Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This "elementary" fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30-40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called "immature chromatin," which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.     

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).     

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.     

Intranuclear crystalline inclusions in spermatozoa ofLumbricus terrestris

Summary In the nuclei of atypical spermatids ofLumbricus terrestris granular or filamentous inclusions are surrounded by dense chromatin. Aggregation and condensation of chromatin in nuclei during spermatid differentiation coincide with increase in density, granularity, and the subsequent crystallization of the intranuclear inclusion. In mature spermatozoa, the crystalline inclusion displaying an irregular shape is composed of parallel repeating units measuring 50–80 Å. The subunits sometimes possess a clear central cavity.Atypical spermatozoa, possessing inclusions that distort their normally cylindrical shape, possess typical acrosomes, middle pieces, and flagella. Spermatozoa bearing intranuclear crystals are rarely observed in the seminal receptacles ofLumbricus.These intranuclear inclusions probably represent proteinaceous material that is not eliminated during nuclear differentiation. Their sole existence in the nuclei of spermatozoa, their transformation into crystalline structures during spermiogenesis, and their similarity to crystals in virus infected plant and animal cells suggest a viral origin.Supported by a training grant (GM-00582-07) from the Public Health Service.     

Chromosome behaviour during meiotic prophase in the solanaceae

The molecular structure of chromatin during dogfish spermiogenesis was examined by electron microscopy after the dispersion of nuclei at low ionic strength. In early and late stages of differentiation (round and elongating spermatids), chromatin is globular, although basic nuclear proteins are different from those present in somatic nuclei. Three protein fractions are complexed with DNA in sperm nuclei. These fractions appear at the end of differentiation (elongated spermatids), subsequently undergoing a modification of their solubilization properties; only one protein fraction remains acid-soluble. Dispersed chromatin from sperm nuclei again shows a beads-on-a-string configuration both in the presence of the three specific sperm proteins and when the acid soluble fraction is extracted. Variations of the mean diameter of chromatin subunits during spermiogenesis appear rather limited compared to extensive modifications of chromatin superstructures.     

Spermiogenesis in the Nile tilapia Oreochromis niloticus with notes on a unique pattern of nuclear chromatin condensation

Spermiogenesis in the Nile tilapia, Oreochromis niloticus, was observed ultrastructurally. The process of spermatid differentiation can be divided into six distinct stages based mainly on changes in the nucleus of spermatids. During the latter half of the process, nuclear chromatin condenses progressively to form many dense globules, which ultimately adhere tightly to pack the head of mature spermatozoa. During chromatin condensation the nucleus diminishes in size, and part of the nuclear envelope and nucleoplasm forms a vesicular structure that is finally discarded from the cells together with an associated thin layer of cytoplasm. The spermatozoon comprises a roundish head, a relatively small midpiece, and a relatively short flagellum consisting of the usual 9+2 axoneme. No acrosomal structure is developed during spermiogenesis.     

Acridine orange-induced precipitation of mouse testicular sperm cell DNA reveals new patterns of chromatin structure

Precipitate resulting from interaction between certain intercalators, such as acridine orange (AO), and nucleic acids can be detected by electron microscopy. Formation of precipitate in nuclei of live cells is modulated by chromatin structure. Susceptibility of in situ DNA to precipitation was studied in mouse testicular germ cells during various stages of sperm maturation. DNA in round spermatid chromatin, similar to somatic cell euchromatin, was rather resistant to precipitation; the electron-dense precipitate was granular and randomly distributed. DNA in elongated spermatids was more susceptible to precipitation; the products were in the form of fibers. At early stages of spermatid maturation these fibers were distributed uniformly throughout the entire nucleus. At later stages, the products appeared as approximately 25-nm-thick fibers arranged longitudinally in arrays within the nucleus. With further cell maturation, fibers in the anterior portion of the nucleus appeared to fuse, forming homogeneously dense product. These fibrous products likely represent AO interactions with DNA in chromatin in which transition proteins had replaced histones. Changing patterns of these precipitated fibers likely reflect progressive stages of chromatin condensation, which starts at the center and anterior portion of the nucleus where the fibers coalesce. Mature sperm cell DNA, known to be complexed with protamines, was more resistant to AO-induced precipitation. The data suggest that precipitation induced by AO and monitored by electron microscopy may be a useful probe of nuclear chromatin structure.     

Organization of chromatin during spermiogenesis: beaded fibers,partly beaded fibers,and loss of nucleosomal structure

The aggregation of chromatin during spermiogenesis in the house cricket and many other animals is an orderly process involving the formation of a series of long, thick, well defined structures. The differentiation of chromatin preliminary to the development of such unusual structures is given attention here. Examination of nuclei after lysis and spreading indicated that fibers with closely spaced nucleosomes, like the fibers of somatic chromatin, make up the chromatin in all stages of early spermiogenesis and most of middle spermiogenesis. The thick structures of late spermatids cannot be formed by aggregation of fibers of this somatic type, however; just before thick structures form, chromatin fibers lose the nucleosomal structure. During the process, fibers with nucleosomes spaced at irregular intervals and with long stretches of smooth thin fiber are found, as if nucleosomes at one site on a fiber are broken down independently of those at adjacent sites. Since prior studies of cricket proteins have indicated that somatic histones persist during the stages when nucleosome structure disappears, the observations imply that the histones which are organized in nucleosomes during early stages must become incorporated into different kinds of nucleoprotein complexes during succeeding stages of spermiogenesis.     

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.     

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.     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

Cytological changes in the testes of vitamin-A-deficient rats. II. Ultrastructural study of the seminiferous tubules.

Ultrastructural study confirmed that, in rats, vitamin A deficiency initially caused the sloughing of some spermatids and spermatocytes into the lumina of the seminiferous tubules around day 3 following the initial decrease of body weight. From days 5 to 10, a considerable number of spermatocytes and spermatids, which still remained in the epithelium, underwent necrosis. Several stages of dying spermatocytes and abnormal spermatids were observed. The latter were distinguished by the presence of chromatin aggregating along the nuclear envelopes and highly vacuolated mitochondria. These cells range from single to multinucleate forms. They were incapable of differentiating further into spermatozoa and ultimately degenerated. Within the same period, Sertoli cells exhibited numerous darkly stained lysosome-like inclusions, and the upper part of their cytoplasm appeared as irregular processes, some of which were broken off and resulted in the thinning of the epithelium. From days 10 to 20, the remaining germ cells comprised mainly spermatogonia and few abnormal spermatocytes. The latter appeared enlarged and were very lightly stained. Their nuclei exhibited unusual blocks of heavily condensed chromatin amidst very highly dispersed chromatin fibers. Though their number was reduced, most of the spermatogonia appeared unaltered. Processes of Sertoli cells became even more irregular and were interrupted at certain sites by large empty spaces. Darkly stained inclusions in their cytoplasm were fewer than observed earlier.     

The three-dimensional structure of in vitro reconstituted <Emphasis Type="Italic">Xenopus laevis</Emphasis> chromosomes by EM tomography

We have studied the in vitro reconstitution of sperm nuclei and small DNA templates to mitotic chromatin in Xenopus laevis egg extracts by three-dimensional (3D) electron microscopy (EM) tomography. Using specifically developed software, the reconstituted chromatin was interpreted in terms of nucleosomal patterns and the overall chromatin connectivity. The condensed chromatin formed from small DNA templates was characterized by aligned arrays of packed nucleosomal clusters having a typical 10-nm spacing between nucleosomes within the same cluster and a 30-nm spacing between nucleosomes in different clusters. A similar short-range nucleosomal clustering was also observed in condensed chromosomes; however, the clusters were smaller, and they were organized in 30- to 40-nm large domains. An analysis of the overall chromatin connectivity in condensed chromosomes showed that the 30–40-nm domains are themselves organized into a regularly spaced and interconnected 3D chromatin network that extends uniformly throughout the chromosomal volume, providing little indication of a systematic large-scale organization. Based on their topology and high degree of interconnectedness, it is unlikely that 30–40-nm domains arise from the folding of local stretches of nucleosomal fibers. Instead, they appear to be formed by the close apposition of more distant chromatin segments. By combining 3D immunolabeling and EM tomography, we found topoisomerase II to be randomly distributed within this network, while the stable maintenance of chromosomes head domain of condensin was preferentially associated with the 30–40-nm chromatin domains. These observations suggest that 30–40-nm domains are essential for establishing long-range chromatin associations that are central for chromosome condensation. Electronic supplementary material The online version of this article (doi ) contains supplementary material, which is available to authorized users.     

An ultrastructural study on the occurrence of aberrant spermatids in the testis of the river sculpin,Cottus hangiongensis

The process of spermatogenesis and spermiogenesis in the river sculpin,Cottus hangiongensis, was observed ultrastructurally. During spermatogenesis, some germinal cysts in the seminal lobules were found to contain spermatocytes, which were provided with irregularly shaped nuclei, doughnut-shaped mitochondria, and atypical intercellular bridges with multiple disk-like cisternae. In addition, many cysts containing binuclear spermatids were observed in the testis. Within the condensed chromatin of the paired nuclei of the aberrant spermatids, highly electron-dense granules occurred, becoming the core of successively developing chromatin globules. The chromatin globules increased in size, resulting in an enlargement of the paired nuclei. These cells were finally released from the cyst into the lumen of the seminal lobules and underwent further degeneration, thus appearing as characteristic ‘spermatid masses’ in the mature testes.     

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, <Emphasis Type="Italic">Bandicota indica</Emphasis>

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.     

Interactions of nuclear proteins with DNA,during sperm differentiation in the ram

Ram spermatid nuclei and caput epididymal sperm nuclei were prepared and treated with DTT under conditions avoiding proteolysis. Whole-mount preparations for the electron microscope were made in the presence or absence of the detergent Joy. The chromatin of the less mature, non-round spermatid nuclei displayed a nucleosomal organization that gradually disappeared at the time the histones leave the nuclei (elongating spermatids). Digestion with micrococcal nuclease suggests that polynucleosome arrays are scarcer and more accessible to nuclease in the elongating than in the round nuclei, with increasing amounts of DNA becoming devoid of nuleosomes. In the protamine-containing nuclei (elongated spermatids), only smooth filaments were observed, which formed thick fibers by parallel aggregation. The change from a nucleosomal organization to bundles of smooth filaments appeared to result from a complex process involving the transitory presence of conspicuous knobby fibers that suggest a periodicity in the organization of the spermatidal proteins along the DNA molecules. X-ray diffraction patterns obtained with protamine-containing spermatid nuclei and with sperm nuclei confirm that the DNA is arranged in smoothly bent bundles of parallel molecules. No higher-order reflections that might correspond to nucleosome structures were detected in the 30–200 Å region.