Ethynylestradiol feminizes gene expression partly in testis developing as ovotestis and disrupts asymmetric Müllerian duct development by eliminating asymmetric gene expression in Japanese quail embryos.

In avian embryos, xenoestrogens induce abnormalities in reproductive organs, particularly the testes and Müllerian ducts (MDs). However, the molecular mechanisms remain poorly understood. We investigated the effects of ethynylestradiol (EE2) exposure on gene expression associated with reproductive organ development in Japanese quail embryos. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the left testis containing ovary-like tissues following EE2 exposure highly expressed the genes for steroidogenic enzymes (P450scc, P45017α, lyase, and 3ß-HSD) and estrogen receptor-ß, compared to the right testis. No asymmetry was found in these gene expression without EE2. EE2 induced hypertrophy in female MDs and suppressed atrophy in male MDs on both sides. RNA sequencing analysis of female MDs showed 1,366 differentially expressed genes between developing left MD and atrophied right MD in the absence of EE2, and these genes were enriched in Gene Ontology terms related to organogenesis, including cell proliferation, migration and differentiation, and angiogenesis. However, EE2 reduced asymmetrically expressed genes to 21. RT-qPCR analysis indicated that genes promoting cell cycle progression and oncogenesis were more highly expressed in the left MD than in the right MD, but EE2 eliminated such asymmetric gene expression by increasing levels on the right side. EE2-exposed males showed overexpression of these genes in both MDs. This study reveals part of the molecular basis of xenoestrogen-induced abnormalities in avian reproductive organs, where EE2 may partly feminize gene expression in the left testis, developing as the ovotestis, and induce bilateral MD malformation by canceling asymmetric gene expression underlying MD development.

In ovo o,p'-DDT exposure induces malformation of reproductive organs and alters the expression of genes controlling sexual differentiation in Japanese quail embryo.

In ovo exposure to o,p'-dichloro-diphenyl-trichloroethane (o,p'-DDT) impairs reproduction by inducing malformation of the reproductive organs in birds, although the mechanism remains unclear. Here, we examined the effects of o,p'-DDT on the development of the reproductive organs, the expression of genes controlling sexual differentiation, and the plasma concentrations of testosterone and estradiol in Japanese quail embryos. o,p'-DDT-containing sesame oil was injected into the yolk sac on Embryonic Day (E) 3 at a dose of 500, 2,000, or 8,000 µg per egg. On E15, the reproductive organs were observed; the gonads and Müllerian ducts (MDs) were sampled to measure the mRNA of steroidogenic enzymes, sex steroid receptors, anti-Müllerian hormone (AMH), and AMH receptor 2 (AMHR2); blood samples were collected to assay plasma testosterone and estradiol levels; and the gonads were used for histological analysis. o,p'-DDT dose-dependently increased the prevalence of hypertrophic MDs in females and residual MDs in males. In female MDs, o,p'-DDT dose-dependently decreased estrogen receptor (ER) α, ERß, and AMHR2 mRNA expression. o,p'-DDT dose-dependently induced left-biased asymmetry of testis size, and ovary-like tissue was found in the left testis after exposure to 8,000 µg per egg o,p'-DDT, although asymmetric gene expression did not occur. o,p'-DDT did not affect ovarian tissue but did decrease 17α-hydroxylase/C17-20 lyase mRNA expression and dose-dependently increased ERß mRNA expression. o,p'-DDT decreased plasma testosterone concentrations in females. These findings suggest that o,p'-DDT induces hypertrophy of the MDs and ovarian tissue formation in the left testis. Abnormal MD development may be linked to altered gene expression for sensing estrogens and AMH signals.

Sexually dimorphic expression of sexual differentiation genes in the internal genital organs of Japanese quail embryos.

Japanese quail (Coturnix japonica) is an avian model used to evaluate the reproductive and developmental toxicity of chemicals. The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail, NIES-L, which may be a better model because of its highly inbred characteristics. To understand sexual differentiation of the reproductive organs and the value of using NIES-L quails for avian toxicity assessment, we profiled estradiol and androgen plasma levels by enzyme-linked immunosorbent assay; the mRNA levels of estrogen receptor-α (ERα), ERß, and androgen receptor (AR) in the gonads, Müllerian ducts, Wolffian ducts; and the mRNA levels of steroidogenic enzymes, cholesterol side chain cleavage enzyme (P450scc), 17α-hydroxylase/C17-20 lyase (P45017α, lyase), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and aromatase (P450arom), anti-Müllerian hormone (AMH), and AMH receptor type 2 (AMHR2) in the gonads of NIES-L Japanese quails on embryonic days 9, 12, and 15 using a real-time quantitative PCR method. The plasma estradiol concentration was higher in females than males on these embryonic days, but no sex difference was found in the plasma androgens. The mRNA levels of all examined steroidogenic enzymes were significantly higher in female than male embryos. In particular, the P450arom mRNA levels showed a striking sex difference: P450arom was expressed in female but not male gonads. In contrast, the AMH and AMHR2 mRNA levels in the gonads were higher in males than females. The ERα, ERß, and AR mRNA levels increased in the left female gonad and peaked on embryonic day 15, but not in the left and right male gonads; therefore, there was a female-biased sex difference. The ERα, ERß, and AR mRNA levels in the left Müllerian duct, but not in the right Müllerian duct, of females increased and peaked on embryonic day 15, which resulted in asymmetric mRNA levels. The Wolffian ducts expressed ERα, ERß, and AR in both sexes, and no sex difference or asymmetry of mRNA levels was found. The information obtained from this study helps elucidate the molecular endocrinological basis of sexual dimorphism formation of reproductive organs and clarify the value of NIES-L quails for toxicity assessment.

Estrogenic action by tris(2,6-dimethylphenyl) phosphate impairs the development of female reproductive functions.

Developmental exposure to environmental chemicals with estrogen-like activity is suspected to permanently impair women's health. In this study, a mouse model was used to evaluate whether tris(2,6-dimethylphenyl) phosphate (TDMPP), a chemical with a putative estrogen-like action, impairs sexual differentiation of the brain. Either TDMPP and 17ß-estradiol (E2) as positive controls or sesame oil as a negative control were administered subcutaneously to dams from gestational day (GD) 14 to parturition, and to pups from postnatal day (PND) 0 to 9. Precocious puberty, irregular estrous cycles, and a lowered lordosis response were found in the TDMPP- and E2-treated groups. A certain amount of TDMPP and its metabolites in the perinatal brain and the masculinization of sexual dimorphic nuclei in the hypothalamus of female mice after treatment were also detected. The experimental evidence demonstrates that TDMPP directly enters the fetal and neonatal brain, thereby inducing changes of sex-related brain structures and impairing female reproductive functions.

Strain differences in intermale aggression and possible factors regulating increased aggression in Japanese quail.

The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail (Coturnix japonica) known as NIES-L by rotation breeding in a closed colony for over 35years; accordingly, the strain has highly inbred-like characteristics. Another strain called NIES-Brn has been maintained by randomized breeding in a closed colony to produce outbred-like characteristics. The current study aimed to characterize intermale aggressive behaviors in both strains and to identify possible factors regulating higher aggression in the hypothalamus, such as sex hormone and neuropeptide expression. Both strains displayed a common set of intermale aggressive behaviors that included pecking, grabbing, mounting, and cloacal contact behavior, although NIES-Brn quail showed significantly more grabbing, mounting, and cloacal contact behavior than did NIES-L quail. We examined sex hormone levels in the blood and diencephalon in both strains. Testosterone concentrations were significantly higher in the blood and diencephalon of NIES-Brn quail compared to NIES-L quail. We next examined gene expression in the hypothalamus of both strains using an Agilent gene expression microarray and real-time RT-PCR and found that gene expression of mesotocin (an oxytocin homologue) was significantly higher in the hypothalamus of NIES-Brn quail compared to NIES-L quail. Immunohistochemistry of the hypothalamus revealed that numbers of large cells (cell area>500µm2) expressing mesotocin were significantly higher in the NIES-Brn strain compared to the NIES-L strain. Taken together, our findings suggest that higher testosterone and mesotocin levels in the hypothalamus may be responsible for higher aggression in the NIES-Brn quail strain.

Analyzing the effects of co-expression of chick (Gallus gallus) melanocortin receptors with either chick MRAP1 or MRAP2 in CHO cells on sensitivity to ACTH(1-24) or ACTH(1-13)NH2: Implications for the avian HPA axis and avian melanocortin circuits in the hypothalamus.

In order to better understand the roles that melanocortin receptors (cMCRs) and melanocortin-2 receptor accessory proteins (cMRAP1 and cMRAP2) play in the HPA axis and hypothalamus, adrenal gland and hypothalamus mRNA from 1day-old white leghorn chicks (Gallus gallus), were analyzed by real-time PCR. mRNA was also made for kidney, ovary, and liver. Mrap1 mRNA could be detected in adrenal tissue, but not in any of the other tissues, and mrap2 mRNA was also detected in the adrenal gland. Finally, all five melanocortin receptors mRNAs could be detected in the adrenal gland; mc2r and mc5r mRNAs were the most abundant. To evaluate any potential interactions between MRAP1 and the MCRs that may occur in adrenal cells, individual chick mcr cDNA constructs were transiently expressed in CHO cells either in the presence or absence of a chick mrap1 cDNA, and the transfected cells were stimulated with hACTH(1-24) at concentrations ranging from 10-13M to 10-6M. As expected, MC2R required co-expression with MRAP1 for functional expression; whereas, co-expression of cMC3R with cMRAP1 had no statistically significant effect on sensitivity to hACTH(1-24). However, co-expression of MC4R and MC5R with MRAP1, increased sensitivity for ACTH(1-24) by approximately 35 fold and 365 fold, respectively. However, co-expressing of cMRAP2 with these melanocortin receptors had no effect on sensitivity to hACTH(1-24). Since the real-time PCR analysis detected mrap2 mRNA and mc4r mRNA in the hypothalamus, the interaction between cMC4R and cMRAP2 with respect to sensitivity to ACTH(1-13)NH2 stimulation was also evaluated. However, no effect, either positive or negative, was observed. Finally, the highest levels of mc5r mRNA were detected in liver cells. This observation raises the possibility that in one-day old chicks, activation of the HPA axis may also involve a physiological response from liver cells.

Genetic Divergence in Domestic Japanese Quail Inferred from Mitochondrial DNA D-Loop and Microsatellite Markers.

To assess the genetic diversity of domestic Japanese quail (Coturnix japonica) populations, and their genetic relationships, we examined mitochondrial DNA (mtDNA) D-loop sequences and microsatellite markers for 19 Japanese quail populations. The populations included nine laboratory lines established in Japan (LWC, Quv, RWN, WE, AWE, AMRP, rb-TKP, NIES-L, and W), six meat-type quail lines reimported from Western countries (JD, JW, Estonia, NIES-Br, NIES-Fr, and NIES-Hn), one commercial population in Japan, and three wild quail populations collected from three Asian areas. The phylogenetic tree of mtDNA D-loop sequences revealed two distinct haplotype groups, Dloop-Group1 and Dloop-Group2. Dloop-Group1 included a dominant haplotype representing most of the quail populations, including wild quail. Dloop-Group2 was composed of minor haplotypes found in several laboratory lines, two meat-type lines, and a few individuals in commercial and wild quail populations. Taking the breeding histories of domestic populations into consideration, these results suggest that domestic quail populations may have derived from two sources, i.e., domestic populations established before and after World War II in Japan. A discriminant analysis of principal components and a Bayesian clustering analysis with microsatellite markers indicated that the domestic populations are clustered into four genetic groups. The two major groups were Microsat-Group1, which contained WE, and four WE-derived laboratory lines (LWC, Quv, RWN, and AWE), and Microsat-Group2 consisting of NIES-L, JD, JW, Estonia, NIES-Br, NIES-Fr, NIES-Hn, W, and commercial and wild populations. The remaining two lines (AMRP and rb-TKP) were each clustered into a separate clade. This hierarchical genetic difference between domestic quail populations is attributed to the genetic background derived from two different genetic sources-the pre-war and post-war populations-which is well supported by their breeding histories.

A comparative study of sex difference in calbindin neurons among mice, musk shrews, and Japanese quails.

The medial preoptic nucleus (MPN) and the bed nucleus of the stria terminalis (BNST) of mice contain sexually dimorphic nuclei (SDNs) that are larger and have more neurons expressing calbindin D-28K (CB), a calcium-binding protein, in males than females. However, it is largely unknown whether such SDNs exist in species other than rodents. In this study, we performed an immunohistochemical study of CB in the MPN and BNST of musk shrews and Japanese quails to examine the existence of homologs of SDNs in mice. Like mice, musk shrews had a SDN exhibiting male-biased sex differences in volume and CB-immunoreactive (ir) cell number in the MPN. The BNST of musk shrews also contained a male-biased SDN, but consisted of non-CB neurons. The paratenial thalamic nucleus of musk shrews, but not mice, had more CB-ir cells in males than females. In Japanese quails of both sexes, CB-ir cells in the MPN and BNST were extremely small in number and did not cluster. These results suggest that the distribution of CB neurons differs among these species. Musk shrews may have a homolog of the SDN composed of CB neurons in the MPN of mice.

Avian Test Battery for the Evaluation of Developmental Abnormalities of Neuro- and Reproductive Systems.

Most of the currently used toxicity assays for environmental chemicals use acute or chronic systemic or reproductive toxicity endpoints rather than neurobehavioral endpoints. In addition, the current standard approaches to assess reproductive toxicity are time-consuming. Therefore, with increasing numbers of chemicals being developed with potentially harmful neurobehavioral effects in higher vertebrates, including humans, more efficient means of assessing neuro- and reproductive toxicity are required. Here we discuss the use of a Galliformes-based avian test battery in which developmental toxicity is assessed by means of a combination of chemical exposure during early embryonic development using an embryo culture system followed by analyses after hatching of sociosexual behaviors such as aggression and mating and of visual memory via filial imprinting. This Galliformes-based avian test battery shows promise as a sophisticated means not only of assessing chemical toxicity in avian species but also of assessing the risks posed to higher vertebrates, including humans, which are markedly sensitive to nervous or neuroendocrine system dysfunction.

In utero and Lactational Exposure to Acetamiprid Induces Abnormalities in Socio-Sexual and Anxiety-Related Behaviors of Male Mice.

Neonicotinoids, a widely used group of pesticides designed to selectively bind to insect nicotinic acetylcholine receptors, were considered relatively safe for mammalian species. However, they have been found to activate vertebrate nicotinic acetylcholine receptors and could be toxic to the mammalian brain. In the present study, we evaluated the developmental neurotoxicity of acetamiprid (ACE), one of the most widely used neonicotinoids, in C57BL/6J mice whose mothers were administered ACE via gavage at doses of either 0 mg/kg (control group), 1.0 mg/kg (low-dose group), or 10.0 mg/kg (high-dose group) from gestational day 6 to lactation day 21. The results of a battery of behavior tests for socio-sexual and anxiety-related behaviors, the numbers of vasopressin-immunoreactive cells in the paraventricular nucleus of the hypothalamus, and testosterone levels were used as endpoints. In addition, behavioral flexibility in mice was assessed in a group-housed environment using the IntelliCage, a fully automated mouse behavioral analysis system. In adult male mice exposed to ACE at both low and high doses, a significant reduction of anxiety level was found in the light-dark transition test. Males in the low-dose group also showed a significant increase in sexual and aggressive behaviors. In contrast, neither the anxiety levels nor the sexual behaviors of females were altered. No reductions in the testosterone level, the number of vasopressin-immunoreactive cells, or behavioral flexibility were detected in either sex. These results suggest the possibility that in utero and lactational ACE exposure interferes with the development of the neural circuits required for executing socio-sexual and anxiety-related behaviors in male mice specifically.

The mechanisms underlying sexual differentiation of behavior and physiology in mammals and birds: relative contributions of sex steroids and sex chromosomes.

From a classical viewpoint, sex-specific behavior and physiological functions as well as the brain structures of mammals such as rats and mice, have been thought to be influenced by perinatal sex steroids secreted by the gonads. Sex steroids have also been thought to affect the differentiation of the sex-typical behavior of a few members of the avian order Galliformes, including the Japanese quail and chickens, during their development in ovo. However, recent mammalian studies that focused on the artificial shuffling or knockout of the sex-determining gene, Sry, have revealed that sex chromosomal effects may be associated with particular types of sex-linked differences such as aggression levels, social interaction, and autoimmune diseases, independently of sex steroid-mediated effects. In addition, studies on naturally occurring, rare phenomena such as gynandromorphic birds and experimentally constructed chimeras in which the composition of sex chromosomes in the brain differs from that in the other parts of the body, indicated that sex chromosomes play certain direct roles in the sex-specific differentiation of the gonads and the brain. In this article, we review the relative contributions of sex steroids and sex chromosomes in the determination of brain functions related to sexual behavior and reproductive physiology in mammals and birds.

Next-generation sequencing reveals genomic features in the Japanese quail.

The Japanese quail has several advantages as a laboratory animal for biological and biomedical investigations. In this study, the draft genome of the Japanese quail was sequenced and assembled using next-generation sequencing technology. To improve the quality of the assembly, the sequence reads from the Japanese quail were aligned against the reference genome of the chicken. The final draft assembly consisted of 1.75 Gbp with an N50 contig length of 11,409 bp. On the basis of the draft genome sequence obtained, we developed 100 microsatellite markers and used these markers to evaluate the genetic variability and diversity of 11 lines of Japanese quail. Furthermore, we identified Japanese quail orthologs of spermatogenesis markers and analyzed their expression using in situ hybridization. The Japanese quail genome sequence obtained in the present study could enhance the value of this species as a model animal.

Melatonin receptor genes (mel-1a, mel-1b, mel-1c) are differentially expressed in the avian germ line.

The presence of melatonin receptor transcripts (mel-1a, mel-1b and mel-1c) was investigated in primordial germ cells (PGCs), immature and mature oocytes, and sperm of Japanese quail by reverse transcription--polymerase chain reaction (RT-PCR). The mel-1a transcript was detected in as few as in a thousand PGCs. Significant differences in the expression of melatonin receptor genes were found in differentiating germ cells: in PGCs only the mel-1a receptor was expressed, in blastoderms and immature oocytes all three transcripts (mel-1a, mel-1b, mel-1c) were present, while in mature ovulated oocytes the predominant transcript was mel-1c (with sporadic occurrence of mel-1a and mel-1b). In sperm, mel-1a and mel-1c were present but mel-1b was absent. This indicates that the expression of melatonin receptor genes changes throughout the differentiation of PGCs into adult gametes: during oocyte differentiation two additional transcripts, mel-1b and mel-1c, are synthesized in addition to mel-1a, but at oocyte maturation, mel-1a and mel-1b are degraded and only mel-1c remains. During male line (spermatozoa) differentiation mel-1c is transcribed in addition to mel-1a, with mel-1b being completely absent. Since melatonin and the activities of enzymes participating in melatonin synthesis are present in the avian yolk, it is reasonable to suggest a role for this molecule in early avian development and germ line differentiation. We propose that melatonin may act as a signaling molecule regulating some differentiation processes (e.g., cell proliferation, migration, etc.) before the formation of neural and hormonal systems.