Influência da contagem de folículos antrais e de fatores de crescimento sobre o cultivo in vitro de folículos pré-antrais bovinos

Abstract

The ovarian reserve of preantral follicles is a key determinant of fertility and can be influenced by antral follicle count (AFC), hormones, and growth factors. However, the impact of AFC and the interactions among these factors on initial folliculogenesis remains poorly explored. Thus, this thesis aimed to: (1) evaluate the effect of different AFC classes (low, medium, and high) on morphology, follicle activation, stromal density, collagen distribution in the extracellular matrix (ECM), and gene expression of proliferating cell nuclear antigen (PCNA), growth differentiation factor-9 (GDF-9), and connective tissue growth factor (CCN2) in cultured bovine ovarian cortex fragments; and (2) evaluate the effect of supplementation with follicle-stimulating hormone (FSH), epidermal growth factor (EGF), and GDF-9, alone or in combination, on the activation, survival, and gene expression of PCNA, GDF-9, and CCN2 in bovine preantral follicles with medium AFC. In the first study, ovarian cortex fragments from cows with low (≤15), medium (16–30), and high (≥31) AFC were cultured in alpha-minimum essential medium (α-MEM) for six days. Tissues with medium and high AFC exhibited higher follicular survival than those with low AFC (p < 0.05). Follicular activation was greater in medium AFC (p < 0.05). Medium AFC also preserved a higher percentage of collagen after culture (p < 0.05). Stromal density decreased across all classes, with no significant differences among them (p > 0.05). PCNA expression was not altered by culture, whereas CCN2 was higher in medium AFC in non-cultured tissues; after culture, GDF-9 and CCN2 were more highly expressed in high AFC. It was concluded that medium AFC provides a more favorable microenvironment for culture. In the second study, fragments with medium AFC were cultured in α-MEM supplemented with FSH (100 ng/mL), EGF (10 ng/mL), GDF-9 (200 ng/mL), EGF + GDF-9, or EGF + GDF-9 + FSH. The EGF + GDF-9 combination promoted the highest percentage of morphologically normal follicles among cultured groups, not differing from the non-cultured control (p > 0.05). All treatments increased follicular activation, with the highest rates observed in the EGF + GDF-9 and EGF + GDF-9 + FSH groups. The EGF + GDF-9 combination also showed a higher percentage of collagen, greater stromal density, and higher CCN2 expression. The addition of FSH provided no additional benefits. It was concluded that the EGF + GDF-9 combination is the most effective strategy for in vitro follicle culture using medium AFC. Taken together, these findings demonstrate that medium AFC confers greater responsiveness of ovarian tissue to in vitro culture, and that combined supplementation with EGF and GDF-9 enhances initial folliculogenesis, promoting greater survival, activation, and ECM integrity. These results contribute to the improvement of reproductive biotechniques.