Glucocorticoid receptor-mediated teratogenesis in the chick embryo

The susceptibility of chick embryos to the teratogenic action of intraamniotically injected hydrocortisone increases by several orders during the first four days of incubation. An attempt was made to correlate this phenomenon with the appearance of specific intracellular binding proteins for glucocorticoids. The binding of [3H] corticosterone to the soluble cytoplasmic proteins of the chick embryo was investigated on days 1.5, 2, 3, and 4 of incubation using a gel filtration method. No evidence of high affinity binding was found in embryos on day 1.5. High affinity binding of [3H] corticosterone to the cytosol proteins was first observed in embryos on day 2, but the binding capacity was four times lower than that found in embryos on days 3 and 4. A correlation was obtained between the increasing sensitivity of the chick embryo to hydrocortisone and the appearance of the intracellular binding protein for glucocorticoids. The causal relationship between these two phenomena is further supported by the finding that administration of a nonteratogenic dose of cortexolone completely prevents the teratogenic "cleft beak" action of hydrocortisone, presumably on the basis of competition for binding sites to the glucocorticoid receptor. These findings are consistent with the hypothesis that the teratogenic action of glucocorticoids is mediated by specific cytoplasmic receptors in the chick embryo.     

Malformation spectra and embryolethality following intraamniotic administration of glucocorticoids in the chick embryo

The effects of a natural hormone hydrocortisone and of the synthetic corticoids dexamethasone and triamcinolone on several morphogenetic systems were investigated in 2, 3, 4 and 5 day-old chick embryos. Embryotoxic doses of the glucocorticoids were administered subgerminally on day 2, and intraamniotically on days 3, 4 and 5 of incubation. On the basis of morphological analysis of embryos performed on day 8 the malformation spectrum was determined, which shows nearly identical characteristics in all the corticoids under study. Morphogenetic systems of the body wall and face were affected most frequently; the eye only to a little extent, and the brain only rarely. No malformations of the limbs, trunk and heart were found. Embryolethal effect was observed approximately in a quarter of embryos of the individual experimental groups.     

Behavior of chick primordial germ cells injected into the blood stream of quail embryos

The distribution and behavior of chick primordial germ cells (PGC) injected into quail embryos were examined. PGC from chick embryos at stages 13-14 were injected into the blood stream of quail embryos at stages 15-20. After one day, the quail embryos were examined histologically. The chick PGC in the quail embryos could be readily identified by the histochemical PAS technique, whereas quail PGC were never stained by PAS. When the chick PGC were injected into the quail embryos during stages 15-18, they appeared mostly in the gonadal region of the recipient quail embryos. A few PGC were found at extragonadal sites. When the chick PGC were injected into the quail embryos at stages 19-20, in which the PGC of the recipient quail embryos had finished their migration into the gonads, most of the donor chick PGC were found at ectopic sites, in the head, trunk and limbs. These results indicate that most of the chick PGC, injected at the earlier stages 15-18, migrated to the gonadal anlagen of the recipient, while following later injection (from stage 19), most of the chick PGC migrated to ectopic sites.     

Age Differences in the Effect of Hydrocortisone on the Synthesis of Insoluble Elastin in Aortic Tissue of Growing Chicks

Glucocorticoids have been shown by others to increase the synthesis of elastin both in aortic tissue of embryo chicks and in cells derived from fetal ligamentum nuchae. This report describes the effects of hydrocortisone on the production of elastin in aortic tissue of developing chick embryos and chicks. While the effect of hydrocortisone on elastin synthesis is stimulatory in the 14 day chick embryo and the day-old chick, the same dose of this glucocorticoid has no effect on elastin production in the 7 day old chick and significantly inhibits synthesis of elastin in the 14 day old chick. These age-related alterations in elastin production in response to hydrocortisone cannot be attributed to an effect of the steroid on the pool size of the radioactively labelled amino acid used to measure elastin synthesis.     

Age differences in the effect of hydrocortisone on the synthesis of insoluble elastin in aortic tissue of growing chicks

Glucocorticoids have been shown by others to increase the synthesis of elastin both in aortic tissue of embryo chicks and in cells derived from fetal ligamentum nuchae. This report describes the effects of hydrocortisone on the production of elastin in aortic tissue of developing chick embryos and chicks. While the effect of hydrocortisone on elastin synthesis is stimulatory in the 14 day chick embryo and the day-old chick, the same dose of this glucocorticoid has no effect on elastin production in the 7 day old chick and significantly inhibits synthesis of elastin in the 14 day old chick. These age-related alterations in elastin production in response to hydrocortisone cannot be attributed to an effect of the steroid on the pool size of the radioactively labelled amino acid used to measure elastin synthesis.     

Behavioral analysis of opiate-mediated inhibition in the early chick embryo

Morphine (opiate agonist) produced a dose-dependent decrease in the spontaneous motility of 5- and 9-day chick embryos. Naloxone (opiate antagonist) appeared to reverse competitively the inhibition of motility caused by morphine. The effects of morphine on spontaneous motility in 5-day embryos were also reversed stereospecifically by the opiate antagonist pairs WIN 44441-3/WIN 44441-2 and levallorphan/dextrallorphan. Levorphanol (opiate agonist) also produced a dose-dependent decrease in the motility of 5-day embryos while its inactive (+)-isomer, dextrophan, was not effective. Etorphine (opiate agonist) was more than 1000-fold more effective than morphine in inhibiting the motility of 5-day embryos. The effectiveness of several opiate agonists and antagonists on the spontaneous motility of 5-day embryos was similar to their effectiveness in radioligand-binding studies on isolated membrane receptors from either adult mammalian brain or ileum. Levorphanol was more effective than dextrophan and etorphine was substantially more effective than morphine in decreasing the spontaneous motility of 4-day embryos. WIN 44441-3 was more effective than WIN 44441-2 in reversing the inhibition of motility in 4-day embryos caused by morphine. Morphine inhibited spontaneous hind-limb motility in both thoracic spinal and sham-operated 7-day embryos; the inhibition of motility caused by morphine was reversed by WIN 44441-3 in both thoracic spinal and sham-operated 7-day embryos. [Leu5]enkephalin-like immunoreactivity in the lumbar spinal cord was concentrated in the superficial laminae of the dorsal horn and along the midline rostral to the central canal. A lesser concentration of immunoreactive processes occurred in the medial and lateral motor columns where labelled varicosities appeared to contact motoneurons. Opiate receptors appear to be present at least as early as day 5 (and perhaps as early as day 4) in the chick embryo. Opiate receptors appear to be present in the lumbar spinal cord of the chick embryo at least as early as day 7. The structural requirements for ligand binding to opiate receptors in the 5-day chick embryo are similar to the requirements for ligand binding to opiate receptors in the adult.     

Cleft beak induced by hydrocortisone in the chick is prevented by increased cell division after experimental reduction of amniotic fluid

 Hypoplasia of the medial nasal process has been reported in chick embryos on embryonic day (ED) 5, 24 h after their exposure to hydrocortisone (HC). As a result, the cleft beak occurs in 80–100% of specimens on ED 9. In order to analyze its influence on cell proliferation, HC was injected intra-amniotically into embryos on ED 4, and the mitotic index and number of BrdU-positive cells were evaluated 24 h later, both in the epithelium and mesenchyme of the medial nasal processes, on serial frontal histological sections. Two hours after BrdU administration, there were 50% of labeled mesenchymal cells in the embryos exposed to HC and only 23% in the control group. The mitotic index of mesenchymal cells was significantly lower in the HC group than in the controls. The epithelium showed no significant difference. HC seemed to prevent the mesenchymal cells from entering mitosis. The cleft beak in the embryos exposed to HC on ED 4 was totally eliminated by tearing open the amnion (amniotomia) and allowing fluid to leak out on ED 5. In some of specimens exposed to HC, the mitotic index was investigated at six time intervals from 15 to 120 min after amniotomia. A significant increase in the mitotic index was detected in the mesenchymal cells of the medial nasal processes during the first hour after amniotomia. Such a prompt increase of the mitotic activity may be hypothetically explained by release of the HC from its receptor binding as a consequence of outflow of the amniotic fluid together with the HC pool, and freeing of the mesenchymal cells, blocked in the G₂ phase, to enter mitosis. As a result, the hypoplasia of the medial nasal process might be compensated and the development of the cleft beak prevented. Accepted: 15 October 1996     

The effect of hydrocortisone on extracellular connective tissue fibrils in the early chick embryo

Two series of chick embryos were treated with hydrocortisone in an attempt to demonstrate the influence of this hormone upon microfibrils found free in the tissue space surrounding the notochord. Embryos in series I were explanted on agar-albumen medium and permitted to survive 1–24 hours after Ringer's solution or hydrocortisone had been pipetted onto the area vasculosa. Doses of hydrocortisone ranged from 5 μg to 1 mg. Series II embryos were similarly treated in ovo through a small shell “window” and then incubated 12 or 24 hours. All embryos were sacrificed at 72 hours incubation age. Ringer-treated embryos in both series exhibited a rich tangle of early extracellular connective tissue fibrils (microfibrils) surrounding the notochord. One hour after treatment with hydrocortisone, embryos in series I showed a reduction in perinotochordal microfibrils. At four hours the effect was maximal and by 12 hours, recovery had been initiated, the morphology of which suggested a re-organization of amorphous material and extracellular debris to form fibrillar structures. Twenty-four hours after treatment, recovery was complete and hydrocortisone-treated embryos exhibited perinotochordal fibril populations that were indistinguishable from Ringer-treated specimens. The results of the series II experiments roughly parallelled those seen in series I but were somewhat less predictable. Possible modes of action of hydrocortisone on connective tissues and their relationship to the present study are discussed. It is suggested that the steroid may induce the release of a substance with an enzymatic activity capable of digesting microfibrils. This catabolic activity may be reflected as an inhibition of connective tissue production if it is assumed that microfibrils are precursors of larger, more mature fibrils. It is further suggested that since microfibrils are thought to contain connective tissue proteins, the initiation of microfibrillar reduction by hydrocortisone could indicate that this hormone may act in a similar manner on more mature connective tissue fibrils.     

On the origin and development of the ventrolateral abdominal muscles in the avian embryo. An experimental and ultrastructural study

In avian embryos the formation of ventrolateral abdominal muscles was studied by (1) heterospecific grafting experiments between chick and quail embryos and (2) ultrastructural examinations of cells having part in this process. The results demonstrate that the muscle cells are of somitic origin while the connective tissue derives from the somatopleure. Somatopleural cells do not differentiate into myocytes, and somite cells which have entered the ventrolateral abdominal wall, do not contribute to the connective tissue. It is concluded that both dermatome and myotome cells undergo muscular differentiation. The formation of muscles is found to take place in four characteristic steps. During the 4th day of development, epithelially structured ventral somite buds enter the somatopleure. The light cells of the inner myotome layer are elongated in a cranio-caudal direction and contain randomly distributed microfilaments. On the 5th day, the buds lose their epithelial arrangement and change into compact processes in which cells intermingle. The myotome cells show short bundles of thin and thick microfilaments. The third step can be characterized by the appearance of intercellular spaces and the disaggregation of processes becoming invaded by somatopleural cells. Thus, subdivision in single muscle blastemata begins to occur. In 7-day embryos, the muscle anlagen are distinctly separated and the first myotubes containing regularly arranged myofibrils are found. Coincidentally, signs of cell death are observed. Up to the 10th day, the tendons being of somatopleural origin become plainly outlined and the muscle anlagen move to their definitive positions. It is assumed that the formation of muscle pattern is controlled by the somatopleure.     

Expression and regulation of the decoy bone morphogenetic protein receptor BAMBI in the developing avian face.

Here, we examine the expression and regulation of the gene BAMBI, a kinase-deficient decoy receptor capable of interacting with type I bone morphogenetic protein (BMP) receptors in avian embryos. Initially, expression was limited to the endoderm during neurula and pharyngula stages. From embryonic day 3.5 (stage 20) and onward, BAMBI expression almost perfectly overlapped with known expression patterns for BMP4, particularly in the face and limbs. We performed bead implant experiments in the face to see which signals could be repressing or promoting expression of BAMBI. Our data point to retinoids and BMPs as being major positive regulators of BAMBI expression; however, fibroblast growth factor 2 acts to repress BAMBI. Furthermore, retinoic acid is likely to act directly on BAMBI as induction occurs in the presence of cycloheximide. The data suggested that BAMBI could be used to regulate Bmp signaling during tissue interactions that are an integral part of facial morphogenesis.     

Effect of Naloxone Hydrochloride on Osteogenesis in Chick Embryos

Naloxone hydrochloride, an opioid receptors blocker, was administered to chick embryos. Morphological analysis of femoral bones of embryos showed an appreciable increase in the thickness of the perichondral bone cuff in the tubular bone diaphyses and increased mitotic activity in the zone of proliferating young cartilage of the epiphyseal plate.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 3, pp. 310–312, March, 2005     

Mitotic activity during somite segmentation in the early chick embryo

Summary The mitotic activity of the somites, segmental plate and posterior mesoderm were investigated in colchicine-treated and untreated chick embryos at st. 7-14. The mitotic figures in the somites are restricted to the proximity of the lumen and have their spindles orientated predominantly tangentially to the cavity. In the segmental plate there is no pattern in terms of the position or orientation of the mitotic spindles, but there is a single region, often found close to the cranial end of the segmental plate, with an elevated mitotic index. This may indicate a certain degree of synchrony among groups of segmental plate cells. These results are discussed in relation to the process of somite segmentation.     

Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis

Bub3 is a conserved component of the mitotic spindle assembly complex. The protein is essential for early development in Bub3 gene-disrupted mice, evident from their failure to survive beyond day 6.5-7.5 postcoitus (pc). Bub3 null embryos appear normal up to day 3.5 pc but accumulate mitotic errors from days 4.5-6.5 pc in the form of micronuclei, chromatin bridging, lagging chromosomes, and irregular nuclear morphology. Null embryos treated with a spindle-depolymerising agent fail to arrest in metaphase and show an increase in mitotic disarray. The results confirm Bub3 as a component of the essential spindle checkpoint pathway that operates during early embryogenesis.     

Sympathoadrenal progenitors in embryonic chick sympathetic ganglia show distinct responses to glucocorticoid hormones

Summary The sympathoadrenal cell lineage originates from the neural crest and comprises the neurons of sympathetic ganglia, adrenal and extra-adrenal chromaffin cells, and the so-called small intensely fluorescent cells.In vitro studies using mammalian immature chromaffin cells, adrenal or sympathetic ganglionic progenitors, or ganglionic small intensely fluorescent cells, have suggested that glucocorticoid hormones are essential for inhibiting neuronal differentiation of sympathoadrenal progenitors and promoting the chromaffin cell phenotype. In avian systems, however, the distinct cellular phenotypes in this lineage and the molecular cues underlying their differentiation have not been fully explored. In the chick embryo, early sympathetic ganglion anlagen are populated by granule-containing cells that morphologically resemble small intensely fluorescent cells and chromaffin cell progenitors. These cells subsequently disappear from the ganglia, by death and by transition into fully differentiated sympathetic neurons, as indicated by the appearance of cells that are ultrastructurally intermediate between granule-containing cells and fully differentiated neurons (granule-containing cells in transition). In the present study, we show that treatment of cultured sympathetic cells dissociated from embryonic day (E) 7, 9, or 11 lumbar sympathetic ganglia with the glucocorticoid hormones hydrocortisone or corticosterone has neither an inhibitory nor an inductive effect on phenotypes of granule-containing cells or granule-containing cells in transition. In cell cultures of E15 ganglia, however, glucocorticoid treatment induces a granule-containing cell resembling the granule-containing phenotype. These results suggest that the early granule-containing cells and granule-containing cells in transition in chick sympathetic ganglia are not the counterparts of glucocorticoid-responsive mammalian small intensely fluorescent or chromaffin progenitor cells, despite their morphological similarity. However, E15 sympathetic ganglia apparently contain a glucocorticoid-responsive progenitor population that can differentiate into chromaffin-like cells. These progenitors seem to require a systemic or intraganglionic developmental signal or undergo a temporal switch that renders them susceptible to glucocorticoids.     

Action of estradiol and tamoxifen on the testis-inducing activity of the chick embryonic testis grafted to the female embryo

The implantation of two testes from 13-day-old male chick donor embryos into the extra-embryonic celom of 3-day-old female embryos induces the masculinization of their ovaries up to a total and definitive inversion of their gonadal sex, i.e., the differentiation of testes in the female hosts. Pretreatment of the donors with estradiol (E2) between day 11 and 13 counteracts the testis-inducing activity of the implants, while co-treatment of donors with both tamoxifen (TAM) and E2 at the same stage restores the initially observed activity. The treatment of 3-day-old male donor embryos with E2 causes the differentiation of their left gonad into an ovotestis totally devoid of testis-inducing activity once grafted in the same conditions as above. An additional treatment with TAM of the grafted host embryos does not modify the results obtained when E2-treated male gonads are grafted to normal host embryos. This shows that the lack of testis-inducing activity exhibited by the E2-treated grafts can not be attributed to a protecting action of endogenous estrogens on the gonads of the host. On account of previous work showing the inhibition by E2 of the Müllero-regressive activity of the chick embryonic testis, our present results can be interpreted in terms of E2-down regulation of Anti-Müllerian Hormone (AMH or MIS), which appears to be a good candidate as testis-inducer. The relevance of our results to the phenomenon of gonad differentiation is discussed.     

Contribution of mesenchymal cell death and mitotic alteration to asymmetric limb malformations induced by MNNG

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces fetal asymmetric limb malformations with exposure of pregnant mice to 50 mg/kg on day 11 of gestation. Hindlimbs were more frequently malformed than forelimbs, and a fourfold greater incidence of postaxial ectrodactyly was found in left forelimbs than in right forelimbs, and a two fold excess in left hindlimbs compared to right hindlimbs. The level of cell death and mitotic index were measured in forelimbs and hindlimbs from treated and control embryos at 1, 4, 18, 24, 48, and 72 hr after exposure to ascertain if these parameters could be correlated with the differential teratogenic susceptibility of the limbs. An increase in necrotic index was first detected in treated limbs at 4 hr, increased at 18 hr, peaked at 24 hr, and began declining at 48 hr to reach the control baseline at 72 hr. At 24 hr, the correlation between the level of cell death and susceptibility of malformation was the strongest, with the left hindlimb having a necrotic index of 58%, the right hindlimb 47%, the left forelimb 30% and the right forelimb 12%. In both forelimbs and hindlimbs, MNNG treatment initially depressed mitotic activity followed by an elevation at 48 hr relative to controls. The magnitude of the depression, extent of the elevation, and overall pattern of mitotic activity could not be uniformly related to limb defects. These results indicate that the amount of cell death in limb buds at 24 hr after MNNG exposure may predict target organ susceptibility. Depressions in mitotic activity and alterations in the pattern of mitosis were also observed which were not as clearly correlated with the incidence of malformations as was the amount of cell death.     

Action of estradiol and tamoxifen on the Müllero-regressive activity of the chick embryonic testis assayed in vivo by organotypic grafting

In the chick, the implantation of a testis graft from a 13-day-old male donor embryo into the extra-embryonic coelom of 3-day-old female embryos induces the total regression of their Müllerian ducts because of the anti-Müllerian hormone (AMH or MIS) secreted by the implant. Pre-treatment of the donors with estradiol (E2), between day 12 and day 13, counteracts in a significant way the Müllero-regressive activity of the implant. Cotreatment of donors at the same stage with both Tamoxifen (TAM) and E2 restores the initially observed activity, thus demonstrating the presence of Tamoxifen-sensitive estrogen receptors at the late stage of treatment in the Sertoli cells responsible for AMH secretion. The treatment of 3-day-old male donor embryos with E2 causes the differentiation of their left gonad into an ovotestis which provides implants totally devoid of Müllero-regressive activity. The additional treatment with TAM of the grafted host embryos, does not modify the results obtained when E2-treated male gonads are grafted to host embryos not treated with TAM. This shows that the lack of Müllero-regressive activity exhibited by the E2treated male gonads does not depend on the estrogens they may secrete during the time of the assay, i.e., it cannot be attributed to a protecting action of estrogens on the MDs of the host. Our results therefore favor the idea that E2 down-regulates AMH. The relevance of such a regulation to the phenomenon of Müllerian duct maintenance, either in the E2-feminized male or in the female chick embryo, is discussed.     

Mechanisms of the Analgesic Effect of Corticotropin-Releasing Factor in Conscious Rats

We report here our studies of the contribution of the hypothalamo-hypophyseal-adrenocortical system (HHACS) and opioid system to the analgesic effect of corticotropin-releasing factor (CRF) in conscious rats exposed to thermal stimuli. Two methodological approaches were used: 1) blockade of the functional activity of the HHACS by administration of hydrocortisone at a pharmacological dose one week before experiments; 2) blockade of glucocorticoid receptors with the glucocorticoid receptor antagonist RU38486. The contribution of the opioid system was studied by blockade of opioid receptors with their antagonist naltrexone. Blockade of opioid receptors completely eliminated the analgesic effect of CRF, blockade of the functional activity of the HHACS decreased it, and blockade of glucocorticoid receptors increased the effect. These data provide evidence that the analgesic effect of CRF in conscious rats exposed to a thermal stimulus is mediated by an opioid-associated mechanism. The actions of opioids on pain sensitivity may be modulated by glucocorticoid hormones.     

Effects on biological properties of embryo-adapted Beaudette strain of infectious bronchitis virus of passage through chick embryo trachea organ culture

Two substrains derived from an uncloned 71st embryo (E71) passage and a cloned 71st embryo-10th chick embryo kidney cell (E71CEK10) passage of the Beaudette strain of avian infectious bronchitis virus were back-passaged through organ cultures of chick embryo trachea. The organ culture-passaged viruses had altered biological properties such as absence of or delayed lethality for chick embryos and lack or diminution of plaque-forming ability in chick embryo kidney monolayers but they showed no clear change of pathogenicity for 3-week-old chickens. These viruses regained their lethality for chick embryos, their cytopathic effects, and their plaque-forming ability in chick embryo kidney cells after they had been back-passaged through chick embryos, depending on the passage number. The results of cross neutralisation tests using the constant serum-variable virus method in chick embryos indicated little antigenic difference between the original virus and various host-passaged derivative strains.     

Acetazolamide teratology and its association with carbonic anhydrase inhibition in the mouse

Acetazolamide, an inhibitor of the enzyme carbonic anhydrase (E.C. 4.2.1.1.), causes a unique congenital anomaly characterized by postaxial reduction of the distal portion of the right forelimb. To gain an understanding of the mechanism of teratogenesis, the activity of carbonic anhydrase in sensitive and resistant mouse strains, and its inhibition by acetazolamide, were examined. Differences in teratologic sensitivity were found not to be attributable to differences in maternal or embryonic drug levels. Enzyme inhibition at acetazolamide concentrations ranging between 10(-11) and 10(-5) M did not differ between the mouse strains when adult erythrocytes or day 12 embryos were assayed. However, in day 10 embryos, the period of maximum teratologic susceptibility, a small strain difference was found which suggested that the form of carbonic anhydrase in the susceptible CBA/J strain at this time is somewhat more sensitive to inhibition by acetazolamide than the form found in the resistant SWV strain. The results suggest further that more than one isozyme of carbonic anhydrase may be present in all three samples.