Celina M. Haraguchi

Chromatoid bodies: aggresome-like characteristics and degradation sites for organelles of spermiogenic cells.

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.

Possible Function of Caudal Nuclear Pocket Degradation of Nucleoproteins by Ubiquitin-Proteasome System in Rat Spermatids and Human Sperm

Many temporarily functioning proteins are generated during the replacement of nucleoproteins in the nuclei of late spermatids and seem to be degraded in the nucleus. This study was designed to clarify the involvement of the ubiquitin-proteasome degradation system in the nucleus of rat developing spermatids. Thus, we studied the nuclear distribution of polyubiquitinated proteins (pUP) and proteasome in spermiogenic cells and sperm using postembedding immunoelectron microscopy. We divided the nuclear area of late spermatids into two regions: (1) a dense area composed of condensed chromatin and (2) a nuclear pocket in the neck region. The latter was located in the caudal nuclear region and was surrounded by redundant nuclear envelope. We demonstrated the presence of pUP in the dense area and nuclear pocket, proteasome in the nuclear pocket, and clear spots in the dense area of rat spermatids. Using quantitative analysis of immunogold labeling, we found that fluctuation of pUP and proteasome levels in late spermatogenesis was mostly synchronized with disappearance of histones and transitional proteins reported previously. In the nuclei of human sperm, pUP was detected in the dense area, whereas proteasome was in the nuclear vacuoles and clear spots. These results strongly suggest that pUP occur in the dense nuclear area of developing spermatids and that the ubiquitin-proteasome system is more actively operational in the nuclear pocket than dense area. Thus, the nuclear pocket might be the degradation site for temporarily functioning proteins generating during condensation of chromatin in late spermatids.

Localization of a Mitochondrial Type of NADP-dependent Isocitrate Dehydrogenase in Kidney and Heart of Rat: An Immunocytochemical and Biochemical Study

We studied the subcellular localization of the mitochondrial type of NADP-dependent isocitrate dehydrogenase (ICD1) in rat was immunofluorescence and immunoelectron microscopy and by biochemical methods, including immunoblotting and Nycodenz gradient centrifugation. Antibodies against a 14-amino-acid peptide at the C-terminus of mouse ICD1 was prepared. Immunoblotting analysis of the Triton X-100 extract of heart and kidney showed that the antibodies developed a single band with molecular mass of 45 kD. ICD1 was highly expressed in heart, kidney, and brown fat but only a low level of ICD1 was expressed in other tissues, including liver. Immunofluorescence staining showed that ICD1 was present mainly in mitochondria and, to a much lesser extent, in nuclei. Low but significant levels of activity and antigen of ICD1 were found in nuclei isolated by equilibrium sedimentation. Immunoblotting analysis of subcellular fractions isolated by Nycodenz gradient centrifugation from rat liver revealed that ICD1 signals were exclusively distributed in mitochondrial fractions in which acyl-CoA dehydrogenase was present. Immunofluorescence staining and postembedding electron microscopy demonstrated that ICD1 was confined almost exclusively to mitochondria and nuclei of rat kidney and heart muscle. The results show that ICD1 is expressed in the nuclei in addition to the mitochondria of rat heart and kidney. In the nuclei, the enzyme is associated with heterochromatin. In kidney, ICD1 distributes differentially in the tubule segments.

Ubiquitin Signals in the Developing Acrosome during Spermatogenesis of Rat Testis: An Immunoelectron Microscopic Study

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4–5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6–8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9–11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.

Spatiotemporal Changes of Levels of a Moonlighting Protein, Phospholipid Hydroperoxide Glutathione Peroxidase, in Subcellular Compartments During Spermatogenesis in the Rat Testis

Abstract We studied temporal changes in the subcellular localization and levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase (PHGPx), in spermatogenic cells and mature sperm of the rat by immunofluorescence and immunoelectron microscopy. The PHGPx signals were detected in chromatoid bodies, clear nucleoplasm, mitochondria-associated material, mitochondrial aggregates, granulated bodies, and vesicles in residual bodies in addition to mitochondria, nuclei, and acrosomes as previously reported. Within mitochondria, PHGPx moved from the matrix to the outermost membrane region in step 19 spermatid, suggesting that this spatiotemporal change is synchronized with the functional change of PHGPx in mitochondria. In the nucleus, PHGPx was associated with electron-lucent spots and with the nuclear envelope, and PHGPx in the latter region increased after step 16. In early pachytene spermatids, PHGPx signals were noted in the nuclear material exhibiting a very similar density to chromatoid bodies and in the intermitochondrial cement, supporting the previous proposal that chromatoid bodies originate from the nucleus and intermitochondrial cement. The presence of PHGPx in such various compartments suggested versatile roles for this protein in spermatogenesis. Quantitative immunoelectron microscopic analysis also revealed dynamic changes in the labeling density of PHGPx in different subcellular compartments as follows: 1) Total cellular PHGPx rapidly increased after step 5 and reached a maximum at step 18; 2) mitochondrial labeling density increased after step 1 and achieved a maximum in steps 15–17; 3) nuclear labeling density suddenly increased in steps 12–14 to a maximum; 4) in cytoplasmic matrix, the density remained low in all steps; and 5) the labeling density in chromatoid bodies gradually decreased from pachytene spermatocytes to spermatids at step 18. These spatiotemporal changes in the level of PHGPx during the differentiation of spermatogenic cells to sperm infer that PHGPx plays a diverse and important biological role in spermatogenesis.