On the Diversity of Sperm Histones in the Vertebrates

Sperm histones display great variability in contrast to the conservation of most classes of somatic histones. To study this paradox, this series of papers examines the variation of histone patterns in the testis and sperm of vertebrates, particularly amphibians and reptiles, and attempts to relate such variation to genetically based sex determination as hypothesized by Bloch [Genetics Supplement 61, 93 (1969)]. In the present study we have investigated spermiogenesis in the newt Notophthalmus viridescens viridescens . Cytochemical experiments indicate that the basic nuclear proteins undergo progressive shifts from somatic type histone → very arginine-rich "stable protamine'in the later spermatids → protamine in the mature sperm. Electrophoresis of Notophthalmus histones extracted from chromatin reveals that the pattern of testes-specific basic proteins in the urodele is distinct from the pattern of testicular proteins in the anurans Bufo americanus and Xenopus laevis . Species within the class Amphibia therefore exhibit considerable diversification in their type of basic sperm proteins.     

Common phylogenetic origin of protamine-like (PL) proteins and histone H1: Evidence from bivalve PL genes

Sperm nuclear basic proteins (SNBPs) can be grouped into three main categories: histone (H) type, protamine (P) type, and protamine-like (PL) type. Protamine-like SNBPs represent the most structurally heterogeneous group, consisting of basic proteins which are rich in both lysine and arginine amino acids. The PL proteins replace most of the histones during spermiogenesis but to a lesser extent than the proteins of the P type. In most instances, PLs coexist in the mature sperm with a full histone complement. The replacement of histones by protamines in the mature sperm is a characteristic feature presented by those taxa located at the uppermost evolutionary branches of protostome and deuterostome evolution, while the histone type of SNBPs is predominantly found in the sperm of taxa which arose early in metazoan evolution; giving rise to the hypothesis that protamines may have evolved through a PL type intermediate from a primitive histone ancestor. The structural similarities observed between PL and H1 proteins, which were first described in bivalve molluscs, provide a unique insight into the evolutionary mechanisms underlying SNBP evolution. Although the evolution of SNBPs has been exhaustively analyzed in the last 10 years, the origin of PLs in relation to the evolution of the histone H1 family still remains obscure. In this work, we present the first complete gene sequence for two of these genes (PL-III and PL-II/PL-IV) in the mussel Mytilus and analyze the protein evolution of histone H1 and SNBPs, and we provide evidence that indicates that H1 histones and PLs are the direct descendants of an ancient group of "orphon" H1 replication-dependent histones which were excluded to solitary genomic regions as early in metazoan evolution as before the differentiation of bilaterians. While the replication-independent H1 lineage evolved following a birth-and-death process, the SNBP lineage has been subject to a purifying process that shifted toward adaptive selection at the time of the differentiation of arginine-rich Ps.     

On the diversity of sperm basic proteins in the vertebrates: V. Cytochemical and amino acid analysis in Squamata, Testudines, and Crocodylia

The variability of sperm basic proteins in representatives of three reptilian orders, Squamata, Testudines, and Crocodylia, has been examined by cytochemistry, acid-urea polyacrylamide gel electrophoresis, and amino acid analysis of amidoblack-stained bands. Snakes contain type 3B intermediate sperm basic proteins by cytochemical criteria. Electrophoresis of basic proteins from epididymis chromatin as well as from testis and ductus deferens cell suspensions shows two fast-moving bands in the vicinity of herring protamine. These proteins are triprotamines containing about 27 mol % arginine, along with lysine and histidine. Lizards have type 1 protamines in their sperm nuclei cytochemically and also show a two-banded electrophoretic pattern similar to that of snakes. However, these proteins are triprotamines, similar to those in snakes with 25 mol % arginine. It may be that these are testis-specific proteins of the spermatid stage in lizards since a cytochemical transition can be observed from type 3A intermediate proteins in spermatids of testis to type 1 protamine in mature sperm of ductus deferens. Turtles contain type 3A intermediate sperm basic proteins cytochemically and basic proteins from epididymis chromatin display both a prominent band and a minor band close to, but slightly slower than, the two bands for snakes and lizards. Amino acid analysis of these bands shows that these basic proteins are also triprotamines but with a higher level of arginine, about 48 mol %, than that in snake and lizard sperm proteins. Basic proteins from epididymis chromatin of a single Mississippi alligator show three main bands moving close to the bands of snakes, lizards, and turtles. These proteins have amino acid compositions typical for triprotamines, with 28-39 mol % arginine. The data indicate that the sperm basic proteins of representatives of 25 species in three reptilian orders are very similar, in contrast to the diversity of sperm protein types found in frogs (Kasinsky, Huang, Kwauk, Mann, Sweeney, and Yee: J. Exp. Zool., 203:109-126, '78; Kasinsky, Huang, Mann, Roca, and Subirana: J. Exp. Zool., 234:33-46, '85a). This appears to be part of a macroevolutionary trend from diversity of sperm basic proteins in frogs to relative constancy in reptiles (Kasinsky, Mann, Pickerill, Gutovich, and Byrd, Jr.:J. Cell Biol., 91:1879, '81; Kasinsky, Mann, Lemke, and Huang: In: Chromosomal Proteins and Gene Expression, Plenum Press, New York, pp. 333-352, '85b). We present the hypothesis that one factor for such a trend resides in the fact that fertilization is internal in reptiles but external in anurans.     

Basic Proteins of Plant Nuclei during Normal and Pathological Cell Growth

Histone proteins were studied by microphotometry of plant tissue sections stained with fast green at pH 8.1. For comparative purposes the Feulgen reaction was used for deoxyribose nuclei acid (DNA); the Sakaguchi reaction for arginine; and the Millon reaction for estimates of total protein. Analysis of Tradescantia tissues indicated that amounts of nuclear histone fell into approximate multiples of the gametic (egg or sperm) quantity except in dividing tissues, where amounts intermediate between multiples were found. In differentiated tissues of lily, corn, onion, and broad bean, histones occurred in constant amounts per nucleus, characteristic of the species, as was found also for DNA. Unlike the condition in several animal species, the basic proteins of sperm nuclei in these higher plants were of the histone type; no evidence of protamine was found. In a plant neoplasm, crown gall of broad bean, behavior of the basic nuclear proteins closely paralleled that of DNA. Thus, alterations of DNA levels in tumor tissues were accompanied by quantitatively similar changes in histone levels to maintain the same Feulgen/fast green ratios found in homologous normal tissues.     

Nuclear Basic Proteins of Xenopus laevis Sperm: Their Characterization and Synthesis during Spermatogenesis

Nuclear basic proteins from morphologically and functionally mature sperm of Xenopus laevis were analyzed by acid/urea/Triton X-100 polyacrylamide gel electrophoresis (AUT-PAGE). Six sperm-specific proteins (SP1-6) were identified in addition to somatic histones H3, H4 and smaller amount of H2A and H2B, but not H1. Of these, SP3–6 were unique in containing 33–41% arginine and having very low lysine/arginine ratios, while SP2 was more similar to H3 and H4 in having a lower arginine and higher lysine content. Fractionations of testicular cells at different spermatogenic stages by unit gravity sedimentation showed that primary spermatocytes and acrosomal vesicle spermatids possess typical somatic type histones but no SPs. Injection of [¹⁴C]-arginine into the testis and its tracing by fluorography on AUT-PAGE gels indicated that all somatic histones are synthesized during the stages between spermatogonia and primary spermatocytes, whereas SPs are synthesized at differentially regulated rates during the stages after acrosomal vesicle formation. In indirect immunofluorescence studies with anti-SP3-5 rabbit antiserum, a positive reaction was observed in the last step of spermiogenesis after the commencement of nuclear coiling.     

Preliminary study of sperm chromatin characteristics of the brachyuran crab Maja brachydactyla. Histones and nucleosome-like structures in decapod crustacean sperm nuclei previously described without SNBPs

An interesting characteristic of decapod crustacean sperm nuclei is that they do not contain highly packaged chromatin. In the present study we re-examine the presence of DNA-interacting proteins in sperm nuclei of the brachyuran Maja brachydactyla. Although previous reports have indicated that, unlike the majority of sperm cells, DNA of decapod sperm is not organized by basic proteins, in this work we show that: (1) histones are present in sperm of M. brachydactyla; (2) histones are associated with sperm DNA; (3) histone H3 appears in lower proportions than the other core histones, while histone H2B appears in higher proportions; and (4) histone H3 in sperm nuclei is acetylated. This work complements a previous study of sperm histones of Cancer pagurus and supports the suggestion that decapod crustacean sperm chromatin deserves further attention.     

Analysis of histones in Xenopus laevis. I. A distinct index of enriched variants and modifications exists in each cell type and is remodeled during developmental transitions

Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. We immunoblotted this global collection of histones with many specific post-translational modification antibodies, including antibodies against methylated histone H3 on Lys(4), Lys(9), Lys(27), Lys(79), Arg(2), Arg(17), and Arg(26); methylated histone H4 on Lys(20); methylated H2A and H4 on Arg(3); acetylated H4 on Lys(5), Lys(8), Lys(12), and Lys(16) and H3 on Lys(9) and Lys(14); and phosphorylated H3 on Ser(10) and H2A/H4 on Ser(1). Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status.     

Histone- and chromatin-binding activity of template activating factor-I

Template activating factor-I (TAF-I) is a histone-binding chromatin remodeling factor. We recently found that TAF-I is capable of mediating decondensation of Xenopus sperm chromatin by releasing sperm-specific basic proteins. Here we present evidence that TAF-I preferentially binds to histone H3 among four core histones. Immunofluorescent staining revealed that TAF-I binds to the decondensed sperm chromatin, of which protein components predominantly consist of histones H3 and H4.     

Electrophoretic analysis of liver and testis histones of the frog Rana pipiens

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.     

Purification and characterization of nuclear basic proteins of human sperm

High purified nuclei were obtained from human sperm without protein loss through the use of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), a newly available detergent. The basic protein complement of these nuclei is highly heterogeneous and comprises histones (some of which are testis-specific), protamines and proteins of intermediate basicity and molecular size. The protamines belong to two different classes of protein. Microheterogeneity observed in some of these protamines originates from slight variations in their amino acid composition as well as from post-synthetic modifications. Two of these protamines previously considered as two different proteins as in fact the same protein with different degrees of phophorylation. All these protamines and intermediate basic proteins are characterized by high amounts of arginine and cysteine. Three of the protamines and all five intermediate basic proteins are also histidine-rich.     

Protamine-like sperm nuclear basic proteins in the primitive frog Ascaphus truei and histone reversions among more advanced frogs

Sperm nuclear basic proteins (SNBPs) that condense chromatin are very diverse. In animals, evolution of SNBPs has proceeded from lysine-rich histone H type in sponges to more arginine-rich protamine-like PL and protamine P types. Yet sporadic PL/P to H reversions are known to occur in both protostomes and deuterostomes. To determine why this is the case, we have examined SNBPs in eleven anuran species. We find that sperm of the primitive, internally fertilizing archeobatrachian frog A. truei (family Ascaphidae) has PL/P type (42 mol % arginine), with an electrophoretic profile similar to SNBPs in another archeobatrachian, externally fertilizing Leiopelma hochstetteri (family Leiopelmatidae). Cytochemistry of sperm nuclei in the advanced, externally fertilizing neobatrachian frogs Crinia signifera and C. deserticola (family Myobatrachidae) indicates that they have reverted to H type SNBPs. This is also known to be the case in externally fertilizing Rana (family Ranidae) and Silurana, a subgenus of Xenopus (family Pipidae). Such a trend, from PL/P type SNBPs in two archeobatrachians to sporadic reversions to H type in more advanced frogs, parallels the ultrastructural simplification from complex A. truei introsperm to neobatrachian aquasperm that Jamieson et al. (1993. Herpetologica 49:52-65) attribute as a secondary reversion to external fertilization.     

Histones from embryos of the sea urchin Arbacia lixula

Whole histones and histone fractions of the sea urchin, Arbacia lixula, embryos have been characterized by their appearance during development and by their amino acid composition. Comparison of electrophoretic mobility of the histone fractions from hatching blastula and gastrula stage embryos demonstrates the similarity of the basic proteins at these two stages. Histones F2a1 and F3 of hatching embryos are very similar to those of sperm, including the presence of cysteine in F2a1 from both sources. Both F2a1 and F3 display electrophoretic heterogeneity due to acetylation, not observed in the homologous sperm histones. F2a2 from embryos has different electrophoretic mobility than that from sperm, although their amino acid compositions are very similar. The relative proportion of F2a2 increases whereas that of F3 decreases during gastrulation. Slightly lysine-rich histone F2b could not be recovered from embryos by the standard methods of extraction. The very lysine-rich histone F1 of late embryos is partially phosphorylated and is remarkably different from that of sperm, notably by its higher electrophoretic mobility and lower content in arginine and proline. The significance of these results is discussed with regard to the structure and activity of chromatin.     

Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: involvement of nucleoplasmin

Immunohistochemical studies with antiserum against the protamines of the toad, Bufo japonicus, revealed that the sperm nucleus loses protamines within 5 min after entry into the egg. Likewise, lysolecithin-permeabilized sperm incubated with the egg extract lose the protamines within 1 min, accompanied by nuclear decondensation. The activities that induce both protamine removal and decondensation in sperm nuclei were found in extracts from growing and mature oocytes and pregastrula embryos, but not in postneurula embryos or adult tissues. SDS-PAGE analyses revealed that the egg extract removed not only protamines from the Bufo sperm, but also selectively the sperm-specific basic proteins from sperm nuclei of Xenopus laevis. The protamine-removing activity (PRA) was partially purified from egg extracts as negatively charged macromolecules by anion-exchange chromatography and gel filtration. The PRA was heat-stable (100 degrees C, 10 min) and sensitive to proteinase K, but not to RNase A and DNase I. Immunoblot analysis of the supernatant after incubation of Bufo sperm in the fraction with the PRA revealed that protamines derived from sperm nuclei were associated with a major protein of the fraction. This protein exhibited mobilities of 140 and 36 kDa on native- and SDS-PAGE, respectively, with the isoelectric points in the range 4.2 to 4.5 and possessed an amino acid composition quite similar to that reported for Xenopus nucleoplasmin. We propose that in fertilized eggs the protamines are removed from sperm nuclei by nucleoplasmin by binding to but not by enzymatic degradation of the protamine.     

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.     

Rates of histone synthesis during early development of Rana pipiens

Newly synthesized histones have been extracted from Rana pipiens oocytes or cleaving embryos previously injected with [³H]lysine or [³H]arginine. The radioactive proteins were fractionated by cation-exchange chromatography and electrophoresis on acid/urea or SDS-polyacrylamide gels; histones were identified by coelectrophoresis with authentic markers. From percentage total incorporation in the putative histones, and absolute rates of lysine or arginine incorporation, rates of histone synthesis were estimated. Rates of histone synthesis in two-cell embryos were at least 10-fold higher than in maturing oocytes. Between the two-cell and blastula stages, the rate increased an additional threefold, from about 1200 pg hr^?1 per embryo to about 4500 pg hr^?1 per embryo. While all histone classes are synthesized during cleavage, synthesis of the various classes is not coordinated; histones are not synthesized in the same relative proportions at which they are found in blastula chromatin. The synthesis of histone H4 in particular is barely detectable during cleavage. This, and other observations, suggested the existence of cytoplasmic histone pools. In approaching the possible existence of histone pools, the amount of H4 present in oocytes was determined. Oocytes contain about 74 ng of H4, an amount sufficient to allow development to the blastula stage. These data are compared to those reported by others on histone synthesis during cleavage in Xenopus.     

Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

Somatic histones are components of the perinuclear theca in bovine spermatozoa

The perinuclear theca is a non-ionic detergent-resistant, electron-dense layer surrounding the condensed nucleus of mammalian sperm. The known proteins originating from the perinuclear theca have implicated the structure in a variety of important cellular processes during spermiogenesis and fertilization. Nonetheless, the composition of the perinuclear theca remains largely unexplored. We have isolated a group of low molecular mass (14-19 kDa) perinuclear theca-derived proteins from acrosome-depleted bovine sperm heads by salt (1 M KCl) extraction and have identified them as core somatic histones. N-terminal sequencing and immunoblotting with anti-histone antibodies confirmed the presence of both intact and proteolytically cleaved somatic histones H3, H2B, H2A, and H4. Identical proteins were isolated using 2% SDS or 1 N HCl extractions. Subsequent acid and SDS extractions of intact bovine sperm revealed the presence of all four intact histone subtypes, with minimal proteolysis. Two-dimensional acid/urea/Triton-SDS-PAGE, coupled with immunoblotting analysis, confirmed the somatic nature of these perinuclear theca-derived histones. Estimates of the abundance of perinuclear theca-derived histones showed that up to 0.2 pg per sperm of each histone subtype was present. Immunogold labeling at the ultrastructural level localized all four core somatic histones to the post-acrosomal sheath region of bovine epididymal sperm, when probed with affinity-purified anti-histone antibodies. Little immunoreactivity was detected in residual perinuclear theca structures following the extractions. Taken together, these findings indicate the unprecedented and stable localization of non-nuclear somatic histones in bovine sperm perinuclear theca.     

Core histones of the amitochondriate protist, Giardia lamblia

Genes coding for the core histones H2a, H2b, H3, and H4 of Giardia lamblia were sequenced. A conserved organism- and gene-specific element, GRGCGCAGATTTVGG, was found upstream of the coding region in all core histone genes. The derived amino acid sequences of all four histones were similar to their homologs in other eukaryotes, although they were among the most divergent members of this protein family. Comparative protein structure modeling combined with energy evaluation of the resulting models indicated that the G. lamblia core histones individually and together can assume the same three-dimensional structures that were established by X-ray crystallography for Xenopus laevis histones and the nucleosome core particle. Since G. lamblia represents one of the earliest-diverging eukaryotes in many different molecular trees, the structure of its histones is potentially of relevance to understanding histone evolution. The G. lamblia proteins do not represent an intermediate stage between archaeal and eukaryotic histones.