Spermatid-specific Protein Research Articles

Characterization of a Gene Encoding a Basic Protein of the Spermatid Nucleus, TNP2, and its Close Linkage to the Protamine Genes in the Bull

During elongation and condensation of the spermatid nucleus, histones are replaced by spermatid-specific transition proteins (TNP). TNP1 is well characterized at the cDNA and at the genomic level and was found to be highly conserved during mammalian evolution (similarity between 83 to 98%). We here describe for the first time the nucleotide sequence and organization of the gene for TNP2. The gene was isolated from a bull cosmid library and was found to contain a single intron of 910 bp. The coding sequence consists of 390 bp and has a similarity of about 70% to that of the TNP2 cDNAs of mouse and rat. At the basis of amino-acid sequences, the bull TNP2 is 14 and 15 amino acids longer than that of mouse and rat, respectively, and the similarity is only 45% between bull and mouse and 42% between bull and rat. However, the evolutionary divergence has not occurred at the cost of basic amino acids which are of functional importance in DNA-protein interaction in the condensing spermatid nucleus. The TNP2 gene is closely linked to the protamine genes in the bull genome.

Chromosomal assignment of four rat genes coding for the spermatid-specific proteins proacrosin (ACR), transition proteins 1 (TNP1) and 2 (TNP2), and protamine 1 (PRM1)

The genes for proacrosin, protamines, and transition proteins are exclusively expressed in haploid spermatogenic cells. From the analysis of mouse x rat cell hybrids which segregate rat chromosomes, the rat gene for proacrosin (ACR) was assigned to chromosome 7, that for transition protein 1 (TNP1) to chromosome 9, and the genes for transition protein 2 (TNP2) and protamine 1 (PRM1) to chromosome 10.

Interaction of spermatid-specific protein TP2 with nucleic acids, in vitro. A comparative study with TP1.

TP2 was purified from rat testes employing a gentle method involving differential salt extraction of the sonication-resistant spermatid nuclei. The nucleic acid binding properties of TP2 were studied by fluorescence quenching, thermal denaturation, circular dichroism techniques and compared with those of TP1 (Singh, J., and Rao, M. R. S. (1987) J. Biol. Chem. 262, 734-740). The tyrosine fluorescence of TP2 was quenched upon binding to double-stranded and denatured DNA and poly(rA). The apparent association constants for binding of TP2 to these nucleic acids were calculated from the fluorescence quenching data, obtained at 50 mM NaCl, and found to be 1.63 x 10(5) M-1, 6.5 x 10(5) M-1, and 7.3 x 10(5) M-1, respectively. Thermal denaturation studies of calf thymus DNA and its complexes with TP2 showed that at 1 mM NaCl, TP2 shifted the Tm from 53 degrees C to 62-67 degrees C, while at 50 mM NaCl, the Tm was shifted from 72 to 78 degrees C suggesting that TP2 is a DNA stabilizing protein. Circular dichroism studies of TP1.DNA and TP2.DNA complexes have revealed that TP2 has a better DNA condensing property than TP1. Furthermore, in contrast to TP1, TP2 does not destabilize in vitro the compactness of liver nucleosome core particles. The DNA binding properties of TP1 and TP2 have been discussed in relation to the significance of their transient appearance during mammalian spermiogenesis.

Nuclear basic protein transition during sperm differentiation. Primary structure of the spermatid-specific protein S2 from the dog-fish Scylliorhinus caniculus.

The remodeling of nucleoproteins during dog-fish spermiogenesis involves two successive nuclear protein transitions: the first from somatic-type histones to transition proteins during the nuclear elongation of spermatids and the second leading to protamine-DNA association in mature spermatozoa. The chromatin of elongating spermatids contains two transition proteins called S1 and S2. The amino acid sequence of protein S1, a polypeptide of 87 residues was determined previously [Chauvière, M., Martinage, A., Briand, G., Sautière, P. & Chevaillier, Ph. (1987) Eur. J. Biochem. 169, 105-111]. In the present paper, we report the elucidation of the primary structure of the minor transition protein S2 established by automated Edman degradation of the protein and of its fragments generated by cleavage at methionine and aspartate residues. S2 contains 80 residues and has a molecular mass of 9726 Da. S2 is mainly characterized by a high content of basic amino acids mostly represented by lysine, a relatively high level of hydrophobic residues, the presence of six phosphorylatable residues and the lack of cysteine. Its amino acid sequence shows that the N-terminal half is highly basic, while the acidic residues are located in the C-terminal part of the protein where more diversity in amino acids is noticed. The two transition proteins S1 and S2 share striking structural similarities. Few but significative similarities have been detected with the mammalian transition protein TP1 [Kistler, W. S., Noyes, C., Hsu, R. & Heinrikson, R. L. (1975) J. Biol. Chem. 250, 1847-1853], suggesting similar functions for all these proteins in chromatin remodeling during sperm differentiation. By contrast, the two dog-fish spermatid-specific proteins are structurally unrelated to sperm protamines and cannot be considered as their precursors.

Les protéines nucléaires basiques de transition au cours de la spermiogenèse de la roussette Scylliorhinus caniculus

During dog-fish spermiogenesis, 2 basic nuclear protein transitions occur: the first from histones to spermatid-specific proteins S1 and S2, the second leading to protamines. S1, the most abundant transition protein, is a polypeptide containing 87 residues (Mr = 11,179 Da) whereas S2, the minor transition protein, contains 80 residues (Mr = 9,726 Da). The 2 proteins are mainly characterized by an asymmetry of the molecule, a very high content of basic residues, a relatively high level of hydrophobic residues and a cluster of acidic residues in the carboxy-terminal quarter of the molecule. The 2 proteins are phosphorylated on serine residues and the degree of phosphorylation is relatively important in protein S1. The 2 transition proteins are structurally unrelated to testis histones or sperm protamines and cannot be considered either as their proteolytic degradation products or as their precursors.

Nuclear protein transitions in cuttle-fish spermiogenesis: immunocytochemical localization of a protein specific for the spermatid stage.

The changes in basic nuclear proteins throughout cuttle-fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti-protein T immune serum was found to recognize protein T and not histones from the testis. Immunoperoxidase staining of sections or of smears of testis with anti-protein T antibodies showed that protein T appears in the nuclei of round spermatids, is abundant in elongating spermatid nuclei, but cannot be detected in elongated spermatids. Nuclei from these elongated spermatids were isolated by sonication treatment of testis cells. A protein, named protein Sp, with the characteristic mobility of a protamine, was isolated from elongated spermatid nuclei. This protein has the same mobility as the protamine present in mature spermatozoa. Taken together, the results indicate that in cuttle-fish, nuclear protein transitions involve the replacement of histones by a spermatid-specific protein (protein T), which is replaced at the end of elongation of the nucleus by a protamine (protein Sp). Thus, spermiogenesis of the cuttle-fish (and perhaps of other cephalopods), shows two basic nuclear protein transitions, which are similar to the transitions observed in higher vertebrates such as mammals.

Characterization of the nuclear basic proteins specific of spermiogenesis in cuttlefish, Sepia officinalis

Two highly basic proteins which appear during cuttlefish spermiogenesis have been isolated from testis chromatin and from sperm nuclei. (1) Protein T is a spermatid-specific protein which is transiently associated to DNA and disappears in spermatozoa. It is rich in arginine (approx. 60%) and is phosphorylated at different levels. (2) Protein Sp is a typical protamine which appears in the late stage of spermiogenesis and constitutes the major basic nuclear component in mature spermatozoa. It is very rich in arginine (approx. 77%) and contains only four different amino acids. Phosphorylated in the testis, it is completely dephosphorylated in the spermatozoa. It is suggested that protein T could be a precursor of protein Sp.

Nuclear basic protein transition during sperm differentiation. Amino acid sequence of a spermatid-specific protein from the dog-fish Scylliorhinus caniculus.

During dog-fish spermatogenesis, chromatin undergoes a continuous processing which involves two basic protein transitions: the first from somatic-type histones to spermatid-specific proteins and the second leading to protamines. Two spermatid-specific proteins S1 and S2 were isolated from nuclei of spermatid-enriched testis zone and the amino acid sequence of S1 has been determined. S1 contains 87 amino acids and has a molecular mass of 11179 Da. It is mainly characterized by a high content of basic residues (45%) and the presence of one residue of cysteine. Its primary structure shows that the N-terminal half is highly basic while the hydrophobic residues are preferentially localized in the C-terminal region. Three forms of S1 are present in testis which correspond to di-, mono- and nonphosphorylated molecules. This spermatid-specific protein shares no common structural feature with either histones and dog-fish protamines or rat spermatid-specific protein which has been previously described.

Purification and characterization of two basic spermatid-specific proteins isolated from the dog-fish Scylliorhinus caniculus

In dog-fish spermatid nuclei two intermediate proteins S 1 and S 2 replace histones before the setting down of protamines. These spermatid-specific proteins were isolated by carboxymethyl-cellulose chromatography and purified by high pressure liquid chromatography. S 1 and S 2 are characterized by a high content of basic residues and by the lack of cysteine and phenylalanine. The determination of their amino acid composition and of their N- and C-terminal sequences prove that each protein corresponds to a specific molecule which can be considered neither as a histone hydrolytic product nor as a protamines precursor.

Nuclear reorganization in ram spermatids

Nuclear changes as a function of ram spermatid differentiation have been studied using electron microscopy, cytochemistry, and treatment of isolated nuclei with chemical and mechanical agents. In round nuclei, chromatin consists of intertwined knobby fibers about 210 and 120 A in diameter. In flattening nuclei, these fibers progressively change into smooth filaments at least 25–33 A thick. This change is interpreted as reflecting somatic histone removal known to occur in these nuclei (M. Loir and M. Lanneau, Exp. Cell. Res. 115 , 231, 1978). The packaging of chromatin is carried out by aggregation of the smooth filaments into large (about 300 A) contorted threads which finally coalesce to form a homogeneous mass. In flattened nuclei a small fraction of chromatin (basal knobs) does not undergo complete packaging and retains both cytochemical properties and resistance to disruption similar to those of chromatin in flattening nuclei. Correlations of changes in nuclear morphology, ultrastructure, and resistance to chemical and physical degradation with changes in nucleoproteins suggest that the cystine-containing spermatid-specific proteins could play an important role in chromatin-structure reorganization, shaping and increasing stabilization of the nucleus. The sperm-specific protein promotes terminal packaging and stabilization of chromatin. Other nuclear components considered in this study are: the RNP-containing structures, the nuclear posterior space, and the redundant nuclear envelope; the last two structures are possibly related to the transfer of proteins leaving the chromatin for the cytoplasm.