Sex-role reversal is reflected in the brain of African black coucals (Centropus grillii)

In most bird species males compete over access to females and have elevated circulating androgen levels when they establish and defend a breeding territory or guard a mate. Testosterone is involved in the regulation of territorial aggression and sexual display in males. In few bird species the traditional sex-roles are reversed and females are highly aggressive and compete over access to males. Such species represent excellent models to study the hormonal modulation of aggressive behavior in females. Plasma sex steroid concentrations in sex-role reversed species follow the patterns of birds with "traditional" sex-roles. The neural mechanisms modulating endocrine secretion and hormone-behavior interactions in sex-role reversed birds are currently unknown. We investigated the sex differences in the mRNA expression of androgen receptors, estrogen receptor alpha, and aromatase in two brain nuclei involved in reproductive and aggressive behavior in the black coucal, the nucleus taeniae and the bed nucleus of the stria terminalis. In the bed nucleus there were no sex differences in the receptor or aromatase expression. In the nucleus taeniae, however, we show for the first time, that females have a higher mRNA expression of androgen receptors than males. These results suggest that the expression of agonistic and courtship behavior in females does not depend on elevated blood hormone levels, but may be regulated via increased steroid hormone sensitivity in particular target areas in the brain. Hence, aggression in females and males may indeed be modulated by the same hormones, but regulated at different levels of the neuroendocrine cascade.     

Sex reversal in medaka treated in vitro with 17alpha-methyldihydrotestosterone during oocyte maturation

Using the S-rR strain of the medaka Oryzias latipes, we examined the effect of a non-aromatizable androgen on sex determination. Intrafollicular immature oocytes isolated before breakdown of the germinal vesicle were incubated in the presence of 17alpha-methyldihydrotestosterone (MDHT) for about 10 h during their maturational period. At the end of incubation, mature oocytes were rinsed and then artificially inseminated in regular saline. The fertilized eggs were then allowed to develop in tap water, and the fry were reared on a regular powdered diet until adulthood. Sex reversal of female to male was observed in a manner dependent on the dose of MDHT. In the solvent control group in which intrafollicular oocytes were matured in medium containing no exogenous androgen, no sex reversal was observed. The present finding, that the sex of medakas can be reversed by a single in vitro exposure of immature oocytes to androgen during the preovulatory period, suggests the existence in the oocyte of a sex determinant sensitive to sex steroids. This method for controlling the sex of eggs before fertilization may establish sex-determined eggs as potent material for investigating the mechanism of sex determination in the medaka.     

Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use

Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex‐linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex‐reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress‐induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North‐Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female‐to‐male sex‐reversed adults had similar body mass as normal males. We recorded no events of male‐to‐female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human‐induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex‐reversed individuals surviving to adulthood may participate in breeding.     

Induced sex reversal in Tilapia nilotica (Cichlidae) with methyl testosterone

Gonadally undifferentiated Tilapia nilotica fry were fed with methyl testosterone at the dosage of 50 mg/kg of dry diet. The fry used in the investigation belonged to two age groups with a mean total length of 10 mm and 13.5 mm. Treatment lasted for a minimum of four weeks and a maximum of six weeks. 100% males were obtained in all the various groups under androgen treatment.     

Novel function of the skin in calcium metabolism in female and male chickens (Gallus domesticus)

To study the role of the skin in differential calcium metabolism in White Leghorn chickens, we compared the composition of suction blister fluid (SBF) collected from cutaneous blisters with blood and serum in female and male animals in various physiological states. As an estimate for interstitial fluid (IF), SBF was used as a determinant of local cutaneous metabolism. Sample collection was carried out under ketamine–xylazine anesthesia. Eight chickens of both sexes were raised freely in similar environmental conditions and fed with similar food during their growth from juvenile to sexually mature and fully adult state. SBF, blood and serum were examined for concentrations of ionized Ca²⁺, Na⁺ and K⁺ with ion-selective electrodes (ISEs), and osmolalities by freezing point osmometry. pH and total protein content were also assessed. Our results showed that SBF of chickens is calcium-poor at the juvenile state and that it draws more Ca²⁺ in adult males than laying females of the same age. Interestingly, Ca²⁺ accumulation was observed also in females after laying had ceased. There was a positive correlation between blood and SBF Ca²⁺ in females but a negative one in males. In general, it was found that SBF of chickens was rich in Na⁺ and K⁺, was hypertonic compared to serum at the juvenile state and had a protein content of 36–47% of that in serum. Different from mammals, SBF in adult chickens was alkaline with the mean values of 8.7 ± 0.14 in females and 8.8 ± 0.06 in males. Age- and sex-related variability in cutaneous Ca²⁺ concentrations in chickens, and the differences of SBF composition between that of mammals point to a novel role of skin functions in avians. Possible functions of the skin as a dynamic calcium source balancing the free circulating Ca²⁺ levels and, also, as an excretory organ for Ca²⁺ are discussed.     

Estradiol,progesterone, and sex hormone-binding globulin in female rhesus monkeys (Macaca mulatta)

We measured the concentrations of estradiol, progesterone, and the sex hormone-binding globulin capacity (rhSHBG) in serum of female rhesus monkeys (Macaca mulatta). Although the serum rhSHBG capacity was altered by the removal of ovarian hormones, presumably estradiol, acute changes in serum estradiol and progesterone did not influence SHBG capacity. There appears to be a relatively low threshold for the effect of estradiol on rhSHBG capacity. The threshold must be present for a finite length of time to have that effect.     

Interaction of estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in immature male chickens (Gallus domesticus)

The interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and estrogen was studied in chickens to more clearly define this relationship in an avian species and its role in the enhanced sensitivity of female chickens to TCDD-induced wasting syndrome. Twenty male chickens (7-9 weeks old) were divided evenly into four groups: control (CTL, received the same volume of vehicle); estrogen-treated (E2, 1 mg/kg estradiol cypionate injections on days 1, 2 and 3); TCDD-treated (TCDD, single 50 microg/kg injection on day 4); and estrogen plus TCDD (E2+TCDD, as above), with measurements taken on day 14. The E2 group compared with the CTL group had decreased comb height (24%), comb length (26%) and adipose tissue (AT) lipoprotein lipase (LPL) activity relative to AT mass (51%), while liver mass and body weight gain were each increased by 28%. The TCDD group had increased liver mass (62%), reduced comb length (17%), and reduced AT LPL activity indexed to AT mass (70%) compared with the CTL group. Finally, the E2+TCDD group had 37% lower body weight gain and 30% larger livers relative to body mass compared with the E2 group, but were not significantly different from the TCDD group. These data show that TCDD antagonized several effects of exogenous estrogen in male chickens, while estrogen enhanced TCDD toxicity in a tissue-specific manner.     

Occurrence and HAT-RAPD analysis of gastrointestinal helminths in domestic chickens (Gallus gallus domesticus) in Phayao province,northern Thailand

The present study determined the prevalence and distribution of gastrointestinal helminths in domestic chickens (Gallus gallus domesticus) between November 2012 and August 2013. One hundred and twenty domestic chickens were purchased from villages in four districts of Phayao province; Mae Chai, Dok Khamtai, Chun and Chiang Kham. Morphological differences were used to identify the helminth species, and HAT-RAPD technique was used to differentiate among closely related species. The results revealed that the total prevalence of infection was 99.2%. Cestode and nematode infections showed the highest prevalence in rainy season, while trematode infections were low and only found in hot season. The species and their prevalence were: Ascaridia galli (50.8%), Heterakis gallinarum (86.7%), Prosthogonimus macrorchis (1.7%), Echinostoma revolutum (0.8%), Raillietina echinobothrida (48.3%), Raillietina tetragona (57.5%), Raillietina cesticillus (12.5%), Raillietina sp. (35.8%), Cotugnia chiangmaii (14.2%) and Cotugnia sp. (32.5%). The prevalence of helminth infections did not differ significantly between male and female chickens. HAT-RAPD analysis, the specific fragment of 400 and 250 bp indicated that Raillietina sp. and Cotugnia sp. found, respectively, differ from other closely related species. This study has confirmed that HAT-RAPD technique can be used to differentiate among related species combined with morphological observations.     

Autosomal inheritance of phosphoglycerate kinase in the domestic chicken (Gallus domesticus)

Electrophoretic variants of the glycolytic enzyme phosphoglycerate kinase (PGK) in the domestic chicken, Gallus domesticus, were found to be inherited in an autosomal manner. No sperm-specific isozyme of PGK exists in the chicken. This is in direct contrast to previous results from therian (placental and marsupial) mammals in which there are two isozymes of PGK, an autosomal form largely or wholly confined to sperm and a sex-linked form found in most other tissues. The implications of these and other results for the question of homology between the avian Z chromosome and the mammalian X chromosome are discussed. It is concluded that there is no evidence of homology between the X chromosome and the Z chromosome.This work was supported by the Australian Research Grants Committee and Macquarie University Research Grants.This work forms part of the Honours degree of B.A. presented to Macquarie University by A. E. C.     

A newly developed genetic sex marker and its application to understanding chemically induced feminisation in roach (Rutilus rutilus)

Oestrogenic wastewater treatment works (WwTW) effluents discharged into UK rivers have been shown to affect sexual development, including inducing intersex, in wild roach (Rutilus rutilus). This can result in a reduced breeding capability with potential population level impacts. In the absence of a sex probe for roach it has not been possible to confirm whether intersex fish in the wild arise from genetic males or females, or whether sex reversal occurs in the wild, as this condition can be induced experimentally in controlled exposures to WwTW effluents and a steroidal oestrogen. Using restriction site‐associated DNA sequencing (RAD‐seq), we identified a candidate for a genetic sex marker and validated this marker as a sex probe through PCR analyses of samples from wild roach populations from nonpolluted rivers. We also applied the sex marker to samples from roach exposed experimentally to oestrogen and oestrogenic effluents to confirm suspected phenotypic sex reversal from males to females in some treatments, and also that sex‐reversed males are able to breed as females. We then show, unequivocally, that intersex in wild roach populations results from feminisation of males, but find no strong evidence for complete sex reversal in wild roach at river sites contaminated with oestrogens. The discovered marker has utility for studies in roach on chemical effects, wild stock assessments, and reducing the number of fish used where only one sex is required for experimentation. Furthermore, we show that the marker can be applied nondestructively using a fin clip or skin swab, with animal welfare benefits.     

Multicolour fluorescent detection and mapping of AFLP markers in chicken (Gallus domesticus)

We describe the mapping of amplified restriction fragment polymorphism (AFLP) markers in chicken (Gallus domesticus) using a multi-colour fluorescent detection system. DNA was used from a population consisting of four families with a total of 183 F2 individuals. The enzyme combination EcoRI/TaqI was used for double digestion, and fluorescently labelled fragments were analysed on an ABI PRISM 377 DNA sequencer. Polymorphic signals in the range of 50-500 bp were genotyped with the ABI PRISM Genotyper 2.0 software, which enabled the analysis of both dominant and incomplete dominant markers (with respect to AFLP, often referred to as codominant). In 19 sets consisting of 3 EcoRI/TaqI primer pair combinations each, a total of 475 polymorphic markers was detected. From these polymorphisms 344 markers could be mapped on the Wageningen linkage map. Fourteen markers were length polymorphisms of the same fragment and 28 markers Z-linked and uniformative; 64 AFLP markers appeared to be unlinked and 25 AFLP markers could not be accurately mapped on the basis of the genotyping results. The resulting AFLP/microsatellite linkage map is comprised of 33 linkage groups with a total of 835 loci.     

Effect of estrogen and 2,3,7,8-tetrachlorodibenzo-rho-dioxin (TCDD) on plasma fatty acids of immature male chickens (Gallus domesticus)

This study investigated the effects of 2,3,7,8-tetrachlorodibenzo-rho-dioxin (TCDD) and estrogen on plasma lipids in immature male chickens. Fatty acids were quantified in plasma collected on day 14 from chickens injected with either: Estrogen plus TCDD-1 mg estradiol cypionate /kg body wt. daily for 3 days and 50 microg TCDD/kg body wt. on day 4; Estrogen--1 mg estradiol cypionate/kg body wt. daily for 3 days and vehicle only on day 4; TCDD-vehicle only for 3 days and 50 microg TCDD/kg body wt. on day 4; or Vehicle--same volume of appropriate vehicle for 4 days. TCDD treatment alone increased the plasma concentrations of total triacylglycerides and of the specific fatty acids 14:0, 15:0, 18:0, 18:2n6, 18:3n3, 20:0, 20:1n9, 20:2n6, 20:3n6, 20:5n3 and 22:1n9, compared with vehicle treatment. The concentration of 22:6n3 was increased in all plasma lipid classes of the estrogen group compared with the vehicle group, but was not increased in the estrogen plus TCDD group. Overall, TCDD treatment alone increased plasma lipids, possibly as a result of decreased clearance or utilization; whereas estrogen plus TCDD treatment antagonized estrogen-induced increases in 22:6n3 but did not cause hyperlipidemia.     

The effect of an acute phase response on tissue carotenoid levels of growing chickens (Gallus gallus domesticus)

Plasma, liver and skin carotenoids decrease following infectious disease challenges. Since these challenges often involve substantial host pathology and chronic immune responses, the mechanism underlying altered carotenoid deposition is unclear. Therefore, changes in tissue carotenoid levels were examined during an acute phase response induced by lipopolysaccharide (LPS) or interleukin-1 (IL-1). In two experiments, chicks were hatched from carotenoid-deplete eggs (n=28, n=64, respectively) and fed 0, 8 or 38 mg carotenoids (lutein+canthaxanthin)/kg diet. For chicks fed 38 mg carotenoids, but not those fed 0 or 8 mg, LPS generally reduced plasma lutein, canthaxanthin and total carotenoids (P<0.05), and liver lutein, zeaxanthin, canthaxanthin and total carotenoids (P<0.05). Additionally, LPS reduced thymic total carotenoids (P=0.05) and increased thymocyte lutein (P=0.07), zeaxanthin (P=0.07) and total carotenoids (P=0.07). Finally, LPS increased bursal canthaxanthin (P<0.01), but had no effect on shank carotenoids (P>0.5). In chicks hatched from carotenoid-replete eggs (n=36) and fed dietary lutein (38 mg/kg diet), LPS reduced plasma and liver zeaxanthin and liver total carotenoids (P<0.05); IL-1 reduced plasma and liver lutein, zeaxanthin and total carotenoids (P<0.05). Therefore, an acute phase response plays a role in reduced tissue carotenoids during infectious disease.     

Mitochondrial DNA-based analysis of genetic variation and relatedness among Sri Lankan indigenous chickens and the Ceylon junglefowl (Gallus lafayetti)

Indigenous chickens (IC) in developing countries provide a useful resource to detect novel genes in mitochondrial and nuclear genomes. Here, we investigated the level of genetic diversity in IC from five distinct regions of Sri Lanka using a PCR-based resequencing method. In addition, we investigated the relatedness of IC to different species of junglefowls including Ceylon (CJF; Gallus lafayetti ), a subspecies that is endemic to Sri Lanka, green ( Gallus varius ), grey ( Gallus sonneratii ) and red ( Gallus gallus ) junglefowls. A total of 140 birds including eight CJF were used to screen the control region of the mitochondrial DNA sequence for single nucleotide polymorphisms (SNPs) and other variants. We detected and validated 44 SNPs, which formed 42 haplotypes and six haplogroups in IC. The SNPs observed in the CJF were distinct and the D-loop appeared to be missing a 62-bp segment found in IC and the red junglefowl. Among the six haplogroups of IC, only one was region-specific. Estimates of haplotype and nucleotide diversities ranged from 0.901 to 0.965 and from 0.011 to 0.013 respectively, and genetic divergence was generally low. Further, variation among individuals within regions accounted for 92% of the total molecular variation among birds. The Sri Lankan IC were more closely related to red and grey junglefowls than to CJF, indicating multiple origins. The molecular information on genetic diversity revealed in our study may be useful in developing genetic improvement and conservation strategies to better utilize indigenous Sri Lankan chicken resources.     

Different effects of mating group size as male and as female on sex allocation in a simultaneous hermaphrodite

Sex allocation theory predicts that the optimal sexual resource allocation of simultaneous hermaphrodites is affected by mating group size (MGS). Although the original concept assumes that the MGS does not differ between male and female functions, the MGS in the male function (MGSm; i.e., the number of sperm recipients the focal individual can deliver its sperm to plus one) and that in the female function (MGSf; the number of sperm donors plus one) do not always coincide and may differently affect the optimal sex allocation. Moreover, reproductive costs can be split into “variable” (e.g., sperm and eggs) and “fixed” (e.g., genitalia) costs, but these have been seldom distinguished in empirical studies. We examined the effects of MGSm and MGSf on the fixed and variable reproductive investments in the sessilian barnacle Balanus rostratus. The results showed that MGSm had a positive effect on sex allocation, whereas MGSf had a nearly significant negative effect. Moreover, the “fixed” cost varied with body size and both aspects of MGS. We argue that the two aspects of MGS should be distinguished for organisms with unilateral mating.     

Sex reversal of germ cell gametogenesis in chimeras of Pleurodeles waltl (urodele amphibian): genetic and immunogenetic demonstration using tolerance or rejection of skin grafts

Viable chimeras were constituted with two cranial and caudal complementary pieces of embryos derived from two distinct histocompatible AA and BB strains, which were incompatible with each other. The embryonic gonads of the resulting chimeras constituted two homo- or heterosexual territories. In most heterosexual chimeras, the testicular territory sex reversed the ovarian territory. The offspring analysis of a male chimera conclusively proved that ZW germ cells derived from the posterior female piece differentiated into spermatozoa. Nevertheless, the opposite situation was also demonstrated with a female chimera in which ZZ germ cells derived from the anterior male piece differentiated into oocytes. These gametogenesis reversions were tested by genetic and immunogenetic analyses of chimera offspring. The phenomenon of tolerance or rejection of skin allo- and autograft was used as a marker of origin of the chimera germ cells, which had produced the offspring. Moreover, in the first stage of the study, the origin of the pieces of adult chimeras was determined using skin grafts. During this stage, the embryonic tolerance was confirmed by the acquisition of four pieces of pairs of chimeras, and by the preservation of skin immunogenicity that was derived from each piece of the chimeras.