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D. A. T. NEW

Summary.

The growth and differentiation of post-implantation rat embryos together with their embryonic membranes, when grown in vitro in different blood sera, has been assessed by examination of the final stage of development attained and by the weight of protein synthesized. Rat embryos of early somite stages develop well to early limb bud stages when grown in homologous serum. This medium has some advantages over plasma clots. Maximum growth of the embryos is obtained in 0·5 to 1·0 ml serum/embryo. No advantage is gained by adding more serum, or transferring the embryos to fresh serum after a period in culture. It is immaterial whether the serum is obtained from male rats, pregnant or non-pregnant females, from the same individual as the embryos or some other, or from the same variety of rats as the embryos or some other.

Heterologous sera are unsatisfactory as culture media. Rabbit serum is rapidly lethal to rat embryos, but will support some development if pre-heated at 56 to 57° C for 30 min. Some samples of fowl serum are rapidly lethal to the rat embryos unless pre-heated, but others support some development.

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D. A. T. NEW and P. T. COPPOLA

Summary.

Rat embryos explanted with their membranes at 9½, 10½, and 11½ days' gestation have been grown in homologous serum under different conditions of oxygenation. In circulating serum, maximum growth of the younger embryos was obtained in 5% CO2 in air and of the older embryos in 5% CO2 in 95% O2. In watch glass cultures, the younger embryos developed well in 5% CO2 in 60 to 95% O2, but growth of the older embryos could only be obtained in hyperbaric oxygen. The yolk sac of the older embryos continued to expand normally under oxygen conditions which were inadequate for growth of the embryos, but the heart and blood circulation functioned best under conditions which were optimal for embryonic growth. Although the proportion of O2 required in the gas phase varied greatly with the type of culture and the stage of development of the embryo, the results could be explained on the assumption that embryonic tissues at all stages require an O2 pressure similar to that provided by blood in equilibrium with air.

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D. A. T. New, P. T. Coppola and D. L. Cockroft

Previous work has shown that rat embryos can be grown in culture for periods of 1–3 days at any time during the 2nd week of gestation, i.e. throughout the period of major organogenesis. The cultured embryos undergo extensive growth and differentiation, and many of them compare well with embryos of equivalent age in vivo. But the rate of growth, as determined by assays of total protein, has usually been found to be lower than in vivo (Berry, 1968; Shepard, Tanimura & Robkin, 1970; Payne & Deuchar, 1972; Robkin, Shepard & Tanimura, 1972; New, 1973; Cockroft, 1973, 1976; Steele, 1975) and a higher proportion of the embryos show malformations (Morriss & Steele, 1974).

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D. A. T. NEW, P. T. COPPOLA and S. TERRY

Various methods have been developed in recent years for growing post-implantation rat and mouse embryos in culture. Young embryos—up to early somite stages—grow well in static medium, e.g. simple watch-glass cultures (New, 1966; Steele, 1972). Older embryos require flowing medium, as in the culture systems of New (1967), Tamarin & Jones (1968), Robkin, Shepard & Tanimura (1972) and Cockroft (1973). These methods and some of their applications have been reviewed by New (1973).

Recently, a simpler technique for maintaining embryos in flowing medium has proved useful in this laboratory (New & Brent, 1972; New & Mizell, 1972). It consists of the culture of free-floating embryos in a constantly rotated tube which contains serum and an appropriate gas phase. We give here a short description of the method and a comparison of the results with those of the `circulator' method (New, 1967).

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S. K. L. Ellington and D. A. T. New

Summary. Rat embryos were cultured with the parietal yolk sac and other membranes intact. The development of the parietal yolk sac after 24 h in vitro was assessed by (1) the increase in size, (2) the morphology of the cellular components, and (3) the development of the embryo within the yolk sac. Development in vitro most resembled that in vivo when the embryos and membranes were explanted and cultured in rat serum diluted 1:1 with Hanks' or Waymouth's medium. High levels of oxygen were also beneficial. Culture in undiluted serum or under low oxygen concentrations resulted in very poor expansion of the yolk sac, abnormal trophoblastic giant cell morphology and stunted embryonic development.

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C. LUTWAK-MANN, MARY F. HAY and D. A. T. NEW

Summary.

The action of various agents was investigated on 6-day-old rabbit blastocysts in vivo and in vitro. The blastocysts were examined by the flat-mount technique. The substances studied included antimetabolites (2-deoxyglucose, 2-deoxyglucose-6-phosphate, 2-deoxygalactose, 6-mercaptopurine and 6-mercaptopurine riboside, ethionine, isoniazid, analogues of vitamin B12), enzyme inhibitors (dl-glyceraldehyde, salicylate, bromoacetylcarnitine, p-chloromercuribenzoate, fluoride), antimitotic agents (colcemid, aminopterin), cytostatic agents (actinomycin D, cytochalasin B), metabolites (DNA, glucose-6-phosphate, 2-deoxyribose, galactose), and hormones (polyoestriol phosphate, growth hormone). The influence of anoxia was also investigated, with special reference to temperature.

Agents which, under the experimental conditions laid down in this study, exerted clearly recognizable effects in vivo and in vitro, were 2-deoxyglucose, 6-mercaptopurine riboside, colcemid, isoniazid, aminopterin and DNA. Agents that were found to act in vitro only, were 2-deoxygalactose, glyceraldehyde, salicylate, bromoacetylcarnitine, 2-deoxyglucose-6-phosphate, actinomycin D, cytochalasin B, p-chloromercuribenzoate, and anoxia. An agent inert in vitro, but conducive (with longer exposure) to embryonic death in vivo, was polyoestriol phosphate. Growth hormone too, was inactive in vitro, but produced a slight effect in vivo. No effect was demonstrable in rabbit blastocysts following treatment with ethionine, analogues of vitamin B12, fluoride, glucose-6-phosphate, 2-deoxyribose, galactose.

Blastocysts, obtained from rabbits that had been subjected to embryotoxic agents (2-deoxyglucose, colcemid), were capable of recovery and further growth in vitro when the damage incurred in vivo was of a minor to moderate degree. Blastocysts moderately damaged by maternal 6-mercaptopurine or 6-mercaptopurine riboside treatment did not recover significantly; however, blastocysts more severely affected by these two compounds in vivo deteriorated further after incubation in vitro. Pretreatment of rabbits with polyoestriol phosphate yielded blastocysts which developed subsequently in vitro particularly well.