Dorothy E. Boatman

In Vitro Growth of Non-Human Primate Pre- and Peri- Implantation Embryos

The close phylogenetic relationship between man and non-human primates has made these animals, especially the rhesus monkey, of particular interest for scientific research. However, since the early pioneer work (Hartman and Corner, 1941; Heuser and Streeter, 1941; Lewis and Hartman, 1941), only a few investigators have studied preimplantation embryology in non-human primates. A major reason for this situation is the expense of maintaining these long-lived animals (at least 30 years in captivity), which are largely monotocous and normally yield relatively few embryos in each reproductive season (albeit females may have as many as 18 reproductive seasons [years]). Additionally, some standard animal research procedures that would curtail the fertility of the animals (e. g., the excision of oviducts for ova/embryo flushing) may be contraindicated by the goals of breeding programs that have been established to maintain adequate supplies of these animals. In the past, the scarcity of non-human primate embryos has tended to obviate their clear appropriateness as models for the study of early human development. Recent advances in the techniques of super-stimulation of follicular growth, in vitro fertilization (IVF) and embryo culture have increased the supply of non-human primate embryos (Bavister et al., 1983a; Boatman and Bavister, 1984; Boatman et al., 1986; Balmaceda et al., 1984).

Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo

The intracellular magnesium and calcium ion concentrations of in vivo‐developed 2‐cell hamster embryos were measured using ratiometric fluorometry. Intracellular magnesium and calcium ion concentrations were found to be 0.369 ± 0.011 mM and 129.3 ± 7.5 nM respectively. Culture of 1‐cell hamster embryos for 24 hr to the 2‐cell stage in control medium containing 0.5 mM magnesium and 2.0 mM calcium resulted in approximately a threefold increase to 343.5 ± 8.0 nM in intracellular calcium ion concentration, while magnesium ion levels were not altered (0.355 ± 0.007 mM). Increasing medium magnesium concentrations to 2.0 mM significantly increased intracellular magnesium ion concentrations of cultured 2‐cell embryos with a concomitant reduction in intracellular calcium ion concentrations. Furthermore, increasing the medium magnesium concentration to 2.0 mM significantly increased development of 1‐cell embryos collected at either 3 or 9 hr post‐egg activation to the morula/blastocyst and blastocyst stages. Resultant blastocysts had an increased total cell number and increased development of the inner cell mass. Most important, however, culture with 2.0 mM magnesium increased the fetal potential of cultured 1‐cells twofold. Therefore, because highest rates of development were observed in a medium that resulted in reduced intracellular calcium ion concentrations, it appears that altered calcium homeostasis is associated with impaired developmental competence of 1‐cell embryos in culture. Mol. Reprod. Dev. 50:443–450, 1998.

Kinematics of trophectoderm projections and locomotion in the peri-implantation hamster blastocyst

The behavior of Golden hamster blastocysts was studied in vitro by continuous time‐lapse videomicrography and computer imaging, during and immediately following escape from the zona pellucida. This study revealed numerous small cytoplasmic trophectoderm projections (TEPs) approximately 18 μm long that penetrated the zona pellucida both radially and tangentially and appeared to be actively involved in zona escape in vitro. After escape from their zonae, some blastocysts moved across the culture dish by an endogenous means of locomotion, most likely involving activity of the small TEPs. Several hours after zona escape, embryos expressed large TEPs up to 46 μm long that moved in an undulating manner and showed rapid cycles of extension and retraction; the timing of their appearance suggested that these TEPs are normally involved in attachment to the uterine epithelium. Embryos fixed in utero, during the developmental interval between zona loss and embryo attachment, exhibited large TEPs similar in morphology to those expressed by cultured blastocysts. These observations document for the first time that mammalian blastocysts are capable of endogenous locomotion, confirm TEPs as components of normal blastocyst activity, reveal that there are two kinds of TEPs that differ temporally and morphologically, and extend earlier reports of TEP activity in guinea‐pig embryos to the hamster.

Fertilization in the Rhesus Monkey

The nonhuman primates occupy a unique niche among species suitable for experimental studies on fertilization and early development. Studies with nonhuman primates provide an important link between the huge literature derived from work on rodents and other common laboratory species and the relatively small amount of data on humans. Research with rodent species continues to provide the bulk of basic information on early development, in large part because of the ready availability of oocytes and embryos, but it is difficult to know to what extent information can be extrapolated to primate species, including humans. Information is increasingly available from the numerous human in vitro fertilization (IVF) programs now in operation. However, the amount and quality of basic scientific data on early development that can be obtained from this source are restricted, in part by ethical constraints (Austin, 1990) and in part by conflicting priorities between research and clinical needs for the supply of oocytes and embryos. For example, most “excess” human IVF embryos in the U.S.A. are destined for cryopreservation or for use in a donor program and are thus unsuitable for any research protocol that might compromise their viability. In contrast, there are no prohibitions on the use of fertilized eggs or embryos of nonhuman primates for research, while the reproductive physiology and embryology of the Old-World nonhuman primates are sufficiently similar to humans that direct extrapolation of concepts is possible.

IVF in Nonhuman Primates: Current Status and Future Directions

A number of laboratories during the past two decades have contributed to the current technical status of nonhuman primate IVF. A major rationale for interest in nonhuman primate IVF was, and still is, to provide data on early development that could be directly applicable to humans. However, it is ironic that progress in production of human embryos by IVF has always been somewhat more advanced than that in nonhuman primates. Thus, although the feasibility of IVF in monkeys was demonstrated by the early 1970s, the first documented human IVF took place several years earlier (Table 2.1). In the early years of IVF research in humans and in monkeys, it was difficult to demonstrate much progress beyond fertilization itself or cleavage to 2 cells. Yet the birth of the first human IVF baby (1) preceded by several years the first demonstrations of live births in nonhuman primates derived from IVF eggs (2, 3). Nevertheless, the chronology of these events does not mean that nonhuman primate IVF cannot point the way to significant improvements in human IVF technology nor increase understanding of key areas in human reproduction. Rather, the indication is that IVF technology in nonhuman primates may be best employed to examine specific events, such as oocyte maturation, sperm capacitation, or development of new culture media, that are more difficult to study in the context of human clinical IVF.