▼ Nursing ensures lactocrine delivery of milk-borne bioactive factors to offspring and the lactocrine hypothesis for maternal programming proposes that disruption of lactocrine communication from birth (postnatal day = PND 0) affects both the program and trajectory of porcine uterine development. Establishment of a model system for study of lactocrine-sensitive uterine organizational events shortly after birth in the neonate and for identification of potentially lactocrine-active factors found in porcine colostrum, such as insulin-like growth factor 1 (IGF1), is important. Studies show that the neonatal porcine uterine transcriptome is age- and lactocrine-sensitive on PND 2. However, whether uterine microRNA (miRNA) expression or the uterine miRNA-mRNA interactome is affected similarly is unknown. Lactocrine deficiency from birth, reflected by low serum immunoglobulin immunocrit, was associated with alterations in aspects of the neonatal uterine developmental program and, long-term, was linked to reduced lifetime fecundity in adult gilts. These observations suggested lactocrine effects on programming of adult uterine function. However, whether lactocrine deficiency and disruption of neonatal uterine development is ultimately reflected by effects on patterns of endometrial gene expression during the periattachment period of early pregnancy in adulthood is unknown. Consequently, research objectives were to: (1) determine acute effects of (a) nursing vs milk replacer feeding, and (b) method of feeding a single dose of colostrum at birth, with or without supplemental IGF1, on porcine uterine development at 12 h postnatally; (2) determine short-term effects of age and nursing on porcine uterine (a) miRNA expression between birth and PND 2 and (b) miRNA-mRNA interactions using integrated target prediction analysis; and (3) determine long-term effects of lactocrine-deficiency from birth on adult endometrial (a) mRNA and miRNA expression during the periattachment period of early pregnancy (pregnancy day 13), including identification of affected miRNA–mRNA interactions. Results showed nursing for 12 h from birth supports rapid establishment of a uterine developmental program, and that a single feeding of colostrum at birth increased endometrial cell proliferation at 12 h, regardless of method of feeding. Further, oral IGF1 was sufficient to support endometrial cell proliferation at 12 h in replacer-fed gilts, and supplementation of colostrum with IGF1 further increased endometrial cell proliferation. Between birth and PND 2, novel age- and lactocrine-sensitive uterine miRNAs and miRNA-mRNA relationships associated with porcine neonatal development were identified. On pregnancy day 13, lactocrine deficiency from birth did not affect corpora lutea number, uterine horn length, uterine wet weight, embryo recovery, or uterine luminal fluid estrogen content. However, next-generation sequencing analyses revealed lactocrine-sensitive endometrial mRNAs and miRNAs associated with aspects of solute transport, endometrial receptivity, and… Advisors/Committee Members: Bagnell, Carol A (chair), Bagnell, Carol A. (chair), Roepke, Troy A (internal member), Uzumcu, Mehmet (internal member), Roepke, Troy A. (internal member), Bartol, Frank F (outside member), Bartol, Frank F. (outside member), School of Graduate Studies.

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The obesity epidemic is receiving research attention, but that attention may be targeted incorrectly. The biggest change in American diet during the 20th century was a replacement of saturated and monounsaturated fats with linoleic acid in the form of industrial seed oils. In order to determine whether fatty acid profile is important for producing obesity I fed wild-type C57BL6/J mice high-fat diets with either high concentrations of saturated fat or linoleic acid. In addition to body weight, I performed metabolic assays and collected hypothalamic tissue for measuring gene expression, targeting the mechanism of hypothalamic inflammation. These experimental diets were fed to in males, ovariectomized females with and without estrogen treatment, and to breeder dams to expose their offspring prenatally. I found that in males, linoleic acid contributed to a small but significant increase in body weight compared to saturated fat, but that all high-fat diets produced obese mice. The biggest difference between groups was insulin resistance in the linoleic acid-fed mice. Gene expression evidence of hypothalamic inflammation was unclear. In female mice, estrogen conferred protection from obesity caused by all experimental high-fat diets. Without estrogen, female mice were equally obese from saturated fat and linoleic acid. Glucose metabolism, however, was also impaired by linoleic acid, and expression of hypothalamic genes for metabolism and inflammation were highly variable. In offspring exposed to maternal high-fat diet, females were again protected but not males. Male mice weaned onto a high-fat diet gained more weight when exposed to linoleic acid through maternal feeding than saturated fat. A similar effect on glucose metabolism was seen in male and female offspring as in the first two experiments, where linoleic acid feeding impaired glucose clearance during glucose or insulin challenge. My conclusion is that, in the mouse, linoleic acid is slightly more obesogenic than saturated fat, but effects glucose metabolism much more potently. The effects on obesity, but not glycemia, are partially protected in female mice by estrogen. Due to a lack of clear hypothalamic inflammation biomarkers, these effects are likely occurring in the periphery.

Advisors/Committee Members: Roepke, Troy A (chair), Bello, Nicholas T (internal member), Campbell, Sara C (internal member), Storch, Judith (internal member), Cai, Dongsheng (outside member).

▼ Endocrine-disrupting chemicals (EDCs) are synthetic or natural compounds that can be found in the environment in various forms, such as pesticides [e.g., methoxychlor (MXC)], plasticizers [e.g., bisphenol A (BPA)], or pharmaceutical agents [e.g., diethylstilbestrol (DES), a potent synthetic estrogen that was prescribed to pregnant women between 1940s and 1970s. Previous studies indicated that the ovary is particularly vulnerable to estrogenic EDC exposures during fetal and neonatal periods. To investigate the effects of estrogenic EDCs in two critical developmental windows, we performed two experiments, using timed-pregnant Fisher CDF rats. In Experiment 1, we exposed the dams to 50 µg/kg/day (Low) BPA or 50 mg/kg/day (High) BPA, or vehicle from embryonic day (E) 18 to 21, and the pups from postnatal day (PND) 0 to 7. In Experiment 2, we exposed animals to 0.1µg/kg/day DES, 75 mg/kg/day MXC, 50 mg/kg/day BPA, or vehicle from E11 to PND7. We then examined reproductive parameters, including pubertal age, regularity of reproductive cycles, and follicular composition. Expression of critical ovarian markers, including estrogen receptor 1 (ESR1), Mullerian inhibiting substance (MIS), cytochrome P450 side chain cleavage (P450scc), luteinizing hormone receptor (LHR), and proliferating cell nuclear antigen (PCNA) were also examined. In Experiment 1, we found that there were no changes in age at puberty or estrous cyclicity in the both Low and High BPA-treated females. However, there was a decrease (p < 0.05) in primary follicles in High BPA-treated rats. In addition, there was an increase in atretic follicle numbers (p < 0.05) in the Low and High BPA-treated females suggesting that there was an effect on follicular dynamics. Moreover, a short window of ovarian exposure to BPA did not show any statistically significant effects on expression of ovarian markers except ESR1which was decreased in primary follicles in Low BPA-treated females. In Experiment 2, our results demonstrated that DES- and MXC-treated females had an accelerated onset of puberty and altered estrous cyclicity. Although there was no effect on the litter size, MXC-treated females showed a strong trend towards reduction in litter size (p = 0.07) as compared to control. MXC caused a decrease in steroid hormone levels. DES, MXC, and BPA exposures altered follicular dynamics. There was an increase in atretic follicles in DES and BPA-treated females. In addition, the number of corpora lutea (CL) was reduced (p < 0.01) in MXC-treated females while the total follicle numbers were not altered. There were alterations in ovarian molecular markers. Expression of MIS significantly increased in secondary and pre-antral follicles in MXC-treated group. Furthermore, expression of P450scc decreased in pre- and early-antral follicles in MXC-treated females while the expression increased in CL in BPA-treated females. Overall, the results show that developmental exposure to estrogenic-EDCs affects female reproduction and ovarian follicular dynamics. In addition, DES and MXC… Advisors/Committee Members: Uzumcu, Mehmet (chair), Zama, Aparna M. (internal member), Bagnell, Carol A. (internal member), Roepke, Troy A. (internal member).

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Alternate day intermittent fasting (IMF) has recently gained traction as a popular dietary strategy to produce weight loss in obese individuals. In this dissertation, we investigate the metabolic, neural, and behavioral effects of IMF to further elucidate the mechanisms driving sustained weight loss. To produce diet-induced obesity, male mice were placed on an ad libitum 45% high fat diet (HFD) for 8 weeks. Animals were subsequently placed on one of four experimental diets for 4 weeks as follows: continuation of ad libitum HFD, IMF of HFD (IMF-HFD), switched to a 10% low fat diet (LFD), or IMF of LFD (IMF-LFD). IMF-HFD and IMF-LFD animals consistently lost body weight, 13-27% and 18-32% reduction respectively, compared to HFD animals. Oral glucose tolerance AUC was lower in IMF-HFD (~50%) while insulin tolerance AUC was reduced in IMF-HFD, LFD, and IMF-LFD (~22-42%). Norepinephrine content in the anterior portion of the medial hypothalamus was higher in IMF animals compared to HFD and LFD groups while only IMF-LFD was higher in the posterior portion. Furthermore, relative Npy gene expression was higher in IMF-HFD and IMF-LFD compared to HFD and LFD mice. Neural activation of hypothalamic NPY neurons was measured following an acute glucoprivic challenge with 2-deoxy-D-glucose (2-DG). In the paraventricular nucleus, there was a significant increase in neural activation following 2-DG administration, but there were no differences between treatment groups given 2-DG. There were no differences in the arcuate nucleus nor were there differences in NPY/c-Fos double-labeling in either region. A subset of animals, which also including groups pair-fed to IMF animals (PF-HFD and PF-LFD) were used to evaluate feeding behavior during IMF and after 6 weeks of HFD re-feeding. At the end of the diet period, the first meal of the last feeding day was recorded. The first meal duration was longer in LFD and IMF-LFD mice compared to HFD. Additionally, IMF-HFD had a greater first meal size and faster rate of consumption than HFD animals. Average meal duration at the end of the diet period was longer in the low-fat diet groups compared to the HFD-groups. There were no meal pattern differences at the end of the HFD re-feeding period. In summary, these results suggest that IMF is an effective strategy for weight loss that produces specific alterations in hypothalamic signaling and that meal patterns are only transiently altered in diet-induced obese male mice.

Advisors/Committee Members: Bello, Nicholas T (chair), Roepke, Troy A (internal member), Shapses, Sue A (internal member), Varady, Krista A (outside member), School of Graduate Studies.

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Fetal alcohol exposure has many detrimental effects on the developing brain and can cause fetal alcohol spectrum disorders (FASDs). Many FASDs patients show lifelong stress response abnormalities, demonstrated by an augmented response to stress hormones such as adrenocorticotropin and corticosterone (Lee et al., 2000), which are likely driven by alterations the hypothalamic-pituitary-adrenal (HPA) axis function (Zhang et al., 2005). Using a rat animal model, we have shown that postnatal ethanol exposure reduces the number and function of stress regulatory β-endorphin producing neurons in the hypothalamus, inducing a hyper-stress response (Sarkar et al., 2007; Logan et al., 2015). Microglia are one of the innate immune cells in the CNS and can be categorized as activated or ramified. Activated microglia are associated with an increase in proinflammatory responses and phagocytosis while ramified microglia are associated with maintaining homeostasis through dynamic communication, remodeling of neuronal synapses, and surveying the environment (Bell-Temin et al., 2013). How β-endorphin neurons communicate with microglia to maintain normal homeostasis has yet to be addressed. β-endorphin can also bind to both mu- and delta-opioid receptors and may serve as a form of communication between β-endorphin neurons and microglia. Exosomes are small vesicles (30-150 nm) that play an important role in local and distant communication between cells. They carry unique cargo (proteins, mRNA, miRNA, and other non-coding RNAs) from the cells they originate from that can affect the recipient cell’s homeostasis and induce apoptosis. Additionally, complement proteins, generally known for their role to opsonize foreign pathogens and support phagocytosis of dying cells may also play a role in ethanol-induced β-endorphin neuronal cell death. Here I demonstrate that ethanol-induced apoptosis of β-endorphin neurons is caused by activation of microglia to release proinflammatory cytokines, pro-apoptotic exosomes, and C1q. Furthermore, mu-opioid receptors activation is critical to ethanol-induced activation of microglia to induce apoptosis of β-endorphin neurons and antagonism of mu-opioid receptors attenuated the ethanol effect. Delta-opioid receptors antagonism did not have an effect on ethanol-induced β-endorphin neuronal cell death.

Advisors/Committee Members: Sarkar, Dipak K (chair), Roepke, Troy A (internal member), Bello, Nicholas T (internal member), Wu, Longjun (outside member), School of Graduate Studies.

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Alcohol exposure during gestation increases breast cancer risk in offspring. While the mechanisms that underlie this effect are not fully understood, serum estradiol (E2) is increased in these animals during proestrus, suggesting a role for the estrogen axis. To test this hypothesis, it was necessary to develop a stress-free method of hormone administration that could be used in long-term studies of carcinogen exposure to ovariectomized (OVX) rats. Rats were OVX on post-natal day (PND) 40 then treated with daily peroral E2 with or without P4 by adding hormones to peanut butter. On PND 50 rats were injected with nitrosomethylurea (NMU) to induce mammary tumor development. After 26 weeks, there was no difference in tumor incidence suggesting that E2 alone at a normal physiological level can result in tumor development. These results indicate that this may be a useful method to examine the mechanisms of steroid action in mammary tumorigenesis. In addition to changes in circulating E2, local estrogen signaling may be altered in the mammary gland in response to alcohol in utero. To explore this possibility as well as obtain a global view of changes that occur in the mammary gland transcriptome, pregnant Sprague-Dawley rats were treated with alcohol or a control diet during pregnancy. Serum analysis demonstrated an increase in circulating E2 in alcohol-exposed dams during gestation suggesting that alcohol in utero may act as an endocrine disruptor during early development. Mammary glands from PND 2, 10, and 20 offspring were analyzed by RNASeq to look for changes in the estrogen axis. Initial analysis with cummeRbund using a low read depth of 25 million reads demonstrated limited differences between alcohol-exposed rats and controls at PND 2 and PND 10. An analysis of additional samples at PND 20 using qRTPCR suggested that heterogeneity of the gland may prevent differences from being observed using the approach taken here. Further analyses of changes in the transcriptome from PND 2 to 10 suggested an increase in stromal cells over time that was corroborated by changes in mammary gland morphology. Comparisons were made with triple negative breast cancer (TNBC), a disease characterized by hormone independent growth similar to this period of mammary gland development. These results suggest that this may be a useful model to study TNBC. Future studies using greater read depth or a larger sample size may uncover differences between alcohol-exposed offspring and controls that were not seen with the present analysis.

Advisors/Committee Members: Cohick, Wendie S. (chair), Roepke, Troy A. (internal member), Quadro, Loredana (internal member), Suh, Nanjoo (outside member).

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Central signaling of 17β-estradiol (E2) in the arcuate nucleus (ARC) is important in many homeostatic functions including reproduction, energy balance, and thermoregulation, among others. KNDy neurons that coexpress kisspeptin, neurokinin B, and dynorphin function in control of multiple homeostatic functions and are integrators of reproduction and energy balance. In this dissertation, I characterize ER-mediated and ER-independent signaling in the ARC and identify the impact of energy balance and E2 on KNDy neurons. First, I characterize ER-mediated E2 signaling in the ARC, where E2 binds to estrogen receptor-alpha (ERα), translocates to the nucleus, and regulates gene transcription by binding to the Estrogen Response Element (ERE) on DNA through ERE-independent signaling. However, E2 also functions through ERE-independent signaling, such as interacting with nuclear transcription factors and binding to E2- responsive G-protein coupled receptors. I use three genotypes of mice: 1) wild-type, 2) knock in/knock out (KIKO), which lack ERE-dependent signaling, and 3) ERα total knock out (ERKO) to determine differential E2 signaling in the ARC. I found that multiple genes involved in reproduction and energy balance are controlled by E2 through ERE- dependent and ERE-independent mechanisms and identify novel signaling pathways that are modulated by E2. In the last section, I examined the interactions of E2 and energy balance on KNDy-associated gene expression in the ARC. I determined that in overnutrition and undernutrition, ARC expression of KNDy neuropeptides and receptors is disrupted, leading to downstream changes in gonadotropin (LH and FSH) production. Our previous studies found that in NPY/AgRP neurons, E2 interacted with fasting and diet-induced obesity to regulate genes involved in ghrelin signaling. Overnutrition and undernutrition did not regulate these ghrelin signaling genes in Tac2 neurons. However, E2 augments the expression of growth hormone secretagogue receptor (Ghsr), ghrelin’s receptor, in Tac2 neurons by six- to eight-fold, suggesting that E2 modulates ghrelin signaling in KNDy neurons. In summary, our results suggest differential E2 signaling mechanisms in the ARC are important to maintain energy homeostasis and reproduction and that expression of KNDy genes are regulated by both positive and negative energy balance, leading to downstream changes in reproduction.

Advisors/Committee Members: Roepke, Troy A (chair), Bagnell, Carol (internal member), John-Alder, Henry (internal member), Pang, Zhiping (outside member).

▼ The reproductive steroid hormone, 17β-estradiol (E2), controls feeding and exploratory behaviors associated with mood disorders. The loss of circulating E2 puts menopausal women at an increased risk for developing obesity and mood disorders when compared to premenopausal women. Therefore, it is critically important to understand the role of sex steroids and their receptors in the neuroendocrine control of feeding and mood. The goal of this project is to understand the role of estrogen response Element (ERE)-dependent and ERE-independent ERα signaling on behavior by characterizing feeding patters and exploratory behaviors in male and female mice lacking either total ERα signaling or lacking ERE-dependent ERα signaling. We hypothesize that ERE-independent ERα is partially sufficient to restore feeding and exploratory behaviors that are lost in total ERα knockout mice. We tested three strains of mice: two ERα transgenic models, a total ERα knock out (ERKO) and a novel ERα knock in/knock out (KIKO) that lacks a functional DNA-binding domain) and their wild type (WT) C57 littermates using a real-time feeding behavior monitoring system and series of standard behavior tests (open field tests, elevated plus maze, forced swim test). To test our hypothesis FI and meal patterns were observed while the animals were given ad libitum access to a LFD (Experiment 1a) followed by HFD (Experiment 1b). A separate set of animals the response to fasting was monitored for 24 h after caloric restriction (Experiment 1c). Exploratory, depressive, and locomotor behavior testing was conducted on mice from Experiments 1a/b (open field, elevated plus maze, forced swim test). Each experiment was initially done with intact animals and then again repeated in ovariectomized (OVX) animals split into either an oil treated control group or an E2-treated group. We observed ERE-dependent mechanisms are the main modulator of homeostatic LFD feeding meal patterns while ERE- independent ERα signaling was involved in the control of palatable, high-fat diet food intake. During refeeding, ERE-independent mechanisms contribute to a decreased first meal food intake and slower rate of ingestion. When observed during a series of behavior tests, WT animals explored more, regardless of treatment (differences could be attributed to higher levels of locomotor activity in WT). However, similarities between WT and KIKO females in the EPM indicate that ERE-independent pathways may contribute towards reducing anxiety measures, independent of locomotor activity. WT females were shown to have a decreased free float time, indicating ERE dependent signaling may be influencing despair like tendencies. Collectively, these suggest that both ERE-dependent and -independent ERαsignaling are involved with both feeding and anxiety like homeostatic parameters. Advisors/Committee Members: Roepke, Troy A (chair), Bello, Nicholas T (internal member), Bagnell, Carol A (internal member), Samuels, Benjamin A (outside member), School of Graduate Studies.

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Organophosphate flame retardants (OPFRs) are a class of chemicals applied to clothing, electronics, plastics, furniture, and building materials to reduce the flammability of commercial products. Over the past 15 years, global use of OPFRs has increased significantly, as they replace other persistent bioaccumulative compounds such as polybrominated diphenyl ethers. OPFRs are chemicals of emerging toxicological and environmental concern due to reports of endocrine disruption, neurotoxicity, and reproductive and developmental toxicity in animals; as well as their environmental persistence. Although research on the adverse effects of OPFR exposure has increased over the past decade, there are still several gaps in our understanding of their behavior in vivo and their potential toxicity, especially in mammals. The purpose of this dissertation was to investigate whether OPFRs interact with molecular targets (acetylcholinesterase, AChE), metabolic enzymes (carboxylesterase, CES), and transporters (multi-drug resistance protein 1, MDR1) that influence their overall disposition and potential for neurotoxicity. The findings in this dissertation demonstrate that OPFRs do not behave like the structurally similar organophosphate pesticides in terms of inhibiting enzyme activity and that OPFRs are more effective inhibitors of CES than AChE. Although, the OPFRs did not demonstrate adverse effects on brain and liver enzymatic activity in vitro, the brain and liver, as well as the kidneys, placenta, and fetus, were identified as targets of toxicity due to their preferential accumulation of OPFRs. Finally, MDR1 was shown to potentially influence the in vivo disposition of TPP in the brain suggesting that MDR1 may protect the brain from TPP accumulation. The results of this study will help guide future research into OPFR-induced organ-specific adverse effects, especially in the brain and in the developing fetus.

Advisors/Committee Members: Buckley, Brian T (chair), Aleksunes, Lauren M (internal member), Richardson, Jason R (internal member), Roepke, Troy A (internal member), Lee, Jeehiun (outside member), School of Graduate Studies.