Björn Brunström

Faced with inequality: chicken do not have a general dosage compensation of sex-linked genes

BackgroundThe contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes, and between sex chromosomes and autosomes. In many organisms, dosage compensation has thus evolved to equalize sex-linked gene expression in males and females. In mammals this is achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma.ResultsHere, we use a microarray approach to show that male chicken embryos generally express higher levels of Z-linked genes than female birds, both in soma and in gonads. The distribution of male-to-female fold-change values for Z chromosome genes is wide and has a mean of 1.4–1.6, which is consistent with absence of dosage compensation and sex-specific feedback regulation of gene expression at individual loci. Intriguingly, without global dosage compensation, the female chicken has significantly lower expression levels of Z-linked compared to autosomal genes, which is not the case in male birds.ConclusionThe pronounced sex difference in gene expression is likely to contribute to sexual dimorphism among birds, and potentially has implication to avian sex determination. Importantly, this report, together with a recent study of sex-biased expression in somatic tissue of chicken, demonstrates the first example of an organism with a lack of global dosage compensation, providing an unexpected case of a viable system with large-scale imbalance in gene expression between sexes.

The avian egg as a test system for endocrine disrupters: effects of diethylstilbestrol and ethynylestradiol on sex organ development.

Many environmental contaminants are known or suspected to interfere with hormonal function in animals. In vivo test methods to detect and characterize chemicals that disrupt the endocrine system are therefore urgently needed. In this study, we assessed the usefulness of abnormalities of the reproductive organs as test endpoints for estrogenic activity of xenobiotics in Japanese quail embryos. Two synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE2), were injected into the yolks of embryonated eggs. At a dose as low as 2 ng EE2/g egg, all male embryos became feminized, containing ovary-like tissue in the left testis. The extent of feminization of the testes was determined by measuring the relative area of the ovary-like component. Persistent Müllerian ducts (oviducts) in male embryos, and malformations of the Müllerian ducts in females occurred at 2 ng EE2/g egg and higher doses. DES was approximately one-third to one-tenth as potent as EE2. The morphological changes studied were dose-dependent, indicating that they are useful as test endpoints for estrogenic activity. Feminization of the left testis in males proved to be the most sensitive endpoint. We propose the quail egg as a simple in vivo test system for estrogenic compounds.

Toxicity and EROD-inducing potency of 24 polycyclic aromatic hydrocarbons (PAHs) in chick embryos

The toxicities (embryolethality) of 24 polycyclic aromatic hydrocarbons (PAHs) were determined in chick embryos using a 72-h test. The substances, dissolved in peanut oil, were injected into the air sacs of eggs preincubated for 7 days. LD50 values were determined for the four most toxic of the 24 compounds. Benzo [k] fluoranthene proved to be the most potent, with an LD50 of 14 μg (56 nmol)/kg egg. Dibenz[a,h]anthracene, benz[a]anthracene and benzo[b]naphtho[2,3-d]thiophene were a few times less toxic [LD50=39 μg (140 nmol)/kg, 79 μg (349 nmol)/kg and 82 μg (350 nmol)/kg, respectively]. The LD50 of benzo [k] fluoranthene was only about 5 times higher than that previously found for the most potent coplanar polychlorinated biphenyl (PCB), 3,3′,4,4′,5-pentachlorobiphenyl [LD50=3.1 μg (9.4 nmol)/kg], in the same kind of test. The toxicities of 18 of the PAHs in this study have also been evaluated previously using a 2-week test in chick embryos. Dibenz[a,h]anthracene, which had not been studied earlier in the 2-week test, proved to be almost as toxic as previously found for benzo [k] fluoranthene in that test. Several of the PAHs studied induced EROD activity in chick embryos, and, in general, the most toxic PAHs were also the most potent inducers of EROD. The highest enzyme activities were found after treatment with indeno[1,2,3-c,d]pyrene (12 times the control value) and dibenz[a,h]anthracene (8 times the control value). However, due to the high toxicity of dibenz[a,h]anthracene, the dose used was 7 times lower than that of indeno [1,2,3-c,d]pyrene. Following injection of PAHs on day 7, the EROD activities on day 10 were considerably lower than those obtained after a corresponding treatment with coplanar PCBs in an earlier study. Of the PAHs studied, some exhibited very high embryotoxicity. The most toxic PAHs induced EROD activity, suggesting that their toxicity was at least partly mediated via binding to the Ah receptor.

Toxicity of coplanar polychlorinated biphenyls in avian embryos

The toxicities of various polychlorinated biphenyls (PCBs) were measured in avian embryos. In chick embryos the non-ortho chlorine substituted coplanar PCBs proved to be very toxic (embryolethality and malformations). 3,3′,4,4′,5-Pentachlorobiphenyl (PeCB) had the highest embryotoxicity of the tested chlorobiphenyls and was the most potent inducer of 7-ethoxyresorufin O-deethylase (EROD) in chick embryos. Whereas 3,3′,4,4′-tetrachlorobiphenyl (TeCB) was only a few times less toxic than the pentachlorobiphenyl, the analogs of TeCB chlorinated at one ortho-position were 3–4 orders of magnitude less toxic than PeCB. Embryos of eight other avian species were considerably less sensitive than chick embryos to TeCB. In addition to this tetrachlorobiphenyl the coplanar pentachlorobiphenyl was less toxic in turkey embryos than in chick embryos, indicating that the interspecific differences in sensitivity to TeCB also are valid for other Ah receptor ligands.

Mono-ortho-chlorinated chlorobiphenyls : toxicity and induction of 7-ethoxyresorufin O-deethylase (EROD) activity in chick embryos

Six mono-ortho-chlorinated chlorobiphenyls were compared regarding their toxicity and 7-ethoxyresorufinO-deethylase (EROD)-inducing potency in chick embryos. Three of the tested chlorobiphenyls have a chloro substituent adjacent to theortho-chlorine, and these congeners were about ten times more potent than the three having ameta-hydrogen adjacent to theortho-chlorine. These more toxic mono-ortho-chlorinated congeners were, however, about three orders of magnitude less toxic and less potent as EROD inducers in chick embryos than 3,3′,4,4′,5-pentachlorobiphenyl in a previous similar study. Malformed eyes and beaks, degenerative hepatic lesions and subcutaneous as well as pericardial edema were detected in embryos exposed to the mono-ortho-chlorine-substituted congeners, as was previously found after exposure to the most toxic non-ortho-chlorinated, coplanar chlorobiphenyls. It is concluded that the monoortho-chlorinated chlorobiphenyls are considerably less toxic and less potent as EROD inducers than the most toxic coplanar ones. Owing to their relatively high concentrations in technical preparations of polychlorinated biphenyls (PCBs) the mono-ortho-chlorine-substituted congeners may, however, contribute to the overall toxicity of PCBs.

Ontogenetic Complexity of Sexual Dimorphism and Sex-Specific Selection

Sex-biased gene expression is becoming an increasingly important way to study sexual selection at the molecular genetic level. However, little is known about the timing, persistence, and continuity of gene expression required in the creation of distinct male and female phenotypes, and even less about how sex-specific selection pressures shift over the life cycle. Here, we present a time-series global transcription profile for autosomal genes in male and female chicken, beginning with embryonic development and spanning to reproductive maturity, for the gonad. Overall, the amount and magnitude of sex-biased expression increased as a function of age, though sex-biased gene expression was surprisingly ephemeral, with very few genes exhibiting continuous sex bias in both embryonic and adult tissues. Despite a large predicted role of the sex chromosomes in sexual dimorphism, our study indicates that the autosomes house the majority of genes with sex-biased expression. Most interestingly, sex-specific evolutionary pressures shifted over the course of the life cycle, acting equally strongly on female-biased genes and male-biased genes but at different ages. Female-biased genes exhibited high rates of divergence late in embryonic development, shortly before arrested meiosis halts oogenesis. The level of divergence on female-biased late embryonic genes is similar to that seen in male-biased genes expressed in adult gonads, which correlates with the onset of spermatogenesis. These analyses reveal that sex-specific selection pressure varies over the life cycle as a function of male and female biology.

Sex-dependent gene expression in early brain development of chicken embryos

BackgroundDifferentiation of the brain during development leads to sexually dimorphic adult reproductive behavior and other neural sex dimorphisms. Genetic mechanisms independent of steroid hormones produced by the gonads have recently been suggested to partly explain these dimorphisms.ResultsUsing cDNA microarrays and real-time PCR we found gene expression differences between the male and female embryonic brain (or whole head) that may be independent of morphological differentiation of the gonads. Genes located on the sex chromosomes (ZZ in males and ZW in females) were common among the differentially expressed genes, several of which (WPKCI-8, HINT, MHM non-coding RNA) have previously been implicated in avian sex determination. A majority of the identified genes were more highly expressed in males. Three of these genes (CDK7, CCNH and BTF2-P44) encode subunits of the transcription factor IIH complex, indicating a role for this complex in neuronal differentiation.ConclusionIn conclusion, this study provides novel insights into sexually dimorphic gene expression in the embryonic chicken brain and its possible involvement in sex differentiation of the nervous system in birds.

The Synthetic Progestin Levonorgestrel Is a Potent Androgen in the Three-Spined Stickleback (Gasterosteus aculeatus)

The use of progestins has resulted in contamination of aquatic environments and some progestins have in experimental studies been shown to impair reproduction in fish and amphibians at low ng L(-1) concentrations. The mechanisms underlying their reproductive toxicity are largely unknown. Some progestins, such as levonorgestrel (LNG), exert androgenic effects in mammals by activating the androgen receptor (AR). Male three-spined stickleback (Gasterosteus aculeatus) kidneys produce spiggin, a gluelike glycoprotein used in nest building, and its production is directly governed by androgens. Spiggin is normally absent in females but its production in female kidneys can be induced by AR agonists. Spiggin serves as the best known biomarker for androgens in fish. We exposed adult female sticklebacks to LNG at 5.5, 40, and 358 ng L(-1) for 21 days. Androgenic effects were found at LNG concentrations ≥40 ng L(-1) including induction of spiggin transcription, kidney hypertrophy, and suppressed liver vitellogenin transcription. These are the first in vivo quantitative data showing that LNG is a potent androgen in fish supporting the contention that androgenic effects of certain progestins contribute to their reproductive toxicity.

Improving environmental risk assessment of human pharmaceuticals.

This paper presents 10 recommendations for improving the European Medicines Agencys guidance for environmental risk assessment of human pharmaceutical products. The recommendations are based on up-to-date, available science in combination with experiences from other chemical frameworks such as the REACH-legislation for industrial chemicals. The recommendations concern: expanding the scope of the current guideline; requirements to assess the risk for development of antibiotic resistance; jointly performed assessments; refinement of the test proposal; mixture toxicity assessments on active pharmaceutical ingredients with similar modes of action; use of all available ecotoxicity studies; mandatory reviews; increased transparency; inclusion of emission data from production; and a risk management option. We believe that implementation of our recommendations would strengthen the protection of the environment and be beneficial to society. Legislation and guidance documents need to be updated at regular intervals in order to incorporate new knowledge from the scientific community. This is particularly important for regulatory documents concerning pharmaceuticals in the environment since this is a research field that has been growing substantially in the last decades.

Methods for studying xenoestrogenic effects in birds

The embryonated bird egg provides a simple whole organism test system that allows examination of xenoestrogenic effects at different levels of biological organisation. Test compounds are injected into the yolk, the albumen or the air chamber at defined stages of embryonic development. Bioavailability and embryonic exposure may be determined by autoradiography and image analysis. Females represent the heterogametic sex (ZW) and estrogens determine differentiation into the female phenotype in birds. Xenoestrogenic effects can be examined by markers of gene expression and anatomical or histological characterization of the gonads and tubular sex organs. Chicks may be raised to sexual maturity and examination of sexual behaviour and reproductive physiology performed. The Japanese quail is a suitable test organism due to its small size and early sexual maturation.