Mutagenic selectivity of carcinogenic nitroso compounds I. N-methyl-N-nitrosourethane

The mutagenicity of the carcinogen methylnitrosourethane (MNUr) was examined in Drosophila with a view to the determination of its activity on heterochromatic loci (especially rDNA) relative to those in the euchromatin. Assays were made of the yield of rDNA mutations (bobbed: bb) relative to other X-chromosome recessive lethals and visibles [X(l + v)] in the same male germ cells after treatment with different doses (1–10 mM) and at various stages in spermatogenesis.Dose dependence followed the same pattern for all genic loci and germ cell stages. In all instances, the regression of mutation frequency on injected molar dose was approximately linear, but could better be described by a quadratic dose curve. In contrast, the mutagenicity pattern during spermatogenesis varied according to the target genes. The response of the euchromatic loci reached a peak among the earlier germ cells (probably the spermatocytes), whereas that for the heterochromatic sites (including rDNA) was maximal in mature sperm.Mutagenic selectivity for rDNA with MNUr, as indicated by the percentage bb/X-mutations, was among the highest for the intrinsically reactive carcinogens (alkylating and arylating agents). This correlates with the strong carcinogenicity of MNUr and adds further support to the concept that rDNA mutations might well be a crucial step in cancer initiation.     

Mutagenicity of dimethylnitrosamine in the metabolic process by rat liver microsomes

The mutagenicity of dimethylnitrosamine (DMN) for bacteria was investigated by means of the metabolic activation process of the compound with rat liver microsomes.Three strains of streptomycin (SM)-dependent Escherichia coli having tetracycline (TC)-resistance factor (Sd-E. coli(TC)) were derived for this study. The reverse mutation in these strains from SM dependence to non-dependence was used as the marker for mutagenicity. The drug resistance factor (R factor) which was transferred to these strains was used in order to get around the bacterial contamination throughout the experiments. The study of the mutagenicity of DMN metabolites has been made by incubating DMN with rat liver microsomes and cofactor system in the presence of indicator bacterial cells.The reverse mutation was markedly induced for all of three strains in the complete incubation mixture but it was not observed when the cofactor system was omitted or the liver microsomal suspension was replaced by the kidney cell sap. When the indicator bacterial cells were added to the mixture in which DMN was previously incubated with the microsomes and cofactor system, the mutagenicity was extremely decreased.     

Relative effectiveness of neutrons and X-rays in induction of crossing over in drosophila males

Relative biological effectiveness of neutrons vs. X-rays in inducing crossing-over in males of D. melanogaster was investigated using 812 and 834 rad of neutrons and the same dose of X-rays. Crossing-over was induced in spermatocytes and spermatogonia of adults and pupae. Neutrons were 4 times more effective in spermatocytes of adults and their effectiveness in pupal spermatocytes was even more. Neutrons also induced more exchanges in spermatogonial cells including predefinitive spermatogonia. Higher effectiveness of neutrons can be attributed to their high linear energy transfer.     

DNA Methylation Assessed by SMRT Sequencing Is Linked to Mutations in Neisseria meningitidis Isolates

The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis.We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes.DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles.Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides.These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes.     

Analysis of mutagen specificity Drosophila melanogaster

The previously reported difference between the mutational spectra of hydrazine (HZ) and hydroxylamine (HA) was confirmed for one selected locus (miniature) at which hydrazine produces no mutations in treated late larval spermatogonia or premeiotic spermatocytes sampled by 3 days' progeny. The genetically effective dose was measured in most experiments by the production of v mutants, and in a few by the production of sex-linked lethals. In a total of over 37 000 X-chromosomes (16 000 from previous, and over 21 000 from present experiments) treatment with HZ yielded no m mutation, but 90 v mutations. After treatment with genetically equivalent doses of HA, m and v mutations were about equally frequent. The ratio of visible mutations at the v locus to lethals on the X-chromosomes was exceptionally high after either treatment. So was the ratio of m mutations to lethals after treatment with HA.     

The essential roles of Mps1 in spermatogenesis and fertility in mice

Monopolar spindle 1 (MPS1), which plays a critical role in somatic mitosis, has also been revealed to be essential for meiosis I in oocytes. Spermatogenesis is an important process involving successive mitosis and meiosis, but the function of MPS1 in spermatogenesis remains unclear. Here, we generated Mps1 conditional knockout mice and found that Ddx4-cre-driven loss of Mps1 in male mice resulted in depletion of undifferentiated spermatogonial cells and subsequently of differentiated spermatogonia and spermatocytes. In addition, Stra8-cre-driven ablation of Mps1 in male mice led to germ cell loss and fertility reduction. Spermatocytes lacking Mps1 have blocked at the zygotene-to-pachytene transition in the prophase of meiosis I, which may be due to decreased H2B ubiquitination level mediated by MDM2. And the expression of many meiotic genes was decreased, while that of apoptotic genes was increased. Moreover, we also detected increased apoptosis in spermatocytes with Mps1 knockout, which may have been the reason why germ cells were lost. Taken together, our findings indicate that MPS1 is required for mitosis of gonocytes and spermatogonia, differentiation of undifferentiated spermatogonia, and progression of meiosis I in spermatocytes.Subject terms: Cell division, Spermatogenesis     

Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6: SEQUENTIAL OXIDATION TO ALDEHYDES AND CARBOXYLIC ACIDS AND ANALYSIS OF REACTION STEPS*

Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH₃CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect (^Dk_app ∼ 10), which was highly expressed in a variety of competitive and non-competitive experiments. The ^Dk_app for DEN was ∼3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO₂H and CH₃CO₂H, respectively. In neither case was a lag observed, which was unexpected considering the k_cat and K_m parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde).     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Measurement of genotoxic activity in multiple tissues following inhalation exposure to dimethylnitrosamine

Chemically-induced DNA repair was measured as unscheduled DNA synthesis (UDS) in selected tissues isolated from rats following in vivo exposure to inhaled dimethylnitrosamine (DMN). UDS was evaluated in epithelial cells from rat nasal turbinates and trachea, in hepatocytes and in pachytene spermatocytes from the same treated animal. At nominal concentrations of 500 and 1000 ppm of DMN in air, chemically-induced DNA repair was observed in the epithelial cells of the upper respiratory system. DMN also entered the circulation, as evidenced by a strong DNA-repair response in hepatocytes. No DNA repair was observed in pachytene spermatocytes indicating either that DMN or its active metabolites did not reach the testes in sufficient concentration to induce DNA repair or that the testes lacked the capability to metabolically activate the compound. These results illustrate the potential of this approach to assess the organ-specific genotoxicity of environmental chemicals.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Expression pattern of vasa in gonads of sea cucumber Apostichopus japonicus during gametogenesis and reproductive cycle

The vasa gene is a reliable germline marker to study the origin and development of germ cells and gonads, although the gene product (mRNA or protein) varies between different species. However, there has been little study on vasa genes in holothuroids to date. Here we determined the expression characteristics of the Apostichopus japonicus vasa gene (Aj-vasa) during gametogenesis in the ovary and testis using in situ hybridization and immunohistochemistry. During oogenesis, the expression pattern of Aj-vasa coincided at the mRNA and protein levels. Intensive signals in oogonia decreased gradually with the development of oocytes. Interestingly, the pattern was different during spermatogenesis. The Aj-vasa mRNA level was the highest in spermatogonia, reduced in spermatocytes, low in spermatids and absent in spermatozoa, but the Aj-VASA protein was restricted to spermatogonia and early spermatocytes. These expression characteristics of Aj-vasa persisted in both male and female gonads throughout the reproductive cycle. Our findings show that Aj-vasa mRNA is a good marker for studying the origin and migration of germline cells; moreover, Aj-VASA is a useful tool to identify spermatogonia in A. japonicus. Our findings indicate that Aj-vasa is vital in the development and differentiation of germ cells.     

GAL4 enhancer traps that can be used to drive gene expression in developing Drosophila spermatocytes

The Drosophila testis has proven to be a valuable model organ for investigation of germline stem cell (GSC) maintenance and differentiation as well as elucidation of the genetic programs that regulate differentiation of daughter spermatogonia. Development of germ cell specific GAL4 driver transgenes has facilitated investigation of gene function in GSCs and spermatogonia but specific GAL4 tools are not available for analysis of postmitotic spermatogonial differentiation into spermatocytes. We have screened publically available pGT1 strains, a GAL4‐encoding gene trap collection, to identify lines that can drive gene expression in late spermatogonia and early spermatocytes. While we were unable to identify any germline‐specific drivers, we did identify an insertion in the chiffon locus, which drove expression specifically in early spermatocytes within the germline along with the somatic cyst cells of the testis. genesis 50:914–920, 2012. © 2012 Wiley Periodicals, Inc.     

Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab,Eriocheir sinensis

During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.     

The induction of chromosomal damage in rat hepatocytes and lymphocytes II. Alkylation damage and repair of rat-liver DNA after diethylnitrosamine,dimethylnitrosamine and ethyl methanesulphonate in relation to clastogenic effects

Rat-liver DNA alkylation by diethylnitrosamine (DEN), dimethylnitrosamine (DMN) and ethyl methanesulphonate (EMS) was studied in an attempt to relate chromosome-damaging effects of these agents (the formation of micronuclei in hepatocytes; see preceding paper) to specific alkylation patterns. No correlation was observed between the induction of micronuclei and liver DNA N-alkylation, measured as 3- and 7-alkyl-purines. O⁶-Alkylguanine is probably not involved in micronucleus induction because it is lost from DNA too rapidly to explain the much more persistent clastogenic effects. In contrast, both the initial amounts of alkylphosphotriesters and the persistencies of these products roughly paralleled the respective effects on micronucleus induction. The possible involvement of alkylphosphotriesters or other O-alkylation products of comparable stabilities is discussed. Results with DMN suggest that part of the primary DNA methylation damage is converted into a secondary (DNA) lesion and that both the primary and secondary lesion(s) contribute to the process of micronucleus formation.     

Drosophila male-sterile mutation emmenthal specifically affects the mitochondrial morphogenesis

Proper mitochondrial morphogenesis is crucial for successful development of motile sperm. It was known that recessive Drosophila melanogaster mutation emm caused anomalies in the formation of a mitochondrial derivative—nebenkern and led to male sterility. Here we identified primary mutation effect and showed that emm is required for the formation and maintenance of inner mitochondrial structure starting from early spermatocytes. Abnormal mitochondria structure affects subsequent cellular processes in spermatogenesis such as meiotic cytokinesis and spermatid elongation.     

Recombinational and mutational analyses of subspecific differentiation in Oryza sativa L.

Recombinational and mutational analyses were performed in an attempt to elucidate the nature of the genic differences and the ancestral relationship between the indica and japonica subspecies of Oryza sativa L. by taking representative varieties from each group. The absence of cytological aberrations at meiosis in indica×japonica hybrids suggest that genetic differences are predominantly genic rather than chromosomal. The pattern of variation induced in the key characters, and the frequency and spectra of mutations obtained in each group, failed to support the hypothesis that indica has given rise to japonica, and suggested that the two subspecies have originated from a common ancestor.     

THE FINE STRUCTURE OF DIFFERENTIATING SPERMATOZOA AND SERTOLI CELLS IN THE GONAD OF THE POND SNAIL, LYMNAEA STAGNALIS

The fine structure has been examined, of spermatogonia, spermatocytes,early spermatids, late spermatids and early spermatozoa nestlingagainst Sertoli cells in the gonad of Lymnaea stagnalis. Changes in the Sertoli cells are linked with the phases of spermdifferentiation. Details on differentiation particularly ofthe head of the sperm, are presented. (Received 14 March 1981;