Acetylation of histone H4 in complex structural transitions of spermiogenic chromatin

In spermiogenic nuclei of the cephalopod mollusc Sepia officinalis histones are replaced by a precursor-protamine molecule, which is later converted into protamine. Simultaneously, spermiogenic chromatin undergoes a complex structural change. Somatic-like chromatin belonging to the earliest spermatid is progressively reorganized into: (a) granules of 20 nm diameter, (b) fibres of 30-35 nm, and (c) fibres of 40-50 nm. In the final phases of spermiogenesis these fibres of 40-50 nm join to form larger structures of condensed chromatin, and lastly, the uniformly packed chromatin in the sperm nucleus. Using specific antibodies for mono- and hyperacetylated forms of histone H4, in this work we show that the first structural remodelling of chromatin (from somatic-like organization into 20 nm granules) is given concomitantly with a massive mono-acetylation of H4 (acetylation in lysine 12), whereas the structural remodelling from 30-35 to 40-50 nm fibres is produced simultaneously with hyperacetylation of H4 and the nuclear removal of histones.     

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     

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.     

Chromatin condensation,cysteine-rich protamine,and establishment of disulphide interprotamine bonds during spermiogenesis of Eledone cirrhosa (Cephalopoda)

During spermiogenesis in Eledone cirrhosa a single protamine substitutes for histones in nuclei of developing spermatids. This protein displays a peculiar primary structure. It contains 22.6 mol% cysteine residues (19 cysteines in 84 residues). This makes it the most cysteine-rich protamine known. The proportion of basic residues is relatively low (arginine 36.9 mol%, lysine 19.0 mol%). The protamine of E. cirrhosa condenses spermiogenic chromatin in a pattern which comprises fibres with a progressively larger diameter and lamellae that finally undergo definitive coalescence. We have also performed a study that estimates the number of interprotamine disulphide bonds formed during the process of spermiogenic chromatin condensation by means of sequential disappearance of MMNA (monomaleimido-nanogold) labelling. During the first step of spermiogenesis, protamines are found spread over very slightly condensed chromatin with their cysteines in a reactive state (protamine-cys-SH). From this stage the interprotamine disulphide bonds are established in a progressive way. First they are formed inside the chromatin fibres. Subsequently, they participate in the mechanism of fibre coalescence and finally, in the last step of spermiogenesis, the remaining free reactive -SH groups of cysteine form disulphide bonds, thus promoting a definitive stabilization of the nucleoprotein complex in the ripe sperm nucleus.     

Chromatin organization during spermiogenesis in Octopus vulgaris. I: Morphological structures

In the process of the chromatin remodeling that occurs during spermiogenesis in some animal species, it is possible to distinguish between two separate aspects: the chromatin condensation pattern itself (granular, fibrillar, or lamellar), and the architecture of this pattern, that is to say, its arrangement within the nucleus. In the cephalopod Octopus vulgaris these two aspects are clearly differentiated. The condensation pattern develops from 25 nm fibers to fibers with a tubular aspect and with a progressively increasing diameter (40-60 nm and then to 80 nm), to end finally in the form of very thin fibers (3-5 nm) product of the coalescence and dissolution of the major fibers. The main directive force that governs this process lies in the global change that occurs in the proteins that interact with all (or the major part) of the genomic DNA. The condensation pattern by itself in this species does not present a fixed order: most of the fibers appear without any predominant spatial direction in the spermiogenic nuclei. However, as the nuclei elongate, the chromatin fibers arrange in parallel following the elongation axis. This parallel disposition of the chromatin fibers appears to be mediated by two specific areas, each of which we call a "polar nuclear matrix" (PNM). These matrices differentiate in the basal and apical nuclear poles adjacent to the centriolar implantation fosse and the acrosome, respectively. The areas that constitute the PNM have the following characteristics: (a) they are the only areas where DNA is found anchored to the nuclear membrane; (b) they are the zones from which the chromatin condensation pattern (fibers/tubules) begins; and (c) they are most probably the points through which the mechanical forces originating from nuclear elongation are transmitted to chromatin, causing the chromatin fibers/tubules to adopt an almost perfectly parallel disposition. Finally, we discuss the importance of the architecture of the chromatin condensation pattern, as it is one of the determining factors of the spatial organization of the mature sperm genome and chromosome positioning.     

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).     

Yeast high mobility group protein HMO1 stabilizes chromatin and is evicted during repair of DNA double strand breaks

DNA is packaged into condensed chromatin fibers by association with histones and architectural proteins such as high mobility group (HMGB) proteins. However, this DNA packaging reduces accessibility of enzymes that act on DNA, such as proteins that process DNA after double strand breaks (DSBs). Chromatin remodeling overcomes this barrier. We show here that the Saccharomyces cerevisiae HMGB protein HMO1 stabilizes chromatin as evidenced by faster chromatin remodeling in its absence. HMO1 was evicted along with core histones during repair of DSBs, and chromatin remodeling events such as histone H2A phosphorylation and H3 eviction were faster in absence of HMO1. The facilitated chromatin remodeling in turn correlated with more efficient DNA resection and recruitment of repair proteins; for example, inward translocation of the DNA-end-binding protein Ku was faster in absence of HMO1. This chromatin stabilization requires the lysine-rich C-terminal extension of HMO1 as truncation of the HMO1 C-terminal tail phenocopies hmo1 deletion. Since this is reminiscent of the need for the basic C-terminal domain of mammalian histone H1 in chromatin compaction, we speculate that HMO1 promotes chromatin stability by DNA bending and compaction imposed by its lysine-rich domain and that it must be evicted along with core histones for efficient DSB repair.     

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.     

Order and disorder in 30 nm chromatin fibers.

The tendency of DNA to form fibers upon condensation with counterions is reviewed. It is shown that chromatin fibers may acquire a relatively constant diameter of about 30 nm simply as an optimal size achieved upon neutralization of DNA, without requiring a repetitive internal structure. Thus the size of chromatin fibers would not be determined by any specific spatial interaction between DNA and histones. The driving force for the formation of fibers in chromatin would be similar to that found in proteins when they acquire a compact globular shape.     

Real-time observation of nucleoplasmin-mediated DNA decondensation and condensation reveals its specific functions as a chaperone

Fertilization requires decondensation of promatine-condensed sperm chromatin, a dynamic process serving as an attractive system for the study of chromatin reprogramming. Nucleoplasmin is a key factor in regulating nucleosome assembly as a chaperone during fertilization process. However, knowledge on nucleoplasmin in chromatin formation remains elusive. Herein, magnetic tweezers (MT) and a chromatin assembly system were used to study the nucleoplasmin-mediated DNA decondensation/condensation at the single-molecular level in vitro. We found that protamine induces DNA condensation in a stepwise manner. Once DNA was condensed, nucleoplasmin, polyglutamic acid, and RNA could remove protamine from the DNA at different rates. The affinity binding of the different polyanions with protamine suggests chaperone-mediated chromatin decondensation activity occurs through protein–protein interactions. After decondensation, both RNA and polyglutamic acid prevented the transfer of histones onto the naked DNA. In contrast, nucleoplasmin is able to assist the histone transfer process, even though it carries the same negative charge as RNA and polyglutamic acid. These observations imply that the chaperone effects of nucleoplasmin during the decondensation/condensation process may be driven by specific spatial configuration of its acidic pentamer structure, rather than by electrostatic interaction. Our findings offer a novel molecular understanding of nucleoplasmin in sperm chromatin decondensation and subsequent developmental chromatin reprogramming at individual molecular level.     

Chromatin organization and basic nuclear proteins in the male germ cells of Rana tigerina

The process of chromatin condensation during spermiogenesis in Rana tigerina is similar to the heterochromatization in somatic cells, where 30 nm fibers are coalesced together into a dense mass in spermatozoa without changing their initial size and nucleosomal organization. This conclusion was supported by the finding that the full set of core histones (H2A, H2B, H3, H4) are still present in sperm chromatin, but histone H1 is replaced by its variant, H1V. Rabbit anti-sera were raised against histone H3, H1, H1V, and H5 (H1 variant in chick erythrocyte). Anti-histone H1 antiserum cross-reacted with histone H1V, which implied the presence of a common epitope. Anti-histone H1V and H5 also showed cross-reaction with each other but not with histone H1, which implied the presence of a common epitope not shared by histone H1. Immunocytochemical studies, using the above antibodies as probes, showed that histones H3 is present in all steps of spermatogenic and spermiogenic cells, and somatic cells including red blood cells, Sertoli cells, and Leydig cells, while histone H1 is present in all of the cells mentioned except in spermatozoa where it is replaced by histone H1V. Histone H1V appears in the early spermatids starting from spermatid 1 (St1), and it persists throughout the course of spermatid differentiation into spermatozoa. Histone H1V is also found in chromosomes of metaphase spermatocyte and red blood cells. Thus histone H1V may cause the final and complete condensation of chromatin in Rana spermatozoa, a process which is similar to the heterochromatization occurring in somatic cells such as metaphase chromosome and chick erythrocyte nucleus.     

Electron microscopic study of chromatin in maturing lymphocyte nuclei using ammoniacal silver]

By means of the ammoniacal silver reaction, cytochemical properties of the chromatin in white rat lymph node lymphocytes were investigated at different stages of their maturation. Electron dense granules of the reaction product are shown to be localized over the compact chromatin region. The number of granules increases as the amount of compact chromatin enlarges. A possible role of arginine histones in the process of chromatin condensation is suggested, this suggestion being based on the assumption that ammoniacal silver binds with active arginine groups of histones.     

Chromatin reorganization during spermiogenesis of the mollusc Thais hemostoma (Muricidae): implications for sperm nuclear morphogenesis in cenogastropods

Thais is a cenogastropod mollusc belonging to the Muricidae family. The sperm nuclear morphogenesis of Thais develops in two well-defined and peculiar steps. In the first one, the round early spermatidyl nucleus is penetrated by an endonuclear channel, which arranges as a helix at the inner nuclear surface and organizes the condensing chromatin all around. In the second step, the spiral channel stretches, dragging along the associated chromatin and leading to a definitive cylinder-shaped sperm nucleus. Simultaneously with these changes in nuclear shape, the chromatin is sequentially organized in granules, fibres, lamellae, and, finally, in a very condensed structure, whereas the spermiogenic DNA-associated proteins become more basic and simple. The sperm nucleus contains a small group of protamines consisting of only four types of amino acid (lysine, arginine, glycine, and serine). The most remarkable fact on nuclear spermiogenesis in Thais is that, whereas the chromatin condensation process, the nuclear proteins, and the final shape of sperm nucleus are very similar to those in other muricidae studied, the pathway of nuclear morphogenesis is completely different. We propose an independent genetic control for those two spermiogenic events (chromatin condensation and nucleomorphogenesis). Finally we discuss briefly the main traits of nucleomorphogenesis of muricid molluscs.     

Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.     

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.     

Protamine 3 shows evidence of weak, positive selection in mouse species (genus Mus)--but it is not a protamine

Protamines are short and highly basic sperm-specific nuclear proteins that replace somatic histones during spermiogenesis in a process that is crucial for sperm formation and function. Many mammals have two protamine genes (PRM1 and PRM2) located in a gene cluster, which appears to evolve fast. Another gene in this cluster (designated protamine 3 [PRM3]) encodes a protein that is conserved among mammals but that does not seem to be involved in chromatin condensation. We have compared protein sequences and amino acid compositions of protamines in this gene cluster, searched for evidence of positive selection of PRM3, and examined whether sexual selection (sperm competition) may drive the evolution of the PRM3 gene. Nucleotide and amino acid analyses of mouse sequences revealed that PRM3 was very different from PRM1 and from both the precursor and the mature sequences of PRM2. Among 10 mouse species, PRM3 showed weak evidence of positive selection in two species, but there was no clear association with levels of sperm competition. In analyses from among mammalian species, no evidence of positive selection was found in PRM3. We conclude that PRM3 exhibits several clear differences from other protamines and, furthermore, that it cannot be regarded as a true protamine.     

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.