Polycystic ovary syndrome (PCOS) is the most common endocrinopathy disorder in reproductive age women. The symptoms of this disorder are the androgen excess seen with anovulation/oligoovulation or morphologically ovarian cysts. The aim of the study was to assess the efficacy of public health nutrition intervention designed to enhance healthy eating and lifestyle modification among PCOS patients attended the obstetrics and gynecology clinic at the American University of Beirut Medical Centre (AUB-MC) in Beirut, Lebanon. A prospective hospital based public health nutrition intervention was proposed in which 76 women with PCOS were recruited in the pilot study and 588 women were recruited in the scale-up intervention divided between PCOS and non-PCOS. During the scale up phase non-PCOS women were recruited to study the effect of the nutritional counseling on them as a way to compare the outcome with PCOS women. Recruited population were divided into 8 groups; group A: overweight/obese PCOS patient’s intervention (received weight management program with nutritional guidelines). Group B: overweight/ obese PCOS controls (received the usual heath care by the gynecologist), Group C: lean PCOS controls (received the usual heath care by the gynecologist), Group D: lean PCOS intervention (received weight maintenance program with nutritional guidelines ), Group E: overweight/obese non-PCOS patient’s intervention (received weight management program with nutritional guidelines) ,Group F: overweight/ obese non-PCOS controls, Group G: lean non- PCOS intervention (received weight maintenance program with nutritional guidelines), Group H: lean non-PCOS controls. Data were collected using a pre-validated questionnaire to capture sociodemographic variables, nutritional status, and physical activity, psychological and medical status. Blood analysis was carried out to determine biochemical indices. Assessment of study indicators were carried out at baseline, after 3 and 6 months from inception of intervention (pilot as well scale up). Patients in intervention groups attended a 6 month tailored nutrition counseling/education program (2 sessions per month), to enhance their understanding of their dietary intake and assist them with weight management, physical activity, healthy cooking, lifestyle, and food shopping. Following a six months pilot study intervention results have shown that 7% weight loss was achieved in overweight/ obese intervention groups and weight maintenance in lean intervention groups( Group A,B,C and D). There was a significant reduction in waist (-4.2 cm (±5.6)) and hip circumference (-3.1cm (±3.5)) with P < 0.001. There was no significant biochemical markers change (fasting blood sugar, CRP, LDL-C,HDL-C,TG,total cholesterol, fasting insulin, total testosterone,Vit D), however there was an increase in physical activity (3.1 hours/week (±1.5)) , and decrease in anxiety and depression score ( BDI-II and BAD-7); -0.8 (±0.8) and -0.7 (±0.7) with P < 0,001 compared to interventions. Following six months scale up intervention, the results have shown a weight reduction among overweight/obese PCOS women (group A) who lost, on average, 8.2 kg (P=0.001). Whilst non-PCOS women lost, on average 11.6 kg (P < 0.001)(Group E). Controls gained weight (Group B, D F and H). The biochemical, psychological and reproductive profile showed significant improvements among PCOS women (P < 0.001). Pregnancy rate increased to 70% among women trying to conceive. The results of this study have shown this intervention to be effective in Lebanese women with PCOS, decreasing their initial body weight by 5%- 10% and improving their reproductive, metabolic and endocrine profiles. This suggests the need for a nutritional intervention (nutritional guidelines) for women diagnosed with PCOS patients as a first line treatment. The study results support the effectiveness of lifestyle modification diet for PCOS women.

O objetivo do estudo foi confirmar se a redução da fecundidade dos mosquitos Aedes aegypti infectados por Plasmodium gallinaceum ocorre por morte das células foliculares dos ovários. Mosquitos infectados produzem menos ovos quando comparado aos mosquitos sadios. Uma explicação é a redução da viabilidade celular que ocorre nos ovários de fêmeas 18, 22 e 24 horas após o repasto sanguíneo infectado (RSI) como foi observado pela técnica MTT. Utilizando o acridine orange, não foi possível observar a morte das células foliculares no intervalo de 18 horas, mas 22 e 24 horas após o RSI essas células estão em morte em relação ao mesmo intervalo do repasto sanguíneo controle (RSC). A análise do DNA fragmentado foi realizada através do TUNEL. Ovários de 22 e 24 horas após RSC e RSI foram negativos nas regiões dos cortes histológicos examinados. Sendo assim, podemos concluir que, utilizando esses ensaios foi possível identificar a morte das células foliculares como uma resposta a redução da fecundidade, porém não foi possível determinar que o tipo de morte é apoptose. / The objective of this study was to confirm the hypothesis that the fertility reduction in Plasmodium gallinaceum-infected Aedes aegypti occurs by follicular cells death. A significant reduction in the number of eggs laid by infected mosquitoes was confirmed. It was observed a reduction of viable cells in 18, 22 and 24 hours PBM infected by MTT assay. It was not possible to observe cell death in ovary tissue 18 hours PBM infected, but the follicular cells showed orange color 22 and 24 hours indicating they are in death in relation to the same interval of PBM control. To determine if these cells exhibit apoptosis, we use the TUNEL which mark the fragmented DNA, a characteristic of the apoptosis process. Ovaries 22 and 24 hours PBM infected and control were negative for TUNEL marker from ovary histological preparations. Thus, we conclude that fecundity reduction occurs as a response to follicular cells death caused by P. gallinaceum infection but it was not possible to affirm if the type of follicular cells death is apoptosis.

by Wong, Cheuk-fai. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 95-99). / List of figures --- p.iv / List of tables --- p.v / Abbreviations --- p.vi / Abstract --- p.vii / Chapter I. --- INTRODUCTION --- p.1 / Chapter 1. --- Inhibin a brief review --- p.1 / Chapter 1.1 --- "Definition and nomenclature, including related substances" --- p.1 / Chapter 1.2 --- Structure --- p.2 / Chapter 1.3 --- Historical background of the inhibin concept --- p.4 / Chapter 1.4 --- Actions of inhibin --- p.9 / Chapter 1.5 --- Control of inhibin production --- p.10 / Chapter 1.6 --- Measurement --- p.11 / Chapter 1.6.1 --- Immunoassay --- p.11 / Chapter 1.6.2 --- Bioassay --- p.13 / Chapter 1.6.2.1 --- In vivo methods --- p.13 / Chapter 1.6.2.2 --- In vitro methods --- p.14 / Chapter 1.7 --- Inhibin in clinical studies --- p.15 / Chapter 2. --- Project design --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.2 --- Objectives --- p.20 / Chapter II. --- MATERIALS AND METHODS --- p.21 / Chapter 1. --- Materials --- p.21 / Chapter 1.1 --- Tracer preparation and purification --- p.21 / Chapter 1.2 --- Inhibin RIA --- p.21 / Chapter 1.3 --- Other immunoassays --- p.22 / Chapter 2. --- In-house inhibin RIA development --- p.22 / Chapter 2.1 --- The tracer preparation --- p.22 / Chapter 2.2 --- The radioimmunoassay --- p.25 / Chapter 2.3 --- Optimization of assay parameters --- p.26 / Chapter 2.3.1 --- Optimization of serum content and second antibody titre --- p.26 / Chapter 2.3.2 --- Verification of second antibodies' precipitating activity --- p.27 / Chapter 2. --- INHIBIN-EASIA --- p.27 / Chapter 3. --- Progesterone --- p.28 / Chapter 4. --- "Oestradiol, LH, and FSH" --- p.28 / Chapter III. --- RESULTS --- p.30 / Chapter Part I. --- In-house inhibin assay development --- p.30 / Chapter 1. --- Iodination and purification --- p.30 / Chapter 1.1 --- Step 1:Iodination followed by Sephadex column purification --- p.30 / Chapter 1.2 --- Step 2: Red A gel column purification --- p.33 / Chapter 1.3 --- Step 3: Sephadex column purification --- p.33 / Chapter 2. --- Inhibin RIA: Binding and antibody dilution curve experiment --- p.36 / Chapter 3.1 --- Verification of binding activity of the second antibody --- p.38 / Chapter 3.2 --- Optimization of serum content/ second antibody titre --- p.39 / Chapter 4. --- Discussion and conclusion --- p.41 / Chapter Part II. --- Hormone results of women undergoing in vitro fertilization --- p.42 / Chapter 1. --- Presentation of analytical results --- p.42 / Chapter 2. --- Comparison of hormone profiles of patients in the two GnRH agonist regimes --- p.43 / Chapter 2.1 --- Gonadotropins --- p.43 / Chapter 2.2 --- E2 --- p.56 / Chapter 2.3 --- Progesterone --- p.62 / Chapter 2.4 --- Inhibin --- p.68 / Chapter 3. --- Relationship between hormone --- p.74 / Chapter 3.1 --- Relationship between hormone changes --- p.74 / Chapter 3.2 --- Regression analysis --- p.87 / Chapter IV. --- DISCUSSSION --- p.89 / Chapter 1. --- Hormone profiles --- p.89 / Chapter 2. --- Hormone correlation --- p.91 / Chapter V. --- CONCLUSION --- p.94 / Chapter VI. --- REFERENCES --- p.95 / Chapter VIII. --- Appendix 1 Protocol for study on IVF and inhibin --- p.100 / Chapter IX. --- Appendix 2 Protocol for the management of IVF cycles --- p.101 / Chapter X. --- Appendix 3 Patients' results (table) --- p.103 / Chapter XI. --- Appendix 4 Patients ' results (graph) --- p.110

Orientador: Wilter Ricardo Russiano Vicente / Banca: Francisca Elda Ferreira Dias / Banca: Ivo Walter dos Santos / Banca: Paulo Henrique Franceschini / Banca: Francisco Guilherme Leite / Resumo: O principal objetivo das pesquisas com MIV canina tem sido o desenvolvimento e implementação adequados da fecundação in vitro (FIV) e do cultivo embrionário in vitro (CIV) para a obtenção de embriões das espécies de canídeos em extinção. Concomitantemente à publicação de dados sobre índices de MIV em cadelas, surgiram alguns relatos sobre taxas de maturação completa e indícios de retomada da meiose em oócitos provenientes de folículos pré-ovulatórios. Assim, o presente estudo objetivou investigar a incidência de maturação oocitária intraovariana de cadelas em anestro, estro natural e estro induzido. Os oócitos foram colhidos após fatiamento ovariano e imediatamente corados com bisbenzimida para avaliação dos diferentes graus de configuração cromossômica. Em conclusão podemos afirmar que há retomada e progressão da meiose em oócitos, no ambiente intrafolicular; não há diferença estatística (P<0,05) nas porcentagens das diferentes configurações cromossômicas de acordo com os estádios reprodutivos avaliados neste estudo (anestro/estro e estro induzido). / Abstract: The main canine MIV research goal has been adequate in vitro fecundation develop and implementation (FIV) and the in vitro embryo cultivation (CIV) to obtain in extinction canid species embryos. Concomitantly to data publication about bitch MIV indexes appeared some relates about complete maturation rates and indications of meiosis retaken in oocytes from pre-ovulatory follicles. This way, the present study aimed investigate the anestrus bitches interovarian oocytary maturation incidence, natural estrus and estrus-induced The oocytes were collected after the ovary slicing and immediately colored with bisbenzimide to evaluate the different chromosomic configuration degrees. As a conclusion, can be affirmed there is meiosis progression and resumption in oocytes, in the intra follicular environment. There were no statistical difference (P<0.05) between the different chromosomic configurations percentages according to the reproductive stages evaluated in this study (anestrus/estrus and estrus- induced). / Doutor

Yu, Man Ying Susana. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-117). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of contents --- p.viii / Symbols and abbreviations --- p.xii / Scientific names --- p.xiv / List of figures --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Regulation of ovarian follicle development --- p.3 / Chapter 1.2.1 --- Endocrine regulation --- p.3 / Chapter 1.2.1.1 --- Gonadotropins- FSH and LH --- p.3 / Chapter 1.2.1.2 --- Co-gonadotropin- growth hormone --- p.5 / Chapter 1.2.2. --- Paracrine regulation --- p.6 / Chapter 1.2.2.1 --- Activin --- p.6 / Chapter 1.2.2.2 --- Insulin-like growth factor I (IGF-I) --- p.7 / Chapter 1.3 --- The GH-IGF-I axis --- p.7 / Chapter 1.3.1 --- The somatomedin hypothesis --- p.8 / Chapter 1.3.2 --- "Structure and signaling of GH, GHR" --- p.8 / Chapter 1.3.3 --- Structure and signaling of IGF system --- p.9 / Chapter 1.3.4 --- Role of GH-IGF system in reproduction --- p.11 / Chapter 1.3.5 --- GH action in ovarian functions --- p.12 / Chapter 1.3.6 --- IGF-I action in ovarian functions --- p.13 / Chapter 1.3.7 --- The mini GH-IGF axis within the ovary --- p.14 / Chapter 1.4 --- Objectives of present study --- p.14 / Chapter Chapter 2 --- "Expression Profiles of the GH-IGF System in the Ovary of Zebrafish, Danio rerio" --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Material and Methods --- p.21 / Chapter 2.2.1 --- Animals --- p.21 / Chapter 2.2.2 --- Isolation of tissues and different stages of follicles from the zebrafish --- p.22 / Chapter 2.2.3 --- Separation of somatic follicle layers and oocytes --- p.22 / Chapter 2.2.4 --- Primary follicle cell culture --- p.22 / Chapter 2.2.5 --- Total RNA extraction --- p.23 / Chapter 2.2.6 --- Reverse transcription --- p.23 / Chapter 2.2.7 --- "Validation of semi-quantitative RT-PCR assays for GH (gh), GHR (ghr), IGF-I (igf1), IGF-II (igf2), and IGF-I receptor (igf1r)" --- p.24 / Chapter 2.2.8 --- Data analysis --- p.25 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Validation of semi-quantitative RT-PCR assays --- p.25 / Chapter 2.3.2 --- Spatial expression of GH-IGF in different tissues of zebrafish --- p.26 / Chapter 2.3.3 --- "Localization of gh, ghr, igf1, igf2 and igf1r within the zebrafish follicle" --- p.26 / Chapter 2.3.4 --- Temporal expression profiles of GH-IGF system during folliculogenesis --- p.28 / Chapter 2.4 --- Discussion --- p.28 / Chapter Chapter 3 --- Regulation of the GH-IGF-I System and Its Cross-talk with the Activin System in the Zebrafish Ovary --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Material and methods --- p.45 / Chapter 3.2.1 --- Animals --- p.45 / Chapter 3.2.2 --- Chemicals and hormones --- p.45 / Chapter 3.2.3 --- Primary follicle cell culture --- p.45 / Chapter 3.2.4 --- Preparation of ovarian fragments --- p.45 / Chapter 3.2.5 --- Total RNA extraction --- p.45 / Chapter 3.2.6 --- RT-PCR --- p.47 / Chapter 3.2.7 --- Construction of real-time PCR standards --- p.47 / Chapter 3.2.8 --- Real-time PCR and semi-quantitative RT-PCR --- p.48 / Chapter 3.2.9 --- Data analysis --- p.49 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- "Expression of growth hormone (gh), growth hormone receptors (ghr1 and ghr2\ IGF-I (igf1), IGF-II (igf2), IGF-I receptor (igf1ra and igf1rb), activin subunits (inhba and inhbb) and follistatin (fst) in cultured zebrafish ovarian fragments" --- p.49 / Chapter 3.3.2 --- "Establishment of real-time RT-PCR for zebrafish inhba, inhbb and bactin" --- p.50 / Chapter 3.3.3 --- GH regulation of activin expression in cultured zebrafish follicle cells --- p.50 / Chapter 3.3.4 --- GH regulation of IGF-I in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.5 --- IGF-I regulation of activin expression in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.6 --- Activin regulation of IGF system --- p.52 / Chapter 3.4 --- Discussion --- p.52 / Chapter Chapter 4 --- Production of recombinant zebrafish growth hormone --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Material and Methods --- p.71 / Chapter 4.2.1 --- Animals --- p.71 / Chapter 4.2.2 --- Construction of expression plasmids pPIC9K/zfGH --- p.71 / Chapter 4.2.3 --- Production of recombinant zebrafish GH using Pichia pastoris --- p.73 / Chapter 4.2.4 --- SDS-PAGE and silver staining --- p.74 / Chapter 4.2.5 --- Purification --- p.74 / Chapter 4.2.6 --- Primary follicle cell culture --- p.75 / Chapter 4.2.7 --- Zebrafish hepatic cell culture --- p.76 / Chapter 4.2.8 --- RNA extraction and RT-PCR --- p.76 / Chapter 4.2.9 --- Real-time PCR --- p.77 / Chapter 4.2.10 --- Cell culture and transient transfection --- p.78 / Chapter 4.2.11 --- Luciferase reporter gene assay --- p.78 / Chapter 4.2.12 --- Data analysis --- p.79 / Chapter 4.3 --- Results --- p.79 / Chapter 4.3.1 --- Production of recombinant zebrafish GH --- p.79 / Chapter 4.3.2 --- Effect of recombiant zfGH on the expression of activin β Aand βB in cultured zebrafish follicle cells --- p.80 / Chapter 4.3.3 --- Effect of zfGH on the expression of igf1 in cultured zebrafish hepatic cells --- p.80 / Chapter 4.3.4 --- Luciferase reporter gene assay --- p.81 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- General Discussion --- p.94 / Chapter 5.1 --- Overview --- p.94 / Chapter 5.2 --- Major achievements of the present study --- p.95 / Chapter 5.2.1 --- Demonstration of a local mini-GH-IGF-I axis within the zebrafish ovary --- p.96 / Chapter 5.2.2 --- Differential expression profiles of the GH-IGF system during folliculogenesis --- p.96 / Chapter 5.2.3 --- The inter-relationship of GH-IGF and activin-follistatin systems --- p.96 / Chapter 5.2.4 --- Production of recombinant zebrafish GH --- p.97 / Chapter 5.3 --- Future prospects --- p.97 / References --- p.102 / Symbols and Abbreviations / Symbols / α Alpha / β Beta / Abbreviations / 20β-HSD 20β-hydroxysteroid dehydrogenase / bp Base pair / cAMP Cyclic adenosine monophosphate / cDNA Complementary cDNA / CHO Chinese hamster ovary / "DHP 17α, 20β-dihydroxy-4-prenane-3 -one" / DNA Deoxyribonucleic acid / EGF Epidermal growth factor

Chronic alcohol (ethanol) consumption has been known to affect the major organs of the body and particularly the liver. However, the effects of chronic ethanol consumption on the female reproductive system remain relatively unstudied. A convenient way to study these effects is by analyzing laboratory mice that have been fed an ethanol diet for an extended period of time and comparing them to control mice. In this study, female mice were separated into control and ethanol fed groups. The mice were placed on their specified diets and observed over the course of six weeks. The mice were fed and weighed daily throughout the duration of the experiment. Once a week, vaginal washes were performed on both groups of mice to determine the stage of the estrous cycle for each mouse. At the end of the six weeks, the mice were sacrificed and the ovaries were harvested and fixed in 4% paraformaldehyde. The ovaries were then paraffin embedded and sectioned. Glass microscope slides were then stained using Hematoxylin and Eosin staining procedures for evaluation using standard light microscopy. The tissue’s morphology, follicle development, presence of corpora lutea, and overall appearance were analyzed. Due to the premature deaths of several mice in first group of ethanol fed mice, the experiment was repeated with three more groups of mice to obtain a better representation of data. The data from the control group was compared to that of the ethanol fed group. The mice that received the ethanol fed diet ceased to cycle and arrested in the diestrous phase of the estrous cycle. Our data indicates that the ovarian follicles within the ethanol fed mice show signs of degeneration in the 4b, 5a, 5b, 6, and 7 levels of development. There are also no notable corpora lutea present within the ovaries of the ethanol fed mice. Our findings indicate that chronic alcohol consumption has deleterious effects on ovarian morphology in mice.

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility and the most common endocrinopathy of women of reproductive age. Genome-wide association studies (GWAS) identified a number of loci associated PCOS in different ethnic populations, including women with Asian and European ancestry. Replication studies have confirmed some of these associations. Among the loci identified are those located near the LH receptor gene (LHCGR), a clathrin-binding protein gene (DENND1A) that also functions as a guanine nucleotide exchange factor, and the gene encoding RAB5B, a GTPase and protein involved in vesicular trafficking. The functional significance of one of these GWAS candidates (DENND1A) was supported by our discovery that a truncated protein splice variant of DENND1A termed DENND1A.V2, is elevated in PCOS theca cells, and that forced expression of DENND1A.V2 in normal theca cells increased CYP11A1 and CYP17A1 expression and androgen synthesis, a hallmark of PCOS. We previously proposed that the PCOS GWAS loci could be assembled into a functional network that contributes to altered gene expression in ovarian theca cells, resulting in increased androgen synthesis. Here we demonstrate the localization of LHCGR, DENND1AV.2 and RAB5B proteins in various cellular compartments in normal and PCOS theca cells. hCG and forskolin stimulation affects the distribution and co-localization of DENND1A.V2 and RAB5B in various cellular compartments This cytological evidence supports our PCOS gene network concept, and raises the intriguing possibility that LHCGR activation, via a cAMP-mediated process, promotes the translocation of DENND1A.V2 and RAB5B-containing vesicles from the PCOS theca cell cytoplasm into the nucleus, resulting in increased transcription of genes involved in androgen synthesis.

<p>Proteases of the plasminogen activator (PA) and the matrix metalloproteinase (MMP) enzyme systems are expressed in the ovulatory follicle and in the developing corpus luteum (CL). However, the functional role of these extracellular degrading protease systems in the ovulatory and CL development processes remains elusive. The first aim of this thesis was to develop a mouse model to study gonadotropin-induced CL formation. The second aim was to study the involvement of the PA and the MMP systems in gonadotropin-induced ovulation, and in CL formation and function.</p><p>A mouse model for gonadotropin-induced CL formation was developed in order to control the timing of CL formation. In this model, immature mice were induced to ovulate by administrating gonadotropins and the endogenous prolactin surges were mimicked by administration of prolactin twice daily from day 2 of CL development. We observed that steroidogenic acute regulatory protein (StAR) mRNA was highly expressed at days 3 and day 6 of CL development and the levels remained high until late stages of CL regression.</p><p>Since mice lacking plasminogen (plg-/-) only have a 14% reduction of ovulation efficiency, our hypothesis was that the MMP system could compensate for the loss of plasminogen. When administrating the MMP-inhibitor galardin to gonadotropin-primed ovulating mice, we found that wild-type mice (plg+/+ and C67BL/J6) and heterozygous mice (plg+/-) had an 18-20% reduction in ovulation efficiency as compared to untreated mice.</p><p>Two models for CL formation, the adult pseudopregnant (psp) mouse model and a model whereby immature gonadotropin-primed mice were treated with prolactin, were used to study the formation and function of the CL in plg-/- mice treated with galardin. At day 3 of CL development, we found no alterations other than a slightly lower number of CL in plg-/- mice. This is most likely a secondary effect of the lower ovulation efficiency found in these mice. On the other hand, we found a 54% reduction in serum progesterone levels in plg-/- mice and a 37% reduction in the plg+/- mice as compared to wild type mice. At day 6 of CL development we saw a 45 % reduction of serum progesterone level in the plg-/- mice and a 22 % reduction in the plg+/- mice. A similar trend was observed at day 3 of CL development in immature gonadotropinprimed mice treated with prolactin. Galardin treatment did not alter the results significantly and the CLs were healthy and viable in these mice.</p><p>In conclusion, our data suggest that both plasminogen and MMPs, alone or in combination, are dispensable for ovulation and for the formation of a viable CL under the conditions used in this study. The reduced serum progesterone levels observed in the plg-/- mice did not appear to be a result of defective CL formation. Instead, plasmin may have a novel role in the maintenance of luteal function. StAR expression may also be a good marker for CL development and regression in mice.</p>

The focus of the present thesis was on ultrasonographic, endocrine and molecular characterization of ovarian antral follicular waves in sheep. Transrectal ultrasonography and computer assisted image analysis were used to determine the feasibility of detecting ovulation and the forming corpus luteum (CL) and to non-invasively monitor CL differentiation and growth. High resolution transrectal ultrasonography and hormone measurements were used to assess changes in numbers of small ovarian antral follicles and their relationships to the emergence of follicular waves in cyclic ewes and to correlate pulsatile secretion of gonadotropins with follicular growth in a wave, during the mid to late-luteal phase of the ovine estrous cycle. A series of experiments were conducted, using treatment with injections of ovine follicle stimulating hormone (oFSH) and measurement of serum concentrations of FSH, in cyclic and anestrous sheep, to investigate the existence of follicular dominance. We also evaluated the characteristics of secretory patterns of FSH that are critical for follicular wave emergence, in anestrous ewes. The possible existence of an endogenous rhythm of FSH secretion, independent of ovarian antral follicular dynamics, was studied in ovariectomized ewes. Finally, ovarian antral follicles at defined stages of growth in a follicular wave (based on transrectal ultrasonographic observations) were collected from separate groups of sheep by ovariectomy, to profile the expression patterns of steroidogenic enzymes (3¦Â-HSD, 17¦Á-OH and aromatase) using immunohistochemistry and gray-scale densitometric analysis. <p> The results of the present studies showed that it is possible to detect ovulation and visualize developing CL as early as 12-24 h after ovulation in the ewe. Changes in echotexture of the CL were closely associated with its morphological and functional characteristics, and we concluded that computer assisted image analysis holds promise for the noninvasive monitoring of CL differentiation and growth. Follicles reaching ovulatory diameter (¡Ý 5 mm) emerged and grew in a wave-like pattern in sheep, but without variation in the number of small follicles (1-3 mm in diameter), as seen in cattle. We concluded that all follicles that are recruited to grow beyond 2-3-mm in diameter, to 4-mm diameter in a wave, succeed in reaching an ovulatory diameter of ¡Ý 5 mm in the ewe. The emergence and growth of ovarian antral follicles in follicular waves, in sheep, do not require changes in LH secretion and may perhaps involve changes in the follicular sensitivity to LH. The largest follicle of a wave, in sheep, appears to have limited effects on other small follicles and on the time of emergence of the next follicular wave. Thus, functional dominance, as is present in cattle, may be absent in sheep. An endogenous rhythm for periodic peaks in serum FSH concentrations that is independent of ovarian follicular dynamics may exist in sheep. The expression patterns of steroidogenic enzymes, in the theca and granulosa compartments of antral follicles growing in each follicular wave in the ewe, paralleled serum estradiol concentrations, with the exception of the concentrations of 3¦Â-HSD in granulosa cells, which increased continuously from follicles 3 mm in diameter to the preovulatory follicle after the LH surge. The largest follicle of any follicular wave, irrespective of the stage of the cycle, would appear to be mature enough to ovulate if a gonadotropin surge is provided.

The circadian system provides living organisms a means to adapt their internal physiology to constantly changing environmental conditions that exists on our rotating planet, Earth. Clocks in peripheral tissues are referred to as peripheral which may participate in tissue-specific functions. The first step to investigating the circadian regulation in the peripheral tissues of avians was to examine for the presence of avian orthologs of core components of the molecular clock using Quantitative real time (qRTPCR) assays. We investigated the avian spleen for daily and circadian control of core clock genes and regulation of the inflammatory response by the spleen clock. The core clock genes, bmal1, bmal2, per2, per3 and clock displayed both daily and circadian rhythms. Proinflammatory cytokines TNFα, IL-1β, IL-6 and IL-18 exhibited daily and circadian rhythmic oscillations. A differential expression of proinflammatory cytokine induction was observed in the spleen undergoing lipopolysaccharide (LPS)-induced acute inflammation. Exogenous melatonin administration during inflammation seems to enhance some and repress a few inflammatory cytokines, implying that melatonin is pleiotropic molecule. To compare and contrast the role of peripheral clocks in regulating energy balance and reproduction in layer vs. broiler chicken, the visceral adipose tissue (VAT), ovary and hypothalamus were examined for the presence of core clock genes were investigated in these two lines of poultry birds. Quantitative RT-PCR was employed to examine daily control of core clock genes in these three peripheral tissues over a 24hr period. The layer hens exhibit rhythmic oscillations in the mRNA abundance of the core clock genes in the VAT, ovary and the hypothalamus. The hypothalamus and VAT of the broiler hens exhibit rhythmic mRNA abundance of the core clock genes. However, the clock genes in the ovary of the broiler pullets exhibit marked reduction in their amplitude and rhythms over a 24hr period. The broiler hens are prone to poor energy balance, obesity and reproductive capacity. In summary, these data provide evidence for a functional link between the circadian clock and the ovary by determining clock gene regulation under conditions of disrupted or eliminated reproductive function vs. normal reproductive output.