236 PHYSIOLOGIC GENETICS 



In any case, it will be useful to know the precise stage at which the embryo is 

 exposed to the teratogen. For this reason an agent such as irradiation, that reaches 

 the embryo immediately, may be chosen in preference to one such as cortisone, for which 

 an indeterminate delay occurs between the time the agent is applied to the mother 

 and the time it, or its metabolic consequences, reaches the embryo in effective quantities. 

 Treatment with specific metabolic inhibitors (such as 6-aminonicotinamide) followed 

 shortly afterward by a protective substance (in this case, nicotinamide) may also be 

 used to achieve a short teratogenic episode, precisely timed. 1008 



The use of analogs as teratogens has the further advantage that their metabolic 

 effects can be inferred from their chemical nature, and study of the embryonic effects 

 of specific inhibitions, at particular stages of development, may provide information 

 about the biochemical properties of the embryo. Further factors influencing the 

 choice of teratogens will become apparent later in this discussion. 



DEVELOPMENTAL STAGE AT WHICH THE TERATOGEN IS USED 



In studying the effects of an agent on development, it is obviously useful to know 

 the gestational stage at which the teratogen is applied. This is usually done by 

 applying the teratogen at a given time after mating, but sometimes by counting back 

 from parturition, assuming gestation length is known from previous observations. 

 The latter is an unreliable method. The time of mating can be established, within 

 limits, by placing the female with a male for a known period of time and observing her 

 at the end of this period for signs of insemination, such as a vaginal plug in mice or 

 rats, or the presence of sperm in the vagina. The frequency of successful matings can 

 be increased by exposing the female to the male when she is in the appropriate stage of 

 estrus, as established by vaginal smear or other signs, depending on the species. It 

 may also be useful (in mice, at least) to maintain the females in a room with a regular 

 light-dark cycle, so that their estrus cycles become synchronized, and they can be 

 exposed to the males at the appropriate time in the cycle. 



No matter how precisely the time of mating is known, there is no assurance that 

 all embryos are exposed at the same developmental stage, since within one litter there 

 may be variations in time of ovulation, fertilization, implantation, and post- 

 implantation development, so that no matter how accurately the time from insemina- 

 tion is measured, there is often quite marked variability in developmental stage from 

 one littermate to another. Variation in developmental rate between litters and 

 between strains (or even sublines within strains) must also be taken into account, by 

 using adequate numbers of animals and appropriate controls. 



Some confusion exists in the literature as to the terminology of timing gestational 

 stages. Some authors refer to the day on which signs of mating are observed as the 

 first day of pregnancy, or day 1 . Others refer to it as day 0. Care should be taken 

 to specify which interpretation is meant in any particular case. The latter system is 

 preferable. 



