Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (similar to 0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in alpha-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 mu g/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [alpha-MEM+ (94.59%); 1 mu g/mL PHA (96.43%); 10 mu g/mL PHA (84.85%); 50 mu g/mL PHA (85.29%); 100 mu g/mL PHA (88.57%), and 200 mu g/mL PHA (87.50)], but the presence of 10 mu g/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 +/- 0.1) and PCNA (4.4 +/- 0.2) was also significantly increased in follicles cultured with 10 mu g/mL PHA in relation to those cultured in alpha-MEM+ (1.0 +/- 0.1). In conclusion, supplementation of culture medium with 10 mu g/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.