Nuclear morphogenesis during spermiogenesis in the nudibranch molluscHypselodoris tricolor (Gastropoda,opisthobranchia)

An electron microscope study was carried out on Hypselodoris tricolor spermatids to describe the development of the nuclear morphogenesis and investigate the possible cause(s) of the change in the shape of the spermatid nucleus during spermiogenesis. Three different stages may be distinguished in the course of the nuclear morphogenesis on the basis of the morphology and inner organization of the nucleus. Stage 1 spermatid nuclei are spherical or ovoid in shape and the nucleoplasm finely granular in appearance. Stage 2 nuclei exhibit a disc- or cup-shaped morphology, and the chromatin forms short, thin filaments. During stage 3, a progressive nuclear elongation takes place, accompanied by chromatin rearrangement, first into fibers and then into lamellae, both formations helically oriented. A row of microtubules attached to the nuclear envelope completely surrounds the nucleus. Interestingly, the microtubules always lie parallel to the chromatin fibers adjacent to them. Late stage 3 spermatids show the highest degree of chromatin condensation and lack the manchette at the end of spermiogenesis. Our findings indicate the existence of a clear influence exerted on the chromatin by the manchette microtubules, which appear to be involved in determining the specific pattern of chromatin condensation in Hypselodoris tricolor.     

Chromatin condensation and acrosome development during spermiogenesis of Ensis ensis (Mollusca,Bivalvia)

We describe chromatin condensation and acrosome development during spermiogenesis of Ensis ensis. The overall shape of the mature spermatozoon corresponds to the primitive type. The nucleus is oval and on its superior pole there is an elongated acrosome; the middle piece contains four mitochondria around the centriolar complex. The condensation of the nuclei seems to occur in three steps: first the diameter of chromatin fibers increases slightly from 17 to 20 nm; second, in midspermatids fiber pairs coalesce; and third, the coalescence continues by addition of other fibers until the nuclei become highly compacted. Chromatin changes are related with nuclear protein composition. Small proacrosomal vesicles show two regions of different electron density. At a later stage they fuse to give a single, spherical vesicle in round spermatids, which migrates to the upper pole and transforms into a tapered acrosome (18 μm long) with a central channel filled with finely fibrous material. © 1994 Wiley-Liss, Inc.     

Chromatin fiber structure and plectonemic model of chromosome condensation in Drosophila cells

Reversible permeable cells have been used to isolate chromatin structures during the process of chromosome condensation. Analysis of individual structures slipping out from nuclei after reversal of permeabilization revealed that chromosomes of Drosophila cells consist of small units called rodlets. The fluorescent images of chromatin fibers were subjected to computer analysis allowing the computer-aided visualization of chromatin fibers. The zig-zag array of fibers consisting of 12-15 nucleosomes with a length of 270-330 nm (average 300 nm) showed decondensed extended strings, condensed loops, and coiled condensed loops. Theoretical considerations leading to the plectonemic model of chromatin condensation are based on experimental data, and give an explanation how the 30 chromatin fibers are formed and further condensed to the 300 nm chromatin loops in Drosophila cells.     

An electron microscopic study of mitosis in mouse duodenal crypt cells confirms that the prophasic condensation of chromatin begins during the DNA-synthesizing (S) stage of the cycle

The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H-thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde-formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography. Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: 1) numerous chromatin "specks" ranging in size from about 5 to 70 nm and averaging 47 nm; 2) a few roughly circular or elongated chromatin "packets" measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20-30 or more per nuclear profile; their average diameter is 128 nm. During mid-prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid- and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleoplasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps. Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro-, meta-, ana-, and telophase. Finally, radioautography 20 min after 3H-thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid-prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid-prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid-prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: 1) a slow, moderate condensation of the chromatin packets during early and mid-prophase and 2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes.     

Ultrastructural and chemical study of the chromatin during spermiogenesis of the fish Scyliorhinus caniculus (L.) (author's transl)

Electron microscopic, cytochemical and biochemical techniques were applied to study structural aspects and changes in nuclear components during the spermiogenesis of Scyliorhinus caniculus. Five major stages of nuclear differentiation were recognized and characterized by variations in the organization and chemical properties of chromatin. Stage I is analogous to a somatic nucleus with heterogeneous chromatin. At the second stage, the nuclear content is dispersed but the chromatin fibers are of the same diameter as those of the stage I. The nuclear elongation begins at stage III, the DNP fibers running preferentially parallel to the long axis of the nucleus. During these early modifications of chromatin structure appear two new basic nuclear proteins (S 1 and S 2) which migrate faster than histones but typical histones remain assosciated with these nuclei. In later elongation stage (stage IV), the chromatin fibers organize in a helical form and fuse side by side giving lamellar systems which have a reticular structure. At the end of this stage, the nuclear material has become uniformly compact. These late variations in chromatin organization are parallel to the association of chromatin with new basic nuclear proteins (S 3, S 4, Z 1, Z 2 and Z 3). The cytochemical and electrophoretical properties of one of these proteins (S 4) which appears at the end of spermiogenesis are similar to those of a protamine. In stage V, the chromatin is homogeneous and the nucleus assumes a helical configuration beginning at the posterior end. The deoxyribonucleoproteins of the mature sperm show some novel chemical characters, including the appearance of a stable nuclear acidophilia with the ALFERT and GESCHWIND method and extraction with 0.25 N HCl of one of the basic protein fractions newly appeared in late spermiogenesis (Z 3), two other fractions (Z 1 and Z 2) being extracted with a more drastic procedure. The other fractions described before are no more detectable.     

Structural organization of the sperm chromatin in a fern (Scolopendrium vulgare) studied by spreading methods

To investigate chromatin organization, we applied the spreading techniques to nuclei isolated from Scolopendrium spermatozoids. Well-dispersed chromatin shows three types of fibers: beaded fibers corresponding to a nucleosomal filament with adjacent nucleosomes in close contact, smooth fibers (14 nm in diameter) associated in a complex network, and knobby fibers constituted by local supercoiling of a very thin (4 nm) smooth filament. Along the knobby fibers, beads of variable size are irregularly spaced. The knobby fibers lie parallel and coalesce in thick bundles. The sperms basic proteins identified by electrophoretic analysis probably promote the supercoiling and the side-to-side attachment of the knobby fibers, which are all the more abundant in spread preparations. These results indicate that knobby fibers are probably located in the outer part of the sperm nucleus in which the chromatin is densely packed. As for the nucleosomal and smooth filaments, they may be situated in the inner part.     

Fragmentation of polyploid nuclei in the giant cells of the rat trophoblast. I. The ultrastructure of the nuclear fragments

Electron microscope study of the nuclear fragments in the rat trophoblast has demonstrated that the division of the trophoblast giant nucleus results first in the formation of a multinuclear cell. Each nuclear fragment is covered with its own nuclear envelope made of two membranes with numerous pore complexes. The chromatin in these nuclear fragments is condenced with various degrees of condensation, which depends on the step of placenta development, cell differentiation and the degree of nuclear fragmentation. The nuclear ultrastructure in nuclear fragments also depends on the degree of nuclear fragmentation and on the level of chromatin condensation. The nucleolus has no granular component. On large fragments, with lower chromatin condensation the nucleolus is not homogenous being made of fragments of more and of less electron dense fibrilles. Small light lacunae are seen in the nucleolus where chromatin threads and strands pass on. With a high chromatin condensation in the nucleus, round small nucleoli look homogenous being made of moderately electron dense fibrilles. Products of chromosome activity have been found in the nuclear fragments: accumulations of minute granules (d = 15--20 nm), perichromatinous granules (d = 35--40 nm), and fibrillar nucleolus-like bodies. In the multinuclear cell, made as the result of fragmentation of the initially giant nucleus, all the small nuclei are first arranged very close to each other, so that the contours of the neighbouring nuclei coincide.     

Differential basic nucleoprotein kinetics in the two kinds of lepidoptera spermatids: Nucleate (eupyrene) and anucleate (apyrene)

Normal lepidopteran males produce two kinds of spermatozoa: nucleate (eupyrene) and anucleate (apyrene). Eupyrene spermatozoa have the usual type of elongate nuclei. But in apyrene spermatids, the nuclei never elongate and the chromatin remains in a telophase-like condition until enucleation occurs. The study of the differential nucleoprotein kinetics of the two types of spermatids, using the fluorescent dye sulfoflavine, shows that: (1) In the elongate eupyrene nuclei, lysine-rich nucleoproteins are replaced by arginine-rich ones, while in the non-elongating apyrene nuclei only lysine-rich nucleoproteins are detected. However, nuclear elongation is not causally related to nucleoprotein transitions as transitions occur in the eupyrene spermatids after nuclear elongation. (2) The replacement of the nucleoproteins occurs in the eupyrene nuclei in a polarized manner. This may be correlated with the heterogeneous ultrastructural configuration of the chromatin fibers in elongating spermatid nuclei, as shown in other insect species. (3) Concomitantly with the eupyrene spermatid nucleoprotein transition, the cytoplasm of the head cyst cell shows an increasing amount of cytoplasmic lysine-rich proteins, while no such a phenomenon occurs in apyrene cysts. This differential pattern distribution may reflact functional differences among the two types of cysts and is probably related to the regulation of the dichotomy in lepidopteran spermatogenesis.     

Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks

In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and conclude that the appearance of precursor (intermediate) molecules facilitated the development of complex patterns of condensation and, as a consequence, a great diversity of forms in the sperm cell nuclei     

SPERMIOGENESIS IN THE PULMONATE SNAIL, EUHADRA HICKONIS II. STRUCTURAL CHANGES OF THE NUCLEUS

In accordance with the characteristic shape of the nucleus and degree of condensation of the nuclear substance, spermiogenesis in Euhadra hickonis can be roughly divided into four stages. The chromatin in the highly polymorphic nucleus of the first stage, early spermatid, forms relatively thick (ca. 50 nm) fibrils which associate here and there into irregular clumps. In the next stage, the spermatid nucleus becomes conspicuously spherical, its contents appear more finely homogeneous and the irregular clumps of chromatin are few. In the third stage, the nucleus gradually takes on an ellipsoidal shape as the antero-posterior axis shortens. The anterior part of its envelope becomes structurally modified in preparation for the adherence to it of the developing acrosome, and an implantation fossa forms posteriorly at the center of a second area where the nuclear envelope has been modified. The diameter of the chromatin fibrils again increases and those near the implantation fossa become oriented perpendicular to the nuclear envelope.
As the nucleus elongates in the fourth stage, a concentric sheath of microtubules closely surrounds it. These appear to depolymerize as the nuclear elongation proceeds, so that they are no longer present in the head region of the mature spermatozoon. The diameter of the chromatin fibrils increases to about 10 nm and they become oriented parallel to the long axis of the cell. With the decrease in the nuclear volume the fibrils unite laterally to form longitudinal sheets, and these finally merge in the mature spermatozoon into a mass of very dense chromatin without perceptible internal structure.     

小鼠生精细胞染色质与染色体构筑的扫描电镜研究（简报）

It remains unclear about the intermediate construction of chromosome due to its highly compact nature and the limitation in methods. The present study was designed to investigate the construction of chromatin and mitotic chromosome in situ with scanning electron microscopy. Mouse testes were selected as the material, because of in which the spermatogenic cells divide actively and successively to form the sperm. Such a feature would be able to study the structure of mammalian chromatin and chromosomes along with the change of nuclear cycle. The animal were perfused with 200 ml of 0.075 mol/L KCl hypotonic solution to remove blood and placed for 15-20 min on ice followed by 0.5% glutaraldehyde and 0.5% formaldehyde for fixing. Through treated by the routine process of fractured and freeze dried with t-butyl alcohol, the specimens were then coated with a 3 nm thick platinum and observed with Hitachi S-430 scanning electron microscopy. It was found that the hypotonic treatment with 0.075 mol/L KCl solution was suit for demonstrating the nuclear structure, when the organelles were well preserved. The chromatin fibers of 10-30 nm and 80-125 nm in diameter could be recognized in the interphase nuclei, which were arranged losely at the region of euchromatin, and folded with each other into chromatin masses at the region of heterochromatin, while the chromatin fibers with the diameter of 80-125 nm often could be viewed on the mitotic chromosomes. Since its presence in interphase nuclei and mitotic chromosomes, it was considered that the chromatin fibers with 80-125 nm in diameter might play a role in the condensation of chromosome, serve as a type of the intermediate structure.     

Ultrastructural and cytochemical changes of the head components of human spermatids and spermatozoa

The ultrastructural study of chromatin condensation simultaneously with the evolution of the perinuclear organelles was conducted in the spermatids and epididymal and ejaculated spermatozoa of man with the aid of the “en bloc” alcoholic PTA staining and the EDTA regressive method. The round nuclei of young spermatids (steps 1, 2) were characterized by the persistence of nucleoli that were PTA positive, and the presence of a subacrosomal layer of well-stained peripheral chromatin. In the beginning of the phase of nuclear elongation (step 3), the central chromatin also became dense, like the peripheral chromatin, while the nuclear ring and the associated manchette and the two anlages of the postacrosomal dense lamina and the posterior ring appeared. During steps 4 and 5, the sliding of the nuclear ring and the manchette, the growth of the postacrosomal dense lamina, and the progression of the posterior ring towards the base of the nucleus were seen along with structural and cytochemical modifications of the chromatin. In the flattened nuclei of step 4 spermatids, coinciding with the loss of the nucleolar components, the chromatin achieved maximum compactness in the entire nucleus and was PTA positive. In the spermatids of step 5, the disappearance of peripheral dense chromatin and the specific staining of the chromatin granules marked the beginning of the second stage of transformation of the basic nucleo-proteins. The condensed nuclei of the mature spermatids were partially stained by PTA in step 6 and totally unstained in step 7. The PTA staining revealed the persistence of PTA-positive chromatin areas in the nuclei of certain spermatids otherwise mature. The morphological aspect of the chromatin then remained the same in the nuclei of epididymal and ejaculated spermatozoa. These observations suggest that in man, as in other mammals studied, new proteins accumulate in the elongating nuclei of spermatids and are replaced at the phase of maturation by sperm-specific nucleoproteins. The defects in condensation of the chromatin that occur during spermiogenesis could be related to the modalities of accumulation of intermediate nucleoproteins.     

小鼠生精细胞染色质与染色体构筑的扫描电镜研究(简报)

正常情况下,染色质和染色体在细胞内呈高度致密状态,在光镜和透射电镜下常呈浓染的斑块状。由于方法学上的困难,至今对染色质乃至染色体的微细结构,仍缺乏清楚的了解。特别是关于染色质如何凝缩形成染色体方面,现仍存在有争论。扫描电镜的冷冻割断技术,曾被用于对游离细胞间期核染色质的观察,并取得了较好的     

Cloned calves from chromatin remodeled in vitro

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).     

THE FINE STRUCTURE AND CHEMICAL COMPOSITION OF NUCLEI DURING SPERMIOGENESIS IN THE HOUSE CRICKET : I. Initial Stages of Differentiation and the Loss of Nonhistone Protein

The early stages of nuclear differentiation in spermatids of the house cricket are described with regard to the fine structural elements and chemical components which occur. Particular attention is given to the loss of nonhistone protein from the nucleus and its relation to chromatin structure. Granular elements about 25 to 80 mµ in diameter, and fibers about 8 mµ in diameter occur in the earliest spermatid nucleus. The fibers are found in diffuse and condensed chromatin while granules are found only in diffuse material. DNA and histone parallel the chromatin fibers in distribution, while nonhistone protein and RNA parallel the granules in distribution. The granules and most of the nonhistone protein are lost, simultaneously, after the early spermatid stage. The protein loss occurs without detectable change in the structure of chromatin fibers. Chromatin fibers first show a structural change in mid spermiogenesis, when they become thicker and very contorted. Unusually thin fibers (about 5 mµ) also appear in mid spermatid nuclei; they are apparently composed of nonhistone protein and free of DNA and histone.     

Ultrastructural studies of spermatogenesis in the anthocerotales. III. Gamete morphogenesis: From spermatogenous cell through midstage spermatid

An ultrastructural examination of spermatogenesis in Phaeoceros has shown nucleoli to be present in spermatogenous cells and to persist until the centrioles become associated with nuclei of young spermatids. At the onset of multilayered structure (MLS) formation, well-defined aggregations of osmiophilic strands begin to form in the nuclei of young spermatids and disappear shortly after chromatin condensation starts in the midstage spermatids. When the centrioles in the young spermatids are orientated perpendicular to the nuclear envelope, the nucleoplasm immediately in front of them is densely stained. Where the spline tubules of the MLS extend over the nucleus, the nuclear envelope is devoid of pores, and the inner nuclear membrane is contacted internally by the local deposition of dense staining nucleoplasm. Chromatin condensation begins with strands extending perpendicularly from the dense staining nucleoplasm beneath the spline and continues with the nuclear beak becoming filled with condensed chromatin. As the MLS lamellae disappear acropetally, the rear portion of the anterior mitochondrion (AM) extends back under the nuclear beak which now narrows to a size that approximates the anterior end of the nucleus of a spermatozoid. By the end of the mid-spermatid stage, the nucleus has coiled approximately one gyre of a helix and the five or six central slpine tubules extend over the plastid which is now located beneath the front end of the AM. Several profiles of endoplasmic reticulum confluent with the nuclear envelope are present. Possible factors which might play a role in determining the morphology of the mid-spermatids are discussed.