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

DEVELOPMENTAL AND REPRODUCTIVE TOXICITY STUDY FINDINGS 391spermatogonia is more likely to produce a permanent deficit in fertility than a compound that causesspermatid retention. Interpretation of testicular histopathologic data should be conducted in conjunctionwith assessment of sperm parameters, testicular weights, fertility data, and macroscopicdata. The testicular histopathologic examination may be the most sensitive indicator of toxicity,particularly when a lesion is observed to have been dose responsive. Presence of debris and sloughedcells in the epididymides can also provide indicators of adverse reproductive effects. Rarely areadverse reproductive effects manifested primarily in the prostate or seminal vesicles.Female reproductive organs that are usually weighed include the uterus and the ovaries; nonreproductiveorgans include the brain, pituitary, and thyroid gland. When evaluating uterine weight,it is essential to determine the stage of estrus of the animal at the time of necropsy. This is bestaccomplished by histopathologic examination of the vagina and uterus. A lavage of vaginal contentsmay also be taken prior to necropsy and examined by light microscopy. However, the stagedetermined from the vaginal lavage is approximately one-half day behind the stage determined byhistopathologic examination of the uterus and vagina. Therefore, results of histopathologic examinationsmay not be consistent with vaginal lavage data, particularly if the vaginal lavage isperformed several hours prior to the necropsy. Fluid retention and cellular proliferation vary greatlyin female rats, depending on the stage of estrus. Weights are highest during the estrus stage andlowest during the diestrus stage. Unless uterine weight is correlated to stage of estrus, false positiveand false negative interpretations may result.Reproductive organs subjected to histopathologic examination in the female include the ovaries,uterus, vagina, and mammary glands. Examination of these organs in combination with vaginalcytology data provides a determination as to whether the female is cycling normally, has enteredan anovulatory stage, or has entered premature reproductive senescence.7. Estrus Cyclicity and Precoital IntervalThe normal rat estrous cycle is four or five days in duration. For the original and extensive treatmentof this topic, refer to Long and Evans. 105 The techniques and principles for determining the estrouscycle have remained the same as in this original reference. However, the application and understandingof parameters of the estrous cycle have greatly improved in the last two decades. In theauthors’ laboratory, the estrous cycle is divided into the following four stages: estrus (E), metestrus(M), diestrus (D), and proestrus (P). During E, the majority of the cells present are cornifiedepithelial cells. These cells are irregularly shaped and may or may not have remnants of a nucleus.Some nucleated epithelial cells may be present; however, no leukocytes are present. Estrus usuallylasts 10 to 15 h. During M, nucleated epithelial cells are surrounded by increasing numbers ofleukocytes. Metestrus is the shortest stage, lasting approximately 6 h. At the beginning of D, celltypes are equally distributed among leukocytes, cornified epithelial cells, and nucleated epithelialcells. During the later part of this stage, the nucleated epithelial cells become more spherical inappearance. Diestrus is the longest stage, lasting two to three days. During P, the majority of cellsare nucleated epithelial cells, with some cornified epithelial cells that become more prevalent asthe E stage approaches. The nucleated epithelial cells appear spherical in shape and are usuallygrouped in clusters. Proestrus usually lasts 8 to 12 h.To determine the estrous cycle pattern in rats, vaginal lavages are performed once per day.Female rats will only be receptive for mating on the evening of the P stage. Therefore, animalsthat do not proceed through a cycle will not mate. It is not unusual for animals to occasionallyhave an extended cycle (six or more days); however, repeated extended cycles may be an indicatorof reproductive toxicity.A variety of methods are used to evaluate the estrous cycle. One approach is to determine thepercentage of days that an animal is in each stage of the cycle (i.e., the percentage of days in E,M, D, or P). Another approach is to determine the number of animals that have one or more irregularcycles (i.e., a cycle of six or more days or a cycle of three or fewer days). Lavages are usually© 2006 by Taylor & Francis Group, LLC