Avaliação do efeito citotóxico da frutalina (lectina de Artocarpus incisa L.) no cultivo in vitro de folículos pré-antrais caprinos.

• Ano de defesa: 2017
• Autor(a) principal: Costa, Maria Amélia Araújo Soares
• Orientador(a): Silva, José Roberto Viana
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Frutalina; Cultivo celular; Folículo ovariano caprino; Toxicidade
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/29896

The objective of this work was to evaluate the effect of frutalin (lectin of Artocarpus incisa L.) on the activation, survival, ultrastructure and gene expression of preantral follicles grown in situ and on in vitro culture of isolated goat secondary follicles. For phase I, goat ovarian fragments were cultured for 6 days in α-MEM+ alone or supplemented with frutalin (1, 10, 50, 100 or 200 μg/mL). The uncultured (fresh control) and cultured ovarian fragments were intended for histological, ultrastructural analysis and expression of anti-and pro-apoptotic genes by qPCR. For phase II, secondary follicles were microdissected and cultured for 6 days in α-MEM + medium alone or supplemented with doxorubicin (0.3 μg/mL) or frutalin (0.6, 6 or 60 μg/mL). After culturing, secondary follicles were analyzed for survival, growth, antrum formation and expression of anti-and pro-apoptotic genes by qPCR. In phase I, frutalin at all concentrations tested did not influence the activation of primordial follicles, but reduced follicular survival. Higher concentrations of frutalin (50, 100 or 200 μg/mL) reduced follicular survival when compared to tissues cultured with 1 or 10 μg/mL frutalin. Ultrastructural analysis showed that after cultivation the atretic follicles had retracted oocytes and a large number of vacuoles distributed throughout the cytoplasm. Also, it was found that although a dose-response effect was not observed in the gene expression, when compared to tissue cultured in the control medium, the presence of frutalin increased the mRNA expression of pro-apoptotic genes. In phase II, doxorubicin and frutalin (0.6, 6 and 60 μg / mL) significantly reduced the percentage of normal follicles when compared to α-MEM + medium. Doxorubicin reduced follicular viability when compared to frutalin at all concentrations tested. In addition, the percentage of normal follicles after frutalin (0.6 and 6.0 μg / mL) was higher than in the presence of 60.0 μg / mL (p <0.05). With the exception of follicles cultivated with 0.6 μg / mL frutalin, the follicular growth rate was reduced after cultivation in the presence of doxorubicin or frutalin when compared to the control medium (p <0.05). Approximately 60.0% of follicles cultured in α-MEM+ presented antrum formation, but the presence of doxorubicin or frutalin reduced the formation of antrum (P <0.05). The presence of doxorubicin or frutalin (60.0 μg / mL) increased the mRNA level for CASP3, CASP6, BAX and BCL2 (P <0.05), but the highest levels of transcripts for CASP3, CASP6, BAX were found in follicles cultured with doxorubicin when compared to those grown in frutalin (60.0 μg / mL). On the other hand, follicles cultivated with frutalin had higher levels of mRNA for BCL2 (P <0.05) when compared to those cultured in the presence of doxorubicin. In conclusion, frutalin does not influence the activation of the primordial follicles in vitro, and maintains a high percentage of healthy follicles (> 60%) when used in low concentrations (1 and 10 μg / mL). Also, the ultrastructural characteristics of cultured follicles show that necrosis is the main pathway of cell death. In addition, frutalin has lower toxic effects than doxorubicin in in vitro cultured goat secondary follicles.