Expression and localization of myosin in fertilized eggs and adult tissues of Ilyanassa obsoleta
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NASA Discipline Developmental Biology↗
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Egg-stronautics Systems Engineering for Descent and Landing Workshop
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Systems Engineering↗
Egg-stronautics Systems Engineering for Descent and Landing Workshop
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Entry Descent and Landing↗
Egg-stronautics Systems Engineering for Descent and Landing Workshop
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Astronautics↗
Egg-stronautics Systems Engineering for Descent and Landing Workshop
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Astronautics↗
Amphibian fertilization and development in microgravity
During the year before launch, female frogs will be tested every 3 months for the quantity and quality of eggs produced. Two weeks or more prior to launch, male and female frogs will be transported to the John F. Kennedy Space Center (KSC). During the few weeks before launch, groups will be periodically tested for egg quality to assure that the frogs have adapted to the KSC laboratory environment. About 27 hours before launch, four females will be placed in a damp foam-lined box, called the Adult Frog Box (AFB), through which 100 cc/min of air wil be circulated. The AFB will be lowered into the Spacelab and loaded into the Frog Environmental Unit (FEU) during the final pre-launch preparations. A sperm suspension, for use in flight to fertilize the eggs, will also be prepared and loaded during the pre-launch period. The sperm suspension, together with a kit of syringes containing Human Chorionic Gonadotropin (HCG), will be stored in a refrigerator aboard the shuttle until needed in flight. On the first day of flight, the AFB will be transferred from the FEU to the General Purpose Work Station (GPWS), which is a type of glove box specially designed to allow the crew to use chemicals and biological materials during the flight without contaminating the shuttle/Spacelab environment. Inside the GPWS the four adult frogs will be injected with the HCG hormone and returned to the FEU. Approximately 16 hours after injection, ovulation should have taken place and 15 to 20 eggs from each frog will be placed on each of two egg baskets and covered with sperm for 10 minutes. The egg baskets are inserted into acrylic egg chambers and 50 ml of 'pond water' (20 percent strength Modified Ringers solution (is added. One of the chambers from each frog will be placed on a centrifuge within the FEU and rotated to simulate normal terrestrial gravity (1 g). The remaining chambers are incubated under microgravity conditions within the FEU. Forty minutes after fertilization, the four chambers exposed to 1 g will be removed and observed within the GPWS using a dissecting microscope and camera system. On the basis of egg rotation and egg appearance, the 'best' and 'second best' frogs will be selected to contribute additional eggs. Using the two best frogs, 22 egg chambers will be loaded with eggs and fertilized. Eleven chambers wil be incubated at microgravity and eleven will be incubated on the centrifuge. At various times during the flight, chambers will be removed from the FEU and transferred to the GPWS where a formaldehyde-based fixative will be injected in order to preserve important developmental stages for in depth study following the flight. Five of the 22 chambers plus the eight fertilization test chambers will be returned to Earth with live tadpoles. The swimming behavior of these free swimming tadpoles will be examined within several hours of landing and some will be fixed for a detailed analysis of their inner ear, the otolity, the animals 'balance system.' Lychakov and Vinikov reported an increase in otolith size in tadpoles that developed (but were not fertilized) in space. Live tadpoles from the SL-J flight will be raised through metamorphsis for studies of maturation including an analysis of the effects of space flight on their ability to produce normal progency. Following the flight, the fixed embryos will be serially sectioned and stained using procedures that were developed for this experiment. The embroys fixed at the 2-4 cell stage will be examined for the distribution of cytoplasmic contents, including the various classes of yolk platelets, and the location and shape of the cleavage furrows. The gastrulae will be assessed for the normality of the complex cellular rearrangements that constitute blastopore formation and involution, i.e., the differentiation of the embryo into its specialized body parts. Using a new procedure, the gastrulae will be bleached which will enable the initial sperm entry point (SEP) to be located. The correlation between the SEP and the dorsal lip of the blastopore will be determined. Under normal terrestrial conditions it was shown that the SEP typically is located on the side of the egg opposite the future dorsal side of the embryo. The neurulae will be examined for the normality and completeness of the neural plate and archenteron expansion. The tadpole stages will be used to study the allometry and morphology of the various organ systems.
Souza, Kenneth A.↗
Respiratory gas exchange of high altitude adapted chick embryos
Study of gas exchange by embryos from chickens acclimatized to an altitude of 3800 m. The oxygen partial pressure and carbon dioxide partial pressure differences across the egg shell were measured and found to be less than the values previously reported for sea-level eggs by about a factor of two. Further measurements of embryonic oxygen consumption and shell conductivity to oxygen indicated that, compared to eggs at sea level, oxygen consumption was reduced by a factor of 0.58 while conductivity to oxygen was increased only by a factor of 1.07 in the high-altitude eggs. These independent measurements predict the change in oxygen partial pressure across the egg shell of the high-altitude eggs to be only 0.54 times that of sea-level eggs; the directly measured factor was 0.53. The authors conclude that at high altitude, a major adaptation of the chick embryo is a reduced metabolism which decreases the change in oxygen partial pressure across the egg shell since its gas conductivity remains essentially unchanged.
Wangensteen, O. D.↗
Hypogravity's Effect on the Life Cycle of Japanese Quail
A series of studies were conducted to determine the effect of activities preceding space-flight and during space-flight on quail embryonic development. While the overall development of the quail embryos was evaluated, the report presented herein, focused on calcium utilization or uptake from eggshells by developing embryos during incubation in space and on earth. In the pre-space trials, fertilized quail eggs were subjected to pre-night dynamics including forces of centrifugation, vibration, or a combination of vibration and centrifugation prior to incubation for 6 or 16 days. In another trial, fertile quail eggs were tested for survivability in a refrigerator stowage kit for eggs (RSKE) which was subsequently used to transport the eggs to space. Eggs in the RSKE were subjected to shuttle launch dynamics including G force and random vibration profiles. In the space- flight trials, 48 fertile quail eggs were launched on space shuttle Flight STS-76 and were subsequently incubated in a Slovakian incubator onboard space station, MIR. Two sets of ground controls each with 48 fertile eggs with and without exposure to launch dynamics were initiated 5 days post-launch. There was a laboratory control (incubated in Lyon RX2 incubator at 37.5 C) and a synchronous control (incubated in Lyon RX2 incubator at 39 - 400 C), which simulated the temperature of the space-flight incubator. Following space-flight trials, post-flight trials were conducted where quail eggs were incubated in Lyon RX2 or Slovakian incubators under various temperatures with or without launch dynamics. Eggshells from all study trials were retrieved and analyzed for calcium content to determine if its utilization by developing quail embryos was affected by activities preceding space-flight or during incubation in space under microgravity. Results from the pre-flight and post-flight showed that pre-flight activities and shuttle launch dynamics had no effect on calcium uptake from the eggshell by developing embryos. However, calcium uptake from the eggshell by developing embryos incubated in micro,aravity was impaired by 12.6% when compared to embryos incubated on earth under laboratory control environment. This impairment was unlikely due to factors other than microgravity. In general, calcium utilization by developing embryos increased with age of incubation with the most increase occurring at day 16 of incubation.
Hester, Patricia Y.↗
Twinning of amphibian embryos by centrifugation
In the frog Xenopus laevis, the dorsal structures of the embryonic body axis normally derive from the side of the egg opposite the side of sperm entry. However, if the uncleaved egg is inclined at lg or centrifuged in an inclined position, this topographic relationship is overridden: the egg makes its dorsal axial structures according to its orientation in the gravitational/centrifugal field, irrespective of the position of sperm entry. Certain conditions of centrifugation cause eggs to develop into conjoined twins with two sets of axial structures. A detailed analysis of twinning provided some insight into experimental axis orientation. First, as with single-axis embryos, both axes in twins are oriented according to the direction of centrifugation. One axis forms at the centripetal side of the egg and the other forms at the centrifugal side, even when the side of sperm entry is normal to the centrifugal force vector. Second, if eggs are centrifuged to give twins, but are inclined at lg to prevent post-centrifugation endoplasmic redistributions, only single-axis embryos develop. Thus, a second redistribution is required for high-frequency secondary axis formation. This can be accomplished by lg (as in the single centrifugations) or by a second centrifugation directed along the egg's animal-vegetal axis.
Black, S. D.↗
Effects of silver ions (Ag+) on contractile ring function and microtubule dynamics during first cleavage in Ilyanassa obsoleta
The terminal phase of cell division involves tight constriction of the cleavage furrow contractile ring, stabilization/elongation of the intercellular bridge, and final separation of the daughter cells. At first cleavage, the fertilized eggs of the mollusk, Ilyanassa obsoleta, form two contractile rings at right angles to each other in the same cytoplasm that constrict to tight necks and partition the egg into a trefoil shape. The cleavage furrow contractile ring (CF) normally constricts around many midbody microtubules (MTs) and results in cleavage; the polar lobe constriction contractile ring (PLC) normally constricts around very few MTs and subsequently relaxes without cleavage. In the presence of Ag+ ions, the PLC 1) begins MT-dependent rapid constriction sooner than controls, 2) encircles more MTs than control egg PLCs, 3) elongates much more than control PLCs, and 4) remains tightly constricted and effectively cleaves the polar lobe from the egg. If Ag(+)-incubated eggs are returned to normal seawater at trefoil, tubulin fluorescence disappears from the PLC neck and the neck relaxes. If nocodazole, a drug that depolymerizes MTs, is added to Ag(+)-incubated eggs during early PLC constriction, the PLC is not stabilized and eventually relaxes. However, if nocodazole is added to Ag(+)-incubated eggs at trefoil, tubulin fluorescence disappears from the PLC neck but the neck remains constricted. These results suggest that Ag+ accelerates and gradually stabilizes the PLC constriction by a mechanism that is initially MT-dependent, but that progressively becomes MT-independent.
Non-NASA Center↗
Deep cytoplasmic rearrangements in axis-respecified Xenopus embryos
In fertilized eggs of the frog Xenopus, the vegetal yolk mass rotates away from the future dorsal side (J. P. Vincent and J. Gerhart, 1987, Dev. Biol. 123, 526-539), and a major rearrangement of the deep animal hemisphere cytoplasm produces a characteristic swirl in the prospective dorsal side (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). The relationship between this swirl and determination of the dorsal-ventral axis was further investigated by attempting to experimentally separate the positions of the swirl and the dorsal-ventral axis. Eggs were obliquely oriented in the gravity field to respecify the direction of yolk mass rotation and the position of the dorsal-ventral axis. When yolk mass rotation occurred in the absence of a sperm, as in activated eggs, a swirl pattern formed on the side away from which the yolk mass had rotated. In fertilized eggs tipped with the sperm entry point (SEP) down or to the side, swirl patterns were always found to form on the side away from which the yolk mass was displaced. However, in eggs tipped SEP up, in which the yolk mass was forced to rotate away from the SEP, more complicated rearrangements were observed in addition to the rotation-oriented swirl. Because the direction of yolk mass rotation was found to be influenced by both gravity and the actual position of the SEP in obliquely oriented eggs (SEP to the side), such complicated rearrangement patterns may result from opposing forces generated by both yolk mass rotation and the expanding sperm aster. Thus, except in cases in which the influences of SEP position and unit gravity opposed each other, it was not possible to experimentally separate the position of the deep cytoplasmic swirl from the direction of yolk mass rotation, and therefore the position of the prospective dorsal side.
Non-NASA Center↗
The utilization of Habrobracon and artemia as experimental materials in bioastronautic studies
In the reproductive performance of female braconids striking contrasts were revealed between the results from the actual biosatellite flight and those from experiments when the recovered vehicle was subjected to the forces of simulated launching and recovery. Second week decreases in egg production due to the radiation damage of cells in mitosis were minimized for the females irradiated during space flight. It was demonstrated that females irradiated for two days during orbital flight laid as many eggs during the second week as the unirradiated ground-based controls. After the 10th day their oviposition records exceeded control values. The hatchability of eggs deposited by Biosatellite II females was excellent. Explanations were sought for the space flight's cancellation of the characteristic radiation-induced decrease in egg production, and for the exceptionally good hatchability of eggs derived from most of the cell types in the irradiated ovarioles. Eggs from only two classes of cells showed enhanced embryonic lethality: those poised in meiotic metaphase during their mother's orbital flight, and those from oocytes beginning vitellogenesis.
Grosch, D. S.↗
Launch Conditions Might Affect the Formation of Blood Vessel in the Quail Chorioallantoic Membrane
AS 2 part of the first joint USA-Russian MIR/Shuttle program, fertilized quail eggs were flown on the MIR 18 mission. Post-flight examination indicated impaired survival of both the embryos in space and also of control embryos exposed to vibrational and g-forces simulating the conditions experienced during the launch of Progress 227. We hypothesized that excess mechanical forces and/or other conditions during the launch might cause abnormal development of the blood supply in the chorioallantoic membrane (CAM) leading to the impaired survival of the embryos. The CAM, a highly vascularized extraembryonic organ, provides for the oxygen exchange across the egg shell and is thus pivotal for proper embryonic development. To test our hypothesis, we compared angiogenesis In CAMS of eggs which were either exposed to the vibration and g-force profile simulating the conditions at launch of Progress 227 (synchronous controls), or kept under routine conditions in a laboratory Incubator (laboratory controls). At various time points during Incubation, the eggs were fixed in paraformaldehyde for subsequent dissection. At the time of dissection, the CAM was carefully lifted from the egg shell and examined as whole mounts by bright-field and fluorescent microscopy. The development or the vasculature (angiogenesis) was assessed from the density of blood vessels per viewing field and evaluated by computer aided image analysis. We observed a significant decrease In blood-vessel density in the synchronous controls versus "normal" laboratory controls beginning from day 10 of Incubation. The decrease in vascular density was restricted to the smallest vessels only, suggesting that conditions during the launch and/or during the subsequent Incubation of the eggs may affect the normal progress of angiogenesis in the CAM. Abnormal angiogenesis In the CAM might contribute to the impaired survival of the embryos observed in synchronous controls as well as in space.
Henry, M. K.↗
A critical period for functional vestibular development in zebrafish
We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe during which the vestibular primary afferent neurons are born, innervate their central and peripheral targets, and remodel their central projections. Copyright 2002 Wiley-Liss, Inc.
Non-NASA Center↗
Simulated Microgravity as a Probe for Understanding the Mechanisms of Early Pattern Specification
Early pattern specification (e.g., axial structure morphogenesis, doreal ventral polarity, etc.) is monitored in amphibian eggs which were subjected to microgravity simulation by constant rotation on a horizontal clinostat. In contrast to previous clinostat experiments, rotation is initiated either prior to fertilization or immediately thereafter. Large proportions of clinostated eggs developed normal axial structures. A model which employs a multiple set of signals for specifying early pattern is discussed. Effects of microgravity simulation on the earliest post fertilization pattern specification event dorsal/ventral polarization is analyzed in detail. Other models are developed and they are discussed. As a general mechanism for explaining the manner in which regional developmental patterns emerge from the initial, radially symmetrial egg, the density compartment model is described. The identification of the various zones or compartments of egg cytoplasm using inverted eggs is explained.
Neff, A. W.↗
Amphibian fertilization and development in microgravity
An experiment investigating the effects of gravity on embryonic development in amphibians is proposed. The planned procedures for the preparation of the frog eggs for launching in the Space Shuttle, for the injection of the eggs with gonadotropin, for the insertion of the eggs into egg chambers, for the storage of one of the chambers in a microgravity area and the second into a centrifuge, and for the fertilization of the eggs are described. The later organogenesis, swimming behavior, cytoplasmic components, cellular formation, neural plate and archenteron expansion, and allometry and expansion of the organ systems will be examined. Normal morphology for embryos and tadpoles developing at microgravity and the formation of the neural plate opposite the sperm entry point meridian are predicted.
Souza, K. A.↗
Preliminary results of the Artemia salina experiments in biostack on LDEF
The mosaic egg of the brine shrimp, Artemia salina, resting in blastula or gastrula state represents a system that during further development, proceeds without any further development to the larval stage, the free swimming nauplius. Therefore, injury to a single cell of the egg will be manifest in the larvae. In several experiments, it was shown that the passage of a single heavy ion through the shrimp egg damaged a cellular area large enough to disturb either embryogenesis or further development of the larvae, or the integrity of the adult individual. Emergence from the egg shell was heavily disturbed by the heavy ions as was hatching. Additional late effects, due to a hit by a heavy ion, are delayed of growth and of sexual maturity, and reduced fertility. Anomalies in the body and the extremities could be observed more frequently for the nauplii which had developed from eggs hit by heavy ions.
Graul, E. H.↗
Motility and centrosomal organization during sea urchin and mouse fertilization
It is noted that microfilaments are essential for incorporation of sperm in sea urchins and for pronuclear apposition in mice. The ability of sea urchin sperm to fertilize eggs is lowered by latrunculin, giving evidence that acrosomal microfilaments are of importance to the process of fertilization. Due to the uncertainty regarding the presence of microfilaments in various mammalian sperm, it is interesting that latrunculin does not noticeably affect the ability of mouse sperm to fertilize oocytes. The movements of the sperm and egg nuclei at the time of sea urchin fertilization are dependent on microtubules arranged into a radial monastral array (the sperm aster). In the mouse egg, microtubule activity is also required during pronuclear apposition, but they are arranged by a number of egg cytoplasmic sites. Results of the investigations show that both microtubules and microfilaments are necessary for the successful completion of fertilization in both mice and sea urchins, but at different stages. Also, it is demonstrated that centrosomes are contributed by the sperm in the process of sea urchin fertilization, but in mammals they may be inherited maternally.
Schatten, Heide↗