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

652 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONin the rate of cell proliferation but did accelerate the rate of differentiation. This was manifestedby an increase in the rate of cavitation, which serves as a measure of trophectoderm differentiation.In a study by Oudiz et al., 17 male mice were treated with ethylene glycol monomethyl ether(EGME) or remained untreated and were mated with superovulated females. Cells from the resultingembryos were disaggregated; those from embryos sired by control males were labeled with FITC,and chimeras were formed from the cells obtained from the control and experimental matings.Proliferation ratios calculated using this approach showed that embryos from EGME male matingshad significant proliferation disadvantages compared with embryos obtained entirely from controlmating.Large quantities of unfertilized eggs collected as cumulus masses after superovulation of thenonpregnant animal can also be used for experiments looking at direct toxicity to the gamete ormechanisms involving the process of IVF. The latter approach provides the distinct added advantageof being able to synchronize development for better dose and response fidelity in evaluation ofchemical effects. An example of the use of this approach in developmental toxicity studies wasreported by Kholkute et al. 18 using B6D2F1 mice. They describe the effects of polychlorinatedbiphenyl (PCB) mixtures (Arochlor 1221, 1254, and 1268) and the individual congener (3,3′,4,4′-tetrachlorobiphenyl) added directly to the IVF medium containing capacitated sperm. Direct effectson fertilization and increased incidence of oocyte degeneration and abnormalities were reported.Additional modifications of this approach are possible but have not been thoroughly exploited forthe evaluation of developmental toxicity mechanisms.Success rates of IVF are sometimes low but can be improved considerably through coculturewith oviductal epithelial cells and/or by supplementation from appropriate growth factors. Thisprocedure will yield higher rates of fertilization and more robust embryos because of the expectedcontribution of natural growth factors provided by the epithelial cells. Such an approach mayprovide important insights into the toxicological significance of oviduct response and regulation.The combination of these two in vitro methods may provide an even more relevant environmentfor mechanistic studies of early development. For example, the direct and interactive consequencesof chemical exposure in the oviduct may be addressed by use of primary cultures of oviductepithelium with early preimplantation embryos under IVF conditions.In vitro evaluations of the response of cultured oviduct epithelial cells to modulators of glutathione(GSH) status and a pesticide (lindane) have been completed. They show that oxidativestress and altered cellular thiol status in the oviduct may lead to altered function and subsequentlyinfluence embryonic growth and development. 19,20 This approach has the mechanistic advantage ofallowing evaluations of chemical effects on the developmental environment, including the nutritiveand signaling functions of the oviduct epithelium. Investigations are underway to elucidate functionalchanges elicited in oviduct function and subsequent effects on the process of fertilization invitro. These studies should provide valuable mechanistic information regarding the manner in whichtoxicants exert direct effects on fertilization and early embryogenesis and also regarding theenvironment in which these events take place.Eggs and embryos during the one- and two-cell stages are particularly sensitive to environmentalconditions, especially ambient temperature and oxygen concentration. Several species differ withrespect to the ease with which their embryos undergo cleavage, compaction, and blastocyst formation,and progress through development. Because of a much higher success rate for culturing twocellembryos through to the blastocyst stage, the mouse is generally the species of choice for thesetypes of experiments. A characteristic difficulty in culturing embryos of several species, includingmouse and human, is a developmental two- or four-cell “block,” beyond which the fertilized embryois unable to progress. Usually considered an artifact of in vitro growth and closely associated withoxidative stress, this condition may actually hold relevance to some mechanisms of early reproductivefailure elicited by chemical agents. Indeed, recent analyses have provided evidence thatreactive oxygen species generated in vitro are responsible for the developmental arrest. 21,22© 2006 by Taylor & Francis Group, LLC