Aneuploidy frequencies in semen fractions from ten oligoasthenoteratozoospermic patients donating sperm for intracytoplasmic sperm injection.

OBJECTIVE: To determine aneuploidy frequencies in pellet and swim-up semen fractions from 10 infertile men with severe oligoasthenoteratozoospermia (OAT) who were donating sperm for intracytoplasmic sperm injection and to determine whether the swim-up isolation method would successfully separate aneuploid from haploid sperm. DESIGN: Prospective study. SETTING: Infertility clinic and molecular genetics laboratory. PATIENT(S): Ten patients with severe OAT. INTERVENTION(S): Cytogenetic analyses by fluorescence in situ hybridization to determine aneuploidy frequencies for chromosomes 1, 13, 18, 21, X, and Y in sperm from swim-up and pellet fractions. MAIN OUTCOME MEASURE(S): Gametic aneuploidy was scored in sperm fractions separated by the swim-up technique and clinical results after intracytoplasmic sperm injection were tabulated. RESULT(S): In all cases, chromosome aneuploidy levels in patients were significantly greater than in controls. The type and percentage of aneuploid sperm for all patients with OAT found in both swim-up and pellet fractions were not different, with the exception of diploid sperm, which remained in the pellet fraction. After ET, 2 (20%) of 10 couples achieved successful pregnancies. CONCLUSION(S): The data show significantly higher rates of diploidy, autosomal disomy and nullisomy, sex chromosome disomy and nullisomy, and total aneuploidy in sperm from all separated fractions obtained from all patients with OAT versus controls. This patient population with OAT may be at increased risk of producing aneuploid offspring.

Induction by nitriles of sex chromosome aneuploidy: tests of mechanism.

Genetic and biochemical assays were conducted to determine if nitrile induced adult paralysis and germline aneuploidy in female Drosophila melanogaster requires a biochemical activation mechanism which results in the release of free cyanide. Two nitriles predicted to differ substantially in their susceptibility to enzymatic cyanide release were found to be equally effective inducers of aneuploidy. Regardless of differences in chemical structure, nitriles seem to be affecting a common cellular target as judged by the lack of synergistic effects when two nitriles are presented simultaneously. Mitochondrial respiration was not inhibited by acetonitrile under conditions in which sodium cyanide completely blocked respiration. A sensitive luciferase enzyme inhibition assay suggests that some, but not all, nitriles may affect hydrophobic protein interactions. These results suggest that there is no single biochemical mechanism by which all nitriles induce aneuploidy, although the cellular target disrupted is probably the same for each chemical. The implications of these findings for structural alert based pre-screening of mutagens are discussed.

The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination.

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution.

Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

The RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were cloned by the polymerase chain reaction. DNA sequence analysis revealed an open reading frame of 418 amino acids for the human RAD52 homolog and of 420 amino acid residues for the mouse counterpart. The identity between the two proteins is 69% and the overall similarity 80%. The homology of the mammalian proteins with their counterparts from yeast is primarily concentrated in the N-terminal region. Low amounts of RAD52 RNA were observed in adult mouse tissues. A relatively high level of gene expression was observed in testis and thymus, suggesting that the mammalian RAD52 protein, like its homolog from yeast, plays a role in recombination. The mouse RAD52 gene is located near the tip of chromosome 6 in region G3. The human equivalent maps to region p13.3 of chromosome 12. Until now, this human chromosome has not been implicated in any of the rodent mutants with a defect in the repair of double-strand breaks.

Refractory ceramic fibers (RCFs) induce germline aneuploidy in Drosophila oocytes.

Mineral fibers are ubiquitous in the natural environment and are widely used in industry in such applications as insulators. We have previously shown that asbestos fibers induce aneuploidy in oocytes of Drosophila melanogaster; here we extend those studies by testing refractory ceramic fibers (RCFs) for their mutagenicity to germ cells. The results establish that the tested RCFs are inducers of aneuploidy following feeding of adult females. A subset of the RCFs were also effective following larval feeding. Our results suggest that RCFs, like certain asbestos fibers, may pose a hazard to germ cells.

5-Azacytidine induces sex chromosome loss and interchange in immature germ cells of Drosophila mei-9 males.

5-Azacytidine (5-AZ) profoundly affects gene expression and chromosome structure in higher eukaryotes, presumably by disrupting normal patterns of DNA methylation. The DNA of several eukaryotic species, including Drosophila melanogaster, is virtually devoid of 5-methylcytosine, and the spectrum of mutagenic effects induced by 5-AZ in such organisms is less well characterized. To investigate the mutagenicity of 5-AZ in Drosophila germ cells, DNA repair-deficient (mei-9) Drosophila 72-hr-old larvae were fed on medium containing 5-AZ, and recovered adult males were tested for induced losses and interchanges involving the paternal sex chromosomes. Moderately toxic doses of 5-AZ were found to induce significant rates of apparent complete losses (CL) of the paternal sex chromosomes, partial losses (PL) of one of the arms of the submetacentric Y-chromosome, and interchanges (X-Y) between the X-chromosome and the short arm of the Y-chromosome. The data suggest that, of the stages tested, germ cells in the early primary spermatocyte stage of development are maximally sensitive to 5-AZ-induced sex chromosome loss and rearrangement. X-Y interchanges comprised a substantial fraction of the 5-AZ-induced breakage events involving the Y-chromosome; in contrast with classical clastogens such as X-rays, the pattern of interchange products suggests that 5-AZ acts by enhancing the frequency of pairing-dependent interchanges between the paternal sex chromosomes.

Molecular analysis of chemically-induced mutations at the RpII215 locus of Drosophila melanogaster.

A substantial fraction, perhaps 50% or more, of spontaneous mutations in Drosophila melanogaster have been shown by molecular analyses to be associated with the presence of a transposable element (TE) inserted into the affected gene. We are interested in the molecular structure of induced mutations in Drosophila, in particular whether TEs are also responsible for a significant proportion of chemically-induced mutations. We report here the molecular analysis of 58 mutations at the RpII215 locus induced with EMS or ENU. While we find evidence for moderately sized deletions at this locus (in 3/58, or 5% of the examined mutants), we failed to detect any mutations which were associated with an insertion event. It may be the case that induced mutations are qualitatively different from spontaneous mutations.

Apurinic endonuclease from Drosophila melanogaster: reduced enzymatic activity in excision-deficient mutants of the mei-9 and mus(2)201 Loci.

An endonuclease activity, which is specific for partially depurinated PM2 DNA, has been assayed in extracts prepared from cultured cells and larval brain ganglia of Drosophila melanogaster. Activity detected in repair-proficient cells is stimulated by Mg++ and is inhibited by EDTA. Extracts prepared from established cell cultures of the excision-deficient strain mei-9a and from larval brain ganglia derived from y mei-9a, mei-9L1, sn3 mei-9D2 and sn3 mei-9D4 are partially deficient in this activity. At least one allele of a second excision-deficient strain, cn mus (2)201D1, also shows reduced AP-endonuclease activity in ganglia extracts.

Third-chromosome mutagen-sensitive mutants of Drosophila melanogaster.

A total of 34 third chromosomes of Drosophila melanogaster that render homozygous larvae hypersensitive to killing by chemical mutagens have been isolated. Genetic analyses have placed responsible mutations in more than eleven complementation groups. Mutants in three complementation groups are strongly sensitive to methyl methanesulfonate, those in one are sensitive to nitrogen mustard, and mutants in six groups are hypersensitive to both mutagens. Eight of the ten loci mapped fall within 15% of the genetic map that encompasses the centromere of chromosome 3. Mutants from four of the complementation groups are associated with moderate to strong meiotic effects in females. Preliminary biochemical analyses have implicated seven of these loci in DNA metabolism.