Amera K. Remick

Ovarian Follicle Counts Using Proliferating Cell Nuclear Antigen (PCNA) and Semi-Automated Image Analysis in Rats

Ovarian follicle counting is a method to assess ovarian toxicity in reproductive toxicity studies in rats. Although ovarian follicle counting has been traditionally performed manually on hematoxylin and eosin (H&E)-stained sections, the use of immunohistochemical methods, including human cytochrome P450 1B1 (CYP1B1) and proliferating cell nuclear antigen (PCNA), have been used to enhance the visibility of the primordial and primary follicles to facilitate manual counting. In this study, serial sections from both ovaries from ten 3-month-old female Sprague Dawley rats were stained using routine H&E and immunohistochemistry for PCNA. Counting of primordial and primary follicles was performed manually using these two stains and by semi-automated image analysis of PCNA-stained slides. Although manual counting of PCNA-stained slides is preferable to manual counting of H&E-stained slides, manual counting involves variability between individual counters. Semi-automated image analysis of PCNA-stained slides yields an accurate and consistent count of these primordial/primary follicles and eliminates variability between individual counters.

Postnatal Development of the Testis in the Rat Morphologic Study and Correlation of Morphology to Neuroendocrine Parameters

Histopathologic examination of the testis from juvenile rats is often necessary to characterize the safety of new drugs for pediatric use and is a required end point in male pubertal development and thyroid function assays. To aid in evaluation and interpretation of the immature testis, the characteristic histologic features of the developing rat testis throughout postnatal development are described and correlated with published neuroendocrine parameter changes. During the neonatal period (postnatal day [PND] 3–7), seminiferous tubules contained gonocytes and mitotically active immature Sertoli cells. Profound proliferation of spermatogonia and continued Sertoli cell proliferation occurred in the early infantile period (PND 8–14). The spermatogonia reached maximum density forming double-layered rosettes with Sertoli cells in the late infantile period (PND 15–20). Leptotene/zygotene spermatocytes appeared centrally as tubular lumina developed, and individual tubules segregated into stages. The juvenile period (PND 21–32) featured a dramatic increase in number and size of pachytene spermatocytes with the formation of round spermatids and loss of “infantile” rosette architecture. In the peri-pubertal period (PND 32–55), stage VII tubules containing step 19 spermatids were visible by PND 46. The presented baseline morphologic and endocrinologic information will help pathologists distinguish delayed development from xenobiotic effects, determine pathogenesis when confronted with nonspecific findings, and identify sensitive time points for targeted study design.

Postnatal Ovary Development in the Rat Morphologic Study and Correlation of Morphology to Neuroendocrine Parameters

Histopathologic examination of the immature ovary is a required end point on juvenile toxicity studies and female pubertal and thyroid function assays. To aid in this evaluation and interpretation of the immature ovary, the characteristic histologic features of rat ovary through the developmental periods are described. These histologic features are correlated with published changes in neuroendocrine profiles as the hypothalamic–pituitary–gonadal axis matures. During the neonatal stage (postnatal day [PND] 0–7), ovarian follicle development is independent of pituitary gonadotropins (luteinizing hormone [LH] or follicle-stimulating hormone [FSH]), and follicles remain preantral. Antral development of “atypical” follicles occurs in the early infantile period (PND 8–14) when the ovary becomes responsive to pituitary gonadotropins. In the late infantile period (PND 15–20), the zona pellucida appears, the hilus forms, and antral follicles mature by losing their “atypical” appearance. The juvenile stage (PND 21–32) is the stage when atresia of medullary follicles occurs corresponding to a nadir in FSH levels. In the peripubertal period (PND 33–37), atresia subsides as FSH levels rebound, and LH begins its bimodal surge pattern leading to ovulation. This report will provide pathologists with baseline morphologic and endocrinologic information to aid in identification and interpretation of xenobiotic effects in the ovary of the prepubertal rat.

Histologic Features of Prepubertal and Pubertal Reproductive Development in Female Sprague-Dawley Rats

In response to growing concerns that environmental chemicals may have adverse effects on human health by altering the endocrine system, the Endocrine Disruptor Screening Program (EDSP), under the auspices of the United States Environmental Protection Agency (U.S. EPA), recently instituted a Tier I battery of tests including a female pubertal assay. This assay requires dosing of female rats from postnatal day (PND) 22 through PND 42 (or 43), the period of pubertal development in the rat, to identify test articles that may have estrogenic or antiestrogenic effects, or may alter hormones or neurotransmitters. While certain landmarks in female rat reproductive development are published, little is published on the microscopic appearance of the female reproductive tract during prepubertal and pubertal development. In this study, reproductive tissues from three female Sprague-Dawley rats were collected each day from PND 20 through PND 50, such that tissues from a total of 93 rats were collected throughout the prepubertal and pubertal period. Tissues were formalin-fixed, trimmed, paraffin-embedded, sectioned at 5-µm thickness, and examined microscopically. The major histologic features of the female reproductive tract throughout this critical period were described in detail. This information will help pathologists interpret findings observed in female pubertal assays.

Scientific and Regulatory Policy Committee Points to Consider Review: Inclusion of Reproductive and Pathology End Points for Assessment of Reproductive and Developmental Toxicity in Pharmaceutical Drug Development.

Standard components of nonclinical toxicity testing for novel pharmaceuticals include clinical and anatomic pathology, as well as separate evaluation of effects on reproduction and development to inform clinical development and labeling. General study designs in regulatory guidances do not specifically mandate use of pathology or reproductive end points across all study types; thus, inclusion and use of these end points are variable. The Scientific and Regulatory Policy Committee of the Society of Toxicologic Pathology (STP) formed a Working Group to assess the current guidelines and practices on the use of reproductive, anatomic pathology, and clinical pathology end points in general, reproductive, and developmental toxicology studies. The Working Group constructed a survey sent to pathologists and reproductive toxicologists, and responses from participating organizations were collected through the STP for evaluation by the Working Group. The regulatory context, relevant survey results, and collective experience of the Working Group are discussed and provide the basis of each assessment by study type. Overall, the current practice of including specific end points on a case-by-case basis is considered appropriate. Points to consider are summarized for inclusion of reproductive end points in general toxicity studies and for the informed use of pathology end points in reproductive and developmental toxicity studies.

Juvenile Toxicology: Relevance and Challenges for Toxicologists and Pathologists

The Society of Toxicologic Pathology (STP) Education Committee and the STP Reproductive Special Interest Group held a North Carolina regional meeting entitled, “Juvenile Toxicology: Relevance and Challenges for Toxicologists and Pathologists” on March 13, 2015, at the National Institute of Environmental Health Sciences/National Toxicology Program in Research Triangle Park, North Carolina. The purpose of this regional meeting was to familiarize attendees with the topic of juvenile toxicity testing and discuss its relevance to clinical pediatric medicine, regulatory perspectives, challenges of appropriate study design confronted by toxicologists, and challenges of histopathologic examination and interpretation of juvenile tissues faced by pathologists. The 1-day meeting was a success with over 60 attendees representing industry, government, research organizations, and academia.

Impact of Age on the Male Reproductive System from the Pathologist’s Perspective:

Age, and in particular young age, can significantly impact the response to toxicants in animals and can greatly influence the interpretation of tissue changes by the toxicologic pathologist. Although this applies to multiple organ systems, the current review focuses on the male reproductive system. When performing microscopic evaluation of male reproductive organs, the toxicologic pathologist must be aware of the dynamic changes in histomorphology, predominantly driven by timed hormonal alterations, at various life stages. Specific challenges pathologists face are understanding the appearance of male reproductive tissues throughout the neonatal, infantile, and juvenile developmental periods, recognizing when normal looks abnormal during tissue development, defining sexual maturity, and working with high interanimal variability in maturation rate and histologic appearance in developing large laboratory animals, such as nonhuman primates, dogs, and pigs. This review describes postnatal development of the male reproductive system in the rat, demonstrates how assessing toxicity during a defined window of postnatal development in the rat may improve definition of toxicant timing and targets, and discusses challenges associated with the interpretation of toxicity in immature large animal species. The emphasis is on key age-related characteristics that influence the interpretation of tissue changes by the toxicologic pathologist.

Points to Consider in Designing and Conducting Juvenile Toxicology Studies

In support of a clinical trial in the pediatric population, available nonclinical and clinical data provide input on the study design and safety monitoring considerations. When the existing data are lacking to support the safety of the planned pediatric clinical trial, a juvenile animal toxicity study is likely required. Usually a single relevant species, preferably a rodent, is chosen as the species of choice, while a nonrodent species can be appropriate when scientifically justified. Juvenile toxicology studies, in general, are complicated both conceptually and logistically. Development in young animals is a continuous process with different organs maturing at different rates and time. Structural and functional maturational differences have been shown to affect drug safety. Key points to consider in conducting a juvenile toxicology study include a comparative development of the organ systems, differences in the pharmacokinetics/absorption, distribution, metabolism, excretion (PK/ADME) profiles of the drug between young animal and child, and logistical requirement in the juvenile study design. The purpose of this publication is to note pertinent points to consider when designing and conducting juvenile toxicology studies and to aid in future modifications and enhancements of these studies to enable a superior predictability of safety of medicines in the pediatric population.

Male Reproductive System

Abstract The male reproductive system is composed of the testes, epididymis, vas deferens, and male accessory sex glands, including the prostate and seminal vesicles. Other structures, such as the efferent ducts, bulbourethral gland, and ejaculatory ducts are part of the male reproductive system, but will not be discussed in this atlas since they are not routinely examined in nonclinical toxicology studies. The various segments of the male reproductive tract become histologically mature at various times. The testes are histologically mature by PND 46 when elongated step 19 spermatids are produced and ready for release. The epididymis is histologically mature by PND 35 when all the various epithelial cell types can be recognized, and the vas deferens is histologically mature by PND 21. The seminal vesicles and the prostate gland, being the two accessory glands routinely examined in preclinical safety toxicology, are both histologically mature by PND 28.

Female Reproductive System

Abstract The female reproductive tract, composed of the ovaries, oviducts, uterus, cervix, and vagina, is immature at birth and develops into a fully functional reproductive unit by PND 42. The ovary is considered mature once the rat has experienced at least two ovulatory cycles, and this typically occurs by PND 42. Not all segments of the reproductive tract mature at the same rate as the ovary. The epithelium of the oviduct has all its components by PND 21 and the epithelium of the vagina is hormonally responsive by PND 30. The aim of this chapter is to describe the postnatal histological development of the female reproductive tract tissues and highlight important developmental landmarks in each tissue at the various postnatal ages.