A Practical Approach, Second Edition=Ronald D. Ho.pdf

658 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONIn vitro development7.5–day +24 h +48 h +72 h−Figure 16.3Scanning electron micrographs of the best mouse embryos cultured in one experiment, showingthe extent of growth and morphogenesis that may be achieved in vitro (bar: 100 µm). Day 8.5embryos (plug day = day 0) were cultured in rotating bottles containing DR50 medium (changedafter 24 h of culture) and gassed intermittently with 5% O 2 , 5% CO 2 , and 90% N 2 for the first 38h; they were then cultured in fresh DR50 medium in a gas phase with 20% O 2 , 5% CO 2 and 75%N 2 until 48 h. Embryos were then cultured in rotating bottles gassed continuously with 20% to40% O 2 until 72 h in vitro. (From Sturm, K. and Tam, P.P.L., Methods Enzymol., 225, 164, 1993.With permission.)transformations, including conversion from an essentially anaerobic, glycolysis-based metabolismto an aerobic, citric acid cycle–based dependence. In the mouse, the window of optimal in vitrogrowth and differentiation begins on gestational day (GD) 8 (plug day = GD 0). In the rat, thisperiod begins on GD 9.5. In each case, a period of 48 h in culture is believed to encompass theoptimal period wherein the in vitro conceptus will grow at almost the same rate as if it had remainedin the uterus, although earlier and later stages have also been grown successfully. The entirespectrum of in vitro growth, spanning a period of 72 h, is shown in Figure 16.3. 61While most of the early efforts to perfect whole embryo culture have utilized mouse and ratconceptuses, whole embryo culture of other species, such as the rabbit, is becoming more common.Rabbit whole embryo culture has not been widely used in toxicological investigations but has greatpotential. In the past decade, procedures for rabbit whole embryo culture have been developedlargely by the efforts of Naya and coworkers, 65 Ninomiya and coworkers, 66 and Pitt and Carney. 67,68Criteria for uniform evaluation of rabbit conceptuses have also been developed and used as astandard of reference during in vitro development. 67,68 While the logistics of rabbit whole embryoculture are similar to those for rodent embryo culture (maximum culture time, timing of explantation,periods of organogenesis, gassing conditions, etc.), some physiologic differences requirespecial attention and make rabbit embryo culture unique (Figure 16.4).Rabbit conceptuses require that a portion of the bidiscoid placenta remain attached throughoutthe culture period. Although not critical for conceptual viability, the placental bed serves the functionof keeping the conceptus intact during the course of incubation. A striking difference between therabbit and rodent conceptus is that the rabbit VYS only surrounds the caudal portion of the partiallyrotated embryo, unlike mouse and rat where the VYS is inverted and completely surrounds the© 2006 by Taylor & Francis Group, LLC