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D. G. WHITTINGHAM

Summary.

Intra- and inter-specific transfers of rat and mouse ova showed that native and foreign ova developed to the blastocyst stage within explanted oviducts of the rat and mouse.

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D. G. WHITTINGHAM

Physiological Laboratory, Downing Street, Cambridge CB2 3EG

(Received 13th December 1974)

Survival of mammalian embryos after storage at sub-zero temperatures was first achieved in the mouse in which all the preimplantation stages were shown to survive storage at −196°C when suitably cooled and thawed (Whittingham, Leibo & Mazur, 1972; Wilmut, 1972; Whittingham, 1974a). Similar procedures have demonstrated that blastocysts of cattle (Wilmut & Rowson, 1973), morulae and blastocysts of sheep (Willadsen, Polge, Rowson & Moor, 1974) and four-cell, eight-cell and morulae of rabbits (Bank & Maurer, 1973; Whittingham & Adams, 1974) will survive storage at − 196°C. This communication reports the survival of two-cell, four-cell and eight-cell rat embryos after storage at − 196°C for periods up to 3 months.

Superovulation of immature female rats, 26 to 28 days of age (CFHB random-bred strain, Carworth-Europe), with intraperitoneal injections of 25 i.u. PMSG and 10 i.u. HCG given 44 to 48

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D. G. WHITTINGHAM

The successful preservation of mouse embryos at sub-zero temperatures has been accomplished with suitably slow rates of cooling and thawing and in the presence of the cryoprotective agent, dimethylsulphoxide (DMSO). In the first report, full-term fetuses and live young were obtained from one-cell, two-cell and eight-cell embryos stored at −196°C for up to 8 days (Whittingham, Leibo & Mazur, 1972) and more recently eight-cell embryos were stored for 8 months at −196°C without any deterioration in viability (Whittingham & Whitten, 1974). The viability of frozen-thawed mouse blastocysts has only been demonstrated by their subsequent expansion during 24 hr in culture. When DMSO was added to the blastocysts at 0°C before freezing and diluted out at 0°C on thawing, the percentage surviving (18%) was low (Whittingham et al., 1972) but higher survival rates (up to 86%) were obtained when DMSO was added and diluted in several increments at 20°C (Wilmut, 1972).

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D. G. Whittingham

Summary. Unfertilized mouse oocytes were frozen and stored at –196°C. Immediately after thawing 331 out of 463 MF1 oocytes (71·5%) and 271 out of 410 F1 (C57BL × CBA) hybrid oocytes (66·1%) were morphologically normal. No significant difference was found between the survival of oocytes frozen and thawed with (70%) or without (66%) the surrounding cumulus cells. Fertilization in vitro of frozen–thawed oocytes was significantly lower than that of freshly collected control oocytes. The overall fertilization rate in vitro for MF1 oocytes was lower than with F1 (C57BL x CBA) hybrid oocytes. The presence or absence of cumulus cells during fertilization in vitro did not affect the fertilization rate. Live 14-day fetuses were obtained after transfer of frozen–thawed unfertilized oocytes directly to the oviducts of females mated with fertile males. However, much higher rates of survival (up to 45%) to 14-day fetuses and liveborn were found after the fertilization of frozen–thawed oocytes in vitro and subsequent transfer at the 2-cell or blastocyst stage to pseudopregnant recipients.

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Y. Tsunoda and D. G. Whittingham

Summary. The effects of purified antibody (IgG) to mechanically isolated or detergent-solubilized zonae pellucidae on the development and implantation of mouse embryos were studied in vitro by culture and in utero by transfer of treated embryos. The proportion of 8-celled embryos treated with zona antibody that developed into blastocysts and attached to the culture dish in vitro were not significantly different from those obtained after treatment of embryos with IgG from normal rabbit serum and antiserum raised against mouse liver and kidney. Pregnancy rate, mean number of implantations and live fetuses, and fetal sex ratio after transfer of zona antibody treated embryos were not significantly different from the values obtained after transfer of embryos treated with control serum IgG. It was concluded that treatment with zona antibody did not inhibit the development and implantation of mouse embryos in vitro or in vivo.

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D. G. Whittingham and E. Anderson

Summary. Survival of frozen 8-cell mouse embryos transferred directly upon thawing to the uteri of Day 3 pseudopregnant foster mothers was significantly lower (26%) than the survival of unfrozen 8-cell embryos transferred immediately after collection (73%). When frozen–thawed 8-cell embryos were cultured for 20–24 hr before transfer survival was similar to that of unfrozen 8-cell embryos transferred after 20–24 hr in culture (65% and 73%, respectively). Ultrastructural examination of the frozen–thawed 8-cell embryos revealed no obvious damage to protoplasmic components.

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D. G. WHITTINGHAM and B. D. BAVISTER

Although many workers have fertilized hamster eggs in vitro since the technique was first described by Yanagimachi & Chang (1963), there is only one reported attempt to obtain further development of these eggs in culture (Yanagimachi & Chang, 1964). In this instance, development ceased at the two-cell stage. The present study examines the development in culture of hamster eggs fertilized in vitro and after transfer to recipient foster mothers.

Immature female hamsters (5 to 6 weeks old and 60 to 80 g in weight) were induced to superovulate with intraperitoneal injections of 25 i.u. PMSG and HCG given 48 to 56 hr apart. Following removal of the oviducts 15 to 17 hr after the injection of HCG, they were blotted on sterile filter paper to remove excess blood and immersed in liquid paraffin contained in a Petri dish (35 mm in diameter, Falcon Plastics). The eggs and surrounding cumulus cells

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J. J. G. Brown and D. G. Whittingham

Summary. Preliminary observations showed that one-cell embryos from random-bred MF1 mice avoid cleavage arrest at the two-cell stage ('in vitro two-cell block') when cultured in modified M16 culture medium containing lactate and pyruvate but lacking glucose. The roles of lactate, pyruvate and glucose during preimplantation development of embryos from random-bred mice in vitro were therefore examined. When all three substrates were present continuously during culture, one-cell embryos arrested at the two- to four-cell stages. Improved development to the morula stage after 96 h in culture was obtained in media containing pyruvate alone, lactate and pyruvate, pyruvate and glucose, lactate pyruvate and glucose for the first 24 h, and medium containing lactate and pyruvate for the remaining 72 h. In a second experiment, embryos were cultured in medium containing pyruvate alone, lactate and pyruvate or pyruvate and glucose for the first 24 h, and lactate plus pyruvate medium for the second 24 h. Subsequent transfer to medium containing lactate, pyruvate and glucose supported the morula to blastocyst transition. These results show that developmental arrest in vitro can be overcome by changing the combination of energy substrates at different stages of preimplantation development.

Keywords: preimplantation; embryo; blastocyst; two-cell block; culture; mouse

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R. G. WALES and D. G. WHITTINGHAM

Summary.

The accumulation and utilization of substrate carbon by two-cell mouse embryos incubated in media containing combinations of lactate and pyruvate were studied. The results were consistent with utilization of the two substrates by common metabolic pathways. The intracellular accumulation of substrate carbon was greater when pyruvate as well as lactate was present in the medium. The evidence also suggests that pyruvate is more effective in maintaining the activity of the tricarboxylic acid cycle than lactate alone.

Fractionation of substrate carbon accumulated in embryos during a 30-min incubation period indicated that approximately 10% of this carbon had entered the acid-insoluble (protein) fraction. This accumulation was increased substantially when a combination of lactate and pyruvate, rather than either substrate alone, was present in the incubation medium.

The major organic acids found to accumulate intracellularly from substrate were malate and citrate. The intracellular accumulation of lactate was confined to embryos incubated in medium containing this substrate. Base-containing compounds accounted for a substantial portion of the substrate carbon in embryos. Glutamic acid was the major amino acid identified in this component. Alanine accumulated from pyruvate or its combination with lactate but not from lactate alone. Small amounts of aspartate accumulated from both substrates.

The effect of the interaction of lactate and pyruvate on the development of two-cell mouse embryos to the blastocyst stage was confirmed. Although pyruvate was effective over a limited range of concentration, the effective range of concentration of lactate was not as limited. The present studies suggest that the beneficial effect of combinations of lactate and pyruvate could be due to a stimulation of the metabolism of the embryos and a more favourable level of macromolecule synthesis.

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S. J. Fuller and D. G. Whittingham

Attempts to freeze mouse spermatozoa in liquid nitrogen (−196°C) have met with limited success. In an attempt to identify the factor(s) that damage mouse spermatozoa during cryopreservation, the effect of slow cooling to 4°C was examined. Epididymal spermatozoa were collected into a variety of media at 37°C, cooled slowly to 4°C over 4 h and warmed in a water bath at 37°C for 5 min. Survival of spermatozoa was assessed by motility, membrane integrity and acrosome status. Labelling with chlortetracycline showed that > 80% of spermatozoa were capacitated and had intact acrosomes immediately after warming compared with < 20% of freshly collected (control) spermatozoa. The rate of fertilization in vitro was similar using spermatozoa cooled in Dulbecco's phosphate-buffered saline and then mixed with oocytes immediately after warming and with control spermatozoa incubated for 2 h before mixing with oocytes (85%). Fewer oocytes were fertilized with spermatozoa cooled in either a modified HEPES-buffered Tyrode's medium or a simple HEPES-buffered medium with a high osmolarity (D3), 63% and 58%, respectively. Two-cell embryos were transferred to the oviducts of pseudopregnant recipients. Implantation was similar in all groups 81–88% and 54–74% of embryos formed normal late stage fetuses.