170

Department of Biochemistry and Biotechnology, University of Kuopio, Kuopio, Finland.

IN VITRO CULTURE OF MAMMALIAN PREIMPLANTATION EMBRYOS

T.T. Peura

CONTENTS Embryos of many domestic and laboratory animal species can now be routinely cultured over so called block-stages and to viable offspring. The most common method used in this is a co-culture with somatic cells. Even though helpful in practice, true requirements for early embryo development are difficult to elucidate in a complex co-culture system. During recent years culture of embryos exhibiting a developmental block has also been possible in simple media, giving rise to the assumption that the earlier need for co-culture might have reflected the use of othewise inappropriate and detrimental culture media. Factors such as the presence of glucose and phosphate in the culture medium, gas atmosphere, growth factors and effect of free oxygen radicals are discussed in this paper.

INTRODUCTION Despite the advances made in mammalian preimplantation embryo culture during recent

years the factors controlling early embryo development are still poorly understood. Embryos of several species (bovine, pig, sheep, hamster, mouse) can be readily cultured to morulaes and blastocysts in vitro, but the quality of these embryos is not necesserily as good as that of their in vivo counterparts. The next logical step after reaching this level of progression is to analyse early embryogenesis in more detail using well-defined, controlable culture conditions in order to find out true needs of early embryos, thus making it possible to formulate a new media effectively fulfilling the reguirements.

COCULTURE Major breakthroughs in bovine, pig and sheep embryo cultures were achieved with co-

culture. Most widely used cells in co-culture are oviductal epithelial cells and granulosa cells, although good results have been achieved even with continuous cell lines as BRL-cells (Buffalo Rat Liver-cells)(van lnzen et at. 1992). Embryo development is usually enhanced in many ways; by overcoming developmental blocks, by increasing cell numbers and by improving embryo viability after transfer. The nature of the beneficial effect of co-culture is not known. Especially after discovering that oviductal cell-conditioned medium could support development also, so called "positive conditioning" of medium by embryotrophic factors secreted by these cells were thought to be responsible. Many attempts have been made in order to characterize these factors (for example see Mermillod et al. 1993). After finding out that somatic cells work equally well it has also been postulated whether so called "negative conditioning", removal of detrimental components from the culture environment, could be responsible. However, even though co-culture methods are still perhaps most widely used in the culture of domestic animal embryos, some critical comments about them have been made (Bavister 1992). It seems clear that although co-culture is useful for many practical purposes it can not give us unambiguous answers about the true needs of early embryos.

EFFECTS OF GLUCOSE AND PHOSPHATE Since the work by Schini and Bavister (1988) demonstrating the link between glucose,

phosphate and developmental blocks, several studies with different species have been performed to further elucidate their effects. Ellington et al. (1990) showed that the omission of glucose during the first two days of culture increased bovine blastocyst formation. However, bovine embryos in this experiment were cultured in a complex environment with bovine oviduct epithelial cells or in conditioned medium. Pinyopummintr and Bavister (1991) showed that the presence of either glucose or phosphate alone had no effect on the development of bovine embryos in simple medium, whereas glucose together with phosphate inhibited development. On the other hand Petters et al. (1990) and Misener et al. (1991) did not see inhibitory effect of glucose in pig embryo culture even in the presence of phosphate.

171

SEMI-DEFINED AND DEFINED MEDIA Embryos of several domestic species have been succesfully cultured in relatively simple

culture media with or without protein. Syntethic oviduct fluid with human serum has been used for sheep embryo culture (Walker et al. 1992), hamster embryo culture medium (HECM) without any protein supplementation for bovine embryo culture (Pinyopummintr and Bavister 1991) and CZB- medium (Misener et al. 1991) and modified Krebs'-Ringer-bicarbonate medium with BSA (Petters et al. 1990) for pig embryo cultures, just to mention a few. These studies have shown that even a simple medium can support embryonic development.

The preimplantation period of mammalian development can be roughly divided into two distinguished stages; before and after compaction. The first stage is characterized by not yet fully active embryonic genome and division of undifferentiated cells; the latter stage by cell differentiation, rapid embryonic growth and marked changes in the energy metabolism of embryos (Rieger 1992). During these stages the mammalian preimplantation embryo is exposed to a continually changing environment as it passes through the female reproductive tract. It might be plausible to assume that these differencies might also create different demands for in vitro culture conditions, and our attempts to optimize embryo culture by formulating just one medium to cater for all the requirements of different stages of development might not be satisfactory to embryos. A simple medium for the first few days of culture followed by more complex conditions for the rest of the culture period might indeed give better results.

GAS ATMOSPHERE Several studies with different species have been conducted in order to find out whether

atmosphere with lower oxygen levels (usually 59'0) or high COP-level (usually 10%) are beneficial to embryonic growth. As a summary, in many of the studies these changes have been indeed beneficial (hamster: Carney and Bavister 1987). whereas in as many studies no beneficial effects were found (bovine: van der Westerlaken et al. 1992). The reasons for these discrepancies may be attributable to overall differencies in experimental designs. The presence of co-culture cells seems to be one important factor; when co-culture was used, 20% oxygen gave better development of bovine embryos than 5%, whereas same conditions in the absence of co-culture cells gave exactly the opposite results (Nagao et al. 1993).

Control and maintenance of intracellular pH is an area which has not been thoroughly studied in early embryo in vitro development, and it is possible that the effects of reduced 02- and elevated C02-levels may mediate their beneficial effect by alterating it (Carney and Bavister 1987). Alternatively, reduced formation of free oxygen radicals might explain some of the beneficial results achieved with lower oxygen tension.

GROWTH FACTORS It has become increasingly evident during the past few years that besides the oviduct and

uterus, the embryos themselves also produce growth factors that can stimulate their cellular proliferation and differentiation. These factors act by binding to specific receptors of embryonic cells, although the precise mechanism they act is not known. Some of the growth factors, whose effects on in vitro embryo culture have been studied, include insulin and the insulin-like growth factors (Heyner at al. 1993), Transforming Growth Factor- alpha and -I3 (TGF- , TGF-B), Platelet Derived Growth Factor (PDGF) and Epidermal Growth Factor (EGF). Both TGF- alpha and TGF-13 have been shown to affect the rate of embryo development, especially increasing the number of inner cells (Maquant-Le Guienne et al. 1989) and enhancing blastocyst cavity expansion (Dardik and Schulz 1991). PDGF promoted the completion of 4th cell cycle in bovine, but reduced embryo blastulation (Larson et al. 1992). EGF promoted oocyte maturation and first cleavage of bovine oocytes in simple medium (Park and Lin, 1993). Several growth factors clearly have an important role in early embryogenesis, but the complexity of their actions and interactions with other compounds makes this area difficult to study. However, this is also an area from which new interesting discoveries are likely to be found in the near future.

OXYGEN RADICALS There is evidence that free radical-induced cellular dysfunction might be partly responsible

for developmental blocks and decreased in vitro development of at least mouse embryos (Goto et al. 1992). Reactive oxygen species can cause cell damage by promoting hydroxy radical formation, a reaction catalysed by transitional metals such as iron. One consequence of free hydroxy radical

172

formation is the peroxidative damage of polyunsaturated membrane lipids, which can be further promoted by free ferrous iron directly (Minotti and Aust 1989). Iron chelators such as ethylenediaminetetraacetic acid (EDTA) and transfemn have been shown to help overcome the block stage in mice (Nasr-Esfahani et al. 1990). The effects of antioxidant enzymes such as superoxide dismutase (SOD) have been shown to have both a positive effect (Chun et al. 1992) or no effect at all (Payne et al. 1992) on overcoming the block in mouse embryos. Sometimes observed beneficial effect of low oxygen tension in culture of domestic animal embryos might be due to lowered levels of free radicals and this effect might perhaps be achieved also by addition Of free radical scavengers or antioxidant enzymes.

REFERENCES Bavister, B.D., 1992: Co-culture for embryo development: is it really necessary? Human

Reproduction 1, 1339-1341. Camey, E.W. and B.D. Bavister, 1987: Regulation of Hamster Embryo Development in Vitro by

Carbon Dioxide. Biol. Reprod. 36, 1155-1 163. Chun, Y.S., J.H. kim, H.T. Lee and K.S. Chung, 1992: Role of superoxide dismutase (SOD) on the

development of preimplantation mouse embryos. Congress Proceedings of 12th International Congress on Animal Reproduction, 23-27 August 1992, The Hague, Holland,

Dardik, A. and R.M. Schulz, 1991: Blastocoel expansion in the preimplantation mouse embryo: stimulatory effect of TGF-a and EGF. Development 113, 919-930.

Ellington, J.E., E.W. Camey, P.B. Farrell, M.E. Simkin and R.H. Foote, 1990: Bovine 1-2-Cell Embryo Development Using a Simple Medium in Three Oviduct Epithelial Cell Coculture Systems. Biol. Reprod. 43, 97-104.

Goto, Y., Y. Noda, K. Narimoto, Y. umaoka and T. Mori, 1992: Oxidative stress on mouse embryo development in vitro. Free Radical Biology 8, Medicine, 13, 47-53.

Heyner, S., N. Shah, R.M. Smith, A.J. Watson and G.A. Schulz, 1993: The role of growth factors in embryo production. Theriogenology 39, 151-161.

Larson, R.C., G.G. lgnotz and W.B. Cunie, 1992: Platelet Derived Growth Factor (PDGF) Stimulates Development of Bovine Embryos During the 4th Cell Cycle. Development, 115,

Marquant-Le Guienne, B., M. G-rard, A. Solari and C. Thibault, 1989: In vitro culture of bovine egg fertilized either in vivo or in vitro. Reprod. Nutr. Dev. 29, 559-568.

Mermillod, P., A. Vansteenbrugge, C. Wils, A. Massip & F. Dessy, 1993: Study of the bovine embryotrophic activity of serum free oviduct conditioned medium. Theriogenology s, 267.

Minotti, G. and S.D. Aust, 1989: The role of iron in oxygen radical mediated lipid peroxidation. Chem-Biol. Interactions, a, 1-19.

Misener, M., J.W. Pollard and K. Metzger, 1991: In vitro culture of porcine embryos in CZB medium. Theriogenology 35, 244.

Nagao, Y., K. Saeki, M. Hoshi and H. Kainuma, 1993: Effect of oxygen concentration on the development of in vitro matured and fertilized bovine oocytes cultured in a protein-free medium. Theriogenology 3 ,273.

VO1.3, 1298-1300.

821-826.

Nasr-Esfahani, M., M.H. Johnson and R.J. Aitken, 1990: Human Reproduction, 5, 997-1003. Payne, S.R., R. Munday and J.G. Thompson, 1992: Addition of Superoxide Dismutase and

Catalase does not necesserily overcome Developmental Retardation of One-cell Mouse Embryos during in-vitro Culture. Reprod. Fertil. Dev., 4, 167-174.

Petters, R.M., B.H. Johnson, M.L. Reed and A.E. Archibong, 1990: Glucose, glutamine and inorganic phosphate in early development of the pig embryo in vitro. J. Reprod. Fertil. 89,

Pinyopummintr, T. and B.D. Bavister, 1991 : In Vitro-Maturedlln Vitro-Fertilized Bovine Oocytes Can Develop into MorulaelBlastocysts in Chemically Defined, Protein-Free Culture Media Biol. Reprod. 45, 736-742.

Rieger, D., 1992: Relationships between energy metabolism and development of early ammalian embryos. Theriogenology =,75-93.

Schini, S.A. and B.D. Bavister, 1988: Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose. Biol. Reprod. 3, 1183-1192.

van Inzen. W.G., Y.A. Wurth, C. Verstrate-Voolstra, Th.A.M. Kruip & S.M. Weima, 1992: Culture of bovine embryos to the blastocyst stage in the presence of embryotrophic factors secreted

269-275.

I73

by Buffalo Rat Liver (BRL) cells. Congress Proceedings of 12th International Congress on Animal Reproduction, 23-27 August 1992, The Hague, Holland, Vo1.3, 1360-1362.

van der Westerlaken, L.A.J., J.J. van der Vlugt, A.A.C. de Wit and A. van der Schans, 1992: The effect of oxygen tension on in vitro fertilization and embryonic development. Theriogenology 37,312.

Walker, S.K., T.M. Heard and R.F. Seamark, 1992: In vitro culture of sheep embryos without co- culture: successes and perspectives. Theriogenology 37, 11 1-126.

Address of the author: Department of Biochemistry and Biotechnology, University of Kuopio, P.0.Box 1627, SF-7021 1 Kuopio, Finland.