Participation of androgen and its receptor in sex determination of an amphibian species.

INTRODUCTION: In the Japanese frog Rana (R.) rugosa the androgen receptor (AR) gene on the W chromosome (W-AR) is barely expressed. Previously we showed that incomplete female-to-male sex-reversal occurred in Z-AR transgenic female frogs. To date, however, there is no report showing that AR with androgens can determine genetically programed male sex fate in any vertebrate species. Here, we examined whether AR together with androgens functions as a sex determinant in an amphibian species. METHODS: To examine whether complete female-to-male sex-reversal occurs in R. rugosa frogs, we produced AR-transgenic (Tg) and -knockdown (KD) female R. rugosa frogs by the I-SceI meganuclease-mediated gene trap and CRISPR/Cas9 system, respectively. AR-Tg and -KD tadpoles were reared in water containing testosterone (T) at 0 to 7.1 ng/ml. Frozen sections were prepared from the gonads of metamorphosed frogs and immunostained for laminin, Vasa, Pat1a, CYP17 and AR. We also employed PCR analysis to examine Dmrt1, Pat1a and CYP17 expression in the gonads of KD and placebo-KD female frogs. RESULTS: Complete female-to-male sex-reversal occurred in the AR-Tg ZW female frogs when a low dosage of T was supplied in the rearing water of tadpoles. However, no sex-reversal was observed in AR-KD ZW female frogs when the gonads were treated with dosages of T high enough to induce complete female-to-male sex-reversal even in wild type frogs. DISCUSSION: These results suggest that AR with its androgen ligand functions as a male sex-determinant in the ZW type R. rugosa frogs.

Elevated testosterone during meiotic segregation stimulates laying hens to produce more sons than daughters.

Biases in avian sex ratios have been documented in relation to a variety of social and environmental conditions. Previous studies suggest that treatment with hormones can stimulate females to manipulate offspring sex, and that this effect occurs before ovulation. For example, acute and chronic treatments with testosterone stimulated significant skews towards male offspring. Hormones may act by influencing which sex chromosome is donated by the heterogametic female bird into the oocyte. However, it is difficult to pinpoint when effects of testosterone on offspring sex occurred in previous experiments because testosterone treatments were given either chronically over the entire period of follicular development or many hours before the critical period of chromosome segregation. We injected laying hens with testosterone injections 5 h prior to ovulation to target this critical period and quantified the sexes of the subsequently ovulated eggs. We hypothesized that an injection of testosterone coincident with segregation of sex chromosomes would stimulate hens to produce more male than female offspring. As hypothesized, hens injected with testosterone produced a significant bias towards male offspring compared to controls, nearly 70%. These results suggest that acute testosterone elevation during meiotic segregation may mediate skews in avian primary sex ratios.

Acute corticosterone administration during meiotic segregation stimulates females to produce more male offspring.

Birds have demonstrated a remarkable ability to manipulate offspring sex. Previous studies suggest that treatment with hormones can stimulate females to manipulate offspring sex before ovulation. For example, chronic treatments with corticosterone, the primary stress hormone produced by birds, stimulated significant skews toward female offspring. It has been suggested that corticosterone acts by influencing which sex chromosome is donated by the heterogametic female bird into the ovulated ovarian follicle. However, it is difficult to pinpoint when in developmental time corticosterone affects offspring sex, because in previous studies corticosterone treatment was given over a long period of time. We treated laying hens with acute high-dose corticosterone injections 5 h before the predicted time of ovulation and quantified the sexes of the subsequently ovulated eggs to determine whether mechanisms exist by which corticosterone can skew offspring sex ratios just before ovulation. We hypothesized that an injection of corticosterone coincident with segregation of the sex chromosomes would stimulate hens to produce more female than male offspring. Contrary to our predictions, hens injected with corticosterone produced a significant bias toward male offspring, nearly 83%. These results suggest that acute corticosterone treatment during meiosis I can influence primary sex ratios in birds, potentially through nonrandom chromosome segregation. Furthermore, acute corticosterone exposure, compared with chronic exposure, may act through different mechanisms to skew offspring sex.

Genotoxic effects on human spermatozoa among pesticide factory workers exposed to fenvalerate.

Fenvalerate, a synthetic pyrethroid insecticide, is widely produced and used worldwide. To explore fenvalerate-induced genotoxic effects, particularly numerical chromosome aberration (CA), we firstly examined conventional semen parameters, the progression and motion parameters of the spermatozoa among 12 fenvalerate-exposed workers and 30 donors of the internal and external control groups. Then numerical CA of chromosome X, Y and 18 were investigated by multicolor fluorescence in situ hybridization (FISH). The results showed the significant differences in the percentage of sperm abnormality between fenvalerate-exposed group and the external control group (P = 0.024). In aneuploid parameters, the frequency (mean +/- S.D.) of sex chromosome disomy was 0.742 +/- 0.131% in fenvalerate-exposed group, which was significantly higher than those in the internal (0.563 +/- 0.135%) and external control group (0.386 +/- 0.140%) (P < 0.01), and the frequency of chromosome 18 disomy in fenvalerate-exposed group (0.326 +/- 0.069%) was significantly higher than those in the internal and external control groups (0.195 +/- 0.094% and 0.124 +/- 0.068%), respectively (P < 0.01). We also found the nullisomies of sex chromosomes and chromosome 18 were significantly higher than those in the external control group and two control groups, respectively (P < 0.01). The frequencies of aneuploidy and numerical CA we detected also showed significant differences between exposed group and control groups (P < 0.05 and/or P < 0.01). Moreover, we found the positive correlation not only between nullisomic frequencies of these chromosomes and numerical CA rate (r > 0.70, P < 0.01) but also between disomic frequency of sex chromosomes, aneuploidy rate and sperm abnormality in all donors (r = 0.530 and r = 0.536, P < 0.01). Our findings suggest that fenvalerate or its metabolites induced morphologic abnormality and genotoxic defects of spermatozoa among fenvalerate-exposed workers by causing numerical CA in spermatogenesis as a special and potential genotoxic agent.

Interstrand crosslink-induced radials form between non-homologous chromosomes, but are absent in sex chromosomes.

Fanconi anemia (FA) and cells lacking functional BRCA1 and BRCA2 proteins are hypersensitive to interstrand crosslinking (ICL) agents and show increased numbers of chromosomal breaks and radials. Although radial formation has been used to diagnose FA for more than 30 years, there has been little analysis of these characteristic formations. In this study, radials were analyzed from FA-A and FA-G fibroblasts as well as normal and retrovirally-corrected FA-A fibroblasts treated with high doses of ICLs. Radials were found to only involve non-homologous chromosome interactions and to be distributed nearly randomly along the length of chromosomes. Sites on chromosomes that did show increased frequency of radial involvement did not correlate with known fragile sites or pericentric regions. Hybrid radials were observed between mouse and human chromosomes in human-mouse hybrid cells produced by microcell-mediated chromosome transfer of mouse chromosomes into human FA-A fibroblasts. Both X and Y chromosomes were notably not involved in radials. These observations suggest that ICL repair may involve short stretches of homology, resulting in aberrant radial formation in the absence of FA proteins.

Detection of mitotic recombination and sex chromosome loss induced by adriamycin, chlorambucil, demecolcine, paclitaxel and vinblastine in somatic cells of Drosophila melanogaster in vivo.

The conventional w/w+ eye assay in Drosophila has been used for the last 10 years for genotoxic evaluation of a broad number of chemicals with different mechanisms of action. Although chemicals that induce genotoxic effects by mechanisms other than covalent binding to DNA are difficult to detect. The aim of this study was the parallel detection of both mitotic recombination and X chromosome loss induced by five chemical compounds used worldwide as antineoplastic drugs using the w/w+ somatic assay in Drosophila melanogaster. The compounds tested were the intercalating agent adriamycin (AD), the alkylating compound chlorambucil (CAB) and the spindle poisons demecolcine (DEM), paclitaxel (taxol, TX) and vinblastine (VBL). We used a cross between heterozygous females with a rod-X and a ring-X chromosome mated with ywf males. All four genotypes in the next generation are heterozygous or hemizygous for the w+ reporter gene and were inspected for the occurrence of white clones in their compound eyes. We found differences in the induction of mitotic recombination when compared with chromosome loss. Genotoxic profiles obtained for the antineoplastic drugs studied indicate direct and indirect effects. While AD seems to be clastogenic due to its induction of X chromosome loss in XrX females; DEM, CAB and TX produced both structural chromosome aberrations through clastogenic activities and mitotic recombination through DNA interactions; the cytotoxic VBL induced rX loss only in XrY and intra-chromosomal recombination (XY) males, probably due to sister strand recombination, generating a w+w+ duplication and a w- deletion, forward mutations or small deletions at the white locus.

Effect of hydroquinone on meiotic segregation in Drosophila melanogaster females.

The induction of sex chromosomes meiotic nondisjunction (ND) by hydroquinone (HQ) given orally was investigated in Drosophila melanogaster 2-7, 8-22, 24, 48, 72 and 96 h-old females. ND was assessed by a system where exceptional females (XXY) and only 1/4 of the expected regular progeny are viable. Oocytes were treated at different stages of development. 4% HQ tested only in 72 h-old females induced ND in oocytes sampled in brood I (mostly mature oocytes at metaphase I). 6% HQ increased ND in brood I of 8-22 h-old females, while other broods, (including cells treated at early prophase) were also affected in older flies, the highest significance being attained in the 48 h-old series. Newly hatched females (2-7 h-old) were refractory to the treatment, though oocytes sampled in the first three subcultures are comparable to cells showing enhancement of ND in series run with older females. Toxicity of 2, 4 and 6% HQ increased with concentration and females' age: (a) 2% was not toxic; (b) 4% was toxic only to 72 h-old females; (c) 6% was increasingly toxic to females 24, 48 and 72 h-old. The results indicate that age plays a significant role on both chromosomal segregation and toxicity and suggest that in Drosophila HQ is metabolized to its reactive species. The lack of toxic and aneugenic effect in very young females could reflect a more efficient detoxification due to the known high specific activity of glutathione-S-transferase (GST) after eclosion. The decline in GST activity around day 2 of adult life coincides with the high effect of HQ in 48 h-old females.

Effects of dibutyl phthalate as an environmental endocrine disruptor on gonadal sex differentiation of genetic males of the frog Rana rugosa.

To examine the effects of dibutyl phthalate (DBP) on gonadal sex differentiation, genetically male tadpoles of Rana rugosa were exposed to dilute solutions of DBP at concentrations of 0.1, 1, or 10 microM during days 19-23 after fertilization, which is the critical period of gonadal sex differentiation in R. rugosa. Tadpoles were necropsied on day 40. The genetically male tadpoles were produced from crossings between males (ZZ) of one local population, in which females are the heterogametic sex, and females (XX) of another local population, in which males are the heterogametic sex. As positive control groups, tadpoles were exposed to dilute solutions of 17beta-estradiol (E(2)) at concentrations of 0. 01, 0.1, or 1 microM during the same period. The internal structure of the gonads was histologically examined in a total of 30 control tadpoles, 86 E(2)-treated tadpoles, and 90 DBP-treated tadpoles. The gonads of the control tadpoles all showed the typical structure of testes. In contrast, 0.01, 0.1, and 1 microM E(2) treatments caused the undifferentiated gonads of 18, 63, and 100% of the tadpoles, respectively, to develop into gonads of complete or partial ovarian structure. After 0.1, 1, and 10 microM DBP treatment, 0, 7, and 17% of tadpoles, respectively, were similarly affected. These findings suggest that DBP was about 1,000-fold less potent than E(2). Nevertheless, DBP is an environmentally dangerous hormone that disrupts the pathways of testicular differentiation in genetically male animals.

Experimental condensation inhibition in constitutive and facultative heterochromatin of mammalian chromosomes.

What drives the dramatic changes in chromosome structure during the cell cycle is one of the oldest questions in genetics. During mitosis, all chromosomes become highly condensed and, as the cell completes mitosis, most of the chromatin decondenses again. Only chromosome regions containing constitutive or facultative heterochromatin remain in a more condensed state throughout interphase. One approach to understanding chromosome condensation is to experimentally induce condensation defects. 5-Azacytidine (5-aza-C) and 5-azadeoxycytidine (5-aza-dC) drastically inhibit condensation in mammalian constitutive heterochromatin, in particular in human chromosomes 1, 9, 15, 16, and Y, as well as in facultative heterochromatin (inactive X chromosome), when incorporated into late-replicating DNA during the last hours of cell culture. The decondensing effects of 5-aza-C analogs, which do not interfere with normal base pairing in substituted duplex DNA, have been correlated with global DNA hypomethylation. In contrast, decondensation of constitutive heterochromatin by incorporation of 5-iododeoxyuridine (IdU) or other non-demethylating base analogs, or binding of AT-specific DNA ligands, such as berenil and Hoechst 33258, may reflect an altered steric configuration of substituted or minor-groove-bound duplex DNA. Consequently, these compounds exert relatively specific effects on certain subsets of AT-rich constitutive heterochromatin, i.e. IdU on human chromosome 9, berenil on human Y, and Hoechst 33258 on mouse chromosomes, which provide high local concentrations of IdU incorporation sites or DNA-ligand-binding sites. None of these non-demethylating compounds affect the inactive X chromosome condensation. Structural features of chromosomes are largely determined by chromosome-associated proteins. In this light, we propose that both DNA hypomethylation and steric alterations in chromosomal DNA may interfere with the binding of specific proteins or multi-protein complexes that are required for chromosome condensation. The association between chromosome condensation defects, genomic instability, and epigenetic reprogramming is discussed. Chromosome condensation may represent a key ancestral mechanism for modulating chromatin structure that has since been realloted to other nuclear processes.

Genotoxicity of two arsenic compounds in germ cells and somatic cells of Drosophila melanogaster.

Two arsenic compounds, sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4), were tested for their possible genotoxicity in germinal and somatic cells of Drosophila melanogaster. For germinal cells, the sex-linked recessive lethal test (SLRLT) and the sex chromosome loss test (SCLT) were used. In both tests, a brood scheme of 2-3-3 days was employed. Two routes of administration were used for the SLRLT: adult male injection (0.38, 0.77 mM for sodium arsenite; and 0.54, 1.08 mM for sodium arsenate) and larval feeding (0.008, 0.01, 0.02 mM for sodium arsenite; and 0.01, 0.02 mM for sodium arsenate). For the SCLT the compounds were injected into males. Controls were treated with a solution of 5% sucrose which was employed as solvent. The somatic mutation and recombination test (SMART) was run in the w+/w eye assay as well as in the mwh +/+ flr3 wing test, employing the standard and insecticide-resistant strains. In both tests, third instar larvae were treated for 6 hr with sodium arsenite (0.38, 0.77, 1.15 mM), and sodium arsenate (0.54, 1.34, 2.69 mM). In the SLRLT, both compounds were positive, but they were negative in the SCLT. The genotoxicity of both compounds was localized mainly in somatic cells, in agreement with reports on the carcinogenic potential of arsenical compounds. Sodium arsenite was an order of magnitude more toxic and mutagenic than sodium arsenate. This study confirms the reliability of the Drosophila in vivo system to test the genotoxicity of environmental compounds.

[Synaptonemal complexes of chromosomes of male mice after prolonged administration of neoaquasept]. / Issledovanie sinaptonemnykh kompleksov khromosom samtsov myshei posle dlitel'nogo vvedeniia neoakvasepta.

A long-term (during 4 months) daily peroral injections of neoaquasept (NA) (a drinking water disinfections agent) at a dose corresponding to the normal level of consumption increases the number of spermatocytes in which the sexual bivalent associates with normal autosomal bivalents (up to 18.2%). In single cells a premature desynapsis of sex chromosomes at late pachytene was observed. There was the appearance of single cells with the X0 karyotype. A 3-fold excess of the normal consumption of NA causes an increase in the number of cells with the above disturbance in the SC structure and behaviour and causes the appearance of heteromorphic bivalents. On the 7th day after a single MTD injection of NA male mice displayed a sharp (up to 27.9%) increase in the number of cells in which the sexual bivalent associated with autosomal bivalents, formation of univalents of autosomal and sex chromosomes, SC degeneration.

Chrysotile and amosite asbestos induce germ-line aneuploidy in Drosophila.

Asbestos toxicity is a problem of considerable public concern and debate, however little is known regarding the biological targets of asbestos fibers. Prompted by reports that asbestos induces aneuploidy in cultured mammalian cells, we have investigated whether asbestos induces germ-line aneuploidy in Drosophila melanogaster. Using the ZESTE genetic test system, we have shown that both chrysotile and amosite asbestos induce sex-chromosome aneuploidy in Drosophila oocytes. Chrysotile appeared to be the more effective agent because it induced approximately equal frequencies of chromosome gain and chromosome loss, while amosite induced chromosome loss only. Two other asbestiform minerals, crocidolite and tremolite, were ineffective in this assay system. These results suggest that possible germ-line effects of asbestos should be considered in evaluating its potential impact on human health.

Aneuploidy in Drosophila, IV. Inhalation studies on the induction of aneuploidy by nitriles.

The Drosophila ZESTE system was used to monitor the induction of sex chromosome aneuploidy following inhalation exposure of adult females to four nitriles: acetonitrile, propionitrile, acrylonitrile and fumaronitrile. Acetonitrile and propionitrile were highly effective aneuploidogens, inducing both chromosome loss and chromosome gain following brief exposures to low concentrations of these chemicals, and these nitriles also induced rapid paralysis. Acrylonitrile-induced chromosome loss only but did not induce paralysis. Fumaronitrile, in contrast with the results reported in yeast, was ineffective in inducing chromosome loss or gain. Virtually all exceptional offspring induced by acetonitrile and propionitrile were recovered in the first sampled eggs, corresponding to treated mature oocytes. Additionally, the time interval between treatment and sampling was shown to be important, suggesting rapid loss or detoxification of the nitriles. Genetic analysis demonstrated that most aneuploids resulted from induced segregation errors during the first division of meiosis. Cold treatments were found to be ineffective in enhancing the effects of acetonitrile, suggesting important differences between the Drosophila and yeast aneuploidy detection systems. Possible mechanisms by which nitriles may disrupt chromosome segregation in Drosophila oocytes are considered.

Mutagenic evaluation of the organophosphorus insecticides methyl parathion and triazophos in Drosophila melanogaster.

The possible genotoxic effects of the organophosphorus insecticides methyl parathion and triazophos were evaluated by their ability to induce gene and chromosome mutations in male germ cells of Drosophila melanogaster. Sex-linked recessive lethal (SLRL), total and partial sex-chromosome losses (SCL), and non-disjunction (ND) assays were conducted. The routes of administration included adult feeding, injection, and larval feeding. Methyl parathion was unable to induce point mutations or chromosome mutations, although a small increase in the frequency of non-disjunction was detected after larval treatment. Triazophos induced point mutations when assayed in the SLRL test and induced a weak increase in the non-disjunction frequency, but gave negative results in the SCL test.

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.

The TX;Y test for the detection of nondisjunction and chromosome breakage in Drosophila melanogaster. II. Results of female exposures.

The TX; Y test is a short-term assay for the detection of sex-chromosome nondisjunction and chromosome breakage in Drosophila melanogaster. It has been used in previous work following the exposure of males. In this work, females are exposed. When females are the exposed parent, only chromosome gain can be detected. Positive results for the induction of aneuploidy were obtained following exposures of females to X-rays, 10 degrees C cold shock, and colchicine. No increase in aneuploidy was obtained following exposures of females to DMSO and trifluralin. Comparison with similar work in males reveals no consistent pattern concerning the more appropriate sex to use for aneuploidy testing in Drosophila, as colchicine was found to be positive in females only and DMSO and trifluralin were effective in males only. Further work is necessary to validate the TX; Y test and to understand the relative efficacy of female and male exposures to aneuploidy inducing agents in Drosophila.

Mutagenicity of dimecron studied in 3 mosaic assays and the sex-linked recessive lethal test in Drosophila melanogaster.

The genotoxicity of dimecron, a systemic organophosphate pesticide, has been tested in the wing, eye and germ line mosaic assays and the sex-linked recessive lethal test in Drosophila melanogaster. Larvae heterozygous for recessive marker mutations were fed the compound for various periods of time. On emergence, the wings and eyes of the adults were screened for mosaic spots and the eggs laid by the females were checked for induction of female germ line mosaicism. Dimecron is mutagenic to the somatic and germ line cells of Drosophila and induces a high frequency of sex-linked recessive lethals.

Non-mutagenicity of fenvalerate in Drosophila.

The induction of genetic damage in germ cells of Drosophila melanogaster by the pyrethroid insecticide fenvalerate was studied. Adult feeding, larval feeding and adult injection were the routes of administration used. Our results indicate that, under the conditions of testing, fenvalerate is unable to induce sex-linked recessive lethals, sex-chromosome losses and non-disjunction.

Indication for weak mutagenicity of the organophosphorus insecticide dimethoate in Drosophila melanogaster.

The organophosphorus insecticide dimethoate was tested for induction of genetic damage in male germ cells of Drosophila melanogaster. Sex-linked recessive lethals, sex-chromosome loss and non-disjunction induction were studied following different routes of administration: adult feeding, injection and larval feeding. Our results show that, after injection, dimethoate induces a slight but significant increase in the frequency of point mutations.

Mutagenicity testing of the pyrethroid insecticide cypermethrin in Drosophila.

The pyrethroid insecticide cypermethrin was tested for the induction of genetic damage in male germ cells of Drosophila melanogaster. Sex-linked recessive lethals, sex-chromosome loss and non-disjunction were studied following different routes of administration: adult feeding, injection and larval feeding. Our results show that, after adult injection and larval ingestion, cypermethrin induces a small but significant increase in the frequency of sex-linked recessive lethal mutations. However, no significant increases were observed in the frequency of sex chromosome loss or non-disjunction after exposure of male flies to cypermethrin at concentrations up to 20 p.p.m.