Growth, development, pathogenicity, and transplantation of Echinostoma caproni (Trematoda) on the chick chorioallantois.

Excysted metacercariae of Echinostoma caproni were cultivated on the chorioallantoic membrane (CAM) of 6-day-old domestic chick embryos for 2-13 days postinoculation. There was no significant difference in the body area of fixed and stained preovigerous worms from the CAM versus those grown in domestic chicks. However, ovigerous worms from the CAM were significantly smaller than those from chicks. Worm development, i.e., gonadal differentiation, uterine curling, vitellinogenesis, ovigerousness, and oviposition, took 1 day longer on the CAM than in the chick. Histopathologic studies of worms attached to the CAM were done on cryostat and paraffin sections stained with hematoxylin and eosin. Some worms attached to the CAM by their collar spines and acetabulum, whereas others penetrated the chorionic epithelium and encapsulated in the mesenchyme. Pathogenicity to the CAM included hyperplasia of the chorionic epithelium, hemorrhagia, reduced fibrocytes and blood vessels, but increased lymphocytes and eosinophils in the mesenchyme. Attempts to transplant 11-day-old CAM worms to new CAMs were unsuccessful.     

Cultivation of excysted metacercariae of Echinostoma caproni (Trematoda) to ovigerous adults on the chick chorioallantois

Excysted metacercariae of Echinostoma caproni were cultivated on the chick chorioallantoic membrane (CAM) maintained at 38.5 +/- 1 C and a relative humidity of 60-65%. Of 59 6-day-old embryos, each inoculated with 25 metacercariae, 29 (49.2%) were infected 2-12 days postinoculation. The total number of worms recovered from the infected eggs was 163 or 22.5% of the 725 inoculated metacercariae. Eggs contained from 1 to 12 (average 5.6) worms per CAM. Worm length increased rapidly from an average of 0.5 mm at 2 days to about 3.0 mm at 6 days postinoculation. Ovigerous worms first were seen on day 8 PI, but fluke eggs did not develop embryos. Worm development in ovo lagged about 1 day behind that of in vivo worms. One worm maintained for 17 days on 2 successive CAMs reached 6 mm in length, contained about 100 eggs in its uterus, and laid an additional 100 eggs on the CAM surface.     

Development of calcium reabsorption by the allantoic epithelium in chick embryos grown in shell-less culture

The allantoic sac of the chick embryo functions as a primitive urinary bladder, storing and modifying the excretory fluid produced by the embryo. We have used chick embryos grown in shell-less culture to study the in situ handling of Ca2+ by the allantoic epithelium. Between Days 8 and 13 of incubation (38 degrees C, 5% CO2), the [Ca2+] of the allantoic sac fluid declines from about 1.5 mM to less than 0.3 mM, with most of this Ca2+ reabsorption occurring between Days 10 and 11. In 13-day-old embryos, the allantoic epithelium reabsorbs within 24 hr 85-92% of 45Ca2+ injected into the allantoic sac, while in 9-day-old embryos 45Ca2+ reabsorption is less than 40% by 24 hr. This is evidence for the developmental onset of a Ca2+ reabsorption process in the allantoic epithelium. The allantoic fluid Ca2+ is reabsorbed into the embryo's blood in which the serum [Ca2+] is about 1.5 mM. Also, electrical potential profiles reveal that the serosal (mesenchymal) side of the allantoic epithelium is 15-30 mV positive compared to the mucosal (luminal) side. Thus, by electrochemical criteria this reabsorption process appears to be active.     

Estradiol secretion by the ovary of 19-day-old hypophysectomized and sham-operated chick embryos

The amounts of estradiol released into culture media by ovaries from 19-day-old hypophysectomized (decapitated) and sham-operated chick embryos were determined by RIA. Per single ovary, ovaries from decapitated embryos secreted slightly less estradiol than ovaries from sham-operated ones during a 4-h culture period (837 +/- 413 vs 935 +/- 378 pg), but this difference was not significant. On an ovarian weight basis, ovaries from decapitated embryos secreted slightly more estradiol than ovaries from sham-operated ones (1.15 vs 0.91 ng), but this difference was not significant either. It is concluded from these results that the hypophysis does not control estradiol secretion by the ovary in the 19-day-old chick embryo.     

A method to obtain avian germ-line chimaeras using isolated primordial germ cells.

Primordial germ cells (PGCs), collected from the blood of 2-day-old chick embryos, were concentrated by Ficoll density centrifugation. The blood contained 0.048% PGCs and the concentrated fraction contained 3.9% PGCs in blood cells. The PGCs were picked up with a fine glass pipette, and one hundred were then injected into the terminal sinuses of 2-day-old Japanese quail embryos (24 somites); bubbles were then inserted to prevent haemorrhage. The embryos were further incubated at 38 degrees C for 24 h, and then fixed. Serial sections were stained with the periodic acid-Schiff reagent (PAS) to demonstrate chicken PGCs and with Feulgen stain to identify quail cells. On the basis of the differences in staining properties, 63.6 +/- 5.3 chick PGCs were detected in the quail embryo in the area where the gonads develop. Furthermore, 39.3 +/- 4.5 chick PGCs were incorporated into the quail germinal epithelium within 24 h of the injection. A similar percentage of the host (quail) PGCs had also migrated to the germinal epithelium at the same stage of development. This technique for obtaining germ-line chimaeras will facilitate research on avian germ-line differentiation.     

A RADIOIMMUNOASSAY FOR THE DETECTION OF DELTA CRYSTALLIN IN CHICK LENS DIFFERENTIATION

Delta crystallin was isolated from 10–13 day chick embryo lens fiber cells. The lens fiber cell extract was isoelectrically precipitated at pH 5.1 to remove alpha and beta crystallins. Further purification by filtration through Sephadex G-150 and then acrylamide gel electrophoresis yielded a single, homogeneous preparation of delta crystallin, as characterized by gel electrophoresis. This purified delta crystallin was injected into rabbits to produce a potent antiserum to chick lens delta crystallin. The purified delta crystallin was iodinated with ¹²⁵Iodine, using the chloramine-T procedure. A radioimmunoassay for delta crystallin was then developed, using the principles of competitive protein binding analysis. The radioimmunoassay developed here had a minimum sensitivity of 50 nanograms, and effectively ranged to 1000 nanograms.
Developing lens rudiments from early chick embryos, beginning from 24 hr incubation up to 72 hr were examined at 6 hr intervals. All determinations from 24 hr through the 48 hr sample showed less than 10 nanograms per 100 lens rudiments. This was below the effective minimum detection limits of the assay. The first accumulation of delta crystallin was detected in the 54 hr sample, and increased thereafter.     

Hypophysis and estradiol secretion in the chick embryo

Ovaries from 10- to 18-day-old chick embryos hypophysectomized by partial decapitation were cultured in vitro and their estradiol secretion was compared to that of ovaries from control embryos. The production of estradiol was not less in the decapitated than in the control embryos at 15-18 days, neither per ovary nor on the basis of ovarian weight. However, the difference was significant at 10-11 days. These results suggest that the hypophysis controls estradiol secretion by the chick embryo ovary in the early stages, but not in the later ones.     

Lack of physiological plasticity in the early chicken embryo exposed to acute hypoxia

By exposing chicken embryos to hypoxia (10%) acutely (2, 4, and 6 hr) during early development (2, 3, and 4 days) we tested the hypothesis that hypoxia has an impact on embryonic growth and impairs cardiac development at the time cardiac morphogenesis is taking place. After the hypoxic perturbation, the embryos were allowed to develop until day 9, when embryo mass, heart mass, and rate of oxygen consumption were recorded. Four-day-old embryos exposed to 6 hr of hypoxia showed an increased mortality (38.9% versus 18% for controls), indicating the immediate effect of hypoxia on survivability. While only 8% of the controls displayed morphological abnormalities, 3- and 4-day-old embryos exposed for 6 hr showed more frequent developmental abnormalities (25% and 30% respectively). No significant differences in embryo or heart mass were found except in 4-day-old embryos exposed for 2 hr. Mass-specific oxygen consumption was not different between controls and embryos exposed to hypoxia at 2 or 3 days of development, but it was increased in 4-day-old embryos exposed for 4 hr (P < 0.05). These results suggest that an acute hypoxic episode does not have an impact when occurring very early in development (days 2 or 3). However, when the hypoxic episode occurs on day 4, survivability is largely decreased. Considering the lack of permanent effects on the surviving embryos, we suggest that the early embryo resorts to a simple strategy of death or survival, and the individual capacity for survival must be based on interindividual differences rather than the existence of compensatory mechanisms. J. Exp. Zool. 286:450-456, 2000.     

Plasminogen activator dependent pathways in the dissemination of human tumor cells in the chick embryo

We have previously shown that inhibition of uPA activity of a human tumor-HEp3-results in a drastic reduction of its metastasis in the chick embryo. Using 125IUdR-labeled tumor cells, we have now studied the role of uPA in individual steps of tumor metastasis. We found that, 48 hr after inoculation of tumor cells on the CAM, the organs of the embryos, inoculated with cells in which uPA was inhibited, contained 4-fold less cells than the controls. Neither the initial advance of the tumor mass into the CAM nor the process of extravasation was affected by the inhibition of tumor uPA. However, the infiltration of the CAM mesenchyme by individual tumor cells was blocked when tumor uPA activity or production was inhibited. In addition, indirect evidence implicated uPA as an essential factor in the tumor cell intravasation.     

Chick endogenous lectin enhances chondrogenesis of cultured chick limb bud cells

We have studied the effect of β-d-galactoside-specific lectin purified from 14-day-old chick embryos on the differentiation of the mesenchymal cells dissociated from the limb buds of stage 24 chick embryos, using the micro-mass culture method described previously. When the cells were incubated with the lectin during the initial 12 hr of culture, cell proliferation became slightly activated. The lectin-treated cells formed a greater number of cartilage nodules and incorporated about twice as much as [³⁵S]sulfate per cell than the control cultures. The results of this study show that the chick endogenous lectin promotes cartilage differentiation in vitro and that endogenous lectin may possibly be involved in chondrogenesis in vivo.     

Effect of some antineoplastics on metabolic heme pathway

1. The porphyrinogenic ability of several antineoplastics used in the therapy of the different cancers was evaluated. The action of cyclophosphamide, busulfan and 5-fluorouracil on the amount and nature of the accumulated hepatic porphyrins and on the activity of delta-aminolaevulinate synthase (ALA-S), were estimated at different doses and times of drug treatment in 17-day-old chick embryos. 2. It was observed that cyclophosphamide produces a significant increase in the accumulation of hepatic porphyrins at different doses as well as in the activity of the ALA-S, at all the incubation times. Cyclophosphamide alters the pattern of porphyrins accumulated in the liver, where a coproporphyrin: protoporphyrin ratio higher than in the controls can be observed. 3. Busulfan increased the hepatic porphyrins accumulated in the liver but to a lesser degree than cyclophosphamide. 4. 5-Fluorouracil did not modify the hepatic porphyrin content when it was administered at doses up to 40 mg/embryo. 5. When the embryos were injected with busulfan or 5-fluorouracil no significant differences were observed in the activity of ALA-S up to 11 hr of incubation. 6. These results indicate that cyclophosphamide has a remarkable porphyrinogenic capacity in chick embryo while busulfan, notwithstanding the fact that it alters the haem pathway, it does so to a degree that does not impair the regulation of ALA-S activity. Fluorouracil seems to be non porphyrinogenic in this system, up to 40 mg/embryo.     

Chronic ethanol exposure in the embryonic chick heart: effect on myocardial function and structure

To investigate the effect of chronic ethanol exposure on the embryonic chick heart, chick embryos were exposed daily to one of seven graded doses of ethanol or to saline only (shams) from 0 to 96 hr of incubation. One hour before and after exposure at 72 hr, and 1 hr before and after exposure at 96 hr, embryos were analyzed for changes in heart function, embryo tissue ethanol content, occurrence of anomalies, and embryo weights. At both 71 and 73 hr of incubation (during cardiogenesis), when compared to shams, heart rate (HR) in embryos receiving ethanol doses greater than 0.0375 ml increased significantly (P less than .05) with commensurate increases in injected ethanol. Additionally, at 73 hr, depressed cardiac contractility, measured as shortening fraction, was noted at doses greater than or equal to .0375 when compared to shams. While slight increases in shortening fraction (SF) across dose were noted at 95 and 97 hr, only random doses were statistically significant from shams, with no specific trend in either HR or SF at this postcardiogenesis stage. Within each time group, gas chromatography analysis of embryo tissue ethanol content demonstrated a linear relationship between dose injected and tissue ethanol content retrieved. With increasing dose and stage, viability decreased. Weights of ethanol-injected embryos were not significantly different from shams within each time group. Our studies of the response of the embryonic chick heart to ethanol indicate both dose and stage susceptibility, with greater susceptibility to ethanol injury during active cardiogenesis.     

Influence of Hydrocortisone on Chick Embryo Retina Development

Treatment of chick embryos in ovo with hydrocortisone-21-phosphate (a single dose of 150 micrograms) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65-70%) in 8-10-day-old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 micrograms daily for 2 days) also produced an age-dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 micrograms of metopirone, a compound that prevents the 11 beta-hydroxylation of steroid molecules in the adrenal glands, impeded the decrease in thymidine kinase activity that normally occurs in chick embryo retina after day 9 of development. In addition, metopirone prevented the inhibition exerted by ACTH on thymidine kinase activity but had no effect on the action of hydrocortisone.     

Complexity of nuclear and polysomal RNAs in growing Dictyostelium discoideum cells

The proliferative activity of undifferentiated brain cells from either 5- or 7-day-old chick embryos has been investigated by labeling the cells with a 24-hr pulse label of [¹⁴C]- or [³H]-thymidine during the early stages (0 to 8 days) of culture. As soon as the neurons and the glial cells could be distinguished (after 4, 7, or 14 days of culture), the cultures were prepared and submitted to the activated autoradiographic method. In some experiments a continuous labeling was applied up to 2 weeks. During the first 48 hr of culture, and for both embryonic ages studied, nearly all neuronal precursors were able to proliferate. After 4 days in culture for the 7-day-old embryo and 7 days in culture for the 5-day-old embryo most of the neuronal cells stopped dividing. These two culture periods correspond to the stage of the embryonic life when the end of the mitotic activity of neuroblasts occurs in vivo. Thus, the proliferation and development in culture of most neuroblasts was found to parallel the in vivo evolution of these cells. Some neuroblasts, however, continued to multiply in vitro for a longer period of time. The astroblasts precursors were found to multiply actively from the 3rd day on, or immediately from time zero, for the 5- and 7-day-old chick embryos, respectively. These observations seem to indicate that the astroblast precursors are in a latent stage until they have reached Day 7. Thereafter, they proliferate actively during the first week of culture and therefore remain in an embryonic stage during this culture period. This fact corresponds also to the in vivo situation, where the glial cell precursors multiply actively around the same time period.     

Effect of partial decapitation (hypophysectomy) on 17β-estradioI secretion by the chick embryo ovary

Ovaries of equally matched partially decapitated (hypophysectomized) and sham-operated (at 42 h of incubation) 11 1/2-day-old chick embryos, i.e. embryos as identical as possible as regards body size and other morphological features, were compared with respect to the amounts of 17 beta-estradiol secreted during a 4 h-culture period. The 17 beta-estradiol secretion rate was very significantly lower in the decapitated than in the control embryos. This result suggests that the hypophysis controls 17 beta-estradiol secretion of the ovary in the 11 1/2-day old chick embryo.     

Culturing two- to eight-cell caprine embryos using domestic chicken eggs

Early-stage caprine embryos were placed in the chick embryo amnion to determine if this culture method would support the development of embryos from a farm animal species. Following superovulation and natural mating, two- to eight-cell embryos were surgically collected from crossbred donor goats. Embryos were allotted to in vitro culture treatments across two different experiments (EXP). In EXP-I, embryos allotted to Treatment A (control) were cultured in Ham's F-10 with 10% fetal calf serum and 1% antibiotic-antimycotic (HF-10). Embryos in Treatment B were placed on a bovine fetal uterine fibroblast monolayer in HF-10, embryos allotted to Treatment C were agarose embedded and injected into the amniotic cavity of a day-4 chick embryo and those placed in Treatment D were co-cultured in HF-10 with day-15 caprine trophoblastic vesicles. In EXP II Treatments A, B, and C were the same; however Treatment D was omitted. EXP-I and EXP-II also differed in that chick embryo co-culture was for 72 hr in EXP-I but was extended to 96 hours in EXP-II. Additionally, the monolayer co-culture was limited to 96 hr in EXP-II; whereas, embryos in EXP-I remained on monolayer culture for 96 hr plus an additional 72 hr for subsequent embryo evaluation. Results indicate that the amniotic cavity of the developing chick embryo enhanced the development of two- to eight-cell caprine embryos through to hatching blastocysts when compared with that of the control medium alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathogenicity of avian nephritis virus for embryonating hen's eggs

The pathogenicity of avian nephritis virus (ANV) for embryonating hen's eggs was studied by various routes of inoculation. When inoculated with ANV by the yolk sac route, 6-day-old embryos showed the highest susceptibility and all of them died 3 to 14 days postinoculation (PI). They manifested hemorrhage and edema of the whole body (3 to 6 days PI) and stunting (7 to 14 days PI). The 50% egg-infective dose of the virus by yolk sac inoculation coincided well with the virus titer expressed in plaque-forming units determined on the monolayer of chicken kidney cell cultures. The virus could be passed serially through the chorioallantoic membrane (CAM) of embryonating hen's eggs. In these eggs the CAM presented edematous thickening at the inoculation site, and the embryo stunting. when inoculated by the CAM route, high virus doses killed all embryos, but low virus doses allowed some of the infected embryos to hatch normally. When inoculated by the allantoic cavity route, the virus did not multiply in the allantoic cavity of embryonating eggs, but some of these eggs became infected. Fluorescent antigens were present only in the kidneys and CAM of embryos infected with the virus. The virus was recovered at a low rate from cloacal swabs of chicks from normally hatched eggs inoculated with the virus by the CAM route. These chicks were variable in growth, but had antibodies against the virus and developed nephritis at 36 days of age.     

Developmental changes in collagen glycosyltransferase activities in chick embryos

The developmental pattern of collagen galactosyltransferase and collagen glucosyltransferase activities was determined in chick embryos between the 4th and 21st day of growth. Both enzyme activities increased up to the 16th day and decreased thereafter in whole chick embryos and in most tissues studied. The highest collagen glycosyltransferase activities were found in the leg tendons of the 16-day-old embryos, and the activities found in cartilage were higher than those noted in either skin or skull, indicating that the the activities of the collagen glycosyltransferases may play a part in the regulation of the carbohydrate content of the collagen synthesized by a given tissue. The changes observed in the collagen glycosyltransferase activities agree with previous data on the development of prolyl and lysyl hydroxylase activities and also with findings on collagen turnover in the developing chick embryo.     

The hen's fertile egg screening test (HEST): A comparison between the acute toxicity for chick embryos and rodents of 20 drugs

The possibilities of using developing chick embryos for evaluating drug activities and toxicities were studied by determining LD₅₀ values for 20 drugs with 14 different pharmacological activities. Fifteen-day old chick embryos received drugs through the air cell and deaths were measured at 48 hr after the treatments. The LD₅₀ values were determined and compared to the i.v., i.p., s.c. and p.o. values from mice listed in the Registry of Toxic Effects of Chemical Substance. The systemic toxicity of 15-day-old chick embryos to drugs were similar to those of mice with the following exceptions. The chick embryos seemed to be more sensitive than mice to antineoplastic or antibiotic agents such as actinomycin D and doxorubicin, whereas, LD₅₀ values of cholinergic and cholinergic blocking drugs by this method were 10 to 20 fold of LD₅₀ (i.v.) of mice. These observations are important for applying the hen's fertile screening test (HEST) to the determination of drug activities other than that of embryo toxicity or teratogenic activity.Abbreviations i.v. intravenous administration - i.p. intraperitoneal administration - s.c. subcutaneous administration - p.o. oral administration - LD₅₀ acute median lethal dosage     

DIFFERENTIATION OF THE DIGESTIVE TRACT EPITHELIUM UNDER THE INFLUENCE OF THE HETEROLOGOUS MESENCHYME OF THE DIGESTIVE TRACT IN THE BIRD EMBRYOS

The endoderm of the oesophagus, proventriculus, gizzard or small intestine of the 5-day-old chick or quail embryo was cultivated in combination with homologous or heterologous mesenchyme on a WxxxOLFFyyy and HxxxAFFHNyyy medium for 7 to 21 days or on the chorio-allantoic membrane (CAM) for 8 days. With homologous mesenchyme the epithelium always differentiated homotypically. In association with heterologous mesenchyme, the differentiation of the epithelium was both homotypical and heterotypical depending on the region of the digestive tract. The oesophagus and small intestine differentiate mainly homotypically both in culture and on CAM, but the gizzard and proventriculus show heterotypic differentiation particularly on CAM. Thus, the endoderm of the digestive tract of the 5-day-old chick or quail embryo, though rather "determined", still reacts to the heterologous stimuli of the mesenchyme to some degree.