Advances in in vitro folliculogenesis in domestic ruminants

The in vitro follicle culture (IVFC) represents an outstanding tool to enhance our understanding of the control of folliculogenesis and to allow the future use of a large number of immature oocytes enclosed in preantral follicles (PFs) in assisted reproductive techniques in humans as well as in others mammalian species including the ruminants. So far, the best results of IVFC were reported from mice with the production of live offspring from primordial follicles cultured in vitro. Live birth has been obtained after the in vitro culture of bovine early antral follicles. However, in other ruminant species, these results have been limited to the production of a variable number of mature oocytes and low percentages of embryos after in vitro culture of goat, buffalo and sheep isolated secondary preantral follicles. The present review presents and discusses the main findings, limitations, and prospects of in vitro folliculogenesis in ruminants focusing on bovine, caprine, and ovine species.

Advances in in vitro folliculogenesis in domestic ruminants

The in vitro follicle culture (IVFC) represents an outstanding tool to enhance our understanding of the control of folliculogenesis and to allow the future use of a large number of immature oocytes enclosed in preantral follicles (PFs) in assisted reproductive techniques in humans as well as in others mammalian species including the ruminants. So far, the best results of IVFC were reported from mice with the production of live offspring from primordial follicles cultured in vitro. Live birth has been obtained after the in vitro culture of bovine early antral follicles. However, in other ruminant species, these results have been limited to the production of a variable number of mature oocytes and low percentages of embryos after in vitro culture of goat, buffalo and sheep isolated secondary preantral follicles. The present review presents and discusses the main findings, limitations, and prospects of in vitro folliculogenesis in ruminants focusing on bovine, caprine, and ovine species.(AU)

Control of growth and development of preantral follicle: insights from in vitro culture

The regulation of folliculogenesis involves a complex interaction among endocrine, paracrine and autocrine factors. The mechanisms involved in the initiation of the growth of the primordial follicle, i.e., follicular activation and the further growth of primary follicles up to the pre-ovulatory stage, are not well understood at this time. The present review focuses on the regulation and development of early stage (primordial, primary, and secondary) folliculogenesis highlighting the mechanisms of primordial follicle activation, growth of primary and secondary follicles and finally transition from secondary to tertiary follicles. We also discuss the importance of in vitro follicle culture for the understanding of folliculogenesis during the preantral phase. Studies suggest that follicular development from primordial to early antral stages is primarily controlled by intra-ovarian ligands but it can also be influenced by many extra-ovarian factors. The control of early folliculogenesis is, therefore, extremely complex because several ligands act through distinct signaling pathways that form sophisticated information networks responding to multiple, often opposing, stimuli. The balance among different stimuli determines follicular survival or death as well as quiescence or activation (growth). The distribution of the ligands and their corresponding receptors varies among follicular compartments and species, and significant changes in gene expression pattern among follicular categories have been reported. Knowing that follicular requirements during early folliculogenesis can be stage-specific and speciesspecific, in vitro culture studies offer an alternative to evaluate single and combined factors during a specific period of follicular development. Herewith we summarize the main findings obtained in vitro together with the mechanisms regulating folliculogenesis.(AU)

Control of growth and development of preantral follicle: insights from in vitro culture

The regulation of folliculogenesis involves a complex interaction among endocrine, paracrine and autocrine factors. The mechanisms involved in the initiation of the growth of the primordial follicle, i.e., follicular activation and the further growth of primary follicles up to the pre-ovulatory stage, are not well understood at this time. The present review focuses on the regulation and development of early stage (primordial, primary, and secondary) folliculogenesis highlighting the mechanisms of primordial follicle activation, growth of primary and secondary follicles and finally transition from secondary to tertiary follicles. We also discuss the importance of in vitro follicle culture for the understanding of folliculogenesis during the preantral phase. Studies suggest that follicular development from primordial to early antral stages is primarily controlled by intra-ovarian ligands but it can also be influenced by many extra-ovarian factors. The control of early folliculogenesis is, therefore, extremely complex because several ligands act through distinct signaling pathways that form sophisticated information networks responding to multiple, often opposing, stimuli. The balance among different stimuli determines follicular survival or death as well as quiescence or activation (growth). The distribution of the ligands and their corresponding receptors varies among follicular compartments and species, and significant changes in gene expression pattern among follicular categories have been reported. Knowing that follicular requirements during early folliculogenesis can be stage-specific and speciesspecific, in vitro culture studies offer an alternative to evaluate single and combined factors during a specific period of follicular development. Herewith we summarize the main findings obtained in vitro together with the mechanisms regulating folliculogenesis.

Vitrification of ovarian tissue from non-human primates

Background: One of the strategies to preserve genetic material from nonhuman primates (NHP) consists in the implementation of germplasm banks, for future application in reproductive biotechniques, as well as for biomedical research. Based on the success rates achieved in human, there is a prominent possibility to succeed also with NHP. However, studies with NHP are still scarce, especially regarding the cryopreservation of ovarian tissue.Review: Neotropical non-human primates, especially males, have been used in research related to reproductive biotechniques in Brazil. Regarding research on female reproduction and ovarian tissue preservation, most studies were performed using domestic animals as models. Current concepts and controversies in the restoration of gametes in adult females does not exclude the needs to preserve ovarian tissue. Importantly, ovarian tissue can be collected and preserved even after the death of the donors, being applied when finding dead females. Furthermore, collection of ovarian biopsies is also feasible and will not affect reproductive function. Among the cryopreservation methods, the vitrification has been indicated due to practical logistic, as well as because it will avoid the formation of large intracellular ice crystals, and it is claimed that ovarian stromal damage will be decreased under vitrification. Considering the number of threatened primate species and the needs to preserve their habitat, but also their gametes, development of preservation protocols are needed. Among the procedures, vitrification appears as a practical method to be applied in the near future. Although a low number of studies is reported, most of them were performed in the recent years. In this context, this article reviews recent information on the vitrification of ovarian tissue of non-human primates. Due to the limited number of studies in these species, observed data are compared with the literature in domestic and human mammals.[...]

Vitrification of ovarian tissue from non-human primates

Background: One of the strategies to preserve genetic material from nonhuman primates (NHP) consists in the implementation of germplasm banks, for future application in reproductive biotechniques, as well as for biomedical research. Based on the success rates achieved in human, there is a prominent possibility to succeed also with NHP. However, studies with NHP are still scarce, especially regarding the cryopreservation of ovarian tissue.Review: Neotropical non-human primates, especially males, have been used in research related to reproductive biotechniques in Brazil. Regarding research on female reproduction and ovarian tissue preservation, most studies were performed using domestic animals as models. Current concepts and controversies in the restoration of gametes in adult females does not exclude the needs to preserve ovarian tissue. Importantly, ovarian tissue can be collected and preserved even after the death of the donors, being applied when finding dead females. Furthermore, collection of ovarian biopsies is also feasible and will not affect reproductive function. Among the cryopreservation methods, the vitrification has been indicated due to practical logistic, as well as because it will avoid the formation of large intracellular ice crystals, and it is claimed that ovarian stromal damage will be decreased under vitrification. Considering the number of threatened primate species and the needs to preserve their habitat, but also their gametes, development of preservation protocols are needed. Among the procedures, vitrification appears as a practical method to be applied in the near future. Although a low number of studies is reported, most of them were performed in the recent years. In this context, this article reviews recent information on the vitrification of ovarian tissue of non-human primates. Due to the limited number of studies in these species, observed data are compared with the literature in domestic and human mammals.[...](AU)

Transplante ovariano: Destaques na reprodução de primatas não humanos / Ovarian transplantation: highlight to non-human primates reproduction

Background: Ovarian transplantation in nonhuman primates (NHP) has been used as a strategy for the development of experimental models for biomedical research in the reproductive area. The prospects for application of this technique range from the restoration of female fertility to the conservation of endangered wild animals. However, studies with NHP were performed mostly focusing on the ovarian transplantation of cryopreserved tissue, in order to simulate the reality of human species, as well as aiming to obtain experimental models suitable for comparative studies. On the other hand, ovarian transplantation could be applied also in NHP species preservation.Review: According to the last census (2012-2014) of the International Union for Conservation of Nature (IUCN), currently, more than half of the 633 types of primates known around the world are in danger of disappearing forever due to the continued destruction of their natural habitat by human activities. Thus, there is an interest to expose the possible methods of ovarian preservation followed by transplantation that can be employed to promote the conservation of endangered NHP. Despite the positive results obtained with avascular autograft of fresh ovarian tissue in NHP, it is important to bring in mind the significant loss of follicles as a result of this procedure due to ischemia and reperfusion during the first days after grafting. This phenomenon leads to the production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical, which are responsible by lipid peroxidation, cell membrane damage and subsequent follicular atresia. An alternative to counteract this oxidative stress consists in the application/administration of different sources of antioxidants previously or during grafting, in the grafted tissue or in the animal receiving the transplant. The most used compounds with a claimed radical scavenger activity are catalase, trolox and some vitamins.[...](AU)

Transplante ovariano: Destaques na reprodução de primatas não humanos / Ovarian transplantation: highlight to non-human primates reproduction

Background: Ovarian transplantation in nonhuman primates (NHP) has been used as a strategy for the development of experimental models for biomedical research in the reproductive area. The prospects for application of this technique range from the restoration of female fertility to the conservation of endangered wild animals. However, studies with NHP were performed mostly focusing on the ovarian transplantation of cryopreserved tissue, in order to simulate the reality of human species, as well as aiming to obtain experimental models suitable for comparative studies. On the other hand, ovarian transplantation could be applied also in NHP species preservation.Review: According to the last census (2012-2014) of the International Union for Conservation of Nature (IUCN), currently, more than half of the 633 types of primates known around the world are in danger of disappearing forever due to the continued destruction of their natural habitat by human activities. Thus, there is an interest to expose the possible methods of ovarian preservation followed by transplantation that can be employed to promote the conservation of endangered NHP. Despite the positive results obtained with avascular autograft of fresh ovarian tissue in NHP, it is important to bring in mind the significant loss of follicles as a result of this procedure due to ischemia and reperfusion during the first days after grafting. This phenomenon leads to the production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical, which are responsible by lipid peroxidation, cell membrane damage and subsequent follicular atresia. An alternative to counteract this oxidative stress consists in the application/administration of different sources of antioxidants previously or during grafting, in the grafted tissue or in the animal receiving the transplant. The most used compounds with a claimed radical scavenger activity are catalase, trolox and some vitamins.[...]

Teste de toxicidade e criopreservação de folículos pré-antrais ovinos isolados utilizando Glicerol, Etilenoglicol, Dimetilsulfóxido e Propanodiol / Toxicity test and cryopreservation of sheep isolated preantral follicles using glycerol, ethylen glycol, dimethil sulfoxyde and propanediol

O objetivo deste estudo foi avaliar foliculos pré-antrais (FOPA) ovinos isolados após sua exposição e criopreservação utilizando glicerol (GLI), etilenoglicol (EG), propanodiol (PROH) ou dimetilsulfóxido (DMSO) a 1,5 e 3,0 M. Cada par ovariano de 5 ovelhas sem raça definida foi coletado em abatedouro local e submetido ao isolamento folicular. Da suspensão obtida, uma aliquota foi imediatamente destinada à análise da viabilidade folicular com o auxílio do corante vital azul de trypan. O restante da suspensão foi dividida em 16 aliquotas de 0,9 mL, suspensas (v/v) em MEM+ com EG, DMSO, GLI ou PROH a 1,5 ou 3,0 M, para teste de toxicidade e criopreservação. Após o término de cada tratamento, a viabilidade folicular foi analisada e os FOPA considerados viáveis se não corados ou não viáveis, quando corados. A análise dos dados mostrou que após o teste de toxicidade e criopreservação, em todos os crioprotetores e em ambas as concentrações, a percentagem de FOPA viáveis foi significativamente reduzida quando comparada ao controle. No teste de toxicidade, quando os crioprotetores foram comparados entre si nas mesmas concentrações, foram observadas percentagens signifIcativamente menores de FOPA viáveis no PROH 3,0 M (38,9%), apresentando-se, portanto, mais tóxico quando comparado aos demais crioprotetores. Após criopreservação, obteve-se percentagens significativamente maiores de foliculos pré-antrais viáveis quando o EG e o DMSO foram utilizados. Em conclusão, FOPA ovinos isolados podem ser criopreservados com sucesso utilizando-se D MSO e EG a 1,5 e 3,0 M.(AU)

The aim of this study was to evaluate isolated sheep preantral follicles (PF) after exposure and cryopreservation using glycerol (GLI), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO) at 1.5 and 3.0 M. Each ovarian pair from 5 mixed breed adult sheeps was obtained at a local slaughterhouse and submited to follicular isolation. From the obtained suspension, one aliquot was immediately analysed with trypan blue. The remaining suspension was divided in 16 aliquots of 0.9 mL, suspended in (v /v) in MEM+with EG, DMSO, GLI or PROH at 1.5 or 3.0 M to the toxicity test and cryopreservation. After the end of each treatment, the follicular viability was analysed and the PF were classified as viable if not dyed or not viable if dyed with trypan blue. The analysis of the results showed that after the toxicity test and cryopreservation, using all cryoprotectants and at both concentrations, the percentage of viable PF was significandy reduced when compared to the control. At the toxicity test, when the cryoprotectants were compared at the same concentrations, the lowest percentage of viable preantral follicles was obtained when 3.0 M PRO H (38,9%) was used, being, more toxic when compared to the others cryoprotectants. After cryopreservation, significantly higher percentual of viable PF was observed when the EG and DMSO were used. In conclusion, sheep PF can be cryopreserved successfully using DMSO and EG at 1.5 and 3.0 M.(AU)

Criopreservação de folículos pré-antrais ovinos

Este estudo teve como objetivo desenvolver um método eficiente de criopreservação (congelação lenta e vitrificação) de folículos pré-antrais ovinos. Quando folículos pré-antrais in situ e isolados foram criopreservados no método de congelação lenta apenas na presença de crioprotetores intracelulares, os melhores resultados foram obtidos na presença de dimetilsulfóxido (DMSO) 1,5 M (in situ) e DMSO ou etilenoglicol (EG) 1,5 ou 3,0 M (isolados). No entanto, após cultivo in vitro, somente os folículos preservados inclusos no tecido ovariano foram aptos a ativar e crescer in vitro. Com o intuito de otimizar os protocolos de criopreservação para folículos pré-antrais ovinos, foram utilizados os métodos mais acurados de análise, adição de crioprotetor extracelular (sacarose), adicionado a estudos também de vitrificação. Devido aos resultados não promissores com folículos isolados, o estudo proseguiu com tecido ovariano e observou-se uma alta taxa de viabilidade e atividade enzimática no citoplasma após congelação lenta na presença de DMSO ou EG 1,0 M adicionados de sacarose 0,5 M e após vitrificação utilizando o método de superfície-sólida na presença de DMSO ou EG 4,0 M. Além disso, após detecção da qualidade folicular pos congelação/descongelação, o tecido ovariano foi cultivado por um curto período (24 h) e foi possível detectar a expressão de fatores diretamente relacionados à atividade funcional de tais folículos: GDF-9, BMP-15, KL e c-Kit. Apesar da eficiência dos dois métodos de criopreservação, folículos ovarianos vitrificados não apresentaram a mesma qualidade que os criopreservados pelo método clássico após cultivo in vitro do tecido ovariano por 24 h