Insulina afeta o desenvolvimento de folículos pré-antrais ovinos cultivados in Situ

• Ano de defesa: 2019
• Autor(a) principal: SILVA, Diogo Manoel Farias da
• Orientador(a): WISCHRAL, Aurea
• Banca de defesa: SILVA, Sildivane Valcácia, BARTOLOMEU, Cláudio Coutinho, MELO, Ismaela Maria Ferreira de, ROCHA, Suzana Santino Nunes da, DUARTE, Sandra Silva
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal Rural de Pernambuco
• Programa de Pós-Graduação: Programa de Pós-Graduação em Ciência Veterinária
• Departamento: Departamento de Medicina Veterinária
• País: Brasil
• Palavras-chave em Português: Insulina; Foliculogênse; Esteroidogênese; Ovino
• Área do conhecimento CNPq: CIENCIAS AGRARIAS::MEDICINA VETERINARIA
• Link de acesso: http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/8229

Insulin may have effects on folliculogenesis in in vitro culture, which allowed a greater preantral follicles to be obtained for later embryo production. In this context, the objective of this work was to determine the effect of the hormone insulin on the in vitro culture of ovarian fragments of the ovine species, associated or not with FSH. For ovaries were collected in slaughterhouses and stored in MEM-HEPES medium at 4 ° C and taken to the laboratory. For in vitro culture, fragments of the ovarian cortex (3x3x2mm) and cultured in α-MEM + medium were obtained for 8 days at 39 ° C with 5% CO2. After culture, the fragments were processed and histologically analyzed for morphological evaluation of follicular development (activation, maintenance of the morphology and diameter of the oocyte and follicle), immunohistochemical analysis for localization of the nuclear proliferation antigen and gene expression of mRNA messenger RNA for the apoptotic gene (caspase 3) and steroidogenesis (CYP17 and CYP19). The flow of information allowed to separate the study in two experiments. Experiment 1: In vitro culture of the tissues, evaluating the effects of the hormone insulin at the concentrations of (0, 1, 10, 100, 1.000 and 10.000 ng/mL) on the activation, development and maintenance of morphology. Experiment 2: In vitro culture with hormone insulin (0.1, 100 ng/mL) associated with FSH on the activation, development, maintenance of morphology, nuclear proliferation antigen (PCNA) and expression of caspase 3, CYP17 and CYP19 genes. In the experiment 1 the results showed that: The highest percentages of activation were observed at concentrations of 1 and 100 ng/mL of the hormone (P <0.05), the percentage of morphologically normal follicles was significantly higher (P <0.05) for tissues cultured in 1 and 10 ng / mL insulin compared to tissues cultured only in α-MEM+. Regarding growth, no difference in oocyte and follicle diameter was observed after in vitro culture at any of the insulin concentrations. However, at the concentration of 10,000 ng / mL there was a significant reduction in follicular diameter when compared to tissues cultured only in α-MEM+ (P <0.05). In the experiment 2, the results showed that after 8 days of culture with insulin concentrations (1, 10 and 100 ng / mL) associated with FSH, they presented a greater activation (P<0.05). Likewise, the percentage of morphologically normal follicles was significantly higher at 10 ng / mL concentration with and without FSH (P <0.05). The PCNA labeling by immunohistochemistry showed the presence of the antigen in the oocyte, in all the analyzed groups, and in the granulosa cells, only in the group that was not submitted to the culture. The percentage of pixel for PCNA labeling at all FSH associated insulin concentrations was higher than the non FSH groups (P <0.05). The expression of the CYP17 and CYP19 genes were identified in all groups, however without significant difference. The expression of the caspase 3 gene in the IN1FSH group (P <0.05) than in the IN10, IN100, IN10FSH and IN100FSH groups. In conclusion, insulin in the in vitro culture of ovarian sheep fragments alone or associated with FSH acts in a dosedependent manner on follicular activation, preserving the morphology, but without altering the follicular diameter. At high concentration (10,000 ng/mL) insulin has an inhibitory effect on the initial follicle growth. It is also concluded that insulin can reduce the expression of the gene related to apoptosis (caspase 3), when in intermediate concentrations (10 and 100 ng/mL) especially associated with FSH.