A method for karyotyping mouse blastocyst embryos developing from in vivo and in vitro fertilized eggs

Summary A method for karyotyping blastocyst-staged mouse embryos is described. The use of this protocol results in the recovery of a high percentage (>70%) of readable karyotypes and can be completed rapidly.This research was supported by NIH grants HD-00827 and HD-09753.     

ATP-content in unfertilized and fertilized eggs ofCiona intestinalis

Riassunto Il dosaggio dell'ATP presente nelle uova è stato fatto col sistema luciferin-luciferasi misurando la luminescenza mediante un contatore a scintillazione liquida. É stato rilevato che 10 min dopo la fecondazione il livello di ATP nelle uova si abbassa di ben sette volte. Il risultato è discusso sulla base delle modificazioni morfologiche e biochimiche che intervengono nell'uovo alla fecondazione.     

Steroid metabolism by mouse preimplantation embryos in vitro

Summary Mouse preimplantation embryos were incubated with radioactive pregnenolone, progesterone or dehydroepiandrosterone for various periods of time. These substrates were not converted to metabolites even after incubation of 120 h. We suggest that preimplantation mouse embryo does not possess enzyme activities for steroid metabolism.Acknowledgments. The authors would like to thank Prof. L. Saxén for giving the animals and the facilities of his tissue culture laboratory.     

Steroid metabolism by mouse preimplantation embryos in vitro.

Mouse preimplantation embryos were incubated with radioactive pregnenolone, progesterone or dehydroepiandrosterone for various periods of time. These substrates were not converted to metabolites even after incubation of 120 h. We suggest that preimplantation mouse embryo does not possess enzyme activities for steroid metabolism.     

In vitro fertilization of mouse and hamster eggs after freezing and thawing

Summary Mouse eggs (12–14%) and hamster eggs (87–94%) appear normal upon thawing after having been stored for 30 min to 25 h at –70°C and –196°C. While 89–98% of the normal appearing hamster eggs are capable of fertilization in vitro, only 4–13% of the mouse eggs can be fertilized. The ability of fertilized eggs to develop in culture and their viability upon transfer to pseudopregnant recipients is under investigation.This work was supported by grants from the U.S. Public Health Service and the Ford Foundation. Sincere thanks are due to Prof.Y. Nishikawa of Kyoto University, Japan, for advice and to Mrs.Rose Bartke and Ms.D. M. Hunt for assistance.     

The collagen substratum influences in vitro hatching and attachment of the mouse blastocyst in a serumless medium

Summary When a serumless medium is used for the in vitro growth and development of post-blastocyst mouse embryos, a collagen substratum causes a delay in the hatching from the zona pellucida. However, the collagen substratum is essential for blastocyst attachment and trophoblast cell outgrowth after hatching has taken place.     

The effect of 5-bromodeoxyuridine on mouse embryos during neurulation in vitro

Mouse embryos explanted at various stages during neurulation were cultured for 20-28 h in the presence of 25-900 micrograms/ml of 5-bromodeoxyuridine (BUdR). BUdR strongly inhibited closure of the cranial neural tube, which was found to be stage-dependent. When mouse embryos were exposed to BUdR after development of the concave curvature in the neuroepithelium of the midbrain to the upper hindbrain regions, they became insensitive to the drug-induced open cranial neural tube. Histological observations showed that BUdR interfered with interkinetic migration and cytokinesis of the neuroepithelial cells. These cellular abnormalities were not dependent on the morphological development of the cranial neural folds. The 3H-BUdR experiment confirmed that the label was mostly incorporated into the DNA fraction.     

The effect of 5-bromodeoxyuridine on mouse embryos during neurulation in vitro

Summary Mouse embryos explanted at various stages during neurulation were cultured for 20–28 h in the presence of 25–900 g/ml of 5-bromodeoxyuridine (BUdR). BUdR strongly inhibited closure of the cranial neural tube, which was found to be stage-dependent. When mouse embryos were exposed to BUdR after development of the concave curvature in the neuroepithelium of the midbrain to the upper hindbrain regions, they became insensitive to the drug-induced open cranial neural tube. Histological observations showed that BUdR interfered with interkinetic migration and cytokinesis of the neuroepithelial cells. These cellular abnormalities were not dependent on the morphological development of the cranial neural folds. The³H-BUdR experiment confirmed that the label was mostly incorporated into the DNA fraction.Acknowledgment. This work was supported by Grant-in-Aids for scientific research No. 557469 and 58480391 from the Ministry of Education, Japan.     

Elevated glucose levels influence in vitro hatching,attachment, trophoblast outgrowth and differentiation of the mouse blastocyst

Summary A glucose concentration of 3.5 mg/ml is optimal for in vitro embryo attachment and trophoblastic cell outgrowth. Raising the concentration above 3.5 mg/ml does not improve embryo culture and can at certain concentrations be detrimental to embryo development.     

A method for injection and transplantation of nuclei and cells inDrosophila eggs

Summary Microinjection ofDrosophila eggs using the principle of thermal expansion is described. The eggs, mounted on cover slips with adhesive, are slightly dried to avoid the formation of exudate and then covered with fluorocarbon oil. Injections are made with a specially designed electrically controlled micropipette.     

A simple method of preparing chick eggs for chorio-allantoic grafts

Summary A method of preparing chick hosts for receiving grafts to the chorio-allantoic membrane (CAM) is described, which is quicker and requires less manipulative skill than that in general use.     

The composition of extracted nuclei of developing frog embryos used as template material for RNA synthesis in vitro

Résumé Les quantités de protéines basiques et résiduelles, de phosphoprotéine et ded-RNA se trouvant dans les noyaux extraits d'embryons deRana pipiens diminuent en passant du stade de blastula à celui de la larve. Cette évidence suggère que l'accumulation progressive des protéines ou dud-RNA, en masquant le DNA n'offre pas le contrôle principal de la synthèse de l'RNA in vivo. Une réduction de la quantité de l'endogène RNA polymerase fonctionelle incluse dans la protéine résiduelle explique mieux la réduction de la synthèse dud-RNA dans les stades plus avancés du développement embryonnaire.     

Okinonellins A and B,two novel furanosesterterpenes,which inhibit cell division of fertilized starfish eggs,from the marine spongeSpongionella sp

Cellular and Molecular Life Sciences - Two novel furanosesterterpenes, okinonellins A (1) and B (2), have been isolated from the spongeSpongionella sp. Both compounds inhibit cell division of...     

The chorio-allantoic membrane. A new site for development of mouse embryos

Résumé On rend compte du succès de la transplantation extra-utérine des embryons de souris âgés de 3 jours sur une membrane chorio-allantoïque d'embryon de poulet. Les observations poursuivies jusqu'à 8 jours après transplantation montrent qu'ils subsistent et grandissent sur la membrane.     

Effects of 5-bromodeoxyuridine on in vitro development of mouse embryos in the early somitic stage

Summary The inhibitory effect of 5-bromodeoxyuridine on the early somitic stages of mouse embryos was largely prevented in the presence of excess thymidine but only partially prevented by deoxycytidine.     

A method for time-lapse cinematography of primitive streak stage rat embryos in culture

Zusammenfassung Eine Kammer für die Kultur von Embryonen wird beschrieben, die es erlaubt, Zeitrafferfilme der Entwicklungsvorgänge über sehr lange Zeitspannen herzustellen.     

Hormone content of mouse pancreatic islets subjected to different in vivo and in vitro functional demands

Summary Mice treated for 4 days with tolbutamide displayed decreased serum glucose values with a concomitant decrease of their islet insulin content. Mouse islets cultured for 1 week at a low (3 mM) or a high (28 mM) glucose concentration contained less insulin than non-cultured islets and islets cultured at a medium (11 mM) glucose concentration. All groups of cultured islets contained more glucagon than non-cultured islets. The somatostatin content of high- and medium-glucose cultured islets was higher than that of freshly isolated islets.The skilled technical assistance of Astrid Nordin, Ewa Forsbeck and Eva Törnelius is gratefully acknowledged. Financial support was received from the Swedish Medical Research Council (12X-109; 12X-2297), the Nordic Insulin Fund and the Swedish Diabetes Association.     

A method for perfusing tissue culture with an in vivo system

Riassunto Il lavoro descrive una modificazione della camera di Rose che permette la perfusione delle culture tramite una membrana semipermeabile.     

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.     

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.