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Abstract

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested.

Keywords

chromatoid body spermiogenesis degradation aggresome immunoelectron microscopy

It is well known that throughout spermiogenesis germ cells from a wide variety of mammalian and non-mammalian species contain some type of dense amorphous material. Although the form of this material varies greatly, it has been termed “nuage” because it usually has an amorphous cloud-like texture (André and Rouiller 1957). The most widely studied form of nuage is the chromatoid bodies (CBs) (Fawcett et al. 1970; Russell and Frank 1978). The CBs appear first in the juxtanuclear area of the pachytene spermatocyte (Fawcett et al. 1970). Early studies cited several candidates for the origin of the CBs such as dense materials appearing in the nucleus of pachytene spermatocytes (Comings and Okada 1972; Söderström and Parvinen 1976a; Söderström 1978; Parvinen and Parvinen 1979) and intermitochondrial cement (Eddy 1970; Fawcett et al. 1970; Söderström 1978). However, a direct demonstration showing the origin has yet to be performed. The CBs increase in size until step 5 and move to the cytoplasm around the basis of flagellum in steps 7-8 spermatids (Fawcett et al. 1970). During these steps of differentiation, the CBs are surrounded by many small vesicles that are positive for acid phosphatase activity (Anton 1983), nicotinamide adenine dinucleotide phosphatase (NADPase) (Thorne-Tjomsland et al. 1988), and polysaccharides (Krimer and Esponda 1980). After step 7, the CBs gradually decrease in size, and the CBs with a typical profile disappear after step 16 (Susi and Clermont 1970). It has been shown that many substances such as RNA (Söderström and Parvinen 1976b; Walt and Armbruster 1984; Saunders et al. 1992), subunits of ribonucleoprotein (RNPs) (Biggiogera et al. 1990; Moussa et al. 1994), actin (Walt and Armbruster 1984), histone (Werner and Werner 1995), and cytochrome c (Hess et al. 1993) are present in the CBs. Given the existence of RNA and subunits of RNPs, it was proposed that the CBs synthesized proteins necessary for the late spermiogenic cells, because virtually all transcription activity ceased in late spermatids (Biggiogera et al. 1990; Moussa et al. 1994). However, to date, there is no direct evidence for protein synthesis in the CBs. In a previous study, we have found by immunoelectron microscopy that phospholipid hydroperoxide glutathione peroxidase (PHGPx) is contained in the CBs, although PHGPx was synthesized before its appearance in the CBs. Also, we observed that PHGPx signals were localized to the nuclear material exhibiting a very similar density to the CBs and in the intermitochondrial cement, both previously proposed as the origin of CBs. These observations tempted us to hypothesize that PHGPx is degraded rather than synthesized in the CBs.

On the other hand, it has been reported that when the amount of misfolded protein in a cell exceeds the capacity for degradation, the misfolded proteins accumulate in the cytoplasm to form aggresomes (Johnston et al. 1998). Overexpression of cytosolic protein chimera also causes the formation of aggresomes (García-Mata et al. 1999). Recent studies have shown that a diverse array of human diseases, including amyloidosis and neurodegenerative disorders, are caused by the accumulation of misfolded proteins due to an impaired degradation system (Johnston et al. 2000; Junn et al. 2002; McNaught et al. 2002; Namekata et al. 2002; Riley et al. 2002; Ryan et al. 2002). Thus, during their long evolution process, cells have acquired a mechanism in which cells assemble misfolded or unnecessary proteins to small aggregates and transport to aggresomes where abnormal proteins are degraded by the ubiquitin proteasome system or eventually by autophagy (see review Kopito and Sitia 2000; Kopito 2000; Garcia-Mata et al. 2002; Wójcik and DeMartino 2003). The aggresome pathway functions as a quality control of proteins (Kopito and Sitia 2000).

As the CBs appear in the vicinity of the Golgi apparatus and are membrane-free inclusions containing many proteins, the CBs have a very similar profile to the aggresomes. To elucidate which types of proteins are contained in the CBs, in the present work we studied the localization of lactate dehydrogenase (LDH) and enolase as cytoplasmic protein, PHGPx and ATP synthase subunit α (F₁α) and subunit β (F₁β) as the mitochondrial proteins coded on nuclear DNA, cytochrome oxidase subunit I (COXI) as a mitochondrial protein coded on mitochondrial DNA, and histone H2B, acetylated histone H2B, and acetylated histone H3 as nuclear proteins using a quantitative immunoelectron microscopic method. Furthermore, we investigated the localization of aggresomal markers, including chaperones, ubiquitin, ubiquitin-conjugating enzyme (E2), and proteasome subunits in the CBs, and of lysosomal markers such as cathepsins, lysosome-associated membrane protein 1 (LAMP1), and leucine aminopeptidase (LAP) in vesicles surrounding the CBs. As we found that the CBs contained the proteins of all the subcellular compartments examined as well as several aggresomal markers and the vesicles surrounding the CBs included lysososmal markers, we proposed that the CBs are a site of degradation rather than synthesis.

Materials and Methods

Animals

Nine-week-old male Wistar albino rats, weighing 200-250 g, were fed the appropriate standard diets for each animal type and water ad libitum until used. The animal experiments were performed in accordance with the Guidance for Animal Experiments, University of Yamanashi.

Antibodies

Rabbit anti-ubiquitin antibody was prepared as described (Hershko et al. 1982). Briefly, 5 mg of bovine erythrocyte ubiquitin (Sigma-Aldrich; St Louis, MO) was conjugated with 8 mg of limpet hemocyanin (Sigma-Aldrich) using glutaraldehyde. The conjugates were mixed with 2% SDS and heated for 5 min. After cooling in ice water, SDS was removed by aluminum chloride. The conjugates were then emulsified with the same volume of Freund's complete adjuvant. The emulsion containing 400 μg of ubiquitin was injected four times at intervals of 2 weeks into the back of Japanese white rabbits. Two weeks after the last injection, blood was collected and the immunoreactivity was checked with ubiquitin. Ubiquitin-specific antibody was purified by affinity chromatography using ubiquitin-coupled Sepharose. Antibodies to lysosomal proteins were described previously: rabbit anti-rat cathepsin H (Yokota and Kato 1987); rabbit anti-cathepsin B, D, and L (Yokota et al. 1985, 1988; Yokota and Kato 1987); rabbit anti-PHGPx (Haraguchi et al. 2003); rabbit anti-LAMP1 (Akasaki et al. 1990); and rabbit anti-LAP antibodies (Haraguchi and Yokota 2002). Rabbit anti-LDH was a gift from Dr. Ohsumi (Department of Life Science, Himeji Institute of Technology, Hyogo, Japan). Mouse anti-Hsp70 antibody and rabbit anti-enolase antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); mouse anti-F₁α and F₁β antibodies, mouse anti-COXI, and Alexa Fluor 546 conjugated goat anti-rabbit IgG were from Molecular Probes (Eugene, OR); mouse anti-ER (endoplasmic reticulum) retention signal (KDEL) and rabbit anti-proteasome activator PA700 and PA28α antibodies from Calbiochem-Novabiochem (San Diego, CA); rabbit anti-histone H2B polyclonal antibody was from Chemicon International (Temecula, CA); mouse anti-vimentin antibody was from Sigma-Aldrich; mouse anti-20S proteasome subunit (p52) antibody was from Progen Biotechnik Gmbh (Heidelberg, Germany); goat polyclonal antibody to ubiquitin-conjugating enzyme E2 (UBC3B) was from Abcam Limited (Cambridgeshire, UK); mouse anti-actin antibody was from ICN Pharmaceuticals (Costa Mesa, CA); rabbit anti-bovine DNase I was from Upstate Biotechnology Inc. (Lake Placid, NY); and rabbit anti-bovine RNase A was from Nordic Immunological Laboratories (Tilburg, The Netherlands).

Tissue Preparation

Testes were dissected out from rats and sliced in ice-cold fixative consisting of 4% paraformaldehyde, 0.1-0.2% glutaraldehyde, 0.01% CaCl₂, and 0.2 M Hepes-KOH buffer (pH 7.4). For immunoelectron microscopy, the tissue slices were fixed in the same fixative containing 0.2% glutaraldehyde for 1 hr at 4C and then cut into small tissue blocks. The tissue blocks were dehydrated in a graded ethanol series and embedded in LR White resin at −20C. For immunofluorescence microscopy, the tissue was fixed with the same fixative containing 0.1% glutaraldehyde.

Post-embedding Immunoelectron Microscopy

Thin sections of rat testis embedded in LR White were cut with a diamond knife equipped with a Reichert Ultracut R (Leica; Vienna, Austria), mounted on nickel grids, and incubated overnight with the primary antibodies against lysosomal proteins (2 μg/ml), PHGPx (X1,000), vimentin (X200), Hsp70 (X1000), F₁α and F₁β (X200), COXI (2.5 μg/ml), 20S proteasome subunit (p52) (X5), KDEL (X4000), histone H2B (X500), proteasome activators PA700 and PA28α (X40), enolase (X200), LDH (X500), actin (X1000), UBC3B (2 μg/ml), RNase (X2000), and DNase (X1000) antibodies at 4C. After washing, sections were treated with rabbit anti-mouse IgG (X2000) or anti-goat IgG (X2000), depending on the primary antibody. Finally, the antigens were visualized using protein A-gold probes 15 nm in diameter. For the immunocytochemical control experiment, the incubation of sections with the primary antibody was omitted, followed by the secondary antibody or by protein A-gold probe. Sections were stained with 2% uranyl acetate for 10 min and lead citrate for 30 sec and examined with a Hitachi H7500 electron microscope (Hitachi; Tokyo, Japan) at an acceleration voltage of 80 kV.

Quantitative Analysis of the Labeling Density in the CBs and Cytoplasmic Matrix

After the immunogold staining for 22 antigens with a 15-nm protein A-gold probe, 10 electron micrographs of spermatids in the developing steps 3-6 were taken for each antigen at X15,000 magnification and enlarged fourfold. The steps of spermatids were determined according to the morphological criteria of Russell et al. (1990). For analysis of gold labeling density, the areas of the CBs (excluding their clear spaces) and cytoplasmic matrix of spermatids were estimated using a digitizer tablet and SigmaScan software (Jan-del Scientific; San Rafael, CA) attached to a computer. For lysosomal enzymes, the areas of the CBs including surrounding vesicles were estimated. Gold particles located in the estimated areas were counted. The labeling density was expressed as gold particles/μm² for each compartment. Background labeling density was measured for section area using the immunocytochemical controls in which incubation with the primary antibodies was omitted.

Routine Electron Microscopy

Tissue slices of rat testis were fixed in 2% glutaraldehyde for 1 hr at 4C and cut into small blocks. After a brief wash in phosphate-buffered saline (PBS), the tissue blocks were post-fixed in 1% reduced osmium tetroxide for 1 hr at room temperature. The tissue blocks were dehydrated in a graded ethanol series and embedded in Epon 812. Thin sections were stained with lead citrate and examined with the same electron microscope described above.

Results

Routine Electron Microscopy

In early pachytene spermatocytes at stages IV to VI, the CBs appeared in the juxtanuclear cytoplasm and were composed of loose aggregates of dense material, being accompanied by many small vesicles (Figure 1A). Solitary dense materials were also observed in the cytoplasm (Figure 1A, large arrow). In later pachytene at stages VIII to × and in step 1 spermatids, the core of CBs became compact and homogeneous (Figure 1B). Many tubules and vesicles enclosed the CBs, and some small vesicles invaded into the core region. These tubulovesicular structures were not decorated with ribosomes. In the spermatocytes, after step 10, the CBs moved to the cytoplasm around the neck and decreased in size. In many cases, multivesicular bodies accompanied the CBs.

Immunoelectron Microscopy

Localization of ER and Lysosomal Markers in the Compartments Surrounding the CBs. The ER retention signal (KDEL) was present in the lumen of ER (Figure 2A) but not in the tubulovesicular structures. LAMP1 was detected on the membrane of tubulovesicular structures surrounding the CBs but not in the core of the CBs (Figure 2B), strongly suggesting the lysosomal nature of the tulubovesiclular structures. Lysosomal enzymes, LAP, cathepsin D, and cathepsin H were present in these structures (Figures 3A-3C). Cathepsins B and L were also detected in the tubulovesicular structures.

Localization of Aggresomal Markers in the CBs. The CBs were heavily stained for ubiquitin (Figure 4A). The gold particles were present in the core. Strong ubiquitin staining of the CBs was noted in spermatids of steps 2-6. The gold signals for 20S proteasome sub-unit p52 were closely associated with the CB (Figure 4B), and weak signals were present throughout the cytoplasm. Strong signal for ubiquitin-conjugating enzyme E2 (UBC3B) was detected in the core of the CBs (Figure 4C). Proteasome activators PA700 (data not shown) and PA28α were also localized to the surface of the CB core as well as the cytoplasm (Figure 4D). The CBs were strongly stained for Hsp70 (Figure 4E). The staining intensity tended to decrease as the differentiation progressed. The intermediate filament protein vimentin was present around the CB (Figure 4F).

Figure 1

Routine EM of an early pachytene spermatocyte. (A) Electron-dense materials (arrows) are gathered in the juxtanuclear area together with small clear vesicles. Solitary dense material is seen (large arrow). N: nucleus. (B) A late pachytene spermatocyte. Dense materials are condensed and surrounded by tubulovesicular structures (arrows). Magnification X24,000 (A) and X27,000 (B). Bars = 0.5 μm.

Figure 2

Immunogold staining of ER retention signal (KDEL) in spermatids. (A) A step 4 spermatid. Gold particles showing KDEL are observed in the nuclear envelope and in slender saccules that are apart from CBs. (B) A step 3 spermatid. Staining for lysosomal membrane protein, LAMP1. Gold signals for LAMP1 are present in tubulovesicular structures surrounding a CB (arrows). Magnification X16,000 (A) and X30,000 (B). Bars = 0.5 μm.

Localization of Other Proteins in the CBs. Nuclear DNA-coded Mitochondrial Proteins. ATP synthase subunits α and β were detected in the core of the CB as well as in the mitochondria (Figures 5A and 5B). As the differentiation of spermatids progressed, the labeling intensity in the CBs decreased (data not shown). Heavy signals for PHGPx were found in the CB core (Figure 5C) as described previously (Haraguchi et al. 2003). Their intensity also decreased as the differentiation of spermatids progressed.

Mitochondrial DNA-coded Protein. Cytochrome oxidase subunit I (COXI) was examined. It was detected in the CBs as well as in the intermembrane space of mitochondria (Figure 5D). The labeling density decreased after step 9.

Cytosolic Protein. Lactate dehydrogenase (LDH) was stained in the CBs (Figure 6A). Enolase was detected in the core of the CBs from pachytene spermatocytes to step 9 spermatids (Figure 6B). The labeling intensity for these proteins decreased as spermatids differentiated. The CBs in spermatids after step 9 were almost all negative.

Nuclear Protein. Histone H2B, acetylated histone H2B, and acetylated histone H3 were detected in the CBs of spermatocytes from pachytene to spermatids of step 6 (Figures 7A-7C). The gold signals were mainly present in the core of the CBs. The staining intensity decreased as the spermatids differentiated. Nuclei of early spermatogenic cells, including spermatogonia and pachytene spermatocytes, were stained heavily for histone H2B and acetylated histone H2B but very weakly for acetylated histone H3 (data not shown). Immunocytochemical control sections that were not incubated with the primary antibodies, followed by secondary probes, showed very weak background labeling (Figure 7D).

Figure 3

Immunogold staining of lysosomal enzymes and nucleic acids in spermatids. (A) Localization of leucine amino peptidase (LAP) in the CB of a step 4 spermatid. Gold particles are present in small vesicles surrounding CB (inset). (B) Staining of cathepsin D in a step 3 spermatid. Gold labeling is seen in the vesicles (inset). (C) Staining of cathepsin H of a step 4 spermatid. Gold particles are present in vesicles indicated by dotted lines (inset). (D) Staining of RNase in the CB of a step 4 spermatid. Most of the gold signals are present in the periphery of the dense core. Magnification X22,000 (A), X45,000 (inset), X24,000 (B), X50,000 (inset), X30,000 (C), X45,000 (inset) and X24,000 (D). Bars: A-D = 0.5 μm; insets = 0.2 μm.

Quantitative Analysis of the Gold Labeling in the CBs

In order to know the concentration in the CBs of the examined proteins, we determined the labeling density (gold particles/μm²) for each protein in the CBs and cytoplasmic matrix of spermatids at steps 3 to 6. All the proteins examined were significantly concentrated in the CBs. The results are shown in Table 1. The values shown were subtracted by the background labeling density obtained from immunocytochemical controls. The labeling density for cytosolic proteins such as actin, LDH, and enolase in the CBs was 3.6- to 9.7-fold higher than that in the cytoplasmic matrix. The cytoplasmic labeling of lysosomal and mitochondrial proteins showing background noise of the labeling was significantly lower than that in the CBs. The labeling density for nuclear proteins, histone H2B, and acetylated histone H3 was ~6-fold higher in the CBs than in the cytoplasmic matrix. The labeling density for aggresomal marker proteins and proteasome-related proteins was also higher in the CBs than in the cytoplasmic matrix.

Discussion

In early studies it was considered that the CBs were the site where proteins necessary for the late spermiogenic cells were synthesized, because virtually all transcription activity ceased in late spermatids (Söderström and Parvinen 1976a; Söderström 1978; Parvinen and Parvinen 1979; Saunders et al. 1992). Ribonucleoproteins, mRNA, and mRNA-binding proteins (p48/52) were detected in the CBs (Biggiogera et al. 1990; Saunders et al. 1992; Oko et al. 1996). Also, polysome-like structures were found in isolated CBs (Figueroa and Burzio 1998). These findings support the idea that mRNA molecules transcribed before the occurrence of nuclear condensation should be stored in the CBs until they are required. On the other hand, it was reported that mRNAs of transition protein 1 and protamine 1 and poly (A)+ RNA were not present in the CBs but scattered in the cytoplasm (Morales et al. 1991; Morales and Hecht 1994). Recent studies suggest a potential connection between nuage and the micro-RNA and/or RNAi pathway in Drosophila and Xenopus oocytes (Findley et al. 2003; Bilinski et al. 2004). Also, a possibility was demonstrated that the CBs are involved in intercellular transport of Golgi-derived haploid gene products between early spermatids through the cytoplasmic bridge in rat (Ventelä et al. 2003). Thus, the role(s) of the CBs are still controversial.

Figure 4

Immunogold staining of aggresomal markers in CBs of spermatids. (A) Staining of ubiquitin (UB) in the CB of a step 5 spermatid. Heavy gold labeling is present in the CB core. (B) Staining of proteasome subunit p52 in the CB of a step 3 spermatid. Note that most gold particles are present in the periphery of the core. (C) Staining for ubiquitin-conjugating enzyme UBC3B (E2) in the CB of a step 4 spermatid. (D) Localization of proteasome activator PA28α in the CB of a step 4 spermatid. (E) Labeling of Hsp70 in the CBs of a step 5 spermatid. Heavy gold labeling is observed in the core of CBs and weak labeling in the cytoplasm. (F) Staining of vimentin in the CB of a step 5 spermatid. Gold particles are associated with the periphery of the CB. Magnification X18,000 (A), X30,000 (B,D), X22,000 (C), and X26,000 (E,F). Bars = 0.5 μm.

Figure 5

Immunogold staining of mitochondrial enzymes in CBs of spermatids. (A) Localization of ATP synthase subunit α (F₁α) in a CB and a mitochondrion of a step 5 spermatid. Gold particles are present in the CB and mitochondrion (arrow). (B) Staining of ATPase subunit β (F₁β) in a CB and mitochondria of a step 7 spermatid. Gold labeling is observed in CBs and mitochondria (arrows). (C) Localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in a CB of a step 5 spermatid. Heavy gold labeling is observed in the CB. (D) Staining of cytochrome oxidase I (COXI) in a CB and mitochondria of a step 5 spermatid. Gold particles are present in the CB and mitochondria (arrows). Magnification X31,000 (A), X20,000 (B), X24,000 (C), and X23,000 (D). Bars = 0.5 μm.

Figure 6

Immunogold staining of cytosolic proteins. (A) Staining of LDH in the CB of a step 5 spermatid. (B) Staining of enolase in the CB of a step 4 spermatid. Note that both proteins are present in the CB core. Magnification X38,000 (A) and X20,000 (B). Bars = 0.5 μm.

Figure 7

Immunogold staining of nuclear proteins. (A) Staining of histone H2B in the CB of a step 3 spermatid. (B) Staining of acetylated histone H2B in the CB of a step 4 spermatid. (C) Labeling of acetylated histone H3 in the CB of a step 3 spermatid. Note that heavy gold labeling is present in the CB core. (D) Immunocytochemical control section incubated without the primary antibody, followed by protein A-gold probe. Note that no gold particles are present in the CB. Magnification X3000 (A,C,D) and X15,000 (B). Bars = 0.5 μm.

We reported previously that PHGPx signals were observed in the nuclear material and in the intermitochondrial cement, which were previously proposed as the origin of CBs (Fawcett et al. 1970; Russell and Frank 1978). Also, we have found by immunoelectron microscopy that PHGPx is contained in the CBs, although PHGPx was synthesized before its appearance in the CBs. These observations tempted us to hypothesize that PHGPx is degraded rather than synthesized in the CBs. In this study, we detected three other mitochondrial proteins in addition to PHGPx in the core of the CBs, of which one (COXI) was encoded on the mitochondrial DNA and two (F₁α and F₁β) on the nuclear DNA. Mitochondria have their own protein synthesis system within the compartment, and some of the codons used in mitochondria differ from those used in the nucleus. Thus, if proteins whose genes are on the mitochondrial DNA are synthesized in the CBs, the amino acid sequences of synthesized proteins in the CBs should be different from those in the mitochondria. The COXI gene contains 17 TGA codons used as a stop codon in the universal codon system; therefore, the COXI protein is unable to be synthesized in the CBs. This strongly suggests that the CBs are not a site for protein synthesis.

The present study also confirmed the earlier findings showing that the CBs had no limiting membrane and were surrounded by small tubules and vesicles (Sud 1961; Fawcett et al. 1970; Susi and Clermont 1970; Russell and Frank 1978; Anton 1983; Thorne-Tjomsland et al. 1988). Furthermore, in the present study we showed that these tubules and vesicles contained LAMP1 and lysosomal proteinases as well as DNase and RNase. Enzyme histochemical studies showed that acid phosphatase and NADPase activities were present in these tubulovesicular structures (Anton 1983; Thorne-Tjomsland et al. 1988). Various proteins, including histone H4, actin, cytochrome c, and basic proteins, have been detected in the core of the CBs (Krimer and Esponda 1980; Walt and Armbruster 1984; Hess et al. 1993; Werner and Werner 1995). These results suggest that the CBs have an intracellular garbage-like function.

Table 1

Labeling density

Marker proteins tested	Chromatoid body	Cytosol
Cytosolic proteins
Actin	22.42 ±	2.96	4.55 ± 0.33
LDH 1	6.03 ± 1.01	2.15 ± 0.22
Enolase	46.51 ± 6.88	12.86 ± 1.61
Cathepsin B	34.30 ± 3.83	3.85 ± 0.39
Cathepsin D	13.70 ± 1.02	1.35 ± 0.11
Cathepsin H	19.32 ± 2.05	1.98 ± 0.14
Lysosomal proteins
Cathepsin L	4.62 ± 0.69	1.12 ± 0.15
LAP 2	29.10 ± 7.02	5.91 ± 1.85
DNase	3.68 ± 1.06	0.40 ± 0.17
RNase	9.57 ± 2.39	1.88 ± 0.50
PHGPx 3	53.94 ± 3.20	5.56 ± 0.42
Mitochondrial proteins
F₁α 4	139.80 ± 8.63	11.82 ± 1.12
F₁β 5	65.75 ± 3.77	1.17 ± 0.08
COXI 6	49.70 ± 3.72	4.61 ± 0.93
Histone H2B	51.16 ± 5.61	8.90 ± 1.30
Nuclear proteins
Acetylated histone H3	48.00 ± 6.62	7.34 ± 1.60
Acetylated histone H2B	70.48 ± 20.77	8.23 ± 2.82
Ubiquitin	93.15 ± 4.14	7.84 ± 0.67
Aggresome- and proteasome-related proteins
Vimentin	12.77 ± 2.31	2.01 ± 0.47
Hsp70	98.20 ± 13.80	21.40 ± 9.90
p52 7	21.80 ± 3.37	9.73 ± 1.80
UBC3B 8	78.29 ± 11.01	4.32 ± 0.67
PA700 9	29.22 ± 6.71	8.25 ± 1.81
PA28α 10	90.02 ±6.96	4.45 ± 0.70

Lactate dehydrogenase.

Leucine aminopeptidase.

Phospholipid hydroperoxide glutathione peroxidase.

⁴

ATP synthase subunit α.

⁵

ATP synthase subunit β.

⁶

Cytochrome oxidase subunit I.

⁷

26S proteosome subunit.

⁸

Ubiquitin-conjugating enzyme (E2).

⁹

Proteasome activator.

¹⁰

Proteasome activator.

Recently, it has been shown that cells have an aggresomal pathway by which aggregates of misfolded proteins or mutated proteins are transported to the area around the microtubule organizing center (MTOC) where they form large aggresomes (Johnston et al. 1998; García-Mata et al. 1999; Kopito 2000; Garcia-Mata et al. 2002). Typical features of aggresomes are as follows: they locate in the pericentriolar region, contain ubiquitinated proteins, chaperones such as Hsp70 and Hsp40, and abnormal proteins, and are surrounded by vimentin filament and proteasomes. To examine whether the CBs have these features, we tried to stain these antigens. Strong signals for ubiquitin were noted in the CBs. The ubiquitin molecules present in the CBs are thought to be conjugated to proteins and function as a targeting signal for proteasome degradation. This is supported by the presence of the ubiquitin-conjugating enzyme E2 in the core of the CBs. The CBs were heavily stained for Hsp70, suggesting their binding to proteins or the accumulation of Hsp70 to be degraded. Signals for vimentin and the 20S proteasome subunit were detected around the CBs. Moreover, in pachytene spermatocytes, the CBs were frequently observed in the vicinity of the Golgi apparatus (Tang et al. 1982; Thorne-Tjomsland et al. 1988) and later in differentiation, they moved to the caudal pole of the nucleus where the centrioles are located. These results strongly suggest that the CBs function as aggresomes. The aggresomes are essentially the site of degradation, and the cytoplasmic proteolytic apparatus, the proteasome, is thought to be the main machinery for gathering proteins to the aggresomes.

For the degradation of proteins sequestrated to the aggresome, an autophagic pathway has been proposed (Kopito 2000). In the autophagic process, macroautophagy and microautophagy have been recognized. Macroautophagy is induced by amino acid deprivation in perfused rat liver (Mortimore and Schworer 1977). In microautophagy, lysosomal compartments incorporate the cytoplasm and specific proteins (de Duve and Wattiaux 1966). The autophagic vacuoles are rarely observed in spermatogenic cells, and those containing CBs are hardly encountered, suggesting that aggregated proteins in the CBs are not degraded through the macroautophagic pathway. The present study showed that the CBs were closely surrounded by the tubulovesicular structures that had several lysosomal markers. It is likely that microautophagy occurs between the CBs and these vesicles. It is not clear whether the vesicles take up in bulk the CBs or directly incorporate proteins through a chaperone-mediated process (Dice 1990). The proportion of proteasomes associated with the centrosome is only 1% in unstressed cells (Fabunmi et al. 2000). This amount of proteasomes might be enough to degrade ubiquitinated proteins used in cell-cycle control but not enough for large amounts of aggregated proteins in the CBs. Thus, the CBs may require the autophagic process in addition to the ubiquitin-proteasome system.

Although any conclusion concerning the function of the CBs must wait until the CBs can be isolated and their composition analyzed, we would like to speculate here the possible role of CBs. During spermatogenesis, the CBs appear in the early pachytene stages, maximize their sizes in the spermatids of steps 7-8, and disappear from those of steps 16-18. In the early stages, mitochondria form a clustered structure with the intermitochondrial cement and later dramatically change to the condensed form. This structural change is thought to make mitochondria more active for ATP production. After this process, mitochondria separate, and some of them move to the flagellum at step 7 and finally wind around flagella at step 12. In the present study, immunocytochemical localization of three mitochondrial proteins was confirmed in the CBs. One of the proteins is coded on mitochondrial DNA, suggesting that at least one material contained in the CBs is derived from the mitochondria. It might originate from mitochondria dismantled by means of an unknown mechanism, and the mitochondrial proteins are possibly transported to the CBs for degradation.

On the other hand, somatic histones are replaced with transition proteins beginning at step 9 of spermatogenesis. At step 13, the transition proteins are completely replaced by protamines (Kistler et al. 1996). These replaced nucleoproteins also might be degraded in the CBs within a limited period. We observed nucleoproteins such as histone H2B, acetylated histone H2B, and acetylated histone H3 on the CBs. Thus, the CBs contain specific proteins that became unnecessary during definitive periods of spermiogenesis. Quantitative analysis of labeling showed that the labeling density of all proteins examined was higher in the CBs than in the cytoplasmic matrix, even if it contained nonspecific background labeling. It is likely that of these proteins, lysosomal enzymes and aggresome- and proteasome related proteins are concerned with the function of the CBs. However, cytosolic proteins, mitochondrial proteins, and nuclear proteins examined are gathered to the CBs to be degraded, because the concentration of the latter proteins must be higher in their original locations than in the CBs if the CBs are synthetic sites of these proteins. From these observations, we propose that the CBs might be involved in the degradation of proteins released from the mitochondria and nucleus during this period to control the quality of these organelles and to adjust the function specific for spermatozoon.

Footnotes

Acknowledgments

This work was supported in part by a grant-in-aid (14580693) and the Center of Excellence (COE) program from the Ministry of Education, Science, Culture and Sport (Japan) to SY.

References

Akasaki

Yamaguchi

Ohta

Matsura

Furuno

Tsuji

(1990) Purification and characterization of a major glycoprotein in rat liver lysosomal membrane. Chem Pharm Bull (Tokyo) 38: 2766–2770

André

Rouiller

(1957) L'ultrastructure de la membrane nucléaire des ovocytes del l'araignée (Tegenaria domestica Clark). Proc Eur Conf Electron Microscopy, Stockholm, 1956. Academic Press, New York, 162–164

Anton

(1983) Association of Golgi vesicles containing acid phosphatase with the chromatoid body of rat spermatids. Experientia 39: 393–394

Biggiogera

Fakan

Leser

Martin

Gordon

(1990) Immunoelectron microscopical visualization of ribonucleoproteins in the chromatoid body of mouse spermatids. Mol Reprod Dev 26: 150–158

Bilinski

Jaglarz

Szymanska

Etkin

Kloc

(2004) Sm proteins, the constituents of the spliceosome, are components of nuage and mitochondrial cement in Xenopus oocytes. Exp Cell Res 299: 171–178

Comings

Okada

(1972) The chromatoid body in mouse spermatogenesis: evidence that it may be formed by the extension of nucleolar components. J Ultrastruct Res 39: 15–23

de Duve

Wattiaux

(1966) Function of lysosomes. Annu Rev Physiol 28: 435–492

Dice

(1990) Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem Sci 15: 305–309

Eddy

(1970) Cytochemical observations on the chromatoid body of the male germ cells. Biol Reprod 2: 114–128

10.

Fabunmi

Wigley

Thomas

DeMartino

(2000) Activity and regulation of the centrosome-associated proteasome. J Biol Chem 275 409–413

11.

Fawcett

Eddy

Phillips

(1970) Observations on the fine structure and relationships of the chromatoid body in mammalian spermatogenesis. Biol Reprod 2: 129–153

12.

Figueroa

Burzio

(1998) Polysome-like structures in the chromatoid body of rat spermatids. Cell Tissue Res 291: 575–579

13.

Findley

Tamanaha

Clegg

Ruohola-Baker

(2003) Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage. Development 130: 859–871

14.

García-Mata

Bebök

Sorscher

Sztul

(1999) Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera. J Cell Biol 146: 1239–1254

15.

Garcia-Mata

Gao

Y-S

Sztul

(2002) Hassles with taking out the garbage: aggravating aggresomes. Traffic 3: 388–396

16.

Haraguchi

Yokota

(2002) Immunofluorescence technique for 100-nm-thick semithin sections of Epon-embedded tissues. Histochem Cell Biol 117: 81–85

17.

Haraguchi

Mabuchi

Hirata

Shoda

Yamada

Hoshi

Yokota

(2003) Spatiotemporal changes of levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase, in subcellular compartments during the spermatogenesis in the rat testis. Biol Reprod 69: 885–895

18.

Hershko

Eytan

Ciechanover

(1982) Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells. Relationship to the breakdown of abnormal proteins. J Biol Chem 257: 13964–13970

19.

Hess

Miller

Kirby

Margoliash

Goldberg

(1993) Immunoelectron microscopic localization of testicular and somatic cytochromes c in the seminiferous epithelium of the rat. Biol Reprod 48: 1299–1308

20.

Johnston

Ward

Kopito

(1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143: 1883–1898

21.

Johnston

Dalton

Gurney

Kopito

(2000) Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA 97: 12571–12576

22.

Junn

Lee

Suhr

Mouradian

(2002) Parkin accumulation in aggresomes due to proteasome impairment. J Biol Chem 277: 47870–47877

23.

Kistler

Henriksen

Mali

Parvinen

(1996) Sequential expression of nucleoproteins during rat spermiogenesis. Exp Cell Res 225: 374–381

24.

Kopito

Sitia

(2000) Aggresomes and Russell bodies. Symptoms of cellular indigestion? EMBO Rep 1: 225–231

25.

Kopito

(2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10: 524–530

26.

Krimer

Esponda

(1980) Presence of polysaccharides and proteins in the chromatoid body of mouse spermatids. Cell Biol Int Rep 4: 265–270

27.

McNaught

KSP

Shashidharan

Perl

Jenner

Olanow

(2002) Aggresome-related biogenesis of Lewy bodies. Eur J Neurosci 16: 2136–2148

28.

Morales

Kwon

Hecht

(1991) Cytoplasmic localization during storage and translation of the mRNAs of transition protein 1 and protamine 1, two translationally regulated transcripts of the mammalian testis. J Cell Sci 100: 119–131

29.

Morales

Hecht

(1994) Poly(A)+ ribonucleic acids are enriched in spermatocyte nuclei but not in chromatoid bodies in the rat testis. Biol Reprod 50: 309–319

30.

Mortimore

Schworer

(1977) Induction of autophagy by amino acid deprivation in perfused rat liver. Nature 270: 174–176

31.

Moussa

Oko

Hermo

(1994) The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat. Cell Tissue Res 178: 363–378

32.

Namekata

Nishimura

Kimura

(2002) Presenilin-binding protein forms aggresomes in monkey kidney COS-7 cells. J Neurochem 82: 819–827

33.

Oko

Korley

Murray

Hecht

Hermo

(1996) Germ cell-specific DNA and RNA binding proteins p48/52 are expressed at specific stages of male germ cell development and are present in the chromatoid body. Mol Reprod Dev 44: 1–13

34.

Parvinen

L-M

(1979) Active movement of the chromatoid body. A possible transport mechanism for haploid gene products. J Cell Biol 80: 621–628

35.

Riley

Warrall

Rothnage

Swagell

van Leewen

French

(2002) The Mallory body as an aggresome: in vitro studies. Exp Mol Pathol 72: 17–23

36.

Russell

Frank

(1978) Ultrastructural characterization of nuage in spermatocytes of the rat testis. Anat Rec 190: 79–98

37.

Russell

Ettlin

Sinha Hikim

Clegg

(1990) Histological and Histopathological Evaluation of the Testis. Clearwater, IL, Cache River Press, 41–119

38.

Ryan

Shooter

Notterpek

(2002) Aggresome formation in neuropathy models based on peripheral myelin protein 22 mutations. Neurobiol Dis 10: 109–118

39.

Saunders

PTK

Millar

Maguire

Sharpe

(1992) Stage-specific expression of rat transition protein-2 mRNA and possible localization to the chromatoid body of step 7 spermatids by in situ hybridization using a non-radioactive probe. Mol Reprod Dev 33: 385–391

40.

Söderström

K-O

Parvinen

(1976a) Transport of material between the nucleus, the chromatoid body and Golgi complex in the early spermatids of the rat. Cell Tissue Res 168: 335–342

41.

Söderström

K-O

Parvinen

(1976b) Incorporation of [3H]uridine by the chromatoid body during rat spermatogenesis. J Cell Biol 70: 239–246

42.

Söderström

K-O

(1978) Formation of chromatoid body during rat spermatogenesis. Z Mikrosk Anat Forsch 92: 417–430

43.

Sud

(1961) Morphological and histochemical studies of the chromatoid body and related elements in the spermatogenesis of the rat. Q J Microscop Sci 102: 495–505

44.

Susi

Clermont

(1970) Fine structural modifications of the rat chromatoid body during spermatogenesis. Am J Anat 129: 177–192

45.

Tang

Lalli

Clermont

(1982) A cytochemical study of the Golgi apparatus of the spermatid during spermatogenesis in the rat. Am J Anat 163: 283–294

46.

Thorne-Tjomsland

Clermont

Hermo

(1988) Contribution of the Golgi apparatus components to the formation of the acrosomic system and chromatoid body in rat spermatids. Anat Rec 221: 591–598

47.

Ventelä

Toppari

Parvinen

(2003) Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat: mechanisms of haploid gene product sharing. Mol Biol Cell 14: 2768–2780

48.

Walt

Armbruster

(1984) Actin and RNA are components of the chromatoid bodies in spermatids of the rat. Cell Tissue Res 236: 487–490

49.

Werner

(1995) Immunocytochemical localization of histone H4 in the chromatoid body of rat spermatids. J Submicrosc Cytol Pathol 27: 325–330

50.

Wójcik

DeMartino

(2003) Intracellular localization of proteasomes. Int J Biochem Cell Biol 35: 579–589

51.

Yokota

Tsuji

Kato

(1985) Immunocytochemical localization of cathepsin D in lysosomes of cortical collecting tubule cells of the rat kidney. J Histochem Cytochem 33: 191–200

52.

Yokota

Kato

(1987) Immunocytochemical localization of cathepsins B and H in rat liver. Histochemistry 88: 97–103

53.

Yokota

Nishimura

Kato

(1988) Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques. Histochemistry 90: 277–283

Chromatoid Bodies: Aggresome-like Characteristics and Degradation Sites for Organelles of Spermiogenic Cells

Abstract

Keywords

Materials and Methods

Animals

Antibodies

Tissue Preparation

Post-embedding Immunoelectron Microscopy

Quantitative Analysis of the Labeling Density in the CBs and Cytoplasmic Matrix

Routine Electron Microscopy

Results

Routine Electron Microscopy

Immunoelectron Microscopy

Quantitative Analysis of the Gold Labeling in the CBs

Discussion

Footnotes

Acknowledgments

References