Differential effects of activin-A and FSH on growth, viability and messenger RNA expression in cultured bovine preantral follicles.

摘要

This study evaluated the effect of follicle stimulating hormone (FSH) alone or in combination with activin-A on survival, growth and expression of mRNA for follicle stimulating hormone receptor (FSH-R), proliferating cell nuclear antigen (PCNA), activin receptor type I B (ActR-IB), activin receptor type II B (ActR-IIB), hyaluronan synthase-1 (HAS 1) and hyaluronan synthase-2 (HAS 2) in bovine secondary follicles cultured in vitro for 18 days. Preantral follicles (~0.2 mm) were isolated and individually cultured in absence ( alpha -minimum essential medium ( alpha -MEM+): control) or presence of activin-A, FSH in increasing concentrations or both activin-A and FSH. An increase in follicular diameter was observed after 6 days of culture in all culture treatments compared to control medium; after 12 days in treatment with FSH alone or the mixture of FSH and activin-A (P<0.05); and, after 18 days only in the presence of FSH alone (P<0.05). However, in combination with activin-A, FSH-stimulated follicle growth after 18 days was blocked (P<0.05) and, had significantly lower (P<0.05) levels of mRNA for ActR-IB, ActR-IIB, when compared to alpha -MEM+, and for FSH-R and PCNA, when compared to alpha -MEM+ supplemented with activin-A only. Moreover, follicles cultured in presence of FSH alone had greater (P<0.05) levels of mRNA for HAS 1 and HAS 2 than those cultured in medium supplemented with both activin-A and FSH. Morphological, immunofluorescence and ultrastructural analysis confirmed the integrity of follicles cultured in FSH after 18 days. In conclusion, activin-A exerts a stimulatory effect on in vitro early follicular development for up to 6 days, has no effect after 12 days of culture and blocks the stimulatory growth effect of FSH after 18 days. The reduced mRNA levels in follicles cultured with both activin-A and FSH suggest a decreased sensitivity of follicles for activin-A and FSH and inhibited follicular cell proliferation after long-term in vitro culture of isolated preantral follicles.