v) for 1 h, rinsed in 96% ethyl alcohol, and kept in 70% ethyl alcohol. The fixed preparations were stained with orcein and Fast Green FCF for about 20 min, and then were rinsed a few times in distilled water.

Antheridia were isolated from the control material and from the proteasome inhibitor-treated nodes. Squashed preparations from the isolated antheridia from all stages of spermiogenesis (I-X) were made on dry ice. The samples were embedded in Canada balsam and analyzed by light microscopy to determine the stage of spermiogenesis. The number of antheridia at each stage of spermiogenesis, from both the control and proteasome inhibitor-treated nodes, was then presented as a percentage of the whole pool of antheridia in a representative group regarded as 100%.

Thirty 3-nodal fragments of thallus were examined per each variant (control and all proteasome inhibitors). In this experiment three replicates were performed.

Electron Microscopy

C. vulgaris plants were fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) supplemented with 0.007 M CaCl2 for 3 h at 4°C. The antheridia were isolated and gently squashed in a drop of heated 2% agar in cacodylate buffer and postfixed in 1% OsO4 in the same buffer for 2 h.

After dehydration in an alcohol series, the material was embedded in Epon 812 and Spurr mixture medium. Ultrathin sections were double-stained with uranyl acetate and lead citrate according to Reynolds [44]. The sections were examined and photographed in a JEOL JEM-1010 transmission electron microscope.

Cytochemical Studies of Histones

To detect histones, whole control plants and those treated with the proteasome inhibitors (epoxomicin, MG-132) were fixed in 4% paraformaldehyde/Sörensen phosphate buffer (0.125 M concentration, pH 7.2) for 1 h at room temperature (RT). Alfert and Geschwind's method [45], modified by Sandritter for spermatozoids [46], was used for staining. Following hydration, the material was hydrolyzed in 5% trichloroacetic acid at 95°C for 15 min. The plants were rinsed three times in 70% ethanol. After a short hydration, they were stained in 0.1% Fast Green FCF (BDH Chemicals Ltd.) in Michaelis buffer (pH 8.18) for 30 min at RT; subsequently, they were rinsed in buffer alone and then briefly in water.

Immunocytochemical Studies of Ubiquitin, Histone H3, and Protamines

The isolated antheridia of C. vulgaris, both from the control and proteasome inhibitor-treated nodes, were fixed in 10% formalin and 4-µm paraffin sections were prepared.

The paraffin sections were deparaffinized with xylene, then gradually hydrated in alcohol series and in distilled water. The antigenic sites were unmasked by microwave treatment (700 W, 0.01 M citrate buffer, pH 6.0, 12 min), after which the slides were cooled and rinsed with distilled water.

Immunocytochemical Localization of Ubiquitin

The sections were washed two times for 5 min each with a mixture of Tris/HCl buffered saline (TBS, pH 7.6, DAKO) and 0.2% Tween 20 (the washing buffer) at RT. The sections then were permeabilized with 0.1% Triton X-100 in TBS for 20 min at RT and subsequently washed two times for 5 min each in the washing buffer.

Next, the sections were incubated for 30 min at RT with the primary antibody (rabbit polyclonal antibody [no. Z0458, DAKO] at a 1:200 dilution) diluted in antibody diluent (DAKO) containing 5% BSA and 0.5% Tween 20.

The sections were washed for 10 min with the washing buffer and then were incubated with the secondary antibodies (anti-rabbit IgG conjugated with FITC [Sigma]) diluted 1:70 in antibody diluent (DAKO) containing 5% BSA and 0.5% Tween 20 for 1 h at RT in darkness. The sections were washed three times for 2 min each in the washing buffer. The slides were stained using DAPI (1 µg/1 ml) for 15 min in darkness. The sections then were embedded in phosphate-buffered saline (PBS: 0.14 M NaCl, 3 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4)/glycerol mixture (9:1) with 2.3% DABCO (1,4-diazabicyclo-[2,2,2] octane, Sigma). The cells were analyzed using an Optiphot-2 epifluorescence microscope (Nikon) equipped with UV-2A (excitation - {lambda} = 360–460 nm) for DAPI and with a B-2A blue light filter (excitation - {lambda} = 450–490 nm) for FITC.

Immunocytochemical Localization of Histone H3

Histone H3 antibodies were used because electrophoretic analyses showed that this histone persisted during spermiogenesis for the longest period of time [33].

Sections were placed in Tris/HCl-buffered saline (TBS, pH 7.6, DAKO) for 10 min at RT. They were then permeabilized with 0.1% Triton X-100 in TBS for 14 min at RT. The sections were washed three times for 2 min each with the mixture of TBS and 0.2% Tween 20 (the washing buffer), blocked in 5% BSA in TBS for 1 h, and rinsed again in the washing buffer for 5 min. After this, the sections were incubated overnight at 4°C with the primary antibody to histone H3 (rabbit polyclonal antibody [no. 9715, Cell Signaling] at a 1:25 dilution) diluted in TBS containing 5% BSA and 0.5% Tween 20.

The sections were washed three times for 5 min each with the washing buffer, and then were incubated with the secondary antibodies (anti-rabbit IgG conjugated with FITC, Sigma), diluted 1:70 in TBS containing 5% BSA and 0.5% Tween 20, for 1 h at RT in darkness. The sections were washed for 10 min in the washing buffer and two times for 10 min each in TBS.

The remainder of the procedure followed that described for immunocytochemical localization of ubiquitin.

Immunocytochemical Localization of Protamines

The sections were placed in PBS for 6 min at RT. They were then permeabilized with 0.1% Triton X-100 in PBS for 14 min at RT. The sections were washed three times for 2 min each with the mixture of PBS and 0.2% Tween 20 (the washing buffer) and then blocked for 5 min at RT in the solution containing 10% (w/v) nonfat dried milk diluted in the washing buffer. The sections then were rinsed for 3 min in the washing buffer, and subsequently were incubated for 90 min at RT, with the primary antibody (rabbit polyclonal antibody; provided by the Department of Immunology, Institute Microbiology and Immunology, University of Lódz ,Poland) to the protamines isolated from antheridia of C. tomentosa [32] (at a 1:500 dilution) diluted in PBS containing 1% BSA and 0.5% Tween 20. The slides were washed with the washing buffer three times for 2 min each and then were incubated with the secondary antibodies (anti-rabbit IgG conjugated with FITC [Sigma]) diluted 1:70 in PBS containing 1% BSA and 0.5% Tween 20 for 1 h at RT in darkness. The remainder of the procedure followed that described for immunocytochemical localization of ubiquitin.

To check for nonspecific staining, control probes were run with the primary antibodies to ubiquitin as well as histone H3 and protamines. The first one was prepared with nonimmune rabbit IgG, and the second without pre-immune rabbit IgG. Positive immunosignals were observed only in the cells treated with the specific primary antibodies.

Photography

Images were captured using a color digital camera DXM 1200 (Nikon) attached to a Optiphot-2 epifluorescence microscope (Nikon), with Nikon ACT-1 software.

RESULTS

Influence of the Inhibitors of Proteasome Proteolytic Activity on Relative Duration of C. vulgaris Stages of Spermiogenesis

Comparative analysis of the percentage of spermatids at all stages of spermiogenesis in the control samples and after application of proteasome inhibitors revealed prolongation of the early stages caused by all these inhibitors, since an increase in the number of spermatids was observed.

The changes occurring at the node V samples, 48 h after application of inhibitors, are most characteristic of their influence; lactacystin and epoxomicin proved to be most effective (Fig. 1A and Table 1).


Figure 01
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  		FIG. 1. A) Percentage of C. vulgaris spermatids during consecutive spermiogenesis stages in the control and after 48-h treatment with the proteasome inhibitors, node V. Bars represent ± SD. Statistical results analyzed by Student t-test with the use of Microsoft Excel 2000. Differences in the percentage of spermatids during consecutive spermiogenesis stages between the control and the proteasome-treated plants are statistically significant, P = 0.05. B) Ultrastructure of ten stages of spermiogenesis according to Kwiatkowska and Poplonska [31], modified from Figure 1 with permission from Folia Histochem Cytobiol.


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  		TABLE 1. Percentage of spermatids during stages I–X of C. vulgaris spermiogenesis in the control and after 48-h treatment with the proteasome inhibitors.a

Prolonged incubation with the inhibitors caused stronger effects with the exception of clasto-lactacystin β-lactone, which quickly entered cells and increased the number of spermatids from the early developmental stages after only 24 h.

Ultrastructural Studies of C. vulgaris Spermatids

Ultrastructural analyses of C. vulgaris spermatids demonstrated that after 48 h of treatment with the proteasome inhibitors, epoxomicin and MG-132 produced similar results. During early and mid spermiogenesis (stages I-VII), no distinct differences were observed between the treated (Fig. 2B) and control cells (Fig. 2A). However, during later stages (VIII and IX), drastic changes in the ultrastructure of spermatid nuclei (Figs. 3B and 4B) were observed in comparison to the controls (Figs. 3A and 4A).


Figure 02
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  		FIG. 2. Ultrastructure of C. vulgaris spermatids in the control material (A) and after 48-h treatment with epoxomicin (B) in stage VI of spermiogenesis, net-like condensed chromatin, longitudinal section. f, Flagellum; mm, microtubular manchette; n, nucleus; p, plastid; pl, plasmodesmata. Original magnification x22 000.


Figure 03
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  		FIG. 3. Ultrastructure of C. vulgaris spermatids in the control material (A; longitudinal section) and after 48-h treatment with the proteasome inhibitor epoxomicin (B; oblique section) with the changed structure of spermatid nuclei in stages VIII and IX of spermiogenesis. ag, Aggresomes; n, nucleus. Original magnification x44 000.


Figure 04
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  		FIG. 4. Ultrastructure of C. vulgaris spermatids in the control material (A; cross section) and after 48-h treatment with the proteasome inhibitor MG-132 (B; cross section) with the changed structure of spermatid nuclei in stages VIII and IX of spermiogenesis. ag, Aggresomes; f, flagellum; mm, microtubular manchette. Original magnification x44 000.

After incubation with either inhibitor, instead of parallel chromatin fibrils and lamellae there were strongly contrasted aggregates of tightly adjacent and adhered structures with irregular spirals against a lighter background devoid of fibrilar structures.

Moreover, in the nuclei from material treated with the inhibitors there were clusters of smaller and larger dark spots surrounding spheric homogeneous structures (Figs. 3B and 4B) that looked like aggresomes as described by many authors as a result of proteasome proteolytic activity disturbances. However, analysis of electron micrographs did not reveal changes caused by the inhibitors in the shape of the nucleus or in the structure of cytoplasm, microtubule manchette, or flagellae.

Cytochemical Studies of Histones

In the controls, we observed a positive reaction to histones in the spermatid nuclei of C. vulgaris in early stages (I–IV) of spermiogenesis (Fig. 6a). During later stages (V–VIII; Fig. 6b) the intensity of this reaction was gradually reduced, while during end stages (IX–X; Fig. 6c) no color reaction revealing histone presence was observed.


Figure 06
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  		FIG. 6. Antheridial filament cells of C. vulgaris after cytochemical staining to reveal histones during successive stages of spermiogenesis in the control (a–c) and after treatment with epoxomicin (d–f). Stages I–II (a, d), VI–VII (b, e), and IX–X (c, f). Original magnification x1650.

In these studies two proteasome inhibitors, epoxomicin and MG-132, yielded similar results. After application of the inhibitors, during initial stages of spermiogenesis (Fig. 6d) the same intensive reaction was observed as in the controls. During stages VI and VII (Fig. 6e) this reaction was less intense than in the earlier stages, but more intense than in the controls (Fig. 6b). During stages IX and X of spermiogenesis and after application of the inhibitors, a positive reaction revealing the presence of histone, which was absent in the controls (Fig. 6c), was still observed (Fig. 6f).

Immunocytochemical Studies

Two inhibitors of proteasome proteolytic activity, epoxomicin and MG-132, were used for immunocytochemical analyses and yielded similar results.

Presence of ubiquitin. A positive antigen reaction was observed in C. vulgaris spermatids during all stages of spermiogenesis. During stages I–IV, antigen signals dominated in the cytoplasm (Fig. 7a) but in the nuclei during stages VII–IX (Fig. 7b). Also, in the treated material (Fig. 7i), pronounced spots, which were not noted in the controls (Fig. 7c), were observed in spermatid nuclei in the late stages of spermiogenesis. This observation suggests an accumulation of ubiquitin in the structures, similar to the aggresomes observed in EM (Figs. 3B and 4B).


Figure 07
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  		FIG. 7. Immunocytochemical localization of ubiquitin in the spermatids of C. vulgaris in the control material (a–c) and after 48-h treatment with MG-132 (g–i), secondary antibodies conjugated with FITC. Staining with DAPI (d–f, j–l). Stages I–II (a, g), VI–VIII (b, h), and IX–X (c, i). Original magnficiation x1750.

Presence of H3 histone. In the controls during stages I–IV of spermiogenesis (Fig. 8a), a positive antigen reaction was observed in spermatid nuclei. Later, during stages V–VIII this reaction was weaker (Fig. 8b), and finally (stages IX and X) a negative result was noted (Fig. 8c; nonspecific, dark green color of nuclei). After application of the inhibitors, positive antigen signals persisted until the end of spermiogenesis, indicating H3 histone presence in stages IX and X of spermiogenesis (Fig. 8i).


Figure 08
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  		FIG. 8. Immunocytochemical localization of histone H3 in the spermatids of C. vulgaris in the control material (a–c) and after 48-h treatment with MG-132 (g–i), secondary antibodies conjugated with FITC. Staining with DAPI (d–f, j–l). Stages I–II (a, g), VI–VIII (b, h), and IX–X (c, i). Original magnification x1750.

Immunocytochemical studies confirmed the cytochemical results described above.

Presence of protamines. In the control spermatids during stages I–IV (Fig. 9a) no positive reaction in nuclei was observed, while during stages V–X the antigen signals were present (Fig. 9, b and c). Also, in stage V strong signals in the shape of strands were observed in the cytoplasm. During stages VI and VIII the signals were noted both in the spermatid nuclei and in the cytoplasm, while toward the end of spermiogenesis (stages IX and X), strong signals appeared at the periphery of nuclei and slightly weaker signals inside the nuclei (Fig. 9c).


Figure 09
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  		FIG. 9. Immunocytochemical localization of protamine-type proteins in the spermatids of C. vulgaris in the control material (a–c) and after 48-h treatment with epoxomicin (g–i), secondary antibodies conjugated with FITC. Staining with DAPI (d–f, j–l). Stages I–II (a, g), VI–VIII (b, h), and IX–X (c, i). Original magnification x1650.

Following application of the inhibitor, increased labeling indicating protamine presence during stages V–X was observed, both in the spermatid nuclei and in the cytoplasm (Fig. 9, h and i). During stages IX and X, a positive reaction in the form of spots was observed in the spermatid nuclei (Fig. 9i). The intensity of the reaction was similar to that in the controls. Proteasome inhibitors inhibited degradation of histones but did not influence the protamines accumulated in the nucleus, as protamine synthesis and the transport of protamines to the nucleus is not correlated with proper proteasome functioning.

DISCUSSION

Spermatogenesis in C. vulgaris consists of two steps: a proliferative stage and spermiogenesis. During proliferation, due to the presence of plasmodesmata with specific structure [47], antheridial filaments undergo synchronous mitotic divisions following a shorter and shorter interphase lacking the G1 phase, as the S phase starts already in telophase [48]. The last mitosis does not exhibit initiation of the S phase in telophase, so the resulting spermatids contain 1C DNA [49]. They undergo spermiogenesis, which takes approximately 7 days [50].

Stages I–VI of spermiogenesis, according to previous research, are characterized by high translational activity, which reaches its maximum in newly formed spermatids and in stage V [31]. Transcriptional activity in spermatids is most intensive in stage IV while during stages VI and VII it is hardly visible [51]. Increased metabolic activity of early spermiogenesis clearly corresponds to a transient DNA demethylation, which was revealed in C. vulgaris by nick-translation in situ using methylation-sensitive restriction enzymes [52]. The same phenomenon had been observed earlier during spermiogenesis in mice [53]. According to the authors of the studies cited above, genes that were blocked in somatic cells may be activated at this stage. These genes must be expressed during early spermiogenesis since they are responsible for subsequent differentiation. This hypothesis is in agreement with the data concerning gene expression during animal spermiogenesis, as de novo synthesis is necessary for the development of specific sperm cell morphology [54–56].

In the controls, we found that early spermiogenesis (stages I–IV) was characterized by subtle changes in the structure of spermatids (Fig. 1B), which still resembled somatic cells but exhibited distinct polarization and slightly increasing chromatin condensation; in stage IV, nucleoli disappear [31, 57]. In the present study, the ultrastructure of these cells was not significantly changed by the inhibitors. Possibly they only retarded the expression of gene cascade since the ubiquitin-proteasome system is necessary for this activation.

In controls, mid-spermiogenesis (stage V) is crucial because the synthesis of the protamine-type proteins begins then [32]. During stage VI, chromatin is composed of a distinct net of condensed chromatin. In the present study, the ultrastructure of the spermatids during stages V and VI was not changed by MG-132 or epoxomicin (Figs. 2 and 3).

In controls, spermatid nuclei gradually elongate, becoming lemon-shaped (stage VI) with simultaneous removal of RNA from them [57]. Further, the nucleus becomes semicircular (stage VII), crescent-shaped (stage VIII), single-double coiled (stage IX), and finally double-triple coiled (stage X) [31]. Simultaneously there are changes in the chromatin structure. First (stage VII) the difference between loose and condensed chromatin disappears; the chromatin forms short, randomly positioned fibrils, then (stage VIII) long, parallel fibrils. In stage IX it becomes lamellar and is anchored to a nuclear envelope with lamin [57]. In stage X chromatin is very strongly condensed.

Electron microscopy of C. vulgaris spermatids during stages VII–IX treated with epoxomicin or MG-132 showed distinct changes in the chromatin structure in comparison with the controls, but not in the shape of the nuclei. Positioning of chromatin fibrils was disturbed, which means that transformation of nucleohistones into nucleoprotamines requires ubiquitin-proteasome system involvement. Moreover, in the ultrastructure of nuclei during these stages we observed clusters of smaller or larger spots surrounding a homogeneous center, which resemble aggresomes as described by other authors in different cells treated with proteasome inhibitors [59–64]. Immunocytochemical results suggested that these clusters were aggregates of ubiquitinated histones that were not degraded due to the inhibition of proteasome activity. Moreover, histones were also present throughout the whole area of the nuclei from the inhibitor-treated spermatids. It was shown by electron microscopy that the delicate spirals, probably consisting of DNA connected with protamines (according to the Ward's model [58, 31]) visible in control material (Fig. 5B), were deformed, randomly positioned, and covered with an amorphous substance that seemed to correspond to nondegraded histones (Fig. 5C).


Figure 05
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  		FIG. 5. Electron micrographs of C. vulgaris spermatids. Nucleosomes (arrows in A), DNA conjugated with protamines in the control (arrow in B), and DNA conjugated with protamines after the treatment with epoxomicin (arrow in C). Original magnification x320 000.

On the other hand, the results obtained by the immunocytochemical reaction revealed that protamines were similar for the control and the treated material, which suggests that both synthesis of protamines and their translocation into nuclei were not influenced by the proteasome inhibitors.

The completely different reactions of C. vulgaris spermatids to inhibitors observed in stages I–VI of spermiogenesis seem to reinforce the finding by Meiners et al. [65] in rats and Lundgren et al. [66] in Drosophila melanogaster that MG-132 and epoxomicin induces concerted expression of proteasome genes. After 5 h of treatment, the mRNA level for proteasomes had already reached its maximum. The observed up-regulation is a general phenomenon in mammalian cells, irrespective of cell type [65]. De novo synthesis of proteasomes has also been observed due to a reduced number of mRNA copies encoding the proteasomal subunit of Rpn10/S5a [66]. One may suppose that during early spermiogenesis in C. vulgaris, when transcription and translation were observed, the above-mentioned phenomenon—namely activation of proteasome genes by inhibitors—led to the reproduction of the proteasome pool necessary for spermatid functioning, thus enabling differentiation to progress, although slightly retarded. However, in mid-late spermiogenesis, inhibition of proteasomes did not lead to their de novo synthesis since there was no active transcription or translation, which resulted in significant changes in chromatin remodeling and blockade of histone removal.

The results obtained indicate that the ubiquitin-proteasome system is crucial for efficient progress of early spermiogenesis in C. vulgaris. Successful removal of histones from DNA by this system in mid-late spermiogenesis is a prerequisite for proper chromatin remodeling and its tight packing in the nuclei of mature spermatozoids.

FOOTNOTES

1Supported in part by the National Committee of Scientific Research (MNiSW), grant no. 3 PO4C 033 25, no. 2 PO4C 009 26. Back

Correspondence: 2Agnieszka Wojtczak., Department of Cytophysiology, University of Lódz, Pilarskiego, 14, 90-231 Lódz, Poland. FAX: 48 42 635 45 14; e-mail: wojag{at}biol.uni.lodz.pl

Received: 16 May 2007.

First decision: 22 June 2007.

Accepted: 9 November 2007.

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K. Poplonska, M. Kwiatkowska, A. Wojtczak, and J. Polit
Immunogold Evidence Suggests That Endoplasmic Reticulum Is the Site of Protamine-Type Protein Synthesis and Participates in Translocation of These Proteins into the Nucleus During Chara vulgaris Spermiogenesis
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