Retrospectiva dos principais biomarcadores envolvidos no desenvolvimento folicular e oocitário em mamíferos / Retrospective of the main biomarkers involved in follicular and oocyte development in mammals

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)

Potential use of bacterial pigments as anticancer drugs and female reproductive toxicity: a review / Uso potencial de pigmentos bacterianos como drogas anticâncer e toxicidade reprodutiva feminina: uma revisão

Natural bioactive compounds obtained from microorganisms, have awakened particular interest in the industry nowadays. This attention comes when natural resources depletion is pronounced, and the acquisition of both new plant origin resources and bioactive products, represents a challenge for the next generations. In this sense, prospecting for large-scale production and use of bacterial pigments is a necessary strategy for the development of novel products. A wide variety of properties have been attributed to these substances and, among them, their therapeutic potential against important diseases, such as cancer. There is consensus that available chemotherapy protocols are known to detrimentally affect cancer patients fertility. Hence, considerable part of the deleterious effects of chemotherapy is related to the drugs cytotoxicity, which, in addition to cancer cells, also affect normal cells. Therefore, the intrinsic properties of bacterial pigments associated with low cytotoxicity and relevant cell selectivity, certified them as potential anticancer drugs. However, little information is available about reproductive toxicity of these new and promising compounds. Thus, the present review aims to address the main bacterial pigments, their potential uses as anticancer drugs and their possible toxic effects, especially on the female gonad.

Os compostos bioativos naturais obtidos de microrganismos têm despertado especial interesse da indústria nos últimos anos. Esta atenção ocorre em um momento em que o esgotamento de recursos naturais é pronunciado, e a aquisição de novos insumos e produtos bioativos de origem vegetal representa um desafio para as próximas gerações. Neste sentido, a prospecção para a produção e uso em larga escala dos pigmentos bacterianos tem representado uma importante estratégia para o desenvolvimento de novos produtos. Uma grande variedade de propriedades foi atribuída a estas substâncias, entre elas, o potencial terapêutico contra doenças importantes, como o câncer. Existe um consenso de que os protocolos quimioterápicos disponíveis são conhecidos por afetarem negativamente a fertilidade de pacientes com câncer. Grande parte dos efeitos deletérios da quimioterapia está relacionado à citotoxicidade das drogas usadas para este fim, que além das células cancerosas, afetam as células normais. Nesse sentido, as propriedades naturais atribuídas aos pigmentos bacterianos associadas à baixa citotoxicidade e relevante seletividade, os qualificaram como potenciais drogas anticâncer. No entanto, pouco se tem de informação a respeito da toxicidade reprodutiva destes novos e promissores compostos. Dessa forma, a presente revisão tem o objetivo de abordar os principais pigmentos bacterianos, suas utilizações potenciais como drogas anticâncer, bem como os seus possíveis efeitos tóxicos, sobretudo, sobre a gônada feminina.

Comparative analysis of the hormone production and gene expression profiles in ovine uterus tissue during oestrus cycle synchronized using medroxyprogesterone acetate plus eCG and prostaglandin analogue / Análise comparativa da produção de hormônios e perfis de expressão gênica em tecido uterino ovino durante o ciclo estral sincronizado usando acetato de medroxiprogesterona mais eCG e análogo de prostaglandina

The combination of medroxyprogesterone acetate (MPA) and gonadotropin chorionic (eCG) has been widely used to synchronize oestrus cycle in sheep, but their effects on the gene expression in uterine tissue are yet to be elucidated. To evaluate the effect of MPA + eCG or prostaglandin analogue (PA) treatments on the rate of oestrus cycle synchronization, as well as further hormone production and gene expression profiles in uterine tissue, 14 Santa Inês ewes were randomly selected. The MPA + eCG group (n=7) received intravaginal insertion of MPA-impregnated sponges for 14 days and was administered 350 IU eCG on the day of sponge withdrawal. The PA group (n=7) was administered two doses of 100 µg of PA separated by 12 days. The ewes were assessed for the rate of oestrus cycle synchronization and the serum concentrations of progesterone (P4) and estradiol (E2). Additionally, the expression of estrogen receptor (ERα), progesterone receptor (P4R), and immunolocalization of interferon receptor (IFNAR1) in the uterine tissue samples collected 15th day post-mating were examined. The rate of oestrus cycle synchronization was 100% (n=7/7) and 57.14% (n=4/7) in the MPA + eCG and PA groups, respectively. Moreover, the MPA + eCG group exhibited higher serum concentration of P4 than the PA group (p < 0.05). However, the E2 serum concentration did not differ between the two groups (p > 0.05). The relative expression of P4R and ERα mRNA analyzed using real-time PCR and immunodetection of IFNAR1 were similar between the two groups tested (p > 0.05). Conclusively, MPA + eCG treatment improved the rate of oestrus cycle synchronization and endogenous P4 production; however, it did not affect the expression of sex steroid receptors and IFNAR1 in uterine ovine tissue.(AU)

A combinação de acetato de medroxiprogesterona (MPA) com gonadotrofina coriônica (eCG) é amplamente utilizada para sincronizar o estro de ovelhas, mas seus possíveis efeitos sobre a expressão gênica em tecido uterino não foram elucidados. Para avaliar o efeito dos protocolos MPA + eCG ou análogo de prostaglandina (PA) sobre a taxa de sincronização do estro, bem como na futura produção hormonal e expressão gênica em tecido uterino, 14 ovelhas Santa Inês foram selecionadas. O grupo MPA + eCG (n=7) recebeu a inserção de esponjas impregnadas de MPA por via intravaginal por 14 dias e 350 UI de eCG no dia da retirada da esponja. O grupo PA recebeu duas doses de 100 µg de PA administradas com 12 dias de intervalo. As ovelhas foram avaliadas quanto à taxa de sincronização do estro, concentração sérica de progesterona (P4) e estradiol (E2). Além disso, foram examinadas a expressão do receptor de estradiol (ERα), receptor de progesterona (P4R) e localização do receptor de interferon (IFNAR1), a partir de amostras de tecido uterino coletadas 15 dias após o acasalamento. A taxa de sincronização do estro foi 100% (n = 7/7) e 57,14% (n = 4/7) nos grupos MPA + eCG e PA, respectivamente. Além disso, o grupo MPA + eCG apresentou maior concentração sérica de P4 em comparação com o grupo PA (P < 0,05). No entanto, a concentração sérica de E2 não diferiu entre os grupos testados (P > 0,05). A expressão relativa de RNAm para P4R e ERα analisado por PCR em tempo real e imunodetecção de IFNAR1 foi semelhante entre os grupos testados (P > 0,05). Em conclusão, o tratamento com MPA + eCG melhora a taxa de sincronização do estro e a produção de progesterona endógena; contudo, não afeta a regulação da expressão de receptores de esteroides sexuais e IFNAR1 no tecido uterino de ovinos.(AU)

Avanços na tecnologia do ovário artificial em ruminantes / Advances in artificial ovary technology in ruminants

O cultivo in vitro de folículos (CIVF), também conhecido como Ovário Artificial, representa uma excelente ferramenta para aumentar nossa compreensão a respeito do controle da foliculogênese inicial, que é crucial para otimizar o uso futuro de um grande número de oócitos imaturos inclusos em folículos pré-antrais (FPs) (principalreserva ovariana) em técnicas de reprodução assistida em humanos, bem como em outras espécies de mamíferos, incluindo os ruminantes. Até o momento, os melhores resultados de CIVF foram relatados em camundongos com a produção de crias vivas a partir de folículos primordiais cultivados in vitro. No entanto, em outras espécies como os ruminantes, esses resultados têm se limitado à produção de um número variável de oócitos maturos e baixas porcentagens de embriões após o cultivo in vitro de folículos pré-antrais secundários tardios isolados de ovários de cabras, búfalas e ovelhas. A presente revisão apresenta e discute os principais achados, limitações e perspectivas da foliculogênese in vitro em ruminantes com foco nas espécies bovinas, caprinas e ovinas.

The in vitro follicle culture (IVFC), also known as artificial ovary, represents an outstanding tool to increase our understanding of the control of early folliculogenesis which is crucial to optimize the future use of a large, number of immature oocytes enclosed in preantral follicles (PFs) (main ovarian reserve) in assisted reproductive techniques in humans as well as in others mammalian species including the ruminants. To date, the best results of IVFC were reported from mice with the production of live offspring from primordial follicles cultured in vitro. 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 late 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.

Avanços na tecnologia do ovário artificial em ruminantes / Advances in artificial ovary technology in ruminants

O cultivo in vitro de folículos (CIVF), também conhecido como Ovário Artificial, representa uma excelente ferramenta para aumentar nossa compreensão a respeito do controle da foliculogênese inicial, que é crucial para otimizar o uso futuro de um grande número de oócitos imaturos inclusos em folículos pré-antrais (FPs) (principalreserva ovariana) em técnicas de reprodução assistida em humanos, bem como em outras espécies de mamíferos, incluindo os ruminantes. Até o momento, os melhores resultados de CIVF foram relatados em camundongos com a produção de crias vivas a partir de folículos primordiais cultivados in vitro. No entanto, em outras espécies como os ruminantes, esses resultados têm se limitado à produção de um número variável de oócitos maturos e baixas porcentagens de embriões após o cultivo in vitro de folículos pré-antrais secundários tardios isolados de ovários de cabras, búfalas e ovelhas. A presente revisão apresenta e discute os principais achados, limitações e perspectivas da foliculogênese in vitro em ruminantes com foco nas espécies bovinas, caprinas e ovinas.(AU)

The in vitro follicle culture (IVFC), also known as artificial ovary, represents an outstanding tool to increase our understanding of the control of early folliculogenesis which is crucial to optimize the future use of a large, number of immature oocytes enclosed in preantral follicles (PFs) (main ovarian reserve) in assisted reproductive techniques in humans as well as in others mammalian species including the ruminants. To date, the best results of IVFC were reported from mice with the production of live offspring from primordial follicles cultured in vitro. 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 late 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)

Ultra-diluted Folliculinum 6 cH impairs ovine oocyte viability and maturation after in vitro culture

This study investigated the effect of Folliculinum 6 cH on the oocyte meiosis resumption and viability rates, progesterone production and mitochondrial activity after in vitro maturation of cumulus-oocyte complexes (COCs) in sheep. Sheep ovaries were collected at a local slaughterhouse and COCs were recovered by slicing technique. The selected COCs were maturated in TCM199 (Control treatment), or control medium supplemented with 0.05% ethanol (v/v) (the vehicle of the homeopathic preparation – Ethanol treatment) or with Folliculinum 6 cH. After 24 h of in vitro maturation (IVM), oocytes were mechanically denuded and incubated with Hoechst 33342 and MitoTracker (0.5 μM) Orange CMTMRos for analysis of viability and chromatin configuration, and mitochondrial activity, respectively. The results showed that Folliculinum 6 cH addition increased oocyte degeneration and reduced meiotic resumption compared to the control (P < 0.05). Interestingly, the percentages meiotic resumption and oocyte maturation were lower in the Folliculinum 6 cH treatment compared to its vehicle (Ethanol treatment) (P < 0.05). On the other hand, when the treatments were compared, higher mitochondrial activity was observed in the Ethanol treatment (P < 0.05). In conclusion, contrary to its vehicle, the addition of Folliculinum 6 cH to the IVM medium promoted oocyte degeneration and affected negatively the mitochondrial distribution, impairing meiosis resumption.

Ultra-diluted Folliculinum 6 cH impairs ovine oocyte viability and maturation after in vitro culture

This study investigated the effect of Folliculinum 6 cH on the oocyte meiosis resumption and viability rates, progesterone production and mitochondrial activity after in vitro maturation of cumulus-oocyte complexes (COCs) in sheep. Sheep ovaries were collected at a local slaughterhouse and COCs were recovered by slicing technique. The selected COCs were maturated in TCM199 (Control treatment), or control medium supplemented with 0.05% ethanol (v/v) (the vehicle of the homeopathic preparation Ethanol treatment) or with Folliculinum 6 cH. After 24 h of in vitro maturation (IVM), oocytes were mechanically denuded and incubated with Hoechst 33342 and MitoTracker (0.5 μM) Orange CMTMRos for analysis of viability and chromatin configuration, and mitochondrial activity, respectively. The results showed that Folliculinum 6 cH addition increased oocyte degeneration and reduced meiotic resumption compared to the control (P < 0.05). Interestingly, the percentages meiotic resumption and oocyte maturation were lower in the Folliculinum 6 cH treatment compared to its vehicle (Ethanol treatment) (P < 0.05). On the other hand, when the treatments were compared, higher mitochondrial activity was observed in the Ethanol treatment (P < 0.05). In conclusion, contrary to its vehicle, the addition of Folliculinum 6 cH to the IVM medium promoted oocyte degeneration and affected negatively the mitochondrial distribution, impairing meiosis resumption.(AU)

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)

Powdered coconut water (ACP 406®) as an alternative base culture medium for in vitro culture of goat preantral follicles enclosed in ovarian tissue

This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 μg/mL penicillin and 100 μg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P < 0.05. At day 2 of culture, a greater (P < 0.05) percentage of morphologically normal follicles was observed between ACP+ and ACP treatments. Moreover, at day 2 of culture, no hormonal difference (P < 0.05) was observed between ACP+ and both α-MEM treatments. At day 6 of culture when ACP and α-MEM treatments were compared the percentage of healthy follicles were similar (P > 0.05) among treatments. Overall, all treatments had lower primordial follicles (P < 0.05) accompany by greater developing follicles (P < 0.05) percentages than non-cultured control treatment, indicating primordial follicle activation. However, at day 6 of culture, the percentage of primordial follicle development were similar (P > 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.(AU)

Powdered coconut water (ACP 406®) as an alternative base culture medium for in vitro culture of goat preantral follicles enclosed in ovarian tissue

This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 μg/mL penicillin and 100 μg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P 0.05) among treatments. Overall, all treatments had lower primordial follicles (P 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.

Supplemented powdered coconut water (ACP-406®) promotes growth of goat secondary follicles and oocyte meiotic resumption

The objective of this study was to test the efficiency of powdered coconut water (ACP-406®) base-medium without or with the addition of supplements on in vitro culture of isolated goat secondary follicles. Follicles were cultured for 18 days in α-MEM or in ACP-406®, both without supplements (referred to as α-MEM and ACP, respectively), or both supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine, and ascorbic acid (referred to as α-MEM+ and ACP+). Follicular morphology, antrum formation, follicular and oocyte diameter, levels of glutathione (GSH), and chromatin configuration after in vitro maturation were evaluated. At the end of culture, ACP-406® base-medium (without or with supplements) showed a higher (P < 0.05) percentage of normal follicles than α-MEM (without or with supplements). Antrum formation was similar among α-MEM+, ACP and ACP+, and significantly higher than α-MEM without supplements. The follicular diameter was greater in ACP+ than α-MEM, and similar to other treatments. Moreover, fully and daily grown rates were higher (P < 0.05) in ACP-406® base-medium (without or with supplements) than α-MEM (without or with supplements). Levels of GSH were similar between ACP+ and α-MEM+ treatments. Both ACP+ and α-MEM+ allowed meiotic resumption without a significant difference between the two groups. In conclusion, supplemented ACP-406® base-medium maintained follicular survival and promoted the development as well as meiotic resumption of isolated goat secondary follicles cultured in vitro for 18 days.(AU)

Supplemented powdered coconut water (ACP-406®) promotes growth of goat secondary follicles and oocyte meiotic resumption

The objective of this study was to test the efficiency of powdered coconut water (ACP-406®) base-medium without or with the addition of supplements on in vitro culture of isolated goat secondary follicles. Follicles were cultured for 18 days in α-MEM or in ACP-406®, both without supplements (referred to as α-MEM and ACP, respectively), or both supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine, and ascorbic acid (referred to as α-MEM+ and ACP+). Follicular morphology, antrum formation, follicular and oocyte diameter, levels of glutathione (GSH), and chromatin configuration after in vitro maturation were evaluated. At the end of culture, ACP-406® base-medium (without or with supplements) showed a higher (P < 0.05) percentage of normal follicles than α-MEM (without or with supplements). Antrum formation was similar among α-MEM+, ACP and ACP+, and significantly higher than α-MEM without supplements. The follicular diameter was greater in ACP+ than α-MEM, and similar to other treatments. Moreover, fully and daily grown rates were higher (P < 0.05) in ACP-406® base-medium (without or with supplements) than α-MEM (without or with supplements). Levels of GSH were similar between ACP+ and α-MEM+ treatments. Both ACP+ and α-MEM+ allowed meiotic resumption without a significant difference between the two groups. In conclusion, supplemented ACP-406® base-medium maintained follicular survival and promoted the development as well as meiotic resumption of isolated goat secondary follicles cultured in vitro for 18 days.

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.

Avanços, aplicações, limitações e perspectivas da tecnologia do ovário artificial de ruminantes / Advances, applications, limitations and prospects of the technology of artificial ovary in ruminant

A maioria dos oócitos imaturos nos ovários encontra-se armazenada nos folículos pré-antrais, sendo este a principal reserva ovariana. Entretanto, durante o desenvolvimento folicular uma pequena proporção (0,1%) destes folículos chega ao estágio pré-ovulatório sendo o restante eliminado por atresia. A tecnologia de cultivo folicular in vitro, denominada ovário artificial, representa uma excelente ferramenta para desvendar o controle da foliculogênese nos estágios iniciais de desenvolvimento, bem como poderá assegurar no futuro a utilização de um grande número de oócitos imaturos, previamente crescidos in vitro, em técnicas de reprodução assistida em humanos e em outras espécies, incluindo os ruminantes. Os melhores resultados da tecnologia do ovário artificial foram relatados em camundongos, com a produção de crias vivas a partir de folículos primordiais crescidos in vitro. Entretanto, em outras espécies, incluindo os ruminantes, os resultados têm sido restritos à produção de um pequeno e variável número de oócitos maturos e uma baixa taxa de produção de embriões após o cultivo in vitro de folículos secundários isolados. A presente revisão discute as aplicações, critérios para avaliação de eficiência, estado atual, limitações e perspectivas da tecnologia do ovário artificial em ruminantes, com ênfase nas espécies bovina, caprina e ovina.(AU)

Most immature oocytes in the ovaries are stored in the preantral follicles, which represent the main ovarian reserve. However, during follicular development, a small proportion (0.1%) of these follicles reaches the preovulatory stage, the rest being eliminated by atresia. The in vitro follicle culture technology, called artificial ovary, represents an excellent tool to understand the control of folliculogenesis in the early stages of development, as well as to ensure in the future the use of a large number of immature oocytes, previously grown in vitro, in assisted reproductive technologies in humans and other species, including ruminants. The best results from artificial ovary technology so far were reported in mice, with the production of live offspring from primordial follicles grown in vitro. However, in other species, including ruminants, the results have been limited to the production of a small and variable number of mature oocytes and a low rate of embryo production after in vitro culture of isolated secondary follicles. The present review discusses the applications, criteria for evaluating efficiency, current status, limitations and perspectives of artificial ovary technology in ruminants, with emphasis on bovine, caprine, and ovine species.(AU)

Avanços, aplicações, limitações e perspectivas da tecnologia do ovário artificial de ruminantes / Advances, applications, limitations and prospects of the technology of artificial ovary in ruminant

A maioria dos oócitos imaturos nos ovários encontra-se armazenada nos folículos pré-antrais, sendo este a principal reserva ovariana. Entretanto, durante o desenvolvimento folicular uma pequena proporção (0,1%) destes folículos chega ao estágio pré-ovulatório sendo o restante eliminado por atresia. A tecnologia de cultivo folicular in vitro, denominada ovário artificial, representa uma excelente ferramenta para desvendar o controle da foliculogênese nos estágios iniciais de desenvolvimento, bem como poderá assegurar no futuro a utilização de um grande número de oócitos imaturos, previamente crescidos in vitro, em técnicas de reprodução assistida em humanos e em outras espécies, incluindo os ruminantes. Os melhores resultados da tecnologia do ovário artificial foram relatados em camundongos, com a produção de crias vivas a partir de folículos primordiais crescidos in vitro. Entretanto, em outras espécies, incluindo os ruminantes, os resultados têm sido restritos à produção de um pequeno e variável número de oócitos maturos e uma baixa taxa de produção de embriões após o cultivo in vitro de folículos secundários isolados. A presente revisão discute as aplicações, critérios para avaliação de eficiência, estado atual, limitações e perspectivas da tecnologia do ovário artificial em ruminantes, com ênfase nas espécies bovina, caprina e ovina.

Most immature oocytes in the ovaries are stored in the preantral follicles, which represent the main ovarian reserve. However, during follicular development, a small proportion (0.1%) of these follicles reaches the preovulatory stage, the rest being eliminated by atresia. The in vitro follicle culture technology, called artificial ovary, represents an excellent tool to understand the control of folliculogenesis in the early stages of development, as well as to ensure in the future the use of a large number of immature oocytes, previously grown in vitro, in assisted reproductive technologies in humans and other species, including ruminants. The best results from artificial ovary technology so far were reported in mice, with the production of live offspring from primordial follicles grown in vitro. However, in other species, including ruminants, the results have been limited to the production of a small and variable number of mature oocytes and a low rate of embryo production after in vitro culture of isolated secondary follicles. The present review discusses the applications, criteria for evaluating efficiency, current status, limitations and perspectives of artificial ovary technology in ruminants, with emphasis on bovine, caprine, and ovine species.

Utilização do cultivo in vitro de folículos pré-antrais suínos inclusos em tecido ovariano (in situ) como modelo de avaliação da eficiência de medicamentos homeopáticos na foliculogênese inicial / In situ cultured preantral follicles is a useful model to evaluate the effect of homeopathy medicine on early folliculogenesis

Este trabalho teve por objetivo avaliar o efeito dos medicamentos homeopáticos (Pulsatilla nigricans e hormônio folículo estimulante homeopático - FSH) e um complexo homeopático (Bos Stress Fertilis) na foliculogênese inicial, utilizando o cultivo in vitro de folículos pré-antrais suínos como modelo in vitro. Para tanto, fragmentos ovarianos foram cultivados por um ou sete dias em α-MEM+ na ausência (controle cultivado) ou presença de FSH homeopático (6 cH), Pulsatilla (6 CH), Bos Stress Fertilis (6 CH), álcool cereal (50% - v/v) ou FSH recombinante (50 ng/ml) adicionados diariamente. Os fragmentos ovarianos não cultivados (controle fresco) ou cultivados por um e sete dias foram processados para histologia clássica. Somente o composto homeopático Bos Stress Fertilis foi eficiente em manter o percentual de sobrevivência folicular após sete dias de cultivo semelhante ao controle não cultivado e α-MEM+. Em relação ao crescimento folicular, somente a adição de FSH homeopático aumentou o diâmetro folicular quando comparado ao controle não cultivado e α-MEM+ após um dia de cultivo. Dessa forma, pode-se concluir que a adição dos medicamentos homeopáticos Bos Stress fertilis e FSH homeopático (6 CH) melhoraram, respectivamente, a sobrevivência e o crescimento in vitro de folículos pré-antrais suínos inclusos em fragmentos de tecido ovariano.

This study investigated the effect of homeopathic medicine (Pulsatilla nigricans and homeopathic follicle stimulating hormone - FSH) and the complex (Bos Stress Fertilis) on the initial folliculogenesis, using the in vitro culture of preantral follicles as in vitro model. For this, swine ovarian fragments were cultured for one or seven days in α-MEM+ in the absence (cultured control) or presence of homeopathic FSH (6 cH), Pulsatilla (6 CH), Bos Stress Fertilis (6 CH), grain alcohol (50% v / v) or recombinant FSH (50 ng / ml) added daily. Uncultured ovarian fragments (fresh control) or cultured for one and seven days were processed for classical histology. Only the homeopathic complex Bos Stress Fertilis maintained the percentage of follicular survival after seven days of culture in relation to the uncultured control and α-MEM+. Regarding follicular growth, only the addition of homeopathic FSH increased the follicular diameter when compared to the uncultured control and a-MEM+. Thus, it can be concluded that the addition of the homeopathic remedies Bos Stress fertilis and homeopathic FSH (6 CH) improved, respectively, survival and in vitro growth of swine preantral follicles included in ovarian tissue.

Utilização do cultivo in vitro de folículos pré-antrais suínos inclusos em tecido ovariano (in situ) como modelo de avaliação da eficiência de medicamentos homeopáticos na foliculogênese inicial / In situ cultured preantral follicles is a useful model to evaluate the effect of homeopathy medicine on early folliculogenesis

Este trabalho teve por objetivo avaliar o efeito dos medicamentos homeopáticos (Pulsatilla nigricans e hormônio folículo estimulante homeopático - FSH) e um complexo homeopático (Bos Stress Fertilis) na foliculogênese inicial, utilizando o cultivo in vitro de folículos pré-antrais suínos como modelo in vitro. Para tanto, fragmentos ovarianos foram cultivados por um ou sete dias em α-MEM+ na ausência (controle cultivado) ou presença de FSH homeopático (6 cH), Pulsatilla (6 CH), Bos Stress Fertilis (6 CH), álcool cereal (50% - v/v) ou FSH recombinante (50 ng/ml) adicionados diariamente. Os fragmentos ovarianos não cultivados (controle fresco) ou cultivados por um e sete dias foram processados para histologia clássica. Somente o composto homeopático Bos Stress Fertilis foi eficiente em manter o percentual de sobrevivência folicular após sete dias de cultivo semelhante ao controle não cultivado e α-MEM+. Em relação ao crescimento folicular, somente a adição de FSH homeopático aumentou o diâmetro folicular quando comparado ao controle não cultivado e α-MEM+ após um dia de cultivo. Dessa forma, pode-se concluir que a adição dos medicamentos homeopáticos Bos Stress fertilis e FSH homeopático (6 CH) melhoraram, respectivamente, a sobrevivência e o crescimento in vitro de folículos pré-antrais suínos inclusos em fragmentos de tecido ovariano.(AU)

This study investigated the effect of homeopathic medicine (Pulsatilla nigricans and homeopathic follicle stimulating hormone - FSH) and the complex (Bos Stress Fertilis) on the initial folliculogenesis, using the in vitro culture of preantral follicles as in vitro model. For this, swine ovarian fragments were cultured for one or seven days in α-MEM+ in the absence (cultured control) or presence of homeopathic FSH (6 cH), Pulsatilla (6 CH), Bos Stress Fertilis (6 CH), grain alcohol (50% v / v) or recombinant FSH (50 ng / ml) added daily. Uncultured ovarian fragments (fresh control) or cultured for one and seven days were processed for classical histology. Only the homeopathic complex Bos Stress Fertilis maintained the percentage of follicular survival after seven days of culture in relation to the uncultured control and α-MEM+. Regarding follicular growth, only the addition of homeopathic FSH increased the follicular diameter when compared to the uncultured control and a-MEM+. Thus, it can be concluded that the addition of the homeopathic remedies Bos Stress fertilis and homeopathic FSH (6 CH) improved, respectively, survival and in vitro growth of swine preantral follicles included in ovarian tissue.(AU)

Tecnologia do ovário artificial: aplicações, estado da arte, limitações e perspectivas / Artificial ovary technology: applications, state of art, limitations and prospects

O ovário dos mamíferos contem de milhares a milhões de oócitos imaturos, sendo a maioria (cerca de90%) inclusa nos folículos pré-antrais. Entretanto, in vivo a quase totalidade destes folículos será eliminada porum processo fisiológico denominado de atresia. Portanto, os principais objetivos da tecnologia do ovárioartificial são: 1- Resgatar os folículos pré-antrais dos ovários antes que eles se tornem atrésicos e 2- cultivar invitro estes folículos até a maturação, com o propósito de produzir oócitos aptos a serem utilizados para aprodução in vitro de embriões. Este artigo de revisão descreve as aplicações e estado da arte da tecnologia doovário artificial, bem como discute os principais resultados, limitações e perspectivas do cultivo in vitro defolículos pré-antrais, com ênfase nos pequenos ruminantes.(AU)

The mammalian ovary contains from thousands to millions of immature oocytes being most of them(approximately 90%) enclosed in preantral follicles. However, in vivo the vast majority of follicles will beeliminated by a physiological process named atresia. Therefore, the main goals of the artificial ovary technologyare: 1- to recover preantral follicles from the ovary before they become atretic and 2- in vitro culture thosefollicles up to maturation stages for the purpose of producing oocytes to be used for further in vitro embryoproduction. This review article describes the applications and state of art of the artificial ovary technology aswell as discusses the main results, limitations and prospects of in vitro follicle culture focus on small ruminants.(AU)