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The mammalian uterus plays a central role in the reproductive process. During pregnancy the organ undergoes a process of remodelling that is not restricted to a change in volume; its behaviour transitions from relative quiescence to regular contraction. This progression is incompletely understood; in particular, questions surround the final stages when the organ transitions from a non-labouring state to a labouring state and complications such as pre-term birth can occur. Pre-term birth is associated with an increased chance of morbidity and mortality for the child and is the leading cause of death in children under 5 worldwide. Up to 13% of births in the developed world are pre-term and the annual cost to society of managing this problem is considerable; in 2006 the financial cost was estimated to be over £2.9 billion in England and Wales alone. In this project a physiologically detailed, multi-dimensional, computational model of the rat uterus, which scales from the single-cell up to a full 3D organ, was developed and used to investigate uterine function. The single-cell model incorporates several changes from the previously published Tong et al 2011 model. The present model includes alternative descriptions of the intracellular ion handling and mechanical systems of the uterine cell, in addition to a reformulated L-Type calcium current, voltage-activated potassium current, calcium-activated potassium current and calcium-activated chloride current. The hypothesis that sodium-channel density can act as a trigger mechanism for initiating labour in late pregnancy was explored. The present model is able to accurately predict and reproduce calcium transients based on experimental patch clamp recordings, as well as the effects of drugs and hormones, including oestradiol, oxytocin, wortmannin and nifedipine. The model was used to investigate the adverse effects of using nifedipine as a tocolytic agent on the cardiovascular system and suggest countermeasures that could permit its use in patients with pre-existing heart conditions: using agonists that target the sodium-potassium pump and SERCA to partially restore vascular function, without impeding nifedipine's tocolytic efficacy. A tissue model was then constructed and used to investigate the synchronisation of electrical activity in myometrial tissue. The primary hypothesis was that heterogeneities within the tissue could evoke the spontaneous and sustained activity required for synchronicity. It was shown that spatial heterogeneity in electrical coupling played an important role in sustaining electrical activity. Spontaneous activity was evoked by using a tissue sample containing cells with heterogeneous excitability, resulting from a stochastic element in the activation potential of their sodium-channels. It was shown that at least 3% - 8% of cells must be pacemaking in order to produce stable electrical waves that propagate successfully in tissue. This work represents an advance in comprehensive uterine cell modelling and extends considerations beyond purely deterministic models, towards models that include stochastic elements, which can uncover complex, non-linear behaviours of uterine electrophysiological systems.

Maternal age is a significant risk factor for adverse pregnancy outcomes, and is strongly associated with an increased risk of aneuploidy of the conceptus, as well as a significantly higher frequency of serious pregnancy complications known as the "Great Obstetrical Syndromes", including miscarriage, pre-eclampsia and fetal growth restriction. In the last 40 years average maternal age has increased considerably in many wealthy countries, and in the UK the number of babies born to women aged 35 and over is set to surpass those born to women under 25. The high incidence of aneuploidy in older mothers can be attributed to abnormalities in the oocyte and embryo, however the "Great Obstetrical Syndromes" do not appear to be related to the oocyte and may instead be linked to abnormal development of the placenta. In this thesis, I show that advanced maternal age in the mouse is associated with a drastically increased variability of developmental progression in utero, including developmental delays and growth restriction, severe embryonic abnormalities and higher resorption rates. I find that these embryonic defects are always accompanied by gross morphological and transcriptomic abnormalities in the placenta. Notably, I show that the increased risk of these complications can be rescued by transfer of embryos from aged females to a young surrogate mother, thus implicating the aged maternal uterus as the basis for embryonic and placental defects. Transcriptomic analysis of the decidua compartment in placentas from aged pregnancies revealed abnormal expression of genes involved in the decidualization process, which occurs during early pregnancy and facilitates implantation and development of the conceptus. I show that these defects are already obvious in the peri-implantation window, with endometrial stromal cells from aged females being unable to mount an adequate decidualization response due to a decline in their ability to respond to pregnancy hormones. This blunted decidualization reaction in turn may lead to abnormal development of the placenta. These age-associated decidualization defects are cell-intrinsic and can be recapitulated in vitro. The detected insufficient activation levels and abnormal intracellular distribution of phospho-STAT3, combined with highly variable progesterone receptor expression, may be possible causes of these defects. In addition, I examined the possible effects of ageing on the epigenome as a potential contributor to the decline in endometrial function. My results indicate that ageing of the uterus displays some of the common epigenetic hallmarks of tissue ageing. However, more importantly, decidual cells of aged females exhibit abnormal distributions of the histone modification H3K4me3, and are refractory to the profound DNA methylation remodeling that I find takes place during pregnancy. These age-related changes in the epigenome may underpin, or contribute to, the observed decline in uterine function during pregnancy. Understanding the mechanism underlying these epigenomic and functional changes in the ageing reproductive tract may pave the way for new therapeutic strategies to improve maternal and fetal outcomes of pregnancy in older mothers.

Our limited understanding of the molecular mechanisms driving the onset of normal human parturition makes it difficult to identify ‘what goes wrong’ in conditions such as preterm labour (PTL), preterm prelabour rupture of membranes (PPROM) and postpartum haemorrhage (PPH). This incomplete understanding seriously hampers the development of effective ways to predict, prevent and treat parturition complications, which are a cause of significant neonatal and maternal morbidity. Two principal barriers to improving our understanding are 1) the great complexity of both the molecular interactions initiating parturition and the aetiology of parturition complications, and 2) the difficulty in generating relevant high quality molecular and epidemiological data. To help make sense of this complexity, data should be analysed comprehensively to maximise the amount of useful information gleaned from it. This thesis aimed to explore the use of specialist methods to analyse novel and previously published data to study the molecular mechanisms initiating human parturition and the epidemiology of parturition complications. The molecular mechanisms initiating parturition were explored through a gene expression microarray of labouring and non-labouring myometrial tissue. This is the largest microarray of its kind to date. Functional analysis and a network graph approach were used to reveal genes and molecular pathways associated with labour. The first ever meta-analysis of similar myometrial microarray datasets was also conducted to assess the reliability and generalisability of the results. This work supported the hypothesis that labour is associated with inflammatory events in the myometrium. A computer model of an inflammatory signalling pathway associated with infection-induced PTL was then built to provide proof of concept that such models can be used to study parturition. The model was based on published data and described lipopolysaccharide-induced activation of the transcription factor Nuclear Factor kappa B (NF-κB). This is the first attempt to generate a dynamic kinetic model that has relevance to the molecular mechanisms of PTL, and the first model of this pathway to explicitly include molecular interactions upstream of NF-κB activation. The epidemiology of complications at parturition was explored using three methods. Firstly, a novel approach was developed to use network graphs to visualise and analyse a dataset of nearly 50,000 birth records. The approach provided a quick and effective way to preliminarily explore relationships between exposures and pregnancy outcomes in an unbiased data-driven manner. Secondly, a record-linkage study of two datasets of birth records was conducted to determine risk factors for PPH, including intergenerational transmission of risk. This confirmed several known risk factors of PPH and showed that women whose mothers or grandmothers had PPH do not appear to be at increased risk themselves. Finally, a systematic review and meta-analysis of three randomised controlled trials investigated the effectiveness of fetal assessment methods in improving maternal and neonatal outcomes following PPROM. The review concluded that there is currently insufficient evidence on the benefits and harms of any method of fetal assessment, and further randomised controlled trials are required.

Bacterial contamination of the female genital tract after parturition is common and subsequent infections are associated with infertility. Infections of the uterus or mammary gland in cattle perturb ovarian function, with decreased dominant follicle growth rate and delayed ovulation. Granulosa cells that line ovarian follicles express the molecular machinery necessary to respond to common bacterial pathogen-associated molecular patterns (PAMPs), suggesting that granulosa cells may play a role in ovarian innate immunity. In the present thesis, the effect of bacterial PAMPs on ovarian follicle cells was examined during follicular growth, from emergence through dominance, and the early stages of embryogenesis. Granulosa cells from emerged or dominant bovine follicles increased production of inflammatory mediators, such as IL-6 and IL-8, through Toll-like receptor (TLR)-dependent pathways in response to the PAMPs lipopolysaccharide and the synthetic triacylated lipopeptide Pam3CSK4. The requirement for TLR2 and TLR4 was confirmed by using siRNA, showing activation of MAPK intracellular pathways and inhibiting key signals (p38, MEK or NFkB) in TLR pathways. The endocrine function of granulosa cells was perturbed by PAMPs, with decreased oestradiol and progesterone output. In addition, the endocrine environment affected granulosa cellular responses, with high EGF increasing the cellular response to PAMPs. Bacterial PAMPs also perturbed the oocyte, with increased cumulus expansion and parthenote cleavage rate, as well as changes in key genes involved in oocyte maturation. Finally, a human granulosa cell line was used to examine if PAMPs perturb human as well as bovine ovarian health. Indeed, human cells also expressed TLRs and mounted a cellular response to PAMPs at the mRNA level. This thesis provides a molecular mechanism for the perturbation of ovarian function by an infection at a site distal to the ovary.

In the human uterus prostaglandins (PG) PGE2, PGD2, PGI2, PGF2α and Thromboxane A2 (TXA2), also termed prostanoids, are synthesised and deactivated to 15-keto PGE2, J2 metabolites, 6-keto-PGF1α, 15-keto PGF2α and TXB2 respectively. However, not all metabolites have been analysed simultaneously within the same tissue. The primary objective of this thesis was to determine full uterine prostanoid profiles in human non-pregnancy, pregnancy and parturition, to better understand these processes and find suitable tocolytic targets. In addition, ten cytokines in human cervico-vaginal fluid (CVF) were measured according to interval to labour to test their suitability as labour onset predictors, with a view to developing a test to determine women at risk of preterm labour. Prostanoid analysis was carried out in endometrium (n=9) and myometrium (n=15- 16) donated by non-pregnant women and lower segment myometrium obtained from pregnant women (before (n=14) and after labour onset (n=7)) by liquid chromatography coupled with electrospray ionisation mass spectrometry (LC/ESIMS/ MS). Cytokines produced by CVF collected from pregnant donors (20-41 weeks gestation, n=2-10) were investigated using Enzyme-Linked Immunosorbent Assay (ELISA) or Luminex®. Human endometrium produced greater concentrations of TXB2, PGE2 and PGF2α than myometrium in vitro (p<0.05). Fifteen prostanoids were detected in human myometrium. Production of 6-keto-PGF1α, PGE1 and PGF1α increased whilst 15- keto PGE2 and PGJ2 decreased at term pregnancy (37-41 weeks gestation) versus non-pregnancy (p<0.05). Myometrium from parturient donors synthesised TXB2 and PGE2 more abundantly than the non-labouring equivalent. Cytokine concentration was greatest in CVF sampled the week before labour, in particular Interleukin-6 (IL-6), Macrophage Inflammatory Protein-1α (MIP-1α) and Monocyte Chemotactic Protein-1 (MCP-1) (p<0.05). Endometrial TXB2, PGE2 and PGF2α could aid in proliferation of glandular epithelium prior to ovulation. Prostacyclin may facilitate prolongation of pregnancy to term and thromboxane could contribute to uterine stimulation during labour. Cervical dilation may be influenced by PGE2 in lower segment myometrium. MCP- 1, MIP-1α and IL-6 could mark a short interval to labour onset.

In mammals, development of a new organism requires fertilisation of the female egg by sperm. The resulting zygote develops into the blastocyst stage as it travels towards the uterus. Within the uterus, the blastocyst invades the maternal tissues and establishes access to the maternal blood supply. This process is called implantation and is absolutely essential for the further development of the conceptus and establishment of pregnancy. Successful implantation requires a proper preparation of the uterus and the embryo as well as a molecular dialogue between the embryo and the uterine tissues. Female mice that have a disruption in the Plcβ1 gene are infertile. In the course of this Thesis it became apparent that the main cause of their infertility is their inability to implant their embryos. PLCβ1 protein is a mediator of G-protein coupled receptor (GPCR) signalling and it is involved in the production of second messengers essential for downstream transmission of signals. A host of reproductive functions are under the control of GPCR signalling. In this PhD Thesis the infertile phenotype of Plcβ1 knockout (KO) female mice was investigated to identify the reproductive processes affected by the lack of a functional PLCβ1 protein. A combination of histological, molecular biology and in vivo techniques were utilised to show that at the time of implantation, embryos fail to attach to the uterine epithelium of KO uteri. In addition, it was demonstrated that estrogen signalling and components of the endocannabinoid metabolism, both key processes for successful implantation are severely altered in KO uteri. These observations show that KO uteri fail to prepare for implantation. In addition, the KO reproductive tract exerts a detrimental effect on pre- and peri- implantation embryo development. Currently, failure of implantation is thought to be one of the major causes of infertility in women and up to this date there are no successful treatments. The results of this project expand our current knowledge on the physiology of implantation and provide cues for the development of diagnostic markers and treatments for the women who are unable to conceive.

While premature birth is still one of the major clinical problems worldwide, the exact physiological mechanisms underlying myometrium activity during pregnancy remain unclear. In this thesis, a novel biophysically detailed model was constructed using available experimental data to simulate chemical, electrical and mechanical activity in a late pregnant rat uterine myocycte. The developed model has been used to elucidate the ionic mechanism underlying myometrium functionality, providing better insights in the function of the uterus during pregnancy. The model consisted of 15 membrane currents, intracellular calcium handling process coupled with a sliding actin-myosin filament mechanical model to describe uterine behaviour and contractile activity at the single myocyte level. Each of the ionic currents were modelled using Hodgkin-Huxley-type equations. The simulated current traces and current-voltage curves were validated with experimental recordings and the model was further validated by the ability to produce a bursting action potential (AP) during an external stimulus. The model replicated the effects of estradiol during pregnancy, modulating the amplitude and activation properties of individual Ca2+ and K+ currents, therefore altering the AP configuration to a tonic-like plateau. The model also reproduced the actions of drugs to inhibit certain channels to investigate their roles in myometrium. Sensitivity analysis was performed to examine the model's behaviour to changing parameters. A simple 1-D study was conducted to investigate how electrical signals propagate along strand of cells. Although the model successfully replicated results similar to recordings seen in the experiments, limitations have to be addressed and more studies have to be carried out to further improve the model.

Although prostaglandins (PG) and oxytocin are crucial mediators of uterine contractility, their receptor-mediated effects during the menstrual cycle, pregnancy and labour are not fully understood. The aim of this thesis was to elucidate the functional expression of EP, FP, TP and oxytocin receptors in isolated human myometrium relative to myocyte mRNA and signal transduction pathways. Myometrial samples were obtained from consenting non-pregnant and pregnant donors. Functional techniques were used to determine isometric muscle contractions. Primary uterine myocytes and fibroblasts were cultured at term to identify stimulated changes in calcium (Ca2+), cyclic adenosine monophosphate (cAMP) and mRNA. Myometrial strips exhibited spontaneous contractions, which were most active midcycle under oestrogenic conditions. At this time intrinsic contractility and responsiveness to uterotonins decreased towards the fundus. PGE2 produced bellshaped responses with predominant utero-relaxant effects mediated via the EP2 subtype. Although activity was partially restored by PGE2 through EP3/1 receptors, tissue excitation was more pronounced at FP, TP and oxytocin receptors. Despite high FP mRNA expression, the lower segment uterus was particularly responsive to U46619 and oxytocin at term pregnancy. Even so, Ca2+ mobilisation by oxytocin was greater via principal release from intracellular stores. Incubations with atosiban, progesterone and a rho-kinase inhibitor reduced oxytocin-stimulated Ca2+ transients. EP2 also attenuated oxytocic effects but this appeared to be mediated through cAMP rather than Ca2+ signalling pathways. With advancing labour, intrinsic myogenic activity declined in parallel with oxytocin desensitisation. However, TP-induced contractions were continued in the lower parturient uterus. These findings demonstrate that PG and oxytocin receptor expression are regulated in a hormone-dependent temporal and spatial manner. EP2-mediated cAMP formation appears to promote uterine quiescence, whilst TP receptors may control muscle tonus during parturition. These receptors and their messenger systems represent effective tocolytic targets for uterine hypercontractile disorders, such as dysmenorrhoea and preterm labour.

Timing mechanisms in biology remain poorly understood. As one prime example, little is known about the mechanisms that specify how long the gestating uterus will remain quiescent before entering labor. Our lack of insight into this fundamental question, which applies to all mammalian species, also limits investigation into potential causes of preterm labor, a major human pregnancy complication. My dissertation work provides evidence that gestation length in mice is determined by an epigenetic timer that runs autonomously within the fibroblasts of the pregnant uterus. The timer is set during the peri-implantation period when select loci establish appropriate levels of the repressive histone mark H3K27me3. These loci then progressively lose H3K27me3, thereby scheduling the uterine cell state transitions and associated gene expression changes of late gestation that are the proximal mediators of luteolysis (progesterone withdrawal) and labor onset. Initial overwinding of the timer via genetic ablation of the histone demethylase KDM6B delays these transitions and extends gestation length. My findings also demonstrate requirements for KDM6 demethylases in inflammation-induced preterm labor, and suggest potential requirements for KDM6B in the uterine-intrinsic pathways of parturition that are distinct from luteolysis. These results unexpectedly implicate epigenetic pathways in fibroblasts as a top-level determinant of both normal and pathological parturition mechanisms. We anticipate that further dissection of the ways such fibroblast programming controls gestation length may suggest novel approaches for improving human pregnancy outcomes.

Background: Uterine myomas may resemble uterine sarcomas in some cases. However, the rate of benign myomas appearing as sarcomas at an ultrasound examination is not known. The objective of this study is to determine the percentage of benign myomas that appear suspicious for uterine sarcoma on ultrasound examination. This is a prospective observational multicenter study (June 2019-December 2021) comprising a consecutive series of patients with histologically proven uterine myoma after hysterectomy or myomectomy who underwent transvaginal and/or transabdominal ultrasound prior to surgery. All ultrasound examinations were performed by expert examiners. MUSA criteria were used to describe the lesions (1). Suspicion of sarcoma was established when three or more sonographic features, described by Ludovisi et al. as frequently seen in uterine sarcoma, were present (2). These features are no visible myometrium, irregular cystic areas, non-uniform echogenicity, irregular contour, cooked appearance, and a Doppler color score of 3-4. In addition, the examiners had to classify the lesion as suspicious based on her/his impression, independent of the number of features present. Eight hundred and ten women were included. The median maximum diameter of the myomas was 58.7 mm (range: 10.0-263.0 mm). Three hundred and forty-nine (43.1%) of the patients had more than one myoma. Using the criterion of >3 suspicious features, 40 (4.9%) of the myomas had suspicious appearance. By subjective impression, the examiners considered 40 (4.9%) cases suspicious. The cases were not exactly the same. We conclude that approximately 5% of benign uterine myomas may exhibit sonographic suspicion of sarcoma. Although it is a small percentage, it is not negligible.

Uterine and ruminal bacterial communities are major contributors to reproductive success and feed efficiency by their impact on their respective environments. The first objective is to evaluate the effect of protein supplementation on uterine and ruminal bacterial communities during heifer development. Pre-pubertal Angus heifers were assigned to a level of crude protein supplementation: 10% control, 20%, or 40%. Every 56 days, uterine flushes and rumen content were collected for bacterial DNA extraction and amplification of the 16S gene. Bacterial analyses were performed in R 4.1 and statistical analyses in SAS 9.4. Observed amplicon sequence variants increased overtime and by pubertal status (P < 0.05), each indicating significant clustering by principal coordinate analyses (P < 0.001) in the uterus. Numerous bacterial taxa abundances differed by protein treatment, pubertal status, and day in the uterus and rumen (P < 0.05). The second objective was to evaluate the impact of lipopolysaccharide (LPS) on the bovine uterine endometrium during the follicular and luteal phases. Explants were collected from the endometrium of the contralateral and ipsilateral horns of uteri in mid-luteal or follicular phases. Explants were cultured at standard conditions for 8 hours in 1 mL complete media with or without LPS (1 µg/mL). Extraction of RNA from explants was reverse transcribed to cDNA for targeted RNA sequencing. Normalized total reads counts for gene expression were analyzed in Metaboanalyst 5.0 and SAS 9.4. Seven genes increased in LPS treatment compared to other groups (P < 0.05). Multiple genes were affected by estrous cycle phase and uterine horn, independent of treatment (P < 0.05). Clustering analysis indicated overall gene expression differences with similar clustering between luteal and follicular phase explants of controls, but distinct separate clustering between phases with LPS treatment (P = 0.001). Mucins contributed the most to differences observed between phases by treatment. Therefore, estrous cycle phase resulted in differing overall endometrial gene expression profiles of immune response to LPS treatment. In conclusion, uterine bacterial communities develop through puberty and may be affected by nutrition. Differing immune responses of the endometrium to bacteria between estrous cycle phases may influence uterine environment and pregnancy success.

The bovine uterine microenvironment is a niche biological site in which the early embryo must complete several key developmental events before progressing on towards implantation and a successful pregnancy. From fertilization (Day 0) to hatching from the zona pellucida (~Day 10), embryo development is semi-autonomous. After hatching, the blastocyst stage embryo becomes completely dependent on endometrial secretions for survival and development, as evidenced in vivo by embryo death when the secretory uterine glands are ablated and by the inability to recapitulate embryo elongation in vitro. From hatching to implantation (~Day 30), the bovine embryo undergoes remarkable growth from an ovoid blastocyst 350 _m to a >20 cm filamentous embryo in the span of just ~25 days, driven by the rapid proliferation of mononuclear undifferentiated trophoblast cells. Amongst mammals, this extended period of preimplantation embryo elongation and delayed implantation until almost 1 month of pregnancy is unique and highlights the generative role of endometrial secretions for early ruminant embryo development. Notably, the timeframe for elongation is concurrent with high rates of early embryonic mortality (EEM) amongst high producing dairy cattle breeds such as the Holstein. Still, despite its biological interest to reproductive physiologists and economic importance to the dairy industry, little is known about the early life-sustaining uterine secretome, how the secretions change in profile and abundance across the estrous cycle, or their effects on embryos. My studies span the timeframe in which preimplantation embryo development takes place and address related questions about pregnancy. First, while my study on methods to enrich the low abundant bovine serum proteome was the most successful published to date, it demonstrates the need for antibody based depletion methods for future bovine serum pregnancy biomarker discovery. Second, my uterine research offers a new perspective on the uterine microenvironment by characterizing the cyclical endometrial secretions that compose uterine milk. By analyzing transcriptomics of endometrial biopsies through a secretory bioinformatics pipeline, I have profiled the stage-specific endometrial secretions that define the luminal fluid from sperm transit to embryo elongation. In parallel, I have complemented predicted endometrial protein secretions by characterizing the uterine fluid proteome at the same timepoints. Together, my endometrial transcriptomics and proteomics datasets are complementary to build a framework for understanding uterine derived factors influencing in utero embryo development. In addition to secretions, I have also considered the role of uterine luminal oxygen tension on embryonic development and measured the dissolved oxygen (pO2) in the uterine lumen across the estrous cycle and in bred heifers. From this, I learned that the uterine lumen is quasi-anoxic, and that the embryo develops in the presence of virtually no oxygen, a condition maintained by the uterine spiral artery vasculature. Finally, my research on reproductive physiology has extended to neonatal calf health by examining the postpartum kinetics of colostrum IgG secretion by the Holstein mammary gland demonstrates that 75% of all IgG is secreted after the first milking (within 2 hours postpartum), which in the context of the co-evolution of cow-calf physiology would suggest a second and third colostrum feeding at four and sixteen hours post-partum could impart immunological and developmental benefits to the neonatal calf. In sum, my systems biology research on bovine reproductive physiology improves our understanding of the temporal endometrial secretions that direct preimplantation embryo development in utero, and precisely defines the kinetics of postpartum IgG secretion and provides opportunities for sustained colostrum feeding for optimized neonatal calf health.

Pregnancy loss remains a major source of economic cost to the equine industry. Frequently, the exact causes of pregnancy loss remain unknown. It has been shown, in other species, that increased dietary protein leading to elevated blood urea nitrogen concentrations (BUN) can be a factor in decreased survival of the early embryo. Our studies provided novel information regarding the effects of elevated BUN on endometrium and embryos from mares as well as insights on changes in their gene expression. Our first objective was to develop an experimental model to elevate BUN during diestrus using intravenous urea infusion. We analyzed the effects of an acute elevation in BUN on uterine and vaginal pH along with changes in the endometrial transcriptome of mares with RNA sequencing. There was a significant increase in BUN and a decrease in uterine pH in the urea group compared to the control group. A total of 193 genes were differentially expressed (DEG) between the urea and control groups. The DEG were predicted to be related to cell pH, ion homeostasis, changes in epithelial tissue, fatty acid metabolism, and solute carriers. Our second objective was to evaluate the effects of elevated BUN in the endometrium of mares using a chronic oral urea administration to elevate BUN in mares. Uterine and vaginal pH were evaluated and RNA sequencing of the endometrium was again performed. There was an increase in BUN in the urea-fed mares, but no significant change in uterine or vaginal pH between the groups. A total of 60 DEG were characterized, with prediction of transcriptomic changes in the endometrium of mares related to cell death (necrosis) and cellular movement (invasion of cells). Our third objective was to determine the effects of a high BUN on the transcriptome of day-14 embryos. There was a positive correlation between plasma BUN and blastocoele fluid urea nitrogen concentration. Changes in embryo transcriptome were related to survival of organism, angiogenesis, adhesion, and quantity of cells. Our final objective was to evaluate the correlation between BUN and follicular fluid urea nitrogen and evaluate the survival of embryos collected from donor mares with high BUN concentrations. Urea nitrogen concentration was positively correlated between the plasma and follicular fluid of mares. Additionally, there was a higher pregnancy rate when embryos were collected from mares with lower BUN. Overall, these results further elucidate the mechanisms through which urea affects endometrial and embryonic transcriptome of mares with high BUN, serving to identify effects of a high BUN in the reproductive tract of mares that might lead to decreased fertility.

Reproduction is a critical component of an animal’s fitness, but it is also energetically intensive. In order to maximize reproductive fitness, organisms modulate their reproductive investment or timing in response to environmental cues that provide information about resource availability or other indicators of reproductive success. Physiologic systems that monitor these cues then influence both reproductive readiness and more scaled measures of investment, including number of offspring or offspring provisioning effort. This kind of physiologic modulation is especially salient to mammals, for whom reproduction (gestation and lactation) is exceptionally intensive, and there is extensive documentation of variation in reproductive effort and success under specific environmental and physiological challenges. Still, the underlying mechanisms that coordinate these shifts in investment remain poorly defined and understood. This dissertation identifies novel endocrine and paracrine mechanisms that regulate ovarian and uterine function in the context of reproductive success. Chapter 1 summarizes our current understanding of female reproductive function and modulation, ultimately identifying 4 key areas of modulation for which the molecular mechanisms are poorly understood or defined: (1) evaluating autonomic nervous system regulation of reproductive organs in ecophysiological contexts, (2) identifying new signals regulating ovary and uterine function; (3) determining the extent to which reproductive stage (e.g., pregnancy) alters regulatory networks and sensitivity; and (4) scaling function of specific cell types or organs to organism-level reproductive outcomes. Chapters 2-5 address areas 2-4 by identifying new mechanisms of mammalian reproductive system control in the ovary and uterus and by evaluating the function of established signaling networks in other reproductive states. Chapter 2 interrogates the direct sensitivity of the ovary to physiological cues using mouse ovarian explants. By using pharmacologic inhibition of glucose metabolism, these experiments identify novel glucose sensitivity in the ovary. Chapter 3 examines the effect of neuropeptide gonadotropin inhibitory hormone (GnIH) on ovarian follicle growth, survival, and steroidogenesis in the domestic cat. As has been shown in other mammals to-date, GnIH promotes follicle degradation in vitro. These findings support a conserved role for GnIH in vertebrate ovarian folliculogenesis. Together, these chapters provide new paracrine and endocrine mechanisms of ovarian regulation that adds to our molecular understanding of how reproductive effort and investment are modulated.Chapter 4 explores the sensitivity of feline uterine endometrial epithelial cells to sex steroids. We demonstrate that sex steroids stimulate morphological changes to endometrial epithelial cell 3D growth in vitro, and that morphological changes are associated with functional changes in gene expression. Though often manipulated as part of normal fertility treatment, we do not have a good understanding of how these hormones facilitate uterine preparedness for implantation and pregnancy in felines. Chapter 4 thus identifies specific mechanisms of reproductive modulation in the feline uterus and also beings to flesh out molecular mechanisms underlying reproductive failure at the organism level. Our results have direct application to endangered felid breeding programs, which utilize in vitro follicle maturation and in vivo hormones treatments to promote breeding success of genetically-important individuals. Finally, chapter 5 uses a classic stress physiology paradigm to identify the mechanism(s) by which psychological stress inhibits ovarian steroid synthesis during pregnancy in mice. These experiments demonstrate that stress-dependent inhibition of ovarian function does not occur through classical, hypothalamic inhibition, emphasizing the dynamic shifts in endocrine function across mammalian pregnancy. These studies add to our comparative understanding of reproductive function, which is critical because of its fundamental connection to organismal fitness. Taken together, these studies also highlight the utility of in vitro approaches for experimentally approaching the challenge of connecting molecular variation to organismal function. We summarize the results and their ultimate importance to female reproductive physiology research in Chapter 6.

Preovulatory estradiol has been reported to play a crucial role in pregnancy establishment and maintenance, but the mechanism by which estradiol exerts its effects has not been well characterized. Specifically, the interactions between the maternal uterine environment and the developing conceptus can greatly impact pregnancy success or loss. The objective of this dissertation is to determine the effects of preovulatory estradiol exposure on uterine and trophectoderm transcriptomes, and uterine luminal fluid (ULF) protein composition. Beef cows/heifers were synchronized, artificially inseminated (d 0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed to collect d16 conceptuses either nonsurgically or following slaughter, and endometrial biopsies (n=29) were collected from the ipsilateral uterine horn. Real-Time PCR (RT-PCR) was performed on trophectoderm (TE; n=21) RNA to measure the relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Total cellular RNA was extracted from endometrium for RNA sequencing. ULF pools (n=28) for the following groups: highE2/noconceptus, highE2/conceptus, lowE2/noconceptus, and lowE2/conceptus were analyzed using a 2D LC-MS/MS based 8plex iTRAQ method. RT-PCR data were analyzed using the MIXED procedure in SAS. Transcript abundances in the endometrium were quantified using kallisto, differentially expressed genes (DEGs) were determined using DESeq2 (FDR2), and IPA was used for pathway analysis. Scaffold Q+ was used to quantitate peptide and protein identifications in the ULF. There were no differences in mRNA abundances in TE, but there were 432 DEGs among the highE2/conceptus versus lowE2/conceptus groups, 253 were downregulated (CR2, CDH4, TROAP, COL1A2) and 179 were upregulated (PRSS8, FABP3, IDO1, MUC13, CXCL10) in the highE2/conceptus group. There were 48 differentially expressed proteins (DEPs) among the highE2/conceptus and lowE2/conceptus groups (19 upregulated, 29 downregulated in the highE2/conceptus group), 6 of these were differentially expressed (FDR

Infertility in beef and dairy cattle costs millions of dollars each year to the animal agriculture industry. The microbiome of the reproductive tract has been indicated to have an effect on pregnancy establishment and maintenance in humans, however the bovine reproductive microbiome and its effect on fertility is not well understood. The objective of the current study is to evaluate bacterial communities of the uterus and vagina of postpartum beef cows undergoing estrous synchronization to determine differences in cows who will become pregnant and those who fail to conceive to fixed time artificial insemination (FTAI). Thirty Angus cows at an average of 82 days postpartum were subjected to a 7 Day Co-Synch protocol with a pre-synchronization step 21 days prior to FTAI (d -21). Uterine and vaginal flushes were collected at each day of the protocol for pH detection and bacterial DNA extraction and sequencing targeting the V1-V3 hypervariable region of the 16S rRNA gene. Pregnancy diagnosis occurred on d 30 by transrectal ultrasound where ten non-pregnant and ten pregnant cows were selected for sequencing. Results indicated a significant decrease between d -9 and d -2 in the number of bacterial species in the uterus of pregnant and non-pregnant cows (P < 0.0001). Many significant differences in relative abundance of bacteria phyla and genera were detected between pregnant and non-pregnant cows and over the duration of the protocol. Many bacteria, such as the common pathogenic bacteria Cornyebacterium, had relative abundances greater than 1% at d -2 in the uterus of non-pregnant cows, but present in less than 1% in the uterus of pregnant cows (P < 0.05). When evaluating pH, uterine pH was lower than vaginal pH on average. Although not significant, uterine pH decreased in pregnant cows and increased in non-pregnant cows through the duration of the estrous synchronization protocol. In conclusion, our data suggests the bovine reproductive tract microbiome fluctuates over time and differences in bacterial species abundances may affect reproductive outcomes.

Abstract: Birth is a complex biological event requiring genetic, cellular and physiological changes to the uterus, resulting in a uterus activated for completing the physiological processes of labour. We define the change from the state of pregnancy to the state of parturition as uterine transitioning, which requires the actions of inflammatory mediators and localized paracrine interactions between intrauterine tissues. In the human, the birth cascade involves positive feedback interactions between a series of parallel events integrating both pro-inflammatory and pro-contractile systems, which accumulate until reaching a ‘critical mass’ or threshold that triggers a labouring state. An activated uterus is marked by increased expression of uterine activation proteins (UAPs) resulting in the increased sensitivity of the myometrium to coordinated contraction. In addition to the actions of steroid hormones, key inflammatory mediators, IL-1β and PGF2α, regulate the expression of critical uterine activation proteins (UAPs) FP and COX2 and pro-inflammatory cytokines and chemokines in cultured primary human myometrium smooth muscle cells (HMSMC). We found that in addition to regulation of the PGF2α receptor FP, IL-1β and PGF2α also upregulate the IL-1 receptor system, including IL1R1, IL1R2, IL1RAcP and IL1RAcPBb. HMSMC treated with IL-1β then PGF2α (or vice versa) produce amplified increases in IL-6 and COX-2 mRNA and protein compared to treatment with either agonist alone. These large increases were unique to myometrium and not observed with hFM. Uterine transition not only comprises interactions between different inflammatory pathways, but also paracrine interactions between neighbouring cells and tissues. Few studies have examined the in vitro interactions between fetal and maternal gestational tissues within this pro-inflammatory environment. Thus we designed a co-culture model to address this gap, incorporating primary term human myometrium smooth muscle cells (HMSMC) with human fetal membrane (hFM) explants to study interactions between the tissues. We hypothesized that crosstalk between tissues at term promotes pro-inflammatory expression and uterine transitioning for parturition. Outputs of 40 cytokines and chemokines encompassing a variety of pro-inflammatory roles were measured; all but one increased significantly with co-culture. Eighteen of the 39 cytokines increased to a higher abundance than the sum of the effect of each tissue cultured separately. In addition, COX2 and IL6, but not FP and OTR mRNA abundance significantly increased in both HMSMC and hFM in response to co-culture. These data suggest that synergistic pro-inflammatory upregulation within intrauterine tissues is involved with uterine transitioning. The series of experiments described in this dissertation together present cooperativity between ligands, cells and tissues as a hallmark of human parturition. Our work establishes PGF2α and IL-1β as key triggers or upstream drivers of this process and IL-6 and COX-2 as key targets, promoting inflammatory amplification in the myometrium through positive feedback and synergy. We surmise that, if similar events occur in vivo, this large increase in mediators could reflect a critical event in the transition of the uterus from the state of pregnancy to the state of parturition.

The mare experiences a transient innate immune response to breeding, the resolution of which is crucial for optimal fertility. The majority of mares are able to modulate this inflammation in a timely fashion, but a subpopulation exists which fail to do so and are considered susceptible to persistent breeding-induced endometritis (PBIE). Seminal plasma has been shown to modulate aspects of this inflammation. Recently, two seminal plasma proteins have garnered interest for their immune modulating properties: cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin. These proteins have been found to alter the binding between sperm and neutrophils based on sperm viability in vitro, but minimal work has evaluated their effect on endometrial mRNA expression of cytokines and inflammation in response to breeding. Experiments were performed to analyze the expression of equine CRISP-3. Found to be primarily synthesized in the ampulla of the vas deferens and to a lesser extent in the vesicular gland, CRISP-3 expression was only seen in the postpubertal stallion. Due to the effect of sperm viability on protein function in vitro, varying sperm populations were analyzed for their effect on gene expression in the uterus. It was determined that viable sperm suppressed the gene expression of the inflammatory modulating cytokine interleukin-6 (IL-6) in comparison to dead sperm. Next, the effect of CRISP-3 and lactoferrin on endometrial gene expression in the normal and susceptible mare was investigated. Neither protein had a significant effect on the mRNA expression of inflammatory cytokines in the normal mares at six hours post-breeding. In contrast, lactoferrin was found to significantly suppress the expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α in susceptible mares. Due to this, lactoferrin was further analyzed as an immunomodulant for the treatment of PBIE. Susceptible mares were infused with varying doses of lactoferrin at six hours post-breeding. Although not in a dose-dependent fashion, lactoferrin was found to decrease both fluid retention and neutrophil migration, in addition to suppressing the expression of the pro-inflammatory cytokine interferon gamma (IFNγ) and increasing the gene expression of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RN). In conclusion, CRISP-3 expression occurs in secretory aspects of the male reproductive tract, and appears to be up regulated after sexual maturation. Viability of spermatozoa affects the immune response to breeding and should be taken into consideration for experimental design and interpretation of data. The seminal plasma proteins CRISP-3 and lactoferrin have minimal effect on endometrial gene expression in normal mares, but lactoferrin suppresses the expression of TNF in susceptible mares. Finally, lactoferrin was found to function as a potent anti-inflammatory for the persistent inflammation seen in susceptible mares when administered post-breeding. This protein should be further investigated as a potential therapeutic for the treatment of persistent breeding-induced endometritis.

Infertility among women has become a growing issue in the world requiring a significant number of women to seek treatment by means of assisted reproductive technologies (ART). One suggested reason for the fertility issue is modern diet, leading to diseases such as obesity and type II diabetes. In this study, twenty gilts three weeks in age, were placed on one of five dietary treatments (n=4 gilts per treatment) containing 15% fat (FAT), 35% fructose (FRU), both fat and fructose (HFHF), or two different controls: one standard industry (IND) diet meant to result in optimal lean growth and a second diet to account for the reduced lysine (LYS) intake in the treatment diets. Two experiments were performed to assess the reproductive outcomes of pre-pubertal gilts consuming a high fat and/or high fructose diet and to demonstrate interactions between diet and infertility using pigs as a model. In the first experiment, follicular fluid was collected from these gilts and introduced into porcine in vitro maturation system to determine whether characteristics of the follicular fluid affect oocyte competence and embryo development. The follicular fluid of females consuming high fructose and fat diets did not alter nuclear maturation of oocytes (p>0.10). There were, however, detrimental effects on subsequent development of embryos, especially blastocyst formation, with the gilts having consumed the HFHF diet having reduced day 5 and 6 blastocysts formation when compared to the IVM follicular fluid free (FFF) group (p=0.03 and p=0.01, respectively). In regards to embryo quality, blastocysts from the FAT group had greater cell number when compared to all other groups. In the second experiment, the reproductive tissues; ovary, oviduct, and uterus were analyzed for genes of interest: estrogen receptor alpha (ESR1), estrogen receptor beta (ESR2), insulin like growth factor I (IGFI), insulin like growth factor I receptor (IGFIR), and growth differentiation factor 9 (GDF9). Resulting data was analyzed in three ways: 1) across all 5 treatments, 2) with gilts grouped by whether or not they consumed fat, or 3) with gilts grouped by whether or not they consumed fructose. There were no differences detected between individual treatments for ESR1 and ESR2. In the ovary samples, the fructose diets decreases ESR2 (p=0.05). Also, GDF9 ovarian expression tended to decrease with fructose consumption (p=0.07). Furthermore in the ovary, there was a positive correlation between ESR2 and GDF9 expression (r=0.92 and p

Bisphenol A (BPA) and 17α-ethinyl estradiol (EE) are estrogenic endocrine disrupting chemicals (EDCs) that adversely affect the structure and function of the uterus. Humans are ubiquitously exposed to BPA via consumption of contaminated food and beverage from polycarbonate packaging or food cans. Humans are exposed to EE through the use of oral contraceptives. While human exposure to these EDCs is widespread, little is known how these compounds alter physiological responses that lead to increased incidence of uterine pathology.The purpose of this dissertation was to investigate and characterize uterine pathologies and associated alterations in immune responsiveness and fibrosis. Two mouse strains were used, the CD1 and the C57Bl/6N strains. Exposure during adulthood included mating, parturition, and offspring rearing and mice were exposed for 12-15 weeks. Whole life exposure included placental transfer, as well as direct oral consumption, exposing mice until postnatal day (PND) 90. In order to closely mimic human exposure, mice were orally exposed through diet to known concentrations of control (0 ppm), BPA (0.03, 0.3, 3, 30, or 300 ppm), or EE (0.0001, 0.001, 0.01, 0.1, or 1.3 ppm), which resulted in calculated BPA doses of 0.04, 0.4, 4, 40, and 400 mg/kg/day and EE doses of 0.00002, 0.0002, and 0.001 mg/kg/day. Other exogenous estrogenic compounds from housing, bedding, and water were eliminated. Two distinct immune-related and fibrotic uterine pathologies were identified. Pyometra developed in C57Bl/6N mice exposed to BPA or EE during adulthood. An equine endometrosis-like phenotype, characterized by increased gland nests and stromal and periglandular fibrosis, naturally occurred in control C57Bl/6N mice. In CD1 mice, this fibrotic phenotype was present in the 30 ppm BPA group. Exposure to BPA significantly increased Col1a1 and Col3a1 expression and decreased Mmp2 and Timp2 expression. Expression and activity of matrix metalloproteinases, MMP2 and MMP14, were significantly decreased in both the control C57Bl/6N and 30 ppm BPA-exposed CD1 mice compared to control CD1 mice. However, both strains presented with an increased immune response, quantified as the percentage of F4/80-positive cells. The C57Bl/6N strain had a significant increase in endometrial macrophages at a lower dose of BPA (0.03 ppm) than the CD1 strain (30 ppm) exposed during adulthood. In both exposure models, CD1 mice had increased macrophages in the 30 ppm BPA group.This dissertation research was the first report of BPA altering the immune response or collagen accumulation in the uterus, leading to the induction of pyometra or an equine endometrosis-like phenotype. It was also the first report of strain-specific differences in sensitivity to the actions of BPA on these endpoints of interest. Finally, this research highlighted the potential for exposure to estrogenic compounds such as BPA and EE to impact the development and progression of fibrotic and immune-related uterine diseases. However, based on the differences observed between the two generations, physiological changes that occur during mating, pregnancy, and parturition may be necessary for estrogenic compounds to elicit fibrotic effects. These studies also emphasized the importance of understanding differences in sensitivity to estrogenic compounds between mouse strains.

An effective bidirectional communication between an implantation-competent blastocyst and the receptive uterus is a prerequisite for mammalian reproduction. The blastocyst will implant only when this molecular cross-talk is established. Muscle segment homeobox gene (Msh) family members Msx1 and Msx2, which are two highly conserved genes critical for epithelial-mesenchymal interactions with overlapping function during development, also play crucial roles in embryo implantation. Msx1 is transiently expressed in the pregnant uterus during the window of receptivity from day 3 of pregnancy until the morning of day 4 with rapid downregulation with impending implantation. Uterine inactivation of Msx genes leads to infertility due to implantation failure, which correlates with altered uterine luminal epithelial cell polarity and affects E-cadherin/β-catenin complex formation through the transcriptional regulation of Wnt5a expression. Application of Wnt5a in vitro compromised blastocyst invasion and trophoblast outgrowth by cultured uterine epithelial cells.

Uterine inflammation after breeding is considered necessary for the clearance of excess semen and debris from the uterus. A subpopulation of mares fails to clear the inflammation in a timely fashion, and develops a persistent breeding induced endometritis (PBIE). Experiments were preformed to evaluate correlations of PBIE to endometrial quality and age. Mares of advanced age and poor endometrial quality had a higher incidence of PBIE. In addition, mares fluctuated in susceptibility to PBIE from one season to the next. The uterine inflammatory gene expression in susceptible and resistant mares within the first 24 hours after breeding was investigated. The peak endometrial cytokine gene expression occurred 6 hours after insemination, and susceptible mares were found to have a reduced response of the inflammatory modulating cytokines during this time. Intrauterine accumulation of the inflammatory byproduct nitric oxide (NO) was investigated in resistant and susceptible mares within the first 24 hours after breeding. Susceptible mares had an increase in NO accumulation over time, whereas NO accumulation in resistant mares remained relatively constant. The effects of immunomodulators on uterine inflammatory response and nitric oxide accumulation in susceptible mares was investigated. Immunomodulators decreased expression of the pro-inflammatory cytokine interleukin-1β and nitric oxide accumulation. In conclusion, endometrial quality and age are indicators of susceptibility to PBIE, and susceptibly can change from year to year. Six hours after breeding is a critical time for the development of PBIE, and susceptible and resistant mares have differential endometrial inflammatory gene expression, with susceptible mares appearing to have a defect in the inflammatory modulating immune response. Finally, treatment with immune modulators alters the IL1β gene expression and intrauterine nitric oxide accumulation, which may help to explain how they act to reduce inflammation during PBIE.

Abstract: Rationale: Smooth muscle (SM) contraction results from activation of the cellular contractile machinery by phosphorylation of myosin regulatory light chain (RLC) at S19 (pRLC) by RLC kinase (MLCK). Subsequent phosphorylation at T18 yields diphosphorylated-RLC (ppRLC), which has been shown to enhance the myosin ATPase activity, and therefore might have implications for the ability of the muscle to generate tension. In uterine myocytes, the extent of diphosphorylation is greater than that observed in vascular myocytes. In preliminary experiments using cultured human uterine smooth muscle cells (hUSMC), we observed that rho-kinase (ROK) inhibition caused a marked reduction in ppRLC, but surprisingly, not pRLC. Therefore we pursued a quantitative comparison in ROK-mediated phosphorylation of RLC in hUSMC and human vascular myocytes (hVSMC). Objectives: 1) to develop and validate quantitative methods for assessment of phosphorylated RLC, and 2) to apply these methods in studies comparing ROK-mediated phosphorylation of RLC in hUSMC and hVSMC. Findings: We have developed and validated two assays for quantitative measurements of phosphorylated RLC using phospho-specific antibodies: 1) a high-throughput in-cell western assay, and 2) western blotting-based quantification of RLC-phospho-states after separation with phos-tag SDS-PAGE. These techniques revealed that hUSMC differ from hVSMC in their primary phosphorylation responses to stimulation. hUSMC respond to oxytocin with increased pRLC and ppRLC, whereas hVSMC respond to endothelin-1 with increased pRLC only. In hUSMC, only ppRLC was sensitive to ROK inhibition using a pharmacologic inhibitor, and this occurred at approximately 100-fold lower concentration of ROK inhibitor than that required to achieve an equivalent reduction of pRLC in hVSMC. Conclusions: In hUSMC, ROK phosphorylates RLC at T18 downstream of MLCK-mediated phosphorylation at S19. This sequential phosphorylation of RLC forms ppRLC. This role of ROK activity appears unique to hUSMC and is a fundamental difference from hVSMC. These findings suggest the possibility of a uterine-specific therapeutic avenue for prevention or arrest of preterm labor achieved by targeting ROK to reduce ppRLC levels in hUSMC. Our data also suggest that ROK inhibition might avoid the prohibitive side effects of currently used agents for preterm labour, of which the most common and serious is hypotension that results from unwanted vascular relaxation.

MRI of the body at 7 T has become possible only very recently. High fields bring the advantages of increased signal to noise ratio, resolution gains, faster image acquisition through better parallel imaging, improved and novel types of contrast, and greater spectral dispersion. There are many obstacles to be faced in the advancement to 7 T including field inhomogeneities, transmit inhomogeneities, and increased RF-absorption risk. Inhomogeneous B1+ is possibly the most significant obstacle currently facing 7 T body imaging in consistently producing clinical-quality images. In this work, the first demonstrations of MR imaging and spectroscopy (MRS) of the liver at 7 T are presented and evaluated with special attention given to parallel imaging. Also presented here is the first demonstration of imaging of the uterus at 7 T with particular emphasis on contrast between uterine layers. A comparison was done between 3 T and 7 T, and relaxation rates were mapped including higher rotating frame relaxations.SWIRLY (spatiotemporal-encoding with incremental refocusing along a trajectory) is a novel pulse sequence that employs a frequency and amplitude modulated excitation pulse in the presence of sinusoidal gradients to move a region of resonance through space along a spiral trajectory. The signal can be sequentially refocused and acquired in an entirely spatiotemporal manner such that no Fourier Transform is needed for reconstruction. Because each resonance region can be treated entirely independently, this sequence has incredible potential for addressing problems that are inherently spatial in nature, such as B1+ and B0 inhomogeneities.

Various investigations were performed to examine how androgens might influence reproduction in gilts. The first investigation was designed to begin to address the endocrinology of how androgens increase ovulation rate in gilts when administered during the follicular phase. Gilts were injected with androgen receptor agonists (5α-dihydrotestosterone, DHT or testosterone), antagonist (flutamide) or a combination of both from day 13 of the estrous cycle (day 0= onset of estrus) until the onset of estrus. Hourly blood samples were collected when treatments were initiated until the onset of estrus. Testosterone or DHT treatments increased (PThe second investigation comprised two experiments and was conducted to examine the effects of androgens on neonatal development of the uterus. A total of sixty gilts were utilized. In Experiment 1, twenty four newborn gilts were assigned randomly to increasing dosages of DHT (0, 6, 60 or 600 µg per kg of body weight) from birth (postnatal day, PND 0) to PND 13. Treating gilts with increasing dosages of DHT did not alter any of the components of uterine development examined on PND 14 (wet weights of the uterus, liver, heart or longissimus dorsi, endometrial thickness, height of luminal epithelium, glandular penetration nor myometrial thickness). In Experiment 2, thirty six gilts were utilized to investigate up to 14 days of age the effects of androgens on estrogenic-induced stimulation of uterine development. Gilts were randomly assigned at birth to receive daily injections of vehicle, 50 µg of estradiol valerate, 60 µg of DHT, 10 mg of flutamide, estradiol valerate plus DHT or estradiol valerate plus DHT plus flutamide, per kg of body weight, from PND 0 to PND 13. Treatment of neonatal gilts with estradiol valerate increased uterine wet weight, endometrial thickness, glandular penetration and myometrial thickness but only the estradiol valerate-induced effects on myometrial thickness were blocked by co-treatment with DHT. Results of these experiments indicate that within the first two weeks of age, androgen treatment failed to influence most of the estrogenic-induced stimulatory effect as has been observed in the adult uterus.

Automobile crashes are the largest cause of death for pregnant females and the leading cause of traumatic fetal injury mortality in the United States. The first way to protect the fetus is to protect the mother considering that maternal death has a near 100% fetal loss rate. If the mother survives, protection of the fetus may best be accomplished by preventing placental abruption. Placental abruption, which is the premature separation of the placenta from the uterus, has been shown to account for 50% to 70% of fetal losses in motor vehicle crashes. Since real world crash data for pregnant occupants is limited to a retrospective analysis and pregnant cadaver studies are not feasible, crash test dummies and computational modeling have been utilized to evaluate the risk of adverse fetal outcome. Although pregnant occupant research has progressed with these tools, they are based on limited tissue data. In order to have more accurate research tools, better pregnant tissue material data are needed. Therefore, the purpose of this dissertation is to provide material properties for the placenta and pregnant uterine tissue in dynamic tension.

The effects of lesser concentrations of estradiol prior to ovulation on embryonic development and uterine characteristics at various times during early gestation in cattle were investigated. The first experiment examined the effect of reducing the interval of proestrus prior to inducing the ovulation of follicles of similar diameter on circulating hormone concentrations. Reducing the interval of proestrus decreased concentrations of estradiol prior to ovulation establishing this animal model as a method to manipulate preovulatory estradiol concentrations in animals with ovulatory follicles of similar diameter. This animal model was used to study characteristics of the uterus or embryo that might explain the reduction in fertility associated with suboptimal concentrations of estradiol prior to ovulation. In a second study, cows with reduced preovulatory estradiol concentrations had increased mRNA for oxytocin receptor, estradiol receptor beta, and cyclooxygenase-2 on day 5 of the estrous cycle compared to cows with elevated preovulatory estradiol concentrations. In cows with lesser estradiol concentrations, altered uterine gene expression on day 5 may indicate alterations in uterine function that could contribute to embryonic death later in pregnancy. In the next study, the impact of duration of exposure to elevated estradiol concentrations during proestrus on embryonic development on d 6 of gestation was investigated. Results suggested that reducing estradiol prior to ovulation does not influence early embryonic development. Across treatments however, peak estradiol concentrations were correlated to total number of nuclei per embryo. Lastly, conceptus and uterine characteristics on day 15.5 of gestation were compared between heifers with normal and deficient preovulatory estradiol concentrations. No differences in conceptus size and viability between heifers with differing preovulatory estradiol concentrations were observed. However, heifers with lesser preovulatory estradiol had reduced mRNA for estradiol receptor alpha in the uterine endometrium, nuclear progesterone receptor in the deep glandular epithelium, and amount of secreted phosphoprotein 1 in the uterus. These differences may represent functional changes occurring within the uterus that results in eventual embryonic mortality. Collectively, these experiments indicated that deficient preovulatory estradiol causes changes in the uterus during early gestation and suggests that embryonic mortality occurs after maternal recognition of pregnancy in estradiol deficient animals.

Polychlorinated biphenyls (PCBs) are persistent environmental toxicants that have been associated with decreased gestation length. Phospholipase A 2 (PLA2)-mediated mobilization of arachidonic acid from glycerophospholipids in gestational tissues is thought to be an essential step in the onset of parturition. However, how PLA2 expression patterns change throughout pregnancy and how PLA2 availability influences uterine sensitivity to toxicant-induced stimulation are not known. Because PCB 50 activates PLA 2 to release arachidonic acid from membrane phospholipids, this ortho-substituted PCB congener was used as a model compound to examine the hypothesis that stimulation of late gestation rat uterine and amniotic phospholipase A2 enzyme(s) may pose a threat to pregnancy maintenance by stimulating uterine contractility through the release of arachidonic acid and prostaglandins. Western blot analysis revealed a gestational age-related increase in uterine expression of calcium-dependent secretory (s)PLA 2-IIA and a previously unreported 50 kDa calcium-independent (i)PLA 2-related protein. This 50 kDa iPLA2-related protein was also detected in late gestation amnion tissue, where over ninety percent of PLA2 activity was calcium-independent. Increases in uterine PLA 2 expression were associated with a gestational age-related increase in both calcium-dependent and calcium-independent PLA2 activity. PCB 50 stimulated the force and frequency of uterine contractions in late, but not mid, gestation rat uterine strips in vitro. Furthermore, pharmacologic inhibition of iPLA2 significantly reduced PCB 50-induced stimulation of uterine strips, suggesting that sensitivity to PCB stimulation is dependent on the gestational age-related increase in the uterine 50 kDa iPLA2-related protein. PCB 50 also stimulated the release of arachidonic acid and prostaglandins from late gestation rat uterine myocytes. Arachidonic acid release was attenuated with inhibition of calcium-dependent and calcium-independent PLA2 inhibitors. Additionally, PCB 50 stimulated the release of arachidonic acid and prostaglandins from late gestation rat amnion cells in vitro, an effect that was associated with a significant increase in 85 kDa iPLA2 expression. These data provide evidence of a previously uncharacterized contribution of calcium-independent PLA2 to total rat uterine and amniotic phospholipase activity and support the hypothesis that late gestation rat uterine and amniotic PLA2 mediate PCB-induced stimulation of uterine contractions via arachidonic acid and prostaglandin release.

Connective tissue growth factor (CTGF/CCN2) is an extracellular matrix-associated matricellular protein that regulates diverse cellular activities including adhesion, migration, mitogenesis, differentiation and survival. The broad biological properties of CTGF/CCN2 in many cell types likely reflect its ability to bind to a variety of cell surface molecules (integrins, low density lipoprotein receptor-related protein, heparin sulfate proteoglycans) as well as to other bioactive molecules. CTGF/CCN2 synthesis can be stimulated by a variety of molecules including transforming growth factor b1 (TGF-b1) which appears to play a major role in the transcriptional activation of the CTGF/CCN2 gene in matrigenic and fibrogenic pathways. This dissertation encompasses the relationship between CTGF/CCN2 and organ fibrosis, TGF-b1 action, gene regulation, pediatric desmoplastic tumors as well as its role in uterine function. Recombinant adeno-associated virus delivery of the CTGF/CCN2 gene was attempted to determine whether CTGF/CCN2 was directly linked to the progression of organ fibrosis. However, since little or no protein was generated in vivo using this system, it could not be deduced whether over-expression of CTGF/CCN2 alone could drive fibrosis. In vitro studies with fibroblasts and hepatic stellate cells (HSCs) demonstrated that TGF-b1, alcohol or CTGF/CCN2 itself could up-regulate CTGF/CCN2 transcription. Additionally, after heparin- or EDTA-dependent HSC adhesion to CTGF/CCN2, preliminary gene array experiments demonstrated a series of genes that were transcriptionally up-regulated including, collagen a-1-type I, fibronectin, GRO oncogene, secreted acidic cysteine rich glycoprotein, tenasin C and tissue inhibitor of matrix metalloproteinase. Further, RT-PCR analysis of similar experiments showed that hepatocyte growth factor, a potent mitogen that accelerates recovery from hepatic fibrosis, was down regulated. In addition, two pediatric fibrotic diseases, congenital hepatic fibrosis and desmoplastic small round cell tumor, were shown to produce elevated levels of CTGF/CCN2, consistent with its pro-fibrogenic properties. Finally, studies demonstrated that uterine CTGF/CCN2 gene transcription is regulated by maternal sex steroids and TGF-b1-dependent and –independent mechanisms. Further data indicated that the epithelium is a key source of CTGF/CCN2 in the mammalian uterus and that CTGF/CCN2 likely plays a role in regulating stromal cell function and placental neovascularization.