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.     

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.     

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.     

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.     

Assembly of spermatid acrosome depends on microtubule organization during mammalian spermiogenesis

The acrosome is a secretory vesicle attached to the nucleus of the sperm. Our hypothesis is that microtubules participate in the membrane traffic between the Golgi apparatus and acrosome during the first steps of spermatid differentiation. In this work, we show that nocodazole-induced microtubule depolarization triggers the formation of vesicles of the acrosomal membrane, without detaching the acrosome from the nuclear envelope. Nocodazole also induced fragmentation of the Golgi apparatus as determined by antibodies against giantin, golgin-97 and GM130, and electron microscopy. Conversely, neither the acrosome nor the Golgi apparatus underwent fragmentation in elongating spermatids (acrosome- and maturation-phase). The microtubule network of round spermatids of azh/azh mice also became disorganized. Disorganization correlated with fragmentation of the acrosome and the Golgi apparatus, as evaluated by domain-specific markers. Elongating spermatids (acrosome and maturation-phase) of azh/azh mice also had alterations in microtubule organization, acrosome, and Golgi apparatus. Finally, the spermatozoa of azh/azh mice displayed aberrant localization of the acrosomal protein sp56 in both the post-acrosomal and flagellum domains. Our results suggest that microtubules participate in the formation and/or maintenance of the structure of the acrosome and the Golgi apparatus and that the organization of the microtubules in round spermatids is key to sorting acrosomal proteins to the proper organelle.     

Spermatogenesis in the Watase's shrew, Crocidura watasei--a light electron microscopic study.

Spermatogenesis in the Watase's shrew, Crocidura watasei, was investigated by light and transmission electron microscopy. The cycle of the seminiferous epithelium was divided into 12 stages using the development of spermatids as a main criterion. The steps of spermatids were characterized by morphological changes of the nucleus and acrosomal structure. The relative frequencies of the stages 1 to x 11 were 11.0, 10.3, 6.8, 10.6, 24.0, 6.4, 4.4, 7.9, 6.4, 4.9, 3.7 and 3.6%, respectively. Four types of spermatogonia (A1, A2, In and B) could be discerned by the observation of whole mount samples. The development of spermatids was divided into four phases (Golgi, cap, acrosome and maturation phases), as in other mammals. In Golgi phase of the spermatid, several acrosomal granules were encountered. In cap phase, the acrosome gradually spread over the nuclear surface. In early acrosome phase, the acrosome began to elongate and reached the maximal length in step 8 spermatids. The acrosome of step 8 spermatids was twice as long as that of spermatozoa. In late acrosome phase, the acrosome was on the way of shrinkage. Finally, the fan-shaped acrosome was formed in maturation phase. These findings suggested that the process of acrosomal formation was quite characteristic in the Watase's shrew in that the spermatid acrosome elongated most prominently in the mammals hitherto examined.     

Spermatocyte/spermatid-specific thioredoxin-3, a novel Golgi apparatus-associated thioredoxin, is a specific marker of aberrant spermatogenesis

Mammalian germ cells are endowed with a complete set of thioredoxins (Trx), a class of redox proteins located in specific structures of the spermatid and sperm tail. We report here the characterization, under normal and pathological conditions, of a novel thioredoxin with a germ line-restricted expression pattern, named spermatocyte/spermatid-specific thioredoxin-3 (SPTRX-3). The human SPTRX-3 gene maps at 9q32, only 50 kb downstream from the TRX-1 gene from which it probably originated as genomic duplication. Therefore, human SPTRX-3 protein comprises a unique thioredoxin domain displaying high homology with the ubiquitously expressed TRX-1. Among the tissues investigated, Sptrx-3 mRNA is found exclusively in the male germ cells at pachytene spermatocyte and round spermatid stages. Light and electron microscopy show SPTRX-3 protein to be predominately located in the Golgi apparatus of pachytene spermatocytes and round and elongated spermatids, with a transient localization in the developing acrosome of round spermatids. In addition, increased levels of SPTRX-3, possibly caused by overexpression, are observed in morphologically abnormal human spermatozoa from infertile men. In addition, SPTRX-3 is identified as a novel postobstruction autoantigen. In this report, we propose that SPTRX-3 can be used as a specific marker for diverse sperm and testis pathologies. SPTRX-3 is the first thioredoxin specific to the Golgi apparatus, and its function within this organelle might be related to the post-translational modification of proteins required for germ cell-specific functions, such as acrosomal biogenesis.     

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.     

C-terminal kinesin motor KIFC1 participates in acrosome biogenesis and vesicle transport

We have identified a possible role for the KIFC1 motor protein in formation of the acrosome, an organelle unique to spermatogenesis. KIFC1, a C-terminal kinesin motor, first appears on membrane-bounded organelles (MBOs) in the medulla of early spermatids followed by localization to the acrosomal vesicle. KIFC1 continues to be present on the acrosome of elongating spermatids as it flattens on the spermatid nucleus; however, increasing amounts of KIFC1 are found at the caudal aspect of the spermatid head and in distal cytoplasm. The KIFC1 motor is also found in the nucleus of very immature round spermatids just prior to its appearance on the acrosome. In some cases, KIFC1 appears localized just below the nuclear membrane adjacent to the subacrosomal membrane. We demonstrate that KIFC1 is associated with importin beta and colocalizes with this nuclear transport factor on curvilinear structures associated with the spermatid nuclei. These data support a model in which KIFC1, perhaps in association with nuclear factors, assists in the formation and/or elongation of the spermatid acrosome. This article represents the first demonstration of a direct association of a molecular motor with the spermatid acrosome, the formation of which is essential for fertilization.     

TMF/ARA160 Governs the Dynamic Spatial Orientation of the Golgi Apparatus during Sperm Development

TMF/ARA160 is known to be a TATA element Modulatory Factor (TMF). It was initially identified as a DNA-binding factor and a coactivator of the Androgen receptor. It was also characterized as a Golgi-associated protein, which is essential for acrosome formation during functional sperm development. However, the molecular roles of TMF in this intricate process have not been revealed. Here, we show that during spermiogenesis, TMF undergoes a dynamic change of localization throughout the Golgi apparatus. Specifically, TMF translocates from the cis-Golgi to the trans-Golgi network and to the emerging vesicles surface, as the round spermatids develop. Notably, lack of TMF led to an abnormal spatial orientation of the Golgi and to the deviation of the trans-Golgi surface away from the nucleus of the developing round spermatids. Concomitantly, pro-acrosomal vesicles derived from the TMF^-/- Golgi lacked targeting properties and did not tether to the spermatid nuclear membrane thereby failing to form the acrosome anchoring scaffold, the acroplaxome, around the cell-nucleus. Absence of TMF also perturbed the positioning of microtubules, which normally lie in proximity to the Golgi and are important for maintaining Golgi spatial orientation and dynamics and for chromatoid body formation, which is impaired in TMF^-/- spermatids. In-silico evaluation combined with molecular and electron microscopic analyses revealed the presence of a microtubule interacting domain (MIT) in TMF, and confirmed the association of TMF with microtubules in spermatogenic cells. Furthermore, the MIT domain in TMF, along with microtubules integrity, are required for stable association of TMF with the Golgi apparatus. Collectively, we show here for the first time that a Golgi and microtubules associated protein is crucial for maintaining proper Golgi orientation during a cell developmental process.     

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     

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.     

Role of microtubule-dependent membrane trafficking in acrosomal biogenesis

The role of microtubule-based trafficking in acrosomal biogenesis was examined by studying the effects of colchicine on spermiogenesis. In electron micrographs of untreated cap-phase mouse spermatids, coated vesicles were always seen on the apex and caudal margins of the developing acrosomal cap. The increase in volume and the accumulation of materials in the acrosome during the Golgi and cap phases were observed to occur via fusion of vesicles at various sites on the growing acrosome. By studying the acid phosphatase localization pattern and colchicine-treated spermatids, the role of clathrin-coated vesicles became clear. Coated vesicle formation at the caudal margin of the acrosome appeared to be responsible for the spreading and shaping of the acrosome over the surface of the nucleus and also established distinct regional differences in the acrosome. In colchicine-treated spermatids, the Golgi apparatus lost its typical membranous stack conformation and disintegrated into many small vesicles. Acrosome formation was retarded, and there was discordance of the spread of the acrosomal cap with that of the modified nuclear envelope. Many symplasts were also found because of the breakdown of intercellular bridges. Colchicine treatment thus indicated that microtubule-dependent trafficking of transport vesicles between the Golgi apparatus and the acrosome plays a vital role in acrosomal biogenesis. In addition, both anterograde and retrograde vesicle trafficking are extensively involved and seem to be equally important in acrosome formation. This work was supported by grants 83-0211-B-002-184 and 93-2320-B-320-012 from the National Science Council, Taiwan, Republic of China.     

大鼠精子细胞的糖蛋白合成——电镜放射自显影研究

本实验用电镜放射自显影技术,在注射~3H-岩藻糖后30分钟和1、4、8、24小时示踪大鼠精子细胞合成糖蛋白的情况以及新合成糖蛋白的去路。实验结果表明: 1.在注射~3H-岩藻糖后30分钟到1小时,放射自显影标记最初出现在高尔基体上。岩藻糖分子首先在高尔基体的外周(皮质)部位掺入糖蛋白,随后,新合成的糖蛋白并不直接转运到别处,而在高尔基体中央(髓质)部位作短暂贮存。说明中央部位在功能上是高尔基体的一个重要组成部分。2.~3H-岩藻糖不仅掺入高尔基期和顶帽期精子细胞的高尔基体,而且掺入顶体期精子细胞的高尔基体,说明顶体期的高尔基体仍有合成糖蛋白的功能。3.新合成糖蛋白的去路,在精子细胞发育的不同阶段是不一样的。在高尔基期和顶帽期精子细胞中,新合成的糖蛋白     

Differentiation of binuclear spermatozoa in mice

In an electron microscopy study of abnormal spermatogenesis in mice, we have found that two discrete haploid nuclei may be located in a single spermatid cytoplasm after the second meiotic division. The spermatid continues to differentiate and forms a binucleate spermatozoon with both nuclei separately packaged within the sperm head. The Golgi apparatus of the double spermatid forms a single proacrosome that attaches to both nuclei. Apparently, one acrosomal structure differentiates to cover and compartmentalize the two haploid nuclei within the sperm head. Chromatin condensation appears normal. The head morphology and number of flagella vary in mature spermatozoa produced by this process. This work demonstrates one pathway by which polyploid spermatids continue to differentiate to spermatozoa after failure of cytoplasmic division or possibly cellular fusion.     

RNF17, a component of the mammalian germ cell nuage, is essential for spermiogenesis

Nuages are found in the germ cells of diverse organisms. However, nuages in postnatal male germ cells of mice are poorly studied. Previously, we cloned a germ cell-specific gene named Rnf17, which encodes a protein containing both a RING finger and tudor domains. Here, we report that RNF17 is a component of a novel nuage in male germ cells--the RNF17 granule, which is an electron-dense non-membrane bound spherical organelle with a diameter of 0.5 mum. RNF17 granules are prominent in late pachytene and diplotene spermatocytes, and in elongating spermatids. RNF17 granules are distinguishable from other known nuages, such as chromatoid bodies. RNF17 is able to form dimers or polymers both in vitro and in vivo, indicating that it may play a role in the assembly of RNF17 granules. Rnf17-deficient male mice were sterile and exhibited a complete arrest in round spermatids, demonstrating that Rnf17 encodes a novel key regulator of spermiogenesis. Rnf17-null round spermatids advanced to step 4 but failed to produce sperm. These results have shown that RNF17 is a component of a novel germ cell nuage and is required for differentiation of male germ cells.     

Ultrastructural localization of enzymatic activity during spermiogenesis in two phytophagous bugs (Hemiptera: Pentatomidae)

Ultrastructural cytochemical techniques were used for the localization of phosphatases and oxidases in spermatid and spermatozoon of the phytophagous bugs Acrosternum aseadum and Nezara viridula (Hemiptera: Pentatomidae). Acid phosphatase was found mainly in the trans most portion of the Golgi complex, and in the acrosome of spermatozoon. Glucose-6-phosphatase was located in the endoplasmic reticulum, trans portion of the Golgi complex and in the acrosome of spermatids. The axoneme showed activity of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase. This observation supports the idea that various phosphates may play some role in spermatid differentiation. Indeed, the presence of cytochrome oxidase activity was only shown in the mitochondrial cristae of early spermatids, suggesting also the participation of this enzyme during spermatid differentiation of this insect.