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C Mezquita

Publications and source records attributed to C Mezquita.

At least 19 recordsLinked to original sources
PubMed1991

Characterization of a chicken polyubiquitin gene preferentially expressed during spermatogenesis.

We have previously reported that a chicken polyubiquitin gene (Ub II) not expressed under normal or heat shock conditions in chick fibroblasts is transcribed during spermatogenesis [(1987) Nucleic Acids Res. 15, 9604]. The level of Ub II mRNA is several-fold higher in testis cells than in somatic tissues. The gene Ub II possesses characteristic features not seen in the polyubiquitin gene expressed in heat shock conditions (Ub I). The 5' noncoding region of Ub II shows the consensus cAMP regulatory element (CRE) followed immediately downstream by a CA dinucleotide. It has been proposed that this extended CRE may be involved in the coordinate expression of various genes during spermatogenesis.

Amino Acid Sequence↗
PubMed1991

Histone H1c decreases markedly in postreplicative stages of chicken spermatogenesis.

The relative proportions of four major chicken histone H1 subtypes (referred to as H1a, H1b, H1c and H1d) change markedly in different chicken tissues. The relative amount of H1c is higher in nonreplicating somatic tissues, such as liver, than in replicating immature testis. The proportion of H1c sharply decreases as spermatogenesis proceeds, being much lower in mature than in immature testis. It has been proposed that the relative increment of H1c correlates with low rates of cell division in chicken tissues. It was assumed that the sharp decrease in H1c observed during maturation of chicken testis was a consequence of the intensification of proliferative activity in spermatogonia (Berdnikov et al., 1976). Our results, however, clearly show that the decrease of H1c during maturation is due to the low levels of this protein in postreplicative stages of spermatogenesis, where H1c is barely detectable. These results suggest that the presence of the arginine-rich H1c subtype would neither be compatible with the relaxed structure of acetylated chromatin present in active replicating cells nor with the hyperacetylated chromatin characteristic of postreplicative late spermatids undergoing the nucleohistone nucleoprotamine transition.

Amino Acid Sequence↗
PubMed1989

Chicken spermatogenesis is accompanied by a genomic-wide loss of DNA methylation.

Renaturation kinetics of DNA obtained from chicken testis cell nuclei separated by sedimentation at unit gravity showed that the undermethylation, previously observed in meiotic and postmeiotic cells, is not a peculiarity of repetitive sequences, but is also a feature of unique sequences. The large proportion of slowly renaturing, intermediately renaturing and rapidly renaturing DNAs contain 27, 32 and 31% less methylcytosines in meiotic and postmeiotic cells than the corresponding fractions of premeiotic cells. DNA methyltransferase activity is lower in meiotic cells containing undermethylated DNA than in immature testis, enriched in spermatogonia, with higher levels of DNA methylation.

Animals↗
PubMed1989

DNA topoisomerase II activity in nonreplicating, transcriptionally inactive, chicken late spermatids.

To study a possible differential involvement of type I and type II DNA topoisomerases in the functional and structural changes that chromatin undergoes during spermatogenesis, we have determined both enzymatic activities in chicken testis cell nuclei at successive stages of differentiation. Whereas DNA topoisomerase I varies in parallel with transcriptional activity, DNA topoisomerase II was present in both replicating, transcriptionally active chicken testis cells and nonreplicating, transcriptionally inactive late spermatids. The presence of DNA topoisomerase II activity in late spermatids and, in addition, the relative increment of drug-induced topo-II-mediated DNA cleavage detected in these cells, suggest that DNA topoisomerase II might modulate the topology of DNA during the marked changes that chromatin structure undergoes in the nucleohistone-nucleoprotamine transition at the end of the spermiogenesis and could be involved in the final organization of DNA within the nucleus of the male gamete.

Animals↗
PubMed1988

Cellular content of ubiquitin and formation of ubiquitin conjugates during chicken spermatogenesis.

Ubiquitin was purified from chicken testis and its content, biosynthesis and formation of conjugates was determined in germinal cells at successive stages of spermatogenesis. Free ubiquitin increased markedly during spermatogenesis, reaching its maximum level in early spermatids. High levels of ubiquitin were still present in late spermatids but were not detectable in mature spermatozoa. Biosynthesis of ubiquitin occurred in vitro in a fraction containing meiotic and pre-meiotic cells, and during spermiogenesis, in early and late spermatids. The cellular content of free ubiquitin increased after ATP depletion, especially in early spermatids. Lysates of chicken testis cells, particularly those obtained from spermatids, were able to form nuclear (24 and 27 kDa) and extranuclear (55-90 kDa) ubiquitin conjugates in vitro. The presence of increasing levels of ubiquitin and ubiquitin conjugates in chicken spermatids may suggest a possible involvement of this protein in the marked changes of protein turnover, chromatin structure and cell-cell interactions that spermatids undergo during spermiogenesis.

Adenosine Triphosphate↗
PubMed1988

Haploid expression of the rooster protamine mRNA in the postmeiotic stages of spermatogenesis.

cDNA clones were prepared from poly(A)+ mRNA isolated from a population enriched in postmeiotic rooster testes spermatogenic cells. A series of clones was sequenced at random and two partial sequences corresponding to the C-terminal coding and 3' untranslated region of the chicken protamine mRNA were obtained. The deduced amino acid sequence of this C-terminal coding region corresponds to the sequence previously described at the protein level for the chicken protamine, galline [Nakano, M., Tobita, T., and Ando, T. (1976), Int. J. Peptide Prot. Res. 8, 565-578]. To study the expression of this protamine gene, RNA was prepared from chicken testes at different stages of development, electrophoresed in formaldehyde-agarose gels, transferred to a nylon membrane, and hybridized with a rooster protamine cDNA probe. Two populations of mRNA of sizes ranging between 420 and 465 bases are expressed in postmeiotic rooster testis cells. To determine if there was a differential expression of the two populations of mRNA in the final postmeiotic haploid stages of spermatogenesis, RNA was purified from adult rooster cells separated at unit gravity according to their differences in size by the Staput technique. The RNA was similarly analyzed by Northern blots. The results indicate that round spermatids are enriched in the 465-nucleotide mRNA species, whereas in the final stage of elongated spermatids the 420-nucleotide species is the only one present, suggesting either post-transcriptional processing, the presence of two different sets of genes that are differentially expressed, or a single set of genes with differential promoter usage.

Amino Acid Sequence↗
PubMed1987

Factors affecting nucleosome disassembly by protamines in vitro. Histone hyperacetylation and chromatin structure, time dependence, and the size of the sperm nuclear proteins.

Histone displaced in vitro from nuclei by protamine competition display a higher degree of hyperacetylation than the residual histones. In addition, hyperacetylated core particle pools are disassembled in vitro with a higher efficiency than control or nonacetylated core particles and when analyzed by electron microscopy display an elongated shape (length/width ratio = 1.52 +/- 0.19) instead of the round compact shape of control nucleosomes (length/width ratio = 1.06 +/- 0.06). In the absence of histone hyperacetylation, the fish protamines, salmine and iridine (32-33 residues), are relatively inefficient in disassembling nucleosomal core particles in vitro as compared to the large (65-70 residues), tyrosine-containing protamines from rooster (galline), squid, and cuttlefish which disassemble nucleosomes in a range of protamine concentrations close to physiological. The fact that an artificially cross-linked salmine dimer acquires the ability of the large protamines from rooster, squid, and cuttlefish to disassemble core particles in vitro and also binds more tightly to the DNA, suggests that the size of the sperm nuclear protamines is a critical factor in this process. Even when the core histones of spermatid chromatin are hyperacetylated in the trout testis, the replacement process by iridine or salmine is slow and time-dependent in vitro. However, since spermiogenesis in trout occurs over several weeks, the slow in vitro nucleosome disassembly process by salmine is sufficient to allow complete displacement, thus supporting the hypothesis that a protamine-mediated displacement of the histones from DNA in vivo may take place in the salmonid fishes by a mechanism similar to that in the rooster, squid, and cuttlefish.

Acetylation↗
PubMed1986

Marked differences in the ability of distinct protamines to disassemble nucleosomal core particles in vitro.

In accordance with the results of classical experiments performed in vitro with calf thymus chromatin and the fish protamine salmine, we have observed that this highly basic, small molecular weight protamine cannot cause major displacement of histones from nucleosomal core particles at concentrations several times higher than physiological (arginine/nucleotide ratios 1-8) and that hyperacetylation of histones facilitates nucleosome disassembly. However, the avian protamine galline, with molecular weight and number of arginine residues almost twice those of common fish protamines, is able to displace the nucleosomal core histones from DNA in vitro at concentrations (arginine/nucleotide ratios 0.6-1.2) within the physiological range (0.8). Our results suggest that the binding of the avian protamine galline to chromatin could be directly involved in the rapid disassembly of nucleosomes that takes place during the nucleohistone nucleoprotamine transition in chicken spermiogenesis.

Animals↗
PubMed1985

Poly(ADP-ribosylation) at successive stages of rooster spermatogenesis. Levels of polymeric ADP-ribose in vivo and poly(ADP-ribose) polymerase activity and turnover of ADP-ribosyl residues in vitro.

Rooster testis cells were separated by sedimentation at unit gravity and the in vivo levels of polymeric ADP-ribose were determined both in intact cells and isolated nuclei by fluorescence methods. Poly(ADP-ribose) polymerase activity was assayed after cell permeabilization or after isolation of nuclei. The turnover of ADP-ribosyl residues was determined in isolated nuclei using benzamide. The content of poly(ADP-ribose), the poly(ADP-ribose) polymerase activity, and the turnover of ADP-ribosyl residues, decreased during the differentiation of the germinal cell line, especially at the end of spermiogenesis. Treatment of cells with 1 mM dimethyl sulfate for 1 h resulted in a marked stimulation of poly(ADP-ribose) polymerase activity in meiotic and premeiotic cells and also in round and late spermatids. The enzymatic activity was not detected and could not be induced in mature spermatozoa. These cells, however, still contained polymeric ADP-ribose with a 2% of branched form.

Adenosine Diphosphate Ribose↗
PubMed1984

Hypomethylation of DNA in meiotic and postmeiotic rooster testis cells.

To study whether changes in methylation of DNA are related to the structural and functional changes that chromatin undergoes throughout rooster spermatogenenis, we analyzed, by high-performance liquid chromatography, the 5-methylcytosine content of DNA purified from rooster testis cell nuclei at successive stages of the cell differentiation process. The DNA of meiotic and postmeiotic cells appears partially under-methylated, containing approximately 30% less methylcytosines than the DNA obtained from premeiotic and somatic cells.

5-Methylcytosine↗
PubMed1983

Quantitative changes of high mobility group non-histone chromosomal proteins HMG1 and HMG2 during rooster spermatogenesis.

The quantitative changes of a group of non-histone chromosomal proteins identified by its solubility, electrophoretic mobility and amino acid analysis as the high mobility group proteins HMG1 and HMG2, were studied throughout rooster spermatogenesis. The ratio HMG1/HMG2 remained constant (0.66 +/- 0.04) during the transition from dividing meiotic and premeiotic cells to nondividing spermatids and from transcriptionally active cells (spermatogonia, spermatocytes and early spermatids) to transcriptionally inactive late spermatids. The ratios HMG1/nucleosomal histone and HMG2/nucleosomal histone increased markedly at the end of spermiogenesis during the transition from nucleohistone to nucleoprotamine when nucleosomes are being disassembled. The high mobility group chromosomal proteins HMG1 and HMG2 were not detectable in the nuclei of rooster spermatozoa.

Amino Acids↗
PubMed1983

Changes in nuclear content of protein conjugate histone H2A-ubiquitin during rooster spermatogenesis.

Electrophoretic analysis of acid-soluble chromosomal proteins isolated from rooster testis cell nuclei at different stages of spermatogenesis, revealed that the nuclear content of a protein identified by its solubility, electrophoretic mobility and amino acid analysis as the protein conjugate histone H2A-ubiquitin (uH2A, A24) changed markedly from meiotic cells to late spermatids. The protein was not detectable in tetraploid primary spermatocytes; it was present in 1.7% of the total amount of nucleosomal core histones in early spermatids and reached its maximum level (3.5% and 11%) at the end of spermiogenesis, when histones are replaced by the protamine galline.

Animals↗
PubMed1982

Histone H4 hyperacetylation and rapid turnover of its acetyl groups in transcriptionally inactive rooster testis spermatids.

In order to study the relationship between acetylation of histones, chromatin structure and gene activity, the distribution and turnover of acetyl groups among nucleosomal core histones and the extent of histone H4 acetylation were examined in rooster testis cell nuclei at different stages of spermatogenesis. Histone H4 was the predominant acetylated histone in mature testes. Hyperacetylation of H4 and rapid turnover of its acetyl groups are not univocally correlated with transcriptional activity since they were detected in both genetically active testicular cells and genetically inactive elongated spermatids. During the transition from nucleohistone to nucleoprotamine in elongated spermatids the chromatin undergoes dramatic structural changes with exposition of binding sites on DNA (1). Hyperacetylation of H4 and rapid turnover of its acetyl groups could be correlated with the particular conformation of chromatin in elongated spermatids and might represent a necessary condition for binding of chromosomal proteins to DNA.

Acetates↗
PubMed1982

Cellular content and biosynthesis of polyamines during rooster spermatogenesis.

The natural polyamines spermine and spermidine, and the diamine putrescine, were extracted from rooster testis cells separated by sedimentation at unit gravity, and from vas-deferens spermatozoa. The ratios spermine/DNA and spermidine/DNA were kept relatively constant throughout spermatogenesis, whereas the ratio putrescine/DNA rose in elongated spermatids. The cellular content of spermine, spermidine and putrescine decreased markedly in mature spermatozoa. Two rate-limiting enzymes in the biosynthetic pathway of polyamines, ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, showed their highest activities at the end of spermiogenesis and were not detectable in vas-deferens spermatozoa. A marked reduction in cell volume during spermiogenesis without a parallel decrease in the cellular content of polyamines suggests the possibility that the marked changes in chromatin composition and structure occurring in rooster late spermatids could take place in an ambience of high polyamine concentration.

Adenosylmethionine Decarboxylase↗