Zelieann R Craig

Interests

Teaching

Reproduction, Physiology, Toxicology

Research

Female reproductive physiology, ovarian follicle biology, ovarian toxicology, environmental chemicals and reproduction, infertility

Courses

Scholarly Contributions

Chapters

• Craig, Z. R. (2017). Plastic Compounds. In Encyclopedia of Reproduction. Elsevier, Inc.
• Peretz, J., Craig, Z. R., Bunting, S. L., & Flaws, J. A. (2016). Experimental approaches to evaluate mechanisms of reproductive toxicity. In Developmental and Reproductive Toxicology: A Practical Approach. CRC Press. doi:10.3109/9781841848211-7
• Rasmussen, L. M., Paunil, E., & Craig, Z. R. (2016). In vitro models of ovarian toxicity. In Comprehensive Toxicology. Elsevier.
• Devine, P., Petrillo, S., Cortvrindt, R., Rasmussen, L. M., Paunil, E., & Craig, Z. R. (2010). In Vitro Ovarian Model Systems. In Comprehensive Toxicology. Elsevier Inc. doi:10.1016/B978-0-12-801238-3.10929-8
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  There are a number of detrimental exposures that are known to cause reproductive toxicity, and numerous others that are also likely to affect the reproductive system. In order to identify possible reproductive toxicants and study the mechanism(s) of action of those that are known, in vitro techniques have been and will continue to be important. This article will describe the currently available in vitro methods for examining ovarian toxicity. Examples will be given on how ovarian tissue cultures, cultures of individual ovarian follicles, and cell lines have been used to study exposures that affect the normal functioning of the ovary. The advantages and disadvantages of each technique will be discussed. Results that provide insight into the mechanisms of action of certain ovarian toxicants will be described to demonstrate the utility of each method.

Journals/Publications

• Jauregui, E. J., McSwain, M., Liu, X., Miller, K., Burns, K., & Craig, Z. R. (2023).

  Human-relevant exposure to di-n-butyl phthalate tampers with the ovarian insulin-like growth factor 1 system and disrupts folliculogenesis in young adult mice

  . Toxicological Sciences, 195(1), 42-52. doi:10.1093/toxsci/kfad064
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  Phthalates are compounds used in consumer and medical products worldwide. Phthalate exposure in women has been demonstrated by detection of phthalate metabolites in their urine and ovarian follicular fluid. High urinary phthalate burden has been associated with reduced ovarian reserve and oocyte retrieval in women undergoing assisted reproduction. Unfortunately, no mechanistic explanation for these associations is available. In short term in vivo and in vitro animal studies modeling human-relevant exposures to di-n-butyl phthalate (DBP), we have identified ovarian folliculogenesis as a target for phthalate exposures. In the present study, we investigated whether DBP exposure negatively influences insulin-like growth factor 1 (IGF1) signaling in the ovary and disrupts ovarian folliculogenesis. CD-1 female mice were exposed to corn oil (vehicle) or DBP (10 µg/kg/day, 100 µg/kg/day, or 1000 mg/kg/day) for 20-32 days. Ovaries were collected as animals reached the proestrus stage to achieve estrous cycle synchronization. Levels of mRNAs encoding IGF1 and 2 (Igf1 and Igf2), IGF1 receptor (Igf1r), and IGF-binding proteins 1-6 (Ifgbp1-6) were measured in whole ovary homogenates. Ovarian follicle counts and immunostaining for phosphorylated IGF1R protein (pIGF1R) were used to evaluate folliculogenesis and IGF1R activation, respectively. DBP exposure, at a realistic dose that some women may experience (100 µg/kg/day for 20-32 days), reduced ovarian Igf1 and Igf1r mRNA expression and reduced small ovarian follicle numbers and primary follicle pIGF1R positivity in DBP-treated mice. These findings reveal that DBP tampers with the ovarian IGF1 system and provide molecular insight into how phthalates could influence the ovarian reserve in females.
• Craig, Z. R., & Keating, A. F. (2022). Editorial - Ovarian Toxicants Special Issue. Reproductive Toxicology. doi:10.1016/j.reprotox.2022.05.011
• Craig, Z. R. (2021). An interview with Dr Bernard Robaire, recipient of the 2020 SSR Jansen Distinguished Leadership and Service Award. Biology of reproduction, 104(1), 1-4.
• Craig, Z. R., Rasmussen, L., Jauregui, E. J., & Lock, J. (2021). Mono-n-Butyl Phthalate Distributes to the Mouse Ovary and Liver and Alters the Expression of Phthalate-Metabolizing Enzymes in Both Tissues. Toxicological Sciences, 183(1), 117-127. doi:10.1093/toxsci/kfab085
• Jauregui, E. J., Lock, J., Rasmussen, L., & Craig, Z. R. (2021). Mono-n-Butyl Phthalate Distributes to the Mouse Ovary and Liver and Alters the Expression of Phthalate-Metabolizing Enzymes in Both Tissues. Toxicological sciences : an official journal of the Society of Toxicology, 183(1), 117-127.
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  Humans are exposed to phthalates daily via items such as personal care products and medications. Reproductive toxicity has been documented in mice exposed to di-n-butyl phthalate (DBP); however, quantitative evidence of its metabolite, mono-n-butyl phthalate (MBP), reaching the mouse ovary and its effects on hepatic and ovarian biotransformation enzymes in treated mice is still lacking. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was employed to quantify MBP levels in liver, serum, and ovary from mice treated with a single or repeated exposure to the parent compound, DBP. Adult CD-1 females were pipet fed once or for 10 days with vehicle (tocopherol-stripped corn oil) or DBP at 1, 10, and 1000 mg/kg/day. Tissues and serum were collected at 2, 6, 12, and 24 h after the single or final dose and subjected to LC-MS/MS. Ovaries and livers were processed for qPCR analysis of selected phthalate-associated biotransformation enzymes. Regardless of duration of exposure (single vs repeated), MBP was detected in the tissues of DBP-treated mice. In single dose mice, MBP levels peaked at ≤6 h and fell close to background levels by 24 h post-exposure. Following the last repeated dose, MBP levels peaked at ≤2 h and fell to background levels by 12 h. Hepatic and ovarian expression of Lpl, Aldh1a1, Adh1, Ugt1a6a, and Cyp1b1 were altered in DBP-treated mice in a time- and dose-specific manner. These findings confirm that MBP reaches the mouse liver and ovary after oral exposure to DBP and influences the expression of hepatic and ovarian phthalate-associated biotransformation enzymes.
• Craig, Z. R. (2020). An interview with Dr Bernard Robaire, recipient of the 2020 SSR Jansen Distinguished Leadership and Service Award. Biology of Reproduction, 104(1), 1-4. doi:10.1093/biolre/ioaa162
• Craig, Z. R., Ortiz-Santana, J., & Colon-Diaz, M. (2020). Data on the activity of DNA methyltransferase in the uteri of CD-1 mice exposed to dibutyl phthalate. Data in Brief. doi:10.1016/j.dib.2019.105061
• Barton, J. K., Craig, Z. R., Koevary, J. W., Hoyer, P. B., Rice, P. F., Howard, C. C., Dominguez Cooks, J. P., Hutchens, G. V., Chambers, S. K., & Connolly, D. C. (2019). Comparison of Reproductive Function in Female TgMISIIR-TAg Transgenic and Wildtype C57BL/6 Mice. Comparative Medicine, 69(1), 16-21. doi:10.30802/aalas-cm-18-000008
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  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.
• Colón-Díaz, M., Ortiz-Santana, J., & Craig, Z. R. (2020). Data on the activity of DNA methyltransferase in the uteri of CD-1 mice exposed to dibutyl phthalate. Data in brief, 28, 105061.
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  Phthalates are industrial chemicals used as plasticizers in food packaging, medical devices, and toys, as well as cosmetics used primarily by women. Epidemiological studies in women and animal studies using rodents have reported associations between phthalate exposures and adverse reproductive health outcomes. Epigenetic mechanisms are thought to be involved in the ability of environmental contaminants to influence development of disease but evidence linking exposure to phthalates and uterine DNA methyltransferase activity are lacking. This article reports the activity of DNA methyltransferase (DNMT) enzymes in uteri from CD-1 mice treated with or without dibutyl phthalate (DBP), a phthalate commonly found in the urine of women of reproductive age. CD-1 mice were orally dosed with tocopherol-stripped corn oil (vehicle) or DBP at 10 μg/kg/day, 100 μg/kg/day and 1000 mg/kg/day daily for 10, 20, and 30 days. These dosages were selected based on estimates of human intake previously reported (10 and 100 μg/kg/day) and included a high dose (1000 mg/kg/day) for comparison with classical toxicity studies. At the end of 10, 20 or 30 days of daily oral dosing, animals were euthanized within 1-2 hours after the final dose. DNMT activity was determined by subjecting uterine nuclear extracts to a commercially-available DNMT activity ELISA assay and measuring optical density with a microplate spectrophotometer at a wavelength of 450 nm. Graph Pad Prism 8 was used for data analysis to determine the activity of DNMT enzymes at different time points and doses versus vehicle. The data presented serves as a resource for researchers working in the field of toxicology because it addresses a gap in knowledge of how exposure to environmental factors such as phthalate esters could produce epigenetic alterations in the uterus, which consequently may increase the risk of developing reproductive disease.
• Craig, Z. R., & Liu, X. (2019). Environmentally relevant exposure to dibutyl phthalate disrupts DNA damage repair gene expression in the mouse ovary†. Biology of Reproduction, 101(4), 854-867. doi:10.1093/biolre/ioz122
• Gal, A., Gedye, K., Craig, Z. R., & Ziv-Gal, A. (2019). Propylparaben inhibits mouse cultured antral follicle growth, alters steroidogenesis, and upregulates levels of cell-cycle and apoptosis regulators. Reproductive toxicology (Elmsford, N.Y.), 89, 100-106.
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  Propylparaben is prevalently used in cosmetics, pharmaceuticals, and foods; yet, its direct effects on the mammalian ovary are unknown. We investigated the direct effects of propylparaben on the growth and steroidogenic function of mouse antral follicles. Antral follicles were isolated from the ovaries of Swiss mice (age: 32-42 days) and cultured in media with dimethylsulfoxide vehicle control or propylparaben (0.01-100 μg/mL) for 24-72 h. Follicle diameter was measured every 24 h to assess growth. Follicles and media were collected at 24 and 72 h for gene expression and hormone measurements. Propylparaben (100 μg/mL) significantly inhibited follicle growth (48-72 h). Further, propylparaben exposure increased expression of cell cycle regulators (Cdk4, Cdkn1a), an apoptotic factor (Bax), and a key steroidogenic regulator (Star). In media, propylparaben decreased accumulation of dehydroepiandrosterone-sulfate, but increased testosterone and 17β-estradiol. Overall, our findings suggest that propylparaben disrupts antral follicle growth and steroidogenic function by altering the cell-cycle, apoptosis, and steroidogenesis pathways.
• Hoyer, P. B., Rice, P. F., Howard, C. C., Koevary, J. W., Dominguez Cooks, J. P., Hutchens, G. V., Chambers, S. K., Craig, Z. R., Connolly, D. C., & Barton, J. K. (2019). Comparison of Reproductive Function in Female Transgenic and Wildtype C57BL/6 Mice. Comparative medicine, 69(1), 16-21.
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  Transgenic (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.
• Liu, X., & Craig, Z. R. (2019). Environmentally relevant exposure to dibutyl phthalate disrupts DNA damage repair gene expression in the mouse ovary†. Biology of reproduction, 101(4), 854-867.
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  Phthalates have a history of reproductive toxicity in animal models and associations with adverse reproductive outcomes in women. Human exposure to dibutyl phthalate (DBP) occurs via consumer products (7-10 μg/kg/day) and medications (1-233 μg/kg/day). Most DBP toxicity studies have focused on high supraphysiological exposure levels; thus, very little is known about exposures occurring at environmentally relevant levels. CD-1 female mice (80 days old) were treated with tocopherol-stripped corn oil (vehicle control) or DBP dissolved in oil at environmentally relevant (10 and 100 μg/kg/day) or higher (1000 μg/kg/day) levels for 30 days to evaluate effects on DNA damage response (DDR) pathway genes and folliculogenesis. DBP exposure caused dose-dependent effects on folliculogenesis and gene expression. Specifically, animals exposed to the high dose of DBP had more atretic follicles in their ovaries, while in those treated with environmentally relevant doses, follicle numbers were no different from vehicle-treated controls. DBP exposure significantly reduced the expression of DDR genes including those involved in homologous recombination (Atm, Brca1, Mre11a, Rad50), mismatch repair (Msh3, Msh6), and nucleotide excision repair (Xpc, Pcna) in a dose-specific manner. Interestingly, staining for the DNA damage marker, γH2AX, was similar between treatments. DBP exposure did not result in differential DNA methylation in the Brca1 promoter but significantly reduced transcript levels for the maintenance DNA methyltransferase, Dnmt1, in the ovary. Collectively, these findings show that oral exposure to environmentally relevant levels of DBP for 30 days does not significantly impact folliculogenesis in adult mice but leads to aberrant ovarian expression of DDR genes.
• Helm, S. V., Craig, Z. R., Pollitt, A. M., Pollitt, A. M., Helm, S. V., Curran, M. A., Craig, Z. R., & Barnett, M. A. (2018). Differentiating environmental concern in the context of psychological adaption to climate change. Global Environmental Change-human and Policy Dimensions, 48, 158-167. doi:10.1016/j.gloenvcha.2017.11.012
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  Abstract Despite existing evidence for the threats of climate change facing people living in the U.S., the psychological impacts of this threat have been neglected in public and scientific discourse, resulting in a notable lack in studies on individuals’ adaptation to climate change. Using social-cognitive theory, we examine how three forms of environmental concern—egoistic (e.g., concern for oneself; one’s health or life), social-altruistic (e.g., concern for others; future generations or country), and biospheric (e.g., concern for plants and animals; nature)—influence concurrent ecological stress and ecological coping strategies. Further, we examine how ecological stress and coping are associated with both depressive symptoms and pro-environmental behaviors. In an online survey of 342 U.S. adults we found unique patterns of the three forms of environmental concern. Only individuals higher in biospheric environmental concern perceived ecological stress and engaged in ecological coping. In contrast, individuals higher in social-altruistic concern did not perceive ecological stress, but did engage in ecological coping. Those higher in egoistic concern neither perceived ecological stress, nor engaged in coping. In addition, perceived ecological stress was positively associated with depressive symptoms; ecological coping negatively predicted depressive symptoms, while positively predicting pro-environmental behaviors. In sum, with the exception of those high in biospheric concern, study participants did not seem to perceive climate change threats as having a profound effect on their own or their family’s life. Differentiating three forms of environmental concern provides a nuanced view on their association with ecological stress and coping, and in turn depressive symptoms and pro-environmental behaviors. Results indicate that current public policy approaches that often focus on the natural environment when depicting or explaining the effects of climate change, may limit the effectiveness of interventions to those people who already show high concern for all living creatures, while failing to affect those motivated by egoistic or altruistic concern, increasing the risks associated with delaying climate change adaptation and the potential for large-scale negative mental health effects in our society.
• Helm, S. V., Pollitt, A. M., Barnett, M. A., Curran, M. A., & Craig, Z. R. (2017). Differentiating environmental concern in the context of psychological adaptation to climate change. Global Environmental Change, In Press.
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  RC1Listed as in progress in 2016.
• Helm, S. V., Pollitt, A. M., Barnett, M. A., Curran, M. A., & Craig, Z. R. (2018). RC1 and RA2: Differentiating environmental concern in the context of psychological adaptation to climate change. Global Environmental Change, 158-167.
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  Helm, S. +Pollitt, A., Barnett, M., Curran, M. A., & Craig, Z. (2018). Differentiating environmental concern in the context of psychological adaption to climate change. Global Environmental Change, 48, 158-167. https://doi.org/10.1016/j.gloenvcha.2017.11.012
• Hoyer, P., Rice, P. F., Howard, C. C., Koevary, J. W., Dominguez Cooks, J. P., Hutchens, G. V., Chambers, S. K., Craig, Z. R., Connolly, D. C., & Barton, J. K. (2018). Comparison of Markers of Reproductive Function in Female C57Bl/6 versus TgMISIIR-TAg Transgenic Mice: Effect of VCD exposure on Ovarian Failure.. Comparative Medicine.
• Ziv-gal, A., & Craig, Z. R. (2018). Pretty Good or Pretty Bad? The Ovary and Chemicals in Personal Care Products.. Toxicological sciences : an official journal of the Society of Toxicology, 162(2), 349-360. doi:10.1093/toxsci/kfx285
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  Personal care products (PCP) contain a myriad of chemicals generally formulated to provide a safe and beneficial use. Nonetheless, an increasing amount of laboratory animal and human studies indicate that some chemicals in PCP are associated with decreased hormone production, diminished ovarian reserve, ovarian cancer, and early pregnancy loss. The ovary is key to female fertility by providing the eggs and sex steroid hormones for fertilization and maintenance of reproductive function, respectively. Thus, understanding how chemicals in PCP affect the ovary will shed some light on their potential effects on female fertility. In this review, we provide an overview of: (1) ovarian function as a determinant of fertility in females, (2) the status of knowledge regarding the effects of seven common chemicals in PCP on the ovary, and (3) significant gaps in the literature along with opportunities to eliminate some of the gaps. Findings from the limited existing data suggest that chemicals in PCP such as dibutyl phthalate can reach the ovary in humans and impact its function in animal models. Unfortunately, it is still difficult to assess how relevant findings of experimental studies are to women because of lack of human exposure data for most of these chemicals and the lack of studies that mimic real-life exposures. In contrast to chemicals such as bisphenol A and dioxin, the investigation of the effects of chemicals in PCP on reproductive function is still limited and warrants further investigation to fill existing data gaps.
• Craig, Z. R., & Ziv-Gal, A. (2017). Pretty good or pretty bad? - The ovary and chemicals in personal care products. Toxicological sciences : an official journal of the Society of Toxicology.
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  Personal care products (PCP) contain a myriad of chemicals generally formulated to provide a safe and beneficial use. Nonetheless, an increasing amount of laboratory animal and human studies indicate that some chemicals in PCP are associated with decreased hormone production, diminished ovarian reserve, ovarian cancer, and early pregnancy loss. The ovary is key to female fertility by providing the eggs and sex steroid hormones for fertilization and maintenance of reproductive function, respectively. Thus, understanding how chemicals in PCP affect the ovary will shed some light on their potential effects on female fertility. In this review, we provide an overview of: (1) ovarian function as a determinant of fertility in females, (2) the status of knowledge regarding the effects of seven common chemicals in PCP on the ovary, and (3) significant gaps in the literature along with opportunities to eliminate some of the gaps. Findings from the limited existing data suggest that chemicals in PCP such as dibutyl phthalate can reach the ovary in humans and impact its function in animal models. Unfortunately, it is still difficult to assess how relevant findings of experimental studies are to women because of lack of human exposure data for most of these chemicals and the lack of studies that mimic real-life exposures. In contrast to chemicals such as bisphenol A and dioxin, the investigation of the effects of chemicals in PCP on reproductive function is still limited and warrants further investigation to fill existing data gaps.
• Craig, Z. R., Liu, X., Rasmussen, L. M., & Sen, N. (2017).

  Effects of oral exposure to the phthalate substitute acetyl tributyl citrate on female reproduction in mice.

  . Journal of applied toxicology : JAT, 37(6), 668-675. doi:10.1002/jat.3413
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  Acetyl tributyl citrate (ATBC), is a phthalate substitute used in food and medical plastics, cosmetics and toys. Although systemically safe up to 1000 mg kg-1  day-1 , its ability to cause reproductive toxicity in females at levels below 50 mg kg-1  day-1 has not been examined. This study evaluated the effects of lower ATBC exposures on female reproduction using mice. Adult CD-1 females (n = 7-8 per treatment) were dosed orally with tocopherol-stripped corn oil (vehicle), 5 or 10 mg kg-1  day-1 ATBC daily for 15 days, and then bred with a proven breeder male. ATBC exposure did not alter body weights, estrous cyclicity, and gestational and litter parameters. Relative spleen weight was slightly increased in the 5 mg kg-1  day-1 group. ATBC at 10 mg kg-1  day-1 targeted ovarian follicles and decreased the number of primordial, primary and secondary follicles present in the ovary. These findings suggest that low levels of ATBC may be detrimental to ovarian function, thus, more information is needed to understand better the impact of ATBC on female reproduction. Copyright © 2016 John Wiley & Sons, Ltd.
• Rasmussen, L. M., Sen, N., Vera, J. C., Craig, Z. R., & Liu, X. (2017). Effects of in vitro exposure to dibutyl phthalate, mono-butyl phthalate, and acetyl tributyl citrate on ovarian antral follicle growth and viability†. Biology of Reproduction. doi:10.1095/biolreprod.116.144691
• Rasmussen, L. M., Sen, N., Vera, J. C., Liu, X., & Craig, Z. R. (2016). Effects of in vitro exposure to dibutyl phthalate, mono-butyl phthalate, and acetyl tributyl citrate on ovarian antral follicle growth and viability. Biology of Reproduction.
• Rasmussen, L. M., Sen, N., Vera, J. C., Liu, X., & Craig, Z. R. (2017). Effects of in vitro exposure to dibutyl phthalate, mono-butyl phthalate, and acetyl tributyl citrate on ovarian antral follicle growth and viability. Biology of reproduction, 96(5), 1105-1117.
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  Dibutyl phthalate (DBP) is present in consumer products and the coating of some oral medications. Acetyl tributyl citrate (ATBC) has been proposed as an alternative to DBP because DBP causes endocrine disruption in animal models. Following ingestion, DBP is converted to its main metabolite mono-butyl phthalate (MBP) which has been detected in >90% of human follicular fluid samples. Previous studies show that DBP reduces the number of antral follicles present in the ovaries of mice. Thus, this study was designed to evaluate the effects of DBP, MBP, and ATBC on in vitro growth and viability of mouse ovarian antral follicles. Antral follicles were isolated from CD-1 females (PND32-37) and treated with vehicle, DBP, MBP, or ATBC (starting at 0.001 and up to 1000 μg/ml for DBP; 24-72 h). Follicle diameter, ATP production, qPCR, and TUNEL were used to measure follicle growth, viability, cell cycle and apoptosis gene expression, and cell death-associated DNA fragmentation, respectively. While MBP did not cause toxicity, DBP exposure at ≥10 μg/ml resulted in growth inhibition followed by cytoxicity at ≥500 μg/ml. ATBC increased the number of nongrowing follicles at 0.01 μg/ml and did not affect ATP production, but increased TUNEL positive area in treated follicles. Gene expression results suggest that cytotoxicity in DBP-treated follicles occurs via activation of cell cycle arrest prior to follicular death. These findings suggest that concentrations of DBP ≥10 μg/ml are detrimental to antral follicles and that ATBC should be examined further as it may disrupt antral follicle function at low concentrations.
• Rasmussen, L. M., Sen, N., Liu, X., & Craig, Z. R. (2016). Effects of oral exposure to the phthalate substitute acetyl tributyl citrate on female reproduction in mice. Journal of applied toxicology : JAT.
• Sen, N., Craig, Z. R., & Liu, X. (2015). Short term exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in young CD-1 mice. Reproductive Toxicology. doi:10.1016/j.reprotox.2015.02.012
• Sen, N., Liu, X., & Craig, Z. R. (2014). Short term exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in young CD-1 mice. Reproductive Toxicology.
• Sen, N., Liu, X., & Craig, Z. R. (2015). Short term exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in young CD-1 mice. Reproductive toxicology (Elmsford, N.Y.), 53, 15-22.
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  Di-n-butyl phthalate (DBP) is present in many beauty and medical products. Human exposure estimates range from 0.007-0.01 mg/kg/day in the general population and up to 0.233 mg/kg/day in patients taking DBP-coated medications. Levels of phthalates tend to be higher in women, thus, evaluating ovarian effects of DBP exposure is of great importance. Mice were given corn oil (vehicle) or DBP at 0.01, 0.1, and 1000 mg/kg/day (high dose) for 10 days to test whether DBP causes ovarian toxicity. Estrous cyclicity, steroidogenesis, ovarian morphology, and apoptosis and steroidogenesis gene expression were evaluated. DBP exposure decreased serum E2 at all doses, while 0.1DBP increased FSH, decreased antral follicle numbers, and increased mRNA encoding pro-apoptotic genes (Bax, Bad, Bid). Interestingly, mRNAs encoding the steroidogenic enzymes Hsd17b1, Cyp17a1 and Cyp19a1 were increased in all DBP-treated groups. These novel findings show that DBP can disrupt ovarian function in mice at doses relevant to humans.
• Craig, Z. R., Flaws, J. A., Gupta, R. K., & Singh, J. (2014). Co-treatment of mouse antral follicles with 17β-estradiol interferes with mono-2-ethylhexyl phthalate (MEHP)-induced atresia and altered apoptosis gene expression. Reproductive Toxicology. doi:10.1016/j.reprotox.2014.01.002
• Craig, Z. R., Singh, J., Gupta, R. K., & Flaws, J. A. (2014). Co-treatment of mouse antral follicles with 17β-estradiol interferes with mono-2-ethylhexyl phthalate (MEHP)-induced atresia and altered apoptosis gene expression. Reproductive toxicology (Elmsford, N.Y.), 45, 45-51.
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  Mono-2-ethyhexyl phthalate (MEHP) is a metabolite of a plasticizer found in many consumer products. MEHP inhibits mouse ovarian follicle growth by reducing 17β-estradiol (E2) production. Yet, whether MEHP causes follicle death (atresia) is unclear. We hypothesized that MEHP causes atresia by altering apoptosis gene expression, and that E2 co-treatment blocks these effects. Follicles were exposed to MEHP (0.36-36μM)±E2 for 48-96h to determine the effect of MEHP±E2 on atresia and gene expression. MEHP increased atresia, but this effect was blocked by co-treatment with E2. MEHP increased the expression of the pro-apoptotic gene Aifm1, but decreased that of the pro-apoptotic gene Bok and the anti-apoptotic gene Bcl2l10. E2 interfered with MEHP-induced changes in Aifm1 and Bcl2l10. Our findings suggest that decreased E2 levels are required for MEHP-induced follicle atresia and that Aifm1, Bok, and Bcl2l10 are involved in this process.
• Craig, Z. R., Flaws, J. A., & Hannon, P. R. (2013). Pregnenolone co-treatment partially restores steroidogenesis, but does not prevent growth inhibition and increased atresia in mouse ovarian antral follicles treated with mono-hydroxy methoxychlor. Toxicology and Applied Pharmacology. doi:10.1016/j.taap.2013.08.002
• Craig, Z. R., Flaws, J. A., Ziv-Gal, A., Wang, W., & Hannon, P. R. (2013). Di-n-Butyl Phthalate Disrupts the Expression of Genes Involved in Cell Cycle and Apoptotic Pathways in Mouse Ovarian Antral Follicles1. Biology of Reproduction. doi:10.1095/biolreprod.112.105122
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  Di-n-butyl phthalate (DBP) is present in many consumer products, such as infant, beauty, and medical products. Several studies have shown that DBP causes reproductive toxicity in rodents, but no studies have evaluated its effects on ovarian follicles. Therefore, we used a follicle culture system to evaluate the effects of DBP on antral follicle growth, cell cycle and apoptosis gene expression, cell cycle staging, atresia, and 17β-estradiol (E(2)) production. Antral follicles were isolated from adult CD-1 mice and exposed to DBP at 1, 10, 100, and 1000 μg/ml for 24 or 168 h. Follicles treated with vehicle or DBP at 1-100 μg/ml grew over time, but DBP at 1000 μg/ml significantly suppressed follicle growth. Regardless of effect on follicle growth, DBP-treated follicles had decreased mRNA for cyclins D2, E1, A2, and B1 and increased p21. Levels of the proapoptotic genes Bax, Bad, and Bok were not altered by DBP treatment, but DBP 1000 μg/ml increased levels of Bid and decreased levels of the antiapoptotic gene Bcl2. DBP-treated follicles contained significantly more cells in G(1) phase, significantly less cells in S, and exhibited a trend for fewer cells in G(2). Although DBP did not affect E(2) production and atresia at 24 h, follicles treated with DBP had reduced levels of E(2) at 96 h and underwent atresia at 168 h. These data suggest that DBP targets antral follicles and alters the expression of cell cycle and apoptosis factors, causes cell cycle arrest, decreases E(2), and triggers atresia, depending on dose.
• Craig, Z. R., Hannon, P. R., & Flaws, J. A. (2013). Pregnenolone co-treatment partially restores steroidogenesis, but does not prevent growth inhibition and increased atresia in mouse ovarian antral follicles treated with mono-hydroxy methoxychlor. Toxicology and applied pharmacology, 272(3).
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  Mono-hydroxy methoxychlor (mono-OH MXC) is a metabolite of the pesticide, methoxychlor (MXC). Although MXC is known to decrease antral follicle numbers, and increase follicle death in rodents, not much is known about the ovarian effects of mono-OH MXC. Previous studies indicate that mono-OH MXC inhibits mouse antral follicle growth, increases follicle death, and inhibits steroidogenesis in vitro. Further, previous studies indicate that CYP11A1 expression and production of progesterone (P4) may be the early targets of mono-OH MXC in the steroidogenic pathway. Thus, this study tested whether supplementing pregnenolone, the precursor of progesterone and the substrate for HSD3B, would prevent decreased steroidogenesis, inhibited follicle growth, and increased follicle atresia in mono-OH MXC-treated follicles. Mouse antral follicles were exposed to vehicle (dimethylsulfoxide), mono-OH MXC (10 μg/mL), pregnenolone (1 μg/mL), or mono-OH MXC and pregnenolone together for 96 h. Levels of P4, androstenedione (A), testosterone (T), estrone (E1), and 17β-estradiol (E2) in media were determined, and follicles were processed for histological evaluation of atresia. Pregnenolone treatment alone stimulated production of all steroid hormones except E2. Mono-OH MXC-treated follicles had decreased sex steroids, but when given pregnenolone, produced levels of P4, A, T, and E1 that were comparable to those in vehicle-treated follicles. Pregnenolone treatment did not prevent growth inhibition and increased atresia in mono-OH MXC-treated follicles. Collectively, these data support the idea that the most upstream effect of mono-OH MXC on steroidogenesis is by reducing the availability of pregnenolone, and that adding pregnenolone may not be sufficient to prevent inhibited follicle growth and survival.
• Craig, Z. R., Hannon, P. R., Wang, W., Ziv-Gal, A., & Flaws, J. A. (2013). Di-n-butyl phthalate disrupts the expression of genes involved in cell cycle and apoptotic pathways in mouse ovarian antral follicles. Biology of reproduction, 88(1), 23.
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  Di-n-butyl phthalate (DBP) is present in many consumer products, such as infant, beauty, and medical products. Several studies have shown that DBP causes reproductive toxicity in rodents, but no studies have evaluated its effects on ovarian follicles. Therefore, we used a follicle culture system to evaluate the effects of DBP on antral follicle growth, cell cycle and apoptosis gene expression, cell cycle staging, atresia, and 17β-estradiol (E(2)) production. Antral follicles were isolated from adult CD-1 mice and exposed to DBP at 1, 10, 100, and 1000 μg/ml for 24 or 168 h. Follicles treated with vehicle or DBP at 1-100 μg/ml grew over time, but DBP at 1000 μg/ml significantly suppressed follicle growth. Regardless of effect on follicle growth, DBP-treated follicles had decreased mRNA for cyclins D2, E1, A2, and B1 and increased p21. Levels of the proapoptotic genes Bax, Bad, and Bok were not altered by DBP treatment, but DBP 1000 μg/ml increased levels of Bid and decreased levels of the antiapoptotic gene Bcl2. DBP-treated follicles contained significantly more cells in G(1) phase, significantly less cells in S, and exhibited a trend for fewer cells in G(2). Although DBP did not affect E(2) production and atresia at 24 h, follicles treated with DBP had reduced levels of E(2) at 96 h and underwent atresia at 168 h. These data suggest that DBP targets antral follicles and alters the expression of cell cycle and apoptosis factors, causes cell cycle arrest, decreases E(2), and triggers atresia, depending on dose.
• Ziv-Gal, A., Craig, Z. R., Wang, W., & Flaws, J. A. (2013). Bisphenol A inhibits cultured mouse ovarian follicle growth partially via the aryl hydrocarbon receptor signaling pathway. Reproductive toxicology (Elmsford, N.Y.), 42, 58-67.
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  Bisphenol A (BPA) is an endocrine disruptor that inhibits growth of mouse ovarian follicles and disrupts steroidogenesis at a dose of 438μM. However, the effects of lower doses of BPA and its mechanism of action in ovarian follicles are unknown. We hypothesized that low doses of BPA inhibit follicular growth and decrease estradiol levels through the aryl hydrocarbon receptor (AHR) pathway. Antral follicles from wild-type and Ahr knock-out (AhrKO) mice were cultured for 96h. Follicle diameters and estradiol levels then were compared in wild-type and AhrKO follicles ± BPA (0.004-438μM). BPA inhibited follicle growth (110-438μM) and decreased estradiol levels (43.8-438μM) in wild-type and AhrKO follicles. However, at BPA 110μM, inhibition of growth in AhrKO follicles was attenuated compared to wild-type follicles. These data suggest that BPA may inhibit follicle growth partially via the AHR pathway, whereas its effects on estradiol synthesis likely involve other mechanisms.
• Brannick, K. E., Craig, Z. R., Himes, A. D., Peretz, J. R., Wang, W., Flaws, J. A., & Raetzman, L. T. (2012). Prenatal exposure to low doses of bisphenol A increases pituitary proliferation and gonadotroph number in female mice offspring at birth. Biology of reproduction, 87(4), 82.
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  The pituitary gland is composed of hormone-producing cells essential for homeostasis and reproduction. Pituitary cells are sensitive to endocrine feedback in the adult and can have altered hormonal secretion from exposure to the endocrine disruptor bisphenol A (BPA). BPA is a prevalent plasticizer used in food and beverage containers, leading to widespread human exposure. Although prenatal exposure to BPA can impact reproductive function in the adult, the effects of BPA on the developing pituitary are unknown. We hypothesized that prenatal exposure to low doses of BPA impacts gonadotroph cell number or parameters of hormone synthesis. To test this, pregnant mice were administered 0.5 μg/kg/day of BPA, 50 μg/kg/day of BPA, or vehicle beginning on Embryonic Day 10.5. At parturition, pituitaries from female offspring exposed in utero to either dose of BPA had increased proliferation, as assessed by mKi67 mRNA levels and immunohistochemistry. Coincidently, gonadotroph number also increased in treated females. However, we observed a dichotomy between mRNA levels of Lhb and Fshb. Female mice exposed to 0.5 μg/kg/day BPA had increased mRNA levels of gonadotropins and the gonadotropin-receptor hormone (GNRH) receptor (Gnrhr), which mediates GNRH regulation of gonadotropin production and release. In contrast, mice treated with 50 μg/kg/day of BPA had decreased gonadotropin mRNA levels, Gnrhr and Nr5a1, a transcription factor required for gonadotroph differentiation. No other pituitary hormones were altered on the day of birth in response to in utero BPA exposure, and male pituitaries showed no change in the parameters tested. Collectively, these results show that prenatal exposure to BPA affects pituitary gonadotroph development in females.
• Craig, Z. R., Flaws, J. A., Ziv-Gal, A., Basavarajappa, M. S., & Wang, W. (2012). Mono-(2-ethylhexyl) Phthalate Induces Oxidative Stress and Inhibits Growth of Ovarian Antral Follicles.. Biology of Reproduction. doi:10.1093/biolreprod/87.s1.261
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  Mono-(2-ethylhexyl) phthalate (MEHP) is the active metabolite of the most commonly used plasticizer, di-(2-ethylhexyl) phthalate, and is considered to be a reproductive toxicant. However, little is known about the effects of MEHP on ovarian antral follicles. Thus, the present study tested the hypothesis that MEHP inhibits follicle growth via oxidative stress pathways. The data indicate that MEHP increases reactive oxygen species (ROS) levels and inhibits follicle growth in antral follicles, whereas N-acetylcysteine (NAC; an antioxidant) restores ROS levels to control levels and rescues follicles from MEHP-induced inhibition of follicle growth. To further analyze the mechanism by which MEHP induces oxidative stress and inhibits follicle growth, the expression and activities of various key antioxidant enzymes (copper/zinc superoxide dismutase [SOD1], glutathione peroxidase [GPX], and catalase [CAT]) and the expression of key cell-cycle regulators (Ccnd2, Ccne1, and Cdk4) and apoptotic regulators (Bcl-2 and Bax) were compared in control and MEHP-treated follicles. The data indicate that MEHP inhibits the expression and activities of SOD1 and GPX; does not inhibit Cat expression; inhibits the expression of Ccnd2, Ccne1, Cdk4, and Bcl-2; but increases the expression of Bax compared to controls. Furthermore, NAC blocks these toxic effects of MEHP. Collectively, these data suggest that MEHP induces oxidative stress by disrupting the activities of antioxidant enzymes. This may lead to decreased expression of cell-cycle regulators and antiapoptotic regulators and increased expression of proapoptotic factors, which then may lead to inhibition of follicle growth.
• Craig, Z. R., Raetzman, L. T., Flaws, J. A., Wang, W., Peretz, J., Himes, A. D., & Brannick, K. E. (2012). Prenatal Exposure to Low Doses of Bisphenol A Increases Pituitary Proliferation and Gonadotroph Number in Female Mice Offspring at Birth1. Biology of Reproduction. doi:10.1095/biolreprod.112.100636
• Karman, B. N., Basavarajappa, M. S., Craig, Z. R., & Flaws, J. A. (2012). 2,3,7,8-Tetrachlorodibenzo-p-dioxin activates the aryl hydrocarbon receptor and alters sex steroid hormone secretion without affecting growth of mouse antral follicles in vitro. Toxicology and applied pharmacology, 261(1), 88-96.
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  The persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an ovarian toxicant. These studies were designed to characterize the actions of TCDD on steroidogenesis and growth of intact mouse antral follicles in vitro. Specifically, these studies tested the hypothesis that TCDD exposure leads to decreased sex hormone production/secretion by antral follicles as well as decreased growth of antral follicles in vitro. Since TCDD acts through binding to the aryl hydrocarbon receptor (AHR), and the AHR has been identified as an important factor in ovarian function, we also conducted experiments to confirm the presence and activation of the AHR in our tissue culture system. To do so, we exposed mouse antral follicles for 96 h to a series of TCDD doses previously shown to have effects on ovarian tissues and cells in culture, which also encompass environmentally relevant and pharmacological exposures (0.1-100 nM), to determine a dose response for TCDD in our culture system for growth, hormone production, and expression of the Ahr and Cyp1b1. The results indicate that TCDD decreases progesterone, androstenedione, testosterone, and estradiol levels in a non-monotonic dose response manner without altering growth of antral follicles. The addition of pregnenolone substrate (10 μM) restores hormone levels to control levels. Additionally, Cyp1b1 levels were increased by 3-4 fold regardless of the dose of TCDD exposure, evidence of AHR activation. Overall, these data indicate that TCDD may act prior to pregnenolone formation and through AHR transcriptional control of Cyp1b1, leading to decreased hormone levels without affecting growth of antral follicles.
• Paulose, T., Hannon, P. R., Peretz, J., Craig, Z. R., & Flaws, J. A. (2012). Estrogen receptor alpha overexpressing mouse antral follicles are sensitive to atresia induced by methoxychlor and its metabolites. Reproductive toxicology (Elmsford, N.Y.), 33(3), 353-60.
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  Methoxychlor (MXC) and its metabolites bind to estrogen receptors (ESRs) and increase ovarian atresia. To test whether ESR alpha (ESR1) overexpressing (ESR1 OE) antral follicles are more sensitive to atresia compared to controls, we cultured antral follicles with vehicle, MXC (1-100 μg/ml) or metabolites (0.1-10 μg/ml). Results indicate that MXC and its metabolites significantly increase atresia in ESR1 OE antral follicles at lower doses compared to controls. Activity of pro-apoptotic factor caspase-3/7 was significantly higher in ESR1 OE treated antral follicles compared to controls. ESR1 OE mice dosed with MXC 64 mg/kg/day had an increased percentage of atretic antral follicles compared to controls. Furthermore, pro-caspase-3 levels were found to be significantly lower in ESR1 OE ovaries than controls dosed with MXC 64 mg/kg/day. These data suggest that ESR1 OE ovaries are more sensitive to atresia induced by MXC and its metabolites in vitro and in vivo compared to controls.
• Peretz, J., Craig, Z. R., & Flaws, J. A. (2012). Bisphenol A inhibits follicle growth and induces atresia in cultured mouse antral follicles independently of the genomic estrogenic pathway. Biology of reproduction, 87(3), 63.
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  Bisphenol A (BPA) is an estrogenic chemical used to manufacture many commonly used plastic and epoxy resin-based products. BPA ubiquitously binds to estrogen receptors throughout the body, including estrogen receptor alpha (ESR1) in the ovary. Few studies have investigated the effects of BPA on ovarian antral follicles. Thus, we tested the hypothesis that BPA alters cell cycle regulators and induces atresia in antral follicles via the genomic estrogenic pathway, inhibiting follicle growth. To test this hypothesis, we isolated antral follicles from 32- to 35-day-old control and Esr1-overexpressing mice and cultured them with vehicle control (dimethylsulfoxide [DMSO]) or BPA (1-100 μg/ml). Additionally, antral follicles were isolated from 32- to 35-day-old FVB mice and cultured with DMSO, BPA (1-100 μg/ml), estradiol (10 nM), ICI 182,780 (ICI; 1 μM), BPA plus ICI, or BPA plus estradiol. Follicles were measured for growth every 24 h for 96-120 h and processed either for analysis of estrogen receptor, cell cycle, and/or atresia factor mRNA expression, or for histological evaluation of atresia. Results indicate that estradiol and ICI do not protect follicles from BPA-induced growth inhibition and that estradiol does not protect follicles from BPA-induced atresia. Furthermore, overexpressing Esr1 does not increase susceptibility of follicles to BPA-induced growth inhibition. Additionally, BPA up-regulates Cdk4, Ccne1, and Trp53 expression, whereas it down-regulates Ccnd2 expression. BPA also up-regulates Bax and Bcl2 expression while inducing atresia in antral follicles. These data indicate that BPA abnormally regulates cell cycle and atresia factors, and this may lead to atresia and inhibited follicle growth independently of the genomic estrogenic pathway.
• Wang, W., Craig, Z. R., Basavarajappa, M. S., Gupta, R. K., & Flaws, J. A. (2012). Di (2-ethylhexyl) phthalate inhibits growth of mouse ovarian antral follicles through an oxidative stress pathway. Toxicology and applied pharmacology, 258(2), 288-95.
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  Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer that has been shown to inhibit growth of mouse antral follicles, however, little is known about the mechanisms by which DEHP does so. Oxidative stress has been linked to follicle growth inhibition as well as phthalate-induced toxicity in non-ovarian tissues. Thus, we hypothesized that DEHP causes oxidative stress and that this leads to inhibition of the growth of antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice (age 31-35days) were cultured with vehicle control (dimethylsulfoxide [DMSO]) or DEHP (1-100μg/ml)±N-acetyl cysteine (NAC, an antioxidant at 0.25-1mM). During culture, follicles were measured daily. At the end of culture, follicles were collected and processed for in vitro reactive oxygen species (ROS) assays to measure the presence of free radicals or for measurement of the expression and activity of various key antioxidant enzymes: Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX) and catalase (CAT). The results indicate that DEHP inhibits the growth of follicles compared to DMSO control and that NAC (0.25-1mM) blocks the ability of DEHP to inhibit follicle growth. Furthermore, DEHP (10μg/ml) significantly increases ROS levels and reduces the expression and activity of SOD1 compared to DMSO controls, whereas NAC (0.5mM) rescues the effects of DEHP on ROS levels and SOD1. However, the expression and activity of GPX and CAT were not affected by DEHP treatment. Collectively, these data suggest that DEHP inhibits follicle growth by inducing production of ROS and by decreasing the expression and activity of SOD1.
• Wang, W., Craig, Z. R., Basavarajappa, M. S., Hafner, K. S., & Flaws, J. A. (2012). Mono-(2-ethylhexyl) phthalate induces oxidative stress and inhibits growth of mouse ovarian antral follicles. Biology of reproduction, 87(6), 152.
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  Mono-(2-ethylhexyl) phthalate (MEHP) is the active metabolite of the most commonly used plasticizer, di-(2-ethylhexyl) phthalate, and is considered to be a reproductive toxicant. However, little is known about the effects of MEHP on ovarian antral follicles. Thus, the present study tested the hypothesis that MEHP inhibits follicle growth via oxidative stress pathways. The data indicate that MEHP increases reactive oxygen species (ROS) levels and inhibits follicle growth in antral follicles, whereas N-acetylcysteine (NAC; an antioxidant) restores ROS levels to control levels and rescues follicles from MEHP-induced inhibition of follicle growth. To further analyze the mechanism by which MEHP induces oxidative stress and inhibits follicle growth, the expression and activities of various key antioxidant enzymes (copper/zinc superoxide dismutase [SOD1], glutathione peroxidase [GPX], and catalase [CAT]) and the expression of key cell-cycle regulators (Ccnd2, Ccne1, and Cdk4) and apoptotic regulators (Bcl-2 and Bax) were compared in control and MEHP-treated follicles. The data indicate that MEHP inhibits the expression and activities of SOD1 and GPX; does not inhibit Cat expression; inhibits the expression of Ccnd2, Ccne1, Cdk4, and Bcl-2; but increases the expression of Bax compared to controls. Furthermore, NAC blocks these toxic effects of MEHP. Collectively, these data suggest that MEHP induces oxidative stress by disrupting the activities of antioxidant enzymes. This may lead to decreased expression of cell-cycle regulators and antiapoptotic regulators and increased expression of proapoptotic factors, which then may lead to inhibition of follicle growth.
• Wang, W., Hannon, P. R., Flaws, J. A., & Craig, Z. R. (2012). Dibutyl Phthalate (DBP) Induces Cell Cycle Arrest and Apoptosis in Ovarian Antral Follicles.. Biology of Reproduction, 87(Suppl_1), 250-250. doi:10.1093/biolreprod/87.s1.250
• Basavarajappa, M. S., Craig, Z. R., Hernández-Ochoa, I., Paulose, T., Leslie, T. C., & Flaws, J. A. (2011). Methoxychlor reduces estradiol levels by altering steroidogenesis and metabolism in mouse antral follicles in vitro. Toxicology and applied pharmacology, 253(3), 161-9.
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  The organochlorine pesticide methoxychlor (MXC) is a known endocrine disruptor that affects adult rodent females by causing reduced fertility, persistent estrus, and ovarian atrophy. Since MXC is also known to target antral follicles, the major producer of sex steroids in the ovary, the present study was designed to test the hypothesis that MXC decreases estradiol (E₂) levels by altering steroidogenic and metabolic enzymes in the antral follicles. To test this hypothesis, antral follicles were isolated from CD-1 mouse ovaries and cultured with either dimethylsulfoxide (DMSO) or MXC. Follicle growth was measured every 24 h for 96 h. In addition, sex steroid hormone levels were measured using enzyme-linked immunosorbent assays (ELISA) and mRNA expression levels of steroidogenic enzymes as well as the E₂ metabolic enzyme Cyp1b1 were measured using qPCR. The results indicate that MXC decreased E₂, testosterone, androstenedione, and progesterone (P₄) levels compared to DMSO. In addition, MXC decreased expression of aromatase (Cyp19a1), 17β-hydroxysteroid dehydrogenase 1 (Hsd17b1), 17α-hydroxylase/17,20-lyase (Cyp17a1), 3β hydroxysteroid dehydrogenase 1 (Hsd3b1), cholesterol side-chain cleavage (Cyp11a1), steroid acute regulatory protein (Star), and increased expression of Cyp1b1 enzyme levels. Thus, these data suggest that MXC decreases steroidogenic enzyme levels, increases metabolic enzyme expression and this in turn leads to decreased sex steroid hormone levels.
• Craig, Z. R., Wang, W., & Flaws, J. A. (2011). Endocrine-disrupting chemicals in ovarian function: effects on steroidogenesis, metabolism and nuclear receptor signaling. Reproduction (Cambridge, England), 142(5), 633-46.
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  Endocrine-disrupting chemicals (EDCs) are exogenous agents with the ability to interfere with processes regulated by endogenous hormones. One such process is female reproductive function. The major reproductive organ in the female is the ovary. Disruptions in ovarian processes by EDCs can lead to adverse outcomes such as anovulation, infertility, estrogen deficiency, and premature ovarian failure among others. This review summarizes the effects of EDCs on ovarian function by describing how they interfere with hormone signaling via two mechanisms: altering the availability of ovarian hormones, and altering binding and activity of the hormone at the receptor level. Among the chemicals covered are pesticides (e.g. dichlorodiphenyltrichloroethane and methoxychlor), plasticizers (e.g. bisphenol A and phthalates), dioxins, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons (e.g. benzo[a]pyrene).
• Hannon, P. R., Flaws, J. A., & Craig, Z. R. (2011). Dibutyl Phthalate (DBP) Alters the Levels of Cell Proliferation and Apoptosis Genes in Mouse Granulosa Cells.. Biology of Reproduction, 85(Suppl_1), 276-276. doi:10.1093/biolreprod/85.s1.276
• Hannon, P. R., Flaws, J. A., & Craig, Z. R. (2011). Dibutyl Phthalate (DBP) Suppresses Growth of Isolated Mouse Antral Follicles.. Biology of Reproduction, 85(Suppl_1), 270-270. doi:10.1093/biolreprod/85.s1.270
• Karman, B. N., Flaws, J. A., Craig, Z. R., & Basavarajappa, M. S. (2011). 2,3,7,8-Tetrachlorodibenzo-p-dioxin Activates the AHR Pathway and Alters Estradiol Levels Without Affecting Growth in CD-1 Mouse Antral Follicles In Vitro.. Biology of Reproduction, 85(Suppl_1), 284-284. doi:10.1093/biolreprod/85.s1.284
• Wang, W., Gupta, R. K., Flaws, J. A., Craig, Z. R., & Basavarajappa, M. S. (2011). Di-(2-ethylhexyl) Phthalate Inhibits Growth of Ovarian Antral Follicles Through an Oxidative Stress Pathway.. Biology of Reproduction, 85(Suppl_1), 40-40. doi:10.1093/biolreprod/85.s1.40
• Wang, W., Karman, B. S., Hernandez-ochoa, I., Flaws, J. A., Craig, Z. R., & Basavarajappa, M. S. (2011). Methoxychlor Alters Pro-Apoptotic and Anti-Apoptotic Factors in the Antral Follicles of the Mouse Ovary.. Biology of Reproduction, 85(Suppl_1), 286-286. doi:10.1093/biolreprod/85.s1.286
• Craig, Z. R., Davis, J. R., Marion, S. L., Barton, J. K., & Hoyer, P. B. (2010). 7,12-dimethylbenz[a]anthracene induces sertoli-leydig-cell tumors in the follicle-depleted ovaries of mice treated with 4-vinylcyclohexene diepoxide. Comparative medicine, 60(1), 10-7.
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  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.
• Craig, Z. R., Leslie, T. C., Hatfield, K. P., Gupta, R. K., & Flaws, J. A. (2010). Mono-hydroxy methoxychlor alters levels of key sex steroids and steroidogenic enzymes in cultured mouse antral follicles. Toxicology and applied pharmacology, 249(2), 107-13.
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  Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by decreasing antral follicle numbers and increasing follicular death. MXC is metabolized in the body to mono-hydroxy MXC (mono-OH). Little is known about the effects of mono-OH on the ovary. Thus, this work tested the hypothesis that mono-OH exposure decreases production of 17β-estradiol (E₂) by cultured mouse antral follicles. Antral follicles were isolated from CD-1 mice (age 35-39 days) and exposed to dimethylsulfoxide (DMSO), or mono-OH (0.1-10 μg/mL) for 96 h. Media and follicles were collected for analysis of sex steroid levels and mRNA expression, respectively. Mono-OH treatment (10 μg/mL) decreased E(2) (DMSO: 3009.72±744.99 ng/mL; mono-OH 0.1 μg/mL: 1679.66±461.99 ng/mL; 1 μg/mL: 1752.72±532.41 ng/mL; 10 μg/mL: 45.89±33.83 ng/mL), testosterone (DMSO: 15.43±2.86 ng/mL; mono-OH 0.1μg/mL: 17.17±4.71 ng/mL; 1 μg/mL: 13.64±3.53 ng/mL; 10 μg/mL: 1.29±0.23 ng/mL), androstenedione (DMSO: 1.92±0.34 ng/mL; mono-OH 0.1 μg/mL: 1.49±0.43ng/mL; 1 μg/mL: 0.64±0.31 ng/mL; 10 μg/mL: 0.12±0.06 ng/mL) and progesterone (DMSO: 24.11±4.21 ng/mL; mono-OH 0.1μg/mL: 26.77±4.41 ng/mL; 1 μg/mL: 20.90±3.75 ng/mL; 10 μg/mL: 9.44±2.97 ng/mL) levels. Mono-OH did not alter expression of Star, Hsd3b1, Hsd17b1 and Cyp1b1, but it did reduce levels of Cyp11a1, Cyp17a1 and Cyp19a1 mRNA. Collectively, these data suggest that mono-OH significantly decreases levels of key sex steroid hormones and the expression of enzymes required for steroidogenesis.
• Craig, Z. R., Marion, S. L., Funk, J. L., Bouxsein, M. L., & Hoyer, P. B. (2010). Retaining Residual Ovarian Tissue following Ovarian Failure Has Limited Influence on Bone Loss in Aged Mice. Journal of osteoporosis, 2010.
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  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.
• Paulose, T., Karman, B. N., Gupta, R. K., Flaws, J. A., & Craig, Z. R. (2010). 1,1,1-Trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH methoxychlor) Alters mRNA Expression of Key Steroidogenic Enzymes in Cultured Mouse Antral Follicles.. Biology of Reproduction, 83(Suppl_1), 284-284. doi:10.1093/biolreprod/83.s1.284
• Hoyer, P. B., Barton, J. K., Craig, Z. R., Davis, J. R., & Marion, S. L. (2009).

  Characterization of Carcinogen-Induced Ovarian Neoplasms in Mice Following VCD-Induced Ovarian Failure.

  . Biology of Reproduction, 81(Suppl_1), 501-501. doi:10.1093/biolreprod/81.s1.501
• Hoyer, P. B., Christian, P. J., Craig, Z. R., & Marion, S. L. (2009).

  Estradiol replacement inhibits increased expression of Cyp17a1 mRNA in residual ovarian tissue of VCD-treated follicle-depleted mice

  . The FASEB Journal, 23. doi:10.1096/fasebj.23.1_supplement.807.15
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  Cyp17a1 encodes the protein responsible for production of androstenedione (A) and it has been proposed to be negatively regulated at the mRNA level by estradiol (E2) via estrogen receptor alpha in a local negative feedback loop. Enriched expression of Cyp17a1 mRNA has been observed in residual ovarian tissue of an ovary-intact mouse model of menopause (VCD-treated mouse) when compared to cycling controls. This study was designed to test the hypothesis that increased expression of Cyp17a1 in VCD-treated follicle-depleted ovaries can be reversed by replacement with E2. Female B6C3F1 mice (age d28) were dosed daily (17d; i.p.) with sesame oil (n=5) or VCD (160mg/kg; n=5). On day 19 after the onset of dosing a group of VCD-treated mice received subcutaneous E2 implants (VCD/E2 mice; 180 μg/ml; silastic) which were replaced on d47, 75, 103 and 131. Ovaries and blood were collected on d188 and processed for analysis by real-time PCR and measurement of circulating E2, A and E1 (estrone). Relative to VCD-treated mice, ovarian Cyp17a1 mRNA was lower (VCD/E2: 2.44±0.35/control; VCD: 8.97±0.14/control; P
• Rivera, Z., Christian, P. J., Marion, S. L., Brooks, H. L., & Hoyer, P. B. (2009). Steroidogenic capacity of residual ovarian tissue in 4-vinylcyclohexene diepoxide-treated mice. Biology of reproduction, 80(2), 328-36.
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  Menopause is an important public health issue because of its association with a number of disorders. Androgens produced by residual ovarian tissue after menopause could impact the development of these disorders. It has been unclear, however, whether the postmenopausal ovary retains steroidogenic capacity. Thus, an ovary-intact mouse model for menopause that uses the occupational chemical 4-vinylcyclohexene diepoxide (VCD) was used to characterize the expression of steroidogenic genes in residual ovarian tissue of follicle-depleted mice. Female B6C3F1 mice (age, 28 days) were dosed daily for 20 days with either vehicle or VCD (160 mg kg(-1) day(-1)) to induce ovarian failure. Ovaries were collected on Day 181 and analyzed for mRNA and protein. Acyclic aged mice were used as controls for natural ovarian senescence. Relative to cycling controls, expression of mRNA encoding steroidogenic acute regulatory protein (Star); cholesterol side-chain cleavage (Cyp11a1); 3beta-hydroxysteroid dehydrogenase (Hsd3b); 17alpha-hydroxylase (Cyp17a1); scavenger receptor class B, type 1 (Scarb1); low-density lipoprotein receptor (Ldlr); and luteinizing hormone receptor (Lhcgr) was enriched in VCD-treated ovaries. In acyclic aged ovaries, mRNA expression for only Cyp17a1 and Lhcgr was greater than that in controls. Compared to cycling controls, ovaries from VCD-treated and aged mice had similar levels of HSD3B, CYP17A1, and LHCGR protein. The pattern of protein immunofluorescence staining for HSD3B in follicle-depleted (VCD-treated) ovaries was homogeneous, whereas that for CYP17A1 was only seen in residual interstitial cells. Circulating levels of FSH and LH were increased, and androstenedione levels were detectable following follicle depletion in VCD-treated mice. These findings support the idea that residual ovarian tissue in VCD-treated mice retains androgenic capacity.
• Wright, L. E., Christian, P. J., Rivera, Z., Van Alstine, W. G., Funk, J. L., Bouxsein, M. L., & Hoyer, P. B. (2008). Comparison of skeletal effects of ovariectomy versus chemically induced ovarian failure in mice. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 23(8), 1296-303.
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  Bone loss associated with menopause leads to an increase in skeletal fragility and fracture risk. Relevant animal models can be useful for evaluating the impact of ovarian failure on bone loss. A chemically induced model of menopause in which mice gradually undergo ovarian failure yet retain residual ovarian tissue has been developed using the chemical 4-vinylcyclohexene diepoxide (VCD). This study was designed to compare skeletal effects of VCD-induced ovarian failure to those associated with ovariectomy (OVX). Young (28 day) C57Bl/6Hsd female mice were dosed daily with vehicle or VCD (160 mg/kg/d, IP) for 15 days (n = 6-7/group) and monitored by vaginal cytology for ovarian failure. At the mean age of VCD-induced ovarian failure (approximately 6 wk after onset of dosing), a different group of mice was ovariectomized (OVX, n = 8). Spine BMD (SpBMD) was measured by DXA for 3 mo after ovarian failure and OVX. Mice were killed approximately 5 mo after ovarian failure or OVX, and bone architecture was evaluated by microCT ex vivo. In OVX mice, SpBMD was lower than controls 1 mo after OVX, whereas in VCD-treated mice, SpBMD was not lower than controls until 2.9 mo after ovarian failure (p < 0.05). Both VCD-induced ovarian failure and OVX led to pronounced deterioration of trabecular bone architecture, with slightly greater effects in OVX mice. At the femoral diaphysis, cortical bone area and thickness did not differ between VCD mice and controls but were decreased in OVX compared with both groups (p < 0.05). Circulating androstenedione levels were preserved in VCD-treated mice but reduced in OVX mice relative to controls (p < 0.001). These findings support that (1) VCD-induced ovarian failure leads to trabecular bone deterioration, (2) bone loss is attenuated by residual ovarian tissue, particularly in diaphyseal cortical bone, and (3) the VCD mouse model can be a relevant model for natural menopause in the study of associated bone disorders.

Proceedings Publications

• Craig, Z. R., Zelinski, M. B., & Watanabe, K. H. (2021). Computational Modeling of the Ovary: Applications for Predictive Toxicology. In AAAS.

Presentations

• Craig, Z. R. (2021). Human Phthalate Exposures and Female Reproductive Function. International Conference on Ecotoxicology - Impacts, Assessment, and Mitigation. Virtual: Lady Doak College, Madurai Kamaraj University, India.
• Craig, Z. R. (2021). Human Phthalate Exposures and Female Reproductive Function. Pathobiology Graduate Program Seminar Series. Virtual: Brown University.
• Craig, Z. R. (2021). Modeling Human Relevant Phthalate Exposures in Mice. Annual Scientific RetreatUniversity of Arizona Cancer Center.
• Craig, Z. R. (2021). Modeling Human Relevant Phthalate Exposures in Mice. Interdisciplinary Faculty of Reproductive Biology Group Seminar SeriesTexas A&M University.
• Craig, Z. R. (2021, January). It’s not you, it’s the environment: environmental chemicals and female reproduction. Department of Obstetrics & Gynecology Grand RoundsCollege of Medicine, The University of Arizona.
• Craig, Z. R. (2021, January). Mechanisms of Phthalate-Inducted Reproductive Dysfunction. NIEHS - Reproductive Developmental Biology Group Seminar SeriesNIEHS.
• Craig, Z. R. (2019, June). From Undergrad to Tenured: An NIH-Funded Journey. NIH Advancing Diversity Programs Conference – Early Investigators Panel. Bethesda, MD, USA: National Institutes of Health.
• Craig, Z. R. (2019, September). Environmentally Relevant Exposures to Phthalates and Female Reproduction. Environmental and Occupational Health Seminar Series. Tucson, AZ, USA: Mel & Enid Zuckerman College of Public Health, The University of Arizona.
• Craig, Z. R. (2018, April 8). Environmentally Relevant Phthalate Exposures and Ovarian Function. Research Initiative for Scientific Enhancement (RISE) Speaker Series. Ponce, Puerto Rico: Ponce School of Medicine.
• Craig, Z. R. (2018, November 16). Environmentally Relevant Exposures to Phthalates and Female Reproduction. Reproductive Biology Forum. College Station, TX: Texas A&M University.
• Craig, Z. R. (2018, September 21). Environmentally Relevant Phthalate Exposures and Ovarian Function. Mountain West Society of Toxicology Annual Meeting. Phoenix, AZ: Society of Toxicology/University of Arizona College of Medicine.
• Nunez, F., Liu, X., & Craig, Z. R. (2018, July). Environmentally-relevant exposure to dibutyl phthalate (DBP) results in decreased expression of Brca1 in the adult mouse ovary. 51st Annual Meeting of The Society for the Study of Reproduction. New Orleans, LA: Society for the Study of Reproduction.
• Craig, Z. R. (2017, August). Keynote Speech. 22nd Annual Undergraduate Research Opportunities Consortium (UROC) Poster Session and Closing Ceremony. Tucson, AZ: University of Arizona Graduate College.
• Craig, Z. R. (2017, October). Environmentally Relevant Phthalate Exposures and Reproductive Function. Center for Occupational and Environmental Health Seminar Series. Irvine, CA: University of California - Irvine.
• Craig, Z. R. (2016, July). Mechanisms of Phthalate-Induced Ovarian Follicle Toxicity. 49th Annual Meeting of The Society for the Study of Reproduction. San Diego, CA: Society for the Study of Reproduction.
• Craig, Z. R. (2015, April). Ovarian follicle toxicity by phthalates. NIEHS Center Director's Meeting.
• Craig, Z. R. (2013, October). Mechanisms of phthalate-induced ovarian follicle toxicity. Central States Society of Toxicology Annual Meeting. Ames, IA: Society of Toxicology and Iowa State University.
• Craig, Z. R. (2013, October). My Scientific Journey. SACNAS Student Chapter Meeting. Ames, IA: SACNAS and Iowa State University.

Poster Presentations

• Craig, Z. R., Langlais, P. R., Jaurgui, E., McSwain, M., Liu, X., & Miller, K. (2022, June). Effects of a Phthalate Mixture on Hormone and Ovarian Antral Follicle Protein Abundance  . Gordon Research Conference - Environmental Endocrine Disruptors.
• Beltran-Gastelum, J. G., Nunez, F. M., Jauregui, E. J., & Craig, Z. R. (2020, March). Exploring reproductive toxicological dosing schemes: effects of environmentally relevant dibutyl-phthalate exposures on terminal estrous stage gene expression. Annual Meeting of The Society of Toxicology and ToxExpo. Anaheim, CA, USA: Society of Toxicology.
• Jauregui, E. J., Lock, J. L., Rasmussen, L. M., & Craig, Z. R. (2020, March). Levels of Mono-n-butyl Phthalate in the Tissues of Adult CD-1 Female Mice after Repeated Oral Administration of Di-n-butyl Phthalate. Annual Meeting of The Society of Toxicology and ToxExpo. Anaheim, CA, USA: Society of Toxicology.
• Lock, J. L., Jauregui, E. J., & Craig, Z. R. (2020, January). Generation of Cell-Specific Ribosome-Bound Messenger-RNA in Mouse Ovaries. 31st Annual Undergraduate Biology Research Program Research Conference. Tucson, AZ, USA: The University of Arizona.
• Nunez, F. M., Beltran-Gastelum, J. G., & Craig, Z. R. (2020, March). The effects of oral dibutyl phthalate (DBP) exposure on transcription factor activation in the mouse ovary. Annual Meeting of The Society of Toxicology and ToxExpo. Anaheim, CA, USA: Society of Toxicology.
• Beltran-Gastelum, J. G., Jauregui, E. J., & Craig, Z. R. (2019, July). Environmentally Relevant Exposure to Dibutyl Phthalate and Ovarian Gene Expression: Effects of Terminal Estrous Cycle Stage. Annual Meeting of the Society for the Study of Reproduction. San Jose, CA, USA: Society for the Study of Reproduction.
• Jauregui, E. J., Rasmussen, L. M., & Craig, Z. R. (2019, March). Measurements of Mono-n-butyl Phthalate in the Tissues of Cycling Adult CD-1 Female Mice after the Oral Administration of Di-n-butyl Phthalate. Annual Meeting of The Society of Toxicology. Baltimore, MD: Society of Toxicology.
• Nunez, F. M., Beltran-Gastelum, J. G., & Craig, Z. R. (2019, July). Effects of dibutyl phthalate (DBP) exposure on the expression of transcription factors in the mouse ovary. Annual Meeting of the Society for the Study of Reproduction. San Jose, CA, USA: Society for the Study of Reproduction.
• Rasmussen, L. M., Jauregui, E. J., & Craig, Z. R. (2019, March). Effects of in vitro exposure to di-n-butyl phthalate and mono-n-butyl phthalate on early embryo viability and development. Annual Meeting of The Society of Toxicology. Baltimore, MD: Society of Toxicology.
• Jauregui, E. J., Liu, X., Beltran-Gastelum, J., & Craig, Z. R. (2018, July). Exposure to di-n-butyl phthalate alters IGF1 expression and causes ovarian toxicity in the adult mouse. 51st Annual Meeting of The Society for the Study of Reproduction. New Orleans, LA: Society for the Study of Reproduction.
• Rasmussen, L. M., & Craig, Z. R. (2017, December). Effects of in vivo exposure to di-n-butyl phthalate on ovulation, fertilization, and embryo development in the mature superovulated mouse. Annual Meeting of The Society of Toxicology. San Antonio, TX: Society of Toxicology.
• Rasmussen, L. M., & Craig, Z. R. (2018, July). Effects of in vivo exposure to di-n-butyl phthalate on ovulation, antral follicle counts, and serum progesterone in the mature superovulated mouse. 51st Annual Meeting of The Society for the Study of Reproduction. New Orleans, LA: Society for the Study of Reproduction.
• Craig, Z. R., Rasmussen, L. M., & Sen, N. (2016, June). Effects of in vitro exposure to the di-n-butyl phthalate and the phthalate substitute acetyl tributyl citrate on mouse antral follicles. 49th Annual Meeting of The Society for the Study of Reproduction. San Diego, CA: Society for the Study of Reproduction.
• Sen, N., Vera, J. C., & Craig, Z. R. (2016, June). Cell cycle and apoptotic gene expression in mouse ovarian antral follicles treated with mono-butyl phthalate. 49th Annual Meeting of The Society for the Study of Reproduction. San Diego, CA: Society for the Study of Reproduction.
• Rasmussen, L. M., & Craig, Z. R. (2015, June). Effects of in vitro exposure to the phthalate substitute acetyl tributyl citrate on isolated antral follicle viability. Annual Meeting of the Society for the Study of Reproduction. San Juan, Puerto Rico.
• Sen, N., Liu, X., & Craig, Z. R. (2014, Fall). A 10-day exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in CD-1 mice. 2015 Annual Meeting of the Society of Toxicology. San Diego, CA: Society of Toxicology.
• Vera, J. C., Sen, N., & Craig, Z. R. (2015, June). Evaluation of cytoxicity in isolated antral follicles treated with mono-butyl phthalate and hydroxyurea. Annual Meeting of the Society for the Study of Reproduction. San Juan, Puerto Rico.
• Acuff, A., & Craig, Z. R. (2014, March). A short, low dose exposure to di-n-butyl phthalate (DBP) disrupts 17beta-estradiol production in CD-1 female mice. 53rd Annual Meeting and ToxExpo - Society of Toxicology. Phoenix, AZ: Society of Toxicology.
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  Abstract submitted in October 2013, accepted in December 2013 and scheduled to be presented in March 2014
• Liu, X., & Craig, Z. R. (2014, July 19-23). A 10-day exposure to di-n-butyl phthalate (DBP) disrupts DNA damage and repair gene expression in the ovaries of CD-1 mice. 47th Annual Meeting of the Society for the Study of Reproduction. Grand Rapids, MI: Society for the Study of Reproduction.