Non-steroidal low molecular substance from porcine follicular fluid inhibits aromatase activity

To clarify the underlying mechanism of the inhibitory control of the non-steroidal substance from porcine follicular fluid on estradiol secretion by granulosa cells, the suppression of aromatase activity was studied in vitro. Follicular fluid from porcine small follicles was filtered through an Amicon PM-10 membrane and a filtrate with a molecular weight of less than 10,000 was obtained. The filtrate was eluted on a Sephadex G-25 column (1.5 x 70 cm) using 0.01N CH3COOH, pH 4.0 as the elution buffer. The fraction containing the first peak of peptide (Fraction A) was used for subsequent experiments. Human placental microsomal fraction was used for the aromatase source. Aromatase activity was examined by counting the radio-activity of 3H2O converted from (1 beta, 2 beta 3H)-testosterone. The conversion of 3H2O from (1 beta, 2 beta 3H)-testosterone by rat's granulosa cells cultured in serum-free medium was also examined. Fraction A significantly suppressed aromatase activity of placental microsomal fraction by 57% at 120 min, and by 38% at 180 min, in dose and time-dependent manners. The aromatase activity of cultured rat granulosa cells was also inhibited by Fraction A in a dose-dependent manner. These results demonstrate the presence of a low molecular non-steroidal substance in the follicular fluid which is capable of inhibiting aromatase activity. This substance could provide important insights into the regulatory mechanism of differentiated functions of granulosa cells.