Phosphorylation of sea urchin sperm H1 and H2B histones precedes chromatin decondensation and H1 exchange during pronuclear formation

Immediately following fertilization in the sea urchin, sperm-specific histones Sp H1 and Sp H2B are phosphorylated. Then, in parallel with chromatin decondensation, nearly all phosphorylated Sp H1 is lost from the pronuclear chromatin, with the concurrent assimilation of the egg phosphoprotein CS H1. Chemical cleavage of in vivo labeled Sp H1 and Sp H2B shows that serine phosphorylation occurs in the unusually long N-terminal region of these proteins. These regions contain tandemly repeated tetra- and pentapeptide units each containing serine, proline, and two basic amino acids. It is proposed that sperm chromatin decondensation may require prior phosphorylation of these unusual N-terminal regions, whose function in the mature sperm may be to condense or stabilize its highly compact chromatin.     

Sperm histones and chromatin structure of the "primitive" sea urchin Eucidaris tribuloides

The "primitive" sea urchin Eucidaris tribuloides resembles the advanced sea urchins (euechinoids) in many respects, yet some features of its biochemistry and morphogenesis are more similar to other echinoderms such as starfish or sea cucumbers. Two unique characteristics of the sperm chromatin of all known euechinoids are an extremely long average nucleosomal repeat length and the presence of two male germ-line-specific histone variants, Sp H1 and Sp H2B. Histone composition and nucleosomal repeat length of the sperm chromatin of Eucidaris were compared to those of several euechinoids and a starfish. Eucidaris sperm chromatin contained large H1 and H2B histone variants typical of euechinoids. The H1 was about nine amino acids smaller than Sp H1 of the advanced urchin Strongylocentrotus purpuratus. Its Sp H2B molecules were the same size as in the euechinoids. Peptide maps showed that N-terminal regions of Sp H1 and Sp H2B contained repeating basic amino acid motifs characteristic of euechinoids. The smaller size of Eucidaris H1 is accounted for by a smaller C-terminal region. The repeat length of Eucidaris sperm chromatin was slightly shorter than that of two euechinoids, but significantly larger than starfish, which lacks a large H2B. The Sp H2B gene of Eucidaris was expressed during spermatogenesis in the same cell types as for S. purpuratus. Thus Sp histone subtype expression and chromatin structure in this distantly related echinoid closely resemble the euechinoids. The presence of an Sp H2B and a very long repeat length appear to be characteristic of the echinoids only.     

Linker DNA destabilizes condensed chromatin.

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.     

Packaging and unpackaging the sea urchin sperm genome.

Two species of histones in sea urchin sperm (Sp H1 and Sp H2B) are chimeric molecules whose highly basic amino-terminal domains are dephosphorylated at the last stage of sperm cell differentiation, and rephosphorylated immediately following fertilization. The phosphorylated regions consist largely of repeating tetrapeptides with two basic residues flanking Ser-Pro residues ('SPKK' motifs) and are predicted to have beta-turn secondary structures. Alteration of the charge and structure of the SPKK sites may play a role in the unusually dense DNA packaging of the mature sperm chromatin. The motif resembles the target site of cell-cycle-associated cdc2 kinases and is found in several other proteins whose nucleic acid affinities may be altered during the cell cycle.     

Histone phosphorylation during sea urchin development

Studies on histone phosphorylation during transitions in chromatin structure occurringin vivoduring spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extractsin vitroand evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structurein vivoand provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.     

Isolation of four core histones from human sperm chromatin representing a minor subset of somatic histones

Using high performance liquid chromatography we have successfully purified four core histones from mature human sperm chromatin. The H2A variants present in sperm (H2A.X and limited H2A.Z) have been shown previously to be minor variants in somatic chromatin. The histones are highly modified as evidenced by extensive acetylation and an as yet uncharacterized multicharge modification of H2B. Based on our data, we conclude that histone proteins are a minor component of each mature spermatozoa. Given the unique nature of the histone variants present in sperm, we propose that this chromatin component has a specific function and may possibly facilitate the programming of genes which will be active in early development.     

Histone-DNA interactions and their modulation by phosphorylation of -Ser-Pro-X-Lys/Arg- motifs.

The sea urchin sperm-specific histones H1 and H2B are multiply phosphorylated in spermatids, dephosphorylated in the final stages of spermatogenesis to give mature sperm, and rephosphorylated upon fertilization. Phosphorylation in spermatids, and probably at fertilization, occurs at repeated -Ser-Pro-X-Basic-motifs in the distinctive N-terminal basic domains of both histones and at the end of the much longer C-terminal domain of H1. Here we identify the consequences of multiple phosphorylation through comparison of some physical and biochemical properties of spermatid (phosphorylated) and sperm (dephosphorylated) chromatin and histones. Study of the DNA binding properties of the intact histones and isolated basic domains suggests that phosphorylation at three dispersed sites in the C-terminal tail of H1 has little effect on its overall DNA binding affinity, whereas, strikingly, binding of the N-terminal domains of H2B and H1 is abolished by phosphorylation at four or six tandemly repeated sites respectively. Together with the relative timing of events in vivo, this suggests that phosphorylation/dephosphorylation of the N-terminal (and distal end of the C-terminal) tail of H1, and/or the N-terminal tail of H2B, effectively controls intermolecular interactions between adjacent chromatin filaments, and hence chromatin packing in the sperm nucleus.     

Histones and nucleosomes in Cancer sperm (Decapod: Crustacea) previously described as lacking basic DNA-associated proteins: a new model of sperm chromatin

To date several studies have been carried out which indicate that DNA of crustacean sperm is neither bound nor organized by basic proteins and, contrary to the rest of spermatozoa, do not contain highly packaged chromatin. Since this is the only known case of this type among metazoan cells, we have re-examined the composition, and partially the structure, of the mature sperm chromatin of Cancer pagurus, which has previously been described as lacking basic DNA-associated proteins. The results we present here show that: (a) sperm DNA of C. pagurus is bound by histones forming nucleosomes of 170 base pairs, (b) the ratio [histones/DNA] in sperm of two Cancer species is 0.5 and 0.6 (w/w). This ratio is quite lower than the proportion [proteins/DNA] that we found in other sperm nuclei with histones or protamines, whose value is from 1.0 to 1.2 (w/w), (c) histone H4 is highly acetylated in mature sperm chromatin of C. pagurus. Other histones (H3 and H2B) are also acetylated, though the level is much lower than that of histone H4. The low ratio of histones to DNA, along with the high level of acetylation of these proteins, explains the non-compact, decondensed state of the peculiar chromatin in the sperm studied here. In the final section we offer an explanation for the necessity of such decondensed chromatin during gamete fertilization of this species.     

Poly(ADP-ribose) polymerases PARP1 and PARP2 modulate topoisomerase II beta (TOP2B) function during chromatin condensation in mouse spermiogenesis

To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling.     

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.     

The developmental fate of angiosperm pollen is associated with a preferential decrease in the level of histone H1 in the vegetative nucleus

In angiosperm pollen, the vegetative cell is assumed to function as a gametophytic cell in pollen germination and growth of the pollen tube. The chromatin in the nucleus of the vegetative cell gradually disperses after microspore mitosis, whereas the chromatin in the nucleus of the other generative cell remains highly condensed during the formation of two sperm nuclei. In order to explain the difference in chromatin condensation between the vegetative and generative nuclei, we analyzed the histone composition of each nucleus in Lilium longiflorum Thunb. and Tulipa gesneriana immunocytochemically, using specific antisera raised against histones H1 and H2B of Lilium. We found that the level of histone H1 decreased gradually only in the vegetative nucleus during the development of pollen within anthers and that the vegetative nucleus in mature pollen after anther dehiscence contained little histone H1. By contrast, the vegetative nucleus contained the same amount or more of histone H2B than the generative nucleus. The preferential decrease in the level of histone H1 occurred in anomalous pollen with one nucleus (uninucleate pollen) or with two similar nuclei (equally divided pollen), which had been induced by treatment with colchicine. The nuclei in the anomalous pollen resembled vegetative nuclei in terms of structure and staining properties. The anomalous pollen was able to germinate and extend a pollen tube. From these results, it is suggested that the preferential decrease in level of histone H1 in pollen nuclei is essential for development of the male gametophytic cell through large-scale expression of genes that include pollen-specific genes, which results in pollen germination and growth of the pollen tube. Received: 9 May 1998 / Accepted: 4 June 1998     

Two kinase activities are sufficient for sea urchin sperm chromatin decondensation in vitro

Decondensation of compact and inactive sperm chromatin by egg cytoplasm at fertilization is necessary to convert the male germ cell chromatin to an active somatic form. We studied decondensation of sea urchin sperm nuclei in a cell-free extract of sea urchin eggs to define conditions promoting decondensation. We find that egg cytosol specifically phosphorylates two sperm-specific (Sp) histones in vitro in the same regions as in vivo. This activity is blocked by olomoucine, an inhibitor of cdc2-like kinases, but not by chelerythrine, an inhibitor of protein kinase C (PKC). PKC phosphorylates and solubilizes the sperm nuclear lamina, one requirement for decondensation. Olomoucine, which does not inhibit lamina removal, blocks sperm nuclear decondensation in the same concentration range over which it is effective in blocking Sp histone phosphorylation. In a system free of other soluble proteins, neither PKC nor cdc2 alone elicit sperm chromatin decondensation, but the two act synergistically to decondense sperm nuclei. We conclude that two kinases activities are sufficient for sea urchin male pronuclear decondensation in vitro, a lamin kinase (PKC) and a cdc2-like Sp histone kinase.     

The ubiquitinated histone species are enriched in histone H1-depleted chromatin regions

Bovine thymus and trout testis chromatin were fractionated into regions which differed in their micrococcal nuclease accessibility and solubility properties, and the distribution of the ubiquitinated histone species among these chromatin regions was elucidated. Ubiquitinated (u) species of histones H2A and H2B were enriched in the nuclease-sensitive, low-ionic-strength, soluble fraction of both chromatins. These results indicate that the presence of ubiquitinated histones may alter nucleosome-nucleosome interactions and destabilize higher-order chromatin structures. Bovine thymus chromatin was separated into aggregation-resistant, salt-soluble and aggregation-prone, salt-insoluble chromatin fractions. The aggregation-resistant chromatin fraction depleted in H1 histones was enriched in uH2A and uH2B, with uH2B showing the greater enrichment. The chromatin fragments were also stripped and reconstituted with the H1 histones prior to fractionation. The results were the same as above: uH2A and uH2B were preferentially localized in the aggregation-resistant. H1-depleted chromatin fraction, suggesting that chromatin regions enriched in ubiquitinated histone species have a reduced affinity for the H1 histones. Thus, ubiquitinated histone species may be one of the contributing factors in the differential assembly of various parts of the genome.     

Proteolysis of somatic type histones in transforming rat spermatid chromatin

Elongated rat spermatid nuclei have been isolated on the basis of their resistance to sonication in 0.32 M sucrose containing 1.5 mM CaCl₂. Chemical analyses indicate that approx. 35% of the DNA in these nuclei is associated with somatic type histones, while the remainder represents sperm histone-DNA complex. In contrast to nuclei of somatic cells, when elongated spermatid nuclei are incubated under appropriate conditions, somatic type histones but not sperm histone are rapidly degraded. Differential extraction of elongated spermatid nuclei with 5 mM HCl and then with various concentrations of NaCl followed by 0.2 M HCl has revealed that they contain two kinds of proteases. The protease in the 5 mM HCl extract is acrosin (EC 4.3.21.10). Rapid degradation of somatic type histones is, however, observable upon incubation of elongated spermatid nuclei which have been treated with 5 mM HCl and are therefore free of acrosin or upon incubation of elongated spermatid chromatin where the majority of acrosin is removed, suggesting that the observed proteolysis of somatic type histone is not due to acrosin. Proteases which may represent the enzymes responsible for the histone degradation are extractable from acrosin-free spermatid nuclei with NaCl (0.9 M) and by subsequent treatment of the salt-extracted nuclei with 0.2 M HCl. The proteases in the NaCl and the 0.2 M HCl extract possess identical properties and appear to be the same enyzyme which may exist in spermatid chromatin in two different forms.     

Male mice lacking three germ cell expressed genes are fertile

In recent years, much knowledge about the functions of defined genes in spermatogenesis has been gained by making use of mouse transgenic and gene knockout models. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp-2), proacrosin (Acr), histone H1.1 (H1.1), and histone H1t (H1t), have been generated and analyzed. Tnp-2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues and germ cells during the S-phase of the cell cycle. The histone gene H1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization, two lines of triple null mice (Tnp-2-/-/Acr-/-/H1.1-/- and Tnp-2-/-/Acr-/-/H1t-/-) were established. Both lines are fertile and show normal sperm parameters, which clearly demonstrate the functional redundancy of these proteins in male mouse fertility. However, sperm only deficient for Acr (Acr-/-) are able to compete significantly with sperm from triple knockout mice Tnp-2-/-/Acr-/-/H1.1-/- (70.7% vs. 29.3%) but not with sperm from triple knockout mice Tnp-2-/-/Acr-/-/H1t-/- (53.6% vs. 46.4%). These results are consistent with a model that suggests that some sperm proteins play a role during sperm competition.     

Poly(ADP-ribose) metabolism is essential for proper nucleoprotein exchange during mouse spermiogenesis

Sperm chromatin is organized in a protamine-based, highly condensed form, which protects the paternal chromosome complement in transit, facilitates fertilization, and supports correct gene expression in the early embryo. Very few histones remain selectively associated with genes and defined regulatory sequences essential to embryonic development, while most of the genome becomes bound to protamine during spermiogenesis. Chromatin remodeling processes resulting in the dramatically different nuclear structure of sperm are poorly understood. This study shows that perturbation of poly(ADP-ribose) (PAR) metabolism, which is mediated by PAR polymerases and PAR glycohydrolase in response to naturally occurring endogenous DNA strand breaks during spermatogenesis, results in the abnormal retention of core histones and histone linker HIST1H1T (H1t) and H1-like linker protein HILS1 in mature sperm. Moreover, genetic or pharmacological alteration of PAR metabolism caused poor sperm chromatin quality and an abnormal nuclear structure in mice, thus reducing male fertility.     

Phosphorylation protects sperm-specific histones H1 and H2B from proteolysis after fertilization

At intermediate stages of male pronucleus formation, sperm-derived chromatin is composed of hybrid nucleoprotein particles formed by sperm H1 (SpH1), dimers of sperm H2A-H2B (SpH2A-SpH2B), and a subset of maternal cleavage stage (CS) histone variants. At this stage in vivo, the CS histone variants are poly(ADP-ribosylated), while SpH2B and SpH1 are phosphorylated. We have postulated previously that the final steps of sperm chromatin remodeling involve a cysteine-protease (SpH-protease) that degrades sperm histones in a specific manner, leaving the maternal CS histone variants unaffected. More recently we have reported that the protection of CS histones from degradation is determined by the poly(ADP-ribose) moiety of these proteins. Because of the selectivity displayed by the SpH-protease, the coexistence of a subset of SpH together with CS histone variants at intermediate stages of male pronucleus remodeling remains intriguing. Consequently, we have investigated the phosphorylation state of SpH1 and SpH2B in relation to the possible protection of these proteins from proteolytic degradation. Histones H1 and H2B were purified from sperm, phosphorylated in vitro using the recombinant alpha-subunit of casein kinase 2, and then used as substrates in the standard assay of the SpH-protease. The phosphorylated forms of SpH1 and SpH2B were found to remain unaltered, while the nonphosphorylated forms were degraded. On the basis of this result, we postulate a novel role for the phosphorylation of SpH1 and SpH2B that occurs in vivo after fertilization, namely to protect these histones against degradation at intermediate stages of male chromatin remodeling.     

Transition of basic protein during spermatogenesis of <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck, 1765)

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed.     

H2A.Bbd: an X-chromosome-encoded histone involved in mammalian spermiogenesis

Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals.