Development of Viable Mammalian Embryos In Vitro

CHAPTER 9

DEVELOPMENT OF VIABLE MAMMALIAN EMBRYOS IN VITRO: EVOLUTION OF SEQUENTIAL MEDIA David K. Gardner and Michelle Lane

INTRODUCTION The field of modern embryo culture spans several decades (Biggers et al., 1965; Whitten and Biggers, 1968; Whittingham, 1971; Biggers, 1987), although it was in the final decade of the twentieth century that a resurgence of interest in embryo physiology and metabolism led to significant improvements in this field. Two discrete approaches led to the formulations of different and yet relatively effective types of culture media. The first approach relied heavily on lessons learned from both embryo and maternal physiology and led to the formulation of sequential media. The second approach used a computer model to generate culture media formulations using a process of simplex optimization. The former approach was used extensively in our laboratory, whereas the laboratory of John Biggers (Lawitts and Biggers, 1993) championed the latter. The history of simplex optimization (SOM) began initially with the formulation of SOM, then led to KSOM and finally to KSOMaa; this has been reviewed elsewhere (Lawitts and Biggers, 1993; Biggers et al., 2000). The term “viable embryo” is used deliberately in the title of this Chapter and throughout, because, as we shall demonstrate, although mammalian embryos can be cultured throughout the preimplantation period in a number of different conditions, the resultant viability of the blastocysts can be extremely different. Viability is here defined as the ability of the blastocyst to implant and develop successfully into a fetus. The emphasis of this treatise is on sequential media; data are presented to support the move to more than one medium formulation in order to culture the mammalian preimplantation embryo throughout the preimplantation period to the viable blastocyst stage.

DYNAMICS OF EMBRYO AND MATERNAL PHYSIOLOGY During the preimplantation period of mammalian embryo development, the conceptus undergoes significant changes in its physiology, metabolism, and genetic control. These changes are so dramatic that the starting point of development, the zygote, and the final stage, the blastocyst, can be likened to two very different Principles of Cloning

Copyright 2002, Elsevier Science (USA). All rights reserved.

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Figure 1 Effect of amino acids on cleavage rates from the four-cell to the eight-cell stage (from Lane and Gardner, 1997b, J. Assist. Reprod. Genet., with permission). There were at least 30 embryos per treatment group. Notches represent the interquartile range, therefore including 50% of the data; whiskers represent 5 and 95% quartiles; line across the box represents the mean. NEGLN-mMTF supplemented with nonessential amino acids and glutamine; ESS–mMTF supplemented with essential amino acids without glutamine; 20AA–mMTF supplemented with all 20 amino acids. *, Significantly different from mMTF (P < 0.05); **, significantly different from mMTF (P < 0.01). a, Like pairs significantly different (P < 0.01); b, like pairs significantly different (P < 0.05). Culture with nonessential amino acids stimulated cleavage rates to the eight-cell stage. Addition of essential amino acids to the nonessential amino acids and glutamine group negated this benefit, plausibly due to competition for specific transporters.

somatic cell types. The zygote, like the oocyte from which it was derived, has a low metabolic activity, exhibiting low levels of oxygen consumption and low QO2, therefore being likened to relatively quiescent adult tissue such as brain (Leese, 1991). Rather than using glucose as its primary energy source, the zygote and cleavage stages utilize the carboxylic acids pyruvate and lactate oxidatively (Biggers et al., 1967), with relatively low levels of glycolysis. At this stage the embryo is under the control of the maternal genome, the embryonic genome being sequentially activated from the two- to the eight-cell stage, depending on the species. Significantly, prior to compaction and the generation of the first transporting epithelium, the individual cells of the embryos are only loosely associated and readily disaggregate if the zona is removed. At this time the individual cells exhibit a physiology typical of unicellular organisms and are consequently more susceptible to their environment (Gardner, 1998a,b; Lane, 2001). Prior to compaction, the embryo in culture benefits from the presence of specific amino acids—for example, alanine, aspartate, asparagine, glycine, glutamate, glutamine, proline, and serine (Gardner, 1994; Gardner and Lane, 1993a; Lane and Gardner, 1997a,b; Steeves and Gardner, 1999). These amino acids have been shown to stimulate cleavage rates and compaction in the mouse (Figs. 1 and 2) and cow, and are known to fill several niches in the embryo’s physiology, serving as osmolytes and buffers of pHi (Table 1). A major change in the embryo occurs at the time of compaction due to the formation of an epithelium, with the cells of the embryo beginning to take on a more somatic-cell physiology. With the establishment of an epithelium the embryo is no longer as dependent on specific amino acids to serve as osmolytes (Hammer et al., 2000; Lane, 2001) and buffers of pHi (Edwards et al., 1998a), but rather can better regulate its internal environment (Figs. 3 and 4). With the establishment of basolaterally positioned ATPases (Benos and Biggers, 1981) the embryo actively creates

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Figure 2 Effect of amino acids in the medium on the compaction of eight-cell embryos after 78 hours post-hCG (57 hours of culture) (from Lane and Gardner, 1997b, J. Assist. Reprod. Genet., with permission). Values are mean; n = at least 30 embryos per treatment. NEGLN— mMTF were supplemented with nonessential amino acids and glutamine; ESS–mMTF were supplemented with essential amino acids without glutamine; 20AA–mMTF were supplemented with all 20 amino acids.**, Significantly different from mMTF and ESS (P < 0.01); the letter “a” denotes like pairs that were significantly different (P < 0.05).

a blastocoel, the composition of which is regulated by the epithelium. Significantly, the blastocyst stage has a high oxidative capacity and QO2 (similar to that of active skeletal muscle) and readily uses glucose as its primary energy source, though is able to adapt and use alternative nutrients should the need arise (Gardner and Leese, 1988); i.e., the embryo adapts to the environment in which it is placed. This trait is referred to as embryo plasticity. The blastocyst of all mammalian species studied exhibits a capacity for aerobic glycolysis, defined as the conversion of glucose to lactate even in the presence of sufficient oxygen for its complete oxidation (Wales, 1969; Wales et al., 1987; Gardner and Leese, 1990), although the levels of aerobic glycolysis do vary between species (Hardy et al., 1989; Gardner and Leese, 1990; Rieger et al., 1992; Gardner et al., 1993) and the medium used for metabolic analysis (Lane and Gardner, 1998). At this point the embryo has two distinct cell types, the inner cell mass and trophectoderm, which exhibit different carbohydrate (Hewitson and Leese, 1993) and amino acid (Lane and Gardner, 1997a) requirements. Table 2 highlights the differences between pre- and postcompaction embryos.

Table 1 Functions of Amino Acids during Preimplantation Mammalian Embryo Development Function Biosynthetic precursors Sources of energy Regulators of energy metabolism Osmolytes Buffers of pHi Antioxidants Chelators

Reference Crosby et al., 1988 Rieger et al., 1992 Gardner and Lane, 1993b Van Winkle et al., 1990 Edwards et al., 1998a Liu and Foote, 1995 Lindenbaum, 1973

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Figure 3 Effect of compaction on glycine uptake by mouse embryos when exposed to increasing salt concentrations in the medium. Glycine uptake by mouse embryos at the eight-cell (solid bars) and compacted eight-cell stages (open bars). **, Significantly different from eight-cell embryos (P < 0.01). As the concentration of sodium chloride increases up to 10 mM there is an increase in the uptake of the amino acid glycine in order to counter the effect of high salt concentrations. Data from Lane (2001).

As well as changes in embryo physiology and metabolism, the environment in the female reproductive tract is also under a state of flux, with differences occurring within the same region of the tract due to endocrine changes (Nichol et al., 1992; Gardner et al., 1996), and differences between regions of the tract, specifically the oviduct and uterus (Fischer and Bavister, 1993; Gardner et al., 1996) (Table 3). The mammalian embryo is therefore exposed to gradients of nutrients (both carbohydrates and amino acids), oxygen, and pH as it develops in vivo.

Table 2 Differences in Embryo Physiology Pre- and Postcompaction Precompaction Low biosynthetic activity Low QO2 Pyruvate preferred nutrient Nonessential amino acids Maternal genome Individual cells One cell type

Postcompaction High biosynthetic activity High QO2 Glucose preferred nutrient Nonessential + essential amino acids Embryonic genome Transporting epithelium Two distinct cell types (ICM and trophectoderm)

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Figure 4 Effect of compaction on intracellular pH (pHi) of mouse embryos incubated at increasing DMO (a nonmetabolizable acid) concentrations, showing the pHi of eight-cell embryos (•) and the pHi of compacted embryos (). Data were taken from different experiments from Edwards et al. (1998a) and therefore no statistics are present. However, it is evident that the decrease in pHi was greater in eight-cell embryos than in those that had undergone compaction. See Edwards et al. (1998a) for further experiments that demonstrate that experimental decompaction results in a decrease in the embryo’s ability to regulate pHi.

Table 3 Differences in Oviduct and Uterine Environments Component Glucose concentration Pyruvate concentration Lactate concentration Oxygen concentration Carbon dioxide concentration pH Glycine concentration Alanine concentration Serine concentration a

Gardner et al. (1996). Fischer and Bavister (1993). c Maas et al. (1977). d Garris (1984). e Dale et al. (1998). f Iritani et al. (1971). g Miller and Schultz (1987). b

Oviduct a

0.5 mM 0.32 mM a 10.5 mM a 8% b 12% c 7.5c 2.77 mM f 0.5 mM f 0.32 mM f

Uterus 3.15 mM a 0.10 mM a 5.2 mM a 1.5% b 10% d 7.1e 19.33 mM g 1.24 mM g 0.80 mM g

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METABOLISM OF THE EMBRYO There have been numerous treatises on the metabolism of the mammalian embryo (Biggers and Stern, 1973; Brinster, 1973; Rieger, 1984, 1992; Biggers et al., 1989; Leese, 1991, 1995; Barnett and Bavister, 1996; Gardner, 1998a,b, 1999; Gardner, et al., 2000a). However, historically the majority of experiments on embryo metabolism have been performed in simple culture media (Whittingham, 1971; Brinster, 1973), which lack amino acids. It has subsequently been demonstrated that amino acids are both metabolized and regulate the utilization of carbohydrates (Gardner and Lane, 1993b). Therefore data from early studies using simple culture conditions need to be interpreted with this in mind. In the following section the current understanding of the utilization of carbohydrates and amino acids by the mammalian preimplantation embryo is reviewed.

GLUCOSE There is a misconception that glucose is toxic to the mammalian embryo in culture. This stems from work on the hamster, which showed that in the presence of phosphate, 5.5 mM glucose induced developmental arrest in culture (Schini and Bavister, 1988; Seshagiri and Bavister, 1989). However, such developmental arrest can be attributed to phosphate alone (Seshagiri and Bavister, 1989; Biggers and McGinnis, 2001). Significantly, glucose is present in both oviduct and uterine fluids (Gardner et al., 1996), and the oocyte and embryo have a specific carrier for this hexose (Gardner and Leese, 1988; Hogan et al., 1991; Aghayan et al., 1992; DanGoor et al., 1997). Furthermore, glucose is used by the embryo throughout the preimplantation period for the synthesis of triacylglycerols and phospholipids and to provide precursors for complex sugars of mucopolysaccharides and glycoproteins. Glucose metabolized by the pentose phosphate pathway (PPP) generates ribose moieties required for nucleic acid synthesis and the NADPH required for the biosynthesis of lipids and other complex molecules (Hume and Weidemann, 1979; Reitzer et al., 1980; Morgan and Faik, 1981). The production of nucleic acids is an important biosynthetic role of glucose. NADPH is also required for the reduction of intracellular glutathione, an important antioxidant for the embryo (Rieger, 1992). In the rodent and human it appears that during invasive implantation there is little vasculature in the vicinity of the implantation site for several hours (Rogers et al., 1982a,b). Therefore, during this period, glycolysis (anaerobic) will be the only available means of energy production for the blastocyst. It is conceivable that during this time the blastocyst uses glycogen, its endogenous glucose store. Certainly from the eight-cell stage to the blastocyst there is a loss of viability if the mouse embryo is cultured without glucose (Gardner and Lane, 1996). This suggests that the embryo is forced to use its endogenous glycogen store to generate free glucose for subsequent metabolism, thereby compromising the embryo at implantation. A similar result was subsequently reported for the hamster, when one-cell embryos were cultured to the blastocyst stage in the presence or absence of 0.5 mM glucose. Hamster embryos cultured for the entire preimplantation period in the absence of glucose had a significantly reduced viability compared to those embryos exposed to 0.5 mM glucose (Ludwig et al., 2001). This experiment shows for the first time that rather than being detrimental to the hamster embryo, glucose is actually beneficial. It has been shown that the concentration of glucose in the culture medium affects its rate of consumption by the embryo (Vella et al., 1997). Therefore, increasing the concentration of glucose in the medium can result in increased glucose uptake and utilization. The genes for the glucose transporter are transcribed in the human oocyte and embryo (Hogan et al., 1991; Aghayan et al., 1992; Dan-Goor et al., 1997), and kinetic studies have indicated the presence of the glucose transporter in the mouse embryo throughout development (Gardner and Leese, 1988).

9 Embryo Development in Sequential Media

The maximal activities of several key enzymes required for glucose metabolism have been determined in the mouse and human embryo (Brinster, 1968, 1971; Chi et al., 1988; Martin et al., 1993; Gardner et al., 2000a). In all cases the activities of the three rate-limiting enzymes of glucose metabolism (hexokinase, phosphofructokinase, and pyruvate kinase) have been determined to be higher than the amount of glucose utilized during the preimplantation period. In all probability glucose utilization during the preimplantation period is not regulated by transport across the plasma membrane or the absence of sufficient enzyme activity. Rather substrate availability (concentration) and the specific regulation of enzyme activity appear to control glucose utilization by the preimplantation embryo (Biggers et al., 1989). In a series of experiments, Barbehenn et al. (1974, 1978), attempted to locate the control points in glycolysis in individual mouse embryos by first starving the embryo for 60 minutes and then refeeding the embryo with either glucose alone or glucose with pyruvate. The levels of metabolic intermediates within an embryo were then quantitated using the technique of enzyme cycling (described by Gardner and Leese, 1999). Using this approach it was determined that at least two enzymes between the two-cell and morula stages were potentially rate limiting in the glycolytic pathway—hexokinase and 6-phophofructokinase. Both of these enzymes appear to be present at sufficient levels for glucose metabolism, thus intracellular control of such enzymes should be considered (Gardner, 1998a; Gardner et al., 2000a).

PYRUVATE

AND

LACTATE

Pyruvate readily enters the embryo by means of a facilitated carrier (Gardner and Leese, 1988; Butcher et al., 1998) and is the preferred nutrient of the cleavage-stage embryo of several species (Leese and Barton, 1984; Hardy et al., 1989; Gardner et al., 1993; Thompson et al., 1996). Although lactate is readily taken up, and can be metabolized to some degree, it cannot support the first cleavage division in the mouse (Biggers et al., 1967; Cross and Brinster, 1973). Inside the embryo pyruvate and lactate are interconverted by the enzyme lactate dehydrogenase (LDH) through the following reaction: Pyruvate + NADH + H+

∫ Lactate + NAD+

A primary function of pyruvate conversion to lactate in cells is to regenerate NAD+ for subsequent use in glycolysis when under anaerobic conditions, and therefore this conversion is of greatest significance at the blastocyst stage. This process is required because the cytoplasmic and mitochondrial pools of NADH are not shared. In order to transfer reducing power between these two distinct cellular compartments, a specific shuttle is required. In mammalian cells this is the malate : aspartate shuttle. Studies have revealed that this shuttle has little or no activity at the one-cell stage in the mouse embryo, but that there is significant activity from the two-cell stage onward. Furthermore, a reduction of activity of this shuttle at the blastocyst stage is associated with aberrant levels of lactate production by blastocysts developed in vitro (Gardner et al., 2000a), and therefore the malate : aspartate shuttle is involved in the regulation of embryo metabolism. Lane and Gardner (2000a) demonstrated that the mouse zygote and blastocyst differ in their ability to metabolize pyruvate and lactate, and that such differences could be accounted for only by a change in the intracellular NAD+ : NADH ratio, which in turn can be affected by the ratio of pyruvate : lactate. This example shows that by changing the ratio of certain medium components one can inadvertently change the ratio of intracellular regulators. Gardner and Sakkas (1993) have previously shown that changing the concentration of lactate in the culture medium can have a significant effect on mouse embryo viability and that this effect is stage specific. Such studies bring into question the potential pitfalls of using one of these carboxylic acids in the absence of the other.

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As well as being used as an energy source throughout development, pyruvate is also a powerful antioxidant, being able to reduce intracellular levels of hydrogen peroxide in the embryo (Kouridakis and Gardner, 1995; O’Fallon and Wright, 1995). Its presence in embryo culture medium therefore confers a significant degree of protection as well as serving as a vital energy source. Furthermore, being weak acids, both pyruvate and lactate can reduce the pHi of the embryo when they are present in culture media at high concentrations, i.e., >1 mM (Gibb et al., 1997; Edwards et al., 1998b). This is particularly pertinent for lactate, which can be present in culture media at over 20 mM. Lactate routinely comes in the form of D and L isomers, both of which can decrease pHi (Edwards et al., 1998b). It is therefore important to use only the biologically active form, the L isomer, in order to reduce effects on pHi.

AMINO ACIDS The majority of mammalian embryos can develop in culture in the absence of amino acids and give rise to offspring. This stems from work initially performed during the 1960s on mouse embryos derived from F1 hybrid strains (Whitten and Biggers, 1968). Embryos from such strains are relatively insensitive to culture conditions. As a result of this the significance of amino acids during preimplantation embryo development was not considered for many years. Up to the early 1990s amino acids were conspicuously missing from the media designed to support mammalian preimplantation embryos in culture; this was in spite of the fact that amino acids are abundant in the fluids of the female reproductive tract (Casslen, 1987; Miller and Schultz, 1987; Moses et al., 1997). Specific amino acids such as glycine and taurine are present in millimolar amounts. Furthermore, the oocyte and embryo maintain an endogenous pool of amino acids (Schultz et al., 1981) and possess specific transport systems to take up amino acids from their surroundings (Van Winkle, 1988). The first indication that amino acids had a role in embryonic development came from Gwatkin (1966), who showed that amino acids were required for the attachment and outgrowth of mouse blastocysts. Gwatkin and Haidri (1973) went on to show that glutamine, isoleucine, methionine, and phenylalanine promoted nuclear maturation of the hamster oocyte. Subsequently Juetten and Bavister (1983) initiated research on the effects of this group of amino acids on hamster embryo development. Further studies on the rat (Zhang and Armstrong, 1990), the mouse (Mehta and Kiessling, 1990; Gardner and Sakkas, 1993; Gardner and Lane, 1993a; Lane and Gardner, 1994), and sheep (Gardner et al., 1994) determined that amino acids were not only beneficial during the culture of various stages of development, but also significantly increased the resultant viability of embryos. Subsequent studies have revealed a biphasic requirement for amino acids during the preimplantation period (Lane and Gardner, 1997a; Steeves and Gardner, 1999). The zygote and cleavage-stage embryo benefit from the inclusion of Eagle’s nonessential amino acids and glutamine. Significantly, this group of amino acids bears a striking homology to those present at high levels in the female reproductive tract. Although the terms “nonessential” and “essential” amino acids as defined by Eagle (1959) have nothing to do with the requirements of the mammalian embryo, they serve as convenient groups in which to place amino acids. Eagle’s nonessential amino acids could perhaps be best classified as facilitators of blastomere function. By contrast, after the eight-cell stage the mammalian embryo benefits from the presence of a more complex array of amino acids (Steeves and Gardner, 1999), with Eagle’s essential amino acids being found to stimulate the development of the inner cell mass (ICM) (Lane and Gardner, 1997a; Lane et al., 2001). Significantly, equivalent rates of implantation to in vivo-developed blastocysts were obtained when mouse zygotes were cultured with nonessential amino acids up to the eight-cell stage

9 Embryo Development in Sequential Media

followed by culture to the blastocyst stage in the presence of 20 amino acids (Lane and Gardner, 1997a).

DECREASING INTRACELLULAR STRESS The significance of embryo plasticity is that it enables the mammalian embryo to develop in vitro in a wide variety of culture conditions, which has been fortuitous for those working in embryology. The variety of culture media used in mammalian embryology is quite staggering (Gardner and Lane, 1993c, 1999; Pool et al., 1998). However, adaptation by an embryo to less than optimal culture conditions is not without cost, the cost paid by the embryo being a reduction in viability. One gross manifestation of embryo adaptation that follows from altered patterns of nutrient consumption is reduced energy production (Menke and McLaren, 1970; Gardner and Leese, 1990). Even a transient exposure (6 hours) of an in vivo-developed mouse blastocyst to inappropriate conditions leads to a reduction in viability (Lane and Gardner, 1998). It is evident that those culture conditions that do not place a stress on the developing embryo, and do not cause it to adapt to less than optimum conditions, are required for the development of viable embryos. For culture media to sustain the viability of an embryo it is essential that metabolic, homeostatic, and osmotic stress are minimized (Gardner, 1998a; Gardner et al., 2000a; Lane, 2001; Lane and Gardner, 2000b, 2001a). This can be achieved by the use of carbohydrate gradients and an increasingly complex array of amino acids as development proceeds, reflecting the changes in physiology (Tables 1–3).

EVOLUTION OF SEQUENTIAL EMBRYO CULTURE MEDIA Although different culture conditions have been used in sequence to support mammalian embryos (Bavister, 1999), we have applied the concept of sequential media that are designed specifically with the changing needs of the embryo in mind (Gardner and Leese, 1990). The design of such media focused on the dynamics of embryo physiology and metabolism, and the reduction of intracellular stress. Furthermore, it took into account data obtained on the environment within the female reproductive tract. Two media, G1 and G2, were therefore formulated for the preand postcompacted embryo (Gardner, 1994; Barnes et al., 1995). These media have subsequently been modified (Gardner et al., 1998) (Series II) and their formulations are shown in Table 4. In spite of species differences in embryo physiology and metabolism, because the sequential media G1 and G2 were designed to minimize intracellular stress, thereby facilitating normal cellular function, these media have been able to support the development of viable blastocysts from a wide variety of mammalian species, including humans, mice, and cows (Tables 5–7) (Figs. 5 and 6). Significantly, not all culture conditions support equivalent levels of blastocyst viability, even though the percentage of pronucleate embryos reaching this stage may be similar. This apparent paradox is exemplified in Fig. 7, in which it is evident that the formation of a blastocyst in vitro does not equate to the formation of a viable blastocyst. Unfortunately the vast majority of research on culture media formulations did not culminate in embryo transfer experiments, leaving open the question of whether the resultant embryos were viable. From the data presented it is evident that those conditions that support optimal blastocyst differentiation in culture actually compromise the development of the zygote (see Figs. 1, 2, and 7). Furthermore, those conditions that are favorable for the zygote, such as nonessential amino acids and EDTA, do not support the development of viable blastocysts (Figs. 7 and 8). Experiments have been performed to compare the efficacy of sequential media with a leading single medium formulation, KSOMaa (Biggers et al., 2000), on mouse

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Table 4 Composition of Sequential Culture Media G1 and G2 (Version II) Medium

Component

Concentration (mM)

G1 (cleavage stage development)

Sodium chloride Potassium chloride Sodium phosphate Magnesium sulfate Sodium bicarbonate Calcium chloride Glucose Lactate Pyruvate Alanyl-glutamine Alanine Aspartate Asparagine Glutamate Glycine Proline Serine Taurine EDTA

90.08 5.5 0.25 1.0 25.0 1.8 0.5 10.5 0.32 0.5 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.01

G2 (blastocyst development)

Sodium chloride Potassium chloride Sodium phosphate Magnesium sulfate Sodium bicarbonate Calcium chloride Glucose Lactate Pyruvate Alanyl-glutamine Alanine Aspartate Asparagine Glutamate Glycine Proline Serine Arginine Cystine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine Choline chloride Folic acid Inositol Nicotinamide Pantothenate Pyridoxal Riboflavin Thiamine

90.08 5.5 0.25 1.0 25.0 1.8 3.15 5.87 0.10 1.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.6 0.1 0.2 0.4 0.4 0.4 0.1 0.2 0.4 0.5 0.2 0.4 0.0072 0.0023 0.01 0.0082 0.0042 0.0049 0.00027 0.00296

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Table 5 Efficacy of the Sequential Media G1 and G2 in Supporting Human Blastocyst Development and Implantation Using Donated Oocytesa Parameter

Measurement

Number of patients Number of pronulceate embryos Blastocyst development on day 5 Blastocyst development on day 6 Total blastocyst development Implantation rate (fetal sac) Implantation rate (fetal heart) Clinical pregnancy rate

211 15.2 ± 0.4 (mean ± SEM) 51.7% 8.3% 60.0% 62.1% 60.8% 79.6%

a

Data from Gardner et al. (2002). Significantly, the implantation rate of human blastocysts developed in sequential media is equivalent to that reported by Buster et al. (1985), who were able to obtain human blastocysts developed in vivo and flushed from the uteri of donors. When transferred to recipient patients an implantation rate of 60% was attained. Such data imply that present culture conditions are reaching optimum levels.

embryos obtained from CF1 females mated to CF1 males (Table 6, Fig. 9). This is in contrast to previously published studies on KSOMaa in which CF1 females were mated with hybrid males (B6D2F1/CrlBR) (Biggers et al., 2000). This is an important issue because the genotypes of both the female and the male have a significant impact on embryo development (Shire and Whitten, 1980a,b). When CF1 females are mated with hybrid males, subsequent embryo development is superior to that obtained when the CF1 females are mated with males of the same strain (Table 6) (Lynette Scott, personal communication). To ensure that the two media were treated in a similar way, embryos in medium KSOMaa were transferred to fresh medium after 48 hours of culture at the time when embryos cultured in medium G1 were moved to medium G2. This ensured that there was no extra buildup of ammonium in the KSOMaa medium drops. It can be seen from Fig. 9 and Table 6 that all parameters measured were significantly lower for mouse embryos cultured in KSOMaa compared to those in sequential media. Furthermore, there were clear differences in the morphology of the resultant blastocysts (Fig. 10).

Table 6 Efficacy of Sequential Media G1 and G2 vs. KSOMaa in Supporting CF1 ¥ CF1 Mouse Zygotes in Culture and Their Subsequent Viability a

Culture system Sequential media KSOMaa

Number of 2PN

≥ Eight cells on day 3 (%)

Compaction at 72 hours post-hCG (%)

226

78.2

29.9

220

42.1b**

7.3b**

Hatching at 117 hours post-hCG (% of total)

Implantation (%)

Fetal development (%)

67.7

38.9

75

55.6

45.5b**,c

16.8b

50*

36.1

Blastocyst at 117 hours post-hCG (%)

a All media were supplemented with 5 mg/ml HSA. Number of embryos transferred, 36 per treatment; implantation and fetal development were determined on day 15 of pregnancy, day 1 being the day of copulation plug. b Significantly different from sequential media: *, P < 0.05; **, P < 0.01. c Blastocyst development in KSOMaa (45.5%) is lower than that reported by Biggers et al. (2000), who obtained 82% blastocyst development when CF1 females were mated to hybrid males. In our laboratory, when CF1 females are mated to hybrid males, embryo development is increased by around 30% over embryos derived from a CF1 ¥ CF1 mating. Using this “correction factor” the data obtained in KSOMaa, as given here, are very similar to previously reported data (Biggers et al., 2000).

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Table 7 Efficacy of the Sequential Media G1 and G2 in Supporting Bovine Embryo Development and Viability Culture systema Coculture with B2 G1/G2 KSOMaa

Number of oocytes

Blastocyst/ oocyte (%)

Blastocyst/ cleaved (%)

Total cell numberb

ICM number

Trophectoderm number

ICM (%)

1388

22.3

33.8

169 ± 9a

63 ± 5a

106 ± 6a

37

1557 1497

16.8 16.1

26.1 25.6

207 ± 13b 169 ± 10a

81 ± 7b 61 ± 7a

126 ± 9b 98 ± 6a

39 37

a

The medium for all treatments was renewed after 72 hours of culture. Cell numbers were obtained from expanded blastocysts. Different letters (a or b) following the cell numbers within a column represent a significant difference (P < 0.05). Embryo viability following culture in the sequential media G1/G2 was determined by the transfer of 234 blastocysts to recipients; 51.3% (120/234) of the recipients became pregnant. When grade 1 blastocysts were transferred the pregnancy rate was 56.4% (88/156). These data compare favorably with data obtained from blastocysts developed in vivo, flushed from the uterus, and transferred to recipient females (67% pregnancy for all grades of blastocysts and 76% for grade 1) (Hasler, 1998). When blastocysts were cultured in G1/G2 and subsequently frozen (in 1.5 M ethylene glycol), thawed, and transferred to Heifer recipients, the pregnancy rate was 47.5% (19/40). Viability data are courtesy of Dr. John Hasler. b

A further experiment was performed to compare the rates of mouse embryo development in vivo and in sequential media. Zygotes from naturally ovulated CF1 females mated to CF1 males were collected from half the females and placed into culture, while embryos from the remaining half were collected on the morning of implantation (day 4 post mating). After 72 hours of culture, embryos from both groups were photographed and cell number and allocation were determined. In Fig. 11 the morphology of blastocysts developed in vivo and in vitro can be compared and are seen to be similar. The cell number of expanded blastocysts developed in vivo was 49.8 ± 1.4, compared to 50.8 ± 2.0 for in vitro-developed embryos. In these two groups of blastocysts the percentage of blastomeres in the ICM was equivalent (29.0 ± 1.2 for in vivo, 35.4 ± 1.6 for in vitro). Such data are most

Figure 5 Photomicrograph of human blastocysts cultured in sequential media G1 and G2. Pronucleate embryos were cultured for 48 hours in medium G1 followed by culture for 48 hours in medium G2. Both media were used at 6% CO2, 5% O2, and 89% N2.

9 Embryo Development in Sequential Media

Figure 6 Photomicrograph of bovine blastocysts cultured in sequential media G1 and G2. Putative zygotes were cultured for 72 hours in medium G1 followed by culture for 72 hours in medium G2. Both media were used at 6% CO2, 5% O2, and 89% N2.

encouraging as they indicate that CF1 mouse embryos cultured in sequential media develop at a rate similar to that of embryos in vivo.

FACTORS OTHER THAN MEDIUM FORMULATION THAT IMPACT EMBRYO DEVELOPMENT AND VIABILITY MACROMOLECULES Historically, embryo culture media have been supplemented with protein in the form of either serum albumin or serum. Under stringent culture conditions, including the presence of amino acids, embryos can be cultured to the blastocyst stage in the absence of protein (Bavister, 1995; Gardner et al., 1999). However, the inclusion of protein does facilitate gamete and embryo manipulation in vitro by acting as a surfactant, while also conferring benefit to the embryo by the chelation of potential toxins (Flood and Shirley, 1991). Significantly, albumin is the most abundant protein in the female reproductive tract (Leese, 1988) and has been shown to maintain embryo physiology and metabolism in vitro compared to embryos cultured in the presence of a synthetic macromolecule, polyvinyl alcohol (PVA) (Eckert et al., 1998; Thompson et al., 1998). Unfortunately a problem with serum albumin, serum, or any biological product is the risk of disease transmission and contamination. This alone is reason to consider alternatives. Furthermore, there is considerable variation in the composition of serum albumin from batch to batch (Batt et al., 1991; Gray et al., 1992; McKiernan and Bavister, 1992), making standardization of procedures difficult. Should albumin be used in an embryo culture system, it is important to ensure that it is fatty acid free. As well as the reasons listed above, the inclusion of serum in embryo culture systems, whether it be in media or for use in coculture, can no longer be considered

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Figure 7 Effect of sequential culture media on the development of F1 (C57BL/6 ¥ CBA/Ca) mouse zygotes in vitro. Zygotes were collected at 20 hours post-hCG. All media were supplemented with BSA (2 mg/ml). All embryos were transferred to fresh medium after 48 hours of culture, with the exception of embryos in medium G1; these embryos were transferred to either medium G1 or G2. To compensate for this, twice the number of embryos were originally cultured in medium G1, although only a designated 50% of these embryos were used in the statistical analysis of the data set covering 44 to 52 hours. (A) Embryo cell number after 44, 48, and 52 hours of culture. Values are mean ± SEM; n = 200 embryos/medium. Media: G1 (solid bar); HTF (open bar); Ham’s F-10 (hatched bar). **, Significantly different from other media (P < 0.01). (B) Embryo development after 72 hours of culture; n = 150 embryos/medium; G1/G2, embryos cultured for 48 hours in medium G1 and then transferred to medium G2; blastocyst, solid bar; hatching blastocysts (as a percentage of total blastocysts), open bar. Letters a, c, and d, like pairs are significantly different (P < 0.05); b (P < 0.01). (C) Embryo development after 92 hours of culture; n = 150 embryos/medium; G1/G2, embryos cultured for 48 hours in medium G1 and then transferred to medium G2; blastocyst, solid bar; hatching blastocysts (as a percentage of total blastocysts), open bar. Letters a, c, and d, like pairs

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acceptable. It is important to emphasize that mammalian embryos are not exposed to serum in vivo. Oviduct and uterine fluids are not simple serum transudates. Rather, serum is a pathological fluid and by default contains abundant growth factors, such as platelet-derived growth factor and transforming growth factor-a, released during platelet aggregation, as well as a host of other growth factors. In spite of this, the addition of serum to culture medium does add a certain degree of protection to the embryo via the minimization of transient pH shifts and chelation of potential toxins. It is owing to this ability to confer a degree of robustness to the culture medium that its use has persisted. However, data on the development of sheep and cattle blastocysts in the presence of serum have raised serious issues regarding the use of serum for embryo culture (Gardner, 1994; Gardner and Lane, 1999). Serum can adversely affect the development of embryos at several levels: 1. Precocious blastocoel formation (Thompson et al., 1995; Walker et al., 1992). 2. Sequestration of lipid (Dorland et al., 1994; Thompson et al., 1995). 3. Abnormal mitochondrial ultrastructure (Dorland et al., 1994; Thompson et al., 1995). 4. Perturbations in metabolism (Gardner et al., 1994). 5. Association with abnormally large offspring in sheep (Thompson et al., 1995). Therefore, in attempts to define embryo culture media, Bavister advocated the use of PVA (Bavister, 1981) to replace serum or serum albumin. This approach has worked for the in vitro development of embryos from several mammalian species. However, the use of such synthetic macromolecules cannot be said to be physiological, and as described above PVA is not able to maintain the physiology and metabolism of the embryo. Furthermore, bovine embryos cultured in the presence of PVA did not survive cryopreservation as well as those cultured in the presence of albumin (Eckert et al., 1998). Recombinant human albumin has become available and has been shown to be as effective as blood-derived albumin in supporting embryo development (Gardner and Lane, 2000; Hooper et al., 2000). Significantly, embryos cultured in the presence of recombinant albumin exhibit an increased tolerance to cryopreservation

are significantly different (P < 0.05); **, significantly different from medium G1 and G1/G2 (P < 0.01). (D) Cell allocation in the blastocyst after 92 hours of culture; n = 150 embryos/medium; G1/G2, embryos cultured for 48 hours in medium G1 and then transferred to medium G2; trophectoderm, solid bars; inner cell mass, open bars; *, significantly different from other media (P < 0.05); **, (P < 0.01). (E) Viability of cultured blastocysts; n = at least 60 blastocysts transferred per treatment; G1/G2, embryos cultured for 48 hours in medium G1 and then transferred to medium G2; implantation, solid bar; fetal development per implantation, open bar. Letters a and d, like pairs are significantly different (P < 0.05); b and c (P < 0.01). When mouse embryos were cultured in medium G1 for the entire preimplantation period up to the blastocyst stage, although the embryos formed healthy-looking blastocysts, most implantations were lost, i.e., they did not have a sufficient inner cell mass to form a viable fetus. The lack of adequate inner cell mass development stems from both the lack of sufficient glucose and the presence of EDTA (both affect glycolysis) and the omission of essential amino acids. In contrast, those mouse embryos that were switched to medium G2 after 48 hours of culture formed blastocysts at the same rate and with morphologies equivalent to those in medium G1 for the entire culture period. However, for those blastocysts developed in medium G1 and switched to medium G2, very few implantations were lost due to the development of a large inner cell mass, thereby maintaining a very high pregnancy rate. [From Gardner, D. K., and Lane, M. (1998). Culture of viable human blastocysts in defined sequential serum-free media. Hum. Reprod. 13(Suppl. 3), 148–159. © European Society of Human Reproduction and Embryology. Reproduced by permission of Oxford University Press/Human Reproduction.]

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Figure 8 Effect of EDTA on bovine blastocyst development and differentiation. Percent blastocyst development after 144 hours of culture, solid bars; trophectoderm cell number, hatched bars; inner cell mass cell number, open bars; percent inner cell mass cells of total cells, striped bars. Like pairs of letters are significantly different (a, d, h, and j, P < 0.05; b, c, e, f, g, and i, P < 0.01). For SOFaa/SOFaa, embryos were cultured for 72 hours in medium SOF supplemented with amino acids but lacking EDTA (SOFaa) (Gardner et al., 1994) for 72 hours, and then transferred to fresh SOFaa for a further 72 hours. For EDTA/SOFaa, embryos were cultured for 72 hours in SOFaa and 100 mM EDTA, and then transferred to fresh SOFaa (no EDTA) for a further 72 hours. For EDTA/EDTA, embryos were cultured for 72 hours in SOFaa and 100 mM EDTA, and then transferred to fresh SOFaa and 100 mM EDTA for a further 72 hours. Data from Gardner et al. (2000b). EDTA has been shown to stimulate the cleavage-stage embryo of both the mouse (Abramczuk et al., 1977; Mehta and Kiessling, 1990; Gardner and Lane, 1996) and the cow (Gardner et al., 2000b). It has been determined that EDTA is an inhibitor of glycolytic kinases and helps prevent aberrant levels of glycolysis (Lane and Gardner, 2001b). However, the inclusion of EDTA in the medium for embryos from the eightcell stage onward (medium G2) is not advisable, because the embryo becomes increasingly glycolytic in nature, especially the inner cell mass (Hewitson and Leese, 1993). Therefore, continued exposure to EDTA negatively impacts blastocyst development and impairs inner cell mass formation (Gardner et al., 2000b).

(Gardner et al., 2001). Another macromolecule present in the female reproductive tract is hyaluronan, which in the mouse increases at the time of implantation (Zorn et al., 1995). Hyaluronan is a high-molecular-mass polysaccharide and can be obtained endotoxin and prion free from a yeast fermentation procedure. It has been demonstrated that not only can hyaluronan replace albumin in a mouse and bovine embryo culture system, but that its use for embryo transfer results in a significant increase in embryo implantation (Gardner et al., 1999). Furthermore, similar to

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Figure 9 Blastocyst cell number and differentiation of mouse (CF1 ¥ CF1) embryos cultured in sequential media (solid bars) or KSOMaa (open bars). Significantly different from G1/G2: *, P < 0.05; **, P < 0.01.

results with recombinant albumin, the presence of hyaluronan in the culture medium increases the cryosurvivability of blastocysts (Gardner et al., 2001; Stojkovic et al., 2001). It has been found that the recombinant albumin and hyaluronan confer a synergistic benefit to the embryo (Gardner et al., 1999; Hooper et al., 2000).

AMMONIUM It has been demonstrated that upon incubation at 37°C, amino acids spontaneously deaminate to release ammonium into the culture medium. Furthermore, the embryos actively deaminate amino acids when they are metabolized, leading to a further buildup of ammonium. The significance of this is that it has been shown that ammonium not only retards embryo development in culture (Gardner and Lane, 1993a), but can also induce fetal retardation and neural tube defects in mice (Lane and Gardner, 1994). Interestingly, there appears to be a link between the concentration of ammonium in serum and the induction of fetal oversize in sheep (McEvoy et al., 1997; Sinclair et al., 1998). Therefore it is imperative to renew the culture medium used at least every 48 to 72 hours in order to circumvent the toxicity of ammonium. The main culprit with regard to deamination and ammonium release is glutamine. However, this amino acid can be replaced with the dipeptide alanylglutamine, which is stable at 37°C, and its inclusion significantly reduces ammonium release into the culture medium (Lane et al., 2001).

INCUBATION VOLUMES It is essential to consider the culture system as a whole and not simply focus on the culture media, because all aspects of the system (gas phase, embryo incubation volume and group size, macromolecule supplementation, etc.) interact (Gardner and Lane, 1999, 2001). The culture of mammalian embryos in reduced volumes of

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Figure 10 Photomicrographs of CF1 ¥ CF1 mouse embryos cultured from the pronucleate stage for 96 hours in (A) sequential media G1 and G2 or (B) KSOMaa. Note the increased expansion and hatching of blastocysts cultured in sequential media.

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Figure 11 Photomicrographs of CF1 ¥ CF1 blastocysts. Both groups of embryos came from mice mated at the same time. (A) Embryos developed in vivo. (B) Embryos developed in media G1 and G2. The photographs were taken after 72 h of culture (based on in vitro group time), i.e., the morning of day 4 post mating. Note that the in vivo developed blastocysts are fully expanded and are beginning to hatch from the zona pellucida.

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medium and/or in groups significantly increases blastocyst development (Wiley et al., 1986; Paria and Dey, 1990; Lane and Gardner, 1992; Salahuddin et al., 1995) and blastocyst cell number (Lane and Gardner, 1992). Furthermore, culturing embryos in reduced volumes increases subsequent viability after transfer (Lane and Gardner, 1992). It has been proposed that the benefit of growing embryos in small volumes and/or in groups is due to the production of specific embryo-derived autocrine/paracrine factors that stimulate development. The culture of embryos in large volumes will result in a dilution of such a factor so that it becomes ineffectual (Gardner, 1994). This phenomenon is not confined to the mouse, in which several embryos reside in the female tract at one time, but has also been reported for the sheep and cow, which, like humans, are monovular (Gardner et al., 1994; Ahern and Gardner, 1998). It has been shown in both the mouse and the cow that increasing the embryo : incubation volume ratio specifically stimulates the development of the ICM. This explains the increased viability of embryos cultured in reduced volumes in groups (Fig. 12) (Ahern and Gardner, 1998).

GAS PHASE The concentration of oxygen in the lumen of the rabbit oviduct is reported to be 2–6%, (Mastroianni and Jones, 1965; Ross and Graves, 1974) and 8% in the oviduct of hamster, rabbit, and rhesus monkey (Fischer and Bavister, 1993). Furthermore, the oxygen concentration in the uterus is significantly lower than in the oviduct, ranging from 5% in the hamster and rabbit to 1.5% in the rhesus monkey (Maas et al., 1976; Fischer and Bavister, 1993). Studies on different mammalian species have demonstrated that culture in a reduced oxygen concentration, especially embryos of ruminants, results in enhanced embryo development in vitro. Several studies have shown that a reduced oxygen concentration (between 5 and 8%) enhances development to the blastocyst stage in mice (Quinn and Harlow, 1978; Umaoka et al., 1992; Gardner and Lane, 1996), rabbits (Li and Foote, 1993), sheep (Thompson et al., 1990), goats (Batt et al., 1991), and cows (Thompson et al., 1990). The concentration of CO2 employed in the culture system has a direct impact on medium pH. Although most media work over a wide range of pH (7.2–7.4), it is preferable to ensure that pH does not go over 7.4, considering that intracellular pH is actually 7.2 (Lane and Gardner, 2001a). Therefore it is advisable to use a CO2 concentration of between 6 and 7% (Lawitts and Biggers, 1993; Gardner and Lane, 2001). We routinely culture embryos at 6% CO2/5% O2/89% N2 in groups in microliter drops of medium under oil. For mice we culture embryos in groups of 10 in 20-ml drops and replace the medium after 48 hours. For ruminant embryos we culture embryos in groups of 4 in 50-ml drops and replace the medium after 72 hours. For human embryos we culture embryos in groups of 4 in 50-ml drops and replace the medium after 48 hours. The macromolecules of choice are serum albumin (preferably recombinant albumin) (Gardner and Lane, 2000; Hooper et al., 2000) and hyaluronan (Gardner et al., 1999).

INHERENT PROBLEMS OF COCULTURE Coculture systems were initially developed in an attempt to make culture systems more physiological (Gandolfi and Moor, 1987). However, not only does coculture not accommodate the changing nutrient requirements of the developing embryo, but one is faced with trying to culture two totally different cell types (somatic and embryonic) in the same culture medium. This is not a feasible proposition, because it is not possible to supply the nutrient requirements of two different cell types. Therefore some compromises in cell viability are inevitable. Furthermore, not only do coculture systems typically rely on the use of tissue culture media rather than

9 Embryo Development in Sequential Media

Figure 12 Effect of incubation volume and embryo grouping on embryo development and differentiation [reproduced from Gardner (1998c) with permission of Martin Dunitz Press]. (A) A single embryo cultured in a four-well plate or test tube, any factor produced by the embryo will become ineffectual due to dilution. (B) Culture of embryos in reduced volumes and/or in groups increases the effective concentration of embryo-derived factors, facilitating their action in either a paracrine or an autocrine manner. (C) Effect of embryo grouping on bovine blastocyst development and differentiation. Bovine embryos were cultured either individually or in groups of two or four in 50-ml drops of medium (Ahern and Gardner, 1998). Like pairs of letters are significantly different (P < 0.05). (Reproduced from Gardner, 1998c.)

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embryo culture media, they usually employ serum as the macromolecule (Gardner, 1998b). The perils of including serum in an embryo culture system were discussed previously. The growing body of evidence is showing that coculture is not required to develop viable blastocysts in vitro (Mullaart et al., 2001), irrespective of the species being studied (Gardner, 1998b). Certainly embryo development in culture, and subsequent viability after transfer, are as good if not better following culture in sequential media as compared to coculture (Table 7).

CONCLUSIONS In this chapter we have presented evidence that in order to optimize mammalian embryo development in culture, sequential media are required, each designed to meet the changing requirements of the developing embryo (Gardner and Leese, 1990). Conditions that are good for the early stage are not optimal for blastocyst differentiation. In contrast, those conditions that do support good blastocyst differentiation and maintain embryo viability are not optimal for the development of the zygote (Gardner and Lane, 1998; Lane and Gardner, 1997a). Supplying the embryo with gradients of carbohydrates and amino acids not only provides the changing nutrients required but also reduces intracellular stress (Gardner, 1998a; Gardner et al., 2000a; Lane, 2001; Lane and Gardner, 2000a). The ability to minimize intracellular stress is a significant factor in being able to maintain embryo viability in culture. This premise is supported by the fact that the sequential culture media G1 and G2 can support the development of viable blastocysts of the primate, rodent, and ruminant, in spite of species differences in embryo physiology. Using sequential media, blastocyst developmental rates in vitro and subsequent implantation rates are very close to those observed in vivo (Table 5). Indeed, the ability of sequential media to support the development of viable inner cell masses was fundamental in the establishment of the first human embryonic stem cell lines (Thomson et al., 1998).

REFERENCES Abramczuk, J., Solter, D., and Koprowski, H. (1977). The beneficial effect EDTA on development of mouse one-cell embryos in chemically defined medium. Dev. Biol. 61, 378–383. Aghayan, M., Rao, L. V., Smith, R. M., Jarett, L., Charron, M. J., Thorens, B., and Heyner, S. (1992). Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development. Development 115, 305–312. Ahern, T. J., and Gardner, D. K. (1998). Culturing bovine embryos in groups stimulates blastocyst development and cell allocation to the inner cell mass. Theriogenology 49, 194. Barbehenn, E. K., Wales, R. G., and Lowry, O. H. (1974). The explanation for the blockade of glycolysis in early mouse embryos. Proc. Natl. Acad. Sci. U.S.A. 71, 1056–1060. Barbehenn, E. K., Wales, R. G., and Lowry, O. H. (1978). Measurement of metabolites in single preimplantation embryos; a new means to study metabolic control in early embryos. J. Embryol. Exp. Morphol. 43, 29–46. Barnes, F. L., Crombie, A., Gardner, D. K., Kausche, A., Lacham-Kaplan, O., Suikkari, A. M., Tiglias, J., Wood, C., and Trounson, A. O. (1995). Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum. Reprod. 10, 3243–3247. Barnett, D. K., and Bavister, B. D. (1996). What is the relationship between the metabolism of preimplantation embryos and their developmental competence? Mol. Reprod. Dev. 43, 105–133. Batt, P. A., Gardner, D. K., and Cameron, A. W. (1991). Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro. Reprod. Fertil. Dev. 3, 601–607. Bavister, B. D. (1981). Substitution of a synthetic polymer for protein in a mammalian gamete culture system. J. Exp. Zool. 217, 45–51. Bavister, B. D. (1995). Culture of preimplantation embryos: Facts and artifacts. Hum. Reprod. Update 1, 91.

9 Embryo Development in Sequential Media Bavister, B. D. (1999). Stage-specific culture media and reactions of embryos to them. In “Towards Reproductive Certainty. Fertility and Genetics beyond 1999” (R. Jansen and D. Mortimer, eds.), pp. 367–377. Parthenon, New York. Benos, D., and Biggers, J. D. (1981). Blastocyst fluid formation. In “Fertilization and Embryonic Development In vitro” (L. J. Mastroianni and J. D. Biggers, eds.), pp. 283–297. Plenum Press, New York. Biggers, J. D. (1987). Pioneering mammalian embryo culture. In “The Mammalian Preimplantation Embryo Regulation of Growth and Differentiation in vitro” (B. D. Bavister, ed.), pp. 1–22. Plenum Press, New York. Biggers, J. D., and McGinnis, L. K. (2001). Evidence that glucose is not always an inhibitor of mouse preimplantation development in vitro. Hum. Reprod. 16, 153–163. Biggers, J. D., and Stern, S. (1973). Metabolism of the preimplantation mammalian embryo. Adv. Reprod. Physiol. 6, 1–59. Biggers, J. D., Moore, B. D., and Whittingham, D. G. (1965). Development of mouse embryos in vivo after cultivation from two-cell ova to blastocysts in vitro. Nature 206, 734–735. Biggers, J. D., Whittingham, D. G., and Donahue, R. P. (1967). The pattern of energy metabolism in the mouse oocyte and zygote. Proc. Natl. Acad. Sci. U.S.A. 58, 560–567. Biggers, J. D., Gardner, D. K., and Leese, H. J. (1989). Control of carbohydrate metabolism in preimplantation mammalian embryos. In “Growth Factors in Mammalian Development” (I. Y. Rosenblum and S. Heyner, eds.), pp. 19–32. CRC Press, Boca Raton. Biggers, J. D., McGinnis, L. K., and Raffin, M. (2000). Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium. Biol. Reprod. 63, 281–293. Brinster, R. L. (1968). Hexokinase activity in the preimplantation mouse embryo. Enzymologia 34, 304–308. Brinster, R. L. (1971). Phosphofructokinase activity in the preimplantation mouse embryo. Wilhelm Roux Arch. Entwicklungsmech. Org. 166, 300–302. Brinster, R. L. (1973). Nutrition and metabolism of the ovum, zygote, and blastocyst. In “Handbook of Physiology,” Vol. 2 (S. R. Geiger, ed.), pp. 165–184. Waverly Press, Maryland. Buster, J. E., Bustillo, M., Rodi, I. A., Cohen, S. W., Hamilton, M., Simon, J. A., Thorneycroft, I. H., and Marshall, J. R. (1985). Biologic and morphologic development of donated human ova recovered by nonsurgical uterine lavage. Am. J. Obstet. Gynecol. 153, 211–217. Butcher, L., Coates, A., Martin, K. L., Rutherford, A. J., and Leese, H. J. (1998). Metabolism of pyruvate by the early human embryo. Biol. Reprod. 58, 1054–1056. Casslen, B. G. (1987). Free amino acids in human uterine fluid. Possible role of high taurine concentration. J. Reprod. Med. 32, 181–184. Chi, M. M., Manchester, J. K., Yang, V. C., Curato, A. D., Strickler, R. C., and Lowry, O. H. (1988). Contrast in levels of metabolic enzymes in human and mouse ova. Biol. Reprod. 39, 295–307. Crosby, I. M., Gandolfi, F., and Moor, R. M. (1988). Control of protein synthesis during early cleavage of sheep embryos. J. Reprod. Fertil. 82, 769–775. Cross, P. C., and Brinster, R. L. (1973). The sensitivity of one-cell mouse embryos to pyruvate and lactate. Exp. Cell Res. 77, 57–62. Dale, B., Menezo, Y., Cohen, J., DiMatteo, L., and Wilding, M. (1998). Intracellular pH regulation in the human oocyte. Hum. Reprod. 13, 964–970. Dan-Goor, M., Sasson, S., Davarashvili, A., and Almagor, M. (1997). Expression of glucose transporter and glucose uptake in human oocytes and preimplantation embryos. Hum. Reprod. 12, 2508– 2510. Dorland, M., Gardner, D. K., and Trounson, A. (1994). Serum in synthetic oviduct fluid causes mitochondrial degeneration in ovine embryos. J. Reprod. Fertil. (Abstr. Ser.) 13, 70. Eagle, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432–437. Eckert, J., Pugh, P. A., Thompson, J. G., Niemann, H., and Tervit, H. R. (1998). Exogenous protein affects developmental competence and metabolic activity of bovine pre-implantation embryos in vitro. Reprod. Fertil. Dev. 10, 327–332. Edwards, L. J., Williams, D. A., and Gardner, D. K. (1998a). Intracellular pH of the mouse preimplantation embryo: Amino acids act as buffers of intracellular pH. Hum. Reprod. 13, 3441–3448. Edwards, L. J., Williams, D. A., and Gardner, D. K. (1998b). Intracellular pH of the preimplantation mouse embryo: Effects of extracellular pH and weak acids. Mol. Reprod. Dev. 50, 434–442. Fischer, B., and Bavister, B. D. (1993). Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J. Reprod. Fertil. 99, 673–679. Flood, L. P., and Shirley, B. (1991). Reduction of embryotoxicity by protein in embryo culture media. Mol. Reprod. Dev. 30, 226–231. Gandolfi, F., and Moor, R. M. (1987). Stimulation of early embryonic development in the sheep by coculture with oviduct epithelial cells. J. Reprod. Fertil. 81, 23–28. Gardner, D. K. (1994). Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biol. Int. 18, 1163–1179. Gardner, D. K. (1998a). Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology 49, 83– 102.

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Gardner and Lane Gardner, D. K. (1998b). Embryo development and culture techniques. In “Animal Breeding: Technology for the 21st Century” (J. R. Clark, ed.), pp. 13–46. Harwood Academic Publishers, London. Gardner, D. K. (1998c). Improving embryo culture and enhancing pregnancy rate. In “Female Infertility Therapy: Current Practice” (Z. Shoham, C. Howles, and H. Jacobs, eds.), pp. 283–299. Martin Dunitz, London. Gardner, D. K. (1999). Development of serum-free culture systems for the ruminant embryo and subsequent assessment of embryo viability. J. Reprod. Fertil. (Suppl.) 54, 461–475. Gardner, D. K., and Lane, M. (1993a). Amino acids and ammonium regulate mouse embryo development in culture. Biol. Reprod. 48, 377–385. Gardner, D. K., and Lane, M. (1993b). The 2-cell block in CF1 mouse embryos is associated with an increase in glycolysis and a decrease in tricarboxylic acid (TCA) cycle activity: Alleviation of the 2cell block is assocated with the restoration of in vivo metabolic pathway activities. Biol. Reprod. 48(Suppl. 1), 152. Gardner, D. K., and Lane, M. (1993c). Embryo culture systems. In “Handbook of In Vitro Fertilization” (D. K. Gardner and A. O. Trounson, eds.), pp. 85–114. CRC Press, Boca Raton. Gardner, D. K., and Lane, M. (1996). Alleviation of the “2-cell block” and development to the blastocyst of CF1 mouse embryos: Role of amino acids, EDTA and physical parameters. Hum. Reprod. 11, 2703–2712. Gardner, D. K., and Lane, M. (1998). Culture of viable human blastocysts in defined sequential serumfree media. Hum. Reprod. 13(Suppl. 3), 148–159. Gardner, D. K., and Lane, M. (1999). Embryo culture systems. In “Handbook of In Vitro Fertilization,” 2nd Ed. (A. O. Trounson and D. K. Gardner, eds.), pp. 205–264. CRC Press, Boca Raton. Gardner, D. K., and Lane, M. (2000). Recombinant human serum albumin and hyaluronan can replace blood-derived albumin in embryo culture media. Fertil. Steril. 74(Suppl. 3), 0–086. Gardner, D. K., and Lane, M. (2001). Embryo culture. In “Textbook of Assisted Reproductive Techniques” (D. K., Gardner, A., Weissman, C. Howles, et al. eds.), pp. 203–222. Martin Dunitz, London. Gardner, D. K., and Leese, H. J. (1988). The role of glucose and pyruvate transport in regulating nutrient utilization by preimplantation mouse embryos. Development 104, 423–429. Gardner, D. K., and Leese, H. J. (1990). Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J. Reprod. Fertil. 88, 361–368. Gardner, D. K., and Leese, H. J. (1999). Assessment of embryo metabolism and viability. In “Handbook of In Vitro Fertilization,” 2nd Ed. (A. O. Trounson and D. K. Gardner, eds.), pp. 347–372. CRC Press, Boca Raton. Gardner, D. K., and Sakkas, D. (1993). Mouse embryo cleavage, metabolism and viability: Role of medium composition. Hum. Reprod. 8, 288–295. Gardner, D. K., Lane, M., and Batt, P. (1993). Uptake and metabolism of pyruvate and glucose by individual sheep preattachment embryos developed in vivo. Mol. Reprod. Dev. 36, 313–319. Gardner, D. K., Lane, M., Spitzer, A., and Batt, P. A. (1994). Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: Amino acids, vitamins, and culturing embryos in groups stimulate development. Biol. Reprod. 50, 390–400. Gardner, D. K., Lane, M., Calderon, I., and Leeton, J. (1996). Environment of the preimplantation human embryo in vivo: Metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil. Steril. 65, 349–353. Gardner, D. K., Schoolcraft, W. B., Wagley, L., Schlenker, T., Stevens, J., and Hesla, J. (1998). A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum. Reprod. 13, 3434–3440. Gardner, D. K., Rodriegez-Martinez, H., and Lane, M. (1999). Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer. Hum. Reprod. 14, 2575–2580. Gardner, D. K., Pool, T. B., and Lane, M. (2000a). Embryo nutrition and energy metabolism and its relationship to embryo growth, differentiation, and viability. Semin. Reprod. Med. 18, 205–218. Gardner, D. K., Lane, M. W., and Lane, M. (2000b). EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation. Mol. Reprod. Dev. 57, 256–261. Gardner, D. K., Maybach, J. M., and Lane, M. (2001). Hyaluronan and rHSA increase blastocyst cryosurvival. In “Proceedings of the 17th World Congress on Fertility and Sterility,” p. 226. Melbourne, Australia. Gardner, D. 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