Displacement of histones by sperm-specific proteins at different stages of spermatogenesis of squid.

Changes in the composition of the chromatin basic proteins during spermatogenesis of the squid Illex argentinus were studied. The core histones of I. argentinus slightly differ from those of calf thymus in the subfractional composition of histones H2A and H2B. A similar amino acid composition is revealed in the histones H1 of the squid I. argentinus and calf thymus. Histone H1 of the squid has a lower molecular mass and a special subfractional composition as compared to those of calf thymus, grass carp and carp studied formerly [Kadura et al. (1983) Comp. Biochem. Physiol. 743, 343-350]. Neither the fractional nor subfractional composition of histones changes during spermatogenesis. The two new proteins were revealed in the chromatin composition of squid testes and spermatozoa illexines I1 and I2. Illexine I2 is composed of two subfractions I2-1 and I2-2. Illexine I2 shows a high content of arginine (75 mol/100 mol). Serine (10 mol/100 mol), histidine (3,2 mol/100 mol) and tyrosine residues (2,9 mol/100 mol) are also present. Illexine I1 shows the presence of arginine (45,6 mol/100 mol), lysine (7.6 mol/100 mol), serine (11.4 mol/100 mol), hystidine (2.3 mol/100 mol) and tyrosine residues (2.8 mol/100 mol). Molecular masses of illexines I2 and I1 are approximately 7 kDa and 9 kDa respectively. It is supposed that during spermatogenesis the histones are displaced in two-stage order: histones----I1----I2.

Rearrangements of chromatin structure during spermatogenesis of squid.

A stepwise replacement of somatic histones on sperm-specific proteins (we have termed them illexines I1 and I2) is found to occur during spermatogenesis of squid Illex argentinus [Kadura, S.N. and Khrapunov, S.N. (1988) Eur. J. Biochem. 175, 603-607]. The chromatin from nuclei of squid immature testes has a nucleosomal DNA repeat which corresponds to the nucleosomal repeat of calf thymus chromatin (195 +/- 5 bp). As spermatozoa become mature and illexine I2 accumulates in the chromatin, the nucleosomal structure of the latter disappears and chromatin compacting takes place. The chromatin DNA from squid spermatozoa is highly resistant to micrococcal nuclease action. Spectrophotometry and spectrofluorimetry were to establish that neither illexine I1 nor illexine I2 forms a globular structure in solution under any conditions studied. Illexine I2 (approx. 7 kDa) shows a high affinity to DNA and remains bound to it under conditions when complexes of illexine I1 (approx. 9 kDa) and salmine (approx. 4.5 kDa) with DNA completely dissociate. This fact, allowing for a similar content (about 75%) of arginine in illexine I2 and salmine, suggests high clustering of arginine residues in the composition of illexine I2. It is suggested that the initial stage of histone substitution with illexine I1, which has a more moderate affinity to DNA than illexine I2, prepares chromatin for the formation of a highly packed structure by illexine I2 during squid spermatogenesis.

[3 types of sperm proteins in eukaryotes]. / Tri tipa spermal'nykh belkov éukariot.

Basic spermal proteins of various species of hydrobionts attributed to Pisces and Cephalopoda are studied. It is established that chromatin of nine species referring to two Cypriniformes families includes the somatic histones. Histone H1 of Cypriniformes is attributed to the lysine-rich type histones and contains 35% mol. of lysine and 0.7% mol. of tyrosine. Chromatin of 14 species of fish referring to nine families of the percoid fish superorder includes protamines similar to salmin, a typical protamine of salmon. The amino acidic analysis of protamine from the sandre sperma has shown that it contains 59% mol. of arginine and no tyrosine. Chromatin of three species from squid superorder referring to Cephalopoda includes gametones -- proteins differing from histones and protamines both in the electrophoretic mobility and amino acidic composition (75% mol. of arginine, 3% mol. of tyrosine).

[Structure of illexine I2 and its complexes with DNA]. / Issledovanie struktury illeksina I2 i ego kompleksov s DNK.

Conformational peculiarities of illexine I2 both in the solution and in the complexes with DNA were studied by circular dichroism, UV-spectroscopy and spectrophotometric melting. IIlexine I2 is shown to have an extended left-handed helical conformation of poly-L-proline II type, that are stable in a wide range of experimental conditions. Upon interaction of illexine I2 with DNA, the parameters of conformation are somewhat distorted but the main peculiarities remain. The DNA double helix changes from B- to the divection of C-form at its interaction with illexine I2. The interaction of illexine I2 with DNA at low ionic strength is non-cooperative and is characterized by some specificity to A--T sequences of DNA. Illexine I2 strongly affects the DNA stability by increasing the melting temperature of DNA.

[Electrophoretic characteristics of nuclear proteins in the rat liver after electric stimulation of the hypothalamus]. / Elektroforeticheskaia kharakteristika iadernykh belkov pecheni krys posle élektrostimuliatsii gipotalamusa.

The liver nuclear proteins of 3- and 6-month old rats were studied by the gel-electrophoresis technique after electrostimulation of hypothalamus. In this case new nuclear proteins with low molecular weight were found. The electrophoretic rate of these proteins is higher than that of histone H4. A decrease in the content of histone H2B in the nuclear liver of 6-month old rats was also found after electrostimulation of hypothalamus. The mechanism of this phenomenon is discussed.

[Accumulation of one of the histone H1 subfractions and increase in chromatin resistance to the nuclease effect in the course of carp and grass carp spermatogenesis]. / Nakoplenie odnoi iz subfraktsii gistona H1 i uvelichenie ustoichivosti khromatina k deistviiu nukleaz v khode spermatogeneza karpa i belogo amura.

Chromatin of immature testes and sperma of grass carp and carp was hydrolyzed by micrococcal nuclease and DNA I. The hydrolysis kinetics was studied. Chromatin of sperm maintains the nucleosomal repeat typical to somatic cells and is more resistant to the nuclease effect than chromatin of testes. Histones H1 and H2B of grass carp differ from the respective histones of the calf thymus. The core histones of sperm testes and liver chromatin in grass carp and carp have similar electrophoretic mobility. However the amount of slowly moving subfraction of histone H1 increases in the course of spermatogenesis. The amino acid composition of histone H1 from the carp sperm is characterized by a high lysine (34.6%) and low glycine (4.5%) content. A histone H1 molecule contains one tyrosine. The lysine/arginine ratio is 21.6 which is considerably higher than in H1-like histones of sperma in other species (for instance of Echinodermata).

[Peculiarities of the amino acid composition, spatial organization and interaction with DNA of histones H1 from calf thymus and carp spermatozoa]. / Osobennosti aminokislotnogo sostava, prostranstvennoi organizatsii i vzaimodeistviia s DNK gistonov H1 iz timusa telenka i spermiev karpa.

The amino acid composition of the H1-like histone isolated from carp spermatozoa (H1carp) is characterized by a high content of lysine (34.6%) and a low content of glycine (4.5%) as compared to that of its calf counterpart (H1calf). The Lys/Arg ratio is 21.6, which is much higher than that for the H1-like histones from other species spermatozoa (cf. echinodermata). It was shown that the fluorescence anisotropy and excitation spectra of histones H1carp and H1calf change synchronically. At the same time the final folding of the polypeptide chains of these histones within their ternary structure is different. These differences manifest themselves in a distinct quantum yield of both histones and different accessibility of the single tyrosine residue for fluorescence quenchers. In histone--DNA complexes the tyrosine fluorescence is quenched. An increase in the ionic strength gives rise to a formation of large-sized aggregates in a histone H1--DNA solution which contain structurally heterogenous histones H1 from different sources. Histone H1carp causes DNA aggregation at lower ionic strength values than its calf counterpart. The complexes are dissociated at 0.6 M NaCl.

Changes in chromatin basic proteins during male gametogenesis of grass carp.

1. The composition of basic of chromatin protein fractions has been studied in the course of male spermatogenesis of grass carp (Ctenopharyngodon idella). 2. Electrophoretic mobility of H1, H2B and H2A histones of grass carp is shown to differ from that of corresponding fraction of calf thymus histones. 3. Comparison of chromatin protein fractions from liver, mature and immature testis and sperm of grass carp has revealed subtle tissue specificity of H2B and H2A histones. 4. Maturation of spermatozoa in grass carp is accompanied by a marked accumulation of the TH1 subfraction (TH1-1), disappearance of some acid-soluble non-histone proteins and of a protein similar to the mammalian A24 protein.

Some age-related structural peculiarities of fractionated chromatin. Thermal denaturation and electrophoretic analysis.

Thermal denaturation and electrophoretic mobility of liver and small intestinal mucosa fractionated chromatin and the DNA of chromatin fractions have been studied in young and old rats. During aging, the thermostability of chromatin increased, electrophoretic mobility of mono-, di-, and trinucleosomal fragments decreased at the expense of increased repeated length of nucleosomal DNA.

[Effects of pH and ionic strength on interaction of different histone fractions and protamine with immobilized histone H4]. / Vliianie pH i ionnoi sily sredy na vzaimodeistvie razlichnykh fraktsii gistonov i protamina s immobilizovannym gistonom H4.

The interaction of histone H4 immobilized on Sepharose 4B with protamine and individual and total histone fractions from calf thymus has been studied. In accordance with the strength of their interaction with histone H4, the individual histone fractions can be arranged in the following order: H4 greater than H2a greater than H2b greater than H1 and protamine. The structure of the complexes, which are formed in a solution of a total histone fraction and specifically interact with the immobilized histone H4, depends on the ionic composition and pH of the medium.