Dynamics of inhibin subunit and follistatin mRNA during development of normal and polycystic ovary syndrome follicles.

Abstract

To further explore the developmental dynamics and possible roles of inhibin, activin, and follistatin in the development of human antral follicles as well as the relationship between mRNA and protein levels of these hormones within follicles, quantitative competitive RT-PCR assays were established to determine mRNA levels for the inhibin/activin subunits and both follistatin splice variants. Granulosa cell RNA was obtained by transvaginally aspirating follicles (6-23 mm) from carefully characterized normal women at different times of the follicular phase. alpha- and beta(A)-subunit mRNA levels increased significantly with follicle diameter (r = 0.56; P < 0.01 and r = 0.45; P < 0.05, respectively) and follicle maturity (r = 0.65; P < 0.001 and r = 0.58; P < 0.01, respectively), but beta(B) mRNA levels, which were at least 10-fold lower than levels of the other subunits, showed no relationship to size or maturity. Both follistatin 315 and 288 transcripts were detected in granulosa cells, but neither follistatin transcript varied significantly across the range of follicle sizes analyzed. In addition, granulosa cells contained three follistatin 315 mRNA transcripts for each follistatin 288 transcript, and the follistatin 315/288 ratio did not vary with follicle size. alpha-Subunit mRNA levels were positively associated with dimeric inhibin A protein in human follicular fluid from the same follicle aspirates (r = 0.71; P < 0.001). Similarly, beta(A)-subunit mRNA was associated with inhibin A (r = 0.59; P < 0.01), and beta(B) mRNA was associated with inhibin B (r = 0.67; P < 0.005) in these samples. Thus, the increase in inhibin subunit transcription and protein synthesis with follicle size suggests that inhibin biosynthesis might be important for continued development of the dominant follicle. To explore this hypothesis further, we compared mRNA levels for each of these transcripts in follicles obtained from six polycystic ovary syndrome patients (eight follicles) and compared the results to those from a group (n = 5) of normal follicles matched for mean diameter. Comparisons were also performed for a subset of polycystic ovary syndrome follicles (n = 5) matched for diameter and size range with the normal group. alpha-Subunit mRNA levels were 16-fold lower in both polycystic ovary syndrome follicle groups relative to size-matched normal follicles (P < 0.02), whereas beta(A)-subunit mRNA was significantly lower only when all polycystic ovary syndrome follicles were compared. beta(B)-Subunit and follistatin mRNA levels and the follistatin 315/288 ratio were not statistically different for any group. These results suggest that insufficient production of inhibin alpha and possibly beta(A)-subunits, but not follistatin, is associated with follicular arrest in polycystic ovary syndrome follicles.