Chromatoid Bodies : Aggresome-like Characteristics and Degradation Sites for Organelles of Spermiogenic Cells
Biology Laboratory (CMH,SY), Department of Biochemistry (TM), Department of Obstetrics and Gynecology (SH,TS,KH), University of Yamanashi, Interdisciplinary Graduate School of Medicine and Engineering, Yamanashi, Japan; and Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima, Japan (KA)
Correspondence to: Dr. Sadaki Yokota, Biology Laboratory, University of Yamanashi, Yamanashi, Japan. E-mail: syokota{at}yamanashi.ac.jp
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Summary |
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Key Words: chromatoid body spermiogenesis degradation aggresome immunoelectron microscopy
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Introduction |
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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 (F1
) and subunit ß (F1ß) 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.
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Materials and Methods |
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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-F1
and F1ß 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 PA28
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, 0.10.2% 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), F1 and F1ß (x200), COXI (2.5 µg/ml), 20S proteasome subunit (p52) (x5), KDEL (x4000), histone H2B (x500), proteasome activators PA700 and PA28
(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 36 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 (Jandel 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 postfixed 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.
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Results |
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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 7A7C). 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).
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Discussion |
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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 (F1
and F1ß) 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.
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 78, and disappear from those of steps 1618. 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.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akasaki K, Yamaguchi Y, Ohta M, Matsura F, Furuno K, Tsuji H (1990) Purification and characterization of a major glycoprotein in rat liver lysosomal membrane. Chem Pharm Bull (Tokyo) 38:27662770[Medline]
André J, Rouiller CH (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, 162164
Anton E (1983) Association of Golgi vesicles containing acid phosphatase with the chromatoid body of rat spermatids. Experientia 39:393394[Medline]
Biggiogera M, Fakan S, Leser G, Martin TE, Gordon J (1990) Immunoelectron microscopical visualization of ribonucleoproteins in the chromatoid body of mouse spermatids. Mol Reprod Dev 26:150158[CrossRef][Medline]
Bilinski SM, Jaglarz MK, Szymanska B, Etkin LD, Kloc M (2004) Sm proteins, the constituents of the spliceosome, are components of nuage and mitochondrial cement in Xenopus oocytes. Exp Cell Res 299:171178[CrossRef][Medline]
Comings DE, Okada TA (1972) The chromatoid body in mouse spermatogenesis: evidence that it may be formed by the extension of nucleolar components. J Ultrastruct Res 39:1523[CrossRef][Medline]
de Duve C, Wattiaux R (1966) Function of lysosomes. Annu Rev Physiol 28:435492[CrossRef][Medline]
Dice JF (1990) Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem Sci 15:305309[CrossRef][Medline]
Eddy EM (1970) Cytochemical observations on the chromatoid body of the male germ cells. Biol Reprod 2:114128[Medline]
Fabunmi RP, Wigley WC, Thomas PJ, DeMartino GN (2000) Activity and regulation of the centrosome-associated proteasome. J Biol Chem 275:409413
Fawcett DW, Eddy EM, Phillips DM (1970) Observations on the fine structure and relationships of the chromatoid body in mammalian spermatogenesis. Biol Reprod 2:129153[Medline]
Figueroa J, Burzio LO (1998) Polysome-like structures in the chromatoid body of rat spermatids. Cell Tissue Res 291:575579[CrossRef][Medline]
Findley SD, Tamanaha M, Clegg NJ, Ruohola-Baker H (2003) Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage. Development 130:859871
García-Mata R, Bebök Z, Sorscher EJ, Sztul ES (1999) Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera. J Cell Biol 146:12391254
Garcia-Mata R, Gao Y-S, Sztul E (2002) Hassles with taking out the garbage: aggravating aggresomes. Traffic 3:388396[CrossRef][Medline]
Haraguchi CM, Yokota S (2002) Immunofluorescence technique for 100-nm-thick semithin sections of Epon-embedded tissues. Histochem Cell Biol 117:8185[CrossRef][Medline]
Haraguchi CM, Mabuchi T, Hirata S, Shoda T, Yamada AT, Hoshi K, Yokota S (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 69:885895
Hershko A, Eytan E, Ciechanover A (1982) Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells. Relationship to the breakdown of abnormal proteins. J Biol Chem 257:1396413970
Hess RA, Miller LA, Kirby JD, Margoliash E, Goldberg E (1993) Immunoelectron microscopic localization of testicular and somatic cytochromes c in the seminiferous epithelium of the rat. Biol Reprod 48:12991308[Abstract]
Johnston JA, Ward CL, Kopito RR (1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143:18831898
Johnston JA, Dalton MJ, Gurney ME, Kopito RR (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 97:1257112576
Junn E, Lee AS, Suhr UT, Mouradian MM (2002) Parkin accumulation in aggresomes due to proteasome impairment. J Biol Chem 277:4787047877
Kistler WS, Henriksen K, Mali P, Parvinen M (1996) Sequential expression of nucleoproteins during rat spermiogenesis. Exp Cell Res 225:374381[CrossRef][Medline]
Kopito RR, Sitia R (2000) Aggresomes and Russell bodies. Symptoms of cellular indigestion? EMBO Rep 1:225231
Kopito RR (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10:524530[CrossRef][Medline]
Krimer DB, Esponda P (1980) Presence of polysaccharides and proteins in the chromatoid body of mouse spermatids. Cell Biol Int Rep 4:265270[CrossRef][Medline]
McNaught KSP, Shashidharan P, Perl DP, Jenner P, Olanow CW (2002) Aggresome-related biogenesis of Lewy bodies. Eur J Neurosci 16:21362148[CrossRef][Medline]
Morales CR, Kwon Y, Hecht NB (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 100:119131[Abstract]
Morales CR, Hecht NB (1994) Poly(A)+ ribonucleic acids are enriched in spermatocyte nuclei but not in chromatoid bodies in the rat testis. Biol Reprod 50:309319[Abstract]
Mortimore GE, Schworer CM (1977) Induction of autophagy by amino acid deprivation in perfused rat liver. Nature 270:174176[Medline]
Moussa F, Oko R, Hermo L (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 178:363378
Namekata K, Nishimura N, Kimura H (2002) Presenilin-binding protein forms aggresomes in monkey kidney COS-7 cells. J Neurochem 82:819827[CrossRef][Medline]
Oko R, Korley R, Murray MT, Hecht NB, Hermo L (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 44:113[CrossRef][Medline]
Parvinen M, Parvinen L-M (1979) Active movement of the chromatoid body. A possible transport mechanism for haploid gene products. J Cell Biol 80:621628[Abstract]
Riley HE, Li J, Warrall S, Rothnage JA, Swagell C, van Leewen FW, French SW (2002) The Mallory body as an aggresome: in vitro studies. Exp Mol Pathol 72:1723[CrossRef][Medline]
Russell LD, Frank B (1978) Ultrastructural characterization of nuage in spermatocytes of the rat testis. Anat Rec 190:7998[CrossRef][Medline]
Russell LD, Ettlin RA, Sinha Hikim AM, Clegg ED (1990) Histological and Histopathological Evaluation of the Testis. Clearwater, IL, Cache River Press, 41119
Ryan MC, Shooter EM, Notterpek L (2002) Aggresome formation in neuropathy models based on peripheral myelin protein 22 mutations. Neurobiol Dis 10:109118[CrossRef][Medline]
Saunders PTK, Millar MR, Maguire SM, Sharpe RM (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 33:385391[CrossRef][Medline]
Söderström K-O, Parvinen M (1976a) Transport of material between the nucleus, the chromatoid body and Golgi complex in the early spermatids of the rat. Cell Tissue Res 168:335342[Medline]
Söderström K-O, Parvinen M (1976b) Incorporation of [3H]uridine by the chromatoid body during rat spermatogenesis. J Cell Biol 70:239246[Abstract]
Söderström K-O (1978) Formation of chromatoid body during rat spermatogenesis. Z Mikrosk Anat Forsch 92:417430[Medline]
Sud BN (1961) Morphological and histochemical studies of the chromatoid body and related elements in the spermatogenesis of the rat. Q J Microscop Sci 102:495505
Susi FR, Clermont Y (1970) Fine structural modifications of the rat chromatoid body during spermatogenesis. Am J Anat 129:177192[CrossRef][Medline]
Tang XM, Lalli MF, Clermont Y (1982) A cytochemical study of the Golgi apparatus of the spermatid during spermatogenesis in the rat. Am J Anat 163:283294[CrossRef][Medline]
Thorne-Tjomsland G, Clermont Y, Hermo L (1988) Contribution of the Golgi apparatus components to the formation of the acrosomic system and chromatoid body in rat spermatids. Anat Rec 221:591598[CrossRef][Medline]
Ventelä S, Toppari J, Parvinen M (2003) Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat: mechanisms of haploid gene product sharing. Mol Biol Cell 14:27682780
Walt H, Armbruster BL (1984) Actin and RNA are components of the chromatoid bodies in spermatids of the rat. Cell Tissue Res 236:487490[Medline]
Werner G, Werner K (1995) Immunocytochemical localization of histone H4 in the chromatoid body of rat spermatids. J Submicrosc Cytol Pathol 27:325330[Medline]
Wójcik C, DeMartino GN (2003) Intracellular localization of proteasomes. Int J Biochem Cell Biol 35:579589[CrossRef][Medline]
Yokota S, Tsuji H, Kato K (1985) Immunocytochemical localization of cathepsin D in lysosomes of cortical collecting tubule cells of the rat kidney. J Histochem Cytochem 33:191200[Abstract]
Yokota S, Kato K (1987) Immunocytochemical localization of cathepsins B and H in rat liver. Histochemistry 88:97103[CrossRef][Medline]
Yokota S, Nishimura I, Kato K (1988) Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques. Histochemistry 90:277283[CrossRef][Medline]