Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint,DNA repair and apoptosis

DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer. Received 10 April 2007; accepted 23 April 2007     

Development of rhythmic melatonin synthesis in cultured pineal glands and pineal cells isolated from chick embryo

The chick pineal gland exhibits circadian rhythms in melatonin synthesis under in vivo and in vitro conditions. A daily rhythm of melatonin production was first detectable in pineal glands isolated from chick embryos at embryonic day 16 and incubated under a LD cycle. All pineal glands isolated from 17-day-old and older embryos were rhythmic while no gland isolated at embryonic day 14 and 15 exhibited a daily rhythm in melatonin synthesis. Melatonin production in static cultures of embryonic pineal cells was rhythmic over 48 h if the cells were kept under a LD cycle. When embryonic pineal cells were incubated in constant darkness the rhythm in melatonin production was damped within 48 h. These results suggest that chick pineal cells from embryonic day 16 onwards are photosensitive but that the endogenous component of the melatonin rhythm is not completely developed at that age. A soluble analogue of cAMP stimulated and norepinephrine inhibited melatonin synthesis in cultured embryonic pineal cells. These findings indicate that the stimulatory and inhibitory pathways controlling melatonin synthesis in the mature pineal gland are effective in pineal cells isolated from chick embryos at least 2 days before hatching.     

Role of the inositol 1,4,5-trisphosphate receptor in early embryonic development

There is now considerable literature on the importance of phosphatidylinositol cycle activation in transducing information of various types across the plasma membrane. Though much of the data derives from studies on somatic cells, there is increasing evidence for crucial events related to development, including fertilization, cell cycle progression and dorsoventral axis formation. In this review, focus is directed mainly to the molecular basis of the inositol 1,4,5-triphosphate receptor expressed in oocytes and early embryos of Xenopus. Recent progress in studies concerning the role of this receptor in early embryonic development is discussed.     

A simple method for determination of the proportion of 2C and 4C cells in a dormant embryo

Summary Pea embryos grown in the presence of 2.5 mM hydroxyurea from the beginning of imbibition showed a mitotic peak the height of which was considerably less than that of the control, material. Soon after the mitotic peak, there was a complete cessation of mitosis in the treated material. The extent of suppression of mitosis during the first post-dormancy cell cycle caused by hydroxyurea has been used to obtain an estimate of the relative proportion of 2C and 4C cells in the embryo.     

A procedure for the rapid freezing of whole embryos.

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 subsequent 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.     

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 raised and lowered incubation temperatures on the sexual differentiation in the embryos of Emys orbicularis L. (Chelonien)]

Eggs of Emys orbicularis L. were incubated at 18 and 19 degrees 5C during the period of sexual differentiation of the gonads of the embryos; genital glands of testicular structure and retrogressed Müllerian ducts were observed in all cases, like in the embryos issued from eggs incubated at 25 +/- 1 degrees C and 27,5 +/- 0,5 degrees C. On the contrary, the sexual phenotype is female in all the embryos which developed at 35 degrees C, as it is the case between 29 and 30 degrees C.     

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.     

Organization of the ventral branchial and lumbar horns of the spinal cord in embryos of snake-like and tetrapod reptiles]

In embryos of Snake-like Reptiles (Anguis fragilis) and of Ophidians (Tropidonotus, Python), the lay-out of motor neuroblast in the ventral horns of the spinal cord, in the brachial and the lumbar regions differs from the one observed in the same regions of embryos of tetrapod Reptiles (Lacerta viridis).     

Abortive effect of autologous anti-alpha-fetoprotein antibodies in the rat]

Female Commentry Rats 80--85 days old, were immunized by 5 intramuscular injections of Mouse alphafetoprotein (AFP) and then mated. After fertilization a supplementary injections was administered. The animals were bled at different times and killed immediately after the last bleeding on the 19 or 20th day of gestation. Titers of AFP and of autologous anti-AFP antibodies in the maternal blood were determined as well as the AFP concentration in the pooled amniotic fluids from live embryos of each litter. Compared to non-immunized control series, the total of live and dead embryos per litter in animals immunized with Mouse AFP showed no difference. However, the number of live embryos was on the average 50% lower than that in the control series. The serum titers of AFP and of antibodies to autologous AFP in immunized pregnant Rats bearing dead embryos decreased concomitantly with the number of live embryos. The results reported herein demonstrate that the presence of anti-autologous AFP antibodies in pregnant Rats correlates with the interruption of development in a significant proportions of embryos. This suggests that certain spontaneous abortions in the Rat and perhaps in other mammals can be explained by the rupture of immunological tolerance to autologous AFP.     

Ribosomal bodies specific to both pollen and zygotic embryogenesis inDatura

Summary Induction of pollen embryos in vitro or zygotic embryos in situ is characterized by the formation of RNA-rich cytoplasmic bodies. These are observed in all pollen embryos irrespective of the different pathways of androgenesis followed in vitro.We thank Drs brigitte S. Sangwan-Norreel and A. Kovoor for discussions and Mr J. Schaeffer for technical assistance.     

Imprinting of the Peking duck (Anas platyrhynchos) and dependence on exposure to light during ontogenesis

Embryos of Peking ducks were either incubated in complete darkness up t o hatching or were put into light one week before hatching. Control embryos were incubated under dim light conditions which corresponded broadly to the natural conditions. Under standardized imprinting conditions the controls and both groups of the light deprived ducklings showed the 'following response'. Most of the dark-incubated embryos, however, did not distinguish between imprinting and test objects of different shapes. Since most of the embryos kept in darkness only for 21 days also failed to develop the capacity for shape discrimination, there is apparently a critical period for light influences on the development of this capacity at some time during the early prenatal period.     

Survival of rabbit embryos after rapid freezing and thawing

Summary Frozen storage of rabbit embryos at the 16-cell stage in 2.0 M dimethylsulfoxide (DMSO) in phosphate-buffered saline (PBS) was achieved by a 2-step procedure. After storage for 10 days at–196°C they were revived by rapidly thawing at 500°C/min. On transfer of these embryos to pseudopregnant foster mothers, 50% survived to term. The difference in in vivo survival between frozen-thawed and frozen-thawed-cultured embryos was not significant.Acknowledgment. The authors thank the Director for the facilities. VHR is supported by Council of Scientific and Industrial Research (India).     

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.     

Existence of cholinergic receptors in the very young chick embryo]

The injection of certain cholinergic agents in the yolk sac of young Chick embryos (40 to 48 hrs. of incubation) gives rise to a twisting of the cervical notochord and spinal cord and contraction of the cervical somites. These morphogenetic changes result in spinal column malformations in older embryos. The effects of the cholinergic agents are inhibited by simultaneous treatments with a cholinergic agonist and an antagonist or a cholinergic receptor ligand. The results lead to the assumption that the early embryos already possess cholinergic receptors, probably located in the notochord.     

Comparison of the effects of concanavalin A on cell growth and the transport of 3-O-methylglucose in chick embryo fibroblasts

The proliferative capacity of Chick embryo fibroblasts was modified by Con A treatment. Con A decreased the growth of fibroblasts from young embryos (8 days), whereas the lectin stimulated the growth of fibroblasts from older embryos (16 days). This differential effect of Con A did not result from changes in cellular permeability to thymidine, but rather from Con A induced modifications of hexose transport. Changes in hexose transport would cause, as a response, parallel modifications in glycolysis and hence energy charge, which would alter proliferative capacity.     

La localisation extra-embryonnaire des cellules germinales chez l'embryon de Lézard vivipare (Lacerta vivipara Jacquin)

Summary The surgical ablation of an extra-embryonic posterior cresent that directly encloses the caudal end of young embryos, reduces to a few gonocytes the germinal stock in the genital ridges. The same operation performed upon embryos of 10 somites is without any action. The presence of some gonocytes in the genital ridges of the operated embryos is discussed.     

Postimplantation embryo culture for the assessment of the teratogenic potential and potency of compounds

Whole rat embryos cultured during the early stages of organogenesis were subjected to a panel of selected chemicals. Of seventeen known in vivo teratogens, seventeen also induced specific malformations in embryos grown in culture. Of ten chemicals which were reported to be negative in in vivo rat teratogenicity studies, eight also did not provoke dysmorphogenic effects in vitro. Of five additionally tested retinoids, all induced multiple malformations. However, concentrations used to induce these effects varied considerably, isotretinoin inducing malformations at 10(-5) M and arotinoid at 10(-11) M. The results indicate qualitatively as well as quantitatively a high predictability of this in vitro system and suggest that the postimplantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.