The interaction between X-rays and transposon mobility in Drosophila: hybrid sterility and chromosome loss

Genetic traits associated with P-M hybrid dysgenesis in Drosophila melanogaster were synergistically affected by X-rays. The interaction between damages induced by these two mutator systems was evident when sterility and X/Y chromosome loss were used as endpoints. No interaction was detected in partial chromosome loss, monitored by the loss of BS and y+ markers. The synergism in sterility, measured either as all-or-none or premature sterility (fecundity) was observed when male hybrids derived from different P strains fathers, namely Harwich or II2, were X-irradiated and the effects compared relative to similarly treated non-dysgenic hybrids. Brooding of sperm showed that the effects of ionizing radiation were ionizing radiation were dependent upon the stage of spermatogenesis during which cells were irradiated. The highly synergistic effect on sterility was found when either spermatids or spermatocytes, but not mature sperm, were irradiated with 550 rad of X-rays. These findings were consistent with the higher radiosensitivity of spermatocytes and spermatids to genetic damage and with the correlation between the incidence of sterility and aging of dysgenic hybrids. The latter observation was particularly evident in the case of Harwich P strain-derived male hybrids whose fertility was greatly reduced due to P element mobility. The synergistic effect of X-rays in these dysgenic hybrids resulted in the virtual abolition of the germ line, increasing the sterility from 50% of the untreated 9-10-day old males, to 85% of the treated males when spermatocytes were irradiated. The synergism observed between transposon mobility and ionizing radiation can best the attributed to an interaction between X-ray-induced and P element-induced chromosome breaks. This interpretation is consistent with the more than additive increase in X or Y chromosome loss in irradiated, Harwich P strain-derived hybrid sons. The induction of these events was 1.164% in dysgenic irradiated males as compared to 0.234% in X-irradiated nondysgenic hybrids and 0.40% in dysgenic untreated males. No synergism was observed in X/Y loss in hybrids derived from II2 P strain fathers where the frequency of the events due to P element mobility alone was only one tenth (0.037%) of that found in Harwich-derived hybrids.     

Trenimon-induced sex chromosome loss in Drosophila: different dose-responses for ring-X loss as compared to rod-X loss and Y-marker loss

Drosophila melanogaster males carrying either a ring- or a rod-shaped X-chromosome were injected or fed with Trenimon (triaziquone) at concentrations ranging from 5 X 10(-5) to 2 X 10(-2) mM. The F1 generation was assayed for the occurrence of total sex chromosome loss and of Y-chromosome markers. Sex-linked recessive lethal tests were carried out simultaneously. The data show that significant induction of ring-X loss occurs already at very low treatment concentrations (5 X 10(-5) -10(-4) mM) whereas rod-X loss or Y-marker loss is only seen at 2-5 X 10(-3) mM and higher. Induction of sex-linked recessive lethals is observed from 10(-4) -10(-3) mM on. These results add to existing evidence that loss of ring-X chromosomes, induced by some chemicals, may proceed by a mechanism different from the kind of events leading to chromosome breakage, as measured by rod-X loss and Y-marker loss.     

Influence of metabolic factors on the mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster

This paper describes the influence of changes in metabolic activity on the in-vivo mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster. A dose-dependent increase in mutagenicity was observed until a plateau value is reached which was increased only slightly after enzyme induction with Aroclor 1254, whereas induction with phenobarbital resulted in a decrease, especially when cyclophosphamide was applied by injection. Treatment of the adult males with inhibitors of the monoamine oxidase (MAO, EC 1.4.3.4), such as iproniazid (Ipr), benzimidazole or tryptamine, led to a marked increase of the mutagenic effectiveness of cyclophosphamide especially in spermatocytes. This indicates the importance of metabolic de-activation processes for the limited mutagenicity of cyclophosphamide in Drosophila. The principal active metabolite of cyclophosphamide, phosphoramide mustard, is extensively de-activated by enzymes that can be inhibited by 1-phenylimidazole (PhI), presumably cytochrome P-450 (EC 1.14.14.1), but not by those blocked by MAO inhibitors. Inhibition of the FAD-containing dimethylaniline monooxygenase (FDMAM, EC 1.14.13.8) by N,N-dimethylbenzylamine (N,N-DMB) resulted in some increase in cyclophosphamide mutagenicity only in spermatids. The marginal mutagenicity of cyclophosphamide in Drosophila larvae could not be increased either by cytochrome P-450 induction with phenobarbital or by MAO inhibition with Ipr. In contrast to the failure of cyclophosphamide to induce rod-chromosome loss, a considerable activity was found when a ring-shaped chromosome was used. Similar to the sex-linked recessive lethal (SLRL) test, ring-X loss frequency could be enhanced by simultaneous treatment with MAO inhibitors. The observed ring-X loss frequency declined when males treated with cyclophosphamide were mated to DNA-repair deficient mei-9L1 females. Cyclophosphamide produces chromosome breaks, detected as 2-3 translocations, in Drosophila spermatocytes, the stage in spermatogenesis that is also the most sensitive to the induction of SLRL mutations.     

A genetic study of the effects of the repair-deficient mei-9 mutation in drosophila on spontaneous and X-ray-induced paternal sex chromosome loss

The repair-deficient mutant, mei-9^a in Drosophila melanogaster was investigated regarding its effect on spontaneous and X-ray-induced chromosome loss in male postmeiotic cells. From matings of males carrying a mei-9^a or an ordinary ring-X and a doubly marked Y chromosome (B^SYy⁺) with mei-9^a or ordinary females, the spontaneous frequencies of complete loss, partial loss, and inferred ring-X loss (based on shifts in sex ratio female:male) were significantly higher with mei-9^a than with non-mei-9^a. When males were given 3000 rad X-irradiation, frequencies of induced partial loss, inferred ring-X loss and the reduction in the number of progeny per female were significantly greater with mei-9^a than with non-mei-9^a. The results provide evidence that the mei-9^a is a potentiator of both spontaneous and X-ray-induced chromosome lesions in sperm of the Drosophila male. Evidence is presented which implicates the presence of mei-9^a in the P₁ female and not the male as (at least) largely responsible for the characteristic mei-9^a effects.     

Studies on non-disjunction of the major autosomes in Drosophila melanogaster. I. Methodology and rate of induction by x-rays for the compound second chromosome

The induction of non-disjunction by X-irradiation of the second chromosome in stage-7 oocytes of Drosophila melanogaster has been studied by employing isochromosome stocks. This makes the quantitative recovery possible of progeny resulting from disomic and nullosomic eggs. Determination of egg hatchability has been used to correct for varying degrees of segregation in males carrying different isochromosomes. Even at exposures as low as 250 R the frequency of non-disjunction is significantly higher than in the controls. No evidence has been obtained for the existence of a threshold. In the stage-7 oocytes, the induction of non-disjunction increased linearly with radiation exposure over a range of 250–3000 R and thus seems to reflect a single-hit event. These findings could be of significance for the evaluation of genetic radiation hazards in man. In slightly younger oocyte stages the induction of disomic eggs followed dose-square kinetics. The frequency of nullosomic eggs rises exponentially with radiation exposure, presumably as a consequence of increasing chromosome loss resulting from unrestituted breaks in each of the two maternal isochromosomes. Furthermore, it was observed that the late stage-7 oocytes were more sensitivie to the induction of non-disjunction than earlier stages.     

Strong antimutagenic effects of fluoride on mutation induction by trenimon and 1-phenyl-3,3-dimethyltriazene in Drosophila melanogaster

After fluoride treatment of mature and immature oocytes of Drosophila females, a clear-cut dose-dependent decrease in fertility and fecundity was observed. The hatchability of mature oocytes was reduced by as much as 35%. When immature oocytes were treated, a pronounced dose-dependent reduction in fecundity occurred.

Exposure of mature sperm to NaF resulted in a slight decrease in fertility, comparable to the effect obtained with immature oocytes. Of the criteria used to measure possible mutagenic effects of NaF (sex-linked lethals, partial and total X- and Y-chromosome losses), only the rate of total losses was enhanced significantly.

The slight mutagenic effect of NaF on mature sperm was not related to the strong antimutagenic activity observed, when applied simultaneously with any of the several chemical mutagens. NaF treatment drastically reduced both the Trenimon-induced decrease in fertility and Trenimon-induced increases in recessive lethal mutation frequencies and rates of partial and total chromosome losses. The inhibitory effect of NaF was less pronounced with 1-phenyl-3,3-dimethyltriazene (PDT), a poor chromosome breaker in Drosophila, and absent for A 137, a weak mutagen which so far has failed to induce chromosomal aberrations in Drosophila. Therefore, the data are interpreted as being the result of a specific fluoride inhibition of chemically induced chromosomal breakage.

In mature and immature oocytes, the decreases in fertility and fecundity, and increase in recessive lethal frequency (mature oocytes) produced by Trenimon were also suppressed in the presence of fluoride. However, since Trenimon failed to produce a significant rise in X losses and NDJ in both stages, the effect of NaF on these mutational classes was, of course, not testable.     

Radiation-induced and transposon-induced chromosome damage in Drosophila: translocations and transmission distortion

The possible interaction between X-ray- and transposon-induced chromosome damage was monitored in the P-M system of hybrid dysgenesis in Drosophila melanogaster. One- to two-day-old F1 dysgenic males originating from a cross between M strain females and P strain males were irradiated with 5.5 Gy (550 rad) or used as controls to monitor X-Y translocations and transmission ratio distortion. Two 3-day sperm broods were sampled for the former and two 4-day broods for the latter to detect damage induced in the most radiosensitive cells. F1 nondysgenic males derived from M female to M male crosses (controls) were treated identically. X-Y chromosome translocations induced by P element mobility alone declined sharply with a decrease in temperature (18 versus 21 degrees C) and they were significantly reduced with aging of hybrid males from brood 2, 4-8 days of age, to brood 3, 7-11 days of age. No significant increase in translocations was observed when X irradiation and P-M dysgenesis were combined, showing no interaction between damages induced by the two mutator systems. In contrast, interaction was observed in transmission ratio distortion which was significantly increased by X irradiation of hybrid males derived from both reciprocal M X P and P x M crosses. The preferential elimination of P element-bearing autosomes occurred when either spermatocytes or spermatids were irradiated. An aging effect was also observed, resulting in less distortion in 9- to 14-day-old dysgenic males compared to 5- to 10-day-old hybrids.     

Mutagenicity of hycanthone in drosophila: Additional results and a comparison with some analogs

The data reported in this paper extend earlier results on the effects of hycanthone in Drosophila. The main findings are the following. (1) A refined brood-pattern analysis of hycanthone-induced sex-linked recessive lethals confirmed the specific sensitivity of mid- and late spermatids. Injection of young males (0–20 h old) did not cause a shift in the brood pattern, but tended to produce higher rates of recessive lethals than injection of 4-day-old males, although the difference was not significant. (2) An autosomal recessive lethal test (chromosome 2) similarly showed a low sensitivity of premeiotic stages. (3) Feeding of hycanthone was much less effective than injection. This difference was not observed for the methyl analog lucanthone. From the observation that hycanthone- and lucanthone-induced mutations exhibited different germ-cell-stage sensitivity patterns, it was concluded that lucanthone does not (at least not exclusively) act via metabolic activation to hycanthone. (4) After injection, the hycanthone analogs IA-4-N-oxide and IA-4-N-oxide were marginally mutagenic. (5) It was shown previously that hycanthone was ineffective in producing breakage events, in Drosophila. In this report, hycanthone is shown to be weakly active in inducing ring-X chromosome loss. This emphasizes the relat ive sensitivity of the ring-X-loss test, in comaprison with the tests that etect translocations or dominant lethals.     

Spermatogenesis in pharate adult testes of Drosophila in tissue cultures without ecdysones

The process of spermatogenesis in explanted testicular fragments from pharate adults (48 hr after puparium formation) of Drosophila melanogaster was examined under in vitro conditions without any added ecdysone substances. In the anterior fragments, which contained spermatogonia, no or only slight changes were found. In the middle fragments which contained germ cells at more advanced stages of spermatogenesis, spermatocytes, and spermatids, a slight increase in the number of spermatocytes or spermatids was observed. In the posterior fragments, which contained sperms at early stages of spermiogenesis, there was a marked elongation of the sperm bundles along their long axis.     

Evidence against a (2)n synchronous increase of spermatogonia to produce spermatocytes in drosophila hydei

The numbers of primary spermatocytes within cysts as well as numbers of postmeiotic spermatids in bundles in Drosophila hydei were determined. Within the contents of a single testis the cysts of primary spermatocytes are found to contain 5–11 germ cells. Furthermore, the number of spermatocytes per cyst is age-dependent, in that pupae have a mean of 8.1 cells whereas fertile adult males have a mean of 7.1 cells. Counts of spermatids in section of testes add further support to the view that the primary spermatocytes, from which the spermatids originated, were not formed in a strict geometric progression.     

Sex-ratio meiotic drive in Drosophila simulans is related to equational nondisjunction of the Y chromosome

The sex-ratio trait, an example of naturally occurring X-linked meiotic drive, has been reported in a dozen Drosophila species. Males carrying a sex-ratio X chromosome produce an excess of female offspring caused by a deficiency of Y-bearing sperm. In Drosophila simulans, such males produce approximately 70-90% female offspring, and 15-30% of the male offspring are sterile. Here, we investigate the cytological basis of the drive in this species. We show that the sex-ratio trait is associated with nondisjunction of Y chromatids in meiosis II. Fluorescence in situ hybridization (FISH) using sex-chromosome-specific probes provides direct evidence that the drive is caused by the failure of the resulting spermatids to develop into functional sperm. XYY progeny were not observed, indicating that few or no YY spermatids escape failure. The recovery of XO males among the progeny of sex-ratio males shows that some nullo-XY spermatids become functional sperm and likely explains the male sterility. A review of the cytological data in other species shows that aberrant behavior of the Y chromosome may be a common basis of sex-ratio meiotic drive in Drosophila and the signal that triggers differential spermiogenesis failure.     

Meichroacidin containing the membrane occupation and recognition nexus motif is essential for spermatozoa morphogenesis

Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.     

Pericentromeric structure of human X “isochromosomes”: evidence for molecular heterogeneity

Summary Three different long-arm X isochromosomes and an isodicentric X chromosome were examined by in situ hybridization with X-chromosome-specific -satellite probes and by quantitation of Southern blots hybridized with proximal short-arm probes. Each chromosome had a unique pericentromeric structure. The isodicentric X chromosome was clearly dicentric, showing two distinct -satellite hybridization signals and duplication of short-arm material. Two isochromosomes showed a larger than normal, bifid -satellite signal and also had duplications of different extents of short-arm material. The third X isochromosome could not be distinguished from a classical long-arm isochromosome; it did not have a short-arm duplication and it had a single -satellite signal. These data indicate that rearrangements responsible for X isochromosome formation can occur at numerous locations in the pericentromeric region and that some X isochromosomes may involve duplications of substantial portions of the short arm.     

Characterization of a maize isochromosome 8S*8S.

An isochromosome was found in the maize HiII Parent B line during somatic karyotyping with a multiprobe fluorescence in situ hybridization (FISH) system. Cytological analyses showed that it pairs with the short arm of chromosome 8 during the pachytene stage of meiosis. The chromosome 8 short arm origin of this isochromosome was also confirmed by FISH at mitotic metaphase. Knob heterochromatin signals were present at the short arms of chromosome 8 when subjected to prolonged exposure and also observed at both ends of the isochromosome. This isochromosome can be a univalent or a trivalent by pairing with the normal chromosome 8 short arms during meiosis. At anaphase and telophase, the isochromosome lagged behind other chromosomes. It had a transmission rate of 17%-20% from both male and female gametes. One plant homozygous for the isochromosome contained 2 isochromosomes that differed in the quantity of their CentC centromere repeat sequence. Both variations of the isochromosome were transmitted to the next generation. Because the 2 isochromosomes should be identical by descent, these observations document a radical change in copy number of the centromere repeat array within 1 generation. Plants with 1 isochromosome were not normal as compared with the original HiII Parent B plants. Those that contained a pair of this isochromosome (6 total copies of 8S) were even more abnormal and had reduced fertility. The results indicate the ability of the somatic karyotyping system to recognize and characterize chromosomal aberrations.     

Radiation and transposon-induced genetic damage in Drosophila melanogaster: X-ray dose-response and synergism with DNA-repair deficiency.

The interaction of X-ray-induced and transposon-induced damage was investigated in P-M hybrid dysgenesis in Drosophila melanogaster. The X-ray dose-response of 330-1320 rad was monitored for sterility, fecundity and partial X/Y chromosome loss among F2 progeny derived from the dysgenic cross of M strain females xP strain males (cross A) and its reciprocal (cross B), using a weaker and the standard Harwich P strain subline. The synergistic effect of P element activity and X-rays on sterility was observed only in cross A hybrids and the dose-response was nonlinear in hybrids derived from the strong standard reference Harwich subline, Hw. This finding suggests that the lesions induced by both mutator systems which produce the synergistic effect are two-break events. The effect of increasing dose on the decline of fecundity was synergistic, but linear, in hybrids of either subline. There was no interaction evident and thus no synergism in X/Y nondisjunction and in partial Y chromosome loss measured by the loss of the Bs marker alone or together with the y+ marker. Interaction was detected in the loss of the y+ marker alone from the X and Y chromosomes. The possible three-way interaction of X-rays (660 rad), post-replication repair deficiency and P element mobility was assessed by measuring transmission distortion in dysgenic males derived from the II2 P strain. X-Irradiation of spermatids significantly increased the preferential elimination of the P-element-bearing second chromosome in mei-41, DNA-repair-deficient dysgenic males, but had no effect in their DNA-repair-proficient brothers. These findings indicate that the post-replication repair pathway is required for processing lesions induced by the combined effect of P element mobility and X-rays, and that the unrepaired lesions ultimately lead to chromosome loss.     

The mutagenicity of azathioprine in mice, Drosophila melanogaster and Neurospora crassa.

The chemotherapeutic coumpound azathioprine was tested for possible mutagenicity in Swiss Albino mice, Drosophila melanogaster and Neurospora crassa. Utilizing the dominant-lethal assay it was found that acute oral doses of azathioprine (2 times 25 mg/kg body weight), induced dominant-lethal mutations in mouse spermatocytes. Chronic oral doses of azathioprine (2 times 25 mg/kg body weight/week for 10 weeks) resulted in a greater rate of dominant-lethality. This increase was not permanent, and by week 4 of gamete sampling there was no significant increase in dominant-lethal mutations. Histological sections showed that chronic treatment of male mice with azathioprine caused pyknosis of spermatocyte nuclei and depletion of the spermatid population. Both acute and chronic doses of azathioprine caused a temporary reduction in sperm viability. Oral treatment of male Canton-S, D. melanogaster with azathioprine caused an increase in dominant-lethality in broods assumed to correspond to spermatid and spermaotcyte stages. Azathioprine also increased the rate of non-disjunction of the X and Y chromosomes, loss of the long arm of the Y chromosome, and loss of the X or Y chromosome in treated male R(I)2, vf/BsYy+D. melanogaster. Since sex-ratio deviation did not occur in progeny from treated rod-X (yv/B2Yy+) male D. melanogaster, it was concluded that the observed sex-ration deviation in the treated ring-X stock was the result of induced ring-X lethality. Azathioprine induced recessive-lethal mutations in the ad-3 region of a N. crassa heterokaryon. In the host-mediated assay using this same heterokaryon and male Swiss Albino mice as host, the mutagenic activity of azathioprine did not appear to be potentiated or detoxified by the host. The results show that azathioprine has a deleterious effect on reproduction in mice and probably induces mutational events in mice, D. melanogaster and N. crassa.     

Determining the origin of human X isochromosomes by use of DNA sequence polymorphisms and detection of an apparent i(Xq) with Xp sequences

Summary The parental origin of five X isochromosomes were determined using 11 DnA markers. The isochromosome was derived from a maternal X chromosome in three cases and from a paternal X chromosome in two. Unexpected heterozygosity was detected for the proximal Xp region in one individual in whom the i(Xq) chromosome was paternally derived. This was confirmed by in situ hybridisation. A mode of formation of isochromosomes by breakage and reunion between the sister chromatids of the arms of an X chromosome is proposed to account for this. Sister chromatid breakage and reunion can be considered as a significant mechanism for the origin of i(Xq) chromosomes.     

X-ray induction of dominant lethals in late spermatids and mature spermatozoa of Drosophila melanogaster: the role of oxygenation

The role of oxygenation in determining the sensitivity to the induction of dominant lethals was examined in late spermatids and mature spermatozoa of Drosophila melanogaster. 0–2-h-old or 7-day-old Oregon-K males were irradiated with different X-ray exposures in nitrogen, air or in oxygen and the frequencies of dominant lethals induced in these stages were studied. The results obtained confirm and extend Sobels' earlier observations and the interpretation derived therefrom namely, that under normal conditions in air, mature spermatozoa are characterised by a higher degree of oxygenation than late spermatids and this difference is sufficient to explain the differential response of these stages. Similar Oxygen-Enhancement-Ratios(OERs) (of about 2) were obtained for both the cell stages. The present data also revealed that late spermatids are significantly less sensitive than mature spermatozoa to the X-ray-induction of dominant lethals in a nitrogen atmosphere. A plausible mechanism is suggested to explain this observation.     

Roles for Drp1, a dynamin-related protein, and milton, a kinesin-associated protein, in mitochondrial segregation, unfurling and elongation during Drosophila spermatogenesis

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post-meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and that is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, we used the FLP-FRT mitotic recombination system to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. We found that absence of Drp1 leads to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. Our work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation.