137 results on '"Hoyer PB"'
Search Results
2. 4-Vinylcyclohexene Diepoxide (VCD)-Induced Fibroadenomas: A Novel Rat Model of Mammary Tumorigenesis.
- Author
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Wright, LE, primary, Lukefahr, AL, additional, Frye, JB, additional, Hoyer, PB, additional, Besselsen, DG, additional, and Funk, JL, additional
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- 2010
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3. Menopause Increases NOS Uncoupling and Reduces Heart Function after Myocardial Infarction
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Kumar, S, primary, Sharma, S, additional, Noonepalle, SK, additional, Liu, J, additional, Hu, T, additional, Hoyer, PB, additional, and Black, SM, additional
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- 2010
- Full Text
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4. Comparison of Skeletal Changes in Rat Models of Ovary-Intact vs. Surgical Menopause.
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Lukefahr, AL, primary, Frye, JB, additional, Wright, LE, additional, Marion, SL, additional, Hoyer, PB, additional, and Funk, JL, additional
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- 2010
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5. The effect of diet and cardiovascular risk on ovarian aging in cynomolgus monkeys (Macaca fascicularis).
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Appt SE, Chen H, Goode AK, Hoyer PB, Clarkson TB, Adams MR, Wilson ME, Franke AA, Kaplan JR, Appt, Susan E, Chen, Haiying, Goode, Amanda K, Hoyer, Patricia B, Clarkson, Thomas B, Adams, Michael R, Wilson, Mark E, Franke, Adrian A, and Kaplan, Jay R
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- 2010
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6. Serum antimüllerian hormone predicts ovarian reserve in a monkey model.
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Appt SE, Clarkson TB, Chen H, Adams MR, Christian PJ, Hoyer PB, Wilson ME, Kaplan JR, Appt, Susan E, Clarkson, Thomas B, Chen, Haiying, Adams, Michael R, Christian, Patricia J, Hoyer, Patricia B, Wilson, Mark E, and Kaplan, Jay R
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- 2009
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7. Effect of duration of dosing on onset of ovarian failure in a chemical-induced mouse model of perimenopause.
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Lohff JC, Christian PJ, Marion SL, and Hoyer PB
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- 2006
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8. Molecular analysis of the effects of steroid hormones on mouse meiotic prophase I progression.
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Burks DM, McCoy MR, Dutta S, Mark-Kappeler CJ, Hoyer PB, and Pepling ME
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- Animals, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Female, Fetus, Gene Expression Regulation, Developmental drug effects, Granulosa Cells metabolism, Mice, Inbred C57BL, Oocytes cytology, Oocytes metabolism, Organ Culture Techniques, Ovary embryology, Ovary metabolism, Pachytene Stage drug effects, Pregnancy, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Estradiol pharmacology, Meiotic Prophase I drug effects, Oocytes drug effects, Ovary drug effects, Progesterone pharmacology
- Abstract
Background: Infertility is linked to depletion of the primordial follicle pool consisting of individual oocytes arrested at the diplotene stage of meiotic prophase I surrounded by granulosa cells. Primordial germ cells, the oocyte precursors, begin to differentiate during embryonic development. These cells migrate to the genital ridge and begin mitotic divisions, remaining connected, through incomplete cytokinesis, in clusters of synchronously dividing oogonia known as germ cell cysts. Subsequently, they enter meiosis, become oocytes and progress through prophase I to the diplotene stage. The cysts break apart, allowing individual oocytes to be surrounded by a layer of granulosa cells, forming primordial follicles each containing a diplotene arrested oocyte. A large number of oocytes are lost coincident with cyst breakdown, and may be important for quality control of primordial follicle formation. Exposure of developing ovaries to exogenous hormones can disrupt cyst breakdown and follicle formation, but it is unclear if hormones affect progression of oocytes through prophase I of meiosis., Methods: Fetal ovaries were treated in organ culture with estradiol, progesterone, or both hormones, labeled for MSY2 or Synaptonemal complex protein 3 (SYCP3) using whole mount immunocytochemistry and examined by confocal microscopy. Meiotic prophase I progression was also followed using the meiotic surface spread technique., Results: MSY2 expression in oocytes was reduced by progesterone but not estradiol or the hormone combination. However, while MSY2 expression was upregulated during development it was not a precise marker for the diplotene stage. We also followed meiotic prophase I progression using antibodies against SYCP3 using two different methods, and found that the percent of oocytes at the pachytene stage peaked at postnatal day 1. Finally, estradiol and progesterone treatment together but not either alone in organ culture increased the percent of oocytes at the pachytene stage., Conclusions: We set out to examine the effects of hormones on prophase I progression and found that while MSY2 expression was reduced by progesterone, MSY2 was not a precise diplotene stage marker. Using antibodies against SYCP3 to identify pachytene stage oocytes we found that progesterone and estradiol together delayed progression of oocytes through prophase I.
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- 2019
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9. Comparison of Reproductive Function in Female TgMISIIR-TAg Transgenic and Wildtype C57BL/6 Mice.
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Hoyer PB, Rice PF, Howard CC, Koevary JW, Dominguez Cooks JP, Hutchens GV, Chambers SK, Craig ZR, Connolly DC, and Barton JK
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- Animals, Cyclohexenes toxicity, Estrus drug effects, Female, Mice, Mice, Inbred C57BL, Mice, Transgenic, Ovarian Follicle drug effects, Vinyl Compounds toxicity, Pharmacogenomic Variants, Reproduction drug effects, Reproduction genetics
- Abstract
Transgenic TgMISIIR-TAg (TAg) mice express the oncogenic virus SV40 in Mullerian epithelial cells. Female TAg mice spontaneously develop epithelial ovarian carcinoma, the most common type of ovarian cancer in women. Female TAg mice are infertile, but the reason has not been determined. We therefore investigated whether female TAg mice undergo puberty, demonstrate follicular development, maintain regular cycles, and ovulate. Ovarian cancers in women commonly develop after menopause. The occupational chemical 4-vinylcyclohexene diepoxide (VCD) accelerates follicle degeneration in the ovaries of rats and mice, causing early ovarian failure. We therefore used VCD dosing of mice to develop an animal model for menopause. The purpose of this study was to characterize reproductive parameters in female TAg mice and to investigate whether the onset of ovarian failure due VCD dosing differed between female TAg and WT C57BL/6 mice. As in WT female mice, TAg female mice underwent puberty (vaginal opening) and developed cyclicity in patterns that were similar between the groups. Vehicle-only TAg mice had fewer ovulations (numbers of corpora lutea) than WT animals. VCD exposure delayed the onset of puberty (day of first estrus) in TAg as compared with WT mice. Morphologic evaluation of ovaries revealed many more degenerating follicles in TAg mice than WT mice, and more VCD-dosed TAg mice were in ovarian failure than VCD-dosed WT mice. These results suggest that despite showing similar onset of sexual maturation, TAg mice have increased follicular degeneration and fewer ovulations than WT. These features may contribute to the inability of female TAg mice to reproduce.
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- 2019
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10. Ex Vivo Fetal Whole Ovarian Culture Model: An Essential Tool for Studies in Reproductive Toxicology and Pharmacology.
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Stanley JA, Arosh JA, Hoyer PB, and Banu SK
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- Animals, Cells, Cultured, Female, Gene Expression Profiling, Gene Expression Regulation, Developmental drug effects, Models, Biological, Ovarian Follicle cytology, Ovarian Follicle metabolism, Ovary drug effects, Ovary metabolism, Rats, Reproduction, Cell Culture Techniques methods, Endocrine Disruptors toxicity, Ovary cytology, Ovary embryology
- Abstract
Major limitations in understanding the direct effects of endocrine-disrupting chemicals (EDCs) and cell signaling events in ovarian cellular dynamics in mammals include a lack of proper and simple tools/techniques as well as gaps in knowledge regarding the critical window(s) of vulnerability. Identifying and validating such tools and evaluating the effects of EDCs on molecular dynamics and cellular events during the critical windows of ovarian development are very important to improve the fertility in women and preserve the future health of the developing fetuses. Therefore, we developed a fetal whole ovarian ex vivo culture model. Ex vivo ovary culture models allow varying culture parameters in a highly controlled manner and thus have the potential to allow a more thorough evaluation for reproductive toxicity studies and drug response. This chapter describes clear and thorough details for setting up and maintaining an ex vivo culture system from the rat ovaries and further analyses of mRNA and protein expressions and estimating follicle numbers.
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- 2019
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11. Colorectal tumor prevention by the progestin medroxyprogesterone acetate is critically dependent on postmenopausal status.
- Author
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Meijer BJ, Wielenga MCB, Hoyer PB, Amos-Landgraf JM, Hakvoort TBM, Muncan V, Heijmans J, and van den Brink GR
- Abstract
The large randomized placebo controlled trials of the Women's Health Initiative have shown that the combination of estrogen and progestin medroxyprogesterone acetate (MPA) protects from colorectal cancer in postmenopausal women. No effect was observed in women treated with estrogen alone. This suggests that progesterone, or more specifically the progestin MPA may have chemopreventive activity. The effect of MPA on colorectal carcinogenesis has been difficult to study in animal models. Most models are not affected by either depleting female hormones by ovariectomy or treatment with MPA. Importantly, an ovariectomy fails to reproduce one of the hall marks of the postmenopausal state in women with intact ovaries. That is, the continued production of androgens by the atrophic postmenopausal ovaries. Here we show that adenoma incidence is increased in the vinyl cylcohexene diepoxide (VCD) mouse model of the menopause compared to age matched fertile female mice. Treatment with MPA protected VCD treated mice from adenomagenesis, but had no effect on adenoma numbers in age-matched fertile female mice. Our data show that the protective effect of MPA depends on the postmenopausal state and suggest that MPA monotherapy may be studied as a chemopreventive agent in postmenopausal women., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.
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- 2018
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12. COMPROMISED FERTILITY IN FREE FEEDING OF WILD-CAUGHT NORWAY RATS (RATTUS NORVEGICUS) WITH A LIQUID BAIT CONTAINING 4-VINYLCYCLOHEXENE DIEPOXIDE AND TRIPTOLIDE.
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Witmer GW, Raymond-Whish S, Moulton RS, Pyzyna BR, Calloway EM, Dyer CA, Mayer LP, and Hoyer PB
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- Animals, Animals, Wild, Contraceptive Agents, Female administration & dosage, Epoxy Compounds pharmacology, Female, Male, Pest Control, Rats, Rats, Sprague-Dawley, Contraceptive Agents, Female pharmacology, Cyclohexenes pharmacology, Diterpenes pharmacology, Fertility drug effects, Phenanthrenes pharmacology, Vinyl Compounds pharmacology
- Abstract
Wild rat pests in the environment cause crop and property damage and carry disease. Traditional methods of reducing populations of these pests involve poisons that can cause accidental exposures in other animals and humans. Fertility management with nonlethal chemicals would be an improved method of rat pest population control. Two chemicals known to target ovarian function in female rats are 4-vinylcyclohexene diepoxide (VCD) and triptolide. Additionally, triptolide impairs spermatogenesis in males. A liquid bait containing no active ingredients (control), or containing triptolide (0.001%) and VCD (0.109%; active) was prepared to investigate the potential use of these agents for wild rat pest population control. Liquid bait was made available to male (n = 8 control; n = 8 active) and female (n = 8 control; n = 8 active) Sprague Dawley rats ( Rattus norvegicus ) for oral consumption prior to breeding. Whereas, control bait-treated females produced normal-sized litters (10.0 ± 1.7 pups/litter), treated females delivered no pups. Wild Norway male (n = 20) and female (n = 20) rats ( Rattus norvegicus ) were trapped, individually housed, and one group given free access to control bait, one group to active bait. Following three cycles of treatment-matched mating pairs, females consuming control bait (control) produced normal litter sizes (9.73 ± 0.73 pups/litter). Females who had consumed active bait (treated) produced no litters on breeding cycles one and two; however, 2 of 10 females produced small litters on the third mating cycle. In a fourth breeding cycle, control females were crossmated with treated males, and treated females were crossmated with control males. In both groups, some dams produced litters, while others did not. The differences in response reflect a heterogeneity in return to cyclicity between females. These results suggest a potential approach to integrated pest management by compromising fertility, and could provide a novel alternative to traditional poisons for reducing populations of wild rat pests.
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- 2017
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13. The VCD Mouse Model of Menopause and Perimenopause for the Study of Sex Differences in Cardiovascular Disease and the Metabolic Syndrome.
- Author
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Brooks HL, Pollow DP, and Hoyer PB
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- Animals, Cyclohexenes, Diabetic Nephropathies physiopathology, Female, Humans, Male, Mice, Ovary cytology, Ovary drug effects, Vinyl Compounds, Cardiovascular Diseases physiopathology, Disease Models, Animal, Menopause, Metabolic Syndrome physiopathology, Perimenopause, Sex Characteristics
- Abstract
In females, menopause, the cessation of menstrual cycling, is associated with an increase in risk for several diseases such as cardiovascular disease, osteoporosis, diabetes, the metabolic syndrome, and ovarian cancer. The majority of women enter menopause via a gradual reduction of ovarian function over several years (perimenopause) and retain residual ovarian tissue. The VCD mouse model of menopause (ovarian failure in rodents) is a follicle-deplete, ovary-intact animal that more closely approximates the natural human progression through perimenopause and into the postmenopausal stage of life. In this review, we present the physiological parameters of how to use the VCD model and explore the VCD model and its application into the study of postmenopausal disease mechanisms, focusing on recent murine studies of diabetic kidney disease, the metabolic syndrome, and hypertension., (©2016 Int. Union Physiol. Sci./Am. Physiol. Soc.)
- Published
- 2016
- Full Text
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14. The steroid hormone environment during primordial follicle formation in perinatal mouse ovaries.
- Author
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Dutta S, Mark-Kappeler CJ, Hoyer PB, and Pepling ME
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- 3-Hydroxysteroid Dehydrogenases genetics, 3-Hydroxysteroid Dehydrogenases metabolism, Animals, Animals, Newborn, Animals, Outbred Strains, Aromatase genetics, Aromatase metabolism, Embryo, Mammalian cytology, Embryo, Mammalian enzymology, Embryo, Mammalian metabolism, Embryonic Stem Cells enzymology, Embryonic Stem Cells metabolism, Estradiol blood, Female, Gene Expression Regulation, Developmental, Immunohistochemistry, Mice, Microscopy, Fluorescence, Organ Culture Techniques, Ovarian Follicle embryology, Ovarian Follicle growth & development, Ovarian Follicle metabolism, Ovary cytology, Ovary embryology, Ovary growth & development, Ovary metabolism, Progesterone blood, Stem Cells enzymology, Stem Cells metabolism, Apoptosis, Embryonic Stem Cells cytology, Estradiol metabolism, Oogenesis, Ovarian Follicle cytology, Progesterone metabolism, Stem Cells cytology
- Abstract
Primordial follicle assembly is essential for reproduction in mammalian females. Oocytes develop in germ cell cysts that in late fetal development begin break down into individual oocytes and become surrounded by pregranulosa cells, forming primordial follicles. As they separate, many oocytes are lost by apoptosis. Exposure to steroid hormones delays cyst breakdown, follicle formation, and associated oocyte loss in some species. One model for regulation of follicle formation is that steroid hormones in the maternal circulation keep cells in cysts and prevent oocyte death during fetal development but that late in pregnancy hormone levels drop, triggering cyst breakdown and associated oocyte loss. However, herein we found that, while maternal circulating levels of progesterone drop during late fetal development, maternal estradiol levels remain high. We hypothesized that fetal ovaries were the source of hormones and that late in fetal development their production stops. To test this, mRNA and protein levels of steroidogenic enzymes required for estradiol and progesterone synthesis were measured. We found that aromatase and 3-beta-hydroxysteroid dehydrogenase mRNA levels drop before cyst breakdown. The 3-beta-hydroxysteroid dehydrogenase protein levels also dropped, but we did not detect a change in aromatase protein levels. The steroid content of perinatal ovaries was assayed, and both estradiol and progesterone were detected in fetal ovaries before cyst breakdown. To determine the role of steroid hormones in oocyte development, we examined the effects of blocking steroid hormone production in organ culture and found that the number of oocytes was reduced, supporting our model that steroid hormones are important for fetal oocyte survival., (© 2014 by the Society for the Study of Reproduction, Inc.)
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- 2014
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15. Involvement of a volatile metabolite during phosphoramide mustard-induced ovotoxicity.
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Madden JA, Hoyer PB, Devine PJ, and Keating AF
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- Animals, Antineoplastic Agents pharmacokinetics, Aziridines pharmacology, Cyclophosphamide pharmacokinetics, Dose-Response Relationship, Drug, Female, In Vitro Techniques, Ovarian Follicle drug effects, Phosphoramide Mustards pharmacokinetics, Rats, Aziridines toxicity, Ovary drug effects, Phosphoramide Mustards toxicity
- Abstract
The finite ovarian follicle reserve can be negatively impacted by exposure to chemicals including the anti-neoplastic agent, cyclophosphamide (CPA). CPA requires bioactivation to phosphoramide mustard (PM) to elicit its therapeutic effects however; in addition to being the tumor-targeting metabolite, PM is also ovotoxic. In addition, PM can break down to a cytotoxic, volatile metabolite, chloroethylaziridine (CEZ). The aim of this study was initially to characterize PM-induced ovotoxicity in growing follicles. Using PND4 Fisher 344 rats, ovaries were cultured for 4 days before being exposed once to PM (10 or 30 μM). Following eight additional days in culture, relative to control (1% DMSO), PM had no impact on primordial, small primary or large primary follicle number, but both PM concentrations induced secondary follicle depletion (P<0.05). Interestingly, a reduction in follicle number in the control-treated ovaries was observed. Thus, the involvement of a volatile, cytotoxic PM metabolite (VC) in PM-induced ovotoxicity was explored in cultured rat ovaries, with control ovaries physically separated from PM-treated ovaries during culture. Direct PM (60 μM) exposure destroyed all stage follicles after 4 days (P<0.05). VC from nearby wells depleted primordial follicles after 4 days (P<0.05), temporarily reduced secondary follicle number after 2 days, and did not impact other stage follicles at any other time point. VC was determined to spontaneously liberate from PM, which could contribute to degradation of PM during storage. Taken together, this study demonstrates that PM and VC are ovotoxicants, with different follicular targets, and that the VC may be a major player during PM-induced ovotoxicity observed in cancer survivors., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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16. Acute 7,12-dimethylbenz[a]anthracene exposure causes differential concentration-dependent follicle depletion and gene expression in neonatal rat ovaries.
- Author
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Madden JA, Hoyer PB, Devine PJ, and Keating AF
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- Animals, Animals, Newborn, Autophagy drug effects, Dose-Response Relationship, Drug, Epoxide Hydrolases genetics, Female, Glutathione Transferase genetics, Ovarian Follicle drug effects, Ovary metabolism, Ovary pathology, Oxidative Stress, Phosphatidylinositol 3-Kinases genetics, Rats, Rats, Inbred F344, 9,10-Dimethyl-1,2-benzanthracene toxicity, Gene Expression Regulation drug effects, Ovary drug effects
- Abstract
Chronic exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), generated during combustion of organic matter including cigarette smoke, depletes all ovarian follicle types in the mouse and rat, and in vitro models mimic this effect. To investigate the mechanisms involved in follicular depletion during acute DMBA exposure, two concentrations of DMBA at which follicle depletion has (75 nM) and has not (12.5 nM) been observed were investigated. Postnatal day four F344 rat ovaries were maintained in culture for four days before a single exposure to vehicle control (1% DMSO; CT) or DMBA (12 nM; low-concentration or 75 nM; high-concentration). After four or eight additional days of culture, DMBA-induced follicle depletion was evaluated via follicle enumeration. Relative to control, DMBA did not affect follicle numbers after 4 days of exposure, but induced large primary follicle loss at both concentrations after 8 days; while, the low-concentration DMBA also caused secondary follicle depletion. Neither concentration affected primordial or small primary follicle number. RNA was isolated and quantitative RT-PCR performed prior to follicle loss to measure mRNA levels of genes involved in xenobiotic metabolism (Cyp2e1, Gstmu, Gstpi, Ephx1), autophagy (Atg7, Becn1), oxidative stress response (Sod1, Sod2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Kitlg, cKit, Akt1) 1, 2 and 4 days after exposure. With the exception of Atg7 and cKit, DMBA increased (P < 0.05) expression of all genes investigated. Also, BECN1 and pAKT(Thr308) protein levels were increased while cKIT was decreased by DMBA exposure. Taken together, these results suggest an increase in DMBA bioactivation, add to the mechanistic understanding of DMBA-induced ovotoxicity and raise concern regarding female low concentration DMBA exposures., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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17. A method to study the impact of chemically-induced ovarian failure on exercise capacity and cardiac adaptation in mice.
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Chen H, Perez JN, Constantopoulos E, McKee L, Regan J, Hoyer PB, Brooks HL, and Konhilas J
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- Animals, Female, Menopause drug effects, Mice, Mice, Inbred C57BL, Ovary physiology, Adaptation, Physiological physiology, Cyclohexenes administration & dosage, Heart physiology, Menopause physiology, Models, Animal, Ovary drug effects, Physical Conditioning, Animal, Vinyl Compounds administration & dosage
- Abstract
The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.
- Published
- 2014
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18. Xenobiotic effects in the ovary: temporary versus permanent infertility.
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Hoyer PB and Keating AF
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- Animals, Female, Humans, Menopause, Premature, Ovary drug effects, Amenorrhea chemically induced, Infertility, Female chemically induced, Ovarian Follicle drug effects, Primary Ovarian Insufficiency chemically induced, Xenobiotics toxicity
- Abstract
Introduction: Damage caused by xenobiotic compounds to the ovaries is a subject of concern because of their critical role in reproduction. Female mammals are born with a finite number of germ cells (oocytes) encased in primordial follicles. Xenobiotic-induced damage to primordial follicles can result in early ovarian failure (premature menopause). Alternatively, damage affecting larger growing follicles can prevent ovulation, thereby causing infertility during childbearing years., Areas Covered: This review summarizes information from animal studies and human observations about xenobiotic compound classes known to target the ovary to potentially cause reversible infertility (amenorrhea) as well as early ovarian failure. Toxicological evidence supporting ovotoxicity mechanisms from some of these compounds is presented. The reader will gain an appreciation of how exposures to certain widespread environmental chemicals are of concern as regards impacting a woman's reproductive capabilities and life span., Expert Opinion: Three emerging areas of mechanistic targeting of the ovary by these chemicals are identified. These areas relate to the type of cell death, effects on follicular development and the importance of ovarian metabolism. In each case, the potential translational relevance of these areas to toxicological as well as physiological insight is highlighted. A recommendation to expand upon these three areas is made.
- Published
- 2014
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19. In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis.
- Author
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Watson JM, Marion SL, Rice PF, Bentley DL, Besselsen DG, Utzinger U, Hoyer PB, and Barton JK
- Subjects
- Animals, Female, Granulosa Cell Tumor pathology, Hyperplasia pathology, Mice, Multimodal Imaging, Time-Lapse Imaging, Tomography, Optical Coherence, Carcinogenesis pathology, Ovarian Neoplasms pathology, Ovary pathology
- 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 [SHG]) to image mouse ovaries in vivo at multiple time points. We demonstrate the feasibility of imaging mouse ovaries in vivo during a long-term 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 SHG. These results suggest that a combined OCT-MPM system may be useful for early detection of ovarian cancer.
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- 2014
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20. Postnatal exposure to chromium through mother's milk accelerates follicular atresia in F1 offspring through increased oxidative stress and depletion of antioxidant enzymes.
- Author
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Stanley JA, Sivakumar KK, Nithy TK, Arosh JA, Hoyer PB, Burghardt RC, and Banu SK
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- Animals, Apoptosis drug effects, Female, Follicle Stimulating Hormone blood, Pregnancy, Rats, Rats, Sprague-Dawley, Receptors, FSH genetics, Antioxidants metabolism, Chromium toxicity, Follicular Atresia drug effects, Milk metabolism, Oxidative Stress drug effects
- Abstract
Hexavalent chromium, CrVI, is a heavy metal endocrine disruptor, known as a mutagen, teratogen, and a group A carcinogen. Environmental contamination with CrVI, including drinking water, has been increasing in more than 30 cities in the United States. CrVI is rapidly converted to CrIII intracellularly, and CrIII can cause DNA strand breaks and cancer or apoptosis through different mechanisms. Our previous study demonstrated that lactational exposure to chromium results in a delay or arrest in follicle development and a decrease in steroid hormone levels in F1 female rats, both of which are mitigated (partial inhibition) by vitamin C. The current study tested the hypothesis that lactational exposure to CrIII accelerates follicle atresia in F1 offspring by increasing reactive oxygen species (ROS) and decreasing cellular antioxidants. Results showed that lactational exposure to CrIII dose-dependently increased follicular atresia and decreased steroidogenesis in postnatal day 25, 45, and 65 rats. Vitamin C mitigated or inhibited the effects of CrIII at all doses. CrIII increased hydrogen peroxide and lipid hydroperoxide in plasma and ovary; decreased the antioxidant enzymes (AOXs) GPx1, GR, SOD, and catalase; and increased glutathione S-transferase in plasma and ovary. To understand the effects of CrVI on ROS and AOXs in granulosa (GC) and theca (TC) cell compartments in the ovary, ROS levels and mRNA expression of cytosolic and mitochondrial AOXs, such as SOD1, SOD2, catalase, GLRX1, GSTM1, GSTM2, GSTA4, GR, TXN1, TXN2, TXNRD2, and PRDX3, were studied in GCs and TCs and in a spontaneously immortalized granulosa cell line (SIGC). Overall, CrVI downregulated each of the AOXs; and vitamin C mitigated the effects of CrVI on these enzymes in GCs and SIGCs, but failed to mitigate CrVI effects on GSTM1, GSTM2, TXN1, and TXN2 in TCs. Thus, these data for the first time reveal that lactational exposure to CrIII accelerated follicular atresia and decreased steroidogenesis in F1 female offspring by altering the ratio of ROS and AOXs in the ovary. Vitamin C is able to protect the ovary from CrIII-induced oxidative stress and follicle atresia through protective effects on GCs rather than TCs., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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21. Two-photon excited fluorescence imaging of endogenous contrast in a mouse model of ovarian cancer.
- Author
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Watson JM, Marion SL, Rice PF, Utzinger U, Brewer MA, Hoyer PB, and Barton JK
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- 9,10-Dimethyl-1,2-benzanthracene, Adenocarcinoma chemically induced, Adenocarcinoma metabolism, Aging metabolism, Aging pathology, Animals, Biomarkers, Tumor metabolism, Collagen metabolism, Cyclohexenes, Disease Progression, Elastin metabolism, Female, Image Interpretation, Computer-Assisted, Linear Models, Lipofuscin metabolism, Mice, NAD metabolism, Ovarian Neoplasms chemically induced, Ovarian Neoplasms metabolism, Ovary metabolism, Vinyl Compounds, Adenocarcinoma pathology, Microscopy, Fluorescence, Multiphoton, Ovarian Neoplasms pathology, Ovary pathology
- Abstract
Background and Objective: Ovarian cancer has an extremely high mortality rate resulting from poor understanding of the disease. In order to aid understanding of disease etiology and progression, we identify the endogenous fluorophores present in a mouse model of ovarian cancer and describe changes in fluorophore abundance and distribution with age and disease., Study Design/materials and Methods: A mouse model of ovarian cancer was created by dosing with 4-vinylcyclohexene diepoxide, which induces follicular apoptosis (simulating menopause), and 7,12-dimethylbenz[a]anthracene, a known carcinogen. Imaging of ovarian tissue was completed ex vivo with a multiphoton microscope using excitation wavelength of 780 nm and emission collection from 405 to 505 nm. Two-photon excited fluorescence images and corresponding histologic sections with selective stains were used to identify endogenous fluorophores., Results: The majority of collected fluorescence emission was attributed to NADH and lipofuscin, with additional contributions from collagen and elastin. Dim cellular fluorescence from NADH did not show observable changes with age. Changes in ovarian morphology with disease development frequently caused increased fluorescence contributions from collagen and adipose tissue-associated NADH. Lipofuscin fluorescence was much brighter than NADH fluorescence and increased as a function of both age and disease., Conclusions: Our finding of NADH fluorescence patterns similar to that seen previously in human ovary, combined with the observation of lipofuscin accumulation with age and disease also seen in human organs, suggests that the findings from this model may be relevant to human ovarian disease. Increased lipofuscin fluorescence might be used as an indicator of disease in the ovary and this finding warrants further study., (Copyright © 2013 Wiley Periodicals, Inc.)
- Published
- 2013
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22. Glutathione S-transferase class μ regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries.
- Author
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Bhattacharya P, Madden JA, Sen N, Hoyer PB, and Keating AF
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- Animals, Animals, Newborn, Cells, Cultured, Female, Ovary enzymology, Rats, Rats, Inbred F344, Cyclohexenes toxicity, Glutathione Transferase physiology, MAP Kinase Kinase Kinase 5 metabolism, Ovary drug effects, Ovary metabolism, Vinyl Compounds toxicity
- Abstract
4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2-8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P<0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P<0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P<0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P<0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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23. 7,12-dimethylbenz[a]anthracene-induced malignancies in a mouse model of menopause.
- Author
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Marion SL, Watson J, Sen N, Brewer MA, Barton JK, and Hoyer PB
- Subjects
- Animals, Cyclohexenes, Female, Immunohistochemistry, Mice, Ovarian Neoplasms pathology, Time Factors, Vinyl Compounds, Benz(a)Anthracenes toxicity, Disease Models, Animal, Ovarian Neoplasms chemically induced, Ovary pathology, Postmenopause
- Abstract
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.
- Published
- 2013
24. Localization of ghrelin and its receptor in the reproductive tract of Holstein heifers.
- Author
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Deaver SE, Hoyer PB, Dial SM, Field ME, Collier RJ, and Rhoads ML
- Subjects
- Animals, Cattle, Corpus Luteum chemistry, Corpus Luteum physiology, Duodenum chemistry, Female, Fluorescent Antibody Technique veterinary, Genitalia, Female physiology, Ghrelin analysis, Hypothalamus chemistry, Ovarian Follicle chemistry, Ovarian Follicle physiology, Receptors, Ghrelin analysis, Uterus chemistry, Uterus physiology, Genitalia, Female chemistry, Ghrelin physiology, Receptors, Ghrelin physiology
- Abstract
The aim of this experiment was to localize the mRNA and protein of ghrelin and its active receptor, growth hormone secretagogue 1A (GHS-R1A), within the reproductive tract of dairy cattle. Ghrelin is an orexigenic hormone that has been identified as a potent regulator of energy homeostasis. Recent evidence suggests that ghrelin may also serve as a metabolic signal to the reproductive tract. Ghrelin and GHS-R1A have been identified in the reproductive tract of several species, including humans, mice, and rats. However, ghrelin and GHS-R1A expression have not been described within bovine reproductive tissues. Therefore, the ampulla, isthmus, uterine body, corpus luteum, and follicles were harvested from 3 Holstein heifers (15.91±0.07 mo of age) immediately following exsanguination. Duodenum and hypothalamus were collected as positive controls for ghrelin and GHS-R1A, respectively. Tissues were fixed in 10% formalin and embedded in paraffin for microscopy. Additional samples were stored at -80°C for detection of mRNA. Ghrelin and GHS-R1A mRNA and protein were observed in all tissue types within the reproductive tract of dairy heifers; however, expression appeared to be cell specific. Furthermore, ghrelin protein appeared to be localized to the cytoplasm, whereas GHS-R1A protein was found on the plasma membrane. Within the reproductive tissues, ghrelin mRNA and protein were most abundantly expressed in the ampulla of the oviduct. Concentrations of GHS-R1A were lower than those of ghrelin but differed between tissues. This is one of the first studies to provide molecular evidence for the presence of ghrelin and GHS-R1A within the entire reproductive tract. However, implications for fertility remain to be determined., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
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25. Analysis of second-harmonic-generation microscopy in a mouse model of ovarian carcinoma.
- Author
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Watson JM, Rice PF, Marion SL, Brewer MA, Davis JR, Rodriguez JJ, Utzinger U, Hoyer PB, and Barton JK
- Subjects
- Animals, Female, Mice, Reproducibility of Results, Sensitivity and Specificity, Algorithms, Image Enhancement methods, Image Interpretation, Computer-Assisted methods, Microscopy, Fluorescence, Multiphoton methods, Ovarian Neoplasms pathology
- Abstract
Second-harmonic-generation (SHG) imaging of mouse ovaries ex vivo was used to detect collagen structure changes accompanying ovarian cancer development. Dosing with 4-vinylcyclohexene diepoxide and 7,12-dimethylbenz[a]anthracene resulted in histologically confirmed cases of normal, benign abnormality, dysplasia, and carcinoma. Parameters for each SHG image were calculated using the Fourier transform matrix and gray-level co-occurrence matrix (GLCM). Cancer versus normal and cancer versus all other diagnoses showed the greatest separation using the parameters derived from power in the highest-frequency region and GLCM energy. Mixed effects models showed that these parameters were significantly different between cancer and normal (P<0.008). Images were classified with a support vector machine, using 25% of the data for training and 75% for testing. Utilizing all images with signal greater than the noise level, cancer versus not-cancer specimens were classified with 81.2% sensitivity and 80.0% specificity, and cancer versus normal specimens were classified with 77.8% sensitivity and 79.3% specificity. Utilizing only images with greater than of 75% of the field of view containing signal improved sensitivity and specificity for cancer versus normal to 81.5% and 81.1%. These results suggest that using SHG to visualize collagen structure in ovaries could help with early cancer detection.
- Published
- 2012
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26. Modeling perimenopause in Sprague-Dawley rats by chemical manipulation of the transition to ovarian failure.
- Author
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Frye JB, Lukefahr AL, Wright LE, Marion SL, Hoyer PB, and Funk JL
- Subjects
- Animals, Body Weight, Cyclohexenes pharmacology, Dose-Response Relationship, Drug, Female, Rats, Rats, Sprague-Dawley, Vinyl Compounds pharmacology, Ovary drug effects, Perimenopause, Sexual Maturation
- Abstract
Various age-related diseases increase in incidence during perimenopause. However, our understanding of the effects of aging compared with hormonal changes of perimenopause in mediating these disease risks is incomplete, in part due to the lack of an experimental perimenopause model. We therefore aimed to determine whether manipulation of the transition to ovarian failure in rats via the use of 4-vinylcyclohexene diepoxide (VCD) could be used to model and accelerate hormonal changes characteristic of perimenopause. We examined long-term (11 to 20 mo), dose-dependent effects of VCD on reproductive function in 1- and 3-mo-old female Sprague-Dawley rats. Twenty-five daily doses of VCD (80 or 160 mg/kg daily compared with vehicle alone) depleted ovarian follicles in a dose-dependent fashion in rats of both ages, accelerated the onset of acyclicity, and caused dose-dependent increases in follicle-stimulating hormone that exceeded those naturally occurring with age in control rats but left serum levels of 17β-estradiol unchanged, with continued ovarian production of androstenedione. High-dose VCD caused considerable nonovarian toxicities in 3-mo-old Sprague-Dawley rats, making this an unsuitable model. In contrast, 1-mo-old rats had more robust dose-dependent increases in follicle-stimulating hormone without evidence of systemic toxicity in response to either VCD dose. Because perimenopause is characterized by an increase in follicle-stimulating hormone with continued secretion of ovarian steroids, VCD acceleration of an analogous hormonal milieu in 1-mo-old Sprague-Dawley rats may be useful for probing the hormonal effects of perimenopause on age-related disease risk.
- Published
- 2012
27. Decreased bone mineral density in rats rendered follicle-deplete by an ovotoxic chemical correlates with changes in follicle-stimulating hormone and inhibin A.
- Author
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Lukefahr AL, Frye JB, Wright LE, Marion SL, Hoyer PB, and Funk JL
- Subjects
- Animals, Bone Density drug effects, Disease Models, Animal, Female, Follicle Stimulating Hormone antagonists & inhibitors, Follicle Stimulating Hormone blood, Humans, Inhibins antagonists & inhibitors, Ovary drug effects, Ovary metabolism, Rats, Rats, Sprague-Dawley, Bone Density physiology, Follicle Stimulating Hormone metabolism, Inhibins blood, Osteoporosis, Postmenopausal chemically induced, Osteoporosis, Postmenopausal physiopathology, Ovary physiopathology
- Abstract
Bone loss during perimenopause, an estrogen-sufficient period, correlates with elevated serum follicle-stimulating hormone (FSH) and decreased inhibins A and B. Utilizing a recently described ovotoxin-induced animal model of perimenopause characterized by a prolonged estrogen-replete period of elevated FSH, we examined longitudinal changes in bone mineral density (BMD) and their association with FSH. Additionally, serum inhibin levels were assessed to determine whether elevated FSH occurred secondary to decreased ovarian inhibin production and, if so, whether inhibins also correlated with BMD. BMD of the distal femur was assessed using dual-energy X-ray absorptiometry (DXA) over 19 months in Sprague-Dawley rats treated at 1 month with vehicle or 4-vinylcyclohexene diepoxide (VCD, 80 or 160 mg/kg daily). Serum FSH, inhibins A and B, and 17-ß estradiol (E(2)) were assayed and estrus cyclicity was assessed. VCD caused dose-dependent increases in FSH that exceeded values occurring with natural senescence, hastening the onset and prolonging the duration of persistent estrus, an acyclic but E(2)-replete period. VCD decreased serum inhibins A and B, which were inversely correlated with FSH (r(2) = 0.30 and 0.12, respectively). In VCD rats, significant decreases in BMD (5-13%) occurred during periods of increased FSH and decreased inhibins, while BMD was unchanged in controls. In skeletally mature rats, FSH (r(2) = 0.13) and inhibin A (r(2) = 0.15) correlated with BMD, while inhibin B and E(2) did not. Thus, for the first time, both the hormonal milieu of perimenopause and the association of dynamic perimenopausal changes in FSH and inhibin A with decreased BMD have been reproduced in an animal model.
- Published
- 2012
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28. 4-vinylcyclohexene diepoxide: a model chemical for ovotoxicity.
- Author
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Kappeler CJ and Hoyer PB
- Subjects
- Animals, Apoptosis drug effects, Dose-Response Relationship, Drug, Female, Humans, Mice, Models, Animal, Occupational Diseases metabolism, Occupational Diseases pathology, Occupational Exposure, Ovarian Follicle metabolism, Ovarian Follicle pathology, Phosphorylation, Primary Ovarian Insufficiency metabolism, Primary Ovarian Insufficiency pathology, Proto-Oncogene Proteins c-kit drug effects, Proto-Oncogene Proteins c-kit metabolism, Rats, Risk Assessment, Species Specificity, Tissue Culture Techniques, Cyclohexenes toxicity, Environmental Pollutants toxicity, Occupational Diseases chemically induced, Ovarian Follicle drug effects, Primary Ovarian Insufficiency chemically induced, Toxicity Tests methods, Vinyl Compounds toxicity
- Abstract
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.
- Published
- 2012
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29. Ovarian expressed microsomal epoxide hydrolase: role in detoxification of 4-vinylcyclohexene diepoxide and regulation by phosphatidylinositol-3 kinase signaling.
- Author
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Bhattacharya P, Sen N, Hoyer PB, and Keating AF
- Subjects
- Animals, Cyclohexenes toxicity, Epoxide Hydrolases genetics, Female, Ovary enzymology, Phosphoinositide-3 Kinase Inhibitors, RNA, Messenger analysis, Rats, Rats, Inbred F344, Vinyl Compounds toxicity, Cyclohexenes metabolism, Epoxide Hydrolases physiology, Ovary drug effects, Phosphatidylinositol 3-Kinases physiology, Signal Transduction physiology, Vinyl Compounds metabolism
- Abstract
4-vinylcyclohexene diepoxide (VCD) is a metabolite of 4-vinylcyclohexene (VCH) which has the potential to be formed in the ovary through CYP2E1 activity. VCD specifically destroys primordial and small primary follicles in the rodent ovary. Mouse ovaries exposed to VCD demonstrate increased mRNA and protein expression of microsomal epoxide hydrolase (mEH), and an inactive tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane) can be formed in mouse ovarian follicles, potentially through detoxification action of mEH. In contrast, mEH can bioactivate another ovotoxic chemical, 7,12-dimethylbenz[a]anthracene (DMBA) to a more toxic compound, DMBA-3,4-diol-1,2-epoxide. Thus, the present study evaluated a functional role for mEH during detoxification of VCD. Additionally, because inhibition of the phosphatidyinositol-3 kinase (PI3K) signaling pathway in a previous study protected primordial follicles from VCD-induced destruction, but accelerated DMBA-induced ovotoxicity, a role for PI3K in ovarian mEH regulation was evaluated. Using a post-natal day (PND) 4 Fischer 344 rat whole ovary culture system inhibition of mEH using cyclohexene oxide during VCD exposure resulted in a greater (P<0.05) loss of primordial and small primary follicles relative to VCD-treated ovaries. Also, relative to controls, meh mRNA was increased (P<0.05) on day 4 of VCD (30 μM) exposure, followed by increased (P<0.05) mEH protein after 6 days. Furthermore, inhibition of PI3K signaling increased mEH mRNA and protein expression. Thus, these results support a functional role for mEH in the rat ovary, and demonstrate the involvement of PI3K signaling in regulation of ovarian xenobiotic metabolism by mEH., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2012
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30. Xenobiotic effects on ovarian preantral follicles.
- Author
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Mark-Kappeler CJ, Hoyer PB, and Devine PJ
- Subjects
- Animals, Environmental Exposure adverse effects, Female, Humans, Menstrual Cycle drug effects, Menstrual Cycle physiology, Models, Animal, Occupational Exposure adverse effects, Ovary drug effects, Ovary physiology, Rats, Ovarian Follicle drug effects, Ovarian Follicle growth & development, Xenobiotics pharmacology
- Abstract
Women are born with a finite population of ovarian follicles, which are slowly depleted during their reproductive years until reproductive failure (menopause) occurs. The rate of loss of primordial follicles is determined by genetic and environmental influences, but certain toxic exposures can accelerate this process. Ionizing radiation reduces preantral follicle numbers in rodents and humans in a dose-dependent manner. Cigarette smoking is linked to menopause occurring 1-4 yr earlier than with nonsmokers, and components of smoke, polycyclic aromatic hydrocarbons, can cause follicle depletion in rodents or in ovaries in vitro. Chemotherapeutic agents, such as alkylating drugs and cisplatin, also cause loss of preantral ovarian follicles. Effects depend on dose, type, and reactivity of the drug, and the age of the individual. Evidence suggests DNA damage may underlie follicle loss induced by one common alkylating drug, cyclophosphamide. Occupational exposures have also been linked to ovarian damage. In an industrial setting, 2-bromopropane caused infertility in men and women, and it can induce ovarian follicle depletion in rats. Solvents, such as butadiene, 4-vinylcyclohexene, and their diepoxides, can also cause specific preantral follicle depletion. The mechanism(s) underlying effects of the latter compound may involve alterations in apoptosis, survival factors such as KIT/Kit Ligand, and/or the cellular signaling that maintains primordial follicle dormancy. Estrogenic endocrine disruptors may alter follicle formation/development and impair fertility or normal development of offspring. Thus, specific exposures are known or suspected of detrimentally impacting preantral ovarian follicles, leading to early ovarian failure.
- Published
- 2011
- Full Text
- View/download PDF
31. Inhibition of ovarian KIT phosphorylation by the ovotoxicant 4-vinylcyclohexene diepoxide in rats.
- Author
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Mark-Kappeler CJ, Sen N, Lukefahr A, McKee L, Sipes IG, Konhilas J, and Hoyer PB
- Subjects
- Animals, Animals, Newborn, Antibodies, Blocking metabolism, Antigen-Antibody Reactions drug effects, Cyclohexenes antagonists & inhibitors, Environmental Pollutants antagonists & inhibitors, Female, Follicular Atresia drug effects, Ligands, Molecular Targeted Therapy, Molecular Weight, Organ Culture Techniques, Ovarian Follicle drug effects, Ovarian Follicle growth & development, Ovarian Follicle metabolism, Ovary growth & development, Ovary metabolism, Phosphorylation drug effects, Protein Kinase Inhibitors antagonists & inhibitors, Proto-Oncogene Proteins c-kit agonists, Proto-Oncogene Proteins c-kit chemistry, Proto-Oncogene Proteins c-kit metabolism, Rats, Rats, Inbred F344, Vinyl Compounds antagonists & inhibitors, Cyclohexenes toxicity, Environmental Pollutants toxicity, Ovary drug effects, Protein Kinase Inhibitors toxicity, Protein Processing, Post-Translational drug effects, Proto-Oncogene Proteins c-kit antagonists & inhibitors, Vinyl Compounds toxicity
- Abstract
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.
- Published
- 2011
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32. 4-Vinylcyclohexene diepoxide (VCD) inhibits mammary epithelial differentiation and induces fibroadenoma formation in female Sprague Dawley rats.
- Author
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Wright LE, Frye JB, Lukefahr AL, Marion SL, Hoyer PB, Besselsen DG, and Funk JL
- Subjects
- Animals, Caseins genetics, Caseins metabolism, Estrous Cycle drug effects, Female, Fibroadenoma metabolism, Fibroadenoma pathology, Gene Expression Regulation, Developmental drug effects, Mammary Glands, Animal metabolism, Mammary Glands, Animal pathology, Mammary Neoplasms, Animal metabolism, Mammary Neoplasms, Animal pathology, Prolactin metabolism, Proto-Oncogene Proteins c-kit genetics, Proto-Oncogene Proteins c-kit metabolism, Rats, Rats, Sprague-Dawley, Cyclohexenes toxicity, Environmental Pollutants toxicity, Fibroadenoma chemically induced, Mammary Glands, Animal drug effects, Mammary Neoplasms, Animal chemically induced, Vinyl Compounds toxicity
- Abstract
4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that targets ovarian follicles and accelerates ovarian failure in rodents, was used to test the effect of early-onset reproductive senescence on mammary fibroadenoma formation. One-month female Sprague Dawley rats were dosed with VCD (80 mg/kg or 160 mg/kg) and monitored for 22 months for persistent estrus and tumor development. Only high-dose VCD treatment accelerated the onset of persistent estrus relative to controls. However, both doses of VCD accelerated mammary tumor onset by 5 months, increasing incidence to 84% (vs. 38% in controls). Tumor development was independent of time in persistent estrus, 17 β-estradiol, androstenedione and prolactin. Delay in VCD administration until after completion of mammary epithelial differentiation (3 months) did not alter tumor formation despite acceleration of ovarian senescence. VCD administration to 1-month rats acutely decreased mammary alveolar bud number and expression of β-casein, suggesting that VCD's tumorigenic effect requires exposure during mammary epithelial differentiation., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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33. Inhibition of PIK3 signaling pathway members by the ovotoxicant 4-vinylcyclohexene diepoxide in rats.
- Author
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Keating AF, Fernandez SM, Mark-Kappeler CJ, Sen N, Sipes IG, and Hoyer PB
- Subjects
- Animals, Base Sequence, DNA Primers genetics, Female, Forkhead Box Protein O3, Forkhead Transcription Factors genetics, Forkhead Transcription Factors metabolism, Ovary pathology, Phosphatidylinositol 3-Kinases metabolism, Proto-Oncogene Proteins c-akt genetics, Proto-Oncogene Proteins c-akt metabolism, Proto-Oncogene Proteins c-kit genetics, Proto-Oncogene Proteins c-kit metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Rats, Inbred F344, Signal Transduction drug effects, Cyclohexenes toxicity, Ovary drug effects, Ovary metabolism, Phosphoinositide-3 Kinase Inhibitors, Vinyl Compounds toxicity
- Abstract
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.
- Published
- 2011
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34. Hexavalent chromium-induced apoptosis of granulosa cells involves selective sub-cellular translocation of Bcl-2 members, ERK1/2 and p53.
- Author
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Banu SK, Stanley JA, Lee J, Stephen SD, Arosh JA, Hoyer PB, and Burghardt RC
- Subjects
- Animals, Antioxidants pharmacology, Down-Regulation drug effects, Female, Granulosa Cells metabolism, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Proto-Oncogene Proteins c-bcl-2 metabolism, Rats, Rats, Sprague-Dawley, Tumor Suppressor Protein p53 metabolism, Up-Regulation drug effects, Apoptosis drug effects, Ascorbic Acid pharmacology, Chromium toxicity, DNA Fragmentation drug effects, Granulosa Cells drug effects
- Abstract
Hexavalent chromium (CrVI) has been widely used in industries throughout the world. Increased usage of CrVI and atmospheric emission of CrVI from catalytic converters of automobiles, and its improper disposal causes various health hazards including female infertility. Recently we have reported that lactational exposure to CrVI induced a delay/arrest in follicular development at the secondary follicular stage. In order to investigate the underlying mechanism, primary cultures of rat granulosa cells were treated with 10 μM potassium dichromate (CrVI) for 12 and 24h, with or without vitamin C pre-treatment for 24h. The effects of CrVI on intrinsic apoptotic pathway(s) were investigated. Our data indicated that CrVI: (i) induced DNA fragmentation and increased apoptosis, (ii) increased cytochrome c release from the mitochondria to cytosol, (iii) downregulated anti-apoptotic Bcl-2, Bcl-XL, HSP70 and HSP90; upregulated pro-apoptotic BAX and BAD, (iv) altered translocation of Bcl-2, Bcl-XL, BAX, BAD, HSP70 and HSP90 to the mitochondria, (v) upregulated p-ERK and p-JNK, and selectively translocated p-ERK to the mitochondria and nucleus, (vi) activated caspase-3 and PARP, and (vii) increased phosphorylation of p53 at ser-6, ser-9, ser-15, ser-20, ser-37, ser-46 and ser-392, increased p53 transcriptional activation, and downregulated MDM-2. Vitamin C pre-treatment mitigated CrVI effects on apoptosis and related pathways. Our study, for the first time provides a clear insight into the effect of CrVI on multiple pathways that lead to apoptosis of granulosa cells which could be mitigated by vitamin C., (Published by Elsevier Inc.)
- Published
- 2011
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35. Midkine, a heparin-binding protein, is increased in the diabetic mouse kidney postmenopause.
- Author
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Diamond-Stanic MK, Romero-Aleshire MJ, Hoyer PB, Greer K, Hoying JB, and Brooks HL
- Subjects
- Animals, Cyclohexenes pharmacology, Diabetes Mellitus, Experimental physiopathology, Female, Gene Expression Profiling, Immediate-Early Proteins metabolism, Mice, Midkine, Oligonucleotide Array Sequence Analysis, Perimenopause drug effects, Postmenopause, Up-Regulation, Vinyl Compounds pharmacology, Cytokines metabolism, Diabetic Nephropathies physiopathology
- Abstract
Estrogen is thought to protect against the development of chronic kidney disease, and menopause increases the development and severity of diabetic kidney disease. In this study, we used streptozotocin (STZ) to induce diabetes in the 4-vinylcyclohexene diepoxide (VCD)-treated mouse model of menopause. DNA microarrays were used to identify gene expression changes in the diabetic kidney postmenopause. An ANOVA model, CARMA, was used to isolate the menopause effect between two groups of diabetic mice, diabetic menopausal (STZ/VCD) and diabetic cycling (STZ). In this diabetic study, 8,864 genes of the possible 15,600 genes on the array were included in the ANOVA; 99 genes were identified as demonstrating a >1.5-fold up- or downregulation between the STZ/VCD and STZ groups. We randomly selected genes for confirmation by real-time PCR; midkine (Mdk), immediate early response gene 3 (IEX-1), mitogen-inducible gene 6 (Mig6), and ubiquitin-specific protease 2 (USP2) were significantly increased in the kidneys of STZ/VCD compared with STZ mice. Western blot analysis confirmed that Mdk and IEX-1 protein abundance was significantly increased in the kidney cortex of STZ/VCD compared with STZ mice. In a separate study, DNA microarrays and CARMA analysis were used to identify the effect of menopause on the nondiabetic kidney; VCD-treated mice were compared with cycling mice. Of the possible 15,600 genes on the array, 9,142 genes were included in the ANOVA; 20 genes were identified as demonstrating a >1.5-fold up- or downregulation; histidine decarboxylase and vanin 1 were among the genes identified as differentially expressed in the postmenopausal nondiabetic kidney. These data expand our understanding of how hormone status correlates with the development of diabetic kidney disease and identify several target genes for further studies.
- Published
- 2011
- Full Text
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36. Distribution and responsiveness of rat anti-Müllerian hormone during ovarian development and VCD-induced ovotoxicity.
- Author
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Mark-Kappeler CJ, Sen N, Keating AF, Sipes IG, and Hoyer PB
- Subjects
- Animals, Animals, Newborn, Anti-Mullerian Hormone analysis, Apoptosis drug effects, Apoptosis physiology, Cells, Cultured, Female, Organ Culture Techniques, Ovarian Follicle drug effects, Ovarian Follicle growth & development, Ovarian Follicle metabolism, Ovary metabolism, Rats, Rats, Inbred F344, Tissue Distribution, Anti-Mullerian Hormone metabolism, Cyclohexenes toxicity, Ovary drug effects, Ovary growth & development, Vinyl Compounds toxicity
- Abstract
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<0.05) AMH mRNA (d4-8) and protein (d6-8). Recombinant AMH (100-400 mg/ml) in PND4 ovaries cultured 8 days±VCD (30 μM) caused an increase (P<0.05) in primordial, and a decrease (P<0.05) in small primary follicles, supporting that AMH retarded primordial follicle recruitment. However, no concentration of AMH had an effect on VCD-induced ovotoxicity. Whereas, VCD caused a reduction in expression of AMH (d4-d8), it followed previously reported initial disruptions in Kit signaling induced by VCD (d2). Thus, collectively, these results do not support a mechanism whereby VCD causes ovotoxicity via generalized activation of primordial follicle recruitment, but instead provide further support for the specificity of other intracellular mechanisms involved in VCD-induced ovotoxicity., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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37. Experimental induction of reduced ovarian reserve in a nonhuman primate model (Macaca fascicularis).
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Appt SE, Clarkson TB, Hoyer PB, Kock ND, Goode AK, May MC, Persyn JT, Vail NK, Ethun KF, Chen H, Sen N, and Kaplan JR
- Subjects
- Animals, Anti-Mullerian Hormone blood, Cyclohexenes, Female, Ovarian Follicle pathology, Ovary drug effects, Ovary pathology, Vinyl Compounds, Disease Models, Animal, Macaca fascicularis, Ovarian Diseases chemically induced, Ovarian Follicle drug effects, Sexual Development drug effects
- Abstract
Chronic diseases including coronary heart disease and osteoporosis represent a substantial health burden to postmenopausal women, yet the initiation of these conditions and their relationships with reproductive aging remain poorly understood. This situation is due, in part, to the lack of animal models reflecting ovarian and hormonal characteristics of peri- and postmenopausal women. Ovaries of women approaching menopause are nearly depleted of primordial follicles but retain a pool of larger developing follicles and androgen-producing stroma, a condition known as reduced ovarian reserve (ROR). The long-term goal of the research presented here was to create a monkey model of reproductive aging, beginning with ROR and progressing to perimenopause and finally postmenopause. Here we sought to develop a method to reduce primordial follicles in cynomolgus monkeys (Macaca fascicularis) and document hormonal changes associated with follicle reduction or ROR. At 30 d after surgical placement of a biodegradable fiber containing approximately 200 mg of 4-vinlycyclohexene diepoxide (VCD) next to one ovary in each of 8 monkeys, primordial follicles were reduced by approximately 70%, with a corresponding decrease (83%) in antimüllerian hormone (AMH, a serum marker of ovarian follicle numbers). At 4 mo after VCD-treatment of both ovaries in 29 monkeys (approximately 200 mg VCD per ovary), AMH was reduced 56% from baseline, testosterone was unchanged, and follicular phase estradiol was slightly increased. These data indicate that VCD treatment markedly reduced primordial follicles while preserving larger estradiol- and testosterone-producing follicles and ovarian stroma, a condition that mimics ROR in women.
- Published
- 2010
38. Dual protective role for glutathione S-transferase class pi against VCD-induced ovotoxicity in the rat ovary.
- Author
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Keating AF, Sen N, Sipes IG, and Hoyer PB
- Subjects
- Animals, Female, Glutathione S-Transferase pi genetics, JNK Mitogen-Activated Protein Kinases metabolism, Ovary drug effects, Ovary metabolism, Proto-Oncogene Proteins c-jun metabolism, RNA, Messenger metabolism, Rats, Rats, Inbred F344, Cyclohexenes toxicity, Glutathione S-Transferase pi metabolism, Ovary enzymology, Vinyl Compounds toxicity
- Abstract
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 <0.05) relative to control on d4-d8; whereas GSTp protein was increased (P<0.05) on d6-d8. A GSTp:JNK protein complex was detected by immunoprecipitation and Western blotting in ovarian tissues. Relative to control, the amount of GSTp-bound JNK was increased (P=0.09), while unbound JNK was decreased (P<0.05) on d6 of VCD exposure. The VCD-induced decrease in unbound JNK was preceded by a decrease in phosphorylated c-Jun which occurred on d4. These findings are in support of a possible dual protective role for GSTp in the rat ovary, consisting of metabolism of VCD and inhibition of JNK-initiated apoptosis., (2010 Elsevier Inc. All rights reserved.)
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- 2010
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39. Simultaneous optical coherence tomography and laser induced fluorescence imaging in rat model of ovarian carcinogenesis.
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Hariri LP, Liebmann ER, Marion SL, Hoyer PB, Davis JR, Brewer MA, and Barton JK
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- 9,10-Dimethyl-1,2-benzanthracene, Animals, Cyclohexenes, Female, Lasers, Ovarian Neoplasms chemically induced, Ovarian Neoplasms pathology, Primary Ovarian Insufficiency etiology, Rats, Rats, Inbred F344, Sex Cord-Gonadal Stromal Tumors diagnosis, Vinyl Compounds, Disease Models, Animal, Ovarian Neoplasms diagnosis, Ovary pathology, Spectrometry, Fluorescence methods, Tomography, Optical Coherence methods
- Abstract
Determining if an ovarian mass is benign or malignant is an ongoing clinical challenge. The development of reliable animal models provides means to evaluate new diagnostic tools to more accurately determine if an ovary has benign or malignant features. Although sex cord-stromal tumors (SCST) account for 0.1–0.5% of ovarian malignancies, they have similar appearances to more aggressive epithelial cancers and can serve as a prototype for developing better diagnostic methods for ovarian cancer. Optical coherence tomography (OCT) and laser-induced fluorescence (LIF) spectroscopy are non-destructive optical imaging modalities. OCT provides architectural cross-sectional images at near histological resolutions and LIF provides biochemical information. We utilize combined OCT-LIF to image ovaries in post-menopausal ovarian carcinogenesis rat models, evaluating normal cyclic, acyclic and neoplastic ovaries. Eighty-three female Fisher rats were exposed to combinations of control sesame oil, 4-vinyl cyclohexene diepoxide (VCD) to induce ovarian failure,and/or 7,12-dimethylbenz[a]anthracene (DMBA) to induce carcinogenesis. Three or five months post-treatment, 162 ovaries were harvested and imaged with OCT-LIF: 40 cyclic, 105 acyclic and 17 SCST. OCT identified various follicle stages,corpora lutea (CL), CL remnants, epithelial invaginations/inclusions and allowed for characterization of both cystic and solid SCST. Signal attenuation comparisons between CL and solid SCST revealed statistically significant increases in attenuation among CL. LIF characterized spectral differences in cyclic, acyclic and neoplastic ovaries attributed to collagen, NADH/FAD and hemoglobin absorption. We present combined OCT-LIF imaging in a rat ovarian carcinogenesis model, providing preliminary criteria for normal cyclic, acyclic and SCST ovaries which support the potential of OCT-LIF for ovarian imaging.
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- 2010
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40. Retaining Residual Ovarian Tissue following Ovarian Failure Has Limited Influence on Bone Loss in Aged Mice.
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Craig ZR, Marion SL, Funk JL, Bouxsein ML, and Hoyer PB
- Abstract
Previous work showed that retaining residual ovarian tissue protects young mice from accelerated bone loss following ovarian failure. The present study was designed to determine whether this protection is also present in aged animals. Aged (9-12 months) C57BL/6Hsd female mice were divided into: CON (vehicle), VCD (160 mg/kg; 15d), or OVX (ovariectomized). Lumbar BMD was monitored by DXA and μCT used to assess vertebral microarchitecture. BMD was not different between VCD and CON at any time point but was lower (P < .05) than baseline, starting 1 month after ovarian failure in VCD and OVX mice. Following μCT analysis there were no differences between CON and VCD, but OVX mice had lower bone volume fraction, trabecular thickness, and a trend for decreased connectivity density. These findings provide evidence that retention of residual ovarian tissue may protect aged follicle-depleted mice from accelerated bone loss to a lesser extent than that observed in young mice.
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- 2010
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41. 7,12-dimethylbenz[a]anthracene induces sertoli-leydig-cell tumors in the follicle-depleted ovaries of mice treated with 4-vinylcyclohexene diepoxide.
- Author
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Craig ZR, Davis JR, Marion SL, Barton JK, and Hoyer PB
- Subjects
- Animals, Female, Immunohistochemistry, Inhibins metabolism, Keratin-7 metabolism, Mice, Ovarian Neoplasms metabolism, Sertoli-Leydig Cell Tumor metabolism, 9,10-Dimethyl-1,2-benzanthracene toxicity, Carcinogens toxicity, Cyclohexenes administration & dosage, Ovarian Follicle drug effects, Ovarian Neoplasms chemically induced, Sertoli-Leydig Cell Tumor chemically induced, Vinyl Compounds administration & dosage
- Abstract
Ovarian cancer is associated with high mortality due to its late onset of symptoms and lack of reliable screening methods for early detection. Furthermore, the incidence of ovarian cancer is higher in postmenopausal women. Mice rendered follicle-depleted through treatment with 4-vinylcyclohexene diepoxide (VCD) are a model of ovary-intact menopause. The present study was designed to induce ovarian neoplasia in this model by treating mice with 7,12-dimethylbenz[a]anthracene (DMBA). Female B6C3F1 mice (age, 28 d) received intraperitoneal sesame oil (vehicle; VCD- groups) as a control or VCD (160 mg/kg; VCD+ groups) daily for 20 d to cause ovarian failure. Four months after the onset of dosing, mice from each group received a single injection of DMBA (VCD-DMBA+ and VCD+DMBA+ groups, n = 15 per group) or vehicle control (VCD-DMBA-, n = 15; VCD+ DMBA-, n = 14) under the bursa of the right ovary. Ovaries were collected 3 or 5 mo after injection and processed for histologic evaluation. Immunohistochemistry was used to confirm classification of neoplasms. None of the animals in the VCD-DMBA- and VCD-DMBA+ groups (that is, mice still undergoing estrus) had tumors at either time point. At the 3-mo time point, 12.5% of the VCD+DMBA+ mice had ovarian tumors; at 5 mo, 57.1% of the VCD+DMBA+ and 14.3% of VCD+DMBA- ovaries had neoplasms. Neoplasms stained positively for inhibin alpha (granulosa cells) and negatively for keratin 7 (surface epithelium), thus confirming classification of the lesions as Sertoli-Leydig cell tumors. These findings provide evidence for an increased incidence of DMBA-induced ovarian neoplasms in the ovaries of follicle-depleted mice compared with that in age-matched cycling controls.
- Published
- 2010
42. A longitudinal study of the effect of genistein on bone in two different murine models of diminished estrogen-producing capacity.
- Author
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Reinwald S, Mayer LP, Hoyer PB, Turner CH, Barnes S, and Weaver CM
- Abstract
This experiment was designed to assess the capacity of dietary genistein (GEN), to attenuate bone loss in ovariectomized (OVX) and ovary-intact VCD-treated mice. Pretreatment of mice with 4-vinylcyclohexene diepoxide (VCD) gradually and selectively destroys ovarian follicles whilst leaving ovarian androgen-producing cells largely intact. VCD induces a perimenopause-like condition prior to the onset of reproductive acyclicity. Sixteen-week-old C57BL/6J mice were randomized to five treatment groups: sham(SHM), OVX, SHM + VCD, OVX + GEN, and SHM + VCD + GEN. In vivo, blood samples were drawn for hormone and isoflavone analyses, estrous cycles were monitored, and X-ray imaging was performed to assess changes in bone parameters. Following sacrifice, ovaries were assessed histologically, bone microarchitecture was evaluated via microcomputed tomography, and bone mechanical properties were measured. Some effects of GEN were observed in OVX mice, but GEN effects were not able to be evaluated in VCD-treated mice due to the subtle diminution of bone during the 4 months of this experiment.
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- 2010
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43. Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a]anthracene in neonatal rat ovaries.
- Author
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Keating AF, J Mark C, Sen N, Sipes IG, and Hoyer PB
- Subjects
- Animals, Animals, Newborn, Female, In Vitro Techniques, Ovarian Diseases chemically induced, Ovarian Diseases pathology, Ovarian Follicle pathology, Piperazines pharmacology, Rats, Time Factors, 9,10-Dimethyl-1,2-benzanthracene toxicity, Chromones pharmacology, Cyclohexenes toxicity, Morpholines pharmacology, Ovarian Follicle drug effects, Phosphoinositide-3 Kinase Inhibitors, Vinyl Compounds toxicity
- Abstract
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<0.05) small primary and larger follicles beginning on day 4. VCD caused primordial and small primary follicle loss (P<0.05) beginning on day 6. With PI3 kinase inhibition, VCD did not affect primordial follicles (P>0.05) at any time, but did cause loss (P<0.05) of small primary follicles. DMBA exposure caused primordial and small primary follicle loss (P<0.05) on day 6. Further, DMBA-induced primordial and small primary follicle loss was greater with PI3 kinase inhibition (P<0.05) than with DMBA alone. These results support that (1) PI3 kinase mediates primordial to small primary follicle recruitment, (2) VCD, but not DMBA, enhances ovotoxicity by increasing primordial to small primary follicle recruitment, and (3) in addition to xenobiotic-induced ovotoxicity, VCD is also a useful model chemical with which to elucidate signaling mechanisms involved in primordial follicle recruitment.
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- 2009
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44. Loss of ovarian function in the VCD mouse-model of menopause leads to insulin resistance and a rapid progression into the metabolic syndrome.
- Author
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Romero-Aleshire MJ, Diamond-Stanic MK, Hasty AH, Hoyer PB, and Brooks HL
- Subjects
- Abdominal Fat physiopathology, Animals, Blood Glucose metabolism, Cholesterol blood, Cyclohexenes, Dietary Fats, Disease Models, Animal, Disease Progression, Estrogen Replacement Therapy, Fatty Acids, Nonesterified blood, Female, Insulin blood, Metabolic Syndrome metabolism, Metabolic Syndrome physiopathology, Mice, Primary Ovarian Insufficiency chemically induced, Primary Ovarian Insufficiency complications, Primary Ovarian Insufficiency metabolism, Primary Ovarian Insufficiency prevention & control, Time Factors, Vinyl Compounds, Weight Gain, Insulin Resistance, Menopause, Metabolic Syndrome etiology, Ovary physiopathology, Primary Ovarian Insufficiency physiopathology
- Abstract
Factors comprising the metabolic syndrome occur with increased incidence in postmenopausal women. To investigate the effects of ovarian failure on the progression of the metabolic syndrome, female B(6)C(3)F(1) mice were treated with 4-vinylcyclohexene diepoxide (VCD) and fed a high-fat (HF) diet for 16 wk. VCD destroys preantral follicles, causing early ovarian failure and is a well-characterized model for the gradual onset of menopause. After 12 wk on a HF diet, VCD-treated mice had developed an impaired glucose tolerance, whereas cycling controls were unaffected [12 wk AUC HF mice 13,455 +/- 643 vs. HF/VCD 17,378 +/- 1140 mg/dl/min, P < 0.05]. After 16 wk on a HF diet, VCD-treated mice had significantly higher fasting insulin levels (HF 5.4 +/- 1.3 vs. HF/VCD 10.1 +/- 1.4 ng/ml, P < 0.05) and were significantly more insulin resistant (HOMA-IR) than cycling controls on a HF diet (HF 56.2 +/- 16.7 vs. HF/VCD 113.1 +/- 19.6 mg/dl x microU/ml, P < 0.05). All mice on a HF diet gained more weight than mice on a standard diet, and weight gain in HF/VCD mice was significantly increased compared with HF cycling controls. Interestingly, even without a HF diet, progression into VCD-induced menopause caused a significant increase in cholesterol and free fatty acids. Furthermore, in mice fed a standard diet (6% fat), insulin resistance developed 4 mo after VCD-induced ovarian failure. Insulin resistance following ovarian failure (menopause) was prevented by estrogen replacement. Studies here demonstrate that ovarian failure (menopause) accelerates progression into the metabolic syndrome and that estrogen replacement prevents the onset of insulin resistance in VCD-treated mice. Thus, the VCD model of menopause provides a physiologically relevant means of studying how sex hormones influence the progression of the metabolic syndrome.
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- 2009
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45. Ovarian neoplasm development by 7,12-dimethylbenz[a]anthracene (DMBA) in a chemically-induced rat model of ovarian failure.
- Author
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Hoyer PB, Davis JR, Bedrnicek JB, Marion SL, Christian PJ, Barton JK, and Brewer MA
- Subjects
- Animals, Drug Administration Schedule, Female, Ovarian Follicle drug effects, Ovarian Follicle pathology, Rats, Rats, Inbred F344, 9,10-Dimethyl-1,2-benzanthracene administration & dosage, Carcinogens administration & dosage, Cyclohexenes administration & dosage, Disease Models, Animal, Ovarian Neoplasms chemically induced, Ovarian Neoplasms pathology, Vinyl Compounds administration & dosage
- Abstract
Objectives: The objectives were to determine the time course for ovarian failure in rats caused by 4-vinylcyclohexene diepoxide (VCD) and develop a model for ovarian cancer in which ovarian neoplasms were chemically induced in an animal that was follicle depleted, but retained residual ovarian tissue., Methods: Initially, female Fisher 344 rats were treated with VCD (to induce ovarian failure) or vehicle control (sesame oil). Three or 6 months after treatment, ovaries were collected and processed for histological evaluation for confirmation of ovarian failure. A further set of female rats was assigned to four groups exposed to combinations of vehicle control, VCD and/or DMBA (directly applied to the ovary) in a novel model for examining early stages of ovarian neoplasia., Results: Three and 6 months following VCD dosing there was a significant reduction of ovarian weight and follicle number. Treatment with DMBA subsequent to VCD resulted in tumors in 42% of animals at 3 months and 57% at 5 months. All neoplasms were classified Sertoli-Leydig cell tumors (SLCT). No tumor occurred in animals treated with vehicle or DMBA alone., Conclusions: These studies demonstrate that the VCD-treated rat can be used as a model for peri- and post-menopause. DMBA induction of ovarian neoplasms was greater in those rats treated with VCD. Whether this increase was due to tumor initiation by VCD or was the result of ovarian failure cannot be distinguished from these results. This represents the only animal model to date for sex cord stromal tumors.
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- 2009
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46. Effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague-Dawley rats: ovarian, clinical, and pathologic outcomes.
- Author
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Muhammad FS, Goode AK, Kock ND, Arifin EA, Cline JM, Adams MR, Hoyer PB, Christian PJ, Isom S, Kaplan JR, and Appt SE
- Subjects
- Aging drug effects, Animals, Body Weight drug effects, Disease Models, Animal, Dose-Response Relationship, Drug, Female, Injections, Intramuscular, Injections, Intraperitoneal, Muscle, Skeletal drug effects, Muscle, Skeletal pathology, Myositis chemically induced, Myositis pathology, Ovarian Follicle drug effects, Ovarian Follicle pathology, Ovary pathology, Rats, Rats, Sprague-Dawley, Sexual Maturation drug effects, Apoptosis drug effects, Carcinogens toxicity, Cyclohexenes toxicity, Ovary drug effects, Vinyl Compounds toxicity
- Abstract
Young rats treated daily with intraperitoneal 4-vinylcyclohexene diepoxide (VCD) undergo selective destruction of primordial follicles, resulting in gradual ovarian failure resembling the menopausal transition in women. To determine whether VCD has similar effects on ovaries of older rats, adult and peripubertal Sprague-Dawley rats were injected intraperitoneally daily for 30 d with vehicle or VCD at 40 or 80 mg/kg. Body weight, food intake, complete blood counts, and markers of liver injury and renal function were measured during VCD treatment. Complete gross necropsy and microscopic observations were performed on day 31, and ovarian follicles were counted. At 80 mg/kg, VCD destroyed primordial and primary follicles to a similar extent in both adult and peripubertal animals, although adult rats likely started with fewer follicles and therefore approached follicle depletion. Treatment with VCD did not affect body weight, but food intake was reduced in both adult and peripubertal rats treated with 80 mg/kg VCD. Adult rats treated with 80 mg/kg VCD had neutrophilia and increased BUN and creatinine; in addition, 4 of these rats were euthanized on days 25 or 26 due to peritonitis. VCD treatment did not increase alanine aminotransferase levels, a marker of liver injury, although the 80-mg/kg dose increased liver weights. In conclusion, VCD effectively destroys small preantral follicles in adult Sprague-Dawley rats, making them a suitable model of the menopausal transition of women. However, because adult rats were more sensitive to the irritant properties of VCD, the use of a lower dose should be considered.
- Published
- 2009
47. Evaluation of ovotoxicity induced by 7, 12-dimethylbenz[a]anthracene and its 3,4-diol metabolite utilizing a rat in vitro ovarian culture system.
- Author
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Igawa Y, Keating AF, Rajapaksa KS, Sipes IG, and Hoyer PB
- Subjects
- 9,10-Dimethyl-1,2-benzanthracene metabolism, Animals, Animals, Newborn, Benz(a)Anthracenes metabolism, Biotransformation, Cyclohexenes pharmacology, Dose-Response Relationship, Drug, Enzyme Induction, Enzyme Inhibitors pharmacology, Epoxide Hydrolases antagonists & inhibitors, Epoxide Hydrolases genetics, Female, Ovarian Follicle drug effects, Ovarian Follicle enzymology, Ovary enzymology, Ovary pathology, RNA, Messenger biosynthesis, Rats, Rats, Inbred F344, Time Factors, Tissue Culture Techniques, 9,10-Dimethyl-1,2-benzanthracene toxicity, Benz(a)Anthracenes toxicity, Epoxide Hydrolases biosynthesis, Ovary drug effects
- Abstract
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<0.05) at lower concentrations of DMBA-3,4-diol (12.5 nM - primordial; 75 nM - primary) than DMBA (75 nM - primordial; 375 nM - primary). The temporal pattern of mEH expression following DMBA exposure showed mRNA up-regulation (P<0.05) on day 2, with increased protein (P<0.05) on day 4, the earliest time of observed follicle loss (P<0.05). mEH inhibition prevented DMBA-induced, but not DMBA-3,4-diol-induced ovotoxicity. These results demonstrate a conserved response in mice and rats for ovarian mEH involvement in DMBA bioactivation to its ovotoxic, 3,4-diol-1,2-epoxide form.
- Published
- 2009
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48. Steroidogenic capacity of residual ovarian tissue in 4-vinylcyclohexene diepoxide-treated mice.
- Author
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Rivera Z, Christian PJ, Marion SL, Brooks HL, and Hoyer PB
- Subjects
- Androstenedione blood, Animals, Carcinogens, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Estrous Cycle drug effects, Estrous Cycle genetics, Estrous Cycle physiology, Female, Follicle Stimulating Hormone blood, Gene Expression Regulation, Enzymologic drug effects, Hydroxysteroid Dehydrogenases genetics, Hydroxysteroid Dehydrogenases metabolism, Luteinizing Hormone blood, Mice, Mice, Inbred C57BL, Ovary drug effects, Primary Ovarian Insufficiency blood, Primary Ovarian Insufficiency genetics, Cyclohexenes, Gonadal Steroid Hormones biosynthesis, Ovary metabolism, Primary Ovarian Insufficiency chemically induced, Primary Ovarian Insufficiency metabolism, Vinyl Compounds
- Abstract
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.
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- 2009
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49. Current methods in investigating the development of the female reproductive system.
- Author
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Devine PJ, Hoyer PB, and Keating AF
- Subjects
- Animals, Female, Immunohistochemistry, Oligonucleotide Array Sequence Analysis, Rats, Reverse Transcriptase Polymerase Chain Reaction, Tissue Culture Techniques, Ovary growth & development
- Abstract
The female reproductive system is important as the site for development and fertilization of an oocyte, for implantation and development of an embryo, and for growth and delivery of the fetus. It also produces protein and steroid hormones that help maintain a female's health. Although the female phenotype is the default pathway for the development of the urogenital system, many processes can become disrupted during and after development which may originate from developmental problems. Improper development can be the underlying cause of structural malformations, sub- or infertility, hormonal abnormalities, endometriosis, carcinogenesis, or other detrimental outcomes. Our research programs examine the normal physiology and function of the female reproductive system and how it can become damaged due to pathologies or environmental/therapeutic exposures, with a focus on the ovary, ovarian follicles, and ovarian hormones. This chapter will describe detailed protocols of an in vitro organ culture system and methods to analyze changes in follicle formation, follicle development, and ovarian physiology. These methods can also be applied to the study of other aspects of female reproduction.
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- 2009
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50. Behavioral consequences of ovarian atrophy and estrogen replacement in the APPswe mouse.
- Author
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Golub MS, Germann SL, Mercer M, Gordon MN, Morgan DG, Mayer LP, and Hoyer PB
- Subjects
- Alzheimer Disease drug therapy, Alzheimer Disease metabolism, Alzheimer Disease physiopathology, Amyloid beta-Protein Precursor genetics, Amyloid beta-Protein Precursor metabolism, Animals, Atrophy chemically induced, Atrophy metabolism, Atrophy physiopathology, Behavior, Animal drug effects, Behavior, Animal physiology, Brain metabolism, Brain physiopathology, Cognition Disorders metabolism, Cognition Disorders physiopathology, Disease Models, Animal, Estrogen Replacement Therapy, Estrogens therapeutic use, Female, Longitudinal Studies, Maze Learning drug effects, Maze Learning physiology, Mice, Mice, Transgenic, Neuroprotective Agents therapeutic use, Ovary drug effects, Ovary metabolism, Reproducibility of Results, Aging metabolism, Brain drug effects, Cognition Disorders drug therapy, Estrogens pharmacology, Neuroprotective Agents pharmacology, Postmenopause metabolism
- Abstract
Cognitive performance was evaluated in a longitudinal study of APPswe2576 transgenic mice (APP) and a wildtype (WT) comparison group. Subgroups of the APP mice were treated with the ovarian toxicant 4-vinylcyclo-hexene diepoxide (VCD) at 60-75 days of age to induce ovarian atrophy and/or given estrogen (estradiol, 4 microg/day) continuously by pellet from 76 days of age. APP mice had a generally poorer radial maze performance than WT at 4.5, 7.5, 10.5 and 15 months of age. In separate tests, APP mice had a slight motor impairment, higher incidence of homecage stereotypy, hyperactivity in an open field and reduced object exploration relative to the WT group. Ovarian atrophy led to better maze performance at 7.5 months. The effect of estrogen on maze performance with aging could not be effectively evaluated due to poor survival (30%) of these mice. No effects of ovarian atrophy or estrogen treatment were identified for amyloid-beta accumulation or plaque formation at 15 months. Long-term longitudinal studies in animal models are needed to explore the consequences of menopause and hormone replacement on Alzheimer's disease, but they are complicated by considerations of survival, pre-aging deficits, testing experience and selection of appropriate estrogen treatment levels.
- Published
- 2008
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