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.

Cyclin D1 gene expression is essential for cell cycle progression from the maternal-to-zygotic transition during blastoderm development in Japanese quail.

Embryogenesis proceeds by a highly regulated series of events. In animals, maternal factors that accumulate in the egg cytoplasm control cell cycle progression at the initial stage of cleavage. However, cell cycle regulation is switched to a system governed by the activated nuclear genome at a specific stage of development, referred to as maternal-to-zygotic transition (MZT). Detailed molecular analyses have been performed on maternal factors and activated zygotic genes in MZT in mammals, fishes and chicken; however, the underlying mechanisms remain unclear in quail. In the present study, we demonstrated that MZT occurred at blastoderm stage V in the Japanese quail using novel gene targeting technology in which the CRISPR/Cas9 and intracytoplasmic sperm injection (ICSI) systems were combined. At blastoderm stage V, we found that maternal retinoblastoma 1 (RB1) protein expression was down-regulated, whereas the gene expression of cyclin D1 (CCND1) was initiated. When a microinjection of sgRNA containing CCND1-targeted sequencing and Cas9 mRNA was administered at the pronuclear stage, blastoderm development stopped at stage V and the down-regulation of RB1 did not occur. This result indicates the most notable difference from mammals in which CCND-knockout embryos are capable of developing beyond MZT. We also showed that CCND1 induced the phosphorylation of the serine/threonine residues of the RB1 protein, which resulted in the degradation of this protein. These results suggest that CCND1 is one of the key factors for RB1 protein degradation at MZT, and the elimination of RB1 may contribute to cell cycle progression after MZT during blastoderm development in the Japanese quail. Our novel technology, which combined the CRISPR/Cas9 system and ICSI, has the potential to become a powerful tool for avian-targeted mutagenesis.

Cellular Invasion and Matrix Degradation, a Different Type of Matrix-Degrading Cells in the Cartilage of Catfish (Clarias gariepinus) and Japanese Quail Embryos (Coturnix coturnix japonica).

We previously studied the phenomena of the mesenchymal cell-dependent mode of cartilage growth in quail and catfish. Thus, we selected the two cartilage models in which mesenchymal cells participate in their growth. In such models, cartilage degradation occurred to facilitate cellular invasion. The studies do not explain the nature of the cartilage degrading cells. The current study aims to explore the nature of the cartilage-degrading cells using transmission electron microscopy (TEM) and immunohistochemistry. Samples of cartilage have been isolated from the air-breathing organ of catfish and the cartilage of the prospective occipital bone of quail embryos. Samples have been processed for TEM and immunohistochemistry. We found that two different cell types are involved in cartilage degradation; the macrophage in the cartilage of catfish and mesenchymal cells in the cartilage of the quail. Areas of cellular invasion in both catfish cartilage and quail embryo cartilage had an immunological affinity for MMP-9. In catfish, cartilage-degrading cells had identical morphological features of macrophages, whereas in quail embryos, cartilage-degrading cells were mesenchymal-like cells which had cell processes rich in vesicles and expressed CD117. Further study should consider the role of macrophage and mesenchymal cells during cartilage degradation. This could be valuable to be applied to remove the defective cartilage matrix formed in osteoarthritic patients to improve cartilage repair strategies.

Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail.

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.

Histamine H4 receptor as a novel therapeutic target for the treatment of Leydig-cell tumours in prepubertal boys.

Leydig-cell tumours (LCTs) are rare endocrine tumours of the testicular interstitium, with recent increased incidence. Symptoms include precocious puberty in children; and erectile dysfunction, infertility and/or gynaecomastia, in adults. So far, scientific evidence points to aromatase (CYP19) overexpression and excessive oestrogen and insulin-like growth factor (IGF) -1 production as responsible for Leydig-cell tumourigenesis. LCTs are usually benign; however, malignant LCTs respond poorly to chemo/radiotherapy, highlighting the need to identify novel targets for treatment. Herein, we investigated the potential role of the histamine receptor H4 (HRH4) as a therapeutic target for LCTs using R2C rat Leydig tumour cells, a well-documented in vitro model for Leydigioma. Also, we studied for the first time the expression of CYP19, IGF-1R, oestrogen receptor (ER) α, ERß, androgen receptor (AR) and HRH4 in human prepubertal LCTs versus normal prepubertal testes (NPTs). HRH4 agonist treatment inhibited steroidogenesis and proliferation in R2C cells and also negatively affected their pro-angiogenic capacity in vitro and in vivo, as assessed by evaluating the proliferative activity of human umbilical vein endothelial cells and by means of the quail chorioallantoic membrane assay, respectively. Moreover, E2 and IGF-1 inhibited HRH4 mRNA and protein levels. In human prepubertal LCTs, CYP19, IGF-1R, ERα and ERß were overexpressed compared with NPTs. In contrast, HRH4 staining was weak in LCTs, but moderate/strong and confined to the interstitium in NPTs. Importantly, HRH4 was absent or barely detectable in seminiferous tubules or germ cells. Overall, our results point to HRH4 as a novel therapeutic target in LCTs.

Embryonic cholesterol esterification is regulated by a cyclic AMP-dependent pathway in yolk sac membrane-derived endodermal epithelial cells.

During avian embryonic development, endodermal epithelial cells (EECs) absorb yolk through the yolk sac membrane. Sterol O-acyltransferase (SOAT) is important for esterification and yolk lipid utilization during development. Because the major enzyme for yolk sac membrane cholesteryl ester synthesis is SOAT1, we cloned the avian SOAT1 promoter and elucidated the cellular functions of SOAT1. Treatments with either glucagon, isobutylmethylxanthine (IBMX), an adenylate cyclase activator (forskolin), a cAMP analog (dibutyryl-cAMP), or a low glucose concentration all increased SOAT1 mRNA accumulation in EECs from Japanese quail, suggesting that SOAT1 is regulated by nutrients and hormones through a cAMP-dependent pathway. Activity of protein kinase A (PKA) was increased by IBMX, whereas co-treatment with the PKA inhibitor, H89 negated the increase in PKA activity. Cyclic AMP-induced EECs had greater cholesterol esterification than untreated EECs. By promoter deletion and point-mutation, the cAMP-response element (-349 to -341 bp) was identified as critical in mediating transcription of SOAT1. In conclusion, expression of SOAT1 was regulated by a cAMP-dependent pathway and factors that increase PKA will increase SOAT1 to improve the utilization of lipids in the EECs and potentially modify embryonic growth.

Effect of in ovo injection of threonine on immunoglobulin A gene expression in the intestine of Japanese quail at hatch.

The objective of this study was to investigate the effect of in ovo injection of threonine (THR) on immunoglobulin A (IgA) gene expression of Japanese quail on hatch day. A total of 540 Japanese quail eggs were assigned into nine groups of 60 each and were set in a single-stage incubator. Treatments were as follows: non-injected (control), two diluent levels (0.05 or 0.1 ml saline), two sites of injection (in or under the air sac) and with or without nutrients (0.5 mg/ml THR). Eggs were injected on d 11 of incubation. On hatch day, after euthanizing hatched quail chicks, the intestine was removed and the jejunum was separated. The relative mRNA expression of jejunal IgA increased (p < 0.05) by the injection of 0.05 ml THR under the air sac when compared to the control group or other treatments of injection. Compared to the control group, no differences were imputable to treatments of 0.1-ml injections on IgA gene expression. Differences with other injected groups were not significant. It was concluded that injection of 0.05 ml saline containing 0.5 mg THR/ml under the air sac can improve jejunal IgA mRNA expression in newly hatched Japanese quail chicks.

Immunohistochemical localization of estrogen receptor in the embryonic gonad of male quail embryo during gonadal differentiation / Localización inmunohistoquímica del receptor de estrógeno en la gónada embrionaria en el embrión de codorniz macho durante la diferenciación gonadal

In birds, male embryo the gonads develop bilateral testes, in which both left and right sides produce functional spermatozoa, whereas female embryo, only the left gonad develops into a functional ovary. Estrogen plays a key role in avian sex determination in both sexes by binding to the estrogen receptor (ER). Surprisingly, chicken estrogen receptor (cER) mRNA is expressed in both sexes; moreover; its expression is only expressed in the left male gonad. The present study aimed to localize ER protein in the left gonad of male quail embryo using immunohistochemistry. The 8-day-old male quail embryos whose embryonic sex distinguished by gonadal morphology were studied. Histology of the left male gonad displayed thin cortex containing 1 to 2 layers of the germinal epithelium, while testicular cords were observed in the medulla. ER-immunoreactive cells were only found in the germinal epithelium but not in the medulla. Localization of ER was detected in the nucleus and cytoplasm of the germinal epithelial cells. The number of ER-immunoreactive cells counted in upper, lateral, and lower regions of the germinal epithelium was 18.20±1.892, 17.60±1.887, and 16.20±1.290, respectively. This study shows the first evidence for expression of ER protein in the left male gonad of the avian embryo, indicating that ER plays a role in avian gonadal sex differentiation.

En las aves, la gónada embrionaria en los machos se desarrolla bilateralmente, ambos testículos producen espermatozoides funcionales, mientras que en el embrión hembra, sólo la gónada izquierda se convierte en un ovario funcional. El estrógeno juega un papel clave en la determinación del sexo aviar, en ambos sexos, mediante la unión al receptor de estrógeno (RE). Fuertemente los receptores de estrógenos de pollo (cRE) el ARNm se expresan en ambos sexos; además, su expresión sólo se produce en la gónada izquierda del macho. El objetivo fue localizar proteínas del RE en la gónada izquierda de embriones de codorniz macho mediante inmunohistoquímica. Se estudiaron embriones de codorniz machos a los 8 días de edad, cuyo sexo embrionario se distinguió por la morfología de las gónadas. La histología de la gónada izquierda estuvo representada por la corteza delgada que contiene de 1 a 2 capas del epitelio germinal, mientras que se observaron cordones testiculares en la médula. El RE se encontró en células inmunorreactivas del epitelio germinal, pero no en la médula. Se detectó la localización de RE en el núcleo y el citoplasma de las células epiteliales germinales. El número de células RE-inmunorreactivas en las regiones superior, lateral e inferior del epitelio germinal fue de 18,20±1,892, 17,60±1,887 y 16,20±1,290, respectivamente. Este estudio muestra la primera evidencia de expresión de la proteína de RE en la gónada izquierda del embrión aviar macho, lo que indica que el RE desempeña un papel en la diferenciación sexual de la gónada aviar.

Crescimento vascular em membrana do saco vitelínico e desenvolvimento embrionário de codornas japonesas (Coturnix japonica) expostas a campo magnético de baixa frequência / Vascular growth in yolk sac membrane and embryonic development of Japanese quail (Coturnix japonica) exposed to low frequency magnetic field

O objetivo deste trabalho foi observar a influência do campo magnético (CM) de baixa frequência na membrana do saco vitelínico (MSV) e no desenvolvimento do embrião de codornas japonesas (Coturnix japonica) em 72 horas de incubação. Ovos fertilizados foram expostos a nove horas consecutivas de CM, sendo um grupo a partir das 48 horas e o outro a partir das 63 horas de incubação. A quantificação da vascularização da MSV foi determinada pela obtenção da dimensão fractal por meio dos métodos de box-counting e de dimensão de informação, enquanto o peso corporal e o percentual de comprimento cefálico dos embriões foram utilizados como parâmetros de desenvolvimento embrionário. O CM não causou diferenças significativas na densidade vascular da MSV nem no desenvolvimento embrionário, quando comparados ao grupo controle...

The aim of this study was to observe the influence of the low frequency magnetic field (MF) on the yolk sac membrane (YSM) and embryonic development of Japanese quail (Coturnix japonica) in 72 hours of incubation. Fertilized eggs were exposed to 9 consecutive hours of MF, with a group from 48 hours and the second group from 63 hours of incubation. The evaluation of YSM vascularization was determined by the fractal dimensions obtained through box-counting method and information dimension, while body weight of the embryo and percentage of cephalic length were used as parameters for embryo development. The MF caused no significant differences in vessel density in the YSM, nor in the embryonic development considering the body weight and percentage cephalic length, when were compared to the control group...

Production of functional gametes from cryopreserved primordial germ cells of the Japanese quail.

The Japanese quail (Coturnix japonica) is a valuable bird as both an experimental animal, for a wide range of scientific disciplines, and an agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs would be a feasible strategy for the conservation of both male and female fertility cells in Japanese quail. However, the effects of freeze-thaw treatment on viability, migration ability and germline transmission ability of quail PGCs still remain unclear. In the present study, male and female PGCs were isolated from the blood of 2-day-old embryos, which were cooled by slow freezing and then cryopreserved at -196 C for 77-185 days, respectively. The average recovery rate of PGCs after freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P<0.05) (85.5% vs. 95.1%). Both fresh and Frozen-thawed PGCs that were intravascularly transplanted into recipient embryos migrated toward and were incorporated into recipient gonads, although the number of PGCs settled in the gonads was 48.5% lower in the frozen group than in the unfrozen control group (P<0.05). Genetic cross analysis revealed that one female and two male recipients produced live progeny derived from the frozen-thawed PGCs. The frequency of donor-derived offspring was slightly lower than that of unfrozen controls, but the difference was not significant (4.0 vs. 14.0%). These results revealed that freeze-thaw treatment causes a decrease in viability, migration ability and germline transmission ability of PGCs in quail.

GSM 900 MHz cellular phone radiation can either stimulate or depress early embryogenesis in Japanese quails depending on the duration of exposure.

PURPOSE: Our study was designed to assess the effects of low intensity radiation of a GSM (Global System for Mobile communication) 900 MHz cellular phone on early embryogenesis in dependence on the duration of exposure. MATERIALS AND METHODS: Embryos of Japanese Quails were exposed in ovo to GSM 900 MHz cellular phone radiation during initial 38 h of brooding or alternatively during 158 h (120 h before brooding plus initial 38 h of brooding) discontinuously with 48 sec ON (average power density 0.25 µW/cm(2), specific absorption rate 3 µW/kg) followed by 12 sec OFF intervals. A number of differentiated somites were assessed microscopically. Possible DNA damage evoked by irradiation was assessed by an alkaline comet assay. RESULTS: Exposure to radiation from a GSM 900 MHz cellular phone led to a significantly altered number of differentiated somites. In embryos irradiated during 38 h the number of differentiated somites increased (p < 0.001), while in embryos irradiated during 158 h this number decreased (p < 0.05). The lower duration of exposure led to a significant (p < 0.001) decrease in a level of DNA strand breaks in cells of 38-h embryos, while the higher duration of exposure resulted in a significant (p < 0.001) increase in DNA damage as compared to the control. CONCLUSION: Effects of GSM 900 MHz cellular phone radiation on early embryogenesis can be either stimulating or deleterious depending on the duration of exposure.

Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity.

Exposure of stage 9 quail (Coturnix coturnix japonica) embryos to glyceryl trinitrate (GTN) induces malformations that were associated in previous studies with an increase in protein nitration. Increased nitration suggests metabolism of GTN by the embryo. The goals of this study were to characterize the enzymes and co-factors required for GTN metabolism by quail embryos, and to determine the effects of in ovo treatment with N-acetyl cysteine (NAC), a precursor of glutathione (GSH), on GTN embryotoxicity. GTN treatment of quail embryos resulted in an increase in nitrite, a decrease in total GSH, and an increase in the ratio of NADP(+)/NADPH, indicating that redox balance may be compromised in exposed embryos. Glutathione S-transferases (GSTs; EC 2.5.1.18) purified from the whole embryo (K(m) 0.84 mM; V(max) 36 µM/min) and the embryonic eye (K(m) 0.20 mM; V(max) 30 µM/min) had GTN-metabolizing activity (1436 and 34 nmol/min/mg, respectively); the addition of ethacrynic acid, an inhibitor of GST activity, decreased GTN metabolism. Peptide sequencing of the GST isozymes indicated that alpha- or mu-type GSTs in the embryo and embryonic eye had GTN metabolizing activity. NAC co-treatment partially protected against the effects of GTN exposure. Thus, GTN denitration by quail embryo GSTs may represent a key initial step in the developmental toxicity of GTN.

Time-lapse microscopy of macrophages during embryonic vascular development.

BACKGROUND: Macrophages are present before the onset of blood flow, but very little is known about their function in vascular development. We have developed a technique to concurrently label both endothelial cells and macrophages for time-lapse microscopy using co-injection of fluorescently conjugated acetylated low-density lipoprotein (AcLDL) and phagocytic dye PKH26-PCL. RESULTS: We characterize double-labeled cells to confirm specific labeling of macrophages. Double-labeled cells circulate, roll along the endothelium, and extravasate from vessels. Most observed macrophages are integrated into the vessel wall, showing an endothelial-like morphology. We used transgenic quail that express a fluorescent protein driven by the endothelial-specific promoter Tie1 in conjugation with the phagocytic dye to analyze these cells. Circulating PKH26-PCL-labeled cells are mostly Tie1-, but those which have integrated into the vessel wall are largely Tie1+. The endothelial-like phagocytic cells were generally stationary during normal vascular development. We, therefore, induced vascular remodeling and found that these cells could be recruited to sites of remodeling. CONCLUSIONS: The active interaction of endothelial cells and macrophages support the hypothesis that these cells are involved in vascular remodeling. The presence of phagocytic endothelial-like cells suggests either a myeloid-origin to certain endothelial cells or that circulating endothelial cells/hematopoietic stem cells have phagocytic capacity.

Developmental toxicity of glyceryl trinitrate in quail embryos.

BACKGROUND: Although glyceryl trinitrate (GTN) is used extensively to treat angina and heart failure, little is known about its effects on the conceptus during organogenesis. The goal of these studies was to investigate the effects of GTN in a model organism, the quail (Coturnix coturnix japonica) embryo. METHODS: To identify the effects of GTN on quail embryo development, fertilized quail eggs (n = 10-12 eggs/group) were injected with GTN (0, 4.4, 44, or 440 µM) at Hamburger-Hamilton (HH) stage 0, 9, or 19 and examined 7 days later. Next, HH 9 embryos were injected with GTN (0, 0.88, 4.4, 8.8, 44, 88, and 440 µM, in 20 µL per egg) and examined 24-hours, 48-hours, or 72-hours postinjection. Finally, the developing eye on one side was exposed to GTN (44 µM) ex ovo and the tissue was probed for the presence of nitrated proteins. RESULTS: In ovo GTN exposure induced a dose-dependent increase in the number of malformed viable quail embryos with a maximal effect in HH 9 embryos. Microphthalmia, craniofacial, heart, and neural tube defects were elevated in GTN-exposed embryos. An increase in nitrated proteins was observed in the developing eye region of embryos exposed ex ovo to GTN. CONCLUSIONS: GTN treatment induced a variety of malformations in quail embryos. The presence of nitrated proteins suggests that organic nitrates, such as GTN, generate reactive nitrogen species. We hypothesize that GTN perturbations in the redox status of the embryo may underlie its developmental toxicity.

The origin of neuroendocrine tumors and the neural crest saga.

In this essay, the role of the neural crest in the development of the vertebrate embryo is briefly described. The techniques used to document the neural crest origin of various cell types and the tumors arising from them are discussed, with emphasis on Le Douarin's quail-chick chimera model. The current dogma on the origin of the cells of the diffuse endocrine system is presented, and some personal conjectures based on the microscopic appearances of various types of normal, vestigial and neoplastic human tissues are offered to the reader as 'food for thought.'

An anteroposterior wave of vascular inhibitor downregulation signals aortae fusion along the embryonic midline axis.

Paracrine signals, both positive and negative, regulate the positioning and remodeling of embryonic blood vessels. In the embryos of mammals and birds, the first major remodeling event is the fusion of bilateral dorsal aortae at the midline to form the dorsal aorta. Although the original bilaterality of the dorsal aortae occurs as the result of inhibitory factors (antagonists of BMP signaling) secreted from the midline by the notochord, it is unknown how fusion is later signaled. Here, we report that dorsal aortae fusion is tightly regulated by a change in signaling by the notochord along the anteroposterior axis. During aortae fusion, the notochord ceases to exert its negative influence on vessel formation. This is achieved by a transcriptional downregulation of negative regulators while positive regulators are maintained at pre-fusion levels. In particular, Chordin, the most abundant BMP antagonist expressed in the notochord prior to fusion, undergoes a dramatic downregulation in an anterior to posterior wave. With inhibitory signals diminished and sustained expression of the positive factors SHH and VEGF at the midline, fusion of the dorsal aortae is signaled. These results demonstrate a novel mechanism by which major modifications of the vascular pattern can occur through modulation of vascular inhibitors without changes in the levels of positive vascular regulators.

Novel method of gene transfer in birds: intracytoplasmic sperm injection for green fluorescent protein expression in quail blastoderms.

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

Distribution and sex differences in aromatase-producing neurons in the brain of Japanese quail embryos.

The biochemical properties, neuroanatomical location, and function of aromatase (ARO), the enzyme that converts testosterone to 17beta-estradiol, have been studied extensively in the adult quail brain. Conversely, very little is known about ARO in quail embryos. This study investigated the distribution of ARO in quail prosencephalon at embryonic days (E) 9, 11, and 15 by immunocytochemistry. ARO-immunoreactive cells were observed within the walls of the cerebral ventricles, the ventral striatum, medial preoptic nucleus (POM), medial part of the bed nucleus of the stria terminalis (BSTM), lateral part of the BST, and in the tuberal region. The BSTM and to a lesser extent the POM showed transient, female-biased sex-differences. In the BSTM, the number of the ARO-immunoreactive cells, the fractional area covered by ARO-immunoreactive structures, and the overall extension of ARO-immunoreactivity were greater in females at E9 and E11, but these differences largely disappeared at E15 and post-hatch day 1. The sex differences were confirmed at the transcriptional level by in situ hybridization. In the lateral part of the POM, females showed slightly more ARO-immunoreactivity than males at E11. Treatment of E9 male embryos with estradiol completely feminized ARO-immunoreactivity at E11. The origins and the functional significance of these sex differences remain unknown.

Effects on differentiation of reproductive organs and sexual behaviour in Japanese quail by excessive embryonic ERalpha activation.

Exposure of Japanese quail (Coturnix japonica) embryos to oestrogenic substances disrupts sexual differentiation of the reproductive tract of both sexes and impairs the copulatory behaviour of the adult male. To examine whether these effects can be induced by selective activation of oestrogen receptor alpha (ERalpha), Japanese quail eggs were injected with various doses of the selective ERalpha agonist 16alpha-lactone-oestradiol (16alpha-LE(2)). The natural oestrogen 17beta-oestradiol (E(2)) was used as a positive control. Both 16alpha-LE(2) and E(2) induced formation of an ovary-like cortex in the left testis (ovotestis) and reduced the size of the right testis in male embryos. The asymmetry in testis size remained in sexually mature males. Both substances induced retention and malformation of the Müllerian ducts in embryos of both sexes and malformed oviducts in juveniles. Male copulatory behaviour was suppressed by embryonic exposure to E(2) and the highest dose of 16alpha-LE(2). However, the lower dose of 16alpha-LE(2), which markedly affected development of the reproductive organs, was without effects on behaviour. It can therefore not be excluded that the behavioural demasculinisation at the 100-fold higher dose involved cross-activation of oestrogen receptor beta (ERbeta). In conclusion, our results suggest that oestrogen-induced disruption of reproductive organ development in Japanese quail can be mediated via ERalpha, whereas the role of ERalpha in demasculinisation of copulatory behaviour remains to be clarified.

Endothelial cell transduction in primary cultures from regressing mesonephros.

Loss of renal function during normal aging is associated with vascular alterations. Consequently, new therapeutic approaches, including gene therapy, to protect renal endothelial cells are expected to be greatly beneficial. Quail mesonephros is a transitory embryonic kidney that has been used for the study of vascular development and involution. Vascular alterations in regressing mesonephros are similar to those observed in aging kidney. In the present study, we examined adenovirus-mediated gene transfer to endothelial cells in primary cultures from developing and regressing quail mesonephros. Quail embryos with developing and regressing mesonephros were examined on day 6 (30HH) and day 11 (40HH) of incubation, respectively. The senescence markers, associated beta-galactosidase activity and p16(INK4a), were examined in whole mesonephros. Quail embryos were injected intracardiacally with adenoviral vectors (rAd-CMV-LacZ) and endothelial cell transduction examined. In addition, primary cell cultures from mesonephros were exposed to adenoviral vectors. Endothelial cells in primary cultures were identified as QH1(+), LEP100(-) and acidic phosphatase(-) cells and adenovirus-transduced cells were those positive for bacterial-associated beta-galactosidase activity. We report that endothelial cells in the whole regressing mesonephros and primary cell cultures expressed senescence markers. In addition, we observed that adenoviral vectors were able to transduce endothelial cells in the whole regressing mesonephros, and that cultured endothelial and macrophagic cells from the regressing mesonephros were more efficiently transduced than those derived from the developing mesonephros. Our results suggest that quail mesonephros provides a practical model to assay gene transfer to endothelial cells in regressing/senescent vessels.