Effect of microgravity on primordial germ cells (PGCs) in silk chicken offspring (Gallus gallus domesticus)

Primordial germ cells (PGCs), precursors of germline cells, display a variety of antigens during their migration to target gonads. Here, we used silk chicken offspring (Gallus gallus domesticus) embryos subjected to space microgravity to investigate the influence of microgravity on PGCs. The ShenZhou-3 unmanned spaceship carried nine fertilized silk chicken eggs, named the flight group, returned to Earth after 7 days space flight. And the control group has the same clan with the flight group. PGCs from flight and control group silk chicken offspring embryos were examined during migration by using two antibodies (2C9 and anti-SSEA-1), in combination with the horseradish peroxidase detection system, and using periodic acid-Schiff’s solution (PAS) reaction. After incubation for about 30 h, SSEA-1 and 2C9 positive cells were detected in the germinal crescent of flight and control group silk chicken offspring embryos. After incubation of eggs for 2–2.5 days, SSEA-1 and 2C9 positive cells were detected in embryonic blood vessels of flight and control group silk chicken offspring embryos. After incubation of eggs for 5.5 days, PGCs in the dorsal mesentery and gonad could also be identified in flight and control group silk chicken offspring embryos by using SSEA-1 and 2C9 antibodies. Based on location and PAS staining, these cells were identified as PGCs. Meanwhile, at the stage of PGCs migration and then becoming established in the germinal ridges, no difference in SSEA-1 or 2C9 staining was detected between female and male PGCs in flight and control group silk chicken offspring embryos. Although there were differences in the profiles of PGC concentration between male and female embryos during the special circulating stage, changing profile of PGCs concentration was similar in same sex between flight and control group offspring embryos. We concluded that there is little effect on PGCs in offspring embryos of microgravity-treated chicken and that PGC development appears to be normal.     

空间重粒子击中卤虫卵的定位技术及其生物效应

为了能够估价空间重粒子对单个生物细胞的放射生物学效应,我们设计、制成了卤虫卵-核乳胶夹层式生物包.经搭载我国“8885”卫星飞行数天后回收.用图象分析仪研究了空间重离子在核乳胶上留下的径迹及其分布;并实验观察了卤虫卵的前期发育情况.看到:随星飞行过的卤虫卵与地面对照组相比,其发育能力明显降低,表现为孵化率低且发育时间推迟.实验表明:生物样品板-核乳胶影象定位技术能清楚而比较准确地给出卤虫卵(或其他生物细胞)受重离子作用的信息,是重离子生物效应及其作用机理研究的可行实验手段及分析方法之一.     

“8785”返地卫星搭载对卤虫卵发育影响的研究

将我国所产卤虫(Artemia salina)的卵,由1987年8月5日发射的返地卫星搭载,在空间飞行5天。于飞行完成后第8、21、24、34及66天,随机取卵进行人工孵化及发育观察。见到的主要现象有:(1)飞行卵的早期发育进程显著变慢;(2)随着回收后时间的延长,飞行卵的早期发育速度出现回升的趋势;(3)飞行卵早期发育中的冒出率和孵出率一般都比地面对照组的低,且随着回收后时间的延长,有继续下降的趋势;(4)飞行卵孵出的卤虫,自孵出第1天至接近全部自然死亡的23天内,存活率的下降情况与地面对照组的没有差异。本文提出一种空间飞行因素对卤虫卵损伤的“临界程度”的假设。实验还表明,我国所产的卤虫卵对空间飞行因素的作用是敏感的,是一种空间生物学研究的好材料。      

Influence of cosmic radiation and/or microgravity on development of Carausius morosus.

Eggs of Carausius morosus were exposed to spaceflight conditions in two spaceflight missions, the German 7 day Spacelab Mission D1 and the Soviet 12.56 day Biosatellite Mission "COSMOS 1887". During spaceflight the eggs continued their development. Eggs of five different ages representing different sensitivity to radiation and different capacity to regeneration were used to investigate the influence of cosmic radiation and/or microgravity on insect development. Using the Biostack concept--eggs in monolayers sandwiched between nuclear track detectors--and the 1 g reference centrifuge of BIORACK in D1 we were able to separate effects of heavy ions of the cosmic radiation from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, embryonic and larval growth kinetics and anomaly frequencies were determined. Microgravity leads to a reduced hatching rate of eggs exposed in the early stages of development. Hatching was normal in eggs which were exposed on the 1 g reference centrifuge. Hits by heavy ions caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high frequency of anomalies. These results obtained from the Spacelab Mission D1, were confirmed in an experiment onboard of COSMOS 1887. In addition to the previous analysis, embryonic development before hatching was followed which showed no major difference between flight and the ground control specimens. Since a reconfirmation of reduced hatching rates was observed in COSMOS 1887, too, the above results suggest some microgravity induced functional impairment of the hatching activity, rather than blockage in embryonic development.     

Development of space-fertilized eggs and formation of primordial germ cells in the embryos of Medaka fish.

In the second International Microgravity Laboratory (IML-2) mission in 1994, four small Japanese killifish (Medaka, Oryzias latipes) made a space travel of 15 days aboard a space shuttle. These four adult Medaka fish successfully mated in space for the first time among vertebrate animals. Moreover, the eggs they laid developed normally, at least in their external appearance, hatching as fry (baby fish) in space. Fish mated and laid eggs every day during the first week. Near the end of the mission most of the eggs had a well-developed body with two pigmented eyes. In total, 43 eggs were laid (detected), out of which 8 fry hatched in space, as truly 'space-originated' babies. A further 30 fry hatched within 3 days after landing. This is the normal hatching rate, compared with the ground-based data. Among the 8 space-originated fry, four were killed for histological sections, and germ cells at the gonadal region were counted for each fry. Their numbers were in the range of the germ cells of the normal control fry (ground-kept samples). Thus, as embryos developed normally in their external appearance, inside the embryos the formation of primordial germ cells took place normally in space, and their migration to the genital ridges was not hindered by microgravity. The two of the remaining space-originated fry have grown up and been creating their offspring in the laboratory. This proved that the primordial germ cells formed in space were also normal from a functional point of view. The four space-travelled adult fish re-started mating and laying eggs on the 7th day after landing and continued to do so every day afterward. Fertilization rate and hatchability of these eggs were as high as the eggs laid by the laboratory-kept fish. This fact implies that in gametogenesis of adult fish, there are no specific stages of germ cells extremely susceptible to microgravity.     

Initial ground experiments of silkworm cultures living on different feedstock for provision of high quality animal protein for human in space

Silkworm could be an alternative to provide edible animal protein in Controlled Ecological Life Support System (CELSS) for long-term manned space missions. Silkworms can consume non-edible plant residue and convert plant nutrients to high quality edible animal protein for astronauts. The preliminary investigation of silkworm culture was carried out in earth environment. The silkworms were fed with artificial silkworm diet and the leaves of stem lettuce (Lactuca sativa L. var. angustana Irish) separately and the nutritional structure of silkworm was investigated and compared, The culture experiments showed that: (1) Stem lettuce leaves could be used as food of silkworm. The protein content of silkworm fed with lettuce leaves can reach 70% of dry mass. (2) The protein content of silkworm powder produced by the fifth instar silkworm larvae was 70%, which was similar to the protein content of silkworm pupae. The powder of the fifth instar silkworm larvae can be utilized by astronaut. (3) The biotransformation rate of silkworm larvae between the third instar and the fifth instar could reach above 70%. The biotransformation cycle of silkworm was determined as 24 days. (4) Using the stem lettuce leaves to raise silkworm, the coarse fiber content of silkworm excrements reached about 33%. The requirements of space silkworm culture equipment, feeding approaches and feeding conditions were also preliminarily designed and calculated. It is estimated that 2.2 m³ of culture space could satisfy daily animal protein demand for seven astronauts.     

Activity of the sympathetic-adrenomedullary system in rats after space flight on the COSMOS biosatellites

The indicators of adrenomedullary activity (catecholamine content (CA) and the activity of the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH)) were measured in the adrenal glands of rats living in a state of weightlessness for 18.5–19.5 days on board the biosatellites COSMOS 936 and COSMOS 1129. None of these indicators was significantly changed by space flight, neither in the group living in a state of weightlessness nor in the group living in a centrifuge on board the spacecraft and exposed to artificial gravity of 1 g (COSMOS 936). Animals exposed after space flight to repeated immobilization stress on Earth showed a significant decrease of adrenal adrenaline and an appreciable increase in adrenal TH activity compared to stressed animals which were not in space. These results suggest that a prolonged state of weightlessness during space flight does not by itself represent an intensive stressful stimulus for the adrenomedullary system but potentiates the response of cosmonauts to stress after return to Earth.     

Effects of long duration space flight on rice seed (or embryo) radiation sensitivity and element microlocalizations.

In long duration space experiments Rice caryopses and embryos, which are able to remain alive 10 years (or more) and tolerate extreme physical conditions (temperature, few water content) during irradiation and post-irradiation storage, were used (8, 40, 201 and 457 days on board of Salyut 7, 2107 days on LDEF). In certain experiments (Salyut 7), samples were irradiated either before or after the flight. Effects of the flight and radiosensitivity were observed in Rice seedlings cultivated in in vitro conditions. Statistical results indicate an increase in radiosensitivity when irradiations occur before the flight. Microanalyses were made in different parts of one caryopsis and of one embryo, and the results compared with those of control samples. With caryopses and embryos of the same Rice varieties, but from LDEF, we made the same kinds of experiments to compare results.     

Effects of space flight and IGF-1 on immune function

We tested the hypothesis that insulin-like growth factor-1 (IGF-1) would ameliorate space flight-induced effects on the immune system. Twelve male, Sprague-Dawley rats, surgically implanted with mini osmotic pumps, were subjected to space flight for 10 days on STS-77. Six rats received 10 mg/kg/day of IGF-1 and 6 rats received saline. Flight animals had a lymphocytopenia and granulocytosis which were reversed by IGF-1. Flight animals had significantly higher corticosterone levels than ground controls but IGF-1 did not impact this stress hormone. Therefore, the reversed granulocytosis did not correlate with serum corticosterone. Space flight and IGF-1 also combined to induce a monocytopenia that was not evident in ground control animals treated with IGF-1 or in animals subjected to space flight but given physiological saline. There was a significant increase in spleen weights in vivarium animals treated with IGF-1, however, this change did not occur in flight animals. We observed reduced agonist-induced lymph node cell proliferation by cells from flight animals compared to ground controls. The reduced proliferation was not augmented by IGF-1 treatment. There was enhanced secretion of TNF, IL-6 and NO by flight-animal peritoneal macrophages compared to vivarium controls, however, O₂ secretion was not affected. These data suggest that IGF-1 can ameliorate some of the effects of space flight but that space flight can also impact the normal response to IGF-1.     

Seedling growth and development on space shuttle.

Young pine seedlings, and mung bean and oat seeds were flown on shuttle flights, STS-3 and STS-51F, in March, 1982 and July/August, 1985, respectively. The plant growth units built to support the two experiments functioned mechanically as anticipated and provided the necessary support data. Pine seedlings exposed to the microgravity environment of the space shuttle for 8 days continued to grow at a rate similar to ground controls. Pine stems in flight seedlings, however, averaged 10 to 12% less lignin than controls. Flight mung beans grew slower than control beans and their stems contained about 25% less lignin than control seedlings. Reduced mung bean growth in microgravity was partly due to slower germination rate. Lignin also was reduced in flight oats as compared to controls. Oats and mung beans exhibited upward growing roots which were not observed in control seedlings. Chlorophyll A/B ratios were lower in flight tissues than controls. The sealed PGCs exhibited large variations in atmospheric gas composition but the changes were similar between flight and ground controls. Ethylene was present in low concentrations in all chambers.     

The effect of artificial gravity on plasma and tissue lipids in rats: The Cosmos 936 experiment

Plasma and tissue lipids in male SPF Wistar rats flown for 18.5 days aboard the Cosmos 936 biosatellite were analyzed. One group of rats was subjected to artificial gravity by use of a centrifuge during the flight. An experiment simulating known space flight factors other than weightlessness was done on Earth. An increase of total cholesterol in plasma, of nonesterified fatty acids in plasma and brown adipose tissue, of triacylglycerols in plasma, liver, thymus and bone marrow was noted several hours after biosatellite landing. Smaller changes were observed in the terrestrial control experiment. With the exception of triacylglycerol accumulation in bone marrow, these increases disappeared 25 days after biosatellite landing. Exposing the rats aboard the biosatellite to artificial gravity was beneficial in the sense that such exposure inhibited the phospholipid and triacylglycerol increase in plasma and inhibited the increase of triacylglycerol in liver and especially in bone marrow.     

The response of endocrine system to stress loads during space flight in human subject.

The responses of endocrine system to the exposure to stress-work load and hormonal changes during oral glucose tolerance tests were studied in the Slovak astronaut before (three weeks before flight), during (on the 4th and the 6th days of space flight), and after space flight (1-3 days and 15-17 days after space flight) on board of space station MIR. Blood samples during the tests were collected via cannula inserted into cubital vein, centrifuged in the special appliance Plasma-03, frozen in Kryogem-03, and at the end of the 8-day space flight transferred to Earth in special container for hormonal analysis. Preflight workload produced an increase of plasma norepinephrine and a moderate elevation of epinephrine levels. Plasma levels of insulin, growth hormone, prolactin and cortisol were not markedly changed immediately or 10 min after the end of work load. The higher increases of plasma growth hormone, prolactin and catecholamine levels were noted after workload during space flight as compared to preflight response. The higher plasma glucose and insulin levels were noted during the oral glucose tolerance test in space flight and also in the post flight period. Plasma epinephrine levels were slightly decreasing during glucose tolerance test; however, plasma norepinephrine levels were not changed. The similar patterns of catecholamine levels during glucose tolerance test were found when compared the preflight, in-flight and post flight values. These data demonstrate the changes of the dynamic responses of endocrine system to stress-work and metabolic loads during space flight in human subject.     

Blood and clonogenic hemopoietic cells of newts after the space flight.

Ribbed newts were used for studying the effect of space flight on board of the biosatellite (Cosmos-2229) on blood and clonogenic hemopoietic cells. In blood of newts of the flight group, the relative proportion of neutrophils increased, whereas that of lymphocytes and eosinophils decreased. Space flight did not result in loss of the ability of newt blood cells to incorporate H3-thymidine. Analysis of clonogenic hemopoietic cells was performed using the method of hemopoietic colony formation on cellulose acetate membranes implanted into the peritoneal cavity of irradiated newts. To analyze reconstitution of hemopoiesis after irradiation donor hemopoietic cells from flight or control newts were transplanted into irradiated newts whose hemopoietic organs were investigated. The newt can be considered an adequate model for studying hemopoiesis under the conditions of the space flight.     

The effect of microgravity on the development of plant protoplasts flown on Biokosmos 9.

An experiment using plant protoplasts has been accepted for the IML-1 Space Shuttle mission scheduled for 1991. Preparatory experiments have been performed using both fast and slow rotating clinostats and in orbit to study the effect of simulated and real weightlessness on protoplast regeneration. Late access to the space vehicles before launch has required special attention since it is important to delay cell wall regeneration until the samples are in orbit. On a flight on Biokosmos 9 ("Kosmos-2044") in September 1989 some preliminary results were obtained. Compared to the ground control, the growth of both carrot and rapeseed protoplasts was decreased by 18% and 44% respectively, after 14 days in orbit. The results also indicated that there is less cell wall regeneration under micro-g conditions. Compared to the ground controls the production of cellulose in rapeseed and carrot flight samples was only 46% and 29% respectively. The production of hemicellulose in the flight samples was 63% and 67% respectively of that of the ground controls. In both cases all samples reached the stage of callus development. The peroxidase activity was also found to be lower in the flight samples than in the ground controls, and the number of different isoenzymes was decreased in the flight samples. In general, the regeneration processes were retarded in the flight samples with respect to the ground controls. From a simulation experiment for IML-1 performed in January 1990 at ESTEC, Holland, regenerated plants have been obtained. These results are discussed and compared to the results obtained on Biokosmos 9. Protoplast regeneration did not develop beyond the callus stage in either the flight or the ground control samples from the Biokosmos 9 experiment.     

Possible mechanism of microgravity impact on Carausius morosus ontogenesis.

Experiments aboard "Spacelab-D1" and "Cosmos-1887" revealed an adverse effect of space flight on Carausius morosus embryos. The main influencing factor for stick insect eggs turned out to be microgravity, while the contribution of HZE particles of cosmic radiation was relatively low. Flight experiments indicated an increased vulnerability of stick insect eggs to microgravity at intermediate stages of development, that could support the "convection" hypothesis.     

用果蝇探查火箭飞行因素的生物遗传效应的实验结果

用黑腹果蝇检查T_7A—S1火箭飞行因素的遗传效应。通过两次飞行实验观察,发现亲代雌蝇在飞行后(主要是第4—6天)所产的卵中,无X染色体卵百分率增高而双X染色体卵百分率未见变化。其性质并非个别雌蝇产生了较多的无X染色体卵,而是产生无X染色体卵的雌蝇个数增多。第二次飞行实验结果也呈现,亲代雌蝇在飞行后第4—6天所产的子1代中,三龄幼虫成神经细胞染色体桥频率增高。上述实验结果表明,火箭飞行因素对雌果蝇生殖细胞遗传物质有轻微影响。      

Retinal photoreceptor and related gene expression in normal and clinostat-treated fish embryos

Medaka fish had performed mating behavior successfully in space for the first time among vertebrate, and the eggs which were laid in space developed normally, and hatched during the space travel of 15 days aboard the space shuttle in the second International Microgravity Laboratory (IML-2) mission in 1994 (Ijiri 1994). But there has been few studies whether microgravity affects the development of rather complex tissues in this fish. Investigating this problem, we focused on the organogenetic events in the retina in developing Medaka under normal and simulated microgravity conditions (by a three-dimensional clinostat, 3D-clinostat). Our results showed that both normal and 3D-clinostat-treated Medaka embryos developed on almost equal time course. Moreover, we investigated the development of the retina in normal and 3D-clinostat-treated embryos, but there were no differences in organogenesis of their retina. Lamination of retina occurred almost at equal timing and the expressions of opsin genes in the 3D-clinostat-treated group also began almost at the same time as control. Our observations suggest that there were no definite effects of simulated microgravity on the organizations of a complex tissue such as retina in developing fish embryos.     

The effects of space flight on some rat liver enzymes regulating carbohydrate and lipid metabolism

We have examined, in the livers of rats carried aboard the Cosmos 936 biosatellite, the activities of about 30 enzymes concerned with carbohydrate and lipid metabolism. In addition to the enzyme studies, the levels of glycogen and of the individual fatty acids in hepatic lipids were determined. Livers from flight and ground control rats at recovery (R₀) and 25 days after recovery (R₂₅) were used for these analyses.

For all parameters measured, the most meaningful comparisons are those made between flight stationary (FS) and flight centrifuged (FC) animals at R₀. When these two groups of flight rats were compared at R₀, statistically significant decreases in the activity levels of glycogen phosphorylase, -glycerol phosphate acyl transferase, diglyceride acyl transferase, aconitase and 6-phosphogluconate dehydrogenase and an increase in the palmitoyl CoA desaturase were noted in the weightless group (FS). The significance of these findings was strengthened by the fact that all enzyme activities showing alterations at R₀ returned to normal 25 days postflight. When liver glycogen and total fatty acids of the two sets of flight animals were determined, significant differences that could be attributed to reduced gravity were observed. The weightless group (FS) at R₀ contained, on the average, more than twice the amount of glycogen than did the centrifuged controls (FC) and a remarkable shift in the ratio of palmitate to palmitoleate was noted. These metabolic alterations, both in enzyme levels and in hepatic constituents, appear to be characteristic of the weightless condition. Our data seem to justify the conclusion that centrifugation during flight is equivalent to terrestrial gravity.