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F. H. BRONSON and W. K. WHITTEN

Summary.

Two methods for the assay of a pheromone produced by male mice were compared. Using these methods it was shown that the pheromone was present in urine from males of two inbred strains and from androgenized, spayed females, but not in urine from castrate males. It was present in urine collected directly from the bladder free from any accessory gland secretion.

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JUDITH A. CHOLEWA and W. K. WHITTEN

Mouse embryos developed from the eight-cell stage to expanded blastocysts in a defined medium in which glycine was the only source of fixed nitrogen (Whitten, 1957). These findings were confirmed by Bunim (1960), who also showed that glycine could be replaced by Versene and suggested that the function of the glycine was not for protein synthesis but to chelate ions of copper or zinc which might have contaminated the medium. However, Brinster (1965) found that only an occasional two-cell embryo developed in the presence of single amino acids, though he subsequently demonstrated that 80% formed blastocysts when glutathione was the only source of fixed nitrogen (Brinster, 1968). Incorporation of labelled leucine by early mouse embryos in vitro was reported by Mintz (1964) and by Monesi & Salfi (1967) but neither Greenwald & Everett (1959), nor Weitlauf & Greenwald (1967), could demonstrate uptake of methionine in vivo. It is not clear if this
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D. G. WHITTINGHAM and W. K. WHITTEN

The first mammalian embryos to be successfully frozen and thawed were those of the mouse (Whittingham, 1971a; Whittingham, Leibo & Mazur, 1972; Wilmut, 1972). Live young were obtained from mouse embryos which had been stored for up to 8 days at −196°C, after subsequent thawing and transfer to suitably prepared foster mothers (Whittingham et al., 1972). We now report the first successful transatlantic aerial shipment of two inbred strains of mice as frozen eight-cell embryos and their survival after storage for up to 8 months at −196°C.

Eight-cell mouse embryos were obtained from two inbred strains of mice (C57BL/6J×C57BL/6J and BALB/cWt × BALB/cWt) at the Jackson Laboratory, Bar Harbor, Maine, U.S.A. They were flushed from the oviducts of naturally mated females on the morning of the second day after finding the coital plug.

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W. K. WHITTEN and J. D. BIGGERS

Whitten (1956) showed that 8-cell mouse embryos develop into blastocysts in a simple chemically defined medium containing glucose, and McLaren & Biggers (1958) demonstrated that blastocysts cultured in this way produce normal mice when transferred into uteri of foster mothers. Whitten (1957) also showed that late 2-cell mouse embryos developed into blastocysts if lactate was incorporated in the medium but earlier stages did not cleave under these conditions. Thus the observation that mouse zygotes develop into normal blastocysts in the lumen of oviducts in organ cultures (Biggers, Gwatkin & Brinster, 1962) suggested that special conditions for initial development are provided by the tube. Recently, Whittingham & Biggers (1967) demonstrated that 1-cell embryos cleave to the 2-cell stage in a simple medium containing lactate and

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P. C. HOPPE and W. K. WHITTEN

A modified Krebs-Ringer bicarbonate solution containing bovine serum albumin (BSA) supports fertilization in vitro of mouse eggs by spermatozoa taken from the uterus (Whittingham, 1968; Cross & Brinster, 1970) or the cauda epididymidis (Miyamoto & Chang, 1972; Hoppe & Pitts, 1973). Human, rabbit or equine serum albumin could be substituted for BSA (Miyamoto & Chang, 1973). Though Miyamoto & Chang (1973) noted sperm penetration of low percentages of mouse eggs in vitro in the presence of a chemically defined medium with added lactate or pyruvate, no significant fertilization has been reported to occur in the absence of albumin.

Further investigations of the requirements for albumin, which are presented in this report, indicate that fertilization occurred efficiently in media supplemented with BSA in concentrations lower than those reported by others. Bovine serum albumin essentially free of fatty acids or

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V. M. CHAPMAN, CLAUDE DESJARDINS and W. K. WHITTEN

Summary.

A significant decrease in pituitary lh accompanied by an increase in blood plasma progestins was observed in pregnant females 24 hr after exposure to alien males (Day 2 post-mating). Conversely, no change in pituitary lth was detectable until 48 hr after females were caged with alien males. These data suggest the lh release may play an important rôle in pregnancy block.