Cholinesterase in the amnion of chick embryos

Cholinesterase (ChE) activity in amnion was studied in developing chick embryos and the enzyme's substrate-inhibitory characteristics were established. The enzyme activity increased until the 8th day of incubation and then gradually decreased; on day 12-15 the activity is 40% only of the maximal one. On the basis of substrate-inhibitory analysis the enzyme was referred to propionyl-cholinesterases. Relations between age changes in ChE activity and morphological structure of smooth-muscle amnion tissue, its differentiation during development and functional activity of amnion are discussed.     

Muscarinic acetylcholine receptors of the chick amnion

The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M₁, methoctramine for M₂, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M₃, and tropicamide for M₄ receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (-logIC₅₀), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 μM), activator of adenylate cyclase (AC), was unidirectional with ?-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC-or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M₃ acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M₃ acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M₃-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter.     

The cholinergic reaction of the amnion in the chick embryo

Studies have been made of the spontaneous contractions of the amnion and acetylcholine sensitivity of amniotic membrane in 8--14-day chick embryos. In 12--14-day embryos, the spontaneous rhythmic contractions were rather rare as compared to those in 8--9-day ones, their frequency being also lower. On the basis of kinetic analysis, it was concluded that both the dissociation constant (K) and the value of Pmax do not exhibit significant changes for tonotropic reaction from the 8th to the 14th day and for chronotropic reaction--from the 8th to the 10th day of incubation. After the 10th day of incubation, dose-effect chronotropic reaction not expressed. The spontaneous activity of the amnion and acetylcholine sensitivity of the amniotic membrane depend on the temperature being maximal at 38 degrees C. Possible regulatory mechanisms of contractile activity in chick amnion are discussed.     

Intracellular transmission of the cholinergic signal in the chick amnion

The role of the system of deposited calcium in the mediation of contractile reactions to carbachol in an isolated amnion of 11–13 day old chicken embryo was studied. It was found that thapsigargin (2 μM, 20 min), an inhibitor of the endoplasmic reticulum Ca²⁺-ATPases, decreases the tonic reaction to carbachol by 40 ± 2%. In the presence of U73122 (5–10 μM, 10 min), a phosphoinositide-specific phospholipase C inhibitor, the rhythmic contractile reaction of the amnion to carbachol is blocked, whereas the tonic reaction decreases to 47 ± 9% of the initial one. Ryanodine (10 μM, 5 min) inhibits the spontaneous contractile activity of the amnion and decreases the tonic reaction to carbachol to 36 ± 3% relative to control. In the presense of ryanodine, nifedipine (0.05 μM) completely blocks the tonic reaction to carbachol. Thus, calcium mobilized from intracellular stores via inositol trisphosphate and ryanodine receptors is involved in realization of contractile reactions, mediated by M3 receptors, in the chick amnion.     

Pharmacokinetic analysis of alpha- and beta-adrenoreceptors in the chick embryo amnion

Following a stimulation with acetylcholine, the beta-adrenergic agonists adrenaline (A), noradrenaline (NA), isoproterenol (Iso) and salbutamol (Sal) induced a concentration-dependent decrease in the tone and (or) rate of amnion contraction with EC50 ISO < NA < A < Sal. Metaprolol, a specific beta 1-antagonist, induced a rightward shift in the dose-response curves of Iso, NA and A, whereas beta-antagonist butoxamine was ineffective. pA2 values for beta-antagonists were propranolol 8.3, metoprolol 7.0, butoxamine 5.6. EC50 values of alpha-adrenergic agonists form a sequence: clonidine < NA < methoxamine < phenylephrine. Specific alpha-antagonists yohimbine and idazoxan were found to antagonise competitively the effects of NA. The data obtained characterize the adrenergic receptors mediating stimulation of amniotic contractile activity as alpha 2-adrenergic receptors. Inhibition of contractile receptors in amnion is mainly mediated by beta 1-adrenergic receptor activation.     

Fusion of epithelial sheets as seen in formation of the chick amnion

Summary Fusion of the amniotic folds over the chick embryo provides an example of epithelial fusion in which one group of ectodermal cells appears to contribute to traction and a second group appears to respond by moving to the midline, becoming internalized and degenerating. Neutral red staining of living embryos and transmission electron microscopy indicate that a patch of dying cells is present in the fusion region. Scanning electron microscopy shows that ectoderm cells at the rim of the amniotic folds are cylindrical in shape with long axes oriented parallel to the rim. These cells have a smooth surface, but show increasing amounts of blebbing toward the midline. Characteristically, a globular region of rounded cells showing extreme blebbing is present at the midline. Just anterior to the globular region, flattened cells are present on the surface with many taut cellular processes. Orientation of ectodermal cells around the region of fusion suggests that traction is exerted from these flattened cells. When treated with cytochalasin D, posterior progression of the zone of fusion is blocked. Also, the flattened cells anterior to the globular region round up, revealing many elongated, anteriorly-directed cytoplasmic processes presumably leading to sites of original attachment.     

Role of motility in embryonic development I: Embryo movements and amnion contractions in the chick and the influence of illumination

This study provides a quantitative analysis of the active movements of the chick embryo and of the contractions of the amnion over the entire developmental period of 21 days. Four types of embryo movements are distinguished. The motor activity of the embryo shows two characteristic peaks, with maximum contraction frequencies on the 12th and on the 16th day. In contrast, the amnion activity is higher at earlier stages and decreases as the body activity increases. The amnion activity is largely independent of the body activity. Illumination has a strong influence on embryo movements. It is shown that increases of light intensity affect the patterns of activity of both the embryo and the amnion. While the effect of light on the embryo can be interpreted as being transmitted via the optic system, the mechanism of the amniotic response is unclear. The results suggest that the amnion itself may be sensitive to light. J. Exp. Zool. (Mol. Dev. Evol.) 291:186-194, 2001.     

Catecholamines and DOPA in the amniotic fluid and amnion of the chick embryo

Identification and quantitative fluorimetric assay have been made on the content of DOPA, dopamine, noradrenaline and adrenaline in the amniotic fluid and amnion of the developing chick embryos. Significant increase in the content of DOPA, noradrenaline and adrenaline in the amniotic fluid was observed between the 6th and the 13th days of incubation; dopamine content sharply decreases at the 13th day. The content on amines in the amnion tissue remained essentially constant throughout the investigated period. The role of catecholamine in amniotic fluid in regulation of contractile activity of amniotic membrane in the developing chick embryo is discussed.     

The motor activity of the chick embryo and amnion during embryogenesis

Studies have been made on the motor activity of amnion and chick embryo from the 5th to the 14th day of development. Between the 5th and the 8th day of embryogenesis, when embryonic movements are rather poor, amnion contractions are mainly observed, their frequency being maximum to the 7th day. On further development (8-14 days), with the increase in the mass of the limbs which account for embryonic movements (body extremities), the increase in the intensity of their motor activity is paralleled by the decrease in the frequency of amnion contractions. Therefore, during the intensive growth and development of mainly frontal part of the embryo, the deficiency of motor activity of rather undeveloped body and extremities is presumably compensated by temporal motor activity of the amnion. Between the 8th and the 10th day, synchronous movements of embryo and amnion are observed. Possible mechanisms of synchronization are discussed.     

The effect of temperature on the motor activity of the chick embryo and amnion at 5-14 days of development]

It has been shown that cooling the developing eggs from 37.7 degrees C results cessation of motor activity of the amnion in 5-14-day embryos at 36-33 degrees C, whereas motor activity of the embryo remains unaffected up to 31-26 degrees C. Immobilization of the embryo was observed on cooling up to 22-18 degrees C. The recovery of motor activity after cooling during heating takes place in a reverse order. Embryonic movements are observed at 18-23 degrees C, contractions of the amnion--at 28-33 degrees C. These experiments reveal complete independence of embryonic movements from the amnion. Motor activity of the amnion is related to that of the embryo only between the 8th and the 10th day of incubation.     

The motor activity of the chick embryo amnion in the late stages of development. The role of serotonin and noradrenaline]

Experiments with 13- to 19-day-old chicken embryos maintained at 37 degrees C were conducted using direct registration of embryonic movements and amnion pulsation. It is shown that in the non-innervated and lacking blood vessels non-striated-muscled amnion, motoric activity could be observed nearly up to the end of the embryonic development, not only during 5 to 14 days old interval as it was supposed earlier. In accordance with our previous results indicating important role of biogenic amines (serotonin and noradrenaline) in the regulation of motoric activity of the chicken embryo amnion at earlier and middle ages, this study provides some evidences of the humoral regulatory mechanism even at later embryonic stages. After being injected into amniotic fluid of older embryos, the serotonin stimulates and noradrenaline inhibits amnion motoric activity (the both are taken at final concentration of 10(-7) to 10(-6) M). Serotonin's receptors blocator, the cyprogeptadin, suppress while beta-adrenoreceptors' blocator, the propranolol, activates intact amnion motorics ( both are taken at final concentration of 10(-7) to 10(-5) M).     

Rhythmic contractions in chick amnio-yolk sac and snake amnion during embryogenesis

The rhythmic movements of fetal membranes in chick and reptile embryos were studied to explore the developmental role of the extra-embryonic motor activity. In the snakes Lamprophis fuliginosus and Elaphe radiata, rhythmic contractions of amnion inside the developing egg were recorded from the 11th incubation day until pre-hatching stages (ca. day 60-72). The duration of these contractions averaged 2.02+/-0.27 min. The frequency ranged from 2 to 6 per 10 min and averaged 4.61+/-0.57 per 10 min. A tendency of frequency to increase toward the end of embryogenesis was observed. Lowering the temperature from 28 to 20 degrees C significantly decreased the frequency of amnion contractions to 2.85+/-0.91 per 10 min. The isolated snake amnion retained its capacity for spontaneous contraction. Noradrenaline inhibited, acetylcholine stimulated and serotonin did not affect the rhythmic activity of the isolated snake amnion. Similar effects were found when these agents were applied into the snake amniotic cavity. In the chick, yolk sac rhythmic contractions were recorded from the fifth until the 12th incubation days. The duration of these contractions ranged from 15 to 60 s, their frequency averaged 11.8+/-3.18 per 10 min and depended on temperature. The low temperature threshold was approximately 30 degrees C. After surgical removal of the amnion and embryo, the yolk sac continued contracting inside the egg. The yolk sac rhythmic contractions likely participate in the space movement of the embryo inside the egg during embryogenesis.     

Primary hypoblast development in the chick

Summary Scanning electron microscopy (SEM) indicates that the primary hypoblast forms beneath the area pellucida during the first 8 h of incubation mainly by establishment of contact among cells which move downward out of the epiblast. This movement, polyingression, begins posteriorly and continues antero-laterally during the period of primary hypoblast formation. Polyingression produces many pits and possibly a crescentic fold in the embryo upper surface with corresponding cell clusters and a ridge on the lower surface. Fixationin situ helps prevent formation of artifactual folds and wrinkles facilitating interpretation of the SEM images.Formation of intercellular adhesions which lead to development of an epithelial primary hypoblast proceeds in a posterior to anterior direction along with polyingression. This epithelialization begins with elaboration of numerous filamentous processes by cells as they arrive from the epiblast, and continues with ongoing input of cells, merging of cells and cell clusters, and cell flattening. We have also shown (Weinberger and Brick 1982) that proliferation of ingressing cells provides additional cells for hypoblast development.