Roles of poly (ADP-ribose) polymerase 1 activation and cleavage in induction of multi-oocyte ovarian follicles in the mouse by 3-nitropropionic acid.

3-nitropropionic acid (3-NPA) is known to be a mitochondrial toxin produced by plants and fungi, which may produce DNA damage in cells. However, studies of its reproductive toxicology are lacking. We know that poly(ADP-ribose) polymerase (PARP) plays an important role in a large variety of physiological processes and is involved in DNA repair pathways. The present study was therefore aimed at exploring the involvement of PARP-1 activation and cleavage after 3-NPA stimulation in female mice. We observed an increased number of atretic follicles and multi-oocyte follicles (MOFs) after treatment with 3-NPA and serum concentrations of 17ß-oestradiol and progesterone were significantly reduced. Our results provide evidence that PARP-1 cleavage and activational signals are involved in pathological ovarian processes stimulated by 3-NPA. In addition, total superoxide dismutase, glutathione peroxidase and catalase activities were significantly increased, whereas succinate dehydrogenase was decreased in a dose-dependent manner. Results from our in vitro study similarly indicated that 3-NPA inhibited the proliferation of mouse granulosa cells and increased apoptosis in a dose-dependent manner. In summary, 3-NPA induces granulosa cell apoptosis, follicle atresia and MOFs in the ovaries of female mice and causes oxidative stress so as to disrupt endogenous hormonal systems, possibly acting through PARP-1 signalling.

Histopathologic alterations associated with global gene expression due to chronic dietary TCDD exposure in juvenile zebrafish.

The goal of this project was to investigate the effects and possible developmental disease implication of chronic dietary TCDD exposure on global gene expression anchored to histopathologic analysis in juvenile zebrafish by functional genomic, histopathologic and analytic chemistry methods. Specifically, juvenile zebrafish were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb, and fish were sampled following 0, 7, 14, 28 and 42 d after initiation of the exposure. TCDD accumulated in a dose- and time-dependent manner and 100 ppb TCDD caused TCDD accumulation in female (15.49 ppb) and male (18.04 ppb) fish at 28 d post exposure. Dietary TCDD caused multiple lesions in liver, kidney, intestine and ovary of zebrafish and functional dysregulation such as depletion of glycogen in liver, retrobulbar edema, degeneration of nasal neurosensory epithelium, underdevelopment of intestine, and diminution in the fraction of ovarian follicles containing vitellogenic oocytes. Importantly, lesions in nasal epithelium and evidence of endocrine disruption based on alternatively spliced vasa transcripts are two novel and significant results of this study. Microarray gene expression analysis comparing vehicle control to dietary TCDD revealed dysregulated genes involved in pathways associated with cardiac necrosis/cell death, cardiac fibrosis, renal necrosis/cell death and liver necrosis/cell death. These baseline toxicological effects provide evidence for the potential mechanisms of developmental dysfunctions induced by TCDD and vasa as a biomarker for ovarian developmental disruption.

Engineered nanomaterials: an emerging class of novel endocrine disruptors.

Over the past decade, engineered nanomaterials (ENMs) have garnered great attention for their potentially beneficial applications in medicine, industry, and consumer products due to their advantageous physicochemical properties and inherent size. However, studies have shown that these sophisticated molecules can initiate toxicity at the subcellular, cellular, and/or tissue/organ level in diverse experimental models. Investigators have also demonstrated that, upon exposure to ENMs, the physicochemical properties that are exploited for public benefit may mediate adverse endocrine-disrupting effects on several endpoints of mammalian reproductive physiology (e.g., steroidogenesis, spermatogenesis, pregnancy). Elucidating these complex interactions within reproductive cells and tissues will significantly advance our understanding of ENMs as an emerging class of novel endocrine disruptors and reproductive toxicants. Herein we reviewed the recent developments in reproductive nanotoxicology and identified the gaps in our knowledge that may serve as future research directions to foster continued advancement in this evolving field of study.

Low-dose gold nanoparticles exert subtle endocrine-modulating effects on the ovarian steroidogenic pathway ex vivo independent of oxidative stress.

Gold nanoparticles (GNPs) have gained considerable attention for application in science and industry. However, the untoward effects of such particles on female fertility remain unclear. The objectives of this study were to (1) examine the effects of 10-nm GNPs on progesterone and estradiol-17ß accumulation by rat ovaries ex vivo and (2) to identify the locus/loci whereby GNPs modulate steroidogenesis via multiple-reference gene quantitative real-time RT-PCR. Regression analyses indicated a positive relationship between both Star (p < 0.05, r(2) = 0.278) and Cyp11a1 (p < 0.001, r(2) = 0.366) expression and P4 accumulation upon exposure to 1.43 × 10(6) GNPs/mL. Additional analyses showed that E2 accumulation was positively associated with Hsd3b1 (p < 0.05, r(2) = 0.181) and Cyp17a1 (p < 0.01, r(2) = 0.301) expression upon exposure to 1.43 × 1(3) and 1.43 × 10(9) GNPs/mL, respectively. These results suggest a subtle treatment-dependent impact of low-dose GNPs on the relationship between progesterone or estradiol-17ß and specific steroidogenic target genes, independent of oxidative stress or inhibin.

Gene expression and pathologic alterations in juvenile rainbow trout due to chronic dietary TCDD exposure.

The goal of this project was to use functional genomic methods to identify molecular biomarkers as indicators of the impact of TCDD exposure in rainbow trout. Specifically, we investigated the effects of chronic dietary TCDD exposure on whole juvenile rainbow trout global gene expression associated with histopathological analysis. Juvenile rainbow trout were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb (ngTCDD/g food), and fish were sampled from each group at 7, 14, 28 and 42 days after initiation of feeding. 100 ppb TCDD caused 100% mortality at 39 days. Fish fed with 100 ppb TCDD food had TCDD accumulation of 47.37 ppb (ngTCDD/g fish) in whole fish at 28 days. Histological analysis from TCDD-treated trout sampled from 28 and 42 days revealed that obvious lesions were found in skin, oropharynx, liver, gas bladder, intestine, pancreas, nose and kidney. In addition, TCDD caused anemia in peripheral blood, decreases in abdominal fat, increases of remodeling of fin rays, edema in pericardium and retrobulbar hemorrhage in the 100 ppb TCDD-treated rainbow trout compared to the control group at 28 days. Dose- and time-dependent global gene expression analyses were performed using the cGRASP 16,000 (16K) cDNA microarray. TCDD-responsive whole body transcripts identified in the microarray experiments have putative functions involved in various biological processes including growth, cell proliferation, metabolic process, and immune system processes. Nine microarray-identified genes were selected for QPCR validation. CYP1A3 and CYP1A1 were common up-regulated genes and HBB1 was a common down-regulated gene among each group based on microarray data, and their QPCR validations are consistent with microarray data for the 10 and 100 ppb TCDD treatment groups after 28 days exposure (p<0.05). In addition, in the 100 ppb group at 28 days, expression of complement component C3-1 and trypsin-1 precursor have a more than 10-fold induction from the microarray experiments, and their QPCR validations are consistent and showed significant induction in the 100 ppb group at 28 days (p<0.05). Overall, lesion in nasal epithelium is a novel and significant result in this study, and TCDD-responsive rainbow trout transcripts identified in the present study may lead to the development of new molecular biomarkers for assessing the potential impacts of environmental TCDD on rainbow trout populations.

Differential gene expression associated with dietary methylmercury (MeHg) exposure in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio).

The objective of this study was to identify and evaluate conserved biomarkers that could be used in most species of teleost fish at most life-stages. We investigated the effects of sublethal methylmercury (MeHg) exposure on developing rainbow trout and zebrafish. Juvenile rainbow trout and young adult zebrafish were fed food with MeHg added at 0, 0.5, 5, and 50 ppm. Atomic absorption spectrometry was applied to measure whole body total Hg levels, and pathologic analysis was performed to identify MeHg-induced toxicity. Fish at 6 weeks were sampled from each group for microarray analysis using RNA from whole fish. MeHg-exposed trout and zebrafish did not show overt signs of toxicity or pathology, nor were significant differences seen in mortality, length, mass, or condition factor. The accumulation of MeHg in trout and zebrafish exhibited dose- and time-dependent patterns during 6 weeks, and zebrafish exhibited greater assimilation of total Hg than rainbow trout. The dysregulated genes in MeHg-treated fish have multiple functional annotations, such as iron ion homeostasis, glutathione transferase activity, regulation of muscle contraction, troponin I binding and calcium-dependent protein binding. Genes were selected as biomarker candidates based on their microarray data and their expression was evaluated by QPCR. Unfortunately, these genes are not good consistent biomarkers for both rainbow trout and zebrafish from QPCR evaluation using individual fish. Our conclusion is that biomarker analysis for aquatic toxicant assessment using fish needs to be based on tissue-, sex- and species-specific consideration.

Effect of the IGF-1/PTEN/Akt/FoxO signaling pathway on the development and healing of water immersion and restraint stress-induced gastric ulcers in rats.

Insulin-like growth factor 1 (IGF-1) and Akt [also known as protein kinase B (PKB)] proteins have been reported to exhibit gastroprotective effects by reducing water immersion and restraint stress (WRS)-induced gastric mucosal cellular apoptosis. To confirm whether the IGF-1/PTEN/Akt/FoxO signaling pathway is effective in protecting against gastric ulcers, our current study was conducted to examine the expression and localization of IGF-1, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), Akt and O subfamily of forkhead box (FoxO) proteins, caspase-3 activity and the number of apoptotic cells in gastric mucosa of rats subjected to WRS. Our results demonstrated that WRS induced gastric ulcers by enhancing cell apoptosis in rat gastric mucosa. In addition, in normal rat gastric mucosa, PTEN, total Akt and FoxO1 were found mainly in the cell cytoplasm of fundic glands in the lamina propria close to the muscularis mucosa. In addition, strong staining of IGF-1, FoxO3a and FoxO4 in the gastric mucosa was primarily concentrated in the cell cytoplasm of the fundic glands in whole lamina propria. However, in rat gastric ulcers, IGF-1, total Akt, FoxO3a and FoxO4 were localized in proximity to the base of the ulcer margin and were also present in the granulation tissues of the gastric ulcers. Moreover, in the rat gastric ulcers, the mRNA transcript levels of IGF-1, PTEN, Akt-1, Akt-2, FoxO3 and FoxO4 were upregulated in the gastric ulcer margin, with a peak between Days 4 and 8 following 7 h of WRS. In conclusion, our results imply that the IGF-1/PTEN/Akt/FoxO signaling pathway plays a certain role(s) in the protection against ulceration through the regulation of cellular apoptosis as observed in the development and healing of rat gastric ulcers.

Current status of potential bisphenol toxicity in dentistry.

Bisphenols are chemical components found in dental composites and sealants. Similar compounds also can be found in baby bottles, food can liners, and even drinking water. Bisphenols have gained attention recently because they, like other natural and synthetic compounds, including hormone-based drugs and soybean products, have the capacity to mimic the actions of the hormone estrogen in living cells and animals. Such estrogenic activity has been linked to a variety of health problems, including breast and prostate cancer, metabolic disorders, and reproductive dysfunction. In early 2010, the FDA issued a report stating that there are some concerns about the safety of bisphenols in food products and called for more research on bisphenol toxicity. At present, no regulatory or professional organization has expressed concern about health effects of bisphenols in dental materials.

Involvement of cell proliferation in the process of follicular atresia in the guinea pig.

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs.

Reproductive toxicity of acrylamide-treated male rats.

Acrylamide content is elevated in fried, baked and heat-processed starchy foods. The present experiment was conducted to investigate the reproductive toxicity of oral acrylamide in male rats. Thirty weaned SD male rats of 21-day-old were randomly allotted to three groups, and acrylamide was administered to each group at doses of 0, 5 and 10 mg/kg-d for 8 consecutive weeks. The results indicated that the growth of rats treated with acrylamide was retarded (P<0.05), but relative weights of testes and epididymides compared to body weight were not significantly different (P>0.05). Our results also indicate that the epididymal sperm reserves decreased significantly (P<0.05), suggesting partial depletion of germ cells. In addition, histopathologic lesions were also present in the testes of treated rats. Furthermore, distinct expression patterns of sGC heterodimers were observed in this animal model. This may suggest different physiologic roles for sGC subunits in spermiogenesis and steroidogenesis.

Gold nanoparticles enter rat ovarian granulosa cells and subcellular organelles, and alter in-vitro estrogen accumulation.

Nanoparticle technology refers to research and technology developed at the atomic or molecular level for materials of approximately 1-100 nm in length. Through accidental or involuntary exposure, nanoparticles are potentially toxic to the body, including reproductive organs. Ovarian granulosa cells play a major role in maintaining ovarian function, health, and female fertility. Since these cells are involved in steroidogenesis, we wished to evaluate whether nanoparticles affected them after traversing their membranes. Cells were co-incubated with 10 nm gold particles for up to 24 h. Transmission electron micrographs were taken of GC treated with 10 nm gold particles in order to compare and contrast ultrastructural locations of nanoparticles with treatment. From micrograph comparisons of treated vs. untreated GC at various culture times, it appeared that some intracellular organelles involved in steroidogenesis were infiltrated and/or altered due to the presence of the nanogold particles. Medium samples were taken in order to determine estradiol-17beta (E2) accumulation/secretion by untreated vs. treated cells. GC incubated with 10 nm nanogold particles for 1, 3, or 5 h were found to accumulate significantly increased amounts of estrogen compared with untreated cells. Conversely, at 24 h there was a significant attenuation with respect to controls. The data presented here provide insight into the toxicologic effects gold nanoparticles elicit on ovarian granulosa cells.

Molecular targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within the zebrafish ovary: insights into TCDD-induced endocrine disruption and reproductive toxicity.

TCDD is a reproductive toxicant and endocrine disruptor, yet the mechanisms by which it causes these reproductive alterations are not fully understood. In order to provide additional insight into the molecular mechanisms that underlie TCDD's reproductive toxicity, we assessed TCDD-induced transcriptional changes in the ovary as they relate to previously described impacts on serum estradiol concentrations and altered follicular development in zebrafish. In silico computational approaches were used to correlate candidate regulatory motifs with observed changes in gene expression. Our data suggest that TCDD inhibits follicle maturation via attenuated gonadotropin responsiveness and/or depressed estradiol biosynthesis, and that interference of estrogen-regulated signal transduction may also contribute to TCDD's impacts on follicular development. TCDD may also alter ovarian function by disrupting various signaling pathways such as glucose and lipid metabolism, and regulation of transcription. Furthermore, events downstream from initial TCDD molecular-targets likely contribute to ovarian toxicity following chronic exposure to TCDD. Data presented here provide further insight into the mechanisms by which TCDD disrupts follicular development and reproduction in fish, and can be used to formulate new hypotheses regarding previously documented ovarian toxicity.

Short-term hormone release from adult female rat hypothalamic and pituitary explants is not altered by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2, 3, 7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has adverse effects on reproduction, in part due to direct actions at the ovary. It is unclear whether effects are further mediated by glands that regulate ovarian function. We investigated whether effects of TCDD are mediated via the hypothalamic-pituitary axis. Hypothalamic and pituitary tissues were cultured in medium with and without TCDD. TCDD did not alter GnRH release from hypothalamic samples. It continued to be pulsatile with no differences in the average peak frequency, average peak amplitude, or baseline GnRH release. TCDD did not alter GnRH-induced release of gonadotropins from pituitary samples. There were no differences in average peak amplitude or baseline release. AhR, ARNT or ER alpha mRNA copy numbers in cultured pituitaries were not affected by TCDD. Our data suggest that TCDD effects on ovarian function are not mediated through the hypothalamic or pituitary release parameters tested in this study.

Autoradiographic localization of aromatic hydrocarbon receptor (AHR) in rhesus monkey ovary.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of a large class of manmade pollutants that persist in the environment. TCDD exerts its toxic effects, in part, by binding to its receptor known as the aromatic hydrocarbon receptor (AHR). TCDD is estrogen modulatory and in some systems its receptor associates directly with estrogen receptors via co-activator molecules. TCDD inhibits steroid synthesis in human ovarian granulosa cells and AHR is found in these cells. We have previously shown that AHR is found in whole rhesus monkey ovary, but have yet to establish its location. In the present study, we set out to show that radiolabeled TCDD binds to monkey ovarian follicles and that this binding is receptor mediated. Ovaries from Macaca mulatta were sectioned on a cryostat at 10 micro m; and sections were incubated with either control vehicle, (3)H-TCDD, or (3)H-TCDD plus alpha-naphthoflavone (ANF), a known receptor-blocking agent. Here, we show for the first time specific binding of TCDD to the granulosa cells of antral follicles and other regions of the rhesus monkey ovary. Our data indicate a 60-fold increase in binding with (3)H-TCDD over that of control, and that this binding is reduced to the levels seen in controls with the addition of the competitive antagonist ANF. These findings support the hypothesis that TCDD directly affects primate ovarian function via the AHR.

TCDD increases inhibin A production by human luteinized granulosa cells in vitro.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic of the halogenated dioxins and one of the most poisonous substances known to man. The major toxic effects of TCDD on reproduction are decreased fertility and diminished ability to maintain a pregnancy. Granulosa cells obtained from hormonally stimulated women participating in an in-vitro fertilization program were cultured with 3.1 femtomolar, 3.1 picomolar and 3.1 nanomolar TCDD. While inhibin B production was not altered, inhibin A production increased significantly after 4 hours of exposure to both nanomolar and micromolar TCDD concentrations. By 8 hours of exposure to these concentrations of dioxin, human luteinizing granulosa cells exhibited a pronounced increase in inhibin A, nearly quadrupling secretion from unexposed control cells. TCDD continued to increase inhibin A secretion at the picomolar concentration at 24 and 36 hours. It is conceivable that TCDD may act at the ovary to augment inhibin A secretion, thereby reducing FSH-stimulable estrogen secretion by granulosa cells.

Inhibition of follicular development, vitellogenesis, and serum 17beta-estradiol concentrations in zebrafish following chronic, sublethal dietary exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent endocrine disruptor with the ability to affect several biologic processes, including reproduction. In fish, sublethal exposure to TCDD is known to modulate overall reproductive capacity, but impacts on follicular development and vitellogenesis are unknown. Here we show that chronic, dietary exposure to 0.08, 0.32, or 0.80 ng TCDD female(-1) day(-1) decreased egg production by more than 50% and that spawning success was reduced by as much as 96%. Serum estradiol concentrations were decreased more than twofold, accounting, in part, for observed decreases in serum vitellogenin concentrations by as much as 29%. Our data suggest that decreased egg production is likely the result of TCDD-mediated inhibition of the transition from pre-vitellogenic stage follicles to vitellogenic stage follicles, as well as the induction of follicular atresia. The majority of reproductive toxicity of TCDD is likely due to direct impacts on the ovary, yet histopathologic observations suggest liver toxicity could also contribute to observed impacts on vitellogenesis. Importantly, even when overall egg production is not significantly affected, our data show that subtle physiologic changes induced by TCDD can lead to altered gonadogenesis. This suggests that long-term exposure to very low concentrations of TCDD could greatly affect fecundity and reproductive success in fishes.

Accumulation, tissue distribution, and maternal transfer of dietary 2,3,7,8,-tetrachlorodibenzo-p-dioxin: impacts on reproductive success of zebrafish.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a reproductive toxicant and endocrine disruptor in nearly all vertebrates; however, the mechanisms by which TCDD alters the reproductive system is not well understood. The zebrafish provides a powerful vertebrate model system to investigate molecular mechanisms by which TCDD affects the reproductive system, but little is known regarding reproductive toxic response of zebrafish following chronic, sublethal exposure to TCDD. Here we investigate the accumulation of TCDD in selected tissues of adult female zebrafish and maternal transfer to offspring following dietary exposure to TCDD (0.08-2.16 ng TCDD/fish/day). TCDD accumulated in tissues of zebrafish in a dose- and time-dependent manner, except for brain. Chronic dietary exposure resulting in the accumulation of 1.1-36 ng/g fish did not induce an overt toxic response or suppress spawning activity. The ovosomatic index was impacted with an accumulation of as little as 0.6 ng/g fish, and 10% of the females showed signs of ovarian necrosis following accumulation of approximately 3 ng/g TCDD. Offspring health was impacted with an accumulation of as little as 1.1 ng/g female; thus the lowest observed effect level (LOEL) for reproductive toxicity in female zebrafish is approximately 0.6-1.1 ng/g fish. Maternal transfer resulted in the accumulation of 0.094-1.2 ng/g, TCDD, which was sufficient to induce the typical endpoints of larval TCDD toxicity, commonly referred to as blue sac syndrome. This study provides the necessary framework to utilize the zebrafish model system for further investigations into the molecular mechanisms by which TCDD exerts its reproductive toxic responses.

Effects of intrauterine growth retardation on development of the gastrointestinal tract in neonatal pigs.

BACKGROUND: Intrauterine growth retardation (IUGR) is a common problem in human and other species and increases the risk of death of the fetus and newborn during the perinatal period. OBJECTIVES: This study was conducted to examine the influences of intrauterine growth retardation (IUGR) on development of the gastrointestinal tract in newborn pigs. METHODS: Ten animals from five litters were divided into five piglets with IUGR and five with normal birth-weight (NW). The IUGR category comprised animals with a birth weight 2 SD below the mean birth weight of the total population, while the NW category included animals with a birth weight within one SD of the mean birth weight in the total population. Animals were anesthetized and sampled within 2-4 h after birth and without suckling. The morphological changes of intestine and stomach of IUGR piglets were compared with NW ones. The expressions of IGF-I and receptors for growth hormone and insulin in intestinal mucosa were semiquantified using reverse transcription PCR. RESULTS: The results of our study indicated that the weights of the stomach, small intestine and small intestinal mucosa were significantly lower in IUGR compared with NW piglets (p<0.01). In addition, the lengths of the small intestine and colon in IUGR pigs were also significantly less than those of NW (p<0.05). Furthermore, insulin-like growth factor-I (IGF-I) mRNA level in intestinal mucosa of IUGR piglets was increased significantly (p<0.05), and the expression mRNA levels of insulin receptor and growth hormone (GH) receptor in the mucosa in IUGR piglets showed a tendency to be lower (p=0.17 and p=0.11, respectively) than those of the NW animals. CONCLUSION: We conclude from the data that IUGR affects intestinal growth and morphology and is in associated with altered gene expression of growth-related proteins. We speculate that the morphological change and associated altered endocrine homeostasis contribute to lower growth rates of pigs affected by IUGR.

Regulation of FoxO1 transcription factor by nitric oxide and cyclic GMP in cultured rat granulosa cells.

FoxO1 is a transcription factor implicated in a multitude of physiological processes including cell cycle progression, apoptosis and insulin signaling. Recent findings indicate that FoxO1 is a key regulator during the proliferation and maturation of granulosa cells. Over the past several years, it has become evident that nitric oxide (NO) and cGMP modulate ovarian function. There has been no information, however, about whether NO-cGMP affects FoxO1 expression or about the relationship between NO-cGMP and FoxO1. In the present study, we used immunoblot analysis to determine whether NO and cGMP affect FoxO1 expression in cultured granulosa cells. Our results clearly showed that FSH suppressed FoxO1 expression in a time-dependent manner, and that NO-cGMP stimulated FoxO1 expression in cultured granulosa cells. In addition, this stimulatory effects of NO and cGMP can be blocked by FSH in cultured granulosa cells. These findings demonstrate that NO and cGMP influence FoxO1 expression possibly through antagonizing the action of FSH in cultured granulosa cells. Results of both immunoblot analysis and immunohistochemistry also show that estradiol implantation do not affect the expression of FoxO1 in rat granulosa cells as gonadotrophins do, indicating that mechanism of estradiol on granulosa cells is different from gonadotrophins. Together, our experiments suggest that expression of FoxO1 in rat granulosa cells can be regulated by gonadotrophins and the NO/cGMP signaling pathway.

In utero and lactational exposure of Long-Evans rats to ammonium perchlorate (AP) disrupts ovarian follicle maturation.

Ammonium perchlorate (AP) is a powerful oxidizer manufactured almost exclusively for the aerospace industry. AP salts are also used in airbags, flares, fertilizers, enamels and paints. As a result of widespread industrial use, AP has become a persistent environmental contaminant of drinking water in several U.S. states. AP ion disrupts the trapping of iodide as well as facilitates the discharge of unorganified iodide from the thyroid gland. Such disturbances in thyroid hormone concentrations during critical periods of development are then known to cause profound reproductive and developmental defects, since thyroid hormones modulate both follicular development and steroidogenesis and affect estrogen metabolism and receptor. This study was designed (1) to determine whether exposure to a low or high concentration of AP (LAP, HAP) exerts detrimental effects on follicle maturation in the Long-Evans hooded rat and (2) to determine whether the modulatory effects of AP can be ameliorated by levo-thyroxine sodium (T4) supplementation. Animals were treated via deionized drinking water on GD 7-21 with LAP (0.4 mg/kg/day) or HAP (4.0 mg/kg/day). Half of each group was also given T4 supplements via drinking water on GD 7-21. Female pups were sacrificed on postnatal days 24/25, and the ovaries were excised, fixed for histology and analyzed. The analysis included a count, measurement and classification of preantral and antral follicles in the greatest cross-sectional area of the ovary. The results indicated that treatment with the HAP significantly reduced the number of preantral follicles <50,000 microm2 and the total number of antral follicles in the <50,000, 50-100,000 and >100,000 microm2 size classes. In ovaries treated with the LAP, we observed no significant decrease in preantral follicles of any size class and only a significant reduction in the largest antral follicles. T4 only circumvented the effect on the number of small preantral and antral follicles; however, a significant diminution in the antral follicle number persisted in the mid-sized (HAP) and large (LAP, HAP)-sized classes. These data support the hypothesis that AP reduces the number of preantral and antral follicles in certain size classes in rats exposed during a critical period of development, and that T4 can attenuate the effects of AP on small preantral and antral follicles, but not on medium or large antral follicles. (T35ES007292 & ES08342.).