Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e. hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL(-1) from Days 0 to 6 and 500 ng mL(-1) from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P < 0.05). Alone, 200 ng mL(-1) GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in a-minimum essential medium supplemented with 3.0 mg mL(-1) bovine serum albumin, 10 mu g mL(-1) insulin, 5.5 mu g mL(-1) transferrin, 5 ng mL(-1) selenium, 2 mM glutamine, 2 mM hypoxanthine and 50 mu g mL(-1) ascorbic acid (alpha-MEM+). Comparisons of uncultured (0.2 mm) and alpha-MEM+ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P, 0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.