Nutrients

It might have been imagined that culturing early embryos in vitro would require a complex medium such as that used to maintain somatic cells. However, this turned out not to be the case. Thus, Biggers (1998), summarizing the pioneering work of Whitten in the 1970s, stated that ‘A surprise from Whitten’s work was that the 8‐cell mouse embryo would develop into a blastocyst in a very simple solution based on a physiological saline, described earlier by Krebs and Henseleit supplemented only with a carbon source (glucose) and bovine plasma albumin’. Building on this observation, Biggers, Brinster, Wales, Whitten, and Whittingham established the critical role of pyruvate in supporting the first cleavage division, the importance of glucose in the later stages of development, and the large increase in metabolic activity that coincided with blastocyst formation. A good account of the history of these events has been provided by Baltz (2013).

The basic nutrient requirements of preimplantation embryos discovered in the 1970s have not changed fundamentally, such that Leese (2012), almost 40 years later, was able to provide the following, contemporary account:

The Krebs cycle and oxidative phosphorylation provide the main source of energy throughout the preimplantation period.* Pyruvate is a central energy substrate during the first cleavage in those species in which energy source requirements of the embryo have been examined, although it is not obligatory for all species (e.g. porcine). Other substrates, notably, amino acids, lactate and endogenous fatty acids derived from triglyceride, combine with pyruvate to provide embryos with a range of potential energy sources through to, and including, the blastocyst stage. These nutrients have numerous, overlapping, metabolic roles. Prior to the morula stage, glucose consumption and metabolism is low, although some glucose is necessary for intracellular signalling purposes. With blastocyst formation, large increases in oxygen consumption and the uptake and incorporation of carbon occur and there is a sharp increase in glycolysis, at least in vitro. The embryo goes from a relatively inactive metabolic tissue at ovulation to a rapidly metabolizing tissue at implantation.

Until recently, metabolic research on early embryos tended to focus on the utilization of the exogenous substrates mentioned above (pyruvate, lactate, glucose, and amino acids) (e.g. Brison et al. 2004), while the role of endogenous substrates, especially fat, was largely ignored. This was despite the egg being the largest diameter cell in the female mammal, with a high content of triglyceride, at least in large animals such as the cow, pig, rabbit, and sheep as well as the human. The pioneering work on endogenous nutrients was carried out by Kane and Foote, who pointed out in the early 1970s (summarized by Kane 1987) that ‘the 1‐cell rabbit embryo has sufficient endogenous energy sources to allow up to 3 or more cleavage divisions in culture in the absence of any possible added energy substrate’. This work has been rediscovered in the past few years, with the realization that fatty acid metabolism plays a pivotal role in egg maturation and could potentially sustain the entire energy requirement of the embryo over the preimplantation period (Sturmey et al. 2009; Dunning et al. 2010; Jungheim et al. 2011; McKeegan and Sturmey 2011; Van Hoeck et al. 2011).

The significance of endogenous reserves will be considered further in the context of embryo autonomy and adaptability.

Ions

In comparison with the wealth of data on the uptake and fate of nutrients by mammalian eggs and early embryos, much less is known about the requirement for ions. The major role of ions in somatic cells is in cell volume regulation. The history of this subject in embryos is also described in the account by Baltz (2013) mentioned previously, which considers the control of intracellular pH and the role of organic osmolytes (especially the amino acids glycine and glutamine) in protecting the embryo in vitro against the effects of osmotic stress and changes in cell volume. Certain ions have specific functions in gamete and early embryo survival (Chan et al. 2012); thus, the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the transport of bicarbonate ions necessary for sperm capacitation and embryo development (Chan et al. 2009). The high bicarbonate concentration also contributes to the alkalinity of oviduct fluid. The mechanism of bicarbonate transport is closely linked to the movement of potassium ions, long known to be present at elevated concentrations in oviduct fluid (Keating and Quinlan, 2012). It should also be noted that gametes (at least sperm) and embryos will only be present in the oviduct following mating but that ion and fluid secretion will be required at all times, for example, to keep the surface of the oviduct moist and protect it from potential pathogens.

Other Molecules in Embryo Culture Media

There are numerous proteins in oviduct and uterine fluids (for reviews, see Coy et al. 2012, Ghersevich et al. 2015, and Miller 2015 and the section on Proteins later in this review) but the only one added routinely to culture media is albumin (in various forms summarized by Morbeck et al. 2014b), which is required to act as a macromolecular source, with functions as diverse as ‘stabilizing’ embryo membranes, facilitating handling of embryos in vitro, and acting as a source of energy and of ‘contaminating’ molecules such as fatty acids and growth factors which may act as embryotrophic agents. In clinical in vitro fertilization (IVF), this is added in the form of human serum albumin. Complex protein supplements in human embryo culture media are also considered by Morbeck et al. (2014b). A further protein, granulocyte–macrophage colony‐stimulating factor (GM‐CSF), present at the time of writing in one embryo culture medium only, is considered later.

Summary of the ‘Demand’ for Molecules by the Preimplantation Embryo

It is straightforward to list the minimum requirements of the early embryo in qualitative terms: the ions (Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺, Cl⁻, PO4⁻, HCO3⁻ and nutrients); pyruvate, lactate, glucose, amino acids, plus the macromolecule albumin, with a pH buffered to around 7.2 and an osmolarity in the range 250–290 mOsmol (Brinster 1965). However, specifying quantitative demands is more difficult.

While ion concentrations tend to reflect those in physiological salts solutions with an increase in K⁺ concentration reflecting oviduct fluid, specifying the quantities of nutrients is not straightforward, as discussed by Leese (2003). Although most media are formulated to an osmolarity of 280–290 mOsmol, with a high Na concentration, at least one medium, KSOM (Lawitts and Biggers 1993) was based on empirical observations, with an osmolarity below 260 mOsmol and a correspondingly lower Na concentration.

As another example, one might start by measuring how much of a given nutrient the embryo consumes, but this depends on how much is provided (the higher the concentration the higher the consumption will tend to be) as well as the concentration of other nutrients in the medium. For example, a high lactate concentration will tend to dampen down glucose consumption, and while glutamine as a single amino acid substrate is consumed avidly by early embryos it is only taken up to a minor extent in the presence of all 20 amino acids (these are two of numerous such examples). Adopting an approach to defining quantitative requirements based on the kinetic properties of a given nutrient transport process is similarly flawed (Leese 2003).

In addition, overriding these considerations is the autonomy of the egg and early embryo, first documented by McLaren in 1976, who remarked that: Eggs and embryos are relatively autonomous and have astonishing regulatory powers (McLaren 1976). The most remarkable feature of embryo autonomy is the ability to develop through the whole preimplantation period when removed from the natural environment, while the ‘astonishing regulatory powers’ were well‐illustrated in a review by Lonergan et al. (2001) who summarized the capacity of oviducts from a variety of species to act as surrogate ‘hosts’ for embryos from different species. Thus, the ligated rabbit oviduct supports the development of sheep, cattle, pig, and horse embryos; excised, intact, mouse oviducts support sheep, cattle, pig, and hamster embryos; and the intact sheep oviduct supports embryos from sheep, cattle, pigs, and horses. The lumen environment will differ in each case, but this is obviously not critical to embryo development.

It is important to note that while the oviduct offers a benign, supportive environment to preimplantation embryos at all stages, this is not the case for the uterine environment, which, with the exception of the human and other primates, is hostile to embryos unless they are at the equivalent developmental stage (Hunter 2002).

These examples of embryo autonomy and adaptability are consistent with the high concentration of endogenous reserves mentioned previously, which will provide a buffer against changes in nutrient availability from the external environment.