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SummaryStudies have shown that daily exposure to different products, whether chemical or natural, can cause irreversible damage to women's reproductive health. Therefore it is necessary to use tests that evaluate the safety and efficacy of these products. Most reproductive toxicology tests are performed in vivo. However, in recent years, various cell culture methods, including embryonic stem cells and tissues have been developed with the aim of reducing the use of animals in toxicological tests. This is a major advance in the area of toxicology, as these systems have the potential to become a widely used tool compared with in vivo tests routinely used in reproductive biology and toxicology. The present review describes and highlights data on in vitro culture processes used to evaluate reproductive toxicity as an alternative to traditional methods using in vivo tests.

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The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.

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A criopreservação e transplante do tecido ovariano representa uma técnica em potencial para apreservação e restauração da fertilidade feminina, especialmente de mulheres jovens que se submetem atratamentos gonadotóxicos como os quimioterápicos, usados contra o câncer. Embora, o transplante de tecidoovariano fresco ou criopreservado tenha resultado no nascimento de indivíduos em diferentes espécies, comocamundongos, ovinos e humanos, os riscos de reintrodução de células cancerosas após o tratamento, temlimitado o uso clínico. Desta forma, o cultivo in vitro de folículos pré-antrais após criopreservação do tecidoovariano pode ser uma alternativa promissora para a preservação da fertilidade em casos extremos. Acombinação destas técnicas é capaz de salvaguardar a fertilidade de pacientes que sofrem de câncer, comotambém permite a preservação do germoplasma de animais de alto valor comercial ou sob risco de extinção. Noentanto, muitos desafios ainda necessitam ser superados, especialmente no que se refere à utilização de tecidoovariano previamente criopreservado em animais de produção.(AU)

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A criopreservação e transplante do tecido ovariano representa uma técnica em potencial para apreservação e restauração da fertilidade feminina, especialmente de mulheres jovens que se submetem atratamentos gonadotóxicos como os quimioterápicos, usados contra o câncer. Embora, o transplante de tecidoovariano fresco ou criopreservado tenha resultado no nascimento de indivíduos em diferentes espécies, comocamundongos, ovinos e humanos, os riscos de reintrodução de células cancerosas após o tratamento, temlimitado o uso clínico. Desta forma, o cultivo in vitro de folículos pré-antrais após criopreservação do tecidoovariano pode ser uma alternativa promissora para a preservação da fertilidade em casos extremos. Acombinação destas técnicas é capaz de salvaguardar a fertilidade de pacientes que sofrem de câncer, comotambém permite a preservação do germoplasma de animais de alto valor comercial ou sob risco de extinção. Noentanto, muitos desafios ainda necessitam ser superados, especialmente no que se refere à utilização de tecidoovariano previamente criopreservado em animais de produção.

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This study investigated the effect of androstenedione (A4) alone or in association with different concentrations of bovine recombinant FSH on the IVC of isolated goat preantral follicles. Follicles were mechanically isolated from ovarian tissue and cultured for 18 days in α-minimum essential medium supplemented or not with A4 (10 ng/mL) alone or in association with fixed (A4 + FixFSH: 100 ng/mL) or sequential (A4 + SeqFSH: Day 0, 100 ng/mL; Day 6, 500 ng/mL; Day 12, 1000 ng/mL) concentrations of FSH. After 18 days, the oocytes were recovered for IVM and fluorescence analysis. At Day 18 of culture, only A4 + SeqFSH treatment showed a lower (P < 0.05) rate of intact follicles, survival probability, and meiotic resumption, as well as higher (P < 0.05) percentage of degeneration and/or extrusion after antrum formation. Taken together, these results reported a positive correlation between fast-growing follicles and follicles that degenerated and/or extruded after antrum formation. When compared with control, the addition of A4 alone or in association of FSH did not increase (P > 0.05) the estradiol production or androstenedione levels on Day 6. However, on Day 18, the androstenedione levels were significantly lower in A4 + SeqFSH treatment when compared with A4 alone or to A4 + FixFSH treatments, whereas the estradiol production did not differ (P > 0.05). In summary, this study found that accelerated follicle growth negatively impacted the morphology of caprine preantral follicle cultured in vitro. In addition, the association of androstenedione with increasing concentration of FSH was detrimental to follicular survival and oocyte meiotic resumption.

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Cryoinjuries caused by vitrification of tissues and organs lead to the loss of membrane proteins that mediate intercellular communications, such as connexins 37 (Cx37) and 43 (Cx43). Thus, the present study aimed to evaluate ovine Cx37 and Cx43 gene and protein expressions and developmental competence by in vitro-cultured secondary follicles retrieved from vitrified ovarian tissue. Ovarian fragments for the same ovary pair were distributed into six treatments: (1) fresh ovarian tissue (FOT); (2) vitrified ovarian tissue (VOT); (3) isolated follicles from fresh ovarian tissue (FIF); (4) isolated follicles from vitrified ovarian tissue; (5) isolated follicles from fresh ovarian tissue followed by in vitro culture (CFIF); (6) isolated follicles from vitrified ovarian tissue followed by in vitro culture (CVIF). In all treatments, Cx37 and Cx43 gene and protein expression patterns were evaluated by reverse transcription polymerase chain reaction and immunocytochemistry. In addition, secondary follicles were analyzed according to follicular integrity and growth, apoptosis, and cell proliferation. In vitro-cultured secondary follicles (CFIF and CVIF) were evaluated based on morphology (extruded follicles), antrum formation, and viability. The percentage of intact follicles was higher, whereas antrum formation, oocyte extrusion rate, and follicle viability were lower in CVIF than in CFIF treatment (P < 0.05). Terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphates nick end-labeling assay demonstrated that apoptosis was absent in FIF, whereas follicles from all other treatments showed positive labeling. Cell proliferation index was higher in isolated follicles from vitrified ovarian tissue and CVIF treatments than in follicles from FIF. Expression of Cx43 messenger RNA was lower in CVIF treatment when compared with follicles from all other treatments (P < 0.05). Follicle Cx37 messenger RNA levels did not show alterations in any treatment (P > 0.05). Cx37 and Cx43 immunolabeling was localized mainly on granulosa cells and oocytes, respectively. In conclusion, isolation of ovine secondary follicles could be done successfully after vitrification of ovarian tissue, and the basement membrane integrity remained intact after in vitro culture. Although the gene and protein expression of Cx37 did not change after vitrification of ovarian tissue, Cx43 turned out to be altered in secondary follicles after vitrification and in vitro culture.

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Introduction: Steroid hormones production is a physiological process termed steroidogenesis. An important stage of this process is the conversion of androgens into estrogens through aromatase enzyme. Furthermore, androgens are important in the process of folliculogenesis, promoting follicular growth in different species. Thus, the aim of this review was to present the process of synthesis, mechanism of action, and importance of androgens in folliculogenesis. Additionally, the main results of in vitro culture of ovarian cells in the presence of these hormones were emphasized. Review: Folliculogenesis begins in prenatal life in most of species and can be defined as the process of formation, follicular growth, and oocyte maturation. Preantral follicles represent 95% of the follicular population and assisted reproductive technologies have been developed (e.g., Manipulation of Oocytes Enclosed in Preantral Follicles - MOEPF) in order to avoid the great follicle loss that occurs naturally in vivo by atresia. The MOEPF aim to obtain a large number of competent oocytes from preantral follicles and then subject to in vitro maturation, fertilization, and culture for embryo production. However, the development of an efficient medium to ensure the follicular survival and oocyte maturation is the major challenge of this biotechnology. To achieve the success on in vitro culture, the effects [...](AU)

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Durante a foliculogênese em mamíferos, ocorre um longo e complexo processo no qual o oócito adquire a competência necessária para a fecundação. Nesse processo ocorre uma comunicação metabólica bidirecional entre os oócitos e as células somáticas dentro do folículo que garante substratos para o oócito em desenvolvimento. Essa comunicação é mediada pelas junções celulares (junções comunicantes e junções aderentes) presentes nas projeções transzonais. As junções celulares e moléculas de adesão são responsáveis principalmente por promover a adesão entre as células foliculares; mas podem atuar em vias de sinalização celular e na regulação da transcrição gênica nas células somáticas e oócitos. Além disso, as junções comunicantes (junções gap) são canais intermembranares que intermediam a comunicação entre essas células através da passagem de pequenas moléculas. Essas junções comunicantes são compostas por proteínas denominadas conexinas; as conexinas 37 e 43 são as predominantes nos folículos ovarianos. Dessa forma, o conhecimento acerca das junções celulares é de extrema importância para o estudo da foliculogênese. A presente revisão teve como objetivo abordar os principais tipos de junções celulares existentes entre as células foliculares, com destaque para as junções gap e as principais proteínas de membranas (conexinas) presentes nos diferentes estágios do desenvolvimento folicular.

During the mammalian folliculogenesis, a long and complex process occurs, which the oocyte acquires the necessary competence for fecundation. In this process there is a metabolic bidirectional communication among the oocyte and somatic cells inside the follicle, which provides substrates for the oocyte developmental competence. This communication is mediated by cellular junctions (occlusions, adherens and gap junctions) localized in the transzonal projections. Cellular junctions and adhesion mollecules are responsable mainly for promoving the adhesion among follicular cells, however they can act in cellular signaling pathways and in regulation of genic transcription in the follicular cells and oocyte. Moreover, the communication junctions (gap junctions) are intermembrane channels that intermediate the communication among these cells through the passage of small molecules. These gap junctions are composed by connexins, of which the connexins 37 and 43 are the most frequently found in the ovarian follicle. Thus, knowledge of these cellular junctions are of great importance for studying the folliculogenesis process. The aim of this review was to report the main types of cellular junctions localized among the follicular cells, especially the gap junctions and the main membrane proteins (connexins) found in different stages of the follicular development.

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A homeopatia apresenta­se como uma excelente opção de baixo custo e toxicidade para uso na prática da reprodução tanto humana quanto animal. Entretanto, desperta um alto nível de ceticismo em relação a sua real eficácia, notadamente devido ao possível efeito placebo. O uso de modelos in vitro, como a tecnologia do ovário artificial, apresenta­se como uma ferramenta de grande precisão para dirimir tal controvérsia. Diante disso, esta revisão tem como objetivo fornecer algumas bases sobre a foliculogênese e sua regulação, relatar a importância do cultivo in vitro, com ênfase no hormônio folículo estimulante (FSH), na avaliação do papel dos medicamentos homeopáticos no tratamento de distúrbios reprodutivos ovarianos e no seu uso para melhorar as biotécnicas reprodutivas.

The homeopathy is a low­cost and toxicity alternative to using in the human and animal reproduction. However, the effect homeopathy awakens a skepticism about its effectiveness, because of the possible placebo effect. The in vitro models, as artificial ovary, is a excellent tool to resolve this controversy. Therefore, the aim of this review to provide some basis on folliculogenesis and its regulation, to report the importance of in vitro culture, with an emphasis on follicle stimulating hormone (FSH), in assessing the role of homeopathic medicines in treating ovarian reproductive disorders and its use to improve reproductive biotechnologies.

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Introduction: Steroid hormones production is a physiological process termed steroidogenesis. An important stage of this process is the conversion of androgens into estrogens through aromatase enzyme. Furthermore, androgens are important in the process of folliculogenesis, promoting follicular growth in different species. Thus, the aim of this review was to present the process of synthesis, mechanism of action, and importance of androgens in folliculogenesis. Additionally, the main results of in vitro culture of ovarian cells in the presence of these hormones were emphasized. Review: Folliculogenesis begins in prenatal life in most of species and can be defined as the process of formation, follicular growth, and oocyte maturation. Preantral follicles represent 95% of the follicular population and assisted reproductive technologies have been developed (e.g., Manipulation of Oocytes Enclosed in Preantral Follicles - MOEPF) in order to avoid the great follicle loss that occurs naturally in vivo by atresia. The MOEPF aim to obtain a large number of competent oocytes from preantral follicles and then subject to in vitro maturation, fertilization, and culture for embryo production. However, the development of an efficient medium to ensure the follicular survival and oocyte maturation is the major challenge of this biotechnology. To achieve the success on in vitro culture, the effects [...]

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O processo reprodutivo é composto de uma série complexa de eventos que ocorrem de uma forma ordenada e cronológica. Entretanto, diversos fatores podem interromper o ciclo reprodutivo causando infertilidade ou esterilidade. Neste contexto, o emprego da homeopatia poderia contribuir para a reversão do quadro patológico. Apesar da homeopatia ser uma ciência antiga, desperta ainda uma série de questões e dúvidas quanto à sua eficiência. Diante disso, esta revisão tem como objetivo relatar a importância dos medicamentos homeopáticos na reprodução humana e animal, enfatizando o papel desses medicamentos no tratamento de distúrbios reprodutivos.

The reproduction is composed of several complex events that occur in an orderly and chronological way. However, many factors may disrupt the reproductive cycle, thus causing infertility or sterility. In this context, the use of homeopathy may contribute to reverse the pathological condition. In spite of homeopathy being an ancient science, it arises many questions and doubts about its efficiency. Therefore, this review paper aims to report the importance of homeopathic medicines in animal and human reproduction, emphasizing the role of these drugs in the treatment of reproductive disorders.

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O processo reprodutivo é composto de uma série complexa de eventos que ocorrem de uma forma ordenada e cronológica. Entretanto, diversos fatores podem interromper o ciclo reprodutivo causando infertilidade ou esterilidade. Neste contexto, o emprego da homeopatia poderia contribuir para a reversão do quadro patológico. Apesar da homeopatia ser uma ciência antiga, desperta ainda uma série de questões e dúvidas quanto à sua eficiência. Diante disso, esta revisão tem como objetivo relatar a importância dos medicamentos homeopáticos na reprodução humana e animal, enfatizando o papel desses medicamentos no tratamento de distúrbios reprodutivos.(AU)

The reproduction is composed of several complex events that occur in an orderly and chronological way. However, many factors may disrupt the reproductive cycle, thus causing infertility or sterility. In this context, the use of homeopathy may contribute to reverse the pathological condition. In spite of homeopathy being an ancient science, it arises many questions and doubts about its efficiency. Therefore, this review paper aims to report the importance of homeopathic medicines in animal and human reproduction, emphasizing the role of these drugs in the treatment of reproductive disorders.(AU)

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Este trabalho teve como objetivo avaliar o efeito do hormônio folículo estimulante recombinante (FSHr) sobre o desenvolvimento in vitro de folículos pré-antrais (FOPA) caprinos e ovinos isolados. Folículos pré-antrais (≥ 150 µ m) foram isolados de fragmentos do córtex ovariano de cabras e ovelhas e cultivados individualmente por 24 dias em α-MEM suplementado com 10% de soro fetal bovino (SFB), 1% de insulina-transferrina-selênio (ITS), 1% de antibióticos e 50µg/mL de ácido ascórbico, na ausência (controle) ou presença de FSHr (100 ou 1000 ng/mL), a 39 °C com 5% de CO2 em ar. Durante o cultivo avaliou-se a morfologia, o crescimento folicular e a formação de antro. Foi observado que, na espécie caprina, o cultivo com FSHr apresentou percentuais de folículos morfologicamente normais semelhantes ao controle até o 12º dia de cultivo (P>0,05), ocorrendo uma redução deste parâmetro no 18º dia (P<0,05) somente no tratamento com 1000 ng/mL de FSRr. Na espécie ovina, o percentual de folículos morfologicamente normais foi semelhante em todos os tratamentos, observando-se um decréscimo a partir do 12º dia (P<0,05). Com relação ao desenvolvimento folicular, em caprinos, a adição de FSHr aumentou a taxa de crescimento em relação ao controle (P<0,05). Entretanto, em ovinos, isso só foi observado no tratamento com 1000 ng/mL de FSHr. No que se refere à formação de antro, em ambas as espécies, não houve diferença significativa entre os tratamentos com FSHr e o controle. Quanto à análise da configuração da cromatina, todos os oócitos permaneceram em estádio de vesícula germinativa. Assim, pôde-se concluir que FOPA caprinos e ovinos são capazes de manter a sobrevivência e o crescimento in vitro até o estádio antral na ausência de FSH. Contudo, este hormônio teve um papel importante na taxa de crescimento folicular em ambas as espécies.

The aim of this work was to evaluate the effects of recombinant follicle stimulating hormone (rFSH) on the in vitro development of isolated caprine and ovine preantral follicles. Preantral follicles (≥ 150 micron) were isolated from fragments of goats and sheeps ovarian cortex and individually cultured for 24 days in α-MEM supplemented with 10% fetal calf serum (FCS), 1% insulin-transferrin -selenium (ITS), 1% antibiotics and 50 mg / mL ascorbic acid, in the absence (control) or presence of FSHR (100 or 1000 ng / mL) at 39 ° C with 5% CO2 in air. Follicular morphology, growth and antral cavity formation were assessed throughout the culture. The results showed that addition of rFSH to the medium had no effect on the percentages of caprine morphologically normal follicles until day 12 as compared with the control, but a decrease was observed on day 18 with the use of the hormone (p<0.05) only rFSH (1000 ng/mL). Regarding ovine follicles, percentages of normal follicles were significantly reduced from day 12 onwards in all treatments, which led to similar results. Growth rates were increased in caprine follicles by use of rFSH (100 and 1000 ng/ml) in comparison to the control (p<0,05). In ovine follicles, such effect was observed only when rFSH was used at 1000 ng/ml. With respect to antral cavity formation, no diference among treatments was observed for both species. The analysis of chromatin configuration revealed that all oocytes remained at the germinal vesicle stage. In conclusion, caprine and ovine preantral follicles are capable to survive and develop to the antral stage in vitro without FSH. However, this hormone is important for increasing follicular growth rates in both species.

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The in vitro culture of preantral follicles is an important tool to elucidate the mechanisms involved in controlling early folliculogenesis as well as to improve reproductive efficiency by providing a large number of oocytes for in vitro production of embryos. To this end, improvements in the performance of current culture systems are needed, including changes in the composition of the medium. Supplementation of culture medium with proteins is a great strategy to improve survival and in vitro development of preantral follicles. This review highlights the importance of in vitro culture of preantral follicles with emphasis on the main protein sources used to supplement the culture media.(AU)

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Este trabalho teve como objetivo avaliar o efeito do hormônio folículo estimulante recombinante (FSHr) sobre o desenvolvimento in vitro de folículos pré-antrais (FOPA) caprinos e ovinos isolados. Folículos pré-antrais ( 150 µm) foram isolados de fragmentos do córtex ovariano de cabras e ovelhas e cultivados individualmente por 24 dias em -MEM suplementado com 10% de soro fetal bovino (SFB), 1% de insulina-transferrina-selênio (ITS), 1% de antibióticos e 50 µg/mL de ácido ascórbico, na ausência (controle) ou presença de FSHr (100 ou 1000 ng/mL), a 39 °C com 5% de CO2 em ar. Durante o cultivo avaliou-se a morfologia, o crescimento folicular e a formação de antro. Foi observado que, na espécie caprina, o cultivo com FSHr apresentou percentuais de folículos morfologicamente normais semelhantes ao controle até o 12º dia de cultivo (P>0,05), ocorrendo uma redução deste parâmetro no 18º dia (P<0,05) somente no tratamento com 1000 ng/mL de FSRr. Na espécie ovina, o percentual de folículos morfologicamente normais foi semelhante em todos os tratamentos, observando-se um decréscimo a partir do 12º dia (P<0,05). Com relação ao desenvolvimento folicular, em caprinos, a adição de FSHr aumentou a taxa de crescimento em relação ao controle (P<0,05). Entretanto, em ovinos, isso só foi observado no tratamento com 1000 ng/mL de FSHr. No que se refere à formação de antro, em ambas as espécies, não houve diferença significativa entre os tratamentos com FSHr e o controle. Quanto à análise da configuração da cromatina, todos os oócitos permaneceram em estádio de vesícula germinativa. Assim, pôde-se concluir que FOPA caprinos e ovinos são capazes de manter a sobrevivência e o crescimento in vitro até o estádio antral na ausência de FSH. Contudo, este hormônio teve um papel importante na taxa de crescimento folicular em ambas as espécies.(AU)

The aim of this work was to evaluate the effects of recombinant follicle stimulating hormone (rFSH) on the in vitro development of isolated caprine and ovine preantral follicles. Preantral follicles ( 150 micron) were isolated from fragments of goats and sheeps ovarian cortex and individually cultured for 24 days in-MEM supplemented with 10% fetal calf serum (FCS), 1% insulin-transferrin -selenium (ITS), 1% antibiotics and 50 mg / mL ascorbic acid, in the absence (control) or presence of FSHR (100 or 1000 ng / mL) at 39 ° C with 5% CO2 in air. Follicular morphology, growth and antral cavity formation were assessed throughout the culture. The results showed that addition of rFSH to the medium had no effect on the percentages of caprine morphologically normal follicles until day 12 as compared with the control, but a decrease was observed on day 18 with the use of the hormone (p<0.05) only rFSH (1000 ng/mL). Regarding ovine follicles, percentages of normal follicles were significantly reduced from day 12 onwards in all treatments, which led to similar results. Growth rates were increased in caprine follicles by use of rFSH (100 and 1000 ng/ml) in comparison to the control (p<0,05). In ovine follicles, such effect was observed only when rFSH was used at 1000 ng/ml. With respect to antral cavity formation, no diference among treatments was observed for both species. The analysis of chromatin configuration revealed that all oocytes remained at the germinal vesicle stage. In conclusion, caprine and ovine preantral follicles are capable to survive and develop to the antral stage in vitro without FSH. However, this hormone is important for increasing follicular growth rates in both species.(AU)

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RESUMO

The in vitro culture of preantral follicles is an important tool to elucidate the mechanisms involved in controlling early folliculogenesis as well as to improve reproductive efficiency by providing a large number of oocytes for in vitro production of embryos. To this end, improvements in the performance of current culture systems are needed, including changes in the composition of the medium. Supplementation of culture medium with proteins is a great strategy to improve survival and in vitro development of preantral follicles. This review highlights the importance of in vitro culture of preantral follicles with emphasis on the main protein sources used to supplement the culture media.

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