RESUMO

Abstract This study aimed to investigate the effect of growth and differentiation factor 9 (GDF-9) during the in vitro culture of isolated caprine early antral follicles. The isolated and selected early antral follicles were individually cultured for 18 days, and the following treatments were tested: α-MEM+ (control treatment) or α-MEM+ supplemented with 200 ng/mL GDF-9. The following endpoints were evaluated: follicular growth and morphology, estradiol production, oocyte nuclear maturation, and relative expression of key genes related to steroidogenesis (CYP19A1, CYP17, and insulin receptor) and basement membrane remodeling (MMP-9 and TIMP-2). In both treatments, a decrease was observed in the percentage of morphologically intact follicles with a concomitant increase in the rates of extruded and degenerated follicles (P < 0.05). The GDF-9 treatment showed higher rates of extruded follicles only on day 6 of culture (P < 0.05). Follicle diameter increased progressively throughout the culture period (P < 0.05) with similar diameters between treatments at all culture times (P > 0.05). Growth and differentiation factor 9 increased the daily growth rate from the first to the second third of culture, with higher values (P < 0.05) than control in the second third. Oocyte maturation rate as well as estradiol levels and relative mRNA expression for CYP19A1, CYP17, MMP-9, TIMP-2, and insulin receptor genes were similar between treatments (P > 0.05). This study shows for the first time that GDF-9 added to a culture medium increased the follicle growth rate of goat early antral follicles cultured in vitro.

Resumo Este estudo teve como objetivo investigar o efeito do GDF-9 durante o cultivo in vitro de folículos antrais iniciais caprinos isolados. Os folículos antrais iniciais isolados e selecionados foram cultivados individualmente por 18 dias, e os seguintes tratamentos foram testados: α MEM+ (tratamento controle) ou α-MEM+ suplementado com 200 ng/mL de GDF-9 (tratamento GDF-9). Os seguintes parâmetros foram avaliados: crescimento e morfologia folicular, produção de estradiol, maturação nuclear do oócito e expressão relativa de genes-chave relacionados a esteroidogênese (CYP19A1, CYP17 e receptor de insulina) e remodelamento da membrana basal (MMP-9 e TIMP-2). Em ambos os tratamentos, observou-se diminuição na porcentagem de folículos morfologicamente intactos com aumento concomitante nas taxas de folículos extrusos e degenerados (P < 0,05). O tratamento GDF-9 apresentou maiores taxas de folículos extrusos apenas no 6º dia de cultivo (P < 0,05). O diâmetro do folículo aumentou progressivamente ao longo do período de cultivo (P < 0,05) com diâmetros semelhantes entre os tratamentos em todos os tempos de cultivo (P > 0,05). O GDF-9 aumentou a taxa de crescimento diário do primeiro para o segundo terço de cultivo, sendo maior (P < 0,05) que o controle no segundo terço. A taxa de maturação oocitária assim como os níveis de estradiol e a expressão relativa de RNAm para os genes CYP19A1, CYP17, MMP-9, TIMP-2 e receptor de insulina foram similares entre os tratamentos (P > 0,05). Em conclusão, este estudo mostra pela primeira vez que GDF-9 adicionado a um meio de cultivo aumentou a taxa de crescimento de folículos antrais iniciais caprinos cultivados in vitro.

RESUMO

A análise da expressão das diversas proteínas relacionadas a foliculogênese e oogênese in vivo e in vitro, através da proteômica, tem demonstrado possíveis biomarcadores celulares. A descoberta desses biomarcadores pode ajudar na elucidação de mecanismos complexos como a foliculogênese (entendimento sobre o crescimento folicular e maturação oocitária), no tratamento de doenças relacionadas a infertilidade feminina, bem como, no aprimoramento de biotécnicas reprodutivas como a manipulação de oócitos inclusos em folículos pré-antrais. Desta forma, a presente revisão abordou as principais proteínas relatadas nos mais novos trabalhos referentes aos estudos proteômicos visando melhores esclarecimentos referentes ao desenvolvimento folicular e oocitário in vivo e in vitro. A escolha dos artigos foi realizada com base nas descobertas mais atuais e do impacto que determinadas proteínas poderiam trazer para melhorar o entendimento da foliculogênese e oogênese nas diferentes espécies de animais mamíferos.(AU)

The analysis of the expression of several proteins related to folliculogenesis and oogenesis in vivo and in vitro, through proteomics, has demonstrated possible cellular biomarkers. The discovery of these biomarkers may help in the elucidation of complex mechanisms such as folliculogenesis (understanding of follicular growth and oocyte maturation), in the treatment of diseases related to female infertility, as well as in the improvement of reproductive biotechniques such as the manipulation of oocytes enclosed in preantral follicles. Thus, the present review addressed the main proteins reported in the newest works related to proteomic studies aiming at better clarifications regarding follicular and oocyte development in vivo and in vitro. The choice of articles was based on the most current discoveries and the impact that certain proteins could bring to improve the understanding of folliculogenesis and oogenesis in different species of mammalian animals.(AU)

Assuntos

RESUMO

The present study aimed to evaluate the structure, survival and development of isolated caprine (secondary-SEC and early antral-EANT) follicles, after vitrification in the presence of synthetic polymers and in vitro culture. Additionally, transzonal projections (TZPs) and p450 aromatase enzyme were evaluated. After isolation, SEC and EANT follicles were in vitro cultured for six days or vitrified. After one week, SEC and EANT follicles were warmed and also in vitro cultured for six days. Data revealed that the percentage of morphologically normal follicles was similar between fresh and vitrified follicles in both follicular categories and antrum formation rate was similar between fresh and vitrified SEC follicles. Fluorescence by calcein-AM did not show difference between fresh and vitrified (SEC and EANT) follicles, however, the trypan blue test showed low viability for vitrified follicles. The integrity of TZPs was not affected between fresh and vitrified SEC follicles, however, in vitrified EANT follicles, there were signs of TZPs loss. Regarding steroidogenic function, it was observed a positive staining for p450 aromatase enzyme in fresh and vitrified SEC and EANT follicles. It was concluded that SEC follicles seem to be more resistant to vitrification than EANT follicles, as shown by the trypan blue test and TZPs assay. Future studies may confirm this hypothesis, in order to consolidate the use of SEC and EANT follicles as an alternative to ovary cryopreservation.

Assuntos

RESUMO

The aims of this study were to investigate the effects of different concentrations of Platelet-derived growth factor-BB (PDGF-BB) on the survival, activation, levels of ROS, and growth of goat preantral follicles enclosed in ovarian tissue. For this, ovarian fragments were cultured for 7 days in Alpha Minimum Essential Medium (α-MEM+ ) with or without PDGF-BB (0, 25, 50 and 100 ng/ml). The results showed that both the 25 ng/ml PDGF and the 50 ng/ml PDGF treatments maintained the percentage of morphologically normal follicles from day 1 to day 7. In addition, the 25 ng/ml PDGF treatment showed a significantly higher percentage of morphologically normal follicles when compared to the other treatments. At day 7, greater (P < 0.05) follicular and oocyte diameters were observed in the 25 ng/ml PDGF and the 50 ng/ml PDGF treatments when compared to the cultured control treatment. On day 7 of culture, all the treatments tested had a significant increase in the percentage of developing follicles when compared to the non-cultured control. However, the percentage of follicle activation, as well as ROS production, were similar (P < 0.05) among the treatments, irrespective of culture time. In conclusion, PDGF-BB improved, in a concentration-dependent manner, follicular survival as well as oocyte and follicular diameter after in vitro culture of goat preantral follicle-enclosed in ovarian tissue fragments.

Assuntos

RESUMO

The aims of this study were to investigate the effects of different concentrations of Platelet-derived growth factor-BB (PDGF-BB) on the survival, activation, levels of ROS, and growth of goat preantral follicles enclosed in ovarian tissue. For this, ovarian fragments were cultured for 7 days in Alpha Minimum Essential Medium (α-MEM+ ) with or without PDGF-BB (0, 25, 50 and 100 ng/ml). The results showed that both the 25 ng/ml PDGF and the 50 ng/ml PDGF treatments maintained the percentage of morphologically normal follicles from day 1 to day 7. In addition, the 25 ng/ml PDGF treatment showed a significantly higher percentage of morphologically normal follicles when compared to the other treatments. At day 7, greater (P < 0.05) follicular and oocyte diameters were observed in the 25 ng/ml PDGF and the 50 ng/ml PDGF treatments when compared to the cultured control treatment. On day 7 of culture, all the treatments tested had a significant increase in the percentage of developing follicles when compared to the non-cultured control. However, the percentage of follicle activation, as well as ROS production, were similar (P < 0.05) among the treatments, irrespective of culture time. In conclusion, PDGF-BB improved, in a concentration-dependent manner, follicular survival as well as oocyte and follicular diameter after in vitro culture of goat preantral follicle-enclosed in ovarian tissue fragments.(AU)

Assuntos

RESUMO

Emergency in vitro fertilization followed by embryo vitrification is one feasible fertility preservation option for cancer patients. However, its clinical application has several limitations. Hormonal stimulation delays the initiation of oncotherapy and it is contraindicated in hormone-sensitive cancers or for use in pre-pubertal females. Vitrification of ovarian cortical tissue prior to the start of cancer treatment could be utilized for autotransplantation or for in vitro maturation of follicles enclosed in ovarian tissue. Nevertheless, the main concern associated with autotransplantation is the risk of malignant cell re-introduction to the patient, which is non-existent with the use of follicular in vitro culture. Since obtaining ovarian tissues from women for research is challenging and experimental studies are difficult to complete due to ethical issues, exploring the alternative usage of animal models for fertility preservation may provide beneficial insight into the prospects of follicular culture as an alternative for fertility restoration following ovarian tissue vitrification. Similarities between ewe and human ovary structures, as well as in ovarian follicular development dynamics, make the ewe a possible animal model for the study of female fertility preservation. As vitrification of ovarian tissue has the potential to cryopreserve preantral ovarian follicles, the present review will describe the progress of ovarian tissue vitrification studies completed in ewes.