118 Effects of Epidermal Growth Factor on in Vitro Survival and Antrum Formation of Isolated Ovine Preantral Follicles

Abstract

The aim of this study was to verify the beneficial effects of different concentrations of epidermal growth factor (EGF) on survival and antrum formation of isolated ovine preantral follicles cultured in vitro. Ovine ovaries (n = 50) were collected from a local slaughterhouse and secondary follicles (150–200 μm in diameter), without antral cavities, were mechanically isolated by microdissection using 26-gauge needles. After selection, the follicles were individually cultured in 100-μL droplets of culture medium at 39°C and 5% CO2 in air for 18 days. The basic control medium consisted of α-minimal essential medium (α-MEM) supplemented with BSA; insulin, transferring and selenium; glutamine; hypoxanthine; and ascorbic acid and then referred to as α-MEM+. For the experimental conditions, follicles were cultured in α-MEM+ alone (control) or in different concentrations of EGF (1, 10, or 50 ng mL–1). Every other day, 60 μL of the culture media was replaced with fresh media. The morphological aspects of all ovine follicles were assessed every 6 days using a precalibrated ocular micrometer in a stereomicroscope at 100× magnification. Only those follicles showing an intact basement membrane, with bright and homogeneous granulosa cells and an absence of morphological signs of degeneration, were classified as morphologically normal follicles. The rupture of the basement membrane was also observed and characterised as follicle extrusion. In addition, antral cavity formation was defined as the emergence of a visible translucent cavity within the granulosa cell layers. Data from morphologically normal follicles, extruded follicles, and antrum formation rate during in vitro culture were expressed as percentages and compared by the chi-squared test, and differences were considered significant when P < 0.05. The results showed that the percentage of morphologically normal follicles decreased significantly throughout the culture periods in all the treatments, except in the 50 ng mL–1 EGF group, which maintained the percentage of normal follicles from Day 0 to 6. Considering the same culture period, 50 ng mL–1 EGF treatment significantly increased the percentage of morphologically normal follicles at Day 18 compared with the control group. Moreover, the addition of EGF to the culture medium, at 50 ng mL–1, significantly reduced the precocious extrusion of oocytes and increased the percentage of antrum formation compared with the control and 1 ng mL–1 EGF after 18 days of culture. Notably, there were no significant differences between 10 ng mL–1 EGF, control medium, and 1 ng mL–1 EGF treatments. In conclusion, this study demonstrated that the addition of EGF to the in vitro culture medium, at 50 ng mL–1, increased the proportion of morphologically normal follicles and antrum formation rate of isolated ovine preantral follicles. This work was supported by FACEPE (Process APQ-0705–5.05/10). L. P. Santos is a recipient of a grant from FACEPE (Brazil).