Mammary Gland Evaluation in Juvenile Toxicity Studies: Temporal Developmental Patterns in the Male and Female Harlan Sprague-Dawley Rat.

There are currently no reports describing mammary gland development in the Harlan Sprague-Dawley (HSD) rat, the current strain of choice for National Toxicology Program (NTP) testing. Our goals were to empower the NTP, contract labs, and other researchers in understanding and interpreting chemical effects in this rat strain. To delineate similarities/differences between the female and male mammary gland, data were compiled starting on embryonic day 15.5 through postnatal day 70. Mammary gland whole mounts, histology sections, and immunohistochemically stained tissues for estrogen, progesterone, and androgen receptors were evaluated in both sexes; qualitative and quantitative differences are highlighted using a comprehensive visual timeline. Research on endocrine disrupting chemicals in animal models has highlighted chemically induced mammary gland anomalies that may potentially impact human health. In order to investigate these effects within the HSD strain, 2,3,7,8-tetrachlorodibenzo-p-dioxin, diethylstilbestrol, or vehicle control was gavage dosed on gestation day 15 and 18 to demonstrate delayed, accelerated, and control mammary gland growth in offspring, respectively. We provide illustrations of normal and chemically altered mammary gland development in HSD male and female rats to help inform researchers unfamiliar with the tissue and may facilitate enhanced evaluation of both male and female mammary glands in juvenile toxicity studies.

A single gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin disrupts the adult uterine response to estradiol in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) given as a cotreatment with estrogen exhibits antiestrogenic properties on the rodent adult uterus, but less is understood regarding hormonal responsiveness of the adult uterus from animals having been exposed to TCDD during critical periods of development. We characterized the inhibitory effects of TCDD (T) exposure at gestational day 15 (GD15), 4 weeks, and 9 weeks of age (TTT) on the adult uterus following hormone treatment. TTT-exposed mice in response to hormone treatment exhibited a blunted weight increase, had fewer uterine glands, displayed morphological anomalies, and had marked decreases in the hormonal regulation of genes involved in fluid transport (Aqp3 and Aqp5), cytoarchitectural (Dsc2 and Sprr2A), and immune (Lcn2 and Ltf) regulation. To determine if the 9-week exposure was responsible for the blunted uterine response, due to the 7- to 11-day half-life of TCDD in mice, a second set of experiments was performed to examine exposure to TCDD given at GD15, GD15 only (cross-fostered at birth), only during lactation (cross-fostered at birth), or at GD15 and 4 weeks of age. Our studies demonstrate that a single developmental TCDD exposure at GD15 is sufficient to elicit a blunted adult uterine response to estradiol and is due in part to fewer gland numbers and the reduced expression of forkhead box A2 (FoxA2), a gene involved in gland development. Together, these results provide insight regarding the critical nature of in utero exposure and the potential impact on ensuing uterine biology and reproductive health later in life.

Exposure to diethylstilbestrol during sensitive life stages: a legacy of heritable health effects.

Diethylstilbestrol (DES) is a potent estrogen mimic that was predominantly used from the 1940s to the 1970s by pregnant women in hopes of preventing miscarriage. Decades later, DES is known to enhance breast cancer risk in exposed women and cause a variety of birth-related adverse outcomes in their daughters such as spontaneous abortion, second trimester pregnancy loss, preterm delivery, stillbirth, and neonatal death. Additionally, children exposed to DES in utero suffer from sub/infertility and cancer of reproductive tissues. DES is a pinnacle compound that demonstrates the fetal basis of adult disease. The mechanisms of cancer and endocrine disruption induced by DES are not fully understood. Future studies should focus on common target tissue pathways affected and the health of the DES grandchildren.

The absence of ER-ß results in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles.

Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER)-ß is highly expressed in ovarian granulosa cells, and mice lacking ER-ß are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and, although informative, provides confounding results due to the heterogeneous cell types present including granulosa and theca cells and oocytes and exposure to in vitro conditions. Herein we isolated preovulatory granulosa cells from wild-type (WT) and ERß-null mice using laser capture microdissection to examine the genomic transcriptional response downstream of pregnant mare serum gonadotropin (mimicking FSH) and pregnant mare serum gonadotropin/human chorionic gonadotropin (mimicking LH) stimulation. This allows for a direct comparison of in vivo granulosa cells at the same stage of development from both WT and ERß-null ovaries. ERß-null granulosa cells showed altered expression of genes known to be regulated by FSH (Akap12 and Runx2) as well as not previously reported (Arnt2 and Pou5f1) in WT granulosa cells. Our analysis also identified 304 genes not previously associated with ERß in granulosa cells. LH-responsive genes including Abcb1b and Fam110c show reduced expression in ERß-null granulosa cells; however, novel genes including Rassf2 and Megf10 were also identified as being downstream of LH signaling in granulosa cells. Collectively, our data suggest that granulosa cells from ERß-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation.

Histopathologic changes in the uterus, cervix and vagina of immature CD-1 mice exposed to low doses of perfluorooctanoic acid (PFOA) in a uterotrophic assay.

The estrogenic and antiestrogenic potential of perfluorooctanoic acid (PFOA) was assessed using an immature mouse uterotrophic assay and by histologic evaluation of the uterus, cervix and vagina following treatment. Female offspring of CD-1 dams were weaned at 18days old and assigned to groups of equal weight, and received 0, 0.01, 0.1, or 1mg PFOA/kg BW/d by gavage with or without 17-ß estradiol (E(2), 500µg/kg/d) from PND 18-20 (n=8/treatment/block). At 24h after the third dose (PND 21), uteri were removed and weighed. Absolute and relative uterine weights were significantly increased in the 0.01mg/kg PFOA only group. Characteristic estrogenic changes were present in all E(2)-treated mice; however, they were minimally visible in the 0.01 PFOA only mice. These data suggest that at a low dose PFOA produces minimal histopathologic changes in the reproductive tract of immature female mice, and does not antagonize the histopathologic effects of E(2).