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  • Author: JOSEPH C. DANIEL Jr x
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JOSEPH C. DANIEL Jr

A number of investigators (Fridhandler, 1961; Fridhandler, Hafez & Pincus, 1957; Pincus, 1941; Smith & Kleiber, 1950) have shown that rabbit ova require oxygen in cleavage stages but that the rate of consumption increases markedly at the blastocyst stage (Fridhandler, 1961). Mills & Brinster (1967) show a similar increase beginning at the 8-cell stage of the mouse. Presumably these observations are related to changes being made in respiratory pathways at this time and reflect increasing metabolic activity.

Embryo culturists typically use high concentrations of oxygen: New & Stein (1963) reported improved results when mouse embryos were grown in 60% oxygen, Pavlok (1967) used 95% for mouse ova growing in explanted oviducts and Huff & Eik-Nes (1966) used 95% oxygen to grow 6-day rabbit blastocysts. Conversely, Glenister (1967) feels that high concentrations of oxygen are detrimental to embryonic

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ROBERT W. McGAUGHEY and JOSEPH C. DANIEL Jr.

Summary.

The effect of oestradiol-17β on fertilized rabbit eggs in vitro was studied following its inclusion in the culture medium.

When oestradiol is present at a concentration of 25 μg/ml and several hours remain between the time of exposure and the expected time of first cleavage, single-celled eggs typically fail to cleave and amphimixis appears to be inhibited. If the concentration of oestradiol is reduced to 10 μg/ml, or if the first cleavage is imminent at the time of exposure, the eggs tend to fragment instead of undergoing normal cleavage, presumably an effect on the cell membrane. Most eggs are apparently unaffected by an oestradiol concentration of 5 μg/ml. A minimal exposure time of about 30 min is necessary to achieve an oestradiol response. Two- and four-celled stages are much more resistant to the oestrogen than are single-celled ones.

The degree of fragmentation is reduced when progesterone is added equimolarly with oestradiol, suggesting that some antagonistic inter-action occurs.

Under incubator conditions the concentration of oestradiol remains constant throughout the time of these experiments but that of oestrone appears to decrease.

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JOSEPH C. DANIEL Jr. and JAMES D. LEVY

Summary.

The manner whereby progesterone acts as an inhibitor of cleavage of mammalian ova was studied using rabbit ova in vitro.

Progesterone blocked cleavage when it was present in the culture medium at concentrations of 10 μg/ml or more, and exerted this same effect on all stages up to the morula. The growth of blastocysts was not affected. The action was reversible: ova resumed cleavage within 2 to 3 hr after removal to progesterone-free media. The reversal was timespecific and was used to phase the subsequent cleavage of ova inhibited for varying periods of time. The inhibition was overcome by increasing the concentrations of amino acids or of the serum component in the medium. Supplementing the progesterone media with oestradiol did not reverse the inhibition.

Autoradiographs of sections of ova that were kept in 14C-labelled progesterone for 8 hr, showed silver grains concentrated on the surface of the ovum and the zona pellucida.

It is concluded that progesterone blocks cleavage by limiting the supply of protein or amino acids and thus inhibiting protein synthesis within the ovum. Presumably this is done by the progesterone aggregating on the surface of the cell or its protective coating.

The inhibitory action appears to be timed in relation to some event that precedes mitosis by an hour or two. It is therefore suggested that the protein synthesis involved is related to formation of the mitotic apparatus or to chromosome condensation.

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JOSEPH C. DANIEL Jr and JOHN D. OLSON

Summary.

The amino acids essential for cleavage of the rabbit ovum were determined by appraisal of the growth of eggs in vitro in defined medium deficient in specific amino acids. The first cleavage will occur in amino acid-free medium but the second requires cysteine, tryptophane, phenylalanine, lysine, arginine, and valine. Subsequent cleavage to the morula stage requires the addition of methionine, threonine and glutamine.

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BELA J. GULYAS, JOSEPH C. DANIEL Jr and R. S. KRISHNAN

Summary.

The effects of the rabbit uterine fluid component, blastokinin (BKN), and rabbit serum on nucleic acid synthesis in rabbit and mink blastocysts were examined. [3H] Thymidine incorporation was not enhanced by BKN in rabbit embryos explanted on the 3rd to 5th days post coitum, but thymidine uptake was somewhat greater in 4- and 5-day blastocysts in serum-containing medium than in the controls in defined medium. [3H] Uridine incorporation was inhibited slightly by serum and significantly stimulated by BKN in 5-day rabbit blastocysts, but not in younger ones. Rabbit serum inhibited uridine uptake in mink blastocysts, while BKN had no effect.