Patricia B Hoyer

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• Hoyer, P. B. (2014). A method to study the impact of chemically-induced ovarian failure on exercise capacity and cardiac adaptation in mice. J Vis. Exp..
• Hoyer, P. B. (2014). Acute 7,12-dimethylbenz[a]anthracene exposure causes differential concentration-dependent follicle depletion and gene expression in neonatal rat ovaries. Toxicol. Appl. Pharmacol..
• Hoyer, P. B. (2014). In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis. Cancer Biol. Ther., 15, 42-60.
• Hoyer, P. B. (2014). Involvement of a volatile metabolite during phosphoramide mustard-induced ovotoxicity. Toxicol Appl Pharmacol, 277, 1-7.
• Hoyer, P. B. (2014). Steroid hormone environment during primordial follicle formation in perinatal mouse ovaries. Biol. Reprod..
• Hoyer, P. B. (2014). Xenobiotic effects in the ovary: Temporary versus permanent infertility. Expert Opin. Drug Metab. Toxicol..
• Watson, J. M., Marion, S. L., Rice, P. F., Bentley, D. L., Besselsen, D. G., Utzinger, U., Hoyer, P. B., & Barton, J. K. (2014). In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis. Cancer Biology and Therapy, 15(1), 42-60.
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  Abstract: Identification of the early microscopic changes associated with ovarian cancer may lead to development of a diagnostic test for high-risk women. In this study we use optical coherence tomography (OCT) and multiphoton microscopy (MPM) (collecting both two photon excited fluorescence [TPEF] and second harmonic generation [SH G]) to image mouse ovaries in vivo at multiple time points. We demonstrate the feasibility of imaging mouse ovaries in vivo during a longterm survival study and identify microscopic changes associated with early tumor development. These changes include alterations in tissue microstructure, as seen by OCT, alterations in cellular fluorescence and morphology, as seen by TPEF, and remodeling of collagen structure, as seen by SH G. These results suggest that a combined OCT-MPM system may be useful for early detection of ovarian cancer. © 2014 Landes Bioscience.
• Hoyer, P., Lohff, J. C., Christian, P. J., Marion, S. L., & Hoyer, P. B. (0). Effect of duration of dosing on onset of ovarian failure in a chemical-induced mouse model of perimenopause. Menopause (New York, N.Y.), 13(3).
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  Repeated daily dosing with 4-vinylcyclohexene diepoxide (VCD) causes gradual ovarian failure in mice. As a result, the animal undergoes ovarian failure, but retains residual ovarian tissue. The purpose of this study was to use a mouse model to regulate the induction of a period analogous to perimenopause in women.
• Hoyer, P., Marion, S. L., Watson, J., Sen, N., Brewer, M. A., Barton, J. K., & Hoyer, P. B. (2013). 7,12-dimethylbenz[a]anthracene-induced malignancies in a mouse model of menopause. Comparative medicine, 63(1).
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  Ovarian cancer has a high mortality rate because there are few symptoms in early disease development. The incidence of ovarian cancer increases in women after menopause. Understanding early events in this disease can best be achieved by using animal models. Therefore, the objective of this study was to develop and track the onset of ovarian tumorigenesis in mice mimicking characteristics of postmenopausal epithelial cancer in women. Female B6C3F1 mice (age, 28 d) received 4-vinylcyclohexene diepoxide (VCD, 160 mg/kg IV daily for 20 d) to cause ovarian failure. Four months after VCD treatment, via surgical intervention, each mouse received a single injection of 7,12-dimethylbenz[a]anthracene (DMBA) or vehicle control (sesame oil) under the bursa of the right ovary to cause ovarian neoplasms. The experimental groups were untreated controls (Con-Con), DMBA-treatment only (Con-DMBA), VCD treatment only (VCD-Con), and VCD+DMBA-treated (VCD+DMBA) mice. At 3, 5, 7, and 9 mo after DMBA injection, ovaries were collected for histologic and immunohistochemical evaluation. No tumors developed in Con-Con mice. All VCD-treated mice (with or without DMBA) exhibited ovarian failure. Mice that received both VCD and DMBA exhibited tumors at 3 mo (50%), 5 mo (14%), 7 mo (90%), and 9 mo (57%) after DMBA treatment; 31% of the tumors were epithelial in origin. Our findings confirm that inducing ovarian tumors in mice by chemical means is an effective method for studying early stages of tumor development that may be relevant to epithelial ovarian cancers that arise in postmenopausal women.
• Hoyer, P., Mayer, L. P., Pearsall, N. A., Christian, P. J., Devine, P. J., Payne, C. M., McCuskey, M. K., Marion, S. L., Sipes, I. G., & Hoyer, P. B. (0). Long-term effects of ovarian follicular depletion in rats by 4-vinylcyclohexene diepoxide. Reproductive toxicology (Elmsford, N.Y.), 16(6).
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  4-Vinylcyclohexene diepoxide (VCD) destroys preantral ovarian follicles in rats. Female 28-day Fisher 344 (F344) rats were dosed (30 days) with VCD (80 mg/kg per day, i.p.) or vehicle, and animals were evaluated for reproductive function at subsequent time points for up to 360 days. At each time point animals were killed, and ovaries and plasma collected. VCD reduced (P
• Hoyer, P., Kappeler, C. J., & Hoyer, P. B. (2012). 4-vinylcyclohexene diepoxide: a model chemical for ovotoxicity. Systems biology in reproductive medicine, 58(1).
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  The occupational chemical 4-vinylcyclohexene diepoxide (VCD) has been shown to cause selective destruction of ovarian small pre-antral (primordial and primary) follicles in rats and mice by accelerating the natural, apoptotic process of atresia. Chemicals that destroy primordial follicles are of concern to women because exposure can result in premature ovarian failure (early menopause). Initial studies using in vivo exposure of rats determined that VCD specifically targets primordial and primary (small pre-antral) follicles and that repeated dosing is required. Through a method of isolation of ovarian small follicles, biochemical and molecular studies determined that intracellular pro-apoptotic pathways are activated following VCD dosing in rats. Subsequently an in vitro system using cultured whole neonatal rat ovaries was developed to provide more mechanistic information. That approach was used to demonstrate that the cell survival c-kit/kit ligand signaling pathway is the direct target for VCD-induced ovotoxicity. Specifically, VCD directly interacts with the oocyte-associated c-kit receptor to inhibit its autophosphorylation, and thereby impair oocyte viability. The cellular and molecular approach developed to determine these findings is described in this article.
• Hoyer, P., Keating, A. F., Fernandez, S. M., Mark-Kappeler, C. J., Sen, N., Sipes, I. G., & Hoyer, P. B. (2011). Inhibition of PIK3 signaling pathway members by the ovotoxicant 4-vinylcyclohexene diepoxide in rats. Biology of reproduction, 84(4).
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  4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that specifically destroys primordial and small primary follicles in the ovaries of rats and mice, is thought to target an oocyte-expressed tyrosine kinase receptor, Kit. This study compared the temporal effect of VCD on protein distribution of KIT and its downstream PIK3-activated proteins, AKT and FOXO3. Postnatal Day 4 Fischer 344 rat ovaries were cultured in control media ± VCD (30 μM) for 2-8 days (d2-d8). KIT, AKT, phosphorylated AKT, FOXO3, and pFOXO3 protein levels were assessed by Western blotting and/or immunofluorescence staining with confocal microscopy. Phosphorylated AKT was decreased (P < 0.05) in oocyte nuclei in primordial (39% decrease) and small primary (37% decrease) follicles within 2 days of VCD exposure. After d4, VCD reduced (P < 0.05) oocyte staining for KIT (primordial, 44% decrease; small primary, 39% decrease) and FOXO3 (primordial, 40% decrease; small primary, 36% decrease) protein. Total AKT and pFOXO3 were not affected by VCD at any time. Akt1 mRNA, as measured by quantitative RT-PCR, was reduced (P < 0.05) by 23% on d4 of VCD exposure, but returned to control levels on d6 and d8. VCD exposure reduced Foxo3a mRNA by 26% on d6 (P < 0.05) and by 23% on d8 (P < 0.1). These results demonstrate that the earliest observed effect of VCD is an inhibition of phosphorylation and nuclear localization of AKT in the oocyte of primordial and small primary follicles. This event is followed by reductions in KIT and FOXO3 protein subcellular distribution prior to changes in mRNA. Thus, these findings further support that VCD induces ovotoxicity by directly targeting the oocyte through posttranslational inhibition of KIT-mediated signaling components.
• Hoyer, P., Mark-Kappeler, C. J., Sen, N., Lukefahr, A., McKee, L., Sipes, I. G., Konhilas, J., & Hoyer, P. B. (2011). Inhibition of ovarian KIT phosphorylation by the ovotoxicant 4-vinylcyclohexene diepoxide in rats. Biology of reproduction, 85(4).
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  In vitro exposure of Postnatal Day 4 (PND4) rat ovaries to the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys specifically primordial and primary follicles via acceleration of atresia. Because oocyte-expressed c-kit (KIT) plays a critical role in follicle survival and activation, a direct interaction of VCD with KIT as its mechanism of ovotoxicity was investigated. PND4 rat ovaries were cultured with and without VCD (30 μM) for 2 days. When assessed by Western analysis or mobility shift detection, phosphorylated KIT (pKIT) was decreased (P < 0.05) by VCD exposure, while total KIT protein was unaffected. Anti-mouse KIT2 (ACK2) antibody binds KIT and blocks its signaling pathways, whereas anti-mouse KIT 4 (ACK4) antibody binds KIT but does not block its activity. PND4 rat ovaries were incubated for 2 days with and without VCD with and without ACK2 (80 μg/ml) or ACK4 (80 μg/ml). ACK2 decreased pKIT; however, ACK4 had no effect. Conversely, ACK2 did not affect a VCD-induced decrease in pKIT, whereas ACK4 further reduced it. Because ACK2 and ACK4 (known to directly bind KIT) affect VCD responses, these results support the fact that VCD interacts directly with KIT. The effect of these antibodies on VCD-induced follicle loss was measured after 8 days of incubation. ACK2 further reduced (P < 0.05) VCD-induced follicle loss, whereas ACK4 did not affect it. These findings demonstrate that VCD induces ovotoxicity by direct inhibition of KIT autophosphorylation of the oocyte. The data also further support the vital function of KIT and its signaling pathway in primordial follicle survival and activation, as well as its role in VCD-induced ovotoxicity.
• Hoyer, P., Keating, A. F., Sen, N., Sipes, I. G., & Hoyer, P. B. (2010). Dual protective role for glutathione S-transferase class pi against VCD-induced ovotoxicity in the rat ovary. Toxicology and applied pharmacology, 247(2).
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  The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral follicles in rats and mice via apoptosis. Detoxification of VCD can occur through glutathione conjugation, catalyzed by glutathione S-transferase (GST) enzymes. Further, GST class pi (GSTp) can negatively regulate JNK activity through protein:protein interactions in extra-ovarian tissues. Dissociation of this protein complex in the face of chemical exposure releases the inhibition of pro-apoptotic JNK. Increased JNK activity during VCD-induced ovotoxicity has been shown in isolated ovarian small pre-antral follicles following in vivo dosing of rats (80mg/kg/day; 15days, i.p.). The present study investigated the pattern of ovarian GSTp expression during VCD exposure. Additionally, the effect of VCD on an ovarian GSTp:JNK protein complex was investigated. PND4 F344 rat ovaries were incubated in control medium+/-VCD (30muM) for 2-8days. VCD increased ovarian GSTp mRNA (P
• Hoyer, P., Mark-Kappeler, C. J., Sen, N., Keating, A. F., Sipes, I. G., & Hoyer, P. B. (2010). Distribution and responsiveness of rat anti-Müllerian hormone during ovarian development and VCD-induced ovotoxicity. Toxicology and applied pharmacology, 249(1).
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  Anti-Müllerian hormone (AMH) is produced by granulosa cells in primary to small antral follicles of the adult ovary and helps maintain primordial follicles in a dormant state. The industrial chemical, 4-vinylcyclohexene diepoxide (VCD) causes specific ovotoxicity in primordial and small primary follicles of mice and rats. Previous studies suggest that this ovotoxicity involves acceleration of primordial to primary follicle recruitment via interactions with the Kit/Kit ligand signaling pathway. Because of its accepted role in inhibiting primordial follicle recruitment, the present study was designed to investigate a possible interaction between AMH and VCD-induced ovotoxicity. Protein distribution of AMH was compared in neonatal and adult F344 rat ovaries. AMH protein was visualized by immunofluorescence microscopy in large primary and secondary follicles of the adult ovary, but in small primary follicles in neonatal rat ovaries. In cultured postnatal day (PND) 4 F344 rat ovaries, VCD exposure (30 μM, 2-8 days) decreased (P
• Hoyer, P., Igawa, Y., Keating, A. F., Rajapaksa, K. S., Sipes, I. G., & Hoyer, P. B. (2009). Evaluation of ovotoxicity induced by 7, 12-dimethylbenz[a]anthracene and its 3,4-diol metabolite utilizing a rat in vitro ovarian culture system. Toxicology and applied pharmacology, 234(3).
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  The polycyclic aromatic hydrocarbon 7, 12-dimethylbenz[a]anthracene, (DMBA), targets and destroys all follicle types in rat and mouse ovaries. DMBA requires bioactivation to DMBA-3,4-diol-1,2-epoxide for ovotoxicity via formation of the intermediate, DMBA-3,4-diol (catalyzed by microsomal epoxide hydrolase; mEH). mEH was shown to be involved in DMBA bioactivation for ovotoxicity induction in B6C3F(1) mouse ovaries. The current study compared DMBA and DMBA-3,4-diol mediated ovotoxicity, and investigated mEH involvement in DMBA-3,4-diol bioactivation in Fischer 344 (F344) rat ovary. F344 postnatal day (PND) 4 rat ovaries were cultured in vehicle control or media containing 1) DMBA or DMBA-3,4-diol (12.5 nM - 1 muM; 15 days); 2) DMBA (1 muM; 6 h - 15 days); and 3) DMBA (1 muM) or DMBA-3,4-diol (75 nM)+/-the mEH activity inhibitor cyclohexene oxide (CHO; 2 mM; 4 days). Ovaries were histologically evaluated and mEH mRNA and protein were measured by reverse transcriptase PCR or Western blotting, respectively. Ovotoxicity following 15 days of culture occurred (P
• Hoyer, P., Keating, A. F., J Mark, C., Sen, N., Sipes, I. G., & Hoyer, P. B. (2009). Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a]anthracene in neonatal rat ovaries. Toxicology and applied pharmacology, 241(2).
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  4-vinylcyclohexene diepoxide (VCD) is an ovotoxicant that specifically destroys primordial and small primary follicles in the ovaries of mice and rats. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA) is ovotoxic to all ovarian follicle classes. This study investigated phosphatidylinositol-3 kinase signaling involvement in VCD- and DMBA-induced ovotoxicity. Postnatal day (PND) 4 Fischer 344 (F344) rat whole ovaries were cultured for 2-12 days in vehicle control, VCD (30 microM), or DMBA (1 microM), +/-PI3 kinase inhibitor LY294002 (20 microM) or its inactive analog LY303511 (20 microM). Following culture, ovaries were histologically evaluated, and healthy follicles were classified and counted. PI3 kinase inhibition had no effect on primordial follicle number, but reduced (P
• Hoyer, P., Rivera, Z., Christian, P. J., Marion, S. L., Brooks, H. L., & Hoyer, P. B. (2009). Steroidogenic capacity of residual ovarian tissue in 4-vinylcyclohexene diepoxide-treated mice. Biology of reproduction, 80(2).
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  Menopause is an important public health issue because of its association with a number of disorders. Androgens produced by residual ovarian tissue after menopause could impact the development of these disorders. It has been unclear, however, whether the postmenopausal ovary retains steroidogenic capacity. Thus, an ovary-intact mouse model for menopause that uses the occupational chemical 4-vinylcyclohexene diepoxide (VCD) was used to characterize the expression of steroidogenic genes in residual ovarian tissue of follicle-depleted mice. Female B6C3F1 mice (age, 28 days) were dosed daily for 20 days with either vehicle or VCD (160 mg kg(-1) day(-1)) to induce ovarian failure. Ovaries were collected on Day 181 and analyzed for mRNA and protein. Acyclic aged mice were used as controls for natural ovarian senescence. Relative to cycling controls, expression of mRNA encoding steroidogenic acute regulatory protein (Star); cholesterol side-chain cleavage (Cyp11a1); 3beta-hydroxysteroid dehydrogenase (Hsd3b); 17alpha-hydroxylase (Cyp17a1); scavenger receptor class B, type 1 (Scarb1); low-density lipoprotein receptor (Ldlr); and luteinizing hormone receptor (Lhcgr) was enriched in VCD-treated ovaries. In acyclic aged ovaries, mRNA expression for only Cyp17a1 and Lhcgr was greater than that in controls. Compared to cycling controls, ovaries from VCD-treated and aged mice had similar levels of HSD3B, CYP17A1, and LHCGR protein. The pattern of protein immunofluorescence staining for HSD3B in follicle-depleted (VCD-treated) ovaries was homogeneous, whereas that for CYP17A1 was only seen in residual interstitial cells. Circulating levels of FSH and LH were increased, and androstenedione levels were detectable following follicle depletion in VCD-treated mice. These findings support the idea that residual ovarian tissue in VCD-treated mice retains androgenic capacity.
• Hoyer, P., Fernandez, S. M., Keating, A. F., Christian, P. J., Sen, N., Hoying, J. B., Brooks, H. L., & Hoyer, P. B. (2008). Involvement of the KIT/KITL signaling pathway in 4-vinylcyclohexene diepoxide-induced ovarian follicle loss in rats. Biology of reproduction, 79(2).
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  Repeated daily dosing of rats with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) depletes the ovary of primordial and primary follicles through an increase in the natural process of atresia. Additionally, in vitro exposure of Postnatal Day 4 (PND 4) rat ovaries to VCD causes similar follicular depletion. This study was designed to investigate survival signaling pathways that may be associated with VCD-induced ovotoxicity in small preantral follicles. Female Fischer 344 rats (PND 28) were dosed daily (80 mg/kg/day VCD i.p.; 12 days in vivo), and PND 4 ovaries were cultured (VCD 20 or 30 microM; 8 days in vitro). Microarray analysis identified a subset of 14 genes whose expression was increased or decreased by VCD in both experiments (i.e., via both exposure routes). Particularly, the analysis showed that relative to controls, VCD did not affect mRNA expression of growth and differentiation factor 9 (Gdf9), whereas there were decreases in mRNA encoding bone morphogenic protein receptor 1a (Bmpr1a) and Kit. To confirm findings from microarray, the genes Gdf9, Bmpr1a, and Kit were further examined. When growth factors associated with these pathways were added to ovarian cultures during VCD exposure, GDF9 and BMP4 had no effect on VCD-induced ovotoxicity; however, KITL attenuated this follicle loss. Additionally, there was a decrease in Kit and an increase in Kitl expression (mRNA and protein) following VCD exposure, relative to control. These results support that VCD compromises KIT/KITL signaling, which is critical for follicular survival in primordial and primary follicles.
• Hoyer, P., Keating, A. F., Rajapaksa, K. S., Sipes, I. G., & Hoyer, P. B. (2008). Effect of CYP2E1 gene deletion in mice on expression of microsomal epoxide hydrolase in response to VCD exposure. Toxicological sciences : an official journal of the Society of Toxicology, 105(2).
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  Females are born with a finite number of primordial follicles. 4-Vinylcyclohexene diepoxide (VCD) is a metabolite formed by epoxidation of 4-vinylcyclohexene (VCH) via its two monoepoxides 1,2- and 7,8-4-vinylcyclohexene monoepoxide (VCM). VCD specifically destroys small preantral (primordial and small primary) follicles in the rodent ovary. The phase I enzyme, cytochrome P450 isoform 2E1 (CYP2E1) is involved in ovarian metabolism of VCM to VCD. Further, microsomal epoxide hydrolase (mEH) can detoxify VCD to an inactive tetrol (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane). This study evaluated the effects of VCD-induced ovotoxicity on mEH in CYP2E1+/+ and -/- mice (129S(1)/SvImJ background strain) using a postnatal day 4 mouse whole ovary culture system. The hypothesis of our study is that there is a relationship between CYP2E1 and mEH gene expression in the mouse ovary. Relative to control, VCD exposure caused follicle loss (p < 0.05) in ovaries from both genotypes; however, after 15 days, this loss was greater (p < 0.05) in CYP2E1+/+ ovaries. In a time course (2-15 days), relative to control, VCD (5 microM) caused an increase (p < 0.05) in mEH mRNA by 0.5-fold (day 10) and 1.84-fold (day 15) in CYP2E1-/- but not +/+ ovaries. 7,12-Dimethylbenz[a]anthracene (DMBA) also destroys ovarian follicles but, unlike VCD, is bioactivated by mEH to an ovotoxic 3,4-diol-1,2-epoxide metabolite. Incubation of ovaries in increasing concentrations of DMBA (0.5-1 microM, 15 days) resulted in greater (p < 0.05) follicle loss in CYP2E1-/-, relative to +/+ ovaries. With greater mEH (CYP2E1-/-), increased follicle loss with DMBA (bioactivation) and decreased follicle loss with VCD (detoxification) support that ovarian expression of CYP2E1 and mEH may be linked.
• Hoyer, P., Keating, A. F., Sipes, I. G., & Hoyer, P. B. (2008). Expression of ovarian microsomal epoxide hydrolase and glutathione S-transferase during onset of VCD-induced ovotoxicity in B6C3F(1) mice. Toxicology and applied pharmacology, 230(1).
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  4-vinylcyclohexene diepoxide (VCD) specifically destroys small pre-antral follicles in the rodent ovary. VCD can be detoxified to an inactive tetrol by microsomal epoxide hydrolase (mEH), or by conjugation to glutathione (GSH) by glutathione S-transferase (GST). Formation of VCD-GSH adducts in the mouse ovary 4 h after VCD exposure (0.57 mmol/kg/day) has been demonstrated. Because the mouse ovary expresses both mEH and GST, expression of mEH and GST pi and mu during a time-course of VCD-induced ovotoxicity was evaluated in a neonatal mouse ovarian culture system. Ovaries from postnatal day 4 (PND4) B6C3F(1) mice were incubated with VCD (15 microM) for 2, 4, 6, 8, 10, 12, or 15 days. Following incubation, ovaries were histologically evaluated, or assessed for mRNA or protein expression. VCD did not cause follicle loss (p>0.05) on days 2, 4, or 6 of culture. At days 8, 10, 12, and 15, VCD reduced (p
• Hoyer, P., Wright, L. E., Christian, P. J., Rivera, Z., Van Alstine, W. G., Funk, J. L., Bouxsein, M. L., & Hoyer, P. B. (2008). Comparison of skeletal effects of ovariectomy versus chemically induced ovarian failure in mice. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 23(8).
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  Bone loss associated with menopause leads to an increase in skeletal fragility and fracture risk. Relevant animal models can be useful for evaluating the impact of ovarian failure on bone loss. A chemically induced model of menopause in which mice gradually undergo ovarian failure yet retain residual ovarian tissue has been developed using the chemical 4-vinylcyclohexene diepoxide (VCD). This study was designed to compare skeletal effects of VCD-induced ovarian failure to those associated with ovariectomy (OVX). Young (28 day) C57Bl/6Hsd female mice were dosed daily with vehicle or VCD (160 mg/kg/d, IP) for 15 days (n = 6-7/group) and monitored by vaginal cytology for ovarian failure. At the mean age of VCD-induced ovarian failure (approximately 6 wk after onset of dosing), a different group of mice was ovariectomized (OVX, n = 8). Spine BMD (SpBMD) was measured by DXA for 3 mo after ovarian failure and OVX. Mice were killed approximately 5 mo after ovarian failure or OVX, and bone architecture was evaluated by microCT ex vivo. In OVX mice, SpBMD was lower than controls 1 mo after OVX, whereas in VCD-treated mice, SpBMD was not lower than controls until 2.9 mo after ovarian failure (p < 0.05). Both VCD-induced ovarian failure and OVX led to pronounced deterioration of trabecular bone architecture, with slightly greater effects in OVX mice. At the femoral diaphysis, cortical bone area and thickness did not differ between VCD mice and controls but were decreased in OVX compared with both groups (p < 0.05). Circulating androstenedione levels were preserved in VCD-treated mice but reduced in OVX mice relative to controls (p < 0.001). These findings support that (1) VCD-induced ovarian failure leads to trabecular bone deterioration, (2) bone loss is attenuated by residual ovarian tissue, particularly in diaphyseal cortical bone, and (3) the VCD mouse model can be a relevant model for natural menopause in the study of associated bone disorders.
• Hoyer, P., Haas, J. R., Christian, P. J., & Hoyer, P. B. (2007). Effects of impending ovarian failure induced by 4-vinylcyclohexene diepoxide on fertility in C57BL/6 female mice. Comparative medicine, 57(5).
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  Repeated daily dosing of mice with 4-vinylcyclohexene diepoxide (VCD) causes a gradual onset of ovarian failure, providing a model for perimenopause. Because increasing numbers of women are delaying starting a family, infertility in aging women is of concern. This study was designed to determine the effects of impending ovarian failure on fertility in VCD-treated mice. Female C57BL/6J mice were dosed daily (17 d) with vehicle control or VCD (160 mg/kg, intraperitoneally) to deplete primordial follicles and then were divided into 2 groups. Group 1 was mated soon after dosing; group 2 was mated on day 20 after dosing, during impending ovarian failure. Fertility was evaluated on gestational day 16. In group 1, cycle length, pregnancy rate, and number of live fetuses did not differ between VCD-treated animals and controls, but VCD-treated mice required more matings to become pregnant and had more resorptions. In group 2, VCD-treated mice demonstrated proestrus and copulatory plugs, but only 1 animal became pregnant, and she had no viable fetuses. Ovaries from pregnant and nonpregnant controls contained similar numbers of follicles and corpora lutea. Ovaries from VCD-treated animals contained no follicles, and corpora lutea were seen only in pregnant animals. In VCD-treated mice mated soon after dosing, conception was more difficult and more resorbed fetuses were seen, whereas in those mated closer to impending ovarian failure, no successful pregnancies were achieved. These results demonstrate that VCD-treated mice can be used to model infertility in perimenopausal women.
• Hoyer, P., Rajapaksa, K. S., Cannady, E. A., Sipes, I. G., & Hoyer, P. B. (2007). Involvement of CYP 2E1 enzyme in ovotoxicity caused by 4-vinylcyclohexene and its metabolites. Toxicology and applied pharmacology, 221(2).
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  4-Vinylcyclohexene (VCH) is bioactivated by hepatic CYP 2A and 2B to a monoepoxide (VCM) and subsequently to an ovotoxic diepoxide metabolite (VCD). Studies suggest that the ovary can directly bioactivate VCH via CYP 2E1. The current study was designed to evaluate the role of ovarian CYP 2E1 in VCM-induced ovotoxicity. Postnatal day 4 B6C3F(1) and CYP 2E1 wild-type (+/+) and null (-/-) mouse ovaries were cultured (15 days) with VCD (30 microM), 1,2-VCM (125-1000 microM), or vehicle. Twenty-eight days female CYP 2E1 +/+ and -/- mice were dosed daily (15 days; ip) with VCH, 1,2-VCM, VCD or vehicle. Following culture or in vivo dosing, ovaries were histologically evaluated. In culture, VCD decreased (p
• Hoyer, P., Rajapaksa, K. S., Sipes, I. G., & Hoyer, P. B. (2007). involvement of microsomal epoxide hydrolase enzyme in ovotoxicity caused by 7,12-dimethylbenz[a]anthracene. Toxicological sciences : an official journal of the Society of Toxicology, 96(2).
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  Ovarian follicle disruption in mice caused by 7,12-dimethylbenz[a]anthracene (DMBA) is attributed to its bioactivation by CYP1B1 to a 3,4-epoxide which is then hydrolyzed to form a 3,4-diol by microsomal epoxide hydrolase (mEH). Further epoxidation by CYP1A1 or 1B1 forms the ultimate ovotoxicant, DMBA-3,4-diol-1,2-epoxide. Studies suggest that the mouse ovary expresses these enzymes, and thus, may be capable of bioactivating DMBA to its ovotoxic metabolite. The present study was designed to evaluate the role of ovarian mEH in DMBA-induced ovotoxicity using a novel neonatal mouse ovarian culture system. Ovaries from postnatal day (PND) 4 B6C3F(1) mice were incubated with DMBA (12.5 nM-1 microM) for various lengths of time. Following incubation, ovaries were histologically evaluated or assessed for mEH protein or mRNA. Following 15 days of incubation, DMBA reduced (p < 0.05) healthy follicles at concentrations >or= 12.5 nM. At 1 microM DMBA, follicle loss and increased mEH protein were measured (p < 0.05) by 6 h. mRNA encoding mEH markedly increased after 2 days of incubation, and this increase preceded accelerated follicle loss at 4 days. Furthermore, follicle loss induced by DMBA was prevented when cyclohexene oxide (2mM), an mEH inhibitor, was added to DMBA incubations. These studies suggest that the PND4 mouse ovary is capable of bioactivating DMBA to its ovotoxic form, and that ovarian mEH enzyme activity is likely involved. Furthermore, these observations support the use of a novel ovarian culture system to study ovary-specific metabolism of xenobiotic chemicals.
• Hoyer, P., & Hoyer, P. B. (2005). Damage to ovarian development and function. Cell and tissue research, 322(1).
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  Ovarian function in women can be compromised by exposure to toxic environmental factors. Chemicals that affect ovarian function can act through direct effects on hormone action (ovary) or by interference with steroid hormone action (hypothalamus and/or pituitary). These effects can cause problems in the form of infertility. Alternatively, ovarian toxicants can directly cause ovarian failure by extensive follicular destruction. This targeting can result in loss of ovarian steroid hormones, eventual ovarian failure (menopause), and ultimate disruption of neuroendocrine feedback causing increased levels of FSH and LH. This article provides an overview of chemicals that in animal studies have been identified to cause disrupted ovarian function with a focus on the sites of targeting by which these disruptions occur. In predicting the impact of environmental factors on reproductive function in women, it is critical to gain a better appreciation of the physiological consequences resulting from the potential variety of mechanisms by which toxicants can disrupt ovarian function. This article attempts to provide such a perspective within the context of specific chemicals for which ovarian sites of toxicity have been identified.
• Hoyer, P., Lohff, J. C., Christian, P. J., Marion, S. L., Arrandale, A., & Hoyer, P. B. (2005). Characterization of cyclicity and hormonal profile with impending ovarian failure in a novel chemical-induced mouse model of perimenopause. Comparative medicine, 55(6).
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  4-Vinylcyclohexene diepoxide (VCD) causes early, gradual ovarian failure in mice because it specifically targets small pre-antral ovarian follicles. The period between loss of these follicles and ovarian failure is analogous to perimenopause in women. We sought to characterize the period of onset of ovarian failure in VCD-treated mice in regard to estrous cycle length and hormonal changes. Female C57Bl/6 mice (age, 28 days) were dosed daily for 15 days with VCD (160 mg/kg intraperitoneally) to cause early ovarian failure or with vehicle only (control animals). Cycle length was monitored by vaginal cytology. Plasma levels of 17beta-estradiol (E2), progesterone (P4), and follicle-stimulating hormone (FSH) in control and VCD-treated animals were measured during proestrus of cycles 1 through 12. Cycle length (mean, 5.8 days) did not differ between groups for cycles 1 through 4. In contrast, cycle length during cycles 5 through 12 was increased (mean length, 10.9 days; P < 0.05 versus control) in VCD-treated animals, which also showed an apparent increase in plasma FSH levels. Plasma E2 and P4 at proestrus did not differ between groups during any cycle. Ovarian failure in VCD-treated mice was confirmed by histological evaluation on day 156 after onset of dosing, whereas control animals were still cycling. Therefore, despite compromised cycle length in VCD-treated mice, peak ovarian steroid production in preovulatory follicles at proestrus is adequate. These results demonstrate that the VCD-treated mouse can serve as an appropriate model to mimic hormonal changes during the perimenopausal transition in women.
• Hoyer, P., Thompson, K. E., Bourguet, S. M., Christian, P. J., Benedict, J. C., Sipes, I. G., Flaws, J. A., & Hoyer, P. B. (2005). Differences between rats and mice in the involvement of the aryl hydrocarbon receptor in 4-vinylcyclohexene diepoxide-induced ovarian follicle loss. Toxicology and applied pharmacology, 203(2).
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  Repeated dosing with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively depletes small pre-antral follicles in the ovaries of rats and mice via apoptosis. The aryl hydrocarbon receptor (AhR) plays a role in mediating the effects of several xenobiotics. Therefore, this study was designed to investigate a potential role of the AhR in VCD-induced ovotoxicity. Female F344 rats, C57BL/6 mice, or AhR-deficient (-/-, AhRKO) mice were dosed daily (15 days) with vehicle, VCD (80 mg/kg, i.p.) and/or the AhR antagonist, alpha-naphthoflavone (ANF; 80 mg/kg, i.p.). Compared with controls, VCD caused a 60% reduction (P < 0.05) in primordial and primary follicles in mice and rats. Concurrent dosing with ANF protected against the VCD-induced follicle loss in rats, but not in mice. As with AhR-intact mice and rats, VCD induced a 70% loss (P < 0.05) of small pre-antral follicles in AhRKO mice. AhR mRNA expression was increased (P < 0.05) by VCD dosing in small pre-antral follicles isolated from ovaries of rats but not mice. AhR protein in rats was increased by VCD dosing in oocyte nuclei in primordial and primary follicles when measured by immunofluorescence and confocal microscopy. In rat small pre-antral follicles, apoptosis-associated caspase-3-like activity was increased (P < 0.05) by VCD treatment, decreased (P < 0.05) by ANF treatment, and unaffected by VCD plus ANF treatment. VCD had no effect on expression of GST Ya1 or GST Ya2 mRNA or CYP 1A1 protein in rats. Taken together, these findings demonstrate a difference between rats and mice in the potential involvement of AhR as related to VCD-induced ovotoxicity. Whereas, AhR appears to be involved in rats, no evidence for a similar role in mice was obtained. Overall, these findings point out that there can be mechanistic species differences in ovarian responses to xenobiotic chemicals.
• Hoyer, P., & Hoyer, P. B. (2004). Can the clock be turned back on ovarian aging?. Science of aging knowledge environment : SAGE KE, 2004(10).
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  A basic tenet of reproductive biology is that female mammals are born with a set number of germ cells (oocytes). A new study by Johnson et al. challenges these beliefs by demonstrating that the postnatal mouse ovary contains actively dividing germ cells. These findings have implications for prolonging the reproductive life span of women.
• Hoyer, P., Devine, P. J., Sipes, I. G., & Hoyer, P. B. (2004). Initiation of delayed ovotoxicity by in vitro and in vivo exposures of rat ovaries to 4-vinylcyclohexene diepoxide. Reproductive toxicology (Elmsford, N.Y.), 19(1).
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  Repeated daily dosing of rats with the ovotoxic, occupational chemical 4-vinylcyclohexene diepoxide (VCD, 80 mg/kg, i.p.) selectively depletes primordial and primary ovarian follicles. This study was designed to investigate whether follicle loss can be achieved following a single, acute exposure to VCD. Ovaries removed from postnatal-day-4 female Fischer 344 rats were cultured in the absence or presence of VCD for 15 days. Continuous in vitro exposure to VCD (15 days) caused concentration-dependent loss of primordial and small primary follicles. A single exposure to VCD in vitro (30 microM, 24 h) also caused significant losses of primordial and primary follicles 14 days later. Additionally, 28-day-old female rats were given a single injection of VCD (40-320 mg/kg, i.p.). A single dose at 320 mg/kg resulted in substantial loss of all follicle stages beginning 6 days later. Overall, these results demonstrate that an acute exposure to high concentrations/doses of VCD is sufficient to cause subsequent delayed loss of follicles.
• Hoyer, P., Mayer, L. P., Devine, P. J., Dyer, C. A., & Hoyer, P. B. (2004). The follicle-deplete mouse ovary produces androgen. Biology of reproduction, 71(1).
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  The follicle-depleted postmenopausal ovary is enriched in interstitial cells that produce androgens. This study was designed to cause follicle depletion in mice using the industrial chemical, 4-vinylcyclohexene diepoxide (VCD), and characterize the steroidogenic capacity of cells in the residual ovarian tissue. From a dose-finding study, the optimal daily concentration of VCD was determined to be 160 mg/kg. Female B6C3F(1) immature mice were treated daily with vehicle control or VCD (160 mg kg(-1) day(-1), 15 days, i.p.). Ovaries were removed and processed for histological evaluation. On Day 15 following onset of treatment, primordial follicles were depleted and primary follicles were reduced to about 10% of controls. On Day 46, primary follicles were depleted and secondary and antral follicles were reduced to 0.7% and 2.6% of control, respectively. Seventy-five percent of treated mice displayed disruptions in estrous cyclicity. All treated mice were in persistent diestrus (acyclic) by Day 58. Plasma FSH levels were increased (P < 0.05) relative to controls on Day 37 and had plateaued by Day 100. Relative to age-matched cyclic controls, by Day 127, the significant differences in VCD-treated mice included reduced ovarian and uterine weights, elevated plasma LH and FSH, and reduced plasma progesterone and androstenedione. Furthermore, plasma 17beta-estradiol levels were nondetectable. Unlike controls, immunostaining for LH receptor, and the high density lipoprotein receptor (SR-BI), was diffuse in ovarian sections from VCD-treated animals. Ovaries from Day 120 control and VCD-treated animals were dissociated and dispersed cells were placed in culture. Cultured cells from ovaries of VCD-treated animals produced less LH-stimulated progesterone than control cells. Androstenedione production was nondetectable in cells from cyclic control animals. Conversely, cells from VCD-treated animals produced androstenedione that was doubled in the presence of insulin and LH (1 and 3 ng/ml). Collectively, these data demonstrate that VCD-mediated follicle depletion results in residual ovarian tissue that may be analogous to the follicle-deplete postmenopausal ovary. This may serve as a useful animal model to examine the dynamics of follicle loss in women as ovarian senescence ensues.
• Hoyer, P., Cannady, E. A., Dyer, C. A., Christian, P. J., Sipes, I. G., & Hoyer, P. B. (2003). Expression and activity of cytochromes P450 2E1, 2A, and 2B in the mouse ovary: the effect of 4-vinylcyclohexene and its diepoxide metabolite. Toxicological sciences : an official journal of the Society of Toxicology, 73(2).
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  4-Vinylcyclohexene (VCH), an occupational chemical, causes destruction of small preantral follicles (F1) in mice. Previous studies suggested that VCH is bioactivated via cytochromes P450 (CYP450) to the ovotoxic, diepoxide metabolite, VCD. Whereas hepatic CYP450 isoforms 2E1, 2A, and 2B can metabolize VCH, the role of ovarian metabolism is unknown. This study investigated expression of these isoforms in isolated ovarian fractions (F1, 25-100 microm; F2, 100-250 microm; F3, >250 microm; interstitial cells, Int) from B6C3F1 mice dosed daily (15 days; ip) with vehicle, VCH (7.4 mmol/kg/day) or VCD (0.57 mmol/kg/day). Ovaries were removed and either isolated into specific ovarian compartments for mRNA analysis, fixed for immunohistochemistry, or prepared for enzymatic assays. mRNA and protein for all isoforms were expressed/distributed in all ovarian fractions from vehicle-treated mice. In the targeted F1 follicles, VCH or VCD dosing increased (p < 0.05) mRNA encoding CYP2E1 (645 +/- 14% VCH; 582 +/- 16% VCD), CYP2A (689 +/- 8% VCH; 730 +/- 22% VCD), and CYP2B (246 +/- 7% VCH) above control. VCH dosing altered (p < 0.05) mRNA encoding CYP2E1 in nontargeted F3 follicles (168 +/- 7%) and CYP2A in Int (207 +/- 19%) above control. Immunohistochemical analysis revealed the greatest staining intensity for all CYP isoforms in the Int. VCH dosing altered (p < 0.05) staining intensity in Int for CYP2E1 (19 +/- 2.4% below control) and CYP2A (39 +/- 5% above control). Staining intensity for CYP2B was increased (p < 0.05) above control in granulosa cells of small preantral (187 +/- 42%) and antral (63 +/- 8%) follicles. Catalytic assays in ovarian homogenates revealed that CYP2E1 and CYP2B were functional. Only CYP2E1 activity was increased (149 +/- 12% above control; p < 0.05) by VCH dosing. The results demonstrate that mRNA and protein for CYP isoforms known to bioactivate VCH are expressed in the mouse ovary and are modulated by in vivo exposure to VCH and VCD. Interestingly, there is high expression of these isoforms in the Int. Thus, the ovary may contribute to ovotoxicity by promoting bioactivation of VCH to the toxic metabolite, VCD.
• Hoyer, P., Cannady, E. A., Dyer, C. A., Christian, P. J., Sipes, I. G., & Hoyer, P. B. (2002). Expression and activity of microsomal epoxide hydrolase in follicles isolated from mouse ovaries. Toxicological sciences : an official journal of the Society of Toxicology, 68(1).
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  Microsomal epoxide hydrolase (mEH) is involved in the detoxification of xenobiotics that are or can form epoxide metabolites, including the ovotoxicant, 4-vinylcyclohexene (VCH). This industrial chemical is bioactivated by hepatic CYP450 to the diepoxide metabolite, VCD, which destroys mouse small preantral follicles (F1). Since ovarian mEH may play a role in VCD detoxification, these studies investigated the expression and activity of mEH in isolated ovarian fractions. Mice were given 1 or 15 daily doses (ip) of VCH (7.4 mmol/kg/day) or VCD (0.57 mmol/kg/day); 4 h following the final dose, ovaries were removed, distinct populations of intact follicles (F1, 25-100 microm; F2, 100-250 microm; F3, > 250 microm) and interstitial cells (Int) were isolated, and total RNA and protein were extracted. Real-time polymerase chain reaction and the substrate cis-stilbene oxide (CSO; 12.5 microM) were used to evaluate expression and specific activity of mEH, respectively. Confocal microscopy evaluated ovarian distribution of mEH protein. Expression of mRNA encoding mEH was increased in F1 (410 +/- 5% VCH; 292 +/- 5% VCD) and F2 (1379 +/- 4% VCH; 381 +/- 11% VCD) follicles following repeated dosing with VCH or VCD. Catalytic activity of mEH increased in F1 follicles following repeated dosing with VCH/VCD (381 +/- 11% VCH; 384 +/- 27% VCD). Visualized by confocal microscopy, mEH protein was distributed throughout the ovary with the greatest staining intensity in the interstitial cells and staining in the theca cells that was increased by dosing (56 +/- 0.8% VCH; 29 +/- 0.9% VCD). We conclude that mEH is expressed and is functional in mouse ovarian follicles. Additionally,in vivo dosing with VCH and VCD affects these parameters.
• Hoyer, P., Devine, P. J., Rajapaksa, K. S., & Hoyer, P. B. (2002). In vitro ovarian tissue and organ culture: a review. Frontiers in bioscience : a journal and virtual library, 7.
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  Many investigations have utilized techniques for culturing ovarian tissue or isolated follicles in vitro. Whole ovaries from fetal or neonatal rodents have been incubated in organ culture systems. This has been utilized to understand the sequence of follicle formation and its hormonal requirements, activation of quiescent follicles, follicular growth and development, and acquisition of steroidogenic capabilities. Adaptations of this technique include incubation of ovaries in a chamber continuously perfused with medium or perfusion of medium through the intact vasculature. Late follicular development, ovulation, and steroidogenesis can also be examined in these systems. Another approach has been to culture individual follicles isolated by enzymatic or mechanical dissociation. The majority of this research has focused on improving follicular development in vitro. This review will discuss these various culture techniques and some of the results that have been acquired. Recent results from toxicological studies utilizing whole ovarian cultures performed will also be described. Future applications for ovarian and follicular cultures may include in vitro follicular development for eventual production of offspring from frozen ovarian tissue, mechanistic studies related to the impact of endocrine disruptors and ovotoxicants on ovarian function, and further investigations into follicle activation and development.
• Hoyer, P., Devine, P. J., Sipes, I. G., Skinner, M. K., & Hoyer, P. B. (2002). Characterization of a rat in vitro ovarian culture system to study the ovarian toxicant 4-vinylcyclohexene diepoxide. Toxicology and applied pharmacology, 184(2).
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  Repeated daily dosing of rats with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) causes selective depletion of the smallest preantral ovarian follicles (primordial and primary). These targeted populations are difficult to study because they comprise very little of the overall mass of ovarian tissue. Additionally, they are randomly distributed throughout the ovary. Therefore, a neonatal rat ovarian culture system containing predominantly primordial and primary follicles was developed and its susceptibility to VCD was assessed. The in vivo sensitivity of neonatal rats to VCD dosing was first confirmed by daily injection of VCD (80 mg/kg/day ip) on postnatal days (PND) 4-19. On PND 19, depletion of primordial and small primary follicles was evident. Ovarian cultures were then established utilizing a floating organ culture system to treat ovaries from PND 4 Fischer 344 rats in vitro. Initial follicle populations in cultured ovaries consisted of primordial (81%) and small primary (19%) follicles, whereas larger-sized preantral follicles had developed after 15 days in culture (67% primordial, 31% small primary, and 2% large primary). Cultured rat ovaries were sensitive to follicle depletion by incubation with VCD (> or =30 microM), and follicle loss occurred in a time-dependent manner (8-15 days). Evidence for apoptosis in VCD-exposed ovaries, as demonstrated in vivo, was obtained using immunohistochemistry. There was significantly more staining for apoptosis-associated active caspase-3 and TUNEL in ovaries incubated with VCD (30 microM, 15 days) compared with matched controls. These results demonstrate that small preantral follicles of cultured neonatal rat ovaries are sensitive to VCD exposure. The similarities between VCD's effects in vitro and in vivo demonstrate the usefulness of this system for future mechanistic studies related to ovarian follicle loss induced by VCD or other ovotoxic chemicals.
• Hoyer, P., Hu, X., Flaws, J. A., Sipes, I. G., & Hoyer, P. B. (2002). Activation of mitogen-activated protein kinases and AP-1 transcription factor in ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats. Biology of reproduction, 67(3).
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  Previous studies have demonstrated that ovotoxicity induced in small preantral (primordial and primary) ovarian follicles by 4-vinylcyclohexene diepoxide (VCD) in rats is likely via acceleration of the normal process of atresia (apoptosis). This acceleration is associated with increased activities of caspase cascades, changes in subcellular distribution of Bcl-2 family members, and alteration of estrogen receptor-mediated signaling pathways. The present study was designed to investigate possible effects of VCD dosing on the mitogen-activated protein kinases (MAPK)/AP-1 signaling pathways in rat ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg i.p., 1 day--a time when ovotoxicity has not been initiated) or dosed daily for 10 or 15 days (80 mg/kg i.p.; 10 days--a time when the earliest signs of impending follicular destruction is seen, 15 days--a time when significant ovotoxicity is underway). Four hours following the final dose, ovaries and livers were collected. Ovarian small (25-100 microm) and large (100-250 microm) preantral follicles were isolated, and cytosolic or nuclear extracts were prepared from follicles and livers for analyses. Activities of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal protein kinase (JNK), and p38 kinase, were determined in follicular and liver cytosolic extracts, and AP-1 DNA binding activity was determined in follicular and liver nuclear extracts. Compared with control, a single dose of VCD caused a decrease in JNK activity and an increase of AP-1 binding activity in isolated small ovarian follicles. After repeated daily dosing with VCD for 10 or 15 days, JNK and p38 kinase activities in small ovarian follicles were increased (p38 kinase: 1.64 +/- 0.14 for 10 days, 1.48 +/- 0.11 for 15 days, VCD/control, P < 0.01; JNK: 1.44 +/- 0.11 for 10 days, 1.37 +/- 0.06 for 15 days, VCD/control, P < 0.01) and AP-1 binding activity in small ovarian follicles was decreased (10 days, 0.29 +/- 0.04; 15 days, 0.51 +/- 0.04, VCD/control, P < 0.01). VCD did not affect any of these measurements in large preantral follicles or liver. Phosphorylation status of c-Jun protein as measured by Western blotting was increased (1.22 +/- 0.1, VCD/control, P < 0.05) after the 15-day daily dosing with VCD, but total c-Jun protein levels were unaffected. Using antibodies against c-Jun or phospho-c-Jun for supershift DNA binding assay, c-Jun and phospho-c-Jun were identified in the AP-1-DNA binding complex, and the binding activity was reduced in tissues with increased phospho-c-Jun protein levels. Taken together, these data provide evidence that accelerated atretic signals induced by VCD is associated with MAPK/AP-1 signaling pathways and phosphorylation of c-Jun plays a significant role in transmitting the apoptotic signals.
• Hoyer, P., Thompson, K. E., Sipes, I. G., Greenstein, B. D., & Hoyer, P. B. (2002). 17beta-estradiol affords protection against 4-vinylcyclohexene diepoxide-induced ovarian follicle loss in Fischer-344 rats. Endocrinology, 143(3).
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  Repeated dosing with 4-vinylcyclohexene diepoxide (VCD) accelerates atresia via apoptosis in primordial and primary follicles in ovaries of rats. The mechanisms that control atresia and VCD-induced toxicity are unknown; however, they could involve 17beta-E2. Atresia slows as animals enter puberty, whereas circulating E2 levels increase with the the onset of cyclicity. This inverse relationship suggests that E2 may be involved in the control of atresia. Therefore, this study was designed to determine whether treatment of immature rats with E2 could protect follicles normally destroyed by VCD-induced apoptosis. Female F344 rats were treated daily with E2, ER analogs, and/or VCD for 15 d. VCD alone caused a 50% reduction in primordial and primary follicles. Coinjection of E2 (0.1 mg/kg) and VCD (80 mg/kg) selectively protected primary follicles from VCD-induced follicle loss. This protection was mimicked by an ER agonist, genistein (0.1 mg/kg), and prevented by an ER antagonist, 4-hydroxytamoxifen (2 mg/kg). VCD treatment increased caspase-3-like activity, whereas concurrent treatment with genistein and VCD restored caspase-3-like activity to control levels. VCD treatment had no effect on circulating E2 levels, uterine weight, or E2 binding to the ER, nor could it directly displace E2 from ERbeta. These observations support the idea that ER-mediated protection against VCD-induced follicle toxicity is obtained by reducing apoptosis in small preantral follicles, although VCD does not appear to directly interact with ER.

Presentations

• Hoyer, P. B. (2015, Sept 23). Free feeding of wild-caught norway rats with a non-toxic liquid bait results in compromised fertility. 10th European Vertebrate Pest Management Conferencce. Universidad de Sevilla, Spain.
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  oral presentation
• Hoyer, P. B. (2014, Oct 29). Endocrine and Reproductive Toxicology Lectures. Course in Toxicology. Baltimore MD: Federal Drug Administration.
• Hoyer, P. B. (2014, Oct. 17). VCD as a tool for understanding ovotoxicity and modeling menopause. Interdisciplinary Environmental Toxicology Open House. Urbana Illinois: University of Illinois - Urbana.
• Hoyer, P. B. (2014, Sept 5). Development of a novel mouse model for peri- and post-menopause. Sex and Gender DIfferences Symposium. University of Arizona: Women's Health Research.