O zebrafish (Danio rerio) tem se destacado na pesquisa biomédica por sua homologia fisiológica e genética aos humanos. No entanto, há poucos relatos sobre a criopreservação ovariana desta espécie. Assim, pesquisamos a utilização de um recipiente de metal na vitrificação de tecido ovariano de zebrafish. O objetivo foi avaliar a sobrevivência e o desenvolvimento in vitro de folículos de zebrafish após a vitrificação de fragmentos ou ovários inteiros usando a cápsula de metal. Primeiro, testamos quatro soluções de vitrificação (VS1 – 1,5 M metanol + 4,5 M propilenoglicol; VS2 – 1,5 M metanol + 5,5 M Me2SO; VS3 – 1,5 M metanol + 4,5 M propilenoglicol + 0,5 M sacarose; VS4 – 1,5 M metanol + 5,5 M Me2SO + 0,5 M sacarose) e cinco estágios de desenvolvimento folicular utilizando o teste de coloração supravital iodeto de propídio combinado com diacetato de fluoresceína. Estes resultados mostraram que os folículos em estágio I, imaturos, apresentaram as maiores taxas de sobrevivência celular e que VS1 foi a melhor solução em termos de viabiidade. No Experimento 2, utilizou-se VS1 para vitrificar o tecido ovariano em diferentes dimensões (fragmentos ou ovários inteiros) e em dois diferentes recipientes (palheta de plástico ou cápsula de metal). Para avaliar a sobrevivência e o crescimento folicular dos folículos em estádio I, o diâmetro dos folículos foi mensurado antes e depois de cultivo in vitro por 24 horas. A morfologia folicular foi analisada por microscopia de luz após vitrificação utilizando a cápsula de metal. Os dados mostraram que a morfologia de folículos imaturos foi bem preservada após a criopreservação. A taxa de sobrevivência folicular foi maior (P <0,05) em fragmentos vitrificados, quando comparados com a vitrificação de ovários inteiros. Não houve diferenças significativas na sobrevivência e crescimento folicular entre os dois recipientes de vitrificação, palheta de plástico ou cápsula de metal. No entanto, a cápsula de metal diminui os riscos de contaminação, pois é hermeticamente fechada evitando contato com nitrogênio líquido e poder ser esterilizada, em vista que é manufaturada em aço inoxidável. Por essas razões, acreditamos que a cápsula de metal tem um uso potencial em reprodução humana para a vitrificação em grau clínico de tecido ovariano. / Zebrafish (Danio rerio) has excelled in biomedical research for its physiological and genetic homology to humans. However, there are few reports on ovarian cryopreservation of this specie. Thus, we studied the use of a metal capsule to vitrify zebrafish ovarian tissue. The aim of this study was to assess the survival and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the metal capsule. First, we tested four vitrification solutions (VS1 - 1.5 M methanol + 4.5 M propylene glycol; VS2 - 1.5 M methanol + 5.5 M Me2SO; VS3 - 1.5 M methanol + 4.5 M propylene glycol + 0.5 M sucrose; VS4 - 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose) and five follicular developmental stages using fluorescein diacetate and propidium iodide supravital staining test. These results showed that immature follicles, stage one, presented the highest survival rates and VS1 the best vitrification solution in terms of viability. In Experiment 2, we used VS1 to vitrify ovarian tissue in different dimensions (fragments or whole ovaries) and tested two different carriers (plastic straw or metal capsule). To evaluate follicular survival and growth of stage I, we measured follicle diameter before and after twenty-four-hour in vitro culture. The follicular morphology was analyzed by light microscopy after vitrification using the metal capsule. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P<0.05) on vitrified fragments, when compared to whole ovaries. There were no significant differences on follicular survival and growth between the two vitrification devices, plastic straw or metal capsule. However, the metal capsule being tightly sealed and manufactured in stainless steel avoids contact with liquid nitrogen and can be sterilized reducing contamination risk. These reasons lead us to believe that the metal capsule has a potential use in human reproduction for the clinical grade vitrification of ovarian tissue.

Protocolos de sucesso para a maturação in vitro de oócitos de peixe são importantes, uma vez que é necessário para garantir uma fertilização bem sucedida, formação do zigoto, crescimento do embrião e seu completo desenvolvimento. Em algumas espécies, a eficiência deste processo ainda é muito baixa ou restrita a poucas substâncias que podem ser utilizadas. Assim, pesquisou-se a utilização de hormônios alternativos ao protocolo já existente para maturação in vitro de ovócitos de zebrafish. O objetivo foi avaliar a eficiência do extrato de hipófise de carpa (EHC), dos hormônios folículo estimulante (FSH) e luteinizante (LH) para fazer a maturação dos ovócitos estádio III de zebrafish. Os oócitos estádio III foram colocados em meio de cultivo Leibovitz modificado, suplementado com soro fetal bovino e adicionado o hormônio correspondente a seu tratamento (T1-controle; T2-16 μg/ml de EHC; T3- 32 μg/ml de EHC; T4- 48 μg/ml de EHC; T5- 64 μg/ml de EHC; T6- 80 μg/ml de EHC; T7- 0,5 μg/ml de FSH; T8- 0,5 μg/ml de LH e T9- 0,5 μg/ml de FSH e 0,5 μg/ml de LH). A taxa de maturação foi avaliada através da visualização da quebra da vesícula germinal (GVBD). Em todos os tratamentos houve maturação, embora o EHC tenha demonstrado taxas de maturação muito baixas (T2= 12,8%; T3=24,8%; T4=27%; T5=22,7%; T6=9,7%) e inferiores em relação a maior eficiência dos hormônios gonadotrópicos (T7=16%; T8=35%; T9=50%). Além disso foi possível verificar a viabilidade dos oócito através da fertilização in vitro do melhor tratamento (T9) com uma taxa de eclosão e desenvolvimento em larva de 60%. Os resultados da maturação in vitro utilizando estes indutores hormonais em oócitos estádio III de zebrafish mostraram-se promissores, e reforçam as perspectivas para o aprimoramento e uso desta técnica para produção in vitro de embriões viáveis. / Successful protocols for maturation of oocytes are important, as it is necessary for ensuring successful fertilization, zygote formation, embryo growth and full development. In some species the efficiency of in vitro maturation is still very low or is still restricted to a little amount of substances which can be used for the matter. Thus, we studied the use of alternative hormones to the existing protocol for in vitro maturation of zebrafish oocytes. The aim of this study was to evaluate the efficiency of the use of carp pituitary extract (CPE), the follicle stimulating hormone (FSH) and luteinizing hormone (LH) to oocyte maturation stage III of zebrafish. Oocytes stage III were placed in modified Leibovitz culture medium, suplemented with fetal bovine serum and added to the correnponding hormone treatment (T1-control; T2-16 g / ml of CHE; T3 32 g / ml of CHE, T4 - 48 g / ml of CHE; T5- 64 g / ml of CHE; T6- 80 g / ml of CHE; T7- 0.5 g / ml of FSH, T8 0.5 mg / ml of LH and T9- 0.5 g / ml of FSH and 0.5 mg / ml LH). The maturation rate was assessed by the germinal vesicle break down (GVBD). In all cases there was maturation, though the EHC has demonstrated fairly low maturation rate (T2= 12,8%; T3=24,8%; T4=27%; T5=22,7%; T6=9,7%) and lower in relation of the high efficiency presented by the gonadotropic hormones (T7=16%; T8=35%; T9=50%). In addition it was possible to verify the viability of the oocyte through IVF of the best treatment (T9) with a result of 60% of hatching and larvae development rate. The results of maturation in turn using this hormones in stage III oocytes of zebrafish proved promising, and enhance the prospects for improvement and use of this technique for in vitro production of viable embryos.

O zebrafish (Danio rerio) tem se destacado na pesquisa biomédica por sua homologia fisiológica e genética aos humanos. No entanto, há poucos relatos sobre a criopreservação ovariana desta espécie. Assim, pesquisamos a utilização de um recipiente de metal na vitrificação de tecido ovariano de zebrafish. O objetivo foi avaliar a sobrevivência e o desenvolvimento in vitro de folículos de zebrafish após a vitrificação de fragmentos ou ovários inteiros usando a cápsula de metal. Primeiro, testamos quatro soluções de vitrificação (VS1 – 1,5 M metanol + 4,5 M propilenoglicol; VS2 – 1,5 M metanol + 5,5 M Me2SO; VS3 – 1,5 M metanol + 4,5 M propilenoglicol + 0,5 M sacarose; VS4 – 1,5 M metanol + 5,5 M Me2SO + 0,5 M sacarose) e cinco estágios de desenvolvimento folicular utilizando o teste de coloração supravital iodeto de propídio combinado com diacetato de fluoresceína. Estes resultados mostraram que os folículos em estágio I, imaturos, apresentaram as maiores taxas de sobrevivência celular e que VS1 foi a melhor solução em termos de viabiidade. No Experimento 2, utilizou-se VS1 para vitrificar o tecido ovariano em diferentes dimensões (fragmentos ou ovários inteiros) e em dois diferentes recipientes (palheta de plástico ou cápsula de metal). Para avaliar a sobrevivência e o crescimento folicular dos folículos em estádio I, o diâmetro dos folículos foi mensurado antes e depois de cultivo in vitro por 24 horas. A morfologia folicular foi analisada por microscopia de luz após vitrificação utilizando a cápsula de metal. Os dados mostraram que a morfologia de folículos imaturos foi bem preservada após a criopreservação. A taxa de sobrevivência folicular foi maior (P <0,05) em fragmentos vitrificados, quando comparados com a vitrificação de ovários inteiros. Não houve diferenças significativas na sobrevivência e crescimento folicular entre os dois recipientes de vitrificação, palheta de plástico ou cápsula de metal. No entanto, a cápsula de metal diminui os riscos de contaminação, pois é hermeticamente fechada evitando contato com nitrogênio líquido e poder ser esterilizada, em vista que é manufaturada em aço inoxidável. Por essas razões, acreditamos que a cápsula de metal tem um uso potencial em reprodução humana para a vitrificação em grau clínico de tecido ovariano. / Zebrafish (Danio rerio) has excelled in biomedical research for its physiological and genetic homology to humans. However, there are few reports on ovarian cryopreservation of this specie. Thus, we studied the use of a metal capsule to vitrify zebrafish ovarian tissue. The aim of this study was to assess the survival and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the metal capsule. First, we tested four vitrification solutions (VS1 - 1.5 M methanol + 4.5 M propylene glycol; VS2 - 1.5 M methanol + 5.5 M Me2SO; VS3 - 1.5 M methanol + 4.5 M propylene glycol + 0.5 M sucrose; VS4 - 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose) and five follicular developmental stages using fluorescein diacetate and propidium iodide supravital staining test. These results showed that immature follicles, stage one, presented the highest survival rates and VS1 the best vitrification solution in terms of viability. In Experiment 2, we used VS1 to vitrify ovarian tissue in different dimensions (fragments or whole ovaries) and tested two different carriers (plastic straw or metal capsule). To evaluate follicular survival and growth of stage I, we measured follicle diameter before and after twenty-four-hour in vitro culture. The follicular morphology was analyzed by light microscopy after vitrification using the metal capsule. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P<0.05) on vitrified fragments, when compared to whole ovaries. There were no significant differences on follicular survival and growth between the two vitrification devices, plastic straw or metal capsule. However, the metal capsule being tightly sealed and manufactured in stainless steel avoids contact with liquid nitrogen and can be sterilized reducing contamination risk. These reasons lead us to believe that the metal capsule has a potential use in human reproduction for the clinical grade vitrification of ovarian tissue.

O zebrafish (Danio rerio) tem se destacado na pesquisa biomédica por sua homologia fisiológica e genética aos humanos. No entanto, há poucos relatos sobre a criopreservação ovariana desta espécie. Assim, pesquisamos a utilização de um recipiente de metal na vitrificação de tecido ovariano de zebrafish. O objetivo foi avaliar a sobrevivência e o desenvolvimento in vitro de folículos de zebrafish após a vitrificação de fragmentos ou ovários inteiros usando a cápsula de metal. Primeiro, testamos quatro soluções de vitrificação (VS1 – 1,5 M metanol + 4,5 M propilenoglicol; VS2 – 1,5 M metanol + 5,5 M Me2SO; VS3 – 1,5 M metanol + 4,5 M propilenoglicol + 0,5 M sacarose; VS4 – 1,5 M metanol + 5,5 M Me2SO + 0,5 M sacarose) e cinco estágios de desenvolvimento folicular utilizando o teste de coloração supravital iodeto de propídio combinado com diacetato de fluoresceína. Estes resultados mostraram que os folículos em estágio I, imaturos, apresentaram as maiores taxas de sobrevivência celular e que VS1 foi a melhor solução em termos de viabiidade. No Experimento 2, utilizou-se VS1 para vitrificar o tecido ovariano em diferentes dimensões (fragmentos ou ovários inteiros) e em dois diferentes recipientes (palheta de plástico ou cápsula de metal). Para avaliar a sobrevivência e o crescimento folicular dos folículos em estádio I, o diâmetro dos folículos foi mensurado antes e depois de cultivo in vitro por 24 horas. A morfologia folicular foi analisada por microscopia de luz após vitrificação utilizando a cápsula de metal. Os dados mostraram que a morfologia de folículos imaturos foi bem preservada após a criopreservação. A taxa de sobrevivência folicular foi maior (P <0,05) em fragmentos vitrificados, quando comparados com a vitrificação de ovários inteiros. Não houve diferenças significativas na sobrevivência e crescimento folicular entre os dois recipientes de vitrificação, palheta de plástico ou cápsula de metal. No entanto, a cápsula de metal diminui os riscos de contaminação, pois é hermeticamente fechada evitando contato com nitrogênio líquido e poder ser esterilizada, em vista que é manufaturada em aço inoxidável. Por essas razões, acreditamos que a cápsula de metal tem um uso potencial em reprodução humana para a vitrificação em grau clínico de tecido ovariano. / Zebrafish (Danio rerio) has excelled in biomedical research for its physiological and genetic homology to humans. However, there are few reports on ovarian cryopreservation of this specie. Thus, we studied the use of a metal capsule to vitrify zebrafish ovarian tissue. The aim of this study was to assess the survival and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the metal capsule. First, we tested four vitrification solutions (VS1 - 1.5 M methanol + 4.5 M propylene glycol; VS2 - 1.5 M methanol + 5.5 M Me2SO; VS3 - 1.5 M methanol + 4.5 M propylene glycol + 0.5 M sucrose; VS4 - 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose) and five follicular developmental stages using fluorescein diacetate and propidium iodide supravital staining test. These results showed that immature follicles, stage one, presented the highest survival rates and VS1 the best vitrification solution in terms of viability. In Experiment 2, we used VS1 to vitrify ovarian tissue in different dimensions (fragments or whole ovaries) and tested two different carriers (plastic straw or metal capsule). To evaluate follicular survival and growth of stage I, we measured follicle diameter before and after twenty-four-hour in vitro culture. The follicular morphology was analyzed by light microscopy after vitrification using the metal capsule. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P<0.05) on vitrified fragments, when compared to whole ovaries. There were no significant differences on follicular survival and growth between the two vitrification devices, plastic straw or metal capsule. However, the metal capsule being tightly sealed and manufactured in stainless steel avoids contact with liquid nitrogen and can be sterilized reducing contamination risk. These reasons lead us to believe that the metal capsule has a potential use in human reproduction for the clinical grade vitrification of ovarian tissue.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Este trabalho tem como objetivo 1) avaliar o efeito da BMP-15 e do FSH, sozinhos ou em diferentes associaÃÃes sobre o crescimento, sobrevivÃncia e formaÃÃo de antro em folÃculos secundÃrios bovinos e 2) avaliar os nÃveis de expressÃo do RNAm para os receptores de BMP-15 (BMPR-IB e BMPR-II) em folÃculos crescidos in vitro. Com relaÃÃo ao cultivo, folÃculos secundÃrios foram microdissecados e cultivados por doze dias em &#945;-MEM+ sozinho, ou suplementado com BMP-15 e/ou FSH, sozinhos ou em diferentes associaÃÃes. ApÃs o cultivo, foram avaliados o diÃmetro folicular, a viabilidade e a taxa de formaÃÃo de antro. A anÃlise de expressÃo gÃnica foi realizada por PCR em tempo real, utilizando folÃculos cultivados nos diferentes tratamentos, para comparaÃÃes dos nÃveis de RNAm. ApÃs o perÃodo de cultivo verificou-se que a utilizaÃÃo da BMP+FSH (0-12) estimulou um aumento significativo no diÃmetro folicular em relaÃÃo aos demais tratamentos (p<0,05), bem como estimulou a formaÃÃo da cavidade antral de forma mais efetiva que o controle (MEM) e a BMP-15 sozinha (p<0,05). No entanto, em relaÃÃo à sobrevivÃncia, apÃs12 dias o tratamento BMP+FSH (0-12) reduziu significativamente a viabilidade folicular em relaÃÃo aos demais tratamentos (p<0,05). Os resultados mostraram que os nÃveis de RNAm para o receptor de BMP-15 do tipo I (BMPR-IB) foram significativamente maiores nos folÃculos cultivados com MEM quando comparado aos demais tratamentos, enquanto que os nÃveis de RNAm para o receptor do tipo II (BMPR-II) nÃo diferiram entre os tratamentos. Os maiores nÃveis de RNAm para o receptor de FSH foram detectados em folÃculos cultivados com BMP-15 sozinha. Em conclusÃo, este estudo demonstrou que a combinaÃÃo de BMP-15 + FSH durante todo o perÃodo de cultivo (D0-D12) acelera o crescimento de folÃculos prÃ-antrais bovinos cultivados por 12 dias, mas este efeito reduz a viabilidade folicular, afetando a integridade ultra- estrutural dos folÃculos. AlÃm disso, a adiÃÃo de BMP isoladamente aumentou os nÃveis de expressÃo de RNAm para FSHR apÃs 12 dias de cultivo,mostrando que BMP-15 e FSH podem contribuir significativamente para o desenvolvimento in vitro de folÃculos prÃ-antrais cultivados por 12 dias nesta espÃcie. / This study aims to 1) evaluate the effect of BMP-15 and FSH, alone or in different combinations on the in vitro development, survival and antrum formation of bovine secondary follicles and 2) quantify the levels of mRNA expression for BMP-15 receptors (BMPR-IB and BMPR-II), FSH receptor and Kit Ligand in follicles grown in vivo and in vitro. With regard to culture, secondary follicles were microdissected and cultured for twelve days in &#945;- MEM+ alone, or supplemented with BMP-15 and/or FSH, alone or in various associations. After culture, follicular diameter were evaluated, viability and rate of antrum formation. For the analyzes gene expression, by real time PCR, were used follicles cultured in the different treatments for comparisons of the levels of mRNA. After the culture period it was found that the association of BMP+FSH (0-12) stimulated a significant increase in follicular diameter in relation to other treatments (p<0.05), as well as stimulated the formation of antral cavity more effectively the control (MEM) and BMP-15 alone (p<0.05). However, for survival, after 12 days treatment BMP+FSH (0-12) significantly reduced follicular viability when compared to other treatments (p<0.05). The results showed that the levels of mRNA for BMP receptor type I (BMPR-IB) were significantly higher in follicles cultured with MEM when compared to other treatments, while levels of mRNA for the receptor type II (BMPR-II) did not differ between treatments. The highest levels of mRNA for the FSH receptor were detected in follicles cultured with BMP-15 alone. In conclusion, this study demonstrated that combination of BMP-15+FSH (D0-D12) accelerates the growth of bovine preantral follicles cultured for 12 days, but this effect reduces the follicular viability, affecting the ultrastructural integrity of the follicles. Furthermore, the addition of BMP alone increased expression levels of RNAm for FSHR after 12 days of culture , showing that BMP-15 and FSH may contribute significantly to the in vitro development of preantral follicles cultured for 12 days in this specie.

Some plasticizers are endocrine-disrupting chemicals that cause reproductive toxicity in both males and females. Several chemicals already approved by the Food and Drug Administration have been proposed as substitutes for some of these plasticizers, one example is acetyl tributyl citrate (ATBC). However, no studies have tested whether ATBC causes direct toxicity to the ovary. Ovarian antral follicles are essential for female fertility because they are the major producers of ovarian steroids and are the only follicle type that can ovulate in response to gonadotropin stimulation. Previous studies have used in-vitro ovarian follicle culture as a screening tool to demonstrate that EDCs can cause direct ovarian toxicity. Therefore, we designed this study to test whether exposure to in vitro treatment with ATBC causes ovarian toxicity in CD-1 mice. We mechanically isolated antral follicles from the ovaries of adult CD-1 mice (35-39 days old) and individually exposed them (n=5 cultures with n≥8 follicles per treatment) to supplemented media alone (NT), dimethyl sulfoxide (DMSO, vehicle for ATBC), and ATBC (0.001-100 µg/mL) for 24-72 h. Follicle growth and survival were monitored by measuring follicle diameter and cytotoxicity (compromised membrane integrity; CellTox Green) every 24 h, and assessing number of metabolically active cells (ATP concentration; Promega CellTiterGlo) at the end time point. The DNA synthesis inhibitor hydroxyurea (HU, 100 mM) was used as a positive control for the viability assays. Exposure to ATBC did not affect the ability of antral follicles to increase their diameter over time at all concentrations tested. When stratified by growth pattern, there was not a significant difference in the proportion of follicles growing normally, growing slowly, or not growing following ATBC exposure at all concentrations tested. ATBC treatment did not cause compromised membrane integrity and did not inhibit ATP production at any of the concentrations tested. The positive control, HU, inhibited follicle growth (24-72 h), decreased follicle cell membrane integrity (72 h), and inhibited ATP production (24-72 h). The purpose of this experiment is to evaluate the effects of oral exposure to ATBC in female CD-1 mice. For the in vivo experiments, the female mice (n=22; PND 81) were randomly divided into treatment groups and dosed according to daily body weight with one of the following treatments: corn oil (vehicle, n=7), 5 mg/kg/day ATBC (n=8), or 10 mg/kg/day (n=7) ATBC for 15 consecutive days. Vaginal smears were performed and analyzed daily to measure any change in estrous cyclicity. After the 15th day of dosing the female mice were introduced into an individually housed proven breeder male’s cage. Daily body weight measurements continued and plug checks were performed every morning. Pregnancy and time to conception data did not statistically differ from the vehicle (oil) for all ATBC treatments (days to conception: vehicle 2.43 ± 0.65, 5 mg/kg/day ATBC 2 ± 0.33, 10 mg/kg/day ATBC 2.5 ± 0.22; gestation length: vehicle 19.71 ± 0.18, 5 mg/kg/day ATBC 20 ± 0.19, 10 mg/kg/day ATBC 20 ± 0.00). On the day of parturition the dams and pups were sacrificed; organ weight and gross morphology data was collected for: uterus, kidneys, adrenals, spleen, liver, and ovaries. The data analyzed includes: estrous cyclicity, pre-dosing body weight % gain, dosing body weight % gain, pregnant body weight % gain, organ weight, gestation length, litter size (live vs. dead), litter weight, implantation sites, time to conception, and pup sex ratio. Interestingly, there was an increase in spleen weight at the 5 mg/kg/day treatment when compared to vehicle control-treated spleen weights (spleen weight, Oil: n=7; 5 mg/kg ATBC: n= 8). Treatment with 5 mg/kg/day ATBC caused a significant decrease in average estrous cycle length in days compared to the pre-dosing average estrous cycle length. Animals exposed orally to 10 mg/kg/day ATBC showed a significant decrease in total follicle number (10 mg/kg/day ATBC, 313 ± 20.37) when compared to vehicle (oil) treated mice (vehicle, 433.71 ± 34.85). Also treatment with 10 mg/kg/day ATBC resulted in significant reduction in secondary (101.67 ± 5.7) and late antral (4.17 ± 1.45) follicle numbers when compared to the vehicle-treated mice (secondary follicles: 141.14 ± 14.79, late antral follicles: 7.00 ± 1.54). ATBC treatment with 10 mg/kg/day showed a significantly decreased mean body weight percent gain during pregnancy on days 3, 5, and 14 when compared to animals treated with vehicle (oil), while animals treated with 5 mg/kg/day ATBC showed a significant decrease in weight gained on only day 13 when compared to the vehicle (oil). These novel findings show that ATBC could disrupt ovarian function in mice when exposed to low-dose ATBC.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-03-30T14:58:19Z No. of bitstreams: 1 LuãBM_DISSERT.pdf: 1732522 bytes, checksum: 8ab1e4a3686c8d21f9bc08fcc449f393 (MD5) / Made available in DSpace on 2017-03-30T14:58:20Z (GMT). No. of bitstreams: 1 LuãBM_DISSERT.pdf: 1732522 bytes, checksum: 8ab1e4a3686c8d21f9bc08fcc449f393 (MD5) Previous issue date: 2017-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Leptin, a cytokine produced by adipose cells, is the target of the scientific community for believing that it has an impact on the reproduction of the animals promoting puberty, folliculogenesis and oogenesis, estrous cycle and aiding in fertilization. The understanding of the mechanisms controlling the reproductive activity of Spix's Yellow-toothed Cavy (Galea spixii) plays a relevant role in the preservation of the species. Thus, the present work proposed to analyze the immunolocalization of leptin receptors (Ob-R) in the ovary of cavies. Ovaries from 20 adult, non-pregnant, healthy females were collected. The samples were fixed in 4% phosphate buffered paraformaldehyde, embedded in paraffin and sectioned for immunohistochemistry. The sections were photomicrographs and intensity of the reaction was measured. Strong immunoreaction was observed in oocyte and theca cells, moderate in ovarian stromal cells and large luteal cells and weak stained in granulosa, endothelial, perivascular and small luteal cells. When compared to receptor expression along follicular development it was observed that the oocyte and the theca cells remained with expression at the same intensity. However, the granulosa cells presented strong stained in the preantral stages, whereas in the antral follicles it presented low intensity. We conclude that in the ovaries of Galea spixii there is the presence of Ob-R in the main structures of the ovary sugesting that this hormone plays a fundamental role in the reproduction of this species / A leptina, uma citocina produzida pelas células adiposas, possui ação na reprodução dos animais promovendo a puberdade, foliculogênese e oogênese, ciclo estral e auxiliando na fecundação. A compreensão dos mecanismos que controlam a atividade reprodutiva de preás (Galea spixii) possui papel relevante para a preservação da espécie. Desta forma, o presente trabalho propôs analisar a imunolocalização dos receptores de leptina (Ob-R) no ovário de preás. Coletaram-se os ovários de 20 fêmeas adultas, não prenhes e saudáveis. As amostras foram fixadas em paraformaldeído a 4% em tampão fosfato, incluídas em parafina e seccionadas para a realização de imunohistoquímica. As secções foram fotomicrografadas e avaliadas quanto à intensidade da reação. Observou-se forte imunorreação no oócito e nas células da teca, moderada nas células do estroma ovariano e nas células luteínicas grandes e fracamente coradas nas células da granulosa, endoteliais, perivasculares e células luteínicas pequenas. Quando comparado a expressão de receptores ao longo do desenvolvimento folicular foi observado que o oócito e as células da teca se mantiveram com expressão na mesma intensidade. Entretanto, as células da granulosa apresentaram forte marcação nos estádios pré-antrais enquanto que nos folículos antrais apresentou fraca intensidade. Concluímos que em ovários de Galea spixii existe a presença de Ob-R nas principais estruturas do ovário sugerindo que este hormônio desempenhe papel fundamental na reprodução desta espécie / 2017-03-30

La cryoconservation de cortex ovarien est la seule technique envisageable pour les patientes pré-pubères et les femmes dont la pathologie nécessite l’administration d’un traitement hautement gonadotoxique dont l’initiation ne peut être différée. L’autotransplantation est jusqu’à présent la seule méthode disponible de réutilisation du tissu ovarien cryoconservé, et a permis d’obtenir plus de 130 naissances dans le monde, dont trois au CHRU de Besançon. Toutefois, en cas de pathologie néoplasique à risque de localisation métastatique ovarienne, cette technique peut présenter un risque de réintroduction de cellules malignes susceptibles d’être présentes dans le greffon. Des méthodes alternatives à l’autogreffe de tissu ovarien cryopréservé fondées sur l’utilisation de follicules ovariens isolés sont actuellement en développement. L’objectif de cette thèse a été de développer un protocole permettant l’isolement et la qualification de follicules ovariens qui pourront être utilisés en thérapie cellulaire à usage humain. Dans un premier temps, une validation de la technique d’isolement de follicules ovariens a été réalisée à partir de la dissociation de fragments de cortex ovarien, issus de patientes ayant subit une résection percœlioscopique, à l’aide d’une collagénase NB6 produite selon les bonnes pratiques de fabrication. Les follicules ainsi obtenus ont été analysés en termes de viabilité (immédiate et après culture in vitro), rendement, morphologie et état prolifératif. Dans un deuxième temps, la sécurité carcinologique des suspensions folliculaires obtenues après isolement a été évaluée par cytométrie en flux multicouleurs à l’aide d’une modélisation ayant consisté en la contamination de suspensions folliculaires avec des cellules leucémiques issues de patients souffrant de leucémies aigües myéloïdes (LAM) ou lymphoblastiques (LAL) d’immunophénotype connu. La collagénase NB6 a permis l’isolement d’un grand nombre de follicules vivants, principalement au stade primordial, et dont la majorité était non activée, même après trois jours de culture in vitro dans un gel de fibrine. La technique d’isolement suivie de trois lavages a permis d’éliminer les cellules leucémiques préalablement ajoutées aux suspensions folliculaires dans 23 cas sur 24, sans endommager les follicules isolés. La cytométrie en flux multicouleurs est une technique d’analyse efficace pour évaluer la contamination, par des cellules leucémiques, de suspensions contenant des follicules ovariens isolés. Le protocole d’isolement de follicules ovariens humains, ayant été réalisé avec la collagénase NB6 de grade clinique, peut être envisagé pour une reconstruction ovarienne à visée thérapeutique humaine. / The cryopreservation of ovarian cortex is the only technique available for prepubertal girls and women when their pathology requires the administration of a highly gonadotoxic treatment whose initiation cannot be delayed. Autotransplantation has so far been the only available method to re-use cryopreserved ovarian tissue, and has resulted in more than 130 births worldwide, including three at the Besançon Hospital. However, in cases of neoplastic disease with a risk of ovarian metastatic localization, this technique may present a risk of reintroducing malignant cells likely to be present in the graft. Alternative methods to cryopreserved ovarian tissue autograft based on the use of isolated ovarian follicles are currently under development. The aim of this thesis was to develop a protocol allowing the isolation and the qualification of ovarian follicles that can be used in cell therapy for human purposes. In a first step, a validation of the technique to isolate ovarian follicles was carried out from the dissociation of cortical ovarian fragments, taken from patients undergoing a laparoscopic ovarian drilling, using collagenase NB6 which is produced according to good manufacturing practices. Follicles thus obtained were analyzed in terms of viability (before and after in vitro culture), yield, morphology and proliferative state. In a second step, the carcinologic safety of follicular suspensions obtained after isolation was evaluated by multicolor flow cytometry using a model involving the contamination of follicular suspensions with leukemic cells from patients suffering from acute myeloid leukemia (AML) or acute lymphoblastic leukemia (LAL) with known immunophenotype. Collagenase NB6 has allowed the isolation of a large number of viable follicles, mostly at the primordial stage and the majority of which were unactivated even after three days of in vitro culture in a fibrin matrice. Our isolation technique followed by three washes has allowed the elimination of leukemic cells previously added to follicular suspensions, in 23 out of 24 cases, without damaging isolated follicles. Multicolor flow cytometry is an effective analytical technique for assessing leukemic cell contamination of suspensions containing isolated ovarian follicles. The protocol to isolate human ovarian follicles, performed with the clinical grade collagenase NB6, may be considered for an ovarian reconstruction to human therapeutic purposes.

In response to LH surge, the selected dominant follicle undergoes a series of complex events viz., meiotic maturation of the oocyte, terminal differentiation of follicular cells, cumulus expansion and follicular rupture leading to ovulation (release of fertilizable oocyte) and formation of corpus luteum. These complex set of events are mediated by dramatic changes in the temporo-spatial transcriptional regulation of a large number of genes in the preovularory follicle. In the present thesis, attempts have been made to delineate the transcriptional changes occurring in the periovulatory follicle in response to gonadotropin surge in monovulatory species (bonnet monkeys and buffalo cows). Further, attempts have also been made to investigate effects of increased circulating IGF-I and glucose on the periovulatory events. Chapter I provides a review of available literature on regulation and factors influencing various aspects of growth and development of ovarian follicles. Chapter II describes initial studies carried out in the bonnet monkeys to examine expression patterns of various genes considered as markers of ovulation and luteinization in granulosa cells of multiple preovulatory-like follicles before and after exposure to luteinizing dose of hCG. Chapter III describes the standardization and validation of an induced ovulation model system in buffalo cows involving determination of the presence of a large follicle by ultrasonography before administering exogenous PGF2αon day 7 of the cycle to induce luteolysis and administration of hCG 36 h post PGF2αto mimic LH surge-like stimulus. Attempts made to characterize the responsiveness of buffalo cows to a range of exogenous doses of GnRH in terms of LH secretion during summer and winter seasons and determination of the exogenous dose of GnRH necessary to elicit a surge-like increase in circulating LH levels during breeding season in buffalo cows are described in Chapter IV. Chapter V describes the global changes in the gene expression pattern in the periovulatory follicle of buffalo cows before and at various time points after the onset of gonadotropin surges. IGF-I is known to regulate the proliferation and survival of follicular cells and play an important role in the selection of dominant follicle however, its role during the periovulatory events remains to be delineated. Chapter VI describes the effects of increased circulating and intrafollicular IGF-I levels on the periovulatory events. An ongoing study to delineate the effects of transient increase in circulating glucose levels on the periovulatory events are described in Appendix I. Thus, this thesis aims at delineating (i) changes in the gene expression patterns regulating periovulatory events and (ii) effects of increased circulating IGF-I and glucose on the periovulatory events.

The role oxygen plays in the developing ovarian follicle is of interest not only to the field of developmental biology but also to in-vitro fertilisation (IVF) technologists, as oxygenation of the oocyte is considered to be a potential determinant of oocyte competence. Oxygen transport through the developing ovarian follicle, and practical aspects of the analysis of oxygen in human follicular fluid were investigated in this work. Mathematical modelling of oxygen transport in the pre-antral, and antrallpreovulatory follicle revealed a number of interesting findings, Contrary to previous conclusions (Gosden & Byatt-Smith, 1986), oxygen can reach the oocyte in the small pre-antral follicle. Improved estimates of diffusion coefficients through the granulosa cell layer and the inclusion of fluid voidage in this layer showed that oxygen can also reach the oocyte in large pre-antral follicles. The amount of oxygen that reaches the oocyte in the pre-antral follicle is a function of its size and degree of vascularisation. Symmetrically distributed vascularisation is superior in achieving a well oxygenated follicle. However, the large pre-antral follicle will eventually reach a size beyond which it cannot grow without anoxic regions developing. The size at which this occurs is consistent with the size at which antrum formation is observed in human follicles. The model predicts that the follicle can avoid an anoxic state through antrum formation, and shows that the follicle develops in a way that is consistent with overcoming mass transport limitations. The oxygen status of the follicle during the antrallpre-ovulatory phase of growth requires that the volume of granulosa cells be balanced by the volume of follicular fluid. Further predictions suggest that oocyte respiration becomes sub-maximal at follicular fluid volumes below approximately 4m1, vascularisation levels below 38%, or fluid i dissolved oxygen levels below 5.1 ~01%. These values are consistent with observations in the literature. It was also shown that the measurement of follicular fluid dissolved oxygen levels could provide a simple measure of the respiratory status of the oocyte, and this may be superior to the measurement of follicular vascularisation which requires knowledge of more parameters. Methodology for the analysis of follicular fluid oxygen solubility and diffusivity was developed using a Clark oxygen electrode. Analysis of these parameters showed that they are similar to human plasma, and allowed the predictive uncertainty of the model to be reduced. Experimental studies into the effects of IVF aspiration on follicular fluid were carried out. Aspiration results in significant changes in the properties of follicular fluid. Dissolved oxygen levels rose 5 * 2 vol%, pH increased by 0.04 * 0.01 pH units, and temperature dropped by 7.7 * 1.3 "C. Mathematical modelling of blood contaminated follicular fluid also showed that contamination results in significant changes in the dissolved oxygen of the fluid. This suggests that if the composition of follicular fluid is to be determined (particularly dissolved oxygen), sampling andlor measurement of fluid must take place before the collection vial of the aspiration kit, and blood contamination must be eliminated. Based on this result, the design and testing of devices capable of reliable sampling andlor rneasurement of oxygen levels of follicular fluid was considered. This presents a continuing challenge, including the integration of routine follicular fluid oxygen measurement into clinical practice.