Justin D. Vidal

Selected Background Findings and Interpretation of Common Lesions in the Female Reproductive System in Macaques.

The authors describe a selection of normal findings and common naturally occurring lesions in the reproductive system of female macaques, including changes in the ovaries, uterus, cervix, vagina, and mammary glands. Normal features of immature ovaries, uteri, and mammary glands are described. Common non-neoplastic lesions in the ovaries include cortical mineralization, polyovular follicles, cysts, ovarian surface epithelial hyperplasia, and ectopic ovarian tissue. Ovarian neoplasms include granulosa cell tumors, teratomas, and ovarian surface epithelial tumors. Common non-neoplastic uterine findings include loss of features of normal cyclicity, abnormal bleeding, adenomyosis, endometriosis, epithelial plaques, and pregnancy-associated vascular remodeling. Hyperplastic and neoplastic lesions of the uterus include endometrial polyps, leiomyomas, and rarely endometrial hyperplasia and endometrial adenocarcinoma. Vaginitis is common. Cervical lesions include endocervical squamous metaplasia, polyps, and papillomavirus-associated lesions. Lesions in the mammary gland are most often proliferative and range from ductal hyperplasia to invasive carcinoma. Challenges to interpretation include the normal or pathologic absence of menstrual cyclicity and the potential misinterpretation of sporadic lesions, such as epithelial plaques or papillomavirus-associated lesions. Interpretation of normal and pathologic findings is best accomplished with knowledge of the life stage, reproductive history, and hormonal status of the animal. Competing Interests: This article was sponsored by Covance Inc. and Schering-Plough. Gerhard F. Weinbauer and Eberhard Buse are employed by Covance Inc. Eveline P. C. T. de Rijk and Eric Van Esch are employed by Schering-Plough. No other competing interests were declared.

Biochemical Assessment of Limits to Estrogen Synthesis in Porcine Follicles

Abstract Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17α-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.

Standard morphologic evaluation of the heart in the laboratory dog and monkey

The nonrodent species most commonly utilized in preclinical safety studies are the purpose-bred beagle dog and cynomolgus macaque (Macaca fascicularis). Potential effects of a new chemical entity (NCE) on the heart pose serious concerns; consequently in vivo testing is focused on detection of functional alterations as well as morphological changes. Macroscopic and microscopic evaluation of the heart is based on a standard survey of key structures to properly assess presence of spontaneous and potential drug-induced lesions. Evaluation of historical controls to determine type and frequency of background change is valuable, as studies with non-rodent species generally have a small sample size. Archived control dog and monkey data were retrospectively reviewed, including terminal body weight (BW), heart weight (HW), and archival glass slides of heart. Control dogs had minimal background changes that included myxomatous or cartilagenous change in the cardiac skeleton and a variable degree of vacuolation in Purkinje fibers. Control monkey hearts commonly contained inflammatory cell infiltrates, myocyte anisokaryosis, and handling artifacts, while myocyte degeneration, squamous plaques, pigment, and intimal plaques were occasionally observed. These findings highlight the utility of consistently recorded and readily accessible archived control data when attempting to discern background spontaneous changes and artifacts from test-article induced changes.

Recommendations from the INHAND Apoptosis/Necrosis Working Group

Historically, there has been confusion relating to the diagnostic nomenclature for individual cell death. Toxicologic pathologists have generally used the terms “single cell necrosis” and “apoptosis” interchangeably. Increased research on the mechanisms of cell death in recent years has led to the understanding that apoptosis and necrosis involve different cellular pathways and that these differences can have important implications when considering overall mechanisms of toxicity, and, for these reasons, the separate terms of apoptosis and necrosis should be used whenever differentiation is possible. However, it is also recognized that differentiation of the precise pathway of cell death may not be important, necessary, or possible in routine toxicity studies and so a more general term to indicate cell death is warranted in these situations. Morphological distinction between these two forms of cell death can sometimes be straightforward but can also be challenging. This article provides a brief discussion of the cellular mechanisms and morphological features of apoptosis and necrosis as well as guidance on when the pathologist should use these terms. It provides recommended nomenclature along with diagnostic criteria (in hematoxylin and eosin [H&E]-stained sections) for the most common forms of cell death (apoptosis and necrosis). This document is intended to serve as current guidance for the nomenclature of cell death for the International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Groups and the toxicologic pathology community at large. The specific recommendations are: Use necrosis and apoptosis as separate diagnostic terms. Use modifiers to denote the distribution of necrosis (e.g., necrosis, single cell; necrosis, focal; necrosis, diffuse; etc.). Use the combined term apoptosis/single cell necrosis when There is no requirement or need to split the processes, or When the nature of cell death cannot be determined with certainty, or When both processes are present together. The diagnosis should be based primarily on the morphological features in H&E-stained sections. When needed, additional, special techniques to identify and characterize apoptosis can also be used.

Yolk Coelomitis in Fiji Island Banded Iguanas (Brachylophus fasciatus)

Abstract Yolk coelomitis is a major cause of death in captive sexually mature female Fiji Island banded iguanas (Brachylophus fasciatus) maintained by the Zoological Society of San Diego. The medical records, breeding histories, and pathology archival materials from this group were reviewed to study this health problem. From 1987 through 2004, deaths of nine of 21 adult females were due to yolk coelomitis. Most iguanas had a history of reproduction-related problems, which included reproductive failure, episodes of lethargy associated with ovarian activity, folliculostasis, ovostasis, and behavioral abnormalities. Most affected iguanas either were found dead or presented moribund and subsequently died or were euthanized. Clinical signs were nonspecific and included lethargy, cutaneous discoloration, and coelomic effusion. Yolk leakage in most cases was associated with the presence of large vitellogenic follicles undergoing atresia and resulted in coelomitis characterized by florid mesothelial proliferation.

Morphologic manifestations of testicular and epididymal toxicity

Histopathologic examination of the testis is the most sensitive means to detect effects on spermatogenesis; however, the complexity of testicular histology, interrelatedness of cell types within the testis, and long duration of spermatogenesis can make assessment of a testicular toxicant challenging. A thorough understanding of the histology and morphologic manifestations of response to injury is critical to successfully identify a testicular effect and to begin to understand the underlying mechanism of action. The basic patterns of response to xenobiotic-induced injury to the testis and epididymis are detailed and discussed.

Evaluation of the Cynomolgus Monkey Stomach: Recommendations for Standard Sampling Procedures in Nonclinical Safety Studies

The cynomolgus macaque is the most commonly used nonhuman primate in nonclinical toxicity testing, but the macroscopic and microscopic anatomy of the stomach in the cynomolgus macaque is poorly described. To develop a reliable sampling method for histologic evaluation of the cynomolgus macaque stomach in regulatory toxicity studies, the stomachs of control animals were prospectively evaluated using an extensive sectioning pattern. The stomach of the cynomolgus macaque differs from that described for the human stomach and has a prominent fundus that lacks parietal cells. A description of the macroscopic and microscopic anatomy is presented along with a recommended sectioning pattern for nonclinical toxicity studies and discussion of species differences. A thorough understanding of normal anatomy and species comparisons are critical to interpretation of potential toxicity findings and assessment of risk in humans.

Estrogen replacement therapy induces telomerase RNA expression in the macaque endometrium

OBJECTIVE To evaluate the effects of hormonal therapies on the expression of telomerase RNA (TRNA) in the endometrium of ovariectomized female cynomolgus macaques (Macaca fascicularis). DESIGN Randomized long-term experimental trial. SETTING Animal study at an academic research institution. PATIENT(S) Surgically postmenopausal cynomolgus macaques. INTERVENTION(S) Treatments were given in the diet for three years and included conjugated equine estrogens (CEE), CEE + medroxyprogesterone acetate (MPA), and tamoxifen, at clinically relevant doses. MAIN OUTCOME MEASURE(S) Expression of TRNA within the basal glands, basal stroma, superficial glands, and superficial stroma of the endometrium by radiolabeled in situ hybridization. RESULT(S) Conjugated equine estrogens increased glandular TRNA expression, and the addition of MPA decreased this effect. Tamoxifen induced glandular TRNA expression to a lesser degree. Both CEE + MPA and tamoxifen increased stromal TRNA expression. The expression of TRNA in the endometrial glands was always greater than TRNA expression in the stroma. Treatment groups with greater proliferation and progesterone receptor expression also had elevated TRNA; within-group correlations were not significant. No statistically significant difference occurred between the basal and superficial endometrial layers. CONCLUSION(S) These results show for the first time a cell-specific hormonal regulation of TRNA in the primate endometrium, with up-regulation of TRNA by treatments associated with increased risk of endometrial cancer in women.

A Monograph on Female Reproductive Pathophysiology in Macaques

the reproductive system, we have chosen to include a manuscript detailing hormonal changes across the menstrual cycle with particular reference to the experimental setting. We have also covered methods related to collection, processing, sectioning, and staining of female reproductive system tissues. Hopefully, topics covered will serve as a practical guide to evaluation of the female reproductive system in macaques and to the use of this evaluation in relating findings to those in women.

Oviduct, Uterus, and Vagina

Abstract The oviduct, uterus, and vagina are part of the female reproductive system and are specialized organs that serve to transport oocytes to the site of fertilization and implantation, to support fetal growth and nourishment, and to connect the growing fetus to the external environment (outside of the body) at the time of birth. The oviduct, uterus, and vagina are responsive to and produce steroid and protein hormones that may act locally or impact organs at distant sites. With the advent of more frequent testing of suspected endocrine disrupting compounds, the organs of the female reproductive system are more often being identified as targets of xenobiotic-induced changes. In rats, both spontaneous and compound-induced alterations can be observed in the female reproductive system and may occur due to direct effects on the oviduct, uterus, and vagina or secondarily as a result of effects on the hypothalamic–hypophyseal–gonadal axis. This chapter describes commonly observed spontaneous and compound-related, non-neoplastic and neoplastic changes of the oviduct, uterus, and vagina.