Cytochemical and immunocytochemical studies of the localization of histones and protamine-type proteins in spermatids of Chara vulgaris and Chara tomentosa

Spermiogenesis in Chara algae, which has been divided into 10 phases (sp I-X), is similar to spermiogenesis in animals. The most important process during spermiogenesis in animals is remodeling of chromatin leading to "sleeping genome", being the result the exchange of histone proteins into protamine-like proteins. Cytochemical studies showed in both Chara species (C. vulgaris, C. tomentosa) that at spI-IV phases only histones were present, at spV-VIII phases--the amount of nuclear protamine-type proteins progressively increased and that of histones decreased while at spIX-X only pro-tamine-type proteins were present. This was also confirmed with capillar electrophoresis. In order to localize more precisely both histones and protamines the immunocytochemical studies with the use of anti-protamine antibodies (protamine-type proteins were obtained from C. tomentosa antheridia) and anti-histone H3 antibodies, have been carried out. More specific immunocytochemical studies confirmed cytochemical results including the exchange of histones into protamine-type during spermiogenesis (spV-VIII) in both Chara species. At phase V spermiogenesis these strong strand-like anti-protamine signals were observed in cytoplasm which might suggest that protamine synthesis took place in ER.     

Occurrence of ubiquitin during spermiogenesis in Chara vulgaris

Experiments with an anti-ubiquitin antibody proved the presence of ubiquitin in spermatids at all spermiogenesis stages in Charta vulgaris. Its level increased before marked ultrastructural changes of spermatids correlated with disappearance of somatic proteins (histones) and appearance of protamine-type generative proteins. The obtained results seem to confirm our earlier hypotheses concerning a significant role of ubiquitin-proteasome system in Chara spermatozoid differentiation.     

Occurrence of calreticulin during the exchange of nucleohistones into protamine-type proteins in Chara vulgaris spermiogenesis

During spermiogenesis of an alga Chara vulgaris, which resembles that of animals, nucleohistones are replaced by protamine-type proteins. This exchange takes place in a spermatid nucleus during the key V spermiogenesis stage, in which rough endoplasmic reticulum is the site of protamine-type protein synthesis and is also the pathway guiding the proteins to their destination, nucleus. In the present work, it was shown that a chaperon protein, calreticulin (CRT), abundantly present at this significant V stage of spermiogenesis in a few cellular compartments, i.e., a nucleus, lumen of cisternae, and vesicles of significantly swollen ER as well as outside these structures, e.g., in Golgi apparatus, could have taken part in the process of exchange of nuclear proteins. Colocalization of two proteins, protamine-type proteins, crucial for reproduction, and CRT, was especially visible in a nucleus, mainly on its peripheries where condensed chromatin was present. Localization of protamine-type proteins and CRT in nucleus is in agreement with our previous results showing that protamine-type proteins were twofold more labelled in the peripheral area in comparison to the nucleus center occupied by noncondensed chromatin. The role of CRT in the reproduction of both plants and animals is also discussed.     

The influence of epoxomicin,inhibitor of proteasomal proteolytic activity,on spermiogenesis in Chara vulgaris

The influence of 48-h treatment with epoxomicin, an inhibitor of proteolytic activity of proteasomes, at the concentration 10 microM, on spermiogenesis in algae Chara vulgaris was examined. In the presence of the inhibitor, the frequency of early spermiogenesis phases significantly increased, the number of spermatids in mid-phases decreased and disappearance of late phases was observed. A hypothesis has been put forward that epoxomicin stops spermiogenesis during the period of preparation to further deep reorganisation of spermatids by blocking proteolysis of short-lived regulatory proteins which are responsible among others for triggering the exchange of nucleohistones into nucleoprotamines.     

Chara tomentosa Antheridial Plasmodesmata at Various Stages of Spermatogenesis

Chara tomentosa antheridial plasmodesmata are described during proliferation and spermiogenesis. In antheridial filament cells which are cycling completely synchronously, unplugged plasmodesmata are filled with light cytoplasm. The same plasmodesmata are observed after cessation of mitotic division followed by the onset of synchronous spermiogenesis. Walls separating cells at different cell cycle stages and dividing antheridial filaments into asynchronous domains are plugged with a dense osmophilic substance. Similarly plugged plasmodesmata are present between antheridial cells of different types, e.g., capitular cells and antheridial filaments. In mid spermiogenesis when abundant endoplasmic reticulum (ER) appears temporarily it penetrates into plasmodesmata enabling cell-to-cell transport via ER cisternae. In late spermiogenesis there are no cisternae in plasmodesmata. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunocytochemical and ultrastructural analyses of the function of the ubiquitin-proteasome system during spermiogenesis with the use of the inhibitors of proteasome proteolytic activity in the alga, Chara vulgaris

Spermiogenesis in Chara vulgaris and in animals share many common features, including exchange of nucleohistones into nucleoprotamines, remodeling and extreme condensation of chromatin, formation of flagellae and of microtubule manchette, and decrease in cytoplasm volume. In C. vulgaris, spermiogenesis is not preceded by meiosis since this alga is a haplobiont. In the present work we showed that in early spermiogenesis characterized by a significant metabolic activity of spermatids, the inhibitors of proteasomes did not visibly change their ultrastructure but significantly prolonged this process. At late stages of spermiogenesis, MG-132 and epoxomicin dramatically changed the structure of nuclei: regular fibrillar and lamellar structure of chromatin was disturbed and clusters of grains corresponding to aggresomes appeared, but the nucleus shape and cytoplasm structure were the same as in the controls. Immunocytochemical studies revealed that these inhibitors blocked disappearance of histones from nuclei while the structures corresponding to aggresomes were clusters of undegraded ubiquitinated histones, since they gave positive immunosignals indicating the presence of ubiquitin and histones.     

Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis

Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.     

Further ultrastructural research of Chara vulgaris spermiogenesis: endoplasmic reticulum,structure of chromatin, 3H-lysine and 3H-arginine incorporation

On the basis of morphological features, 10 consecutive structural phases of spermatids were identified in Chara vulgaris spermiogenesis. They were schematically presented. In early and middle spermiogenesis, i.e. during the period preceding formation of fibrillar structure of mature spermatozoid nucleus, a slight remodelling of chromatin, accompanied by proplastid transformation into an amyloplast as well as by development of 2 flagella and a microtubular manchette, is observed. First, condensed chromatin concentrates around the nuclear envelope (phases III-V) and then it transforms into a network-like structure (phase VI). This change in chromatin structure is preceded by nucleolar extrusion to the cytoplasm where nucleoli become degraded (phase IV) and by a dynamic development of rough endoplasmic reticulum (RER) (phase V) which is continuous with the nuclear envelope and with RER of the adjacent spermatids via plasmodesmata. The inner membrane of the nuclear envelope invaginates into the nucleoplasm in which "nuclear reticulum" appears. It all happens during increased 3H-arginine and 3H-lysine incorporation into proteins which are rapidly translocated into the nucleus. In medium-late spermiogenesis (phases VI-VIII), network-like condensed chromatin disappears. Next, the structure of the nucleus changes dramatically. Short, randomly positioned fibrils (phase VII) appear and gradually become longer (phase VIII), thicker (phase IX) and more distinct, lying parallel to the surface of elongating and curling nucleus. Membranes of the nuclear envelope become closer to each other and a distinct dark layer--probably lamin--appears adhering to the inner membrane of the nuclear envelope. Towards the end of spermiogenesis (phase X), very densely packed parallel helices, ca 2 nm in diameter, are visible. The surfaces of flagella and the spermatozoid are covered with diamond-shaped larger and smaller scales, respectively. Helically coiled spermatozoids are liberated from antheridial filament cells through earlier created (phase VIII) "liberation pores" with pads of unknown nature.     

Cytochemical studies on the antheridial mucilage and changes in its concentration and amount during the spermatogenesis in Chara vulgaris L.

The internal space of the antheridium in Chara vulgaris L. is filled with the PAS-positive mucilage which is of pectic nature. Morphometric and cytophotometric measurements on the semithin sections indicate that the concentration and amount of PAS-positive polysaccharides: 1) increase during the time of antheridial growth accompanying the phase of antheridial filament divisions, 2) these parameters have the maximum after spermatid formation and at the beginning of their differentiation, i.e. spermiogenesis, 3) both concentration and amount of this substance decrease at the end of spermiogenesis. A decrease in mucilage concentration is also observed in the young antheridia after 3 days of continuous darkness. The results suggest that PAS-positive mucilagenous material is a nutritive substance, accumulated in the first phase of antheridial development and utilized mainly in spermiogenesis. These substances may also be used up in the young antheridia during the lack of energy supply. The autoradiographic studies with the use of a 3H-glucose and 3H-galactose mixture seem to confirm these suggestions.     

Brg1 subunit of chromatin remodelling complex is present during Chara vulgaris (Charophyceae) spermatogenesis

ABSTRACT

During spermatogenesis, cells developed as a result of numerous mitotic and meiotic divisions transform into mature spermatozoids. In spermatids, remodelling of chromatin structure takes place which is connected with nuclear protein exchange, DNA double strand breaks and epigenetic modifications. Chromatin remodelling complexes, which have mostly been studied in animals, also participate in this process. The Brg1 protein, a functional homologue of the yeast Swi2/Snf2 catalytic subunit of the SWI/SNF complex, is engaged in regulation of cell proliferation and highly expressed in round spermatids in mammals. Immunocytochemical studies with the anti-Brg1 antibody revealed positive reactions in nuclei of the green alga Chara vulgaris at the 64-cell proliferative stage and in spermatid nuclei at the I/II–VII spermiogenesis stages. The most intensive reaction was observed at the early spermiogenesis stages (I/II–III), while at the initial stages of a proliferative phase (4-, 8- and 16-cell) the reaction was not observed, and at 32-cell and VII stages the immunosignals were very weak. Ultrastructural studies with the immunogold technique confirmed the results of the immunocytochemical studies. The highest numbers of gold grains were observed at stages I/II and III of spermiogenesis, and together they constituted above 48% of the total number of gold grains. A much lower, but still substantial, amount of these grains was observed at the 64-cell stage and IV stage (>15% and 17%), respectively. Percentage analysis revealed the lowest number of gold particles at stage VII (3.72%). The significant presence of Brg1 protein at early spermiogenesis stages is correlated with acetylation of the H4K12 histone. It may also be hypothesized that in C. vulgaris the Brg1 subunit participates in processes important for proper chromatin condensation and facilitates maintenance of the correct shape of the spermatid nucleus. On the basis of earlier and current studies it seems that chromatin remodelling in spermatids of this model alga proceeds according to the model presented for mammals.     

A comparative study of sperm morphology, cytology and activation in Caenorhabditis elegans, Caenorhabditis remanei and Caenorhabditis briggsae

Studies of sterile mutants in Caenorhabditis elegans have uncovered new insights into fundamental aspects of gamete cell biology, development, and function at fertilization. The genome sequences of C. elegans, Caenorhabditis briggsae and Caenorhabditis remanei allow for informative comparative studies among these three species. Towards that end, we have examined wild-type sperm morphology and activation (spermiogenesis) in each. Light and electron microscopy studies reveal that general sperm morphology, organization, and ultrastructure are similar in all three species, and activation techniques developed for C. elegans were found to work well in both C. briggsae and C. remanei. Despite important differences in the reproductive mode between C. remanei and the other two species, most genes required for spermiogenesis are conserved in all three. Finally, we have also examined the subcellular distribution of sperm epitopes in C. briggsae and C. remanei that cross-react with anti-sera directed against C. elegans sperm proteins. The baseline data in this study will prove useful for the future analysis and interpretation of sperm gene function across nematode species.     

Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris. II. Capitular cells

In young antheridia, the structure of capitular cells is typical of meristematic cells. The cytoplasm is characterized by poorly developed ER system, numerous free ribosomes, active Golgi apparatus and plastids at the stage of proplastids. In the period of mitotic divisions, i.e. during formation of the initial cells of antheridial filaments, the nuclei of capitular cells have a changing structure. When capitular cells stop budding leading to the formation of successive antheridial filaments. DNA content in the nucleus is at 2C-4C level. The nucleolus with nucleolonema-like structure becomes gradually smaller in the course of the development of the anteheridium. During spermiogenesis capitular cells are vacuolated, cytoplasm contains numerous polysomes, mitochondria assume condensed structure, the incorporation of 3N-uridine and of labelled aminoacids increases. It has been suggested that capitular cells collaborate with other antheridial cells in the regulation of the course of spermiogenesis.     

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     

Ultrastructural examination of spermiogenesis within the testis of the ground skink, Scincella laterale (Squamata, Sauria, Scincidae)

Although the events of spermiogenesis are commonly studied in amniotes, the amount of research available for lizards (Sauria) is lacking. Many studies have described the morphological characteristics of mature spermatozoa in lizards, but few detail the ultrastructural changes that occur during spermiogenesis. The purpose of this study was to gain a better understanding of the subcellular events of spermiogenesis within the temperate ground skink (Scincella laterale). The morphological data presented here represent the first complete ultrastructural study of spermiogenesis within the Scincidae clade. Samples of testes from 20 specimens were prepared using standard techniques for transmission electron microscopy. Many of the ultrastructural changes occurring during spermiogenesis within the ground skink are similar to that of other saurians. However, there were a few unique characteristics that to date have not been described during spermiogenesis in other lizards. For example, during early round spermatid development within the ground skink testis, proacrosomal granules begin to form within the acrosomal vesicle before making contact with the apex of the nucleus. Also, a prominent microtubular manchette develops during spermiogenesis; however, the circular component of the manchete is absent in this species of skink. This developmental difference in manchette formation may lead to the more robust and straight mature spermatozoa that are common within the Scincidae family. These anatomical character differences may be valuable nontraditional sources that along with more traditional sources (i.e., mitochondrial DNA) may help elucidate phylogenetic relationships, which are historically considered controversial at best, among species within Scincidae and Sauria.     

Barriers to diffusion of plasma membrane proteins form early during guinea pig spermiogenesis.

The plasma membrane of the mature guinea pig sperm is segregated into at least four domains of different composition. Previous studies have shown that some proteins localized within these domains are free to diffuse laterally, suggesting that barriers to protein diffusion are responsible for maintaining the nonuniform distribution of at least some surface proteins in mature sperm. The different membrane domains appear sequentially during sperm morphogenesis in the testis and during later passage through the epididymis. To determine when diffusion barriers become functional during sperm development, we examined the diffusion of two proteins that are expressed on the cell surface of developing spermatids and become segregated to different plasma membrane domains during the course of spermiogenesis. Both proteins exhibited rapid lateral diffusion throughout spermiogenesis, even after they become localized to specific regions of the surface membrane. These results suggest that barriers to membrane diffusion form concomitantly with membrane domains during spermiogenesis.     

Developmental expression of spermatid-specific thioredoxin-1 protein: transient association to the longitudinal columns of the fibrous sheath during sperm tail formation

The mammalian sperm tail presents a complex organization in which a number of additional structures, namely outer dense fibers and fibrous sheath, surround the central axoneme and are thought to regulate flagellar motility. We have previously described a novel member of the thioredoxin family of proteins with a spermatid specific expression pattern, spermatid-specific thioredoxin-1 (Sptrx-1). We report here the developmental analysis of Sptrx-1 expression during murine spermiogenesis. Immunocytochemical analysis of Sptrx-1 through the different steps of spermiogenesis in rat seminiferous tubule sections showed that its expression begins at step 9, gets progressively stronger until steps 14-16 (where a peak is reached), and then diminishes in steps 17 and 18 until practically no immunolabeling is detected in step 19 spermatid. During its transient expression in spermiogenesis, Sptrx-1 is most concentrated in the periaxonemal compartment of the tail of the elongating spermatid, except in the very last steps (steps 17-19), when periaxonemal labeling disappears and a residual buildup of Sptrx-1 occurs in the shrinking cytoplasmic lobe. Electron microscopic analysis by immunogold labeling pinpointed the localization of Sptrx-1 to the assembling longitudinal columns of the fibrous sheath, whereas the forming ribs of the fibrous sheath were unlabeled. Immunoblotting of isolated fibrous sheath and tails obtained from epididymal or ejaculated sperm of rat and human confirmed our immunocytochemical observation: Sptrx-1 is no longer a component of the mature fibrous sheath. To our knowledge, this is the first report of a protein that specifically associates to the fibrous sheath during development but does not become a permanent structural component. The expression pattern of Sptrx-1 during rat spermiogenesis suggests that it could be part of a nucleation center for the formation of the longitudinal columns and transverse ribs that bridge the latter.     

Characterization of chromatin-condensing proteins during spermiogenesis in a neogastropod mollusc (Murex brandaris)

During the process of chromatin cndensation in the spermiogenesis of the neogastropod mollusc Murex brandaris, the nuclear protein complement undergoes a complex series of changes. These changes lead to the appearance of three small protamines in the ripe sperm nuclei. We have characterized this system electrophoretically and at the compositions with antibodies elicited against a specific spermatozoan protamine. Our results indicate that the complex pattern of chromatin condensation during spermiogenesis in this species (M. brandaris) may be modulated by a series of post-translational (and intranuclear) modifications of DNA-interacting proteins, such as precursors to the sperm protamines. The amino acid composition of each sperm protamine is remarkably simple (lys + arg + gly ≥96 mol%). This system of spermiogenic/spermatozoal proteins in the neogastropod M. brandaris clearly differs from that in patellogastropods and archaeogastropods, and it may be helpful in understanding evolutionary changes in the chromatin condensation pattern during the spermiogenesis of gastropod molluscs. © 1994 Wiley-Liss, Inc.     

Synthesis and assembly of connecting-piece proteins as revealed by radioautography

The synthesis and assembly of connecting-piece proteins have been studied during spermiogenesis in the rat by electron microscopy and radioautography following intratesticular injection of radiolabeled amino acids [3H]proline and [3H]cystine. Early in spermiogenesis (steps 1-7) the two centrioles that give rise to the connecting piece are essentially unmodified. During the 6.5-day period between steps 8 and 15, the major elements of the connecting piece (striated columns and capitulum) gradually become assembled from an electron-dense material that is deposited around the walls of the centrioles; throughout this period, protein molecules containing proline and cystine are synthesized by the step 8-15 spermatids and incorporated into the developing neck region. These proteins subsequently become permanent structural components of the connecting piece. Following completion of the major elements in step 15, few additional proteins are added to the connecting piece during the final steps 16-19 of spermiogenesis.     

Chromatin organization during spermiogenesis in Octopus vulgaris. II: DNA-interacting proteins

In this article we study the proteins responsible for chromatin condensation during spermiogenesis in the cephalopod Octopus vulgaris. The DNA of ripe sperm nuclei in this species is condensed by a set of five different proteins. Four of these proteins are protamines. The main protamine (Po2), a protein of 44 amino acid residues, is extraordinarily simple (composed of only three different amino acid types: arginine (R), serine (S), and glycine (G). It is a basic molecule consisting of 79.5 mol% arginine residues. The rest of the protamines (Po3, Po4, Po5) are smaller molecules (33, 28, and 30 amino acid residues, respectively) that are homologous among themselves and probably with the main Po2 protamine. The ripe sperm nucleus of O. vulgaris also contains a small quantity of a molecule (Po1) that is similar to Po2 protamine. This protein could represent a Po2 protamine-precursor in a very advanced step of its processing. We discuss the characteristics of these proteins, as well as the relation between the complexity of chromatin condensation and the transitions of nuclear proteins during spermiogenesis in O. vulgaris.