El modelo de ovario artificial como prueba celular para evaluar la seguridad de los extractos de Spirulina maxima y Kéfir: un estudio preliminar / The model of artificial ovary as a cellular test to evaluate the safety of Spirulina máxima and Kefir extracts: a preliminary study

RESUMEN S. maxima y Kefir son conocidos y utilizados por sus propiedades antioxidantes e inmunoestimulantes. El objetivo en este estudio fue evaluar los extractos naturales de estos dos agentes con la técnica de manipulación de ovocitos incluidos en folículos preantrales (MFP), porque la técnica puede reemplazar el uso de animales de laboratorio y también podría armonizar las leyes que intentan reducir y mejorar el uso de animales para el estudio de nuevos fármacos y para integrar a las buenas prácticas de laboratorio. Los extractos naturales estudiados fueron obtenidos a partir de S. maxima y Kéfir, las dos sustancias son conocidas en el mercado por su actividad antioxidante e inmunoestimulante. Los dos extractos fueron evaluados en suspensiones de 10, 50, 100 and 200 µg.mL-1. Los resultados muestran que Spirulina produjo disminución en la sobrevivencia, el desarrollo y el diámetro folicular. Mientras que Kéfir no mostró influencias positivas o negativas sobre el crecimiento y desarrollo de los folículos preantrales, sólo la concentración de 200 µg.mL-1 disminuyó la sobrevivencia folicular. La técnica MFP demostró encajar en la política de las 3R (reemplazo, reducción y refinamiento) y permitió evaluar la citotoxicidad mostrando que la técnica puede ser usada como una prueba de seguridad en extractos naturales.

SUMMARY S. maxima and Kéfir are known and used for their antioxidant and immunostimulant properties. This study aimed to evaluate the natural extracts of these two agents with the technique of manipulation of oocytes included in preantral follicles (MFP), because the technique can replace the use of laboratory animals and could also harmonize the laws that try to reduce and improve the use of animals for the study of new drugs and to integrate good laboratory practices. The natural extracts studied were obtained from S. maxima and Kefir, both substances are known in the market for their antioxidant and immunostimulating activity. Both were evaluated in suspensions of 10, 50, 100 and 200 µg.mL-1. The results show that Spirulina produced a decrease in survival, development, and follicular diameter. While Kefir did not show positive or negative influences on the growth and development of preantral follicles, only the concentration of 200 µg.mL-1 decreased follicular survival. The MFP technique proved to fit into the 3R policy (replacement, reduction, and refinement) and allowed to evaluate cytotoxicity, showing that the technique can be used as a safety test in natural extracts.

Impacto dos agentes antineoplásicos sobre os folículos ovarianos e importância das biotécnicas reprodutivas na preservação da fertilidade humana / Impact of antineoplastic agents on the ovarian follicles and importance of reproductive biotechnologies in the preservation of human fertility

A preservação da fertilidade de jovens mulheres em idade reprodutiva tem se tornado um dos grandes desafios da medicina, já que a maioria dos tratamentos contra o câncer pode causar insuficiência ovariana prematura, devido à toxicidade ovariana dos agentes quimioterápicos.Com base nessa sensibilidade e com vistas a reverter ou minimizar os danos às células reprodutivas decorrentes da quimioterapia, cada vez se tornam mais frequentes os estudos e a busca tanto por opções para preservar a fertilidade feminina quanto por tratamentos para células cancerosas que sejam mais seletivos. Este artigo tem como objetivo apresentar uma visão geral sobre os danos causados pelos quimioterápicos à função ovariana e as possíveis opções para a preservação da fertilidade feminina em pacientes com câncer e as perspectivas em oncofertilidade. Foi feita uma pesquisa no banco de dados National Library of Medicine (PubMed), em que foram recuperados artigos publicados entre 1967 e 2015 sobre agentes quimioterápicos e seus danos à fertilidade feminina.

The preservation of fertility in young women of reproductive age has become a major challenge in medicine, since most cancer treatments can cause premature ovarian failure due to ovarian toxicity of chemotherapeutic agents. Based on this sensitivity it is necessary to revert or minimize damage to reproductive cells resulting from chemotherapy. It have become more frequent studies and researches for alternatives to preserve female fertility and treatments that are more selective for cancer cells. Currently there are several options for preservation of fertility in women undergoing chemotherapy treatments.This article aims to present an overview of the damage caused by chemotherapy ovarian function and possible options for preserving fertility in female cancer patients and prospects in oncofertilidade. A search on databases were searched for relevant articles: the National Library of Medicine (PubMed) and the Virtual Health Library. Articles published between 1967 and 2015 on chemotherapeutic agents and their damage on female fertility was held.

Transforming growth factor-β (TGF-β) maintains follicular ultrastructure and stimulates preantral follicle growth in caprine ovarian tissue cultured in vitro / Fator de crescimento transformante-β (TGF-β) mantém a ultraestrutura folicular e estimula o crescimento de folículos pré-antrais caprinos inclusos em tecido ovariano cultivado in vitro

The objectives of this study were to investigate whether TGF-β affect the survival, activation and further growth of goat primordial follicles enclosed in ovarian cortex after in vitro culture. Goat ovaries were collected from an abattoir and pieces of ovarian tissues were cultured for one or seven days in a supplemented alpha Minimum Essential Medium, alone or containing TGF-β (1, 5, 10 or 50ng/mL). Ovarian tissues from the fresh control as well as those cultured were processed for histological and ultrastructural studies. The results showed that when compared with fresh control, there was decrease in the percentages of histologically normal follicles in all treatments only after seven days culture. TGF-β did not affect the activation of preantral follicles regardless of its concentration, however, larger follicles diameter (P<0.05) was observed using 10ng/mL TGF-β than in the fresh control and other treatments. Moreover, this concentration maintained the normal ultrastructure after seven days of culture. In conclusion, TGF-β showed additional effect on the follicle growth and the maintenance of ultrastructural integrity of goat preantral follicles enclosed in ovarian tissue when used at 10ng/mL during seven days of culture...

O objetivo desse estudo foi investigar se o TGF-β afeta a sobrevivência, ativação e crescimento de folículos primordiais caprinos inclusos no córtex ovariano após o cultivo in vitro. Ovários de cabras foram coletados em abatedouro e fragmentos de tecido ovariano foram cultivados por um e sete dias em meio essencial mínimo alfa (α-MEM+) sozinho ou suplementado com TGF-β (1, 5, 10 ou 50ng/mL). Fragmentos ovarianos não cultivados e cultivados foram processados para análise histológica e ultraestrutural. Os resultados mostraram que, comparado ao controle fresco, houve diminuição no percentual de folículos morfologicamente normais em todos os tratamentos somente após sete dias de cultivo. O TGF-β não afetou a ativação folicular independente da concentração testada, contudo, o diâmetro folicular foi superior (P<0.05) no tratamento com 10ng/mL de TGF-β quando comparado ao controle fresco e aos demais tratamentos. Além disso, essa mesma concentração manteve a ultraestrutura normal dos folículos após sete dias de cultivo. Em conclusão, o TGF-β apresentou efeito adicional no crescimento folicular e na manutenção da integridade ultraestrutural de folículos pré-antrais caprinos inclusos no tecido ovariano quando utilizado na concentração de 10ng/mL durante sete dias de cultivo...

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 microm) were isolated by micro-dissection and cultured in 0 (control), 10(-3), 10(-5), 10(-7), and 10(-9) M SNP. To examine the reversible effect of SNP, PFs were cultured with 10(-5) M SNP + 1 mM Nomega-nitro-L-arginine methyl ester (L-NAME) or 1.0 microg hemoglobin (Hb). The results showed that greater concentrations of SNP (10(-3), 10(-5), 10(-7) M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10(-9) M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.

Effects of alpha-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured / Efeitos dos antioxidantes alpha-tocoferol e ternatina na morfologia e na ativação de folículos pré-antrais caprinos cultivados in vitro

The effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15µM, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2 percent and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture.

Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folículos pré-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle não-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial Mínimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15µM, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folículos pré-antrais normais no controle não-cultivado foi de 73,2 por cento, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle não-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural não mostrou folículos pré-antrais íntegros após cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α-tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular após o cultivo in vitro.

Característica histológica, ultra-estrutural e produção de nitrito de folículos pré-antrais caprinos cultivados in vitro na ausência ou presença de soro / Histological and ultrastructural feature and nitrite production of caprine preantral follicles in vitro cultured in the presence or absence of serum

Avaliou-se o efeito da adição de diferentes tipos e concentrações de soro sobre o desenvolvimento e a sobrevivência de folículos ovarianos pré-antrais (FOPA) caprinos in vitro. Além disso, verificou-se a relação entre as concentrações de nitrito presentes no meio de cultivo e a viabilidade folicular. Cada par ovariano foi dividido em 29 fragmentos, sendo um destinado ao controle. Os fragmentos foram cultivados por um ou sete dias em meio essencial mínimo suplementado (MEM+) ou MEM+ com diferentes concentrações (10 ou 20 por cento) de soro fetal bovino (SFB), soro de cabra em estro (SCE) ou soro de cabra em diestro (SCD). Na análise morfológica após sete dias, apenas o tratamento com 10 por cento de SFB apresentou percentual de FOPA normais similar ao MEM+ (P>0,05). A análise ultra-estrutural dos folículos cultivados por sete dias com MEM+ ou MEM+ com 10 por cento de SFB mostrou danos oocitários, porém células da granulosa normais. A análise do meio de cultivo revelou correlação positiva entre a viabilidade folicular e a produção de nitrito. A suplementação com soro não melhorou a viabilidade de FOPA e a concentração de nitrito no meio de cultivo funcionou como um indicador da viabilidade das células da granulosa de FOPA caprinos cultivados in vitro.

The effect of the addition of different types and concentrations of sera on the viability and development of caprine preantal follicles (PAF) in vitro cultured was analyzed. In addition, it was evaluated the correlation between nitrite concentrations in culture medium and folicular viability. Each ovarian pair was divided in 29 fragments and one was used as control. The fragments were cultured for one or seven days in minimal essential medium (MEM+) or MEM+ with different concentrations of (10 or 20 percent) bovine fetal serum (BFS), estrous goat serum (EGS), or diestrous goat serum (DGS). After seven days, the morphological analysis showed that only the treatment with 10 percent BFS maintained the percentage of normal PAF similar to MEM+ (P>0.05). The ultrastructural analysis of follicles cultured for seven days in MEM+ or MEM+ with 10 percent BFS showed some oocyte damage, although the granulosa cells were normal. Analysis of culture medium revealed a positive correlation between follicular viability and nitrite production. Supplementation with serum did not improve the viability of PAF and nitrite levels in culture medium served as an indicator of viability of granulose cells from caprine PAF in vitro cultured.

Conservação de folículos pré-antrais bovinos em solução salina 0,9 por cento ou TCM 199 / Preservation of bovine preantral follicles in 0.9 percent saline solution or TCM 199

Investigou-se a eficiência da solução salina 0,9 por cento (SS) e TCM 199 na conservação de folículos pré-antrais (FOPA) bovinos in situ em diferentes temperaturas e tempos de incubação. Cada par ovariano foi dividido em 25 fragmentos. Um fragmento foi escolhido aleatoriamente e fixado imediatamente após a coleta (controle). Os demais foram distribuídos em tubos contendo SS ou TCM 199 a 4, 20 ou 39ºC por 2, 4, 12 ou 24h. A análise histológica mostrou que a conservação a 4ºC em ambas as soluções manteve a porcentagem de FOPA normais similar ao controle. A conservação em SS a 20ºC por 12 ou 24h, TCM 199 a 20ºC por 24h e em ambas as soluções a 39ºC a partir de 2h aumentou (P<0,05) a porcentagem de FOPA degenerados comparada à porcentagem de folículos-controle. Em ambas as soluções, independente do tempo de incubação, a porcentagem de folículos normais, após conservação a 39ºC, foi (P<0,05) menor que a obtida com 4 e 20ºC. FOPA bovinos podem ser conservados eficientemente a 4ºC por até 24h em ambas as soluções, e a 20ºC por 4 e 12h em SS e TCM 199, respectivamente.

The efficiency of 0.9 percent saline solution (SS) and TCM 199 on the preservation of bovine preantral follicles (PF) in situ at different temperatures and incubation times was investigated. Each ovarian pair was divided into 25 fragments. One fragment was taken randomly and immediately fixed (control). The other fragments were distributed in tubes containing SS or TCM 199 at 4, 20 or 39ºC for 2, 4, 12 or 24h. The histological analysis showed that the storage at 4ºC in both solutions kept the percentage of normal follicles similar to control values. Preservation in SS at 20ºC for 12 or 24h, TCM 199 at 20ºC for 24h and in both solutions at 39ºC from 2 h onward (P<0.05) increased the percentage of degenerated follicles when compared with control. In both solutions, independent of incubation time, the percentage of normal follicles observed at 39ºC was (P<0.05) lower them those observed at 4 and 20ºC. Bovine PF can be preserved efficiently at 4ºC for up to 24h in both solutions, at 20ºC for 4 and 12h in SS and TCM 199, respectively.

In vitro Follicular Growth and Ovulation of Mouse Preantral Follicles Cryopreserved by Vitrification / 대한불임학회지

OBJECTIVE: To define an appropriate vitrification condition of preantral follicle that yields high survival and to evaluate growth and ovulation rate of mouse follicles during in vitro culture after vitrification. METHODS: Preantral follicles were isolated mechanically from mouse ovaries that were surgically recovered from mice aged 14 days. Retrieved preantral follicles were placed in EG (Ethylene Glycol) for 2, 5, 10 minutes and transferred to EFS-40 (40% EG, 18% Ficoll-70, 0.5 M sucrose) for 0.5, 1, 2 minutes. And then, preantral follicles were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing was carried out at room temperature. After defining the most appropriate vitrification condition that yields high survival, in vitro growth and ovulation rate of follicles were evaluated. RESULTS: Appropriate vitrification condition that yield high survival rate (83.2+/-2.1%) of preantral follicle was EG for 5 minutes and EFS-40 for 0.5 minutes. In vitro survival rate of the vitrified preantral follicles were 85.5+/-0.5%, 67.9+/-0.8% and 40.2+/-0.5% on day 2, 6 and 10. And in vitro growth of the vitrified preantral follicles were 107.1+/-16.1 micrometer, 117.1+/-18.4 micrometer, 178.4+/-45.6 micrometer and 325.4+/-54.4 micrometer on day 0, 2, 6 and 10. Although in vitro survival rate and growth of vitrified preantral follicles were lower than that of non-vitrified preantral follicles, the patterns of survival and growth were similar in vitrified and non-vitrified preantral follicles. The ovulation rate of antral follicles that was grown from vitrified preantral follicles was 32.6+/-1.2%. CONCLUSION: Vitrified preantral follicles could be grown to antral sizes, and mature oocytes that can be used for IVF-ET programs were produced successfully. These data suggest that cryopreservation of preantral follicle by vitrification can be used for the preservation of the fertility.

Optimization of In Vitro Culture System of Mouse Preantral Follicles / 대한불임학회지

No abstract available.