AbstractFactor VIII activity was studied in fifteen white laboratory rats, strain CFE, which were given subcutaneous doses of a solution containing estrogenic substances and progesterone over a six week period. A modified version of the thromboplastin generation test was used in conjunction with a factor VIII-deficient plasma to test for factor VIII activity.The rat population consisted of three groups, One group received a high concentration dosage level of the hormone The other group received a lower concentration dosage level of the hormone compound. The third group served as the control and received no hormone treatment. Each of the above groups contained five test animals.Factor VIII activity increased in all the experimental animals except one, as exhibited by a reduction in the coagulation time. No increase in factor VIII activity was observed in the control group. A significant statistical difference was observed between the experimental rats and those of the control. The 0.05 level of statistical significance was chosen for this study.Ball State UniversityMuncie, IN 47306

Evidence from an abundant number of studies suggests that human female reproductive functions have become impaired over the past half century and that there might be a relationship between endocrine disrupting chemicals (EDCs) and reduced fertility. It is, however, not known by what mechanisms EDCs affect different reproductive functions such as endometrial receptivity, embryo implantation and placentation. The endometrium is continuously changing its morphological and functional properties, responding to cyclic changes of oestrogen and progesterone levels during the menstrual cycle. These changes include monthly preparation for embryo implantation through changed endometrial angiogenic activity and consequent changes in endometrial vasculature. Use of primary human endometrial endothelial cells (HEECs) in this work was evaluated as a possible screening tool for effects caused by EDCs on human endometrial vasculature and subsequently on various endometrial functions. In this study HEEC and endometrial stromal cells were isolated. HEECs were grown in monocultures, and together with stromal cells in co-cultures, and exposed to endocrine active substances. These were cadmium, which has oestrogenic properties, tamoxifen, with anti-oestrogenic effects, mifepristone, which is an anti-progestin, and bisphenol A, with oestrogenic properties. The effects were evaluated by using proliferation and viability assays, migration and tube formation assays, quantitative PCR (qPCR), immunohistochemistry and western blot. Cadmium affected the expression of angiogenesis-related genes, and caused different effects in HEECs cultured alone vs. HEECs co-cultured with stromal cells. Tamoxifen altered the expression of angiogenesis-related genes and reduced HEEC migration, thus having an anti-angiogenic effect. Mifepristone caused reduced formation of tubular structures in tube-formation assays involving HEECs co-cultured with stromal cells. Bisphenol A promoted tube formation in co-cultured HEECs which was related to changes in the expression of several angiogenesis-related genes as well as up-regulated expression of VEGF-D protein. In conclusion, we showed that EDCs have the ability to induce changes in endometrial angiogenic activity in vitro and may thus disturb normal endometrial functions related to fertility and pregnancy. HEECs grown in vitro may provide valuable information on the effects of EDCs on human endometrial functions. However, this model is not suitable as a large-scale screening tool.

<p>The progesterone receptor (PR) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. The steroid hormone progesterone binds to PR and induces a conformational change that enables the receptor to bind DNA, recruit cofactors, and directly regulate the transcription of target genes. In addition, extra-nuclear PR can indirectly regulate gene expression by rapidly activating other signaling pathways such as Src/MAPK. Although the direct and indirect functions of PR have been well studied in isolation, it is important to understand the molecular mechanisms by which these pathways can cross talk and integrate to ultimately impact gene expression.</p><p>Towards this end, we initiated studies to assess the overall impact of MAPK inhibition on PR transcriptional activity in T47D breast cancer cells treated with the synthetic progestin R5020. During the course of microarray and biochemical analyses that were undertaken to address this issue, we discovered a subset of PR target genes that are enriched for E2F binding sites. Subsequently, we determined that PR-B is a component of several distinct pathways that function both directly and indirectly to positively up-regulate E2F1 expression in T47D breast cancer cells. Firstly, PR directly regulates E2F1 transcription by binding to proximal and distal enhancer sites located near E2F1. Secondly, progestin induces the hyperphosphorylation of Rb, which results in increased recruitment of E2F1 to its own promoter, thereby activating a positive feedback loop that further amplifies its transcription. Finally, PR induces expression of Krüppel-like factor 15 (KLF15) and potentially other Sp/KLF family members, which can bind to GC-rich DNA within the E2F1 promoter and further activate transcription. Together, these results suggest a paradigm for multimodal PR gene regulation that entails cooperation between direct and indirect pathways of PR signaling to achieve the desired downstream transcriptional cascade.</p><p>In the breast and other tissues of the female reproductive system, progesterone plays an important role in normal development and function. Therefore, synthetic PR modulators (PRMs) are widely used to manipulate the downstream biology of PR for purposes including contraception and hormone replacement therapy (HRT). However, progestins and PR have also been implicated in disease pathologies such as breast cancer. While the molecular mechanisms by which PR regulates breast tumor growth have not been fully elucidated, recent studies highlight the fact that progestins may have a dose-dependent role in breast cancer progression. Consequently, we undertook studies to identify and characterize any differential effects of low-dose versus high-dose progestins on the downstream activities of PR. Specifically, we found that treatment of breast cancer cells with low-dose progestins can induce maximal transcriptional activation of a subset of PR target genes, including the cell cycle regulators cyclin D1 and E2F1. Furthermore, low-dose and high-dose progestins have differential effects on the phosphorylation of PR and subsequent receptor turnover. Cumulatively, these findings underscore the importance of establishing the effects of a wide range of progestin concentrations on target gene expression and other PR actions, so that we are able to accurately predict the potential consequences of PRMs on downstream PR signaling pathways and biology.</p> / Dissertation

This research was conducted to test the hypothesis that select nutrients including glucose, leucine, arginine and glutamine stimulate conceptus development by activating mTOR (mammalian target of rapamycin; HGNC approved gene name: FRAP1, FK506 binding protein 12-rapamycin associated protein 1) signaling pathway. First, temporal changes in quantities of select nutrients (glucose, amino acids, glutathione, calcium, sodium and potassium) in uterine lumenal fluid from cyclic (Days 3 to 16) and pregnant (Days 10 to 16) ewes were determined. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium and sodium was greater in uterine fluid of pregnant compared to cyclic ewes between Days 10 and 16 after onset of estrus. Of note were remarkable increases in glucose, Arg, Leu and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Second, effects of the estrous cycle, pregnancy, progesterone (P4) and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3 and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters, cationic amino acid transporters (SLC7A1, SLC7A2 and SLC7A3), neutral amino acid transporters (SLC1A4, SLC1A5, SLC3A1, SLC6A14, SLC6A19, SLC7A5, SLC7A6, SLC7A8, SLC38A3, SLC38A6 and SLC43A2) and acidic amino acid transporters (SLC1A1, SLC1A2 and SLC1A3) in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. Among these genes, SLC2A3 and SLC7A6 were detectable only in trophectoderm and endoderm of conceptuses. The abundance of mRNAs for SLC2A1, SLC2A4, SLC5A1, SLC5A11, SLC7A1, SLC7A2, SLC1A4, SLC1A5, SLC43A2 and SLC1A3 changed dynamically in ovine uterine endometria according to day of the estrous cycle and early pregnancy. Expression of mRNAs for SLC2A1, SLC5A11 and SLC7A1 in endometria was induced by P4 and further stimulated by IFNT with shortterm treatment (12 days), while expression of SLC7A1 and SLC1A5 in endometria required long-term treatment (20 days) with P4 and IFNT. Third, effects of the estrous cycle, pregnancy, P4 and IFNT on expression of nitric oxide synthase (NOS1, NOS2 and NOS3), GTP cyclohydrolase (GCH1), ornithine decarboxylase 1(ODC1), insulin-like growth factor II (IGF2), FRAP1 complexes (FRAP1, LST8, MAPKAP1, RAPTOR, RICTOR), regulators (TSC1, TSC2, RHEB) and an effector (EIF4EBP1) of FRAP1 signaling in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. All of these genes were expressed in ovine uterine endometrium and conceptuses. Among these genes, expression of NOS1, IGF2, RHEB and EIF4EBP1 changed dynamically due to day of the estrous cycle and early pregnancy. Progesterone stimulated NOS1 and GCH1 expression while IFNT inhibited NOS1 expression in uterine endometria, and P4 and IFNT stimulated expression of RHEB and EIF4EBP1 in uterine endometria. Collectively, these results indicate that: 1) the availability of select nutrients in the ovine uterine lumen increases to support the rapid growth and elongation of the conceptus during the peri-implantation stage of pregnancy; 2) P4 and/or IFNT stimulate(s) glucose and amino acid transporters to facilitate their transport from maternal tissues and/or blood into the uterine lumen during early pregnancy; 3) the FRAP1 cell signaling pathway mediates interactions between the maternal uterus and peri-implantation conceptus and both P4 and IFNT affect this pathway by regulating expression of RHEB and EIF4EBP1. Expression of NOS, ODC1 and IGF2 appear to be linked to FRAP1 signaling in both uteri and peri-implantation conceptuses.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on December 29, 2009). Thesis advisor: Dr. Matthew C. Lucy and Rodney D. Geisert. Vita. Includes bibliographical references.

Thesis (M.A.)--University of North Carolina at Wilmington, 2004. / Includes bibliographical references (leaves : [109]-111).

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2009. / Härtill 4 uppsatser.

The goal of the current research was to examine the expression, signaling and function of the membrane progestin receptors (mPRs) in the ovarian follicular cells of the Atlantic croaker (Micropogonias undulatus) and in human breast cancer cells. Multiple studies have examined the role of mPRs in the germ cells of several vertebrate classes, yet few studies have examined the role of the mPRs in the somatic cells of reproductive tissues. Therefore this research examines the mechanism of mPR action and its function in somatic cells of female reproductive tissues. Results from studies on the expression, localization and signaling of the mPR[alpha] in co-cultures of granulosa and theca cells from the croaker suggest that the mPR[alpha] is localized to the plasma membrane of both cell types and that the mPR[alpha] is associated with and signals via pertussis toxin-sensitive inhibitory G proteins to decrease intracellular cAMP and activate ERK. In addition, exposure of follicular co-cultures to progestins that activate the mPR[alpha] results in a decrease in serum starvation-induced cell death which is not replicated by progestins which activate the nuclear progestin receptor (nPR), indicating mPR mediation. Similar studies in two immortalized human breast cancer cell lines, MDA-MB-468 and SKBR3, suggest that the mPR[alpha] is also present in the membranes of these cells and signals in human breast cancer cell lines via activation of a pertussis toxin-sensitive G protein to significantly decrease in intracellular cAMP and activate ERK. Progesterone exposure also decreased serum starvation-induced cell death in SKBR3 cells which are nPR positive and in MDA-MB-468 cells which are nPR negative. Synthetic progestins which activate the nPR but not the mPR were ineffective in inhibiting death in either cell type suggesting that the mPR is the mediator of this progestin action. mPR[alpha], mPR[beta] and mPR[gamma] expression analysis of paired normal and malignant breast tissue biopsies from thirteen women revealed that at least one mPR isoform was upregulated in the malignant tissue of 70% of the women. In addition the expression of mPR[gamma] was positively correlated with the expression of the nPR and CK19, a breast epithelial cell marker. / text

Introduction: Premenstrual Dysphoric Disorder (PMDD) is a condition that affects about 2-6% of women of reproductive age. The relation to ovarian steroids is apparent as symptoms are absent during anovulatory cycles. Neuroactive steroids like allopregnanolone have effect in the brain and on brain function and have been proposed to play an important role for the symptomatology of premenstrual symptoms and in the interaction between the GABAA receptor and alcohol. High doses of alcohol elevate allopregnanolone levels both in rats and humans. Allopregnanolone is a positive modulator of the GABAA receptor with sedative, anxiolytic and anticonvulsant effect in both human and animals. Aims: The aim was to investigate if a low dose (100μg) of GnRH agonist (buserelin) is effective for the treatment of PMDD and if allopregnanolone serum levels during treatment are associated to symptom severity. Furthermore, the studies aimed at investigating the effect of a low dose of alcohol upon saccadic eye movements in women with PMDD, and control subjects in different phases of the menstrual cycle, and to evaluate if there was a difference in response to alcohol between men and healthy women. We also wanted to see if this low dose of alcohol could have an effect on serum allopregnanolone levels in women with PMDD and control subjects in the follicular and luteal phases of the menstrual cycle. Methods: The effect of low dose (100μg) of GnRH agonist (buserelin) on premenstrual symptoms was evaluated in a randomized, placebo controlled, double-blinded cross-over trial. 27 PMDD patients were randomized to either GnRH agonist intranasally once a day or placebo for two months before the crossover. The main outcome measure was the daily symptom ratings for mood and physical symptoms made by the patients. In a subgroup of 12 women, grouped as buserelin responders and placebo responders, luteal phase serum progesterone, allopregnanolone, and pregnanolone was measured together with daily ratings for mood and physical symptoms. Alcohol responsiveness was measured in PMDD patients, female control subjects and men by comparing the effect of a low dose (0.2g/kg) of intravenous alcohol or placebo infusion upon saccadic eye movements. Blood samples for measurement of allopregnanolone and cortisol were taken throughout the alcohol/placebo challenges. Results: Low dose GnRH agonist was effective as treatment of premenstrual irritability and depression. Anovulatory cycles were confirmed in 56% of the subjects, particularly in older women. Buserelin as well as placebo responders displayed decreased allopregnanolone and progesterone levels in parallel with symptom improvement. PMDD patients displayed blunted saccadic eye movement response to alcohol infusion, especially in the luteal phase. Control subjects did not change their response to alcohol between cycle phases. We found no difference in saccadic eye movement sensitivity to alcohol between males and females. Allopregnanolone levels significantly decreased in the luteal phase following the alcohol infusion. Conclusions: Low dose GnRH agonist is effective in treatment of premenstrual depression and irritability but is likely to induce anovulation with increasing age. Independent of whether buserelin or placebo treatment was given decreased levels of allopregnanolone appear to be related to symptom improvement. Women with PMDD have altered saccadic eye movement sensitivity in response to alcohol, particularly in the luteal phase. The low dose of alcohol did not induce any difference in saccade measurements between males and females. Low dose of alcohol does not result in increased peripheral levels of allopregnanolone.

It is the first time in Lithuania that practical application of semen filtration by Sephadex G-15 for the needs of artificial insemination industry was assessed. The efficacy of semen filtration was assessed applying a battery of sperm quality assays to study bovine spermatozoa quality before and after filtration, as well as after cryopreservation. The estimation of effect of different concentrations of progesterone, oestradiol, heparin separately or in combination, on different functional parameters of bull spermatozoa after thawing was performed. These are the first published studies applying integrated approach to study the effects of sperm challenge with several biologically active substances.