20 research outputs found
In vitro development of primordial follicles after long-term culture of goat ovarian tissue
This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro
Progesterona e hormĂ´nio folĂculo-estimulante interagem e promovem a sobrevivĂŞncia e o desenvolvimento in vitro de folĂculos prĂ©-antrais caprinos
Este trabalho veri_icou os efeitos da progesterona e do hormonio foliculo-estimulante (FSH) na sobrevivencia e no crescimento de foliculos pre-antrais caprinos. Fragmentos de tecido ovariano foram cultivados por 1 ou 7 dias em Meio Essencial Minimo (MEM) sozinho ou contendo progesterona (1, 2.5, 5, 10 ou 20ng/mL), FSH (50ng/mL) ou a combinacao entre esses dois hormonios. O tecido fresco (controle nao-cultivado) e o cultivado foram processados para analise histologica e ultra-estrutural. Apos 7 dias a adicao de FSH a todas as concentracoes de progesterone manteve o percentual de foliculos normais similar ao controle fresco. No dia 7 de cultivo, um alto percentual de foliculos em desenvolvimento foi observado somente no tratamento com 2,5ng/ml de progesterona associada ao FSH ou com 10ng/ml de progesterona sozinha, em relação ao controle fresco. Do dia 1 para o dia 7 de cultivo, um aumento signi_icativo no percentual de foliculos em desenvolvimento foi observado no MEM sozinho e adicionado de 2,5ng/ml de progesterona + FSH. Alem disso, apos 7 dias, em todos os tratamentos, houve um aumento signi_icativo no diametro folicular em relacao ao controle, exceto nos tratamentos com MEM sozinho, 5ng/ml de progesterona + FSH ou 10ng/ml de progesterona sozinha. A analise ultra-estrutural con_irmou a integridade follicular apos 7 dias de cultivo no tratamento com 2,5ng/ml de progesterona + FSH. Em conclusao, este estudo demonstrou que a interação entre progesterona e FSH mantem a integridade ultra-estrutural, estimula a ativacao de foliculos primordiais e o posterior crescimento de foliculos pre-antrais caprinos cultivados in vitro.We investigated the effects of progesterone and follicle stimulating hormone (FSH) on survival and growth of caprine preantral follicles. Pieces of ovarian tissue were cultured for 1 or 7 days in minimum essential medium (MEM) alone or containing progesterone (1, 2.5, 5, 10 or 20ng/mL), FSH (50ng/mL) or the interaction between progesterone and FSH. Fresh (non-cultured control) and cultured ovarian tissues were processed for histological and ultrastructural studies. After 7 days the addition of FSH to all progesterone concentrations maintained the percentage of normal follicles similar to fresh control. At day 7 of culture, a higher percentage of developing follicles was observed only in 2.5ng/ml of progesterone associated with FSH or 10ng/ml of progesterone alone when compared with control. From day 1 to day 7 of culture, a signi_icant increase in the percentage of developing follicles was observed in MEM and 2.5ng/ml of progesterone + FSH. In addition, after 7 days, in all treatments, there was a signi_icant increase in follicular diameter when compared with control, except for MEM alone and in 5ng/ml of progesterone + FSH or 10ng/ml of progesterone alone. Ultrastructural studies con_irmed follicular integrity after 7 days of culture in 2.5ng/ml of progesterone with FSH. In conclusion, this study demonstrated that the interaction between progesterone and FSH maintains ultrastructural integrity, stimulates primordial follicles activation and further growth of cultured caprine preantral follicles
Sphingosine 1-phosphate promotes activation of aprine preantral follicle in vitro
Este estudo descreve o efeito da esfingosina 1-fosfato (S1P) no desenvolvimento de folĂculos prĂ©-antrais, portanto da ativação e viabilidade de folĂculos caprinos cultivados in vitro. Fragmentos de ovários foram cultivados por um ou sete dias em meio essencial mĂnimo com diferentes concentrações de S1P (0, 1, 10, 50, 100 ou 200ng/mL). Os fragmentos de ovário foram processados para análise histolĂłgica em microscopia Ăłptica, microscopia eletrĂ´nica e microscopia de fluorescĂŞncia. O tratamento usando 1ng/mL de S1P foi capaz de manter a porcentagem de folĂculos normais durante o perĂodo de cultivo de sete dias. Ao final do perĂodo de cultivo, houve uma redução significativa (p<0,05) na porcentagem de folĂculos primordiais em todos os grupos tratados com S1P, comparados com os grupos controle (FC e CC), seguida por um aumento do nĂşmero de folĂculos ativados (intermediários, primários e secundários). Adicionalmente, na cultura por sete dias com meio suplementado com S1P (1ng/mL), houve preservação da ultraestrutura das organelas e manteve-se a viabilidade dos folĂculos prĂ©-antrais avaliados por microscopia de fluorescĂŞncia. Em conclusĂŁo, apĂłs sete dias de cultura, o meio suplementado com 1ng/mL de S1P ativa o desenvolvimento de folĂculos prĂ©-antrais de caprino, cultivados in situ e mantĂ©m as viabilidades oocitária e folicular.This study describes the effect of sphingosine 1-phosphate (S1P) for development of preantral follicle, therefore the activation and follicular viability of caprine follicles cultured in vitro. Ovarian fragments were cultured for 1 or 7 days in Minimum Essential Medium with different S1P concentrations (0, 1, 10, 50, 100 or 200ng/mL). All ovarian fragments were processed for histological analysis in optical microscopy, transmission electron microscopy and fluorescence analysis. The treatment using 1ng/mL of S1P was able to maintain the percentage of normal follicles with the progression of the culture from day 1 to 7. At end of the 7-day culture period there was a significant reduction (P<0.05) in the percentage of primordial follicles in all groups treated with S1P, compared with fresh control (FC) and Control Culture (CC), which was followed by an increase of activated follicles (intermediary, primary and secondary). In addition, the culture for 7 days with media supplemented with S1P with 1ng/mL preserved the ultrastructure of organelles and kept the preantral follicular viability when evaluated by fluorescence microscopy. In conclusion, after 7 days of culture, the 1ng/mL of S1P activates the development of preantral caprine follicles, cultured in situ and maintains the oocitary and follicular viability
Diferentes origens do HormĂ´nio FolĂculo Estimulante (FSH) influenciam a viabilidade e o desenvolvimento de folĂculos prĂ©-antrais caprinos
The aim of this study was to evaluate the effects of pituitary (pFSH) or recombinant (rFSH) FSH on the survival and growth of caprine preantral follicles. Caprine ovarian tissues were in vitro cultured for one or seven days in Minimum Essential Medium (MEM) alone or containing 10, 50, 100 and 1000 ng/ml of pFSH or rFSH. Control tissues (non-cultured) and those cultured were processed for histological and ultrastructural studies. In addition, follicular and oocyte diameter were analysed. After seven days of culture, only 50 ng/ml of rFSH maintained the percentage of normal follicles similar to control. Moreover, 10 ng/ml of pFSH and all the concentrations of rFSH promoted primordial follicles activation. In addition, the presence of 50 ng/ml of rFSH promoted the highest follicular diameter at day seven of culture. In conclusion, 50 ng/ml of rFSH maintained the ultrastructural integrity of caprine preantral follicles, promoted primordial follicles activation and further growth of cultured follicles.O objetivo deste estudo foi avaliar os efeitos do FSH pituitário (pFSH) ou recombinante (rFSH) sobre a sobrevivĂŞncia e o crescimento de folĂculos prĂ©-antrais caprinos. O tecido ovariano foi cultivado in vitro por um ou sete dias em Meio Essencial MĂnimo (MEM) sozinho, ou contendo 10, 50, 100 e 1000 ng/ml de pFSH ou rFSH. O grupo controle (nĂŁo cultivado) e aqueles cultivados foram processados para análises histolĂłgica e ultra-estrutural. AlĂ©m disso, os diâmetros folicular e oocitário foram avaliados. ApĂłs sete dias de cultivo, apenas 50 ng/ml de rFSH manteve o percentual de folĂculos normais semelhante ao controle. AlĂ©m disso, 10 ng/ml de pFSH e todas as concentrações de rFSH promoveram ativação de folĂculos primordiais. A presença de 50 ng/ml de rFSH promoveu o maior diâmetro folicular apĂłs sete dias de cultivo. Em conclusĂŁo, 50 ng/ml de rFSH manteve a integridade de folĂculos prĂ©-antrais caprinos e promoveu a ativação e o crescimento dos folĂculos cultivados
Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles
This study evaluates the effects of ascorbic acid and its interaction with follicle-stimulating hormone (FSH) on the morphology, activation, and in vitro growth of caprine preantral follicles. Ovarian fragments were cultured for 1, 7, or 14 d in minimum essential medium (MEM) containing ascorbic acid (50 or 100 ÎĽg/mL), FSH (50 ng/mL), or both of these substances. Ovarian tissue that was either fresh (control) or cultured for 1, 7, or 14 d was processed for histological and ultrastructural evaluation. The results showed that after 14 d of culture, medium supplemented with 50 ÎĽg/mL of ascorbic acid alone or combined with FSH showed higher rates of follicular survival compared with MEM. After 7 d of culture, FSH, ascorbic acid at 50 ÎĽg/mL with or without FSH, and ascorbic acid at 100 ÎĽg/mL increased the percentage of follicular activation compared to fresh control. In addition, FSH alone significantly increased the percentage of growing follicles after 14 d. The combination of 50 ÎĽg/mL of ascorbic acid and FSH promoted a significant increase in oocyte and follicular diameter after 7 d of culture. Ultrastructural and fluorescent analysis confirmed the integrity of follicles cultured with 50 ÎĽg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50 ÎĽg/mL of ascorbic acid and FSH maintained follicular integrity and promoted follicular activation and growth after long-term in vitro culture of caprine preantral follicles
Effects of α-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured
Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folĂculos prĂ©-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle nĂŁo-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial MĂnimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15M, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folĂculos prĂ©-antrais normais no controle nĂŁo-cultivado foi de 73,2%, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle nĂŁo-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural nĂŁo mostrou folĂculos prĂ©-antrais Ăntegros apĂłs cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α -tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular apĂłs o cultivo in vitro. ______________________________________________________________________________________________________________ ABSTRACTThe effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15M, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2% and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture
Histological and ultrastructural feature and nitrite production of caprine preantral follicles in vitro cultured in the presence or absence of serum
Avaliou-se o efeito da adição de diferentes tipos e concentrações de soro sobre o desenvolvimento e a sobrevivĂŞncia de folĂculos ovarianos prĂ©-antrais (FOPA) caprinos in vitro. AlĂ©m disso, verificou-se a relação entre as concentrações de nitrito presentes no meio de cultivo e a viabilidade folicular. Cada par ovariano foi dividido em 29 fragmentos, sendo um destinado ao controle. Os fragmentos foram cultivados por um ou sete dias em meio essencial mĂnimo suplementado (MEM+) ou MEM+ com diferentes concentrações (10 ou 20%) de soro fetal bovino (SFB), soro de cabra em estro (SCE) ou soro de cabra em diestro (SCD). Na análise morfolĂłgica apĂłs sete dias, apenas o tratamento com 10% de SFB apresentou percentual de FOPA normais similar ao MEM+ (P>0,05). A análise ultra-estrutural dos folĂculos cultivados por sete dias com MEM+ ou MEM+ com 10% de SFB mostrou danos oocitários, porĂ©m cĂ©lulas da granulosa normais. A análise do meio de cultivo revelou correlação positiva entre a viabilidade folicular e a produção de nitrito. A suplementação com soro nĂŁo melhorou a viabilidade de FOPA e a concentração de nitrito no meio de cultivo funcionou como um indicador da viabilidade das cĂ©lulas da granulosa de FOPA caprinos cultivados in vitro. ______________________________________________________________________________________________________________ ABSTRACTThe effect of the addition of different types and concentrations of sera on the viability and development of caprine preantal follicles (PAF) in vitro cultured was analyzed. In addition, it was evaluated the correlation between nitrite concentrations in culture medium and folicular viability. Each ovarian pair was divided in 29 fragments and one was used as control. The fragments were cultured for one or seven days in minimal essential medium (MEM+) or MEM+ with different concentrations of (10 or 20%) bovine fetal serum (BFS), estrous goat serum (EGS), or diestrous goat serum (DGS). After seven days, the morphological analysis showed that only the treatment with 10% BFS maintained the percentage of normal PAF similar to MEM+ (P>0.05). The ultrastructural analysis of follicles cultured for seven days in MEM+ or MEM+ with 10% BFS showed some oocyte damage, although the granulosa cells were normal. Analysis of culture medium revealed a positive correlation between follicular viability and nitrite production. Supplementation with serum did not improve the viability of PAF and nitrite levels in culture medium served as an indicator of viability of granulose cells from caprine PAF in vitro cultured
A ProteĂna MorfogenĂ©tica Ă“ssea-6 (BMP-6) induz a atresia em folĂculos primordiais caprinos cultivados in vitro
O presente estudo investigou os efeitos da proteĂna morfogenĂ©tica Ăłssea-6 (BMP-6) no desenvolvimento in vitro de folĂculos primordiais caprinos. Amostras de cĂłrtex ovariano de cabras foram cultivados por 1 ou 7 dias em Meio Essencial MĂnimo (meio controle) suplementado com diferentes concentrações de BMP-6. As taxas de sobrevivĂŞncia, ativação e crescimento foram avaliadas por histologia clássica e microscopia eletrĂ´nica de transmissĂŁo (MET). ApĂłs 7 dias de cultivo, a análise histolĂłgica demonstrou que a BMP-6 aumentou o percentual de folĂculos primordiais degenerados no dia 7 quando comparados ao controle fresco (D0). AlĂ©m disso, houve um aumento significativo do diâmetro folicular e oocitário em ambos os perĂodos de cultivo em todos os tratamentos na presença de BMP-6. Com a progressĂŁo do cultivo do dia 1 para o dia 7, nos tratamentos com 1 ou 50ng/ml de BMP-6, foi observado um aumento significativo no diâmetro folicular. Entretanto, contrário ao observado no meio controle, a MET revelou que os folĂculos cultivados nesses tratamentos apresentavam sinais evidentes de atresia. Em conclusĂŁo, esse estudo demonstrou que a BMP-6 afeta negativamente a sobrevivĂŞncia e a ultra-estrutura de folĂculos primordiais caprinos.This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles