Presence of an aromatase inhibitor, possibly heat shock protein 90, in dominant follicles of cattle

In cattle, it has been suggested that follicular fluid has direct modulator y effects on follicular growth and maturation. In the first part of this st udy, an in vitro test using aromatase activity of follicular wall fragments as an end point was validated for cattle follicles and was used to test wh ether follicular fluid (from dominant or non-dominant follicles) modulates aromatase activity. Fluid from dominant follicles at a concentration of 24 or 12% (obtained during the luteal and follicular phases, respectively) sig nificantly inhibited aromatase activity. Inhibitory activity was low or abs ent in fluid from nondominant follicles. FSH-stimulated aromatase activity was also reduced by fluid from dominant follicles, but not to a greater ext ent than in basal conditions. Finally, charcoal-treated fluid from dominant follicles retained its inhibitory activity. Ln contrast, ovarian venous se rum draining a dominant follicle had no activity at the three concentration s tested (6, 12 and 24%). Ln the second part of the study, identification o f the compounds involved in this modulatory activity was attempted using SD S-PAGE. Comparison of the fluorographs from de novo synthesized proteins st ored in follicular fluid (inhibitory medium) with those secreted in incubat ion medium (inactive medium) demonstrated that one protein (90 kDa, pI 5.8) was significantly (P < 0.05) more abundant in fluid from dominant follicle s (2.0 +/- 0.09%) than in the culture medium (1.3 +/- 0.1% of the total pro teins). This protein had characteristics similar to those of heat shock pro tein 90 (hsp 90). Therefore, in the final part of the study, the presence o f hsp 90 in ovarian cells and follicular fluid was investigated using immun ohistochemistry and western blot analysis. After immunohistochemistry, a po sitive signal was detected mainly in the granulosa cells of larger follicle s and to a smaller extent in thecal cells and oocytes. Western blot analysi s also demonstrated the presence of hsp 90 in follicular wall fragments and fluid. When blotting was achieved on a sample of follicular fluid resolved by two-dimensional PAGE, the spot detected had a similar location to that at 90 kDa and pI 5.8. Addition of purified hsp 90 to bovine follicles in vi tro depressed aromatase activity by altering the Km value (and possibly the V-max value) of the enzyme. It is proposed that hsp 90 is a functional reg ulator of follicular maturation through its action on aromatase.