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J. D. Biggers

Extensive experimental work on the developmental biology of the mammalian embryo (Burki, 1986; Hogan et al., 1986; Rossant & Pedersen, 1986) was made possible by the discovery of usable culture methods that were perfected about 20 years ago for the mouse and, to some extent, the rabbit (Biggers, 1987). This work on preimplantation embryo development has far outstripped the study of the microenvironments encountered by an embryo as it moves along the female genital tract to the uterus. This fact is perhaps surprising, since the culture media used to study development are substitutes for these environments. There are three reasons for this lack of attention to the composition of the genital tract secretions during the initial stages of pregnancy. Firstly, the available culture media are adequate for much of the developmental work to proceed. Secondly, the paucity of the secretions makes it very difficult to obtain samples for biochemical analysis. For example, the composition of the uterine secretions has received relatively little attention, although those interested in animals which implant superficially had speculated for years that the histotrophe produced in these species has a nutritional role (Amoroso, 1952). Thirdly, the oviduct has been traditionally thought of as a superfluous organ that acts only as a conduit for the embryo as it passes from the site of fertilization to the uterus. This view was particularly fostered by the results of the Estes operation, which was discussed by Hunter (1977), and later by Adams (1979) at the last Symposium held by the Society for the Study of Fertility on the milieu of the ovum. This operation, first described by Dudley in 1900 and later by Estes in 1909, was the method sometimes used until the 1960s to treat sterility due to tubal occlusion (Biggers, 1984). The ovary, attached to its pedicle, was moved into the uterine cavity where it was hoped ovulation would occur. It was argued that because a few pregnancies occurred following this operation the passage of the preimplantation embryo through the oviduct was unnecessary and that the oviduct is not involved in the nurture of the embryo. Biggers (1979) has pointed out that this clinical evidence is particularly weak to reach such a conclusion.

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J. D. Biggers

Introduction

About 100 years ago, on 27 April 1890, Walter Heape transferred rabbit embryos from one mother to another. One animal became pregnant and delivered young from the transferred embryos. Thus the first mammalian embryo transfer experiment was successfully completed. In this paper, I describe these experiments in some detail and then analyse why they were done. I place this embryo transfer work in perspective as it relates to other contributions of this pioneer in reproductive biology.

The Heape family has been traced back to the eleventh century (Heape & Heape, 1905). Walter Heape's father, Benjamin Heape, an entrepreneur, moved from England to Melbourne in the then Australian colonies as a young man and founded the very successful general merchant company of Heape and Grice. He returned with his wealth to Manchester, England, to take over the family textile business. Benjamin Heape eventually became a Director and Vice-Chairman of the

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J. A. Lawitts and J. D. Biggers

Summary. Culture media were developed for pronuclear-stage mouse embryos using simplex optimization, which has the benefit of being able to optimize several components simultaneously. Initially, several different media were generated. All media contained the same components, yet each medium was characterized by having a different component at a high concentration. The simplex procedure identified 4 components (NaCl, pyruvate, KH2PO4 and glucose) which at high concentrations were detrimental to embryo development, compared to the other components tested. For example, all embryos cultured in a medium with high NaCl blocked at the 2-cell stage. The optimization method then adjusted each medium by lowering the concentration of the component or removing it entirely, which resulted in a significant increase in development. In an experiment comparing 8 media generated from the simplex optimization, along with 7 other media, removal of KH2PO4 resulted in the largest increase in development; 88% of embryos were ≥4 cells on Day 3 after hCG, and 53% developed into blastocysts by Day 5.

Another experiment compared 4 of the best media generated from the simplex optimization. In 3 out of the 4 media, 90% or more of the embryos were ≥4 cells on Day 3. In 3 of the media, approximately 60% or more of the embryos developed into blastocysts. The simplex optimization procedure is an efficient method for developing culture media and determining requirements for development in vitro.

Keywords: Simplex; culture; mouse; embryo

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R. L. BRINSTER and J. D. BIGGERS

In 1959, Chang provided the first irrefutable proof of fertilization of mammalian ova in vitro. Since that time, fertilization of rabbit ova in vitro has been done by a number of workers. More recently, Yanagamachi & Chang (1963, 1964) have succeeded in fertilizing in vitro the eggs of the golden hamster. This was established microscopically and by the cleavage of some of the ova to the twocell stage. No one has yet succeeded in fertilizing mouse ova in vitro. This report describes a method for fertilizing mouse ova in vitro and obtaining the successive cleavage stages up to the blastocyst.

The unfertilized ova were obtained from random-bred Swiss mice which were superovulated by an intraperitoneal injection of 10 i.u. of pregnant mare serum gonadotrophin (Gestyl, Organon), followed 48 hr later by an intraperitoneal injection of 10 i.u. of human chorionic gonadotrophin (Pregnyl, Organon). Since ovulation occurs 11 to 14 hr

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R. F. S. CREED and J. D. BIGGERS

Summary.

A description is given of some of the gross anatomical characteristics of the placenta of representatives of five carnivore species of the families Procyonidae and Mustelidae. Emphasis is laid on the occurrence of well-defined haemophagocytic structures in these placentae, which have not, hitherto, been investigated.

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R. G. WALES and J. D. BIGGERS

Summary.

The level of radio-isotope in 2- and 8-cell mouse embryos was measured following a 30-min incubation in isotopically labelled malate. Although there was very little accumulation of substrate carbon and no output of radio-active carbon dioxide by 2-cell embryos, 8-cell embryos accumulated substrate carbon during the incubation and oxidized some of the malate to carbon dioxide. A change in the concentration of malate in the medium and the addition of ouabain had little effect on the accumulation of substrate in 8-cell embryos. On the other hand, its accumulation was suppressed during incubations at 5° C.

The evidence suggests that there is little or no uptake of malate by 2cell embryos, and that an active uptake of the compound occurs at a later stage of development.

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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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E. W. Overström, D. J. Benos and J. D. Biggers

Summary. The rates of incorporation of [35S]methionine into Na+/K+ ATPase, actin (β- and γ-isoforms), and total protein of the preimplantation rabbit blastocyst were determined between Days 4 and 7 of development. Blastocyst proteins were metabolically radiolabelled with [35S]methionine and subsequently analysed by co-isolation with purified Na+/K+ ATPase using two-dimensional polyacrylamide gel electrophoresis, immunoprecipitation, immunoblotting, fluorography, and liquid scintillation spectroscopy. The rate of [35S]methionine incorporation into acid-soluble total protein increased 24-fold between Days 4 and 6 post coitum (p.c.), then diminished ∼79% on Day 7. In-vitro incorporation of [35S]methionine was linear at each stage of blastocyst development. [35S]methionine incorporation rates were unaffected by low free intracellular methionine concentration (< 0·06 mm) and stage-related differences in blastocoele volume. Analysis of β- and γ-actin synthesis revealed patterns of [35S]methionine incorporation rates which were similar to those of total protein. In contrast, synthesis of blastocyst Na+/K+ ATPase was characterized by a 90-fold increase (P < 0·001) in the rate of [35S]methionine incorporation between Days 4 and 6 p.c. The results demonstrate that Na+/K+ ATPase is actively synthesized at a high and increasing rate during preimplantation development in the rabbit at a period which is characterized by rapid fluid accumulation by the blastocyst.

Keywords: rabbit; blastocyst; protein synthesis; Na+/K+ ATPase

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J. D. Biggers, J. F. Baskar and D. F. Torchiana

Summary. The intrauterine injection of 7-oxa-13-prostynoic acid, 18,18,20-trimethyl PGE-2, and meclofenamic acid in mice at the expected times of implantation significantly reduced the number of implantation sites. Indomethacin was ineffective possibly because it was exposed to high pH during the preparation of the solutions for injection. It is suggested that these prostaglandin antagonists exert their antifertility action at multiple sites involving both the embryo and mother.

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L. L. Nielsen, D. J. Benos and J. D. Biggers

Summary. Plasma aldosterone, corticosterone, and cortisol were measured during the first week of pseudopregnancy or pregnancy in New Zealand White rabbits to determine whether any sustained elevations of adrenal steroids occur. There were no pregnancy-specific alterations in circulating adrenal steroid concentrations during the preimplantation stages of embryonic development. Elevation of plasma aldosterone in vivo did not induce amiloride-sensitive Na+ transport across the embryonic trophectoderm. It therefore seems unlikely that an increase in maternal adrenal steroid concentrations is necessary for the development of amiloride-sensitive Na+ transport in rabbit blastocysts.

Sodium efflux from Day 6 post coitum (p.c.) blastocysts was lower than Na+ influx. By day 7p.c. Na+ efflux was equivalent in magnitude to the component of Na+ influx not inhibited by amiloride. This suggests that between Days 6 and 7p.c. the amiloride-sensitive component of Na+ influx becomes essential for blastocyst expansion.