Human testis-specific histone TH2B: Fractionation and peptide mapping

The presence of the nonionic detergent Triton X-100 was shown to improve the resolution of the human TH2B on gel electrophoresis and on gel filtration. Total histones of human testis, including TH2B, were resolved by electrophoresis in 15% polyacrylamide gels containing 0.4% Triton X-100, 1.5 m urea, and 0.9 n acetic acid. Gel filtration on Bio-Gel P-200 in 0.4% Triton X-100, 5.0 m urea, and 0.01 n HCl permitted the purification of human TH2B from human testis and sperm in preparative amounts. The structure of human TH2B so prepared was compared to that of rat TH2B, human H2B, and rat H2B by tryptic peptide mapping. The results showed some similarities between all four proteins, but closer similarity was observed within the germ cell histone (TH2B) group and within the somatic histone (H2B) group than between histones of the same species. In addition, human TH2B and rat TH2B each contained one unique peptide absent from other histones.     

Acetylation of rat testis histones H2B and TH2B

The in vivo acetylation of rat testis histones H3 and H4 has been demonstrated in previous studies. In this study, analysis of purified histone fractions revealed the in vivo acetylation of histone H2B, the testis histone variant designated TH2B, and two or more of the histone H2A variants. These findings are quite significant, because it is possible that all of the core histones are acetylated in elongating spermatids at the time of removal of the entire histone complement for replacement by basic spermatidal transition proteins (S.R. Grimes and N. Henderson, 1983, Arch. Biochem. Biophys. 221, 108-116).     

Isolation of rat testis histone TH2B-x, and interaction of TH2B-x antiserum with histones and mononucleosomes

A method is reported for the isolation of histone TH2B-x from rat testis by affinity chromatography on an agarose-p-chloromercurianilino column. This purified TH2B-x was used to raise antibodies in the rabbit, and the antiserum was assayed by an enzyme-linked double-antibody procedure. At low concentration the antiserum cross-reacts with histone H2B and with histones TH1-x + H1 to the extent of 11-14% of the interaction with TH2B-x. Antiserum preincubated in three successive H2B-coated tubes still retains 80-89% of the original anti-TH2B-x activity when assayed subsequently in TH2B-x-coated tubes, but cross-reaction with H2B is practically zero. The anti-TH2B-x antibodies also interact with tubes coated with mononucleosomes isolated from nuclei of seminiferous epithelial cells (SEC) of rat testis, but the interaction with mononucleosomes from rat liver nuclei is almost zero. The data suggest that in nucleosomes some of the antigenic determinants which are unique to TH2B-x are accessible, while those determinants which are common to H2B and TH2B-x are not accessible for interaction with antibodies. Competition by mononucleosomes, both from rat testis SEC and rat liver (to a lesser degree), in solution is detected by the reduction of binding of enzyme-labeled IgG to TH2B-x-coated tubes. However, an attempted competition by histones TH2B-x or H2B in solution resulted in an increase in the binding of the enzyme-labeled IgG to the mononucleosome-coated tubes. The interpretation of this type of competition assay is complicated by possible interaction of added histones with the coating mononucleosomes, followed by binding of antibodies to the histones. This TH2B-x antibody should be useful in studying changes in structure and function of chromatin during spermatogenesis and in the isolation of TH2B-x mRNA.     

The nuclear basic proteins of human testes and ejaculated spermatozoa

Human testicular nuclei were fractionated into two fractions according to their sedimentation in a sucrose density gradient. The nuclear basic proteins isolated from these two fractions were similar and also resembled electrophoretic mobilities and amino acid composition of the liver histones. Only quantitative differences among histone electrophoretic bands were observed. The nuclear basic proteins of ejaculated spermatozoa differed totally from those of the testes. The proteins could be divided into two categories on the basis of their electrophoretic mobilities, molecular weights and amino acid compositions. One group (SpH) was similar to testicular histones; another (HP) group was smaller, with nearly twice the electrophoretic mobility and a much higher arginine content. These proteins (HP) represent a new type of nuclear basic protein found in human tissues.     

Acetylation of histones during spermatogenesis in the rat

Acetate was actively incorporated into rat testis histones when testis cells were prepared by the trypsinization technique in the presence of [³H]acetate. The acetylation was enhanced by 10 mm sodium butyrate. Although histones H3 and H4 were the only histones which incorporated high levels of acetate, the testis-specific histones TH2B and TH3 also appeared to incorporate acetate. This was shown by electrophoresis of the histones on polyacrylamide gels containing Triton X-100. Results, obtained from analysis of histones by two-dimensional gel electrophoresis, confirmed a recent report (P. K. Trostle-Weige, M. L. Meistrich, W. A. Brock, K. Nishioka, and J. W. Bremer, (1982) J. Biol. Chem.257, 5560–5567) that TH2A was a testis-specific histone. The results also confirmed the H2A nature of a testis-enriched histone band, previously designated X2. When histones from populations of cells enriched in specific testis cell types, representing various stages of spermatogenesis, were examined, the patterns of acetylation varied dramatically. Very high levels of acetate were incorporated into multiacetylated species of histone H4 from a population of cells enriched in transition stage spermatids (steps 9–12) compared to the levels of acetate incorporated into H4 from round spermatids (steps 1–8) and earlier stages of spermatogenesis, where acetate was incorporated primarily into the monoacetylated species of H4. Thus, a striking correlation exists between the time of hyperacetylation of histone H4 and the time of removal of histones for their replacement by the basic spermatidal transition proteins designated TP, TP2, and TP4. Hyperacetylation of histone H4 may facilitate the removal of the entire histone complement during the protein transition. In any case, it must be an obligatory step in the dramatic process.     

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.     

Histones are the major chromosomal protein components of the sperm of the nemerteans Cerebratulus californiensis and Cerebratulus lacteus

We have characterized for the first time the sperm nuclear basic proteins (SNBP) from two species of nemerteans: Cerebratulus californiensis and Cerebratulus lacteus. Gel electrophoretic and chromatographic (RP-HPLC) analysis of the nuclear sperm extracts indicate that histones are the major protein components which are present. The linker histones (histones of the H1 family) exhibit a rather unusual composition and some of them contain cysteine. Several histone H1 isoforms are present, one of which has a composition similar to that of other H1 histones found in the sperm chromatin of other groups of lower invertebrates.     

Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis

We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.     

Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.     

Localization of testis-variant histones in rat testis chromatin.

Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.     

Increased accessibility of the N-terminus of testis-specific histone TH2B to antibodies in elongating spermatids

Changes in chromatin structure during spermatogenesis were investigated using a monoclonal antibody that immunoreacts with the N-terminus of the testis-specific histone TH2B. This monoclonal antibody, which had been raised against rat tyrosine hydroxylase (TH), cross-reacted with TH2B because of sequence homology at the N-termini of TH and TH2B. The epitope was localized to the N-terminus of TH2B as trypsin-digested chromatin which lacked the N-terminal tail did not react with anti-TH and preincubating anti-TH with a synthetic peptide made from the homologous sequence between TH2B and TH inhibited its binding to TH and TH2B. In histological sections of rat testis, the primary spermatocytes and round spermatids immunoreacted weakly, whereas elongating spermatids at steps 10–12 immunoreacted intensely with anti-TH. Increased staining of elongating spermatids was also observed in mouse and hamster by immunohistochemistry. However, immunoblotting proteins extracted from separated rat testis cells showed no increase in the TH2B content of these late steps of spermatids. The apparent increase in the immunohistochemical staining corresponds to increased accessibility of the epitope in the elongating spermatids. This indicated that the N-terminus of TH2B is less tightly bound to DNA or to other proteins at this time in preparation for the removal of TH2B and other histones. © 1995 wiley-Liss, Inc.     

Structural organization of the meiotic prophase chromatin in the rat testis

Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4. The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin. However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions. Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels. While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones. A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles. Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA.     

A new procedure for the purification of rat testis-specific histone TH2B involving affinity-related chromatography

A new procedure for the isolation of rat testis-specific histone TH2B has been devised. First, rat testis chromatin fragments were applied to a hydroxylapatite column in 0.5 m NaCl, 0.1 m potassium phosphate buffer, pH 6.7, and histones were selectively stripped off the bound DNA in groups (H1/TH1, H2A/H2B/TH2B, and H3/H4). The fraction containing H2A, H2B, and TH2B, but lacking H3, was reduced, desalted, and applied to a p-chloromercuribenzoyl-aminoethyl-Sepharose 4B-CL column in 8 m urea, 0.1 m Tris-HCl, pH 8.1. After washing with the same buffer to remove H2A and H2B, covalently bound TH2B was eluted out with 10 mm dithiothreitol in the same buffer. No contaminants were detectable in the purified TH2B either by polyacrylamide gel electrophoresis in 0.4% Triton X-100, 2.5 m urea, 0.9 n acetic acid, or by N-terminal analysis with dansyl chloride.     

On the diversity of sperm histones in the vertebrates: IV. Cytochemical and amino acid analysis in Anura

The variability of sperm histones in frogs has been studied by cytochemical and amino acid analyses. Cytochemically, Rana sperm proteins fall into Bloch's ('69, '76) type 4 somatic-like histone category, while Xenopus and Bufo have type 3 intermediate sperm histones. Extractability in 5% trichloroacetic acid (TCA) at different temperatures splits this type 3 category into two groups: type 3B intermediate sperm histones of Bufo are extractable at 85-90 degrees C, while Xenopus intermediate type 3A sperm histones require temperatures of 95-100 degrees C for extraction. Amino acid analysis confirms that Rana sperm histones are of the nucleosomal type, with a testis-specific, very lysine-rich H1 histone. The sperm protein in Bufo is richer in arginine than the proteins in Xenopus. Both of these genera contain lysine and histidine as well as arginine in their sperm proteins. These results confirm earlier electrophoretic data (Kasinsky et al., '78) and indicate that sperm histones in the order Anura can vary markedly between different genera.     

Histone variants in rat spermatogonia and primary spermatocytes

The levels and synthesis of histone variants have been directly measured in spermatogonia and in various stages of primary spermatocytes purified from the rat testis. These measurements were made possible by the development of a procedure, employing centrifugal elutriation and density gradient centrifugation, to separate highly enriched populations of such cells from immature rat testes at the early stages of spermatogenesis. The results show a difference in regulation of the synthesis and accumulation of testis-specific histones H1t, TH2A, TH2B, and TH3. TH3 is present and actively synthesized in A and B spermatogonia. The testis-enriched variants, H2A.X and H1a, are also present at their maximal levels in A spermatogonia. No detectable amounts of H1t, and at most, low levels of TH2A and TH2B could be found in spermatogonia. While TH2A and TH2B are already present and actively synthesized in early primary spermatocytes (around the preleptotene stage), H1t does not accumulate until the pachytene stage.     

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.