Active movements of the chromatoid body. A possible transport mechanism for haploid gene products

Recent data indicate that the chromatoid body typical of rat spermatogenesis may contain RNA synthesized in early spermatids by the haploid genome. Analyses of living step-1 and step-3 spermatids by time-lapse cinephotomicrography have shown that the chromatoid body moves in relation to the nuclear envelope in two different ways. Predominantly in step 1, the chromatoid body moves along the nuclear envelope on a wide area surrounding the Golgi complex and has frequent transient contacts with the latter organelle. In step 3, the chromatoid body was shown to move perpendicular to the nuclear envelope. It was seen located very transiently at the top of prominent outpocketings of the nuclear envelope with apparent material continuities through nuclear pore complexes to intranuclear particles. The rapid movements of the chromatoid body are suggested to play a role in the transport of haploid gene products in the early spermatids, including probably nucleocytoplasmic RNA transport.     

Transport of material between the nucleus,the chromatoid body and the golgi complex in the early spermatids of the rat

Summary The movement and transport of material between intranuclear dense particles, the chromatoid body and the Golgi complex have been studied in early spermatids of the rat. The analyses involved observation of living accurately identified cells, time-lapse cinemicrography and electron microscopy.The chromatoid body establishes transient contacts with intranuclear material during early spermiogenesis. The chromatoid body also makes contacts with the Golgi complex. It is suggested that the chromatoid body receives material from the nucleus during the postmeiotic period and participates in the early formation of the acrosomic system.This work was supported by the Finnish National Research Council for Medical Sciences. The authors are grateful to Mrs. Marita Aaltonen and Mrs. Raija Andersen for their skilful technical assistance     

Incorporation of (3H)uridine by the chromatoid body during rat spermatogenesis

The in vitro incorporation of tritiated uridine into RNA by the spermatogenic cells of the rat has been analyzed by high-resolution autoradiography. Special attention has been focused on the unique cytoplasmic organelle, the chromatoid body. After a short labeling time (2 h), this organelle remains unlabeled in the vast majority of the early spermatids although the nuclei are labeled. When the 2-h incubation with (3H)uridine is followed by a 14-h chase, the chromatoid body is seen distinctly labeled in all spermatids during early spermiogenesis from step 1 to step 8. Very few grains are seen elsewhere in the cytoplasm of these cells. When RNA synthesis in the spermatid ceases, the chromatoid body also remains unlabeled. It is likely that the chromatoid body contains RNA which is synthesized in the nuclei of the spermatids. The function of this RNA as a stable messenger RNA needed for the regulation of late spermiogenesis is discussed.     

Polysome-like structures in the chromatoid body of rat spermatids

A procedure for isolating the chromatoid body from the testis of 40-day-old rats was developed. Electron-microscopical analysis indicated that about 70% of the isolated organelles were chromatoid bodies, while the remaining structures corresponded to dense bodies and probably to satellites. Negative staining of the isolated organelles revealed the presence of polysome-like structures in about 20% of the chromatoid bodies suggesting that the polysomes were not due to contamination with cytoplasmic polysomes. Moreover, the presence of RNA in the stroma of the chromatoid body was confirmed by RNAse-gold staining. Preliminary electrophoretic analysis of the RNA extracted from the organelles revealed the presence of a complex population of RNAs including 5.8 and 5 S ribosomal RNAs but no tRNA.     

The relationship between the nuage and the chromatoid body during spermatogenesis in the Rat

Summary Cytoplasmic structures ultrastructurally similar to the nuage are present in the cytoplasm of all spermatogenic cells in adult rats. The nuage is a discrete organelle which should not be confused with the chromatoid body. In step 7–8 spermatids transient contact is established between the nuage and the chromatoid body. This indicates a very specific recognition of the nuage by the chromatoid body. It is suggested that the nuage and the chromatoid body are separate cell organelles the functions of which are somehow related to each other.     

Further study of the chromatoid body in rat spermatocytes and spermatids

Ultrastructure of the chromatoid body in rat spermatocytes and spermatids was studied by transmission electron microscopy. The following was found: 1. electron dense granules, 72.1 +/- 14.73 (SD) nm, that appeared to be both primary (assembling) and end (disassembling) structural subunits in the biogenesis of the chromatoid body, 2. relationship between chromatoid body and cytoplasmic microtubules, 3. ribbon-like structures and aggregates of 25 nm granules. The discussion focuses on a) a probable sequence of formation and breakdown of the chromatoid body, and b) the chromatoid body as an example of a common cellular design involving an interrelationship of dense material-smooth membranes-microtubules.     

黑脊倒刺Ba生精细胞拟染色体的形成过程

用电子显微镜观察了黑脊倒刺Ba生精细胞中拟染色体的形成过程。拟染色体在初级精原细胞中形成。在初级精原细胞的细胞核中,拟染色体前体物质先附着于核膜内侧,该处核膜崩溃。并在拟染色体前体物质的内侧,新核膜形成。新核膜将拟染色体前体物质分隔出细胞核之外。新核膜呈凹陷状。拟染色体前体物质集中于该凹陷中,并聚集成拟染色体。新核膜上有较大的空隙。核内还有少量拟染色体前体物质通过该空隙进入核表面的凹陷中,并结合到拟染色体上。黑脊倒刺Ba生精细胞拟染色 体的形成方式与通常认为的核内物质通过核孔排出核外的方式不同,似可表明核内物质向外运输存在着另一种机制。拟染色体形成后不久就与线粒体结合,并离开核凹。在以后的发育过程中,拟染色体又与线粒体分离。     

线粒体可能参与鲫鱼精子发生中拟染色体的形成

运用电子显微镜观察了鲫鱼生精细胞发育过程中拟染色体的形成和解体,以及拟染色体和线粒体的关系。在精子细胞阶段之前的各期生精细胞中都存在拟染色体。仅在精原细胞中观察到拟染色体的形成过程。拟染色体的形成方式与其它鱼类中拟染色体的形成方式相似。在生精细胞的发育过程中,线粒体的形态和数量发生变化。在初级精原细胞阶段,线粒体较大,多为球形,嵴少,基质电子密度低。随着生精细胞的发育,线粒体逐渐变小,多为长条状,嵴多,基质的电子密度升高。拟染色体形成后往往与线粒体结合。与拟染色体结合的线粒体往往解体,部分或全部的外膜和内膜破裂以至消失。线粒体解体后,其中的物质可能会转移到拟染色体中[动物学报52(2):328-334,2006]。     

Morphogenesis and fate of the residual body in human spermiogenesis

Summary In the human testis the formation of the residual body of the spermatid and its morphological changes during and after spermiation were studied by means of electron microscopy. The caudal cytoplasmic mass of the late spermatid contains a Golgi complex, mitochondria, annulate lamellae, a chromatoid body, flower-like structures, ribosomes, a few large vacuoles, myelin-like membrane profiles and sporadic lipid droplets. When, by detachment of the caudal cytoplasm from the free spermatozoon, the residual body is formed, the chromatoid body has disappeared; the mitochondria are clustered peripherally; the ribosomes appear as a single complex in contact with a large vacuole containing granular material; in place of the Golgi complex aggregations of vesicles are present. The lipid droplets remain unchanged. The residual bodies or their fragments are either extruded via the seminiferous tubular lumen into the excurrent ducts or they are engulfed by Sertoli cells where in the supranuclear region the successive steps of decomposition can be observed. The participation of the various constituents in the disintegration of the residual body is discussed. In contrast to other mammalian species, in man the sporadic lipid droplets seem to be of minor importance in the fate of the residual body.     

Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes

A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed. This evaluation confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the gold particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-gold complexes at the electron microscope level.     

The chromatoid body: a germ-cell-specific RNA-processing centre

The chromatoid body, a unique cloud-like structure of male germ cells, moves dynamically in the cytoplasm of haploid spermatids, but its function has remained elusive for decades. Recent findings indicate that microRNA and RNA-decay pathways converge to the chromatoid body. This highly specialized structure might function as an intracellular focal domain that organizes and controls RNA processing in male germ cells.     

Labelling of the chromatoid body by [H]uridine in rat pachytene spermatocytes

In this study it is shown that a cytoplasmic cell organelle, the chromatoid body, becomes labelled with [³H]uridine in the pachytene spermatocytes. The chromatoid body becomes labelled when the cells are first labelled for 2 h in the presence of [³H]uridine and thereafter chased for 9 h in the presence of unlabelled uridine. This labelling is inhibited by the specific RNA polymerase II inhibitor α-amanitin. Based on this it is suggested that part of the RNA synthesized in the pachytene spermatocytes is stored in the chromatoid body and transported to the postmeiotic spermatids where it is used in the differentiation of the spermatids.     

Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues: comparative evaluation of the different labelings

Several cytochemical techniques, such as sodium tungstate, acid hydrolysis phosphotungstic acid (HAPTA), ethylenediaminetetraacetic acid (EDTA), RNase-gold, and osmium-ammine, have been applied for the ultrastructural demonstration of nucleic acids on sections of tissues fixed in glutaraldehyde postfixed with osmium tetroxide and embedded in Epon. In order to obtain specific results, the sections had to be treated with sodium metaperiodate prior to performing the labeling protocol. The results for each method were identical to those obtained on nonosmicated tissues; the main difference being the enhancement in the ultrastructural preservation, which allowed for higher resolution. In addition to these techniques, and for comparative evaluations, DNA was also revealed by the DNase-gold approach on nonosmicated tissue sections. The consistency in the results, obtained over the nucleus with either EDTA or the RNase-gold complex for revealing RNA and those obtained with either osmium-ammine or DNase-gold for revealing DNA, supports the high specificity of the RNase-gold, DNase-gold, and osmium-ammine techniques. Furthermore, these results demonstrate the possibility of performing various cytochemical techniques on tissues processed for routine electron microscopy.     

Labelling of the chromatid body by [3H]uridine in rat pachytene spermatocytes

In this study it is shown that a cytoplasmic cell organelle, the chromatoid body, becomes labelled with [³H]uridine in the pachytene spermatocytes. The chromatoid body becomes labelled when the cells are first labelled for 2 h in the presence of [³H]uridine and thereafter chased for 9 h in the presence of unlabelled uridine. This labelling is inhibited by the specific RNA polymerase II inhibitor α-amanitin. Based on this it is suggested that part of the RNA synthesized in the pachytene spermatocytes is stored in the chromatoid body and transported to the postmeiotic spermatids where it is used in the differentiation of the spermatids.     

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

Proteins homologous to the Xenopus oocyte mRNA binding proteins mRNP₃₊₄ and designated p48/52 have been identified in male mouse germ cells (1993: Dev Biol 158:90–100). Western and Northwestern blots of extracts from testes and isolated germ cells indicate that p48/52 are present during meiosis but reach their highest levels postmeiotically at a time when many mRNAs are stored. Here we analyze the cellular and subcellular distribution of p48/52 in rat and mouse testes by LM and EM immunocytochemistry using an anti-mRNP₃₊₄ antibody. Immunolabeling was found to be predominantly cytoplasmic and specific to germ cells at certain periods during their development. p48/52 were first detected in early pachytene spermatocytes at stage V of the seminiferous cycle and progressively increased during the remainder of meiotic prophase to a post-meiotic peak in steps 1–8 round spermatids; thereafter, labeling gradually declined as elongated spermatids underwent nuclear condensation and elongation. A proportionally higher concentration of cytoplasmic immunolabeling was found within the lacunae of the anastomotic granulofilamentous network of the chromatoid body. The pattern of synthesis of these mRNA binding proteins together with their association with the chromatoid body suggests a role as germ cell-specific mRNA stabilizing and/or storage proteins. © 1996 Wiley-Liss, Inc.     

Actin and RNA are components of the chromatoid bodies in spermatids of the rat

Chromatoid bodies present in spermatocytes and spermatids of the rat show directed movements around spermatid nuclei during differentiation. This transient organelle contains RNA and establishes contact to intranuclear material and to the acrosomal complex. In order to determine possible components of motility and to verify the presence of RNA, we used a recently developed low-temperature embedding resin combined with protein A-gold and enzyme-gold techniques for studies at the ultrastructural level. All chromatoid bodies analyzed display high concentrations of gold particles over the electron-dense regions when labeled with antiactin. In contrast, RNase-gold particles were localized mainly in the electron-translucent areas. Corresponding controls were always negative. The results suggest a relationship between the impressive motility of the chromatoid body and actin present in the organelle. In addition, specific localization of RNA supports earlier findings that consider the chromatoid body an essential element for differentiation during spermiogenesis.     

Inhibitor induced alterations of chromatoid bodies in male germ line cells of Xenopus laevis

The action of inhibitors of protein synthesis on the structure of cytoplasmic inclusions found in the male germ cell line of the anuran, Xenopus laevis, has been studied by light and electron microscopy. Results indicate that one such inclusion, the chromatoid body, is sensitive to treatment with either chloramphenicol or puromycin. These drugs administered in vivo or in vitro cause up to a thirty-fold increase in the volume of the chromatoid body in all stages where it is normally present. Maximum size increase obtainable is the same for either drug, but is different and characteristic for each germ cell stage. Drug action is dose dependent, with "chromatoid body syndrome" occurring over a relatively narrow concentration range. Cyclohexamide, in contrast to chloramphenicol or puromycin, does not produce a clear increase in the size of chromatoid bodies, and is capable of blocking the action of the other drugs at normally effective concentrations. Results obtained in this investigation suggest that primary spermatogonia contain enough chromatoid body material to account for the total amount present in all subsequent germ cell stages. This fact, coupled with other studies where chromatoid-like bodies have been observed, suggests the hypothesis that the chromatoid body represents at least in part an aggregation stage of materials associated with the microtubule population of the germ cell line. Alternately, or in addition, ribonucleoprotein may contribute to the structure of the chromatoid body.     

Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat: mechanisms of haploid gene product sharing

Stable cytoplasmic bridges (or ring canals) connecting the clone of spermatids are assumed to facilitate the sharing of haploid gene products and synchronous development of the cells. We have visualized these cytoplasmic bridges under phase-contrast optics and recorded the sharing of cytoplasmic material between the spermatids by a digital time-lapse imaging system ex vivo. A multitude of small (ca. 0.5 microm) granules were seen to move continuously over the bridges, but only 28% of those entering the bridge were actually transported into other cell. The average speed of the granules decreased significantly during the passage. Immunocytochemistry revealed that some of the shared granules contained haploid cell-specific gene product TRA54. We also demonstrate the novel function for the Golgi complex in acrosome system formation by showing that TRA54 is processed in Golgi complex and is transported into acrosome system of neighboring spermatid. In addition, we propose an intercellular transport function for the male germ cell-specific organelle chromatoid body. This mRNA containing organelle, ca. 1.8 microm in diameter, was demonstrated to go over the cytoplasmic bridge from one spermatid to another. Microtubule inhibitors prevented all organelle movements through the bridges and caused a disintegration of the chromatoid body. This is the first direct demonstration of an organelle traffic through cytoplasmic bridges in mammalian spermatogenesis. Golgi-derived haploid gene products are shared between spermatids, and an active involvement of the chromatoid body in intercellular material transport between round spermatids is proposed.