Day-old Embryos Research Articles

Mapping the lipidomic secretome of the early equine embryo.

The lipidomic secretions of embryos provide a unique opportunity to examine the cellular processes of the early conceptus. In this study we profiled lipids released by the early equine conceptus, using high-resolution mass spectrometry to detect individual lipid species. This study examined the lipidomic profile in embryo-conditioned media from in vivo-produced, 8-9 day-old equine embryos (n = 3) cultured in vitro for 36 h, analyzed over 3 timepoints. A total of 1,077 lipid IDs were recorded across all samples, containing predominantly glycerolipids. Seventy-nine of these were significantly altered in embryo conditioned-media versus media only control (p < 0.05, fold-change >2 or < 0.5). Fifty-five lipids were found to be released into the embryo-conditioned media, of which 54.5% were triacylglycerols and 23.6% were ceramides. The sterol lipid, cholesterol, was also identified and secreted in significant amounts as embryos developed. Further, 24 lipids were found to be depleted from the media during culture, of which 70.8% were diacylglycerols, 16.7% were triacylglycerols and 12.5% were ceramides. As lipid-free media contained consistently detectable lipid peaks, a further profile analysis of the various components of non-embryo-conditioned media consistently showed the presence of 137 lipids. Lipid peaks in non-embryo-conditioned media increased in response to incubation under mineral oil, and contained ceramides, diacylglycerols and triacylglycerols. These results emphasize the importance of a defined embryo culture medium and a need to identify the lipid requirements of the embryo precisely. This study sheds light on early embryo lipid metabolism and the transfer of lipids during in vitro culture.

In vitro study of the toxic and teratogenic effects of prednisolone, azathioprine and mycophenolate mofetile on embryological development of rats

This study aimed to evaluate the effects of the glucocorticoid prednisolone, the mycophenolic acid prodrug, azathioprine, and the fungi fermentation end product, mycophenolate mofetile on the embryological development of rats. Nine day-old rat embryos were cultured in rat serum containing prednisolone at varying concentrations (5–30 µg/ml) for 48 h. The test groups were cultured separately in rat serum containing 0.3–10 µg/ml azathioprine and 1–10 µg/ml mycophenolate mofetile. Embryonic development parameter effects of both drugs in combination with prednisolone (20 µg/ml) were studied using morphological methods, with special attention given to the incidence of malformations. The genotoxic effects of agents evaluated with the TUNEL test revealed that prednisolone is not a cause of developmental toxicity. The maximum safe dose of prednisolone that could be used in combination with other immunosuppressive agents was determined to be 20 µg/ml. Azathioprine was found to be toxic and teratogenic for the rat embryos beginning at a dose of 1 µg/ml. Dose-dependent toxic and teratogenic effects of mycophenolate mofetile were detected at doses lower than normal clinical ones.

HIGH RESISTANCE OF CHICKEN EMBRYO RED BLOOD CELLS TO ACID ACTION

In this paper, the results of the comparative studies of the resistance of Gallus gallus chicken embryo and human erythrocytes are presented to acid action.MethodsRed blood cells from decapitated 10–12 day‐old chicken embryos (5±0.6 g) and human donor blood were used in the experiments. The cells were isolated by centrifugation and washed three times with 145 μmol/l NaCl solution. The hemolysis was performed using: a) Mcllvein buffer solution (MBS) (0.31 g/l Na2HPO4 and 1.87 g/l citric acid, pH = 2.5); b) 0.1 N HCl. The optical density (D) was registered by using photocolorimeter at λ=540 nm. The part of the experiments was made by using isotonic 145 μmol/l choline chloride solution. The experiments were carried out at 18–20°C; the reliability of the differences was evaluated with the Van der Waerden test.ResultsThe hemolytic resistance of the chick embryo erythrocytes was higher than of human erythrocytes. The lag phase value, the time to 50% hemolysis (t50) and the time to complete hemolysis increased twice (p&lt;0.05). The D value remained high after the completion of hemolysis. We believe that the effect of acid on the erythrocyte nucleus led to the decondensation and swelling of chromatin, causing an increase in optical density. The replacement of NaCl with choline chloride did not affect the resistance of human erythrocytes and led to an increase in the chick embryo resistance (p&lt;0.05). In particular, the hemolytic start time (t0) and the end time (tend) were increased by 16% (p &lt;0.05) and 21% (p &lt;0.05) respectively. The results of these experiments showed that Na+/H+ exchange couldn't lead to an increase in the acid resistance of chick embryo erythrocytes. The calculations indicated that at the anion exchanger density of 2760 copies/μm2 and 8,300 copies/μm2 in chicken and human erythrocytes, respectively. The amount of anion exchangers was 1.2 × 106 copies per human erythrocyte and 6.3 × 105 copies per chick embryo erythrocyte. This is a in good agreement with the 2‐fold increased resistance of the chicken embryo erythrocytes compared to the human erythrocytes.Thus, the high resistance of chicken erythrocytes in comparison with human erythrocytes can be associated with a lower density of anion exchangers in the chicken red blood cells membrane.Support or Funding InformationThe study was supported by project № AAAA‐A17‐117012310152‐2This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular evaluation of M2 protein of Iranian avian influenza viruses of H9N2 subtype in order to find mutations of adamantane drug resistance

Background: The H9N2 subtype of influenza A viruses is considered to be widespread in poultry industry. Adamantane is a group of antiviral agents which is effective both in prevention and treatment of influenza A virus infections. These drugs inhibit M2 protein ion channel which has role on viral replication. OBJECTIVES: The main objective of this study is to evaluate M gene of avian influenza viruses (AIVs) of H9N2 subtype in order to find adamantane drug resistance mutations. METHODS: Over 100 suspected samples were collected from different geographical regions of Iran during 2012-2013. Samples were injected via allantoic sac of 9-11 day-old chicken embryos. A total of 11 out of 100 were AIV. The H9N2 subtype was confirmed by specific RT-PCR. The RT-PCR was conducted for full length M gene. PCR amplified products were purified and then conducted for commercial direct sequencing. Finally, sequences were checked for possible sites of adamantane resistance mutations. RESULTS: Overall, 8 out of 11 viruses harbored the adamantane resistance-associated mutations. Of which, four viruses were isolated in 2013 and four viruses in 2012. Two different resistance-associated mutations were observed during different years. CONCLUSIONS: The present study provided clear evidence concerning resistance AIVs of H9N2 subtype that were circulating in Iranian poultry sector. This concern is always present as M segment might be introduced into human influenza viruses by reassortment phenomenon.

Chicken Immune Response after In Ovo Immunization with Chimeric TLR5 Activating Flagellin of Campylobacter jejuni.

Campylobacter jejuni is the main cause of bacterial food-borne diseases in developed countries. Chickens are the most important source of human infection. Vaccination of poultry is an attractive strategy to reduce the number of C. jejuni in the intestinal tract of chickens. We investigated the immunogenicity and protective efficacy of a recombinant C. jejuni flagellin-based subunit vaccine with intrinsic adjuvant activity. Toll-like receptor activation assays demonstrated the purity and TLR5 stimulating (adjuvant) activity of the vaccine. The antigen (20–40 μg) was administered in ovo to 18 day-old chicken embryos. Serum samples and intestinal content were assessed for antigen-specific systemic and mucosal humoral immune responses. In ovo vaccination resulted in the successful generation of IgY and IgM serum antibodies against the flagellin-based subunit vaccine as determined by ELISA and Western blotting. Vaccination did not induce significant amounts of flagellin-specific secretory IgA in the chicken intestine. Challenge of chickens with C. jejuni yielded similar intestinal colonization levels for vaccinated and control animals. Our results indicate that in ovo delivery of recombinant C. jejuni flagellin subunit vaccine is a feasible approach to yield a systemic humoral immune response in chickens but that a mucosal immune response may be needed to reduce C. jejuni colonization.

Greater rhea (Rhea americana) external morphology at different stages of embryonic and fetal development

Knowledge of wild species embryonic development is important for their maintenance in captivity or the wild. The objective of the present study was to characterize the external morphology and define the biometry of greater rhea embryos and fetuses at different stages of development. A total of 41 embryos and fetuses were analyzed to describe their external morphology using a stereoscopic microscope. The crown-rump (CR), total length (TL), cephalocaudal length (CCL), biparietal diameter (BPD), beak, humerus and tibio-tarsal lengths were measured by digital pachymeter, millimetric scale ruler and cotton thread. The weight of the embryos and fetuses was measured on digital scales. The greater rhea embryos at 5, 6 and 7 days incubation presented a “C” shape. At 9, 10 and 11 days the eyes were big and pigmented. At 11, 12 and 13 days the eyelid covered more than half the eye, resulting in an oval slit. In 14 and 15 day-old embryos, the skin was still thin and the ribs evident, but at 18 days this structure was thicker. In embryos at 21 and 27 days of development closed eyelids were observed forming an eyelid slit, and the eye ball was less pronounced at 27 days. Weight gain presented an exponential growth curve, while measurements such as TL, DBP, beak, humerus and tibio-tarsal length had linear growth over time. Thus it was possible to characterize the greater rhea embryos and fetuses at several incubation ages using their external morphology and morphometric analyses.

Investigation of conception rates achieved with the transfer of sexed and unsexed bovine embryos

The objective of this study was to investigate the conception rates achieved with the transfer of in-vivo-derived sexed and unsexed Holstein bovine embryos to appropriate recipients and to determine the accuracy of nonelectrophoretic PCR sexing. Seven- day-old embryos were derived in vivo by the nonsurgical flushing of the uterus. Before being vitrified and frozen some of the embryos obtained were sexed, while others were not sexed and were maintained as the control group. After thawing, the sexed and unsexed embryos were transferred to 23 and 21 bovine recipients, respectively. The conception rates achieved with the transfer of the sexed and unsexed embryos were 30.4% (7/23) and 42.9% (9/21), respectively. The difference between conception rates achieved in the 2 groups was not statistically significant (P > 0.05). For the sexed embryos the conception rates achieved with the transfer of male and female embryos were 27.2% (3/11) and 33.3% (4/12), respectively. The accuracy of embryo sexing with nonelectrophoretic PCR was 66.6% for male embryos and 100% for female embryos postdelivery. The mean rate of accuracy determined for embryo sexing at the end of the study was 83.33%.

The influence of sustained dual-factor presentation on the expansion and differentiation of neural progenitors in affinity-binding alginate scaffolds

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co-transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate-sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor-2 (bFGF) were affinity-bound to alginate-sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium-crosslinked alginate. NPCs isolated from 18 day-old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10-14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ.

Effects of copper exposure on the hatching status and antioxidant defense at different developmental stages of embryos and larvae of goldfish Carassius auratus

This study aims to assess the effects of copper exposure on hatching status and antioxidant defense at different stages of embryos and larvae of goldfish Carassius auratus. In this study, day-old embryos were randomly grouped after fertilization and then exposed to copper concentrations of 0, 0.1, 0.4, 0.7, and 1.0mgL−1. Copper-exposed fish embryos were sampled every 24h to determine superoxide dismutase (SOD), and catalase (CAT) activities, as well as malondialdehyde (MDA) content. In addition, cumulative mortality and larval deformity were also investigated. The findings showed that cumulative mortality and larval deformity rate increased gradually with copper concentration increase. SOD and CAT activities were inhibited at higher copper concentrations. At a lower concentration (0.1mgL−1), SOD activity increased in larvae, whereas CAT activity showed no significant change (p>0.05). MDA, as the lipid peroxidation product, gradually accumulated in embryos and larvae with increasing copper concentration and the extension of exposure time. At 0.4mgL−1 and more, copper toxicity was shown in embryos and larvae. In conclusion, copper-exposed effects on hatching status and antioxidant defense in C. auratus embryos and larvae showed concentration- and time-dependent patterns. The biochemical parameters in this study can be used as effective indicators for evaluating the responses of copper-exposed fish embryos. In addition, this study demonstrates that 0.4mgL−1 copper (corresponding to 1mgL−1 copper sulfate), used to kill parasites in aquaculture, is not safe concentration, because it can result in toxicity to larvae. Therefore, the copper concentration to kill pathogen should be less than 0.4mgL−1 for C. auratus.

Responses of acid/alkaline phosphatase, lysozyme, and catalase activities and lipid peroxidation to mercury exposure during the embryonic development of goldfish Carassius auratus

This study assessed the impact of mercury exposure on goldfish (Carassius auratus) embryos based on the dynamic characteristics of chemical parameters. Day-old embryos were exposed to different Hg2+ concentrations (0, 0.2, 1, 5, and 10μg/L). Subsequently, the embryos were sampled every 24h during embryonic development to measure acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LSZ), and catalase (CAT) activities, as well as malondialdehyde (MDA) content. The results revealed that the responses of ACP and AKP to mercury exposure presented in dose-dependent and time-dependent manners. The enzyme activities were significantly induced with increased concentrations and extended exposure (at 5μg/L after 72h and 10μg/L after 48h; p<0.05 or p<0.01). LSZ was not sensitive to lower Hg2+ concentrations, whereas LSZ significantly increased at higher concentrations and longer exposure (at 5μg/L at 120h and 10μg/L after 72h; p<0.05 or p<0.01). CAT activities were significantly inhibited at different periods of embryonic development, particularly at 5 and 10μg/L (p<0.05 or p<0.01). Reduced CAT activities were observed at 72, 96, and 120h at 1μg/L (p<0.05 or p<0.01), whereas a decline at 0.2μg/L was evident at 96h (p<0.01). MDA content significantly increased at various stages of embryonic development, particularly at 10μg/L (p<0.05 or p<0.01), and increased further at 72, 96, and 120h at 5μg/L (p<0.05 or p<0.01). At 96h, MDA content was only increased by exposure to 0.2 and 1μg/L (p<0.01). The activities of ACP, AKP, and LSZ remarkably increased at 120h in contrast to 96h (p<0.05 or p<0.01). Therefore, 96h is an important shifting period of embryonic development because the activity of enzyme has been enhanced at this time. Thus, the increased ACP, AKP, and LSZ activities revealed an enhanced ability of the embryo to synthesize more enzymes and attenuate mercury damage. CAT activity negatively correlates with MDA accumulation. The enhanced enzyme activities after specific embryonic stages are used to strengthen the ability to cope with mercury stress and attenuate mercury damage. The biochemical parameters, except LSZ, exhibited sensitivity to mercury, suggesting that they may act as potential biomarkers in assessing the environmental mercury risk on C. auratus embryos.

Effects of 50 Hz electromagnetic fields on the histology, apoptosis, and expression of c-Fos and β-Catenin on the livers of preincubated white leghorn chicken embryos

Reports have demonstrated occurrences of abnormalities in the early stages of chicken embryonic development due to the exposure to electromagnetic fields (EMFs). This article was designed to investigate the effects of sinusoidal EMF on the histopathology, apoptosis, and expressions of c-Fos and β-Catenin genes of the livers of preincubated White Leghorn chicken embryos, based on our published experiments. 300 healthy, fresh fertilized eggs were divided into control (n = 70), sham (n = 70), and four experimental (1–4,days 13, 14, 5, and 19, n = 40) groups. Experimental eggs were exposed to the most effective intensity in a coil with 7.32 mT density, and sham groups were also located in the same coil with no exposure, both for 24 h before incubation. Control, sham, and experimental groups were then incubated in an incubator (37°C, humidity 60%) for 13,14,15, and 19 days. Livers of 13–15 and 19 day-old chicken embryos were removed by C-section and fixed in formalin (10%), stained with Hematoxylin-Eosin and TUNEL for histopathological and apoptosis studies. Others were used for investigating c-Fos and β-Catenin expressions, using RT-PCR. Results showed extensive hemorrhages all over the chicken embryos' bodies and livers, more lymphoid tissues, disturbed parenchymal tissues, sinusoid denaturation, vesiculizad cytoplasm, an increase in the number of apoptotic cells, and a decrease on the levels of expressions of c-Fos and β-Catenin genes in experimental groups of 1–4, comparing control and sham groups.

Bordetella avium causes induction of apoptosis and nitric oxide synthase in turkey tracheal explant cultures

Bordetellosis is an upper respiratory disease of turkeys caused by Bordetella avium in which the bacteria attach specifically to ciliated respiratory epithelial cells. Little is known about the mechanisms of pathogenesis of this disease, which has a negative impact in the commercial turkey industry. In this study, we produced a novel explant organ culture system that was able to successfully reproduce pathogenesis of B. avium in vitro, using tracheal tissue derived from 26 day-old turkey embryos. Treatment of the explants with whole cells of B. avium virulent strain 197N and culture supernatant, but not lipopolysaccharide (LPS) or tracheal cytotoxin (TCT), specifically induced apoptosis in ciliated cells, as shown by annexin V and TUNEL staining. LPS and TCT are known virulence factors of Bordetella pertussis, the causative agent of whooping cough. Treatment with whole cells of B. avium and LPS specifically induced NO response in ciliated cells, shown by uNOS staining and diaphorase activity. The explant system is being used as a model to elucidate specific molecules responsible for the symptoms of bordetellosis.

Embryo vaccination of chickens using a novel adjuvant formulation stimulates protective immunity against Eimeria maxima infection

Our previous study demonstrated that chickens immunized subcutaneously with an Eimeria recombinant profilin protein vaccine emulsified in a Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant developed partial protection against experimental avian coccidiosis compared with animals immunized with profilin alone. Because in ovo vaccination is presently used in commercial applications worldwide throughout the poultry industry, the current study was undertaken to investigate chicken embryo vaccination with profilin plus QCDC adjuvant. Eighteen day-old embryos were immunized with isotonic saline (control), profilin alone, QCDC alone, or profilin plus QCDC, and orally challenged with live Eimeria maxima at 7 days post-hatch. Body weight gain, fecal oocyst output, and intestinal cytokine transcript levels were assessed as measures of protective immunity. While immunization with profilin alone or QCDC alone did not alter body weight gain of infected chickens compared with the saline control group, vaccination with profilin plus QCDC increased body weight gain such that it was equal to the uninfected controls. Immunization with profilin plus QCDC also reduced fecal oocyst shedding compared with unimmunized controls, although in this case QCDC failed to provide an adjuvant effect since no difference was observed between the profilin-only and profilin/QCDC groups. Finally, increased levels of transcripts encoding IL-1β, IL-15, and IFN-γ were seen in the intestinal tissues of animals given profilin plus QCDC compared with the profilin-only or QCDC-only groups. In summary, this study demonstrates an adjuvant effect of QCDC on body weight gain and intestinal cytokine responses following in ovo vaccination of chickens with an Eimeria profilin vaccine.

Meiotic and mitotic behaviour of a ring/deleted chromosome 22 in human embryos determined by preimplantation genetic diagnosis for a maternal carrier

BackgroundRing chromosomes are normally associated with developmental anomalies and are rarely inherited. An exception to this rule is provided by deletion/ring cases. We were provided with a unique opportunity to investigate the meiotic segregation at oogenesis in a woman who is a carrier of a deleted/ring 22 chromosome. The couple requested preimplantation genetic diagnosis (PGD) following the birth of a son with a mosaic karyotype.The couple underwent two cycles of PGD. Studies were performed on lymphocytes, single embryonic cells removed from 3 day-old embryos and un-transferred embryos. Analysis was carried out using fluorescence in situ hybridisation (FISH) with specific probe sets in two rounds of hybridization.ResultsIn total, 12 embryos were biopsied, and follow up information was obtained for 10 embryos. No embryos were completely normal or balanced for chromosome 22 by day 5. There was only one embryo diagnosed as balanced of 12 biopsied but that accumulated postzygotic errors by day 5. Three oocytes apparently had a balanced chromosome 22 complement but all had the deleted and the ring 22 and not the intact chromosome 22. After fertilisation all the embryos accumulated postzygotic errors for chromosome 22.ConclusionThe study of the preimplantation embryos in this case provided a rare and significant chance to study and understand the phenomena associated with this unusual type of anomaly during meiosis and in the earliest stages of development. It is the first reported PGD attempt for a ring chromosome abnormality.

Dopamine D4 receptors regulate intracellular calcium concentration in cultured chicken cone photoreceptor cells: Relationship to dopamine receptor-mediated inhibition of cAMP formation

Dopamine is a retinal neuromodulator secreted from amacrine and interplexiform cells. Activation of dopamine D4 receptors on photoreceptor cells reduces a light-sensitive pool of cAMP. The aim of the present study was to evaluate the role of dopamine receptors and cAMP in the regulation of intracellular Ca2+ concentrations ([Ca2+]i) in photoreceptor cells of chick retina. Retinal cells from 6 day-old chicken embryos were isolated and cultured for 5–7 days prior to experiments. Cone photoreceptors were the predominant cell type in these cultures. Dopamine and agonists of dopamine D4 receptors suppressed K+-stimulated uptake of 45Ca2+ and [Ca2+]i, measured with the Ca2+-sensitive fluorescent dye fura-2AM. The effects of the agonists were blocked by dopamine D2/D4 receptor antagonists or by pertussis toxin. 8Br-cAMP, a cell-permeable analog of cAMP, had no effect on inhibition of K+-stimulated 45Ca2+ influx or [Ca2+]i by dopamine D2/D4 receptor agonists. Quinpirole inhibited the increase in cAMP level elicited by K+, which requires Ca2+ influx through voltage-gated Ca2+ channels, but not that induced by the calcium ionophore A23187. Moreover, dopamine had no effect on either forskolin-stimulated or Ca2+/calmodulin-stimulated adenylyl cyclase activity in cell membranes prepared from the cultured cells. These data indicate that the decrease of cAMP elicited by dopamine D4 receptor stimulation may be secondary to decreased [Ca2+]i.

Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision

The elucidation of mechanisms underlying telencephalic neural development has been limited by the lack of knowledge regarding the molecular and cellular aspects of the ganglionic eminence (GE), an embryonic structure that supplies the brain with diverse sets of GABAergic neurons. Here, we report a comprehensive transcriptomic analysis of this structure including its medial (MGE), lateral (LGE) and caudal (CGE) subdivisions and its temporal dynamics in 12.5 to 16 day-old rat embryos. Surprisingly, comparison across subdivisions showed that CGE gene expression was the most unique providing unbiased genetic evidence for its differentiation from MGE and LGE. The molecular signature of the CGE comprised a large set of genes, including Rwdd3, Cyp26b1, Nr2f2, Egr3, Cpta1, Slit3, and Hod, of which several encode cell signaling and migration molecules such as WNT5A, DOCK9, VSNL1 and PRG1. Temporal analysis of the MGE revealed differential expression of unique sets of cell specification and migration genes, with early expression of Hes1, Lhx2, Ctgf and Mdk, and late enrichment of Olfm3, SerpinE2 and Wdr44. These GE profiles reveal new candidate regulators of spatiotemporally governed GABAergic neuronogenesis.

Diagnostic prénatal et diagnostic pré-implantatoire : arbre décisionnel, nouvelles pratiques ?

Preimplantation genetic diagnosis (PGD) purpose is to assess the genetic status of 3 day-old embryos. PGD offers thus to couples "at-risk" of a genetic disorder an earlier option to prenatal diagnosis (PND). At the beginning, PGD's indications, patients and law were very closed to PND, but PGD specificities are gradually raising. Particularly, indications vary considerably in countries where the absence of law authorizes all the practices. Some of these applications are moreover raising serious ethical issues. Even in France, where this activity is particularly supervised, the recent modification to the law marks this evolution.

Vasculogenesis and angiogenesis in the mouse embryo studied using quail/mouse chimeras

Using quail/chick chimeras, we have previously shown that different embryonic territories are vascularized through two distinct mecanisms, angiogenesis and vasculogenesis. Angiogenesis occurs in tissues of somatopleural origin, vasculogenesis occurs in territories of splanchnopleural origin. The aim of this work was to establish if these modes of vascularization were conserved in the mammalian embryo. Since in vivo manipulations with mammalian embryos are difficult to perform, we used a quail/mouse chimera approach. Mouse limb buds of somatopleural origin, and visceral organ rudiments of splanchnopleural origin, were grafted into the coelomic cavity of 2.5 day-old quail embryos. After four to seven days, the hosts were killed and the origin of the endothelial cells in the mouse tissues was determined by double staining with the quail endothelial and hematopoietic cell-specific marker, QH1 and mouse-specific VEGFR2 and VEGFR3 probes. Our findings show that the great majority of vessels which developed in the mouse limbs was QH1+, indicating that these tissues were vascularized by angiogenesis. Conversely, visceral organs were vascularized through the vasculogenesis process by mouse endothelial cells which differentiated in situ. These results demonstrate for the first time that in the mouse embryo, as previously shown in avian species, the tissues from somatopleural origin are vascularized by angiogenesis, while rudiments of a splanchnopleural origin are vascularized by vasculogenesis, both at vascular and lymphatic levels.

Effect of ciclosporin A on the organogenesis and function of embryonic metanephroi allografted into adult rats

To evaluate the effect of ciclosporin A (CsA) on the organogenesis and function of embryonic metanephroi allografted into adult rats. The whole metanephroi from the 15, 16 and 17 embryonic day-old (E15, E16, E17) embryos of Sprague-Dawley (SD) rat were allografted into the omenta of SD adult rats with their left kidneys resected, which were divided into 2 categories: 3 CsA-treated groups of 10 rats (E15CsASD, E16CsASD, and E17CsASD) and 3 non-CsA-treated groups of 10 rats (E15SD, E16SD, and E17SD). Thirty SD rats without kidney resection were divided into 6 equal groups and underwent allografting embryonic metanephroi in the same manner as mentioned above. The E15 metanephroi of Lewis rats were allografted into the omenta of Thirty adult Brown Norway (BN) rats with one kidney resected. Were divided into 6 equal groups, received allografting of embryonic metanephroi, and injected with CsA of normal saline in the manner as mentioned above (E15CsABN and E15BN). Two to 4 weeks after implantation, the metanephroi allografted in host rats were removed for histopathological examination or anastomosed for renal function measurement 4 weeks later. (1) Four weeks after implantation, the E17SD and E16SD metanephroi showed signs of acute rejection as hypercellular glomeruli and mononuclear cell infiltration in the interstitium. The E16CsASD and E17CsASD metanephroi formed mature nephrons and collecting ducts with few lymphocytic infiltrates. After CsA was discontinued, the E16CsASD and E17CsASD metanephroi were rejected fully within 21 days. (2) 4 weeks after implantation, the E15SD metanephroi were enlarged, became vascularized, and developed mature tubules and glomeruli; however, they were rejected by 100 days after implantation. The E15 Lewis metanephroi were fully rejected within two weeks in the BN adult rats. With CsA administrated, the E15 Lewis metanephroi developed normal mature nephrons and collecting ducts within the adult BN rats. If CsA was discontinued, the E15CsABN metanephroi were rejected. (3) The E15CsASD metanephroi had significantly lower values of wet weight (P = 0.006) and higher values of creatinine clearances (P = 0.007) than the E15SD metanephros transplants, but were identical to those of the E16CsASD metanephroi (P = 0.948, P = 0.840). (4) The metanephroi did not grow or differentiate in the rats without host kidney resection. (1) Cyclosporin A may suppress graft rejection, thus normalizing the growth and function of fetal metanephroi in the omenta of host rats. (2) A variety of factors affect the growth and development of allografted metanephroi, whereas rejection remains the major one.

Organizational effects of diethylstilbestrol on brain vasotocin and sexual behavior in male quail

In Japanese quail, we previously described a sexual dimorphism of the parvocellular vasotocin system of the limbic region that, as the reproductive behavior, is steroid-sensitive and is organized during embryonic life by the exposure to estradiol. We verified in this study whether diethylstilbestrol, a chemical xenoestrogen, has analogous organizational effects on the vasotocin system of limbic regions and on copulatory behavior of male Japanese quail. We injected in the yolk sac of 3 day-old quail embryos diethylstilbestrol or estradiol benzoate (a treatment which suppresses male copulatory behavior in adulthood and reduces vasotocin innervation), or sesame oil (control). No further hormonal manipulations were performed after hatching. Sexual behavior was recorded in males at the age of 6 weeks. Estradiol- and diethylstilbestrol-treated males exhibited a total suppression of copulatory behavior. After behavioral tests, all males were sacrificed and brain sections processed for vasotocin immunocytochemistry. Significant decrease in the density of vasotocin immunoreactivity was detected in the medial preoptic nucleus, in the bed nucleus of stria terminalis, and in the lateral septum of diethylstilbestrol-treated males. The magnocellular vasotocin neurons were, in contrast, not affected. In conclusion, the present data demonstrate that embryonic treatment with diethylstilbestrol induces a full sex reversal of behavioral phenotype as well as a significant decrease of vasotocin expression in the preoptic-limbic region in male Japanese quail. Therefore, the parvocellular vasotocin system could represent an optimal model to investigate the effects of pollutants on neural circuits controlling reproductive functions.