Ectopia cordis thoracalis with craniofacial defects resulting from early amnion rupture

A 33-week-gestation fetus was evaluated as having ectopia cordis thoracalis, midline sternal cleft, frontonasal dysgenesis, a midfacial cleft, and amniotic bands. Fibrous bands were attached to the apex of the heart, face, and the brain. There were no associated limb defects or scoliosis. The findings in this fetus and those in the literature suggest that acute amnion rupture in the third week of gestation may be the cause of these defects. Mechanical teratogenesis by tissue bands adherent to the heart may lead to ectopia cordis. The changes seen in the thorax, face, and brain suggest pressure necrosis, incomplete morphogenesis, tearing and tethering by amnion, and compression as a cause for these defects. A spectrum of defects is found following amnion rupture with the abnormalities being dependent upon the timing of the event. Ectopia cordis with amniotic bands appears to have an etiology distinct from isolated ectopia cordis. This suggests several different etiologies for ectopia cordis.     

Teratogenic effects of dilantin on thoraco-abdominal organs of developing chick embryos

Congenital anomalies on some viscera like heart, liver and kidney have been investigated in chick embryos after a single injection of dilantin (3 mg/egg), a known antiepileptic drug, on 4th day of incubation. On 19th day of incubation, chick embryos were collected to observe the gross malformations and histological changes in heart, liver and kidney. On gross examination, visceroptosis (29%), thin anterior abdominal wall (28%), ectopia cordis (10%) and dextrocardia (1%) were observed. Histological examination of the kidney revealed glomerular degeneration in kidney while in liver, dilated central veins with degenerated hepatocytes were present. Longitudinal section of the heart showed thicker musculature specially of ventricles with a narrower lumen in comparison to that of the control. The results indicate teratogenicity of dilantin in developing chick embryos.     

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 effects of simulated microgravity on avian embryonic development.

Based on the few reports available, microgravity (MG) can have adverse effects on the early development of vascularised extra-embryonic membranes in avian eggs. Whether gravity or oxygen availability is the stimulus for development of the blood vessels in the chorioallantoic membranes (CAM) remains unclear. Under gravity the blastoderm forms on top of the yolk sac, closest to the oxygen rich region beneath the shell membranes, and from there the CAM buds from an abdominal extension subsequently to form a close contact with shell membranes. Then as the embryo develops it spreads beneath the eggshell surface to maximise the surface area of the CAM vascular bed available for O2 uptake. To investigate how simulated MG influences development of the CAM and embryo we conducted experiments on chicken embryos during incubation in a 3D-clinostat (control or continuous MG treatment at 5 rpm). Further, to determine if CAM angiogenesis is directed towards regions of high O2 tension or gravity we investigated the effects of wax treatment (50% shell surface area) on development in MG. We found that clinostat MG caused embryonic failure between day 0-5 by preventing normal development of CAM-shell membrane complex. Thereafter acute MG promoted increases in CAM mass, but did not affect embryo mass. Preliminary findings suggest that combined acute MG and wax treatment did not significantly affect embryonic growth in either MG or control groups, but retarded CAM growth in control embryos only. Finally, we will present evidence to show that acute and prolonged exposure to MG does not prevent normal growth and hatching, but might have more subtle effects on hatchling physiology, including reduced heart mass.     

The importance of perivitelline fluid convection to oxygen uptake of Pseudophryne bibronii eggs

The ciliated epithelium of amphibian embryos produces a current within the perivitelline fluid of the egg that is important in the convective transfer of oxygen to the embryo's surface. The effects of convection on oxygen uptake and the immediate oxygen environment of the embryo were investigated in Pseudophryne bibronii. Gelatin was injected into the eggs, setting the perivitelline fluid and preventing convective flow. Oxygen consumption rate (M(.)o?) and the oxygen partial pressure (Po?) of the perivitelline fluid were measured in eggs with and without this treatment. M(.)o? decreased in eggs without convection at Gosner stages 17-19 under normoxia. The lack of convection also shifted embryos from regulators to conformers as environmental Po? decreased. A strong Po? gradient formed within the eggs when convection was absent, demonstrating that the loss of convection is equivalent to decreasing the inner radius of the capsule, an important factor in gas exchange, by 25%. M(.)o? also declined in stage 26-27 embryos without cilia-driven convection, although not to the extent of younger stages, because of muscular movements and a greater skin surface area in direct contact with the inner capsule wall. This study demonstrates the importance of convective flow within the perivitelline fluid to gas exchange. Convection is especially important in the middle of embryonic development, when the perivitelline space has formed, creating a barrier to gas exchange, but the embryos have yet to develop muscular movements or have a large surface area exposed directly to the jelly capsule.     

Ectopia cilia with pedigree analysis: Second case report in the world

We present a case of ectopia cilia in a 28-year-old male patient. Ectopia cilia was were seen in the outer third of left upper eyelid. The patient''s maternal grandfather also had ectopia cilia of the left upper eyelid as reported by the patient''s mother. Ectopia cilia is a rare condition seen in humans. Only 12 cases of ectopic cilia in humans have been reported so far in the world. The present case of ectopia cilia is the second case report in the world with pedigree analysis.     

In vitro and in vivo association of an oviduct estrus-associated protein with bovine zona pellucida

The interaction between the bovine egg zona pellucida and a 97 kDa estrus-associated protein produced by the oviduct was examined in vitro and in vivo. In vitro matured bovine eggs were incubated with oviduct fluid recovered throughout the estrous cycle from separate indwelling cannulae placed in the ampulla and isthmus of the same oviduct. Immunofluorescence techniques and a polyclonal antiserum against the 97 kDa protein were used to localize this protein on washed eggs previously incubated with oviduct fluid. Intensity and distribution of immunofluorescence varied with stage of cycle and to a lesser degree with region of oviduct from which the oviduct fluid was obtained. The most intense fluorescence was observed on the zonae pellucidae of eggs incubated with oviduct fluid pooled from days near estrus and ovulation compared to fluid pooled from luteal stage days. The immunofluorescence of isthmus-derived oviduct fluid was more intense than was ampulla-derived oviduct fluid collected near estrus. The zonae pellucidae of 7-day-old embryos flushed from the uterus displayed immunofluorescence comparable to that observed on the zonae pellucidae of eggs incubated in vitro with peri-estrus oviduct fluid. No immunofluorescence was observed associated with the perivitelline space, egg cytoplasm, or blastomeres. The apparent uptake of a 97 kDa estrus-associated protein by the zonae pellucidae of eggs in vitro and embryos in vivo may indicate that this protein functions in fertilization and/or early embryo development.     

Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine

Cryopreservation can cause changes to the genetic material of cells, but the mechanism and significance of these changes are still unknown. It has been suggested that some damage to the sperm genome could be repaired by the DNA repair system of the oocyte after fertilisation. Caffeine has been reported to be an inhibitor of such repair processes. In this study the effect of caffeine on the repair system of Loach (Misgurnus fossilis) oocytes was investigated. Loach eggs were fertilised using cryopreserved sperm. Embryos derived from cryopreserved sperm were exposed to 2.6mM caffeine for 1h after fertilisation. The experiments were carried out using 32313 embryos from four females and eight males. Embryo survival was evaluated for 46 h until the hatching stage. Reduction in embryo survival after 20th stage is generally believed to result from the failure in the genome function of embryos. Cryopreservation of sperm significantly decreased embryo survival (53.4+/-2.8% compared to 68.4+/-2.8% of control) after the 20th stage. However, the addition of caffeine to the embryos derived from cryopreserved sperm, in contrast to our expectation, significantly increased survival of loach embryos (70.9+/-2.8% compared to 53.4+/-2.8% of embryos derived from cryopreserved sperm in the absence of caffeine). The effect of individual donors of sperm and eggs on overall embryo survival was also studied. Whilst no significant differences were observed between males, the effect of individual females on embryo survival was significant. The analysis of embryo survival at different developmental stages showed that embryo survival both before and after 20th stage decreased with embryo development. When fresh sperm were used the decline of embryo survival with development was more pronounced compared with those embryos derived from cryopreserved sperm. Possible explanations of these effects are presented.     

Sodium balance in eggs and dechorionated embryos of the Atlantic salmon Salmo salar L. exposed to zinc, aluminium and acid waters

Ionic regulation of eyed eggs of Atlantic salmon was studied using intact eggs and embryos from which the chorion and perivitelline fluid had been removed, a preparation which remained in good condition for at least 2 weeks. Both intact eggs and embryos in freshwater showed similar Na+ influx rates (0.0064 and 0.01 microM g-1 hr-1, respectively) but Na+ efflux rates (0.0024 and 0.0064 microM g-1 hr-1, respectively) were greater in embryos, suggesting that the perivitelline fluid which is known to bind cations has an important function in preventing Na+ loss from the yolk and embryo. Over 90% of the egg Na+ is located in the yolk and embryo but only about 10% is exchangeable while the chorion contains about 8% Na+ which is non-exchangeable. Both eggs and embryos in acid water at pH 4, in Zn2+ 5 mM l-1 and in aluminium 1 mM l-1 showed greatly reduced Na+ uptake but eggs in 10 microM l-1 aluminium or 100 microM l-1 Zn2+ showed normal Na+ balance while embryos were normal in 10 microM l-1 aluminium but showed reduced Na+ uptake in 100 microM l-1 Zn2+. It is concluded that the chorion and perivitelline fluid have a capacity to protect the embryo from metal ions and acid water.     

Embryonic behaviour in the lizard, Lacerta vivipara

The behaviour of embryos of the lizard Lacerta vivipara has been studied in cultured eggs, removed from the mother. Spontaneous movements begin at a time when the embryo is still unresponsive to touch, and about two days before the appearance of reflex responses. The earliest movements consist of lateral flexion which gradually gives place to dorsiventral flexion. Embryonic movements are not necessarily related to contractions of the amnion. The activity of the embryo, as measured by the number of times movements were initiated and the amount of time spent in activity during a half hour period, rises to a plateau and then decreases sharply as the time of hatching approaches. Activity of the tail is prominent, particularly at times when other movements occur in rapid succession. Serpentine movements were not observed in embryos younger than stage 39; some prematurely born animals showed such activity in response to contact with the substratum. During embryonic life any part of the animal may move individually or in combination with any other part, such combined movements being unco-ordinated. In these respects the spontaneous motility of the lizard embryo closely resembles that of the chick. Statistical analysis of the embryonic movements provides evidence of rhythmic activity within the developing central nervous system.     

Egg Turning During Incubation has no Effect Upon the Growth of Embryos of Alligator mississippiensis

Alligator eggs are not turned during incubation, instead the embryo adheres to the top inside of the shell. Turning is alleged to shear off the embryo and kill it. Avian egg turning allegedly facilitates embryonic development by stimulating growth of the area vasculosa and minimizing the effects of unstirred yolk and albumen layers. From day 10 to day 45 of incubation, alligator eggs were experimentally turned, gently, through ± 60° in an hourly cycle. This turning regime killed only 6 out of 25 embryos. Compared with unturned controls, no significant effects were observed on the growth, production of extraembryonic fluids or utilization of albumen and yolk for those embryos that survived turning. The protein concentration of amniotic fluid at various stages of alligator development was examined in eggs incubated at 30 and 33°C. The fluid contained very little protein (max <8 mg) at any time: the protein concentration did not change consistently as development progressed. Differences in response to egg turning in birds and reptiles may be associated with the length of the incubation period, the protein content of the albumen and the mechanism of albumen utilization.     

Catecholaminergic and cholinergic regulation of swimming motility development in free embryos of Cichlasoma Nigrofasciatum

The divergence of progeny from the same spawners of Cichlasoma nigrofasciatum into two groups by duration of embryogenesis and the level of motor activity was studied close to the end of the embryonic period. Free embryos were also studied. During the study, eggs were treated with agents, modifying the activity of catecholaminergic and cholinergic systems. 3-Hydroxytyramine and L-Tyrosine were found to exert a weak influence on embryonic motility. After hatching, these substances modify swimming performance of free embryos, approximating movements of fish at later stages. 6-Hydroxydopamine and, still more, alpha-Bungarotoxin, decrease embryonic motility and postpone the hatching. The influence of these substances on the development of embryo motility increases during early ontogenesis, as indicated by decreased concentration of the substance, necessary for adequate reaction. Neither L-Tyrosine nor 6-Hydroxydopamine influenced the divergence of the progeny into two groups. Injection of the perivitelline fluid with high concentration of hatching enzyme from pre-hatching embryos into the perivitelline space of earlier embryos was found to induce the appearance of rotation movements, typical for more advanced embryos. Changes of correlation between the miogenic and neurogenic motor activity during early development of fish are discussed.     

Introductory class exercises in experimental embryology using Xenopus laevis

Abstract

This paper describes classical experiments of experimental embryology which may be repeated at class level using the early embryos of Xenopus laevis. The experiments demonstrate the effects of lithium on the early development of the nervous system, and the use of hypertonic salt solutions to produce exogastrulation. Both experiments involve the removal of the jelly coats around the embryo, and for this purpose a rapid chemical method using a cysteine hydrochloride/papain solution is described. Both experiments are theoretically important: the effects of lithium are used as evidence for the existence of an anterior-posterior gradient in the early embryo, while exogastrulation provides a convenient system for interfering with the primary inductive effect of the chordo-mesoderm, necessary for ectodermal neuralization.

The procedure for obtaining fertile eggs from mature Xenopus specimens by injection of mammalian chorionic gonadotrophin is described.     

Entamoeba histolytica: genetic control of susceptibility in chicken eggs.

Five strains of axenized Entamoeba histolytica were established in eggs from chickens of different genetic backgrounds. The eggs of outbred and inbred chickens varied in the proportion of embryos from which the strains of E. histolytica were recovered and there were variations in the numbers of trophozoites recovered from individual positive eggs. It was possible, however, to find individual, single-mated hens which laid only uniformly susceptible or resistant eggs. Susceptibility and resistance to E. histolytica could be demonstrated in embryonated eggs derived both from outbred heterogenous and in highly inbred chickens but eggs from a genetically stable source were relatively constant in the distribution of susceptible and resistant embryos. Except for gross hydrocephalus, tissue reactions were not observed in the chick embryos or in the chorioallantoic, amniotic, or yolk sac membranes. The yolk sac inoculation route and 37 C incubation temperature provided the optimal survival conditions for the amoebae. Infection with avian leukosis virus and interferon production did not seem to play a role in susceptibility and resistance. Neither the presence of E. histolytica cross-reacting, maternal antibody from the hen transmitted transovarially via the yolk sac, or other antiamoebic substances appeared to play a role in the resistance observed in nonsusceptible eggs. Multiple alternating passages of trophozoites in hen's eggs/TPS-1 broth led to an increase in the number of positive embryos and a decrease in embryo deaths.     

EMBRYO AND ORGAN CULTURES OF THE HORSESHOE CRAB,TACHYPLEUS TRIDENTATUS*

This paper deals with some preliminary technical experiments on cultures of the embryos and organs of Tachypleus tridentatus. In embryo culture, using the embryos at various stages of development, embryonic areas with hardly any yolk remaining in them were cultured in various media and by various methods (Series A); hemispheres of embryos containing the embryonic area (Series B) and whole embryos without egg membranes (Series C) were cultured in sterilized sea water. The suitability of the culture media and methods is described; the medium containing calf serum or 199, and the hanging-drop method were found to be the most appropriate of those used in respective series of experiments. The changes in external and internal structures of the explants from embryos at various developmental stages cultured in various media and by various methods are described; dorsal closure, secretion of inner egg membrane, thickening of the cell layer and the elongation of limb buds were all observed in the Series A experiments. In Series B and C, coelom formation and also normal development of the embryos cultured after the 2nd embryonic moulting were observed. In organ culture, fragments of the embryonic heart were cultured in various media and by various methods. The hanging-drop method resulted in longer survival than the method using agar media.     

Taste sensitivity in the embryo of the domestic fowl

Two experiments were carried out to investigate the sense of taste in embryos of the domestic fowl. In the first, four taste substances; NaCl, HCl, glucose and SOA were diluted with distilled water and the response was compared with that to distilled water alone. No significant effects of taste were found. In the second experiment five taste substances: HCl, fructose, NaCl, KCl and quinine were diluted with fluids normally imbibed by the embryo: amniotic and/or allantoic fluid taken from other eggs. These solutions and also distilled water were compared with egg-fluid alone. A highly significant effect of the five solutions was found showing that the taste system becomes functional before the time of hatching. Distilled water produced on an unexpectedly large response in the embryo; possible reasons for this are discussed.     

Embryo transfer as a means of controlling the transmission of viral infections. X. The in vivo exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus

Two experiments involving the transfer of embryos from donors infected with swine vesicular disease virus (SVDV) to "clean" recipients were carried out. In Experiment 1, 47 embryos were collected from 4 SVDV-infected donors and transferred to 2 recipients that subsequently produced 10 piglets. All of the recipients and piglets remained seronegative for SVDV. In addition to the transfers, 10 embryos and 58 unfertilized eggs from the infected donors were assayed in vitro and found to be negative for SVDV infectivity. A fifth donor was also inoculated with SVDV in this experiment, but it could not be demonstrated that infection had occurred. This SVDV-exposed donor provided two embryos for transfer and one embryo and two unfertilized eggs for in vitro assay. In Experiment 2, 158 embryos from 9 infected donors were transferred to 7 recipients, resulting in 12 piglets. A total of 7 embryos and 37 unfertilized eggs were assayed in vitro. The recipients, piglets, and embryos/eggs were all negative for SVDV infectivity. Although a final conclusion on the safety of using embryo transfer for the control of swine vesicular disease (SVD) is not possible, the results obtained justify additional studies.     

Periodic cooling of bird eggs reduces embryonic growth efficiency

For many bird embryos, periodic cooling occurs when the incubating adult leaves the nest to forage, but the effects of periodic cooling on embryo growth, yolk use, and metabolism are poorly known. To address this question, we conducted incubation experiments on eggs of zebra finches (Taeniopygia guttata) that were frequently cooled and then rewarmed or were allowed to develop at a constant temperature. After 12 d of incubation, embryo mass and yolk reserves were less in eggs that experienced periodic cooling than in controls incubated constantly at 37.5 degrees Celsius. Embryos that regularly cooled to 20 degrees Celsius had higher mass-specific metabolic rates than embryos incubated constantly at 37.5 degrees Celsius. Periodic cooling delayed development and increased metabolic costs, reducing the efficiency with which egg nutrients were converted into embryo tissue. Avian embryos can tolerate periodic cooling, possibly by adjusting their physiology to variable thermal conditions, but at a cost to growth efficiency as well as rate of development. This reduction in embryo growth efficiency adds a new dimension to the fitness consequences of variation in adult nest attentiveness.     

Lectin teratogenesis: defects produced by concanavalin A in fetal rabbits.

Concanavalin A (con A) is teratogenic to rabbit embryos during gestational days 12--15. Intracoelomic injections of 40 microliter con A solution (4 microgram/microliter) were performed on rabbit embryos during gestational days 10--15. Control embryos received either 40 microliter of saline, sham injection or no treatment. Con A caused increased fetal resorptions on days 10 and 11, but malformation levels did not differ from controls. On days 12--15, con A produced craniofacial, trunk and limb anomalies. The highest percentage of malformation occurred on day 14. The defects were classified into four groups: (1) malformations of limbs including paw and digital dysplasias as well as fusions of the limbs to the head or body wall; (2) "closure" defects such as umbilical hernia, encephalocoele, exencephaly or ectopia cordis; (3) "contracture" defects such as club paws, extended knees, or clenched digits, which exhibited normal osseous and cartilaginous skeletons; and (4) miscellaneous, non-specific anomalies including fused or dysplastic sternebrae or ribs. Histologic analysis of selected 12-day embryos 4 to 18 hours post-injection was performed to ascertain potential sites of teratogenic action. At 12 hours ectodermal necrosis was observed in the limb buds adjacent to the apical ectodermal ridge. By 18 hours, the ectoderm had eroded, exposing the basal lamina to the amniotic fluid. Focal areas of mesenchymal necrosis were observed in association with the ectodermal erosion. The potential roles of amniocentesis and limb bud repair in the genesis of the malformations are discussed.     

Environmental stress linked to consumption of maternally derived carotenoids in brown trout embryos (Salmo trutta)

The yellow, orange, or red colors of salmonid eggs are due to maternally derived carotenoids whose functions are not sufficiently understood yet. Here, we studied the significance of naturally acquired carotenoids as maternal environmental effects during embryo development in brown trout (Salmo trutta). We collected eggs from wild females, quantified their egg carotenoid content, fertilized them in vitro in full‐factorial breeding blocks to separate maternal from paternal effects, and raised 3,278 embryos singly at various stress conditions until hatching. We found significant sire effects that revealed additive genetic variance for embryo survival and hatching time. Dam effects were 5.4 times larger than these sire effects, indicating that maternal environmental effects play an important role in determining embryo stress tolerance. Of the eight pigment molecules that we targeted, only astaxanthin, zeaxanthin (that both affected egg redness), and lutein were detected above our confidence thresholds. No strong link could be observed between carotenoid content in unfertilized eggs and embryo mortality or hatching timing. However, the consumption of carotenoids during our stress treatment was negatively correlated to embryo survival among sib groups and explained about 14% of the maternal environmental variance. We conclude that maternally derived carotenoids play a role in the ability of embryos to cope with environmental stress, but that the initial susceptibility to the organic pollution was mainly determined by other factors.