A procedure for the rapid freezing of whole embryos

Summary A procedure for the rapid freezing of whole chick embryos for histochemical treatment is described. The problems of deformation during preparation for quenching and orientation for sectioning have been largely overcome by placing embryos inside lengths of chicken trachea. The subsequenct disorientation of tissues that follows cracking and shattering due to the rapid freezing of whole embryos is avoided. The method permitted a more precise identification of the position and time of appearance of formaldehyde-induced fluorescence and myosin antibody immunofluorescence in serially sectioned embryos.We gratefully thank Dr Ute Gröschel-Stewart for preparing sections for immunofluorescence.     

Effects of low temperatures on survival of frozen-thawed mouse embryos

Summary Almost no damage to mouse morulae was observed between 0 and –40°C, and freezing damage to embryos in DMSO, glycerol or ethylene glycol occurred after exposure to –60, –50 or –50°C, respectively. Cooling embryos in DMSO or glycerol to temperatures below –50 to –60°C increased freezing damage. To the contrary, in the presence of ethylene, glycol, no more damage occurred after exposure to temperatures below –50°C.     

Eine neue Methode zum schnellen Einfrieren von Geweben

Summary A new unit for the rapid freezing of tissues in liquid nitrogen is described.     

Schnelles Einfrieren von Geweben und Flüssigkeiten

Summary A new unit for rapid freezing of tissues and liquids in liquid nitrogen is described.     

Effects of retinoic acid on embryonic development of mice in culture

The effects of all-trans-retinoic acid (RA) (tretinoin) on the craniofacial development of mouse embryos were examined using whole embryo culture. In day 8 embryos cultured for 48 h, embryonic growth was inhibited concentration-dependently by all-trans-RA treatment. Most of the treated embryos exhibited hypoplasia of the primary palatal processes and a reduction in the development of the first visceral arches. In day 10 embryos cultured for 48 h, although embryonic growth was not inhibited at any concentrations of all-trans-RA, median cleft lip (93%), hypoplasia of the primary palatal processes (37%) and limb reduction deformities (48%) occurred commonly. Furthermore, RA treatment greatly reduced the size of the secondary palatal processes. The incorporation of 3H-thymidine in the treated maxillary processes was decreased to 65% of the control value at 1.0 x 10(-7) M all-trans-RA. These findings indicate that all-trans-RA is teratogenic in mouse whole embryo culture.     

Effects of retinoic acid on embryonic development of mice in culture

Summary The effects of all-trans-retinoic acid (RA) (tretinoin) on the craniofacial development of mouse embryos were examined using whole embryo culture. In day 8 embryos cultured for 48 h, embryonic growth was inhibited concentration-dependently by all-trans-RA treatment. Most of the treated embryos exhibited hypoplasia of the primary palatal processes and a reduction in the development of the first viscreral arches. In day 10 embryos cultured for 48 h, although embryonic growth was not inhibited at any concentrations of all-trans-RA, median cleft lip (93%), hypoplasia of the primary palatal processes (37%) and limb reduction deformities (48%) occurred commonly. Furthermore, RA treatment greatly reduced the size of the secondary palatal processes. The incorporation of³H-thymidine in the treated maxillary processes was decreased to 65% of the control value at 1.0×10^–7 M alltrans-RA. These findings indicate that all-trans-RA is teratogenic in mouse whole embryo culture.     

Freezing and transplantation of brain tissue in rats

Summary Neocortical tissue obtained from rat embryos was frozen and stored at –70°C for 6 h prior to transplantation into the cerebellum of neonatal rats. Growth, differentiation, and integration of this tissue within the host brain was comparable to that obtained from freshly dissected and transplanted tissue. It is suggested that freezing to low temperatures does not adversely effect the viability or transplantability of the neural tissue.Supported by N.I.H. research grants Nos NS-08817 and CA-14650.     

Rapid freezing of biologic tissue. Measurement of temperature and rate of freezing by thin-layer thermocouple]

An apparatus for rapid freezing of biological tissues by contact with a copper block cooled by liquid helium has been devised to reduce the contamination of copper block surface. It prevents the precooling of the specimen while going through the layers of cold helium gas surrounding the copper block and reduces the quantity of helium necessary for freezing. It also enables one to obtain easily reproducible results from freezing by immersion in liquid coolants. Freezing rates are measured directly, related to the specimen thickness, by a thin film thermocouple; its low thermal inertia gives speed measurements of the order of 100,000 degrees C/s.     

A simple method for counting nuclei in the preimplantation mouse embryo

An easy and rapid method of counting the number of cells in the preimplantation mouse embryo is described. The procedure increases the speed with which large numbers of embryos can be processed using a simple squash technique. Cell numbers are determined by exposing the embryos to the fluorescent DNA-binding dye, Hoechst 33258, removing the zona pellucida and simply squashing the embryo and counting the number of fluorescent nuclei. An increase in fluorescent intensity and maintenance of nuclear conformation of the squashed preparations are greatly improved by the use of the non-ionic detergent Triton X-100. Viability of dye-treated fertilized one-cell and blastocyst stage embryos is maintained at least up to day 13 of pregnancy following transfer of the embryos to the uteri of pseudopregnant recipients. Additional uses for this staining technique are discussed.     

A simple method for counting nuclei in the preimplantation mouse embryo

Summary An easy and rapid method of counting the number of cells in the preimplantation mouse embryo is described. The procedure increases the speed with which large numbers of embryos can be processed using a simple squash technique. Cell numbers are determined by exposing the embryos to the fluorescent DNA-binding dye, Hoechst 33258, removing the zona pellucida and simply squashing the embryo and counting the number of fluorescent nuclei. An increase in fluorescent intensity and maintenance of nuclear conformation of the squashed preparations are greatly improved by the use of the non-ionic detergent Triton X-100. Viability of dye-treated fertilized one-cell and blastocyst stage embryos is maintained at least up to day 13 of pregnancy following transfer of the embryos to the uteri of pseudopregnant recipients. Additional uses for this staining technique are discussed.Acknowledgment. Financial support was provided by NIH Grant HDD-06210 (KME) and by Basil O'Connor Starter Research Grant No. 5-379 from the March of Dimes Birth Defects Foundation (VEP). We thank Steven Halpern for help with the photography and Jon Flax for expert technical assistance.     

Comparaison de l'effet de congélations et de lyophilisations sur la viabilité ultérieure d'embryons d'Evonymus vulgaris Miller (E. europaeus L.p.p)

Summary A comparison is made between the effects of freezing, freeze drying (by vacuum sublimation), and drying (by vacuum) on isolated embryos ofEvonymus vulgaris after a short period of physical hydration.     

Effects of the temperature of ice-seeding on survival of frozen-and-thawed mouse morulae

Summary Mouse morulae were frozen to –196°C in the presence of 1.2 M ethylene glycol or 1 M glycerol. After iceseeding in embryo samples at –10°C or below, the survival rates in vitro were lower than those obtained by ice-seeding between –4° and –8°C. The proportion morulae developing into live young in vivo after ice-seeding at –13°C and freezing was lower than that of embryos seeded at –4°C.     

Micromanaging freeze tolerance: the biogenesis and regulation of neuroprotective microRNAs in frozen brains

When temperatures plummet below 0 °C, wood frogs (Rana sylvatica) can endure the freezing of up to?~?65% of their body water in extracellular ice masses, displaying no measurable brain activity, no breathing, no movement, and a flat-lined heart. To aid survival, frogs retreat into a state of suspended animation characterized by global suppression of metabolic functions and reprioritization of energy usage to essential survival processes that is elicited, in part, by the regulatory controls of microRNAs. The present study is the first to investigate miRNA biogenesis and regulation in the brain of a freeze tolerant vertebrate. Indeed, proper brain function and adaptations to environmental stimuli play a crucial role in coordinating stress responses. Immunoblotting of miRNA biogenesis factors illustrated an overall reduction in the majority of these processing proteins suggesting a potential suppression of miRNA maturation over the freeze–thaw cycle. This was coupled with a large-scale RT-qPCR analysis of relative expression levels of 113 microRNA species in the brains of control, 24 h frozen, and 8 h thawed R. sylvatica. Of the 41 microRNAs differentially regulated during freezing and thawing, only two were significantly upregulated. Bioinformatic target enrichment of the downregulated miRNAs, performed at the low temperatures experienced during freezing and thawing, predicted their involvement in the potential activation of various neuroprotective processes such as synaptic signaling, intracellular signal transduction, and anoxia/ischemia injury protection. The predominantly downregulated microRNA fingerprint identified herein suggests a microRNA-mediated cryoprotective mechanism responsible for maintaining neuronal functions and facilitating successful whole brain freezing and thawing.     

Hensen's node — The ’Organizer’ of the amniote embryo

Conclusion If the hundred years of study on theHensen's node — i.e. on gastrulation and early determination of the embryos of amniote vertebrates — teach anything, they teach in the first place how limited and fragmentary our knowledge is about one of the most central problems of the whole developmental biology. We know that the events in early amniote development — or early avian development, on which our data and ideas are nearly all based — in many ways resemble those in early Amphibian development, which is only slightly better understood, but we also know that direct extrapolations from anamniotes to amniotes cannot be made without proper reservations and without studying the amniote embryos themselves. And we have practically no idea of what is really going on in the cells of the blastoderm when they move, invaginate, induce or are induced, interact, become determined and begin their differentiation. We know that at the stages of gastrulation, the node, and indeed the whole blastoderm, is in a very labile state and can be regulated in many ways to produce a harmonious whole — or a monster — although we only understand very poorly the modes of this regulation. The progress made during the decades, and particularly in recent years, shows, however, that useful information is accumulating to produce a coherent picture, and there is no reason to be pessimistic.Dedicated to ProfessorEtienne Wolff on the occasion of his retirement.     

三圈冻结法在淤泥质土深基坑工程的应用

以往的冻结法在淤泥质土及特殊工程土体中,由于所需的冻结温度低、冻结扩展速度慢、形成冻结壁的强度低等原因,在施工时容易出现片帮、底鼓、突水、涌砂等事故.轻则影响工程进度,重则造成严重的经济损失.针对此类问题,提出了用三圈冻结法优化淤泥质土深基坑开挖的方案,并应用大型数值模拟软件ADINA进行模拟分析.对普通冻结法、连续桩及三圈冻结法等围护结构的位移进行了对比,得出三圈冻结法围护结构不仅最大位移小,而且位移随深度变化平稳.     

Modifications in the type distribution of muscle fibers induced by repeated local freezings the rat sciatic nerve]

In the Rat, after a localized freezing of the sciatic nerve inducing a complete denervation of the medial head of the gastrocnemius muscle, the reinnervation took place within 16-18 days under our experimental conditions. After only one freezing, a limited and stable "type-grouping" of muscle fibres is observed from 30 to 360 days. After 2 to 5 freezing repeated every three weeks, the muscular changes observed one month after the last freezing are much more pronounced than when only one freezing is performed. These changes consist of a progressive increase in the number of type I and type II C fibres. This transformation is not stable: 3 months after the 3rd freezing, the muscle pattern does not differ from that which is noted after a single freezing.     

Primary asymmetry in the distribution of primordial germ cells during colonization of gonadal buds in chick embryo]

Analysis of the numerical data obtained from a population of 529 chick embryos, selected according to their stage of development, shows that the primary distribution of PGC is asymmetric with a bias towards the left side. The kinetics of the colonisation process are in conformity with the law of logistic growth: the number of PGC settling in the germinal epithelia tends to an upper limit not exceeding the number of PGC identifiable in the anterior germinal crescent. Taking the embryo population as a whole, the degree of primary asymmetry at the end of colonisation is such that 56% of the total number PGC fixed in the gonadial primordia are found in the left epithelia.     

Titers of ecdysone, 20-hydroxyecdysone and juvenile hormone III throughout the life cycle of a hemimetabolous insect,the ovoviviparous cockroachNauphoeta cinerea

Summary Titers of ecdysone, 20-hydroxyecdysone and juvenile hormone III were measured in whole body extracts or hemolymph of embryos, first, penultimate and last stadium nymphs, and adult females ofNaupoheta cinerea. We used a gas-chromatography/mass spectrometry method for quantifying juvenile hormone and a radio-immunoassay for ecdysteroid determination. Juvenile hormone III is particularly abundant in the embryonic stage (up to 960 ng/g), at a low level in first and penultimate stadium nymphs (2–10 ng/ml) and almost absent in the last nymphal stadium; in the adult female the juvenile hormone titer rises to 180 ng/ml in hemolymph during rapid oocyte growth. The titers of ecdysone and 20-hydroxyecdysone undergo similar fluctuations in the embryonic and nymphal stages, being highest at the time of cuticle formation in the embryo and a few days before the nymphal and adult molts (around 100–200 ng/ml for exdysone and 2–4 g/ml for 20-hydroxyecdysone).Acknowledgments. We thank Mrs A. Tschan for rearing the cockroaches, Mr M. Kaltenrieder for drawing the graphs, Mr G.C. Jamieson and Mrs C. Reuter for GC/MS analyses. We are also grateful to the Swiss National Science Foundation (grant no. 3.291-0.82 to B. Lanzrein) and the United States National Science Foundation (grant no. PCM 82-08665 to D.A. Schooley) for their financial support.     

Blastomere biopsy influences epigenetic reprogramming during early embryo development,which impacts neural development and function in resulting mice

Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.     

Water or ice?--the challenge for invertebrate cold survival

The ecophysiology of cold tolerance in many terrestrial invertebrate animals is based on water and its activity at low temperatures, affecting cell, tissue and whole organism functions. The normal body water content of invertebrates varies from 40 to 90% of their live weight, which is influenced by water in their immediate environment, especially in species with a water vapour permeable cuticle. Water gain from, or loss to, the surrounding atmosphere may affect animal survival, but under sub-zero conditions body water status becomes more critical for overwinter survival in many species. Water content influences the supercooling capacity of many insects and other arthropods. Trehalose is known to maintain membrane integrity during desiccation stress in several taxa. Dehydration affects potential ice nucleators by reducing or masking their activity and a desiccation protection strategy has been detected in some species. When water crystallises to ice in an animal it greatly influences the physiology of nearby cells, even if the cells remain unfrozen. A proportion of body water remains unfrozen in many cold hardened invertebrates when they are frozen, which allows basal metabolism to continue at a low level and aids recovery to normal function when thawing occurs. About 22% of total body water remains unfrozen from calculations using differential scanning calorimetry (compared with ca 19% in food materials). The ratio of unfrozen to frozen water components in insects is 1:4 (1:6 for foods). Such unfrozen water may aid recovery of freezing tolerant species after a freezing exposure. Rapid changes in cold hardiness of some arthropods may be brought about by subtle shifts in body water management. It is recognised that cold tolerance strategies of many invertebrates are related to desiccation resistance, and possibly to mechanisms inherent in insect diapause, but the role of water is fundamental to them all. Detailed experimental studies are needed to provide information which will allow a more complete and coherent understanding of the behaviour of water in biological systems and aid the cryopreservation of a wide range of biological material.