Vascular Endothelial Growth Factor (VEGF) has been shown to be an absolute requirement for ovarian follicle development. Although VEGF is commonly regarded primarily as an angiogenic factor, granulosa cells are a major site of VEGF synthesis in the follicle and they express VEGF receptors (VEGFR1 and VEGFR2). Further, the development of the dominant follicle is characterised by a substantial increase in granulosa cell expression of VEGF and its receptors. In spite of this, potential non-angiogenic effects of VEGF in these follicles have not been elucidated. The objective of the three studies described in this thesis was to use an in vitro bovine granulosa cell model to investigate the roles of VEGF during development of the dominant follicle. In addition, in light of evidence in other cell types, potential interactions between VEGF signalling and heterotrimeric protein signalling in these follicles were also investigated. In the first study, granulosa cells were obtained from healthy follicles with diameters of 4 to 8 mm (corresponding to just before the selection of a dominant follicle during a follicular wave) or 9 to 14 mm (encompassing all developmental stages of a dominant follicle) and exposed to a range of VEGF concentrations (1 to 100 ng/ml) encompassing concentrations found naturally in bovine dominant follicles. VEGF at 1 ng/ml, but not at higher concentrations (P > 0.1), induced significant proliferation of bovine granulosa cells from 4 to 8 mm follicles (P = 0.024) and increased the proliferative response of these cells to FSH (P = 0.045). VEGF also induced a dose-dependent increase in ERK1/2 activation by granulosa cells from 4 to 8 mm follicles (P < 0.03) but did not have any effect on expression of the steroidogenic enzyme, CYP11A1, by these cells (P > 0.1). VEGF, at a dose of 1 ng/ml (P = 0.003), but not at higher doses (P > 0.1), induced an increase in COX-2 expression by granulosa cells from 9 to 14 mm follicles. In addition, LH stimulation of both ERK phosphorylation (P < 0.05) and COX-2 expression (P < 0.05) in granulosa cells from 9 to 14 mm follicles were prevented (P > 0.1) by specific inhibition of VEGFR2, indicating that VEGF may mediate COX-2 responses to LH in these cells. The second study sought to examine the expression of heterotrimeric G-protein á subunits and PLCâ isoforms by real-time PCR and westen blotting in bovine granulosa cells throughout follicle development to identify specific molecular components of heterotrimeric G-protein pathways that may functionally interact with intracellular VEGF signals. Results showed that GNAS, GNA11 and GNAI2 were all expressed at significantly (P < 0.05) higher levels in granulosa cells of pre-ovulatorysize follicles (10.0 to 13.9 mm) than in cells from smaller follicles (2.0 to 5.9 mm and 6.0 to 9.9 mm). In addition, all PLCB isoforms except PLCB2 were expressed in bovine granulosa cells with PLCB3 being more abundant than PLCB1 and -4. Levels of PLCB3 in granulosa cells from pre-ovulatory-size follicles were much higher (>16-fold; P < 0.005) than in smaller follicles. Immunocytochemical analysis revealed that PLCB3 was located primarily in the cytoplasm, whereas PLCB1 was distributed primarily in the nucleus. These results identified Gs, Gq/11, Gi2 and PLCâ3 as candidates for cross-talk between VEGF and heterotrimeric G-protein signalling during the development of the dominant follicle. The potential involvement of these molecules on VEGF-induced responses in granulosa cells from 9-14 mm follicles was investigated in the third study by determining the effects of specific inhibitors of Gi (pertussis toxin, PTX) or Gq/11 (YM-25489) or PLCB3 siRNAs on VEGF-induced p-ERK. Results showed a 2.3 fold mean increase in p-ERK in response to VEGF in the absence of G protein inhibitors (P < 0.0001) but a VEGF response that was completely or partially abolished, respectively, in the presence of PTX (P > 0.8) or YM-25489 (1.6-fold mean increase relative to untreated controls; P = 0.039). LH induced a 1.6 fold increase in p-ERK1/2 (P < 0.02) and this response was prevented by pre-incubation with PTX (P > 0.4) or YM-25489 (P > 0.5). In contrast, similar EGF-induced phosphorylation of ERK (about 5-fold relative to controls) occurred in the absence (P < 0.003) or presence of PTX (P < 0.003) or YM-25489 (P < 0.003). Transfection of granulosa cells with 3 siRNAs targeting PLCB3 that had been previously validated by western blotting and immunocytochemistry had no effect (P = > 0.7) on phosphorylation of ERK in response to VEGF, LH or EGF in granulosa cells. In conclusion, taken together, these results suggest novel roles of VEGF in stimulating granulosa cell proliferation and expression of COX-2 in bovine dominant follicles and implicate VEGF in synergising and/or mediating the effects of gonadotrophins in these cells. In addition, these results indicate a requirement for Gi2 and Gq/11 in VEGF activation of ERK1/2 and induction of the above responses in granulosa cells.

INTRODUÇÃO: Os programas de preservação de fertilidade baseados na criopreservação de gametas e tecido ovariano têm sido amplamente empregados para salvaguardar o potencial reprodutivo de mulheres em idade reprodutiva. Entretanto,o risco de reintrodução de células malignas após o reimplante do tecido e a dificuldade de aplicação dos protocolos de estimulação ovariana em pacientes estrogênio dependentes, dificultam o acesso destas pacientes aos programas de preservação de fertilidade já estabelecidos. O cultivo de folículos isolados vem se mostrando uma técnica promissora capaz de atender a essas pacientes, com resultados bastante satisfatórios. A recuperação de oócitos fertilizáveis tem se mostrado bem sucedida em camundongos resultando em nascidos vivos; já em outros mamíferos de grande porte,como primatas não humanos e humanos,há escassos relatos da obtenção de oócitos em estagio MII . OBJETIVO: Este estudo teve como objetivo analisar a eficiência do cultivo tridimensional in vitro no desenvolvimento de folículos ovarianos bovinos e a sua influência sobre o metabolismo oxidativo, a geração de estresse oxidativo celular e os níveis de transcritos de genes ligados atividade mitocondrial, apoptose e crescimento e desenvolvimento oocitário. MATERIAL E MÉTODOS: Foi realizado o cultivo in vitro tridimensional de folículos secundários bovinos por 16 dias em meio ?- MEM suplementado, em atmosfera de 5% de CO2 e 20% de O2 (Grupo in vitro - Gvt). Como controles foram incluídos dois grupos: um de folículos secundários isolados frescos não submetidos a cultivo (Grupo imaturos- GI) e outro de folículos extraídos já em estágios pré-antrais mais avançados do desenvolvimentos (Grupo in vivo- Gvv). Para a avaliação do desenvolvimento folicular, foram realizadas análises morfológica e testes de viabilidade, além da expressão por RT- qPCR de genes alvos para a atividade mitocondrial e estresse oxidativo. RESULTADOS: As análises do desenvolvimento dos folículos in vitro mostraram que 81,1% dos folículos secundários apresentaram crescimento, com uma taxa de viabilidade em torno de 84%. Cerca de 50% de oócitos recuperados após o cultivo estavam viáveis pela avaliação morfológica, embora nenhum deles tenha atingido o estágio final de maturação (MII). Além disso, o cultivo não promoveu aumento da expressão de genes ligados a apoptose o que também sugere uma boa viabilidade dos folículos cultivados. O mesmo não ocorreu com o sistema Kit Ligand e c-Kit, considerados como marcadores da capacidade global de crescimento e desenvolvimento folicular, verificou-se uma menor expressão de KL nas células somáticas da parede folicular. Com relação ao metabolismo oxidativo, embora o estado redox, marcado pela produção de NAD e FAD, estivesse bastante aumentado nos folículos produzidos in vitro quando comparado com o controle imaturo fresco e maturado in vivo, não se observou aumento das espécies reativas de oxigênio em oócitos obtidos destes mesmos folículos, podendo sugerir a manutenção da capacidade de controlar o balanço oxidativo por parte destas células. ,Também não houve alteração na expressão de genes ligados à biogênese mitocondrial entre os grupos estudos. CONCLUSÃO: O método de cultivo proposto neste estudo, utilizando o sistema 3D em matriz de gel de alginato, permitiu a maturação parcial de folículos secundários isolados, mantendo a viabilidade e características do metabolismo oxidativo semelhantes a folículos maturados in vivo de mesmo tamanho. Entretanto, há algum grau de comprometimento na saúde global do folículo, especialmente em relação às células somáticas da parede folicular. / The fertility preservation programs based on oocyte and ovarian tissue cryopreservation has been widely employed to safeguard the reproductive potential of these patients. However, the risk of reintroducting malignant cells after ovarian tissue transplantation and the difficulty of applying ovarian stimulation protocols in patients with estrogen dependent diseases hinder the access of these patients to already established fertility preservation programs. Isolated follicle culture appears as a promising technique for these cases, with satisfactory results. The recovery of fertilizable oocytes has been shown to be successful in mice resulting in live births; in other large mammals, such as non-human primates and humans, there are a few reports of the obtention of oocytes in the MII stage. OBJECTIVE: This study aimed to analyze the efficiency of in vitro three-dimensional culture in the development of bovine ovarian follicles and their influence on oxidative metabolism, on cellular oxidative stress generation and on the transcript levels of genes linked to mitochondrial activity, apoptosis and oocyte growth and development. MATERIAL AND METHODS: Three-dimensional in vitro culture of bovine secondary follicles was carried out for 16 days in supplemented ?-MEM medium, in an atmosphere of 5% CO2 and 20% O2 (In vitro Group - Gvt). As controls, two groups were included: one of the fresh none cultured isolated secondary follicles (GI-immature group) and one of follicles isolated in a more advanced preantral stage (in vivo Group-Gvv). For the evaluation of the follicular development, morphological analysis and viability tests were performed, besides the expression by RT-qPCR of target gene for mitochondrial activity and oxidative stress were also evaluated. RESULTS: Analysis of the in vitro follicle development showed that 81.1% of the secondary follicles have grown, with a viability rate of 84%. About 50% of oocytes recovered after culture were viable by morphological evaluation, although none of them had reached the final maturation stage (MII). In addition, the culture did not promote increased on the expression of apoptosis-linked genes which also suggests good viability of the cultured follicles. The same was not observed with the Kit Ligand and c-Kit, considered as markers of the global capacity of follicular growth and development; there was a lower expression of KL in the somatic cells from the follicular wall. In relation to the oxidative metabolism, although the redox state, marked by the production of NAD and FAD, was greatly increased in follicles produced in vitro when compared to the immature controls and the in vivo matured group, there was no increase on the production of reactive oxygen species in the oocytes obtained from these follicles, suggesting that these cells were able to control their oxidative imbalance. Also, there was no alteration in the expression of genes linked to mitochondrial biogenesis between the studied groups. CONCLUSION: The culture method proposed in this study, using the 3D system in an alginate gel matrix, allowed the partial maturation of isolated secondary follicles, maintaining the viability and oxidative metabolism characteristics similar to follicles of the same size matured in vivo. However, there is some degree of impairment in overall health, especially in relation to the somatic cells from the follicular wall.

Lau, Shuk Wa. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 84-102). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.v / Table of content --- p.vi / List of figures --- p.ix / Symbols and abbreviations --- p.x / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Folliculogenesis and its control --- p.2 / Chapter 1.2.1 --- Ovarian follicle growth and development --- p.2 / Chapter 1.2.2 --- Follicle recruitment and regulation --- p.4 / Chapter 1.2.3 --- Oocyte maturation and ovulation --- p.9 / Chapter 1.2.4 --- Intercellular communication between oocytes and somatic cells --- p.10 / Chapter 1.3 --- Overview of proteomics --- p.12 / Chapter 1.3.1 --- Two-dimensional gel electrophoresis --- p.13 / Chapter 1.3.2 --- Mass spectrometry --- p.14 / Chapter 1.4 --- Objectives of the study --- p.15 / Chapter Chapter 2 --- Proteomic Analysis of Folliculogenesis in Zebrafish Ovary --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Animals --- p.21 / Chapter 2.2.2 --- Isolation of ovarian follicles --- p.21 / Chapter 2.2.3 --- Protein extraction and quantification --- p.22 / Chapter 2.2.4 --- Two-dimensional electrophoresis --- p.23 / Chapter 2.2.5 --- Staining --- p.24 / Chapter 2.2.6 --- In-gel digestion --- p.24 / Chapter 2.2.7 --- Mass spectrometry --- p.25 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Establishment of the protein profiles of different follicle stages --- p.25 / Chapter 2.3.2 --- Mass spectrometry analysis on the differentially expressed proteins --- p.26 / Chapter 2.4 --- Discussion --- p.27 / Chapter Chapter 3 --- Characterization of Y-box Binding Protein 1 (YB-1) in Zebrafish --- p.46 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and Methods --- p.49 / Chapter 3.2.1 --- Animals --- p.49 / Chapter 3.2.2 --- Isolation of ovarian follicles --- p.49 / Chapter 3.2.3 --- Protein extraction and quantification --- p.49 / Chapter 3.2.4 --- SDS polyacrylaminde gel electrophoresis (SDS-PAGE) --- p.50 / Chapter 3.2.5 --- Western blot analysis --- p.50 / Chapter 3.2.6 --- RNA isolation and reverse transcription --- p.51 / Chapter 3.2.7 --- Semi-quantitative RT-PCR quantification of expression --- p.51 / Chapter 3.2.8 --- Data analysis --- p.52 / Chapter 3.2.9 --- Immunohistochemistry --- p.52 / Chapter 3.2.10 --- Cloning of full-length ybl cDNA from zebrafish ovary and construction of recombinant plasmid for expressing ybl --- p.53 / Chapter 3.2.11 --- Expression and purification of recombinant zebrafish YB-1 protein --- p.54 / Chapter 3.2.12 --- Immunoprecipitation --- p.55 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- Confirmation of the presence of YB-1 --- p.58 / Chapter 3.3.2 --- Tissue distribution of YB-1 protein and ybl gene expression in zebrafish --- p.58 / Chapter 3.3.3 --- Stage distribution of YB-1 protein and ybl gene expression in ovarian follicles --- p.59 / Chapter 3.3.4 --- Localization of YB-1 protein within the ovarian follicle --- p.59 / Chapter 3.3.5 --- Degradation of YB-1 in the ovary --- p.60 / Chapter 3.3.6 --- Production of recombinant YB-1 (zfYB-1) --- p.60 / Chapter 3.3.7 --- Identification of YB-1 -bound partners --- p.60 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 4 --- General Discussion --- p.77 / References --- p.84

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 2 uppsatser. Även tryckt utgåva.

Baigiamojo darbo tikslas buvo ištirti 3 metų amžiaus jūrų kiaulyčių kiaušidžių folikulų ir jų ovocitų morfometrinius ypatumus. LSMU Patologijos centre buvo atliktas trijų jūrų kiaulyčių preparavimas ir paimta tyrimams reikalinga medžiaga. Anatomijos ir Fiziologijos katedroje buvo atliktas histologinių preparatų paruošimas. Preparatai buvo naudojami atlikti mikroskopines nuotraukas naudojant histologinių vaizdų analizatorių (mikroskopą) „Olympus DP72 BX63“. Naudojant UTHSCSA Image Tool for Windows Version 3 programą buvo atlikta 3 jūrų kiaulyčių, kurių amžius 3 metai, užuomazginių, pirminių ir antrinių kiaušidės folikulų, bei juose esančių ovocitų, ovocitų branduolių, folikulinių ląstelių bei pamatinės membranos morfometriniai matavimai. Statistinė duomenų analizė atlikta naudojant statistinę programą ANOVA (Statistica Version 10, StatSoft inc.). Mūsų tyrimo metu buvo nustatyta, kad 3 metų amžiaus jūrų kiaulyčių kiaušidžių užuomazginių folikulų diametras svyravo nuo 23,22±2,62 µm iki 26,16±3,58 µm, ovocitų diametras svyravo nuo 6,26±1,13 µm iki 7,37±0,51 µm. Pirminių kiaušidžių folikulų diametras svyravo nuo 48,68±1,47 µm iki 61,78±4,33 µm, ovocitų dydis siekė nuo 13,56±5,48 µm iki 14,61±2,79 µm. Antrinių kiaušidžių folikulų diametras svyravo nuo 149,34±29,97 µm iki 209,46±52,54 µm, ovocitų diametras svyravo nuo 41,6±8,35 µm iki 63,59±4,67 µm. Visų tirtų jūrų kiaulyčių užuomazginių kiaušidžių folikulų diametras, plotas, ovocitų diametras ir plotas, ovocitų branduolių... [toliau žr. visą tekstą] / Master ending work includes 43 pages, 35 pictures, to write the work was used 32 literary sources. The aim of this work was to investigate morphometric features of different stages ovarian follicles and oocytes in 3 years old three guinea pigs. 3 guinea pigs‘ preparation was performed in the LSMU Pathology center, was taken required material for the research. Histological preparations laying in were performed in the Anatomy and Physiology department. Preparations were used perform microscopic images using histological image analyzer (microscope) „Olympus DP72 BX63“. Using UTHSCSA Image Tool for Windows Version 3 program was performed 3 guinea pigs, with age 3 years, primordial, primary and secondary ovarian follicles’, oocytes’, oocyte nucleis’, follicular cells’ and basement membranes’ morphometric measurements. Statistical analysis of the data performed using the statistical program ANOVA (Statistical Version 10, Stat Soft inc.). Our research showed, that 3 years old guinea pigs‘ primordial ovarian follicle diameter ranged from 23,22±2,62 µm till 26,16±3,58 µm, oocyte diameter ranged from 6,26±1,13 µm till 7,37±0,51 µm. Primary ovarian follicle diameter ranged from 48,68±1,47 µm till 61,78±4,33 µm, oocyte size was from 13,56±5,48 µm till 14,61±2,79 µm. Secondary ovarian follicle diameter ranged from 149,34±29,97 µm till 209,46±52,54 µm, oocyte diameter ranged from 41,6±8,35 µm till 63,59±4,67 µm. All guinea pigs primordial follicles’ diameter and area, oocytes’ diameter... [to full text]

Tiriamojo darbo sritis – histologiniai kačių kiaušidžių tyrimai. Darbo tikslas buvo ištirti keturių kačių nuo 7 mėn. iki 1,8 metų įvairių stadijų kiaušidžių folikulus ir juos įvertinti.Kačių kiaušidės buvo paimtos ovariohisterektomijos metu.Kiaušidės supjaustytos į 5µm storio pjūvius, pjūviai dažyti hematoksilino-eozino dažais. Folikulai suskirstyti pagal išsivystymą ir matmenis į užuomazginius, pirminius, ankstyvuosius antrinius. Atlikti matavimai, išmatuoti užuomazginių, pirminių, antrinių folikulų diametrai, plotai, folikulų ovocitų diametrai, plotai, jų branduolių diametrai, plotai, folikulinės ląstelės – jų aukštis ir plotis, pamatinė membrana, antrinių folikulų skaidrioji zona. Ištirta, jog užuomazginių, pirminių bei antrinių folikulų, ovocitų ir jų branduolių diametras, plotas, pamatinės membranos storis ir antrinių folikulų ovocitų skaidriosios zonos storis yra panašūs. Užuomazginių folikulų ovocitą gaubia vienas sluoksnis plokščiųjų folikulinių ląsteliu – joms būdinga tai, jog, jų plotis didesnis už aukštį, o pirminių folikulų ovocitą gaubia kūbiškosios ląstelės, kurių tiek aukštis, tiek plotis panašūs.Antrinių ankstyvųjų folikulų ovocitų paviršių gaubia 2-3 sluoksnių folikulinių ląstelių, kurių plotis ir aukštis yra panašūs. Statistinių duomenų patikimumas 95 % arba p<0,05. / Range of investigation is histological analysis of feline ovary. The purpose of this scientific research is to explore various stages ovarian follicles in four cats (age 7 months- 1,8 years).Ovaries were cut into 5 µm sections, than sections were stained by eosin-hematoxylin. Folicles were classified regarding to their development and dimensions into primordial, primary and early secoundary follicles. Were performed measurements.Were measured diameters and areas of primordial, primary and early secoundary follicles, diameters and areas of oocytes and nucleus of oocytes, heigt and width of folliculas cells, thickness of basal lamina .Also were measured zona pellucida of secoundary follicles. Results revealed that diameter and area of primordial, primary and early secoundary follicles, oocytes and nucleus were comparable.Basal lamina thickness were simmilar too, the same thing is about zona pellucida in secoundary follicles. Primordial follicle is surrounded by one layer of squamous cells - basic feature of these cells is that their width is much bigger than height meanwhile primary follicle is surrounded by cuboidal one layer cells with almost equal width and height. Cells of early secoundary follicle are surrounding oocyte at least with two layers and their width and height are almost equal. Calculated statistical data reliability is 95 % or p<0,05.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Submitted by (edna.saturno@ufrpe.br) on 2016-07-21T14:11:28Z No. of bitstreams: 1 Diogo Manoel Farias da Silva.pdf: 910423 bytes, checksum: 04f5adf7d2e8585ac8f1123c2dfff6f9 (MD5) / Made available in DSpace on 2016-07-21T14:11:28Z (GMT). No. of bitstreams: 1 Diogo Manoel Farias da Silva.pdf: 910423 bytes, checksum: 04f5adf7d2e8585ac8f1123c2dfff6f9 (MD5) Previous issue date: 2014-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Factors regulating apoptosis can determine the oocyte viability; however there are few studies about the genes of these factors and their action in the antral follicles cumulus-oocyte complex (COC) development, neither about the relationship with Leptin (LEP) and its receptor (LEPR) in sheep. The aim of this study was evaluate the mRNA expression of LEP, LEPR, and the apoptotic regulating factors (Bcl-2, Bax, p53 and caspase 3), in oocytes (OO) and cumulus cells (CC) from different sizes of antral follicles. Twenty pools of OO (20 unities) and of CC (from 20 COC) harvested from follicles > 3 mm and ≤ 3 mm were evaluated. The gene expression was determined after mRNA extraction and further cDNA amplification by real time PCR. Bcl2, p53 and caspase 3 genes were expressed in all cells and follicles size. Bax expression was observed only in OO. LEP and LEPR were expressed only in CC. The expression of apoptosis promoting factors was higher in OO > 3mm, while Bcl2 (anti-apoptotic) expression was higher in OO ≤ 3 mm. Comparing OO with CC from the same size of follicles, there was higher expression of Bcl2 in OO ≤ 3 mm, and lower expression of p53 in OO > 3mm. LEPR tended to be more expressed in CC > 3mm than ≤ 3 mm. Was observed a positive correlation between p53 and BAX expression in OO (r = 0.66), and between Bax in OOs and LEPR in CCs (r = 0.64). It was concluded that the expression of anti- and pro-apoptotic genes in COC can be related to the follicle and oocyte development, since follicles ≤ 3 mm presented lower expression of factors promoting apoptosis. Additionally, leptin can be a regulatory factor of oocyte development due presence of LEPR in CC. / Fatores reguladores da apoptose podem determinar a viabilidade de oócitos, entretanto há poucos estudos referentes à ação dos genes destes fatores no desenvolvimento do complexo cumulus-oócito (COC) de folículos antrais, nem sua relação com a Leptina (LEP) e seu receptor (LEPR) em ovinos. O objetivo deste estudo foi avaliar a expressão do mRNA da LEP, LEPR, e dos fatores que levam a regulação da apoptose (Bcl-2, Bax, p53 e caspase), em oócitos (OO) e células do cumulus (CC) de folículos antrais ovinos de diferentes tamanhos. Foram avaliados 20 pools de OO (20 unidades) e de CC (de 20 COC) obtidos de folículos > 3 mm e ≤ 3 mm. A expressão gênica foi avaliada pela extração do mRNA e posterior amplificação do cDNA em real time PCR. Bcl2, p53 e caspase 3 foram expressos em todas as categorias de células e tamanho folicular. A expressão da Bax foi observada apenas em oócitos. LEP e LEPR foram expressos apenas em CC. A maior expressão dos fatores promotores da apoptose foi em OO > 3mm, enquanto a Bcl2 (anti-apoptótica) foi mais expressa nos OO ≤ 3 mm. Comparando OO com CC das mesmas categorias de folículos, houve maior expressão de Bcl2 nos OO ≤ 3 mm, e menor de p53 nos OO > 3mm. LEPR foi mais expressa em CC > 3mm. Houve correlação positiva entre a expressão de p53 e BAX em oócitos (r = 0,66), e entre Bax em OO e LEPR em CC (r = 0,64). Conclui-se que a expressão dos genes anti- e pró-apoptóticos em COC pode estar relacionada com o desenvolvimento folicular e oocitário, sendo que os menores (≤ 3 mm) apresentam menor expressão dos fatores promotores da apoptose. Adicionalmente, a leptina pode ser um fator regulatório do desenvolvimento oocitário devido a presença de seus receptores nas CC.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-04-20T12:31:20Z No. of bitstreams: 2 Tese - Eduardo Camelo de Castro - 2015.pdf: 5148354 bytes, checksum: bbedf154e2b63e301d86d884047f4fed (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-04-20T12:34:39Z (GMT) No. of bitstreams: 2 Tese - Eduardo Camelo de Castro - 2015.pdf: 5148354 bytes, checksum: bbedf154e2b63e301d86d884047f4fed (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-04-20T12:34:39Z (GMT). No. of bitstreams: 2 Tese - Eduardo Camelo de Castro - 2015.pdf: 5148354 bytes, checksum: bbedf154e2b63e301d86d884047f4fed (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-01-31 / OBJECTIVES: To evaluate the variability of three-dimensional automatic counting of follicles measuring between 2 - 6 mm and 2 - 10 mm during the menstrual cycle. Considering that this is a standard exam for the early follicular phase, to check if this test can be applied during other phases of the ovarian cycle. METHODS: A Prospective observational study. Serial transvaginal ultrasound scans were performed from 04/20/2013 to 10/30/2014 on infertile patients. Inclusion criteria included age between 18 and 35 years, body mass index 18-25 kg / m2, regular menstrual cycles, no history of ovarian surgery and no hormonal changes to TSH, prolactin, fasting insulin and fasting glucose. Those patients with ovarian cysts or who did not complete one or more days of the serial transvaginal ultrasound scans and who did not undergo three-dimensional (3D) scanning with the researcher, were excluded. The antral follicle count was taken in 3D mode with automated software. Visits were scheduled for early follicular (2 to 5 days), mid-follicular (days 6 - 10), periovulatory (days 12 - 16) and luteal (days 20 - 26) phases of the menstrual cycle. RESULTS: Forty-five women were included. The Friedman test showed that the total number of antral follicles measuring 2-6 mm varied significantly (p=0.001) across the four periods of the menstrual cycle. The Paired Student’s t-test showed a significant increase in follicles count 2-6 mm from the mid-follicular and periovulatory to the luteal phase. We found no significant intracycle variation between the small antral follicles (2-6 mm) in the early follicular, mid-follicular and periovulatory phases. The Friedman test showed that the total number of antral follicles measuring 2-10 mm varied significantly (p=0.003) across the menstrual cycle. Paired Student’s t-test showed a significant increase in count from the early follicular to the mid-follicular phase. CONCLUSIONS: The variation of three-dimensional automatic follicles counting 2-6 mm in the early follicular, mid-follicular and periovulatory phases was not statistically significant. There was a significant variation in the 3D automatic count of ovarian follicles measuring 2-10 mm during the menstrual cycle. The significant variability in the counting of antral follicles measuring 2-10 mm across the menstrual cycle does not permit this examination to be carried out of early follicular phase. / OBJETIVOS: Avaliar a variabilidade da contagem automática tridimensional dos folículos de 2 a 6 mm e de 2 a 10 mm durante o ciclo menstrual. Considerando que este exame esta padronizado para a fase folicular precoce, verificar se este teste pode ser aplicado durante as outras fases do ciclo ovariano. METODOLOGIA: Estudo prospectivo observacional. Foram incluídas todas as pacientes inférteis que fizeram monitorização da ovulação de 20/04/2013 a 30/10/2014, que tinham entre 18 e 35 anos, índice de massa corporal de 18 a 25 kg/m2, ciclos menstruais regulares, sem história de cirurgia ovariana e sem aterações hormonais sugestivas de doenças endócrinas como alteração da dosagem do TSH, prolactina, insulina de jejum e glicemia de jejum. Foram excluídas as pacientes que apresentaram cistos ovarianos, as que faltaram em um ou mais dias da monitorização da ovulação e as que não realizaram a monitorização no ultrassom tridimensional (3D) com o pesquisador. A contagem dos folículos antrais (CFA) foi feita pelo modo 3D com software de automação na fase folicular precoce (2º ao 5º dia), na fase folicular media (6º ao 10º dia), no período periovulatório (12º - 16º dia) e na fase lútea (20º ao 26º dia). RESULTADOS: Quarenta e cinco mulheres foram incluídas. Houve diferença entre as médias das contagens dos folículos que mediram de 2 a 6 mm (p=0,001) e 2 a 10 mm (p=0,003) pelo teste de Friedman que avaliou conjuntamente a CFA das quatro fases do ciclo. Quando se aplicou o teste t-Student pareado, houve um aumento significativo na CFA de 2 a 6 mm quando se comparou a contagem desses folículos na fase folicular média e periovulatória com a contagem da fase lútea. Não houve diferença estatisticamente significante entre a contagem destes folículos pequenos nas fases folicular precoce, folicular média e periovulatória. Houve um aumento significativo na CFA de 2 a 10 mm da fase folicular precoce para a fase folicular média quando se aplicou o teste t-Student pareado. CONCLUSÕES: A variação da contagem automática tridimensional dos folículos de 2 a 6 mm, nas fases folicular precoce, folicular média e periovulatória, não mostrou significância estatística. Houve uma variação significativa da contagem automática 3D dos folículos ovarianos de 2 a 10 mm durante o ciclo menstrual. A variabilidade significativa da contagem dos folículos antrais de 2 a 10 mm durante o ciclo não permite que este exame seja realizado fora da fase folicular precoce.

Alors que le protéome de l'ovocyte isolé, aux stades VG et MII a déjà été étudié, celui du follicule en croissance n'a jamais été décrit.Dans cette étude, nous avons cherché à identifier, comparer et caractériser les profils protéiques de follicules ovariens de souris à trois stades de leur développement in vitro distincts morphologiquement : follicules secondaires en début de culture - stade initial (IS), follicules avec une rupture complète de la membrane de Slavjanski (RMS) et follicules avec une cavité similaire à l'antrum (FA).Après un préfractionnement par IEF et une analyse LC-MS/MS en deux configurations (1D et 2D), 1403 protéines ont pu être identifiées dans le follicule ovarien de souris. 43,4 % (609) des protéines identifiées étaient communes aux trois stades et d'autres ont été identifiées uniquement à un stade : 71 au stade IS, 182 au stade RMS et 193 au stade FA. De plus, on a identifié 365 protéines qui n'avaient pas été décrites antérieurement dans le protéome de l'ovocyte ce qui pourrais indiquer qu'elles sont exprimées dans les cellules somatiques du follicule. Des analyses qualitatives et quantitatives complémentaires ont démontré une surreprésentation pour 44 fonctions biologiques par rapport aux fonctions biologiques des gènes constituant le génome de Mus musculus et mis en évidence des différences d'expression et d'abondance des protéines liées au cycle cellulaire, à la fixation des ions de calcium et à la glycolyse selon le stade de développement. Ces résultats représentent un point de départ pour beaucoup d'autres études de caractérisation moléculaire du développement folliculaire. / Until now only the proteome of isolated oocyte from fully grown follicle were described with the aim of oocyte maturation characterization. However the ability of oocyte to mature is acquired during its development within the follicle. Thus in this study we proposed a protein identification and characterization of whole mice ovarian follicle at three morphological stages during in vitro development: early secondary stage, described as initial stage (IS), follicles with a complete Slavjanski membrane rupture (RMS) and follicles with an antrum like cavity formation (FA). Using an IEF pre fractionation before protein digestion and two configurations of LC-MS/MS analysis (1D and 2D), 1403 proteins were successfully identified in the murine ovarian follicle. From those, 43.4 % (609) were commonly identified in the three stages and some were identified only at one single stage: 71 at IS stage, 182 at RMS stage and 193 at FA stage. Compared to the proteomes of isolated oocyte previously described, 365 proteins were only identified in our samples indicating that those ones were probably expressed in the somatic cells of the follicle. Additional qualitative and quantitative analysis highlighted 44 biological processes over represented in our samples when compared to Mus musculus gene database and different expressions and protein abundance implicated in cell cycle, calcium ion binding and glycolysis, throughout in vitro follicle development. This report represents so far the most complete catalogue of follicle proteins and could be an important milestone in the proteomic study of the follicle metabolism throughout in vitro development.