The production of viable germ cells during human embryonic development determines adult reproductive success. This is particularly true for females, as development of germ cells (GCs) into primordial follicles before birth is imperative for future fertility. During fetal development GCs migrate to the genital ridge to form the gonad, after which several tightly regulated events, including proliferation, differentiation, and association with somatic cells, must occur to form a functional gonad. In the ovary these processes also include the initiation and subsequent arrest of meiosis. These developmental processes are orchestrated by local autocrine and paracrine factors, many of which remain to be identified in the human. In order to decipher further the pathways by which the gonad and GCs develop, potential regulators including prostaglandin (PG) E2, the interleukin (IL)6-type cytokines, and the prokinetecins (PROKs), were examined in the human fetal ovary and PROKs in the human fetal testis. Patterns of gene expression, protein localisation, function, and interaction of the potential mediators throughout human development (8-20 weeks gestation) were determined. Primary fetal tissue was investigated, in addition to immortalized GCs (T-Cam2 cells) and a murine model of fetal ovarian development. PGE2 interacts with known regulators of GC development in non-reproductive organs. It was postulated PGE2 may regulate GC progression by modulating these factors. Examination of PGE2 receptors and precursor enzymes in the fetal ovary revealed that all were present and some were developmentally regulated, with mRNA expression increasing with gestation. These developmentally regulated components were localised to the GCs. The PGE2 receptors were among those differentially expressed, with one localised solely to mature GCs. Culture of human fetal ovary confirmed that PGE2 regulates known regulators of GC development, increasing expression of survival and anti-apoptotic factors. To test the hypothesis that PGE2 is necessary for female GC development, paracetamol, an inhibitor of PGE2 precursor enzymes, was utilised in a murine model of fetal exposure. Fetal ovaries from this experiment displayed disruption of normal development. The IL6-type cytokines are also postulated to be involved in early gonad development, and are known to regulate proliferation and differentiation of mouse embryonic stem and GCs in vitro. A significant increase in transcript levels of the shared receptor components was determined in second trimester human ovaries, as well as developmental increases of several of the IL6-type ligands. Both common receptor components were located specifically in the GCs identifying them as the target of IL6 action in the human fetal ovary. The PROKs regulate cell migration, proliferation and differentiation, and modulate secretion of PGE2 and expression of some IL6-type cytokines. To-date, PROKs have not been examined in the human fetal gonad. Transcript levels were higher in the fetal testis compared to the ovary, with receptor and ligand components increasing with gestation. Most components also increased with gestation in the ovary. However, location of PROK components was strikingly different between the two tissues, with GCs being the primary target of PROK action in the fetal ovary, and Leydig and interstitial cells being the target in the testis. PROKs interaction with other regulators of gonad development was examined utilising a GC line in the case of the ovary and primary interstitial cell cultures in the case of the testis. These studies have identified new factors involved in human fetal gonad development, and how they interact with known regulatory pathways of development.

Hair growth disorders cause significant psychological distress, but are poorly controlled. Since prostaglandin F₂α (PGF₂α) and prostamide F₂α analogue glaucoma treatments cause eyelash growth as side-effects, they may be useful for alopecia. How they function is unknown; possibilities include direct action on hair follicles or stimulating follicular blood flow. It is important to clarify whether scalp follicles can also respond as human follicle response to androgens differ with body site. Therefore, human scalp follicles were grown in vitro in organ culture with PGF₂α, latanoprost, a PGF₂α analogue, and bimatoprost, a prostamide F₂α analogue, with, or without, appropriate antagonists, and the presence of PGF₂α (FP) and prostamide F₂α receptors were investigated using molecular biological and immunohiostochemical methods. Each treatment significantly stimulated follicle growth rate, the percentage of growing follicles, and the amount of hair produced in a dose-responsive manner (10nM-1μM); the receptor antagonists blocked these effects. Immunohistochemistry of frozen scalp sections demonstrated FP protein only in dermal papillae and connective tissue sheaths. RT-PCR identified FP and various prostamide F₂α receptors in anagen follicles and isolated dermal papillae and bulbar connective tissue sheath, but not in bulb matrix or other epithelial tissues. Therefore, isolated human scalp hair follicles can respond biologically to PGF₂α and related pharmaceuticals in organ culture via follicular receptors and express the genes and protein for FP and prostamide F₂α receptors. PGF₂α-related drugs appear to act directly on follicles via receptors in the regulatory dermal papilla. They offer an exciting, novel approach for treating alopecia and merit clinical investigation.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 6 uppsatser.

Thesis (M.Sc.). / Written for the Dept. of Pharmacology and Therapeutics. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references (p. 122-142).

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 5 uppsatser.

Orientador: Marco Antônio Alvarenga / Resumo: Na espécie equina, o embrião se movimenta no útero entre os dias 9 e 16 após a ovulação, o concepto apresenta uma forma esférica e se move constantemente no lúmen uterino mediante contrações miometriais, produzidas por estimulação química da vesícula embrionária. O deslocamento embrionário durante este período é essencial para o reconhecimento materno da gestação. Este longo período de estímulo garante que o embrião produza sinais anti-luteolíticos no endométrio, evitando a luteólise. Após 17 dias, a vesícula embrionária cessa a mobilidade e ocorre a fixação em um dos cornos. Durante a mobilidade, o embrião produz prostaglandinas PGE-2, PGF-2α e PGI-2. Se houver uma falha durante a migração embrionária, o reconhecimento materno da gestação pode ser afetado e consequentemente há lise do corpo lúteo, resultando na perda precoce da prenhes. O uso do anti-inflamatório flunixin meglumine imediatamente após a transferência do embrião é amplamente utilizado para prevenir uma reação inflamatória local, a luteólise e a perda embrionária precoce. No entanto, de acordo com a literatura, o ácido mefenâmico causa menos efeitos sobre a mobilidade embrionária e aumenta as taxas de gestação. O objetivo deste estudo foi elucidar o efeito do ácido mefenâmico sobre a mobilidade embrionária em éguas. Foram selecionadas 10 éguas para o estudo. Após a confirmação da gestação com ultrassom transretal, a mobilidade embrionária foi avaliada por ultrassonografia em série (a cada 5 minutos) durante 1 h... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the equine specie, the embryo moves in the uterus between days 9 and 16 after ovulation, the conceptus presents a spherical shape and moves constantly in the uterine lumen through myometrial contractions, produced by chemical stimulation of the embryonic vesicle. Embryonic displacement during this period is essential for maternal recognition of pregnancy. This long stimulus period ensures that the embryo produces anti-luteolytic signals to the endometrium, avoiding the luteolysis. After 17 days the embryonic vesicle stops the mobility and occurs the fixation in one of the horns. During mobility, the embryo produces prostaglandins PGE-2, PGF, and PGI-2. If there is a failure during embryo migration the maternal recognition of pregnancy may be affected and consequently lysis of corpus luteum, resulting in the early pregnancy loss. The use of the anti-inflammatory flunixin meglumine immediately after the embryo transfer is widely used in order to prevent a local inflammatory reaction, luteolysis, and early embryonic loss. However, according to the literature, mefenamic acid cause less effects on embryonic mobility and increases pregnancy rates. The objective of this study was to elucidate the effect of mefenamic acid on embryonic mobility in mares. There were selected 10 mares for the study. After confirmation of pregnancy with transrectal ultrasound, the embryo mobility was evaluated by serial ultrasonography (every 5 minutes) during 1 hour. This examination was considered t... (Complete abstract click electronic access below) / Mestre

Chronic inflammation has been linked to cancer. Prostaglandin E2 (PGE2) is the most pro-inflammatory lipid and one of the downstream products of 2 isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Although both COX isoforms have similar structure and function, they are encoded by different genes and show distinct expression patterns. COX-1 is expressed in most cells and tissues and remains constant under most physiologic conditions to play a housekeeping role whereas the COX-2 form is inducible and usually only expressed in response to various inflammatory stimuli. COX enzymes may be involved in both tumor establishment and maintenance of existing tumors. PGE2 exerts its effects on target cells by coupling to four subtypes of receptors which have been classified as EP1-4. Ovarian cancer is the most lethal gynecological malignancy and mainly occurs in older women. Prevention may be the best approach to reduce ovarian cancer. Ovarian cancer is the fifth leading cause of cancer death among women and the most lethal gynecological malignancy. There are at least 3 well established risk factors for ovarian cancer: age, family history and environmental factors. Ovarian cancer is mainly seen in older women when their ovaries are not reproductively functional. Close to half of the women with ovarian cancer (48%) are in the age group of 65 or older. Epidemiological and preclinical studies indicate that increased dietary intake of omega-3 fatty acids (OM-3FAs) reduces the incidence and growth of various cancers. Thus, increasing the consumption of OM-3FAs may be a nontoxic way to prevent or suppress ovarian cancer. Flaxseed is the richest vegetable source of omega-3 fatty acids which may be effective in the prevention of ovarian cancer. Fish oil is a source of OM-3FAs which may be effective in prevention of ovarian cancer. The main OM-6FA, Linoleic Acid (LA), is a direct precursor of the Arachidonic Acid (AA). Alpha-linolenic acid (ALA) is the main OM-3FA found in flax oil, whereas eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the OM-3FAs in fish oil. ALA is elongated to form EPA and DHA in the intestine. Celecoxib is a non-steroidal anti-inflammatory (NSAID) drug that selectively inhibits COX-2. There are evidences showing that Celecoxib has some anti-cancer properties. Progress in the treatment and prevention of ovarian cancer has been hampered due to the lack of an appropriate animal model and absence of effective chemo-prevention strategies. The domestic hens spontaneously develop ovarian adenocarcinomas that share similar histological appearance and symptoms such as ascites and metastasis with humans. Our first objective was to investigate the effect of flaxseed supplementation for one year on ovarian cancer and correlate its effects to expression of COX enzymes and concentrations of prostaglandins. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for one year. The severity of ovarian cancer was determined by gross pathology and histology. COX-1 and COX-2 localization and protein and mRNA expression and PGE2 and PGE3 concentrations in ovaries were measured by Immunohistochemistry, western blot, quantitative real-time PCR and LC-MS-MS, respectively. The results demonstrated a significant reduction in late stage ovarian tumors in the flaxseed-fed hens compared with the control diet-fed hens. In correlation with decreased ovarian cancer severity, concentrations of PGE2 and expression of COX-2 were diminished in ovaries of flaxseed-fed hens. PGE3 concentrations were below the level of detection. The results demonstrated that in normal ovaries, COX-1 was localized to the granulosa cell layer surrounding the follicles and ovarian surface epithelium (OSE) whereas COX-2 protein was localized to the granulosa cell layer in the follicle. Extensive COX-1 and COX-2 protein expression was found throughout the ovarian carcinoma. Our findings suggest that the flaxseed-mediated reduction in the severity of ovarian cancer in hens is correlated to the reduction in PGE2 in the ovaries of flaxseed-fed hens. Since no effect on ovarian cancer incidence was detected after feeding the 2. 5 year old hens with 10% flaxseed for 1 year, we designed a long-term study using 6 month old hens. Our objectives were: 1) to examine the expression of COX enzymes and PGE2 levels in ovaries and correlate them to ovarian cancer and aging 2) to determine if long-term consumption of a flaxseed enriched diet decreases ovarian cancer severity and incidence in the laying hen and to investigate its potential correlation with the expression of COX enzymes and PGE2 concentration. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for 4 years. The severity and incidence of ovarian cancer were determined by gross pathology and histology. COX-1 and COX-2 protein and mRNA expression and PGE2 concentrations in ovaries were measured by western blot, quantitative real-time PCR and ELISA, respectively. Our results indicated an increase in ovarian cancer incidence and expression of both COX enzymes in ovaries of older hens. In correlation with ovarian cancer incidence and COX enzymes expression, PGE2 concentrations were elevated with age. Ovaries with tumor had elevated COX-1 expression and PGE2 concentration compared to normal ovaries. Our findings suggest that the up-regulation of COX enzymes with age is the main contributing factor in the age associated increase in PGE2. Furthermore, elevated PGE2 in ovaries of hens concomitant with age suggests its important role in early stages of ovarian carcinogenesis. The results demonstrated that there was a reduction in ovarian cancer severity and incidence in hens fed flaxseed diet. In correlation with decreased ovarian cancer severity and incidence, concentration of PGE2 and expression of COX-2 were diminished in ovaries of hens fed flaxseed. Our findings suggest that the lower levels of COX-2 and PGE2 are the main contributing factors in the chemo-suppressive role of long-term flaxseed consumption in ovarian cancer in laying hens. These findings may provide the basis for clinical trials of dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer. Based on our previous findings, targeting COX expression and prostaglandin biosynthesis by dietary intervention using OM-3FAs and selective COX inhibitor can be an effective approach to prevent or suppress ovarian cancer. Thus, we conducted a series of studies to assess effect of fish oil, flax oil, Celecoxib, fish oil and Celecoxib combined or flax oil and Celecoxib combined on COX-1 and COX-2 expression, PGE2 concentrations, proliferation and apoptosis in normal and cancerous ovaries of laying hens. This study had not been performed in hens before, thus the first step was to find the optimum doses. In order to do so, we utilized one year old hens, divided them to groups of 6 hens, and fed them different doses of fish oil (50, 100, 175, 375 and 700 mg/kg), flax oil (100, 250, 500, 1000 and 1500 mg/kg) or Celecoxib (35, 65 and 100 mg/kg) for three weeks. The OM3-FAs andomega-6 fatty acids contents of egg yolks were determined by gas chromatography. Proliferation, apoptosis,COX-1, COX-2 and prostaglandin receptor subtype 4 (EP4) protein and mRNA expression and PGE2 concentration in ovaries were measured by PCNA, TUNEL, western blot, quantitative real-time qPCR and ELISA, respectively. The results indicated that 100 mg/kg fish oil was the most effective dose in reducing COX enzymes and PGE2, and increased apoptosis and reduced proliferation in ovaries. The lower doses of fish oil incorporated more OM-3FAs into yolks, reduced OM-6FAs and increased the egg laying frequency but did not affect EP4 expression. Unlike fish oil, the highest dose of flax oil (1500 mg/kg) caused the most significant reduction in COX expression and PGE2 concentration. Celecoxib was not perfectly selective in targeting COX-2, however, treating the hens with 65 mg/kg Celecoxib resulted in the most significant amelioration of PGE2 levels in ovaries. Using the optimum doses of fish oil, flax oil and Celecoxib, we aimed to investigate if these components can alter ovarian cancer end-points in normal and cancerous hen ovaries. There is an adverse relation between ovulation and health of ovaries. Thus, 3-4 year old hens were monitored for egg laying frequency and the hens with the least ovulation rate were selected for health assessment. The hens presenting poor health were scanned using ultrasound and if tumor mass and/or ascites were detected, they were chosen for this study. The hens with normal and cancerous ovaries were divided to groups and were fed fish oil, flax oil, Celecoxib, fish oil and Celecoxib combined, or flax oil and Celecoxib combined for 42 days. The results showed that fish oil and flax oil increased the incorporation of OM-3FAs into egg yolks in both normal and cancerous ovaries of hens. Fish oil reduced COX-1 and COX-2 in normal and cancerous ovaries. Fish oil, flax oil and Celecoxib reduced the COX-2 expression in ovaries. Combination of fish oil and Celecoxib and flax oil and Celecoxib decreased COX and PGE2 more than each of these treatments alone. The cancerous ovaries of hens treated with fish oil, flax oil, Celecoxib, and flax oil and Celecoxib combined increased the percentage of apoptotic cells compared to cancerous ovaries of control hens. The cancerous ovaries of hens treated with fish oil and Celecoxib had the highest number of apoptotic cells indicating that the combination of fish oil and Celecoxib is more effective than fish oil or Celecoxib alone. To our knowledge the present study provides the first insight into the efficacy of fish oil, flax oil, Celecoxib, alone or combined on the reduction of COX enzyme expression, PGE2 concentration and apoptosis in the normal and cancerous ovaries and further demonstrates the utility of the hen model for ovarian cancer. Our studies provided new insight into the potential mechanism of action of flaxseed, fish oil, flax oil and Celecoxib in the reduction of ovarian cancer and will establish the foundation for clinical trials to test the efficacy of dietary intervention for the prevention and suppression of ovarian cancer in women.