Previously we identified a fraction of follicular fluid (follicle regulatory protein: FRP) which inhibits granulosa cell aromatase activity. During the course of these studies the question of FRP acting via autocrine as well as paracrine mechanisms arose in addition to the need for a more efficient method of screening for aromatase inhibitory activity during the purification of FRP. Accordingly, we assessed the effects of FRP on aromatase activity in a microsomal assay. Placental microsome preparations were preincubated for 20 minutes with or without FRP prior to a 20 minute incubation with testosterone. Significantly less conversion of testosterone to estrogen occurred with FRP compared to control preincubation. When follicular protein was added without pre-incubation, there was no apparent change in microsomal aromatase activity, whereas after a 20 minute pre-incubation with the follicular protein fraction, significantly less testosterone was converted into estrogen. When various concentrations of FRP were assayed in the placental aromatase assay, a dose-response curve demonstrated a 50% inhibitory dose (ID50) of approximately 400 micrograms/ml. To further purify the aromatase inhibitory activity, 5 mg of the crude follicular fluid preparation was eluted through an anion exchange column via HPLC using a sodium acetate gradient. The fractions in the central elution peak contained aromatase inhibitor activity with ID50 values of 25-160 micrograms/ml. Thus Fractions were further purified by elution through a gel exclusion column via HPLC which demonstrated inhibition of cell free placental aromatase activity in the 15,000-18,000 molecular weight range with an ID50 of 5 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe and compare the use of isoelectric focusing (IEF) in a granulated Sephadex matrix and in polyacrylamide immobilized pH gradients to separate an aromatase inhibitor (follicle regulatory protein: FRP) in preparative amounts from porcine follicular fluid (PFF). The starting material for IEF was derived from pFF after passage through agarose immobilized textile dye Orange A (0.5 KC1 eluent). Before IEF, some Orange A bound (OAB) material was further purified on a FPLC employing a Mono-Q anion exchange column. Previous use of chromatofocusing indicated that aromatase inhibitory activity is largely concentrated in OAB fractions with a pI in the ranges of pH approximately 4.5 and approximately 6.5. The current study revises these findings to provide a more precise measure of the isoelectric points in question to pH 4.73 +/- 0.05 and pH 6.41 +/- 0.06. The use of Sephadex was limited by gradient instability and the selection of pH ranges available. IEF using immobilized pH gradients had several advantages over Sephadex: 1) broader selection of gradients from 0.1 to 7.0 pH units; greater resolving power, and enhanced stability. The principal disadvantage of the immobiline system was the recovery of focused material from the gel matrix. The use of isoelectric focusing with immobilized pH gradients on a preparative scale to purify FRP from OAB resulted in a greater than 50% recovery with a substantial increase in specific activity (from ID50 approximately 300 micrograms/ml to 20 ng/ml).

Further purification of a procine follicular fluid fraction, referred to as follicle regulatory protein, that inhibits granulosa cell aromatase was performed and the results of in vitro bioassays with these highly purified reagents are reported. The 0% to 35% saturated ammonium sulfate extract of porcine follicular fluid was percolated through an orange A dye matrex gel column and the bound fraction was eluted. Further purification of 0% to 35% orange A-bound fraction of porcine follicular fluid was performed by anion exchange chromatography with the use of the Mono Q column. Mono Q eluents containing follicle regulatory protein activity were injected onto a Mono P hydrogen ion-exchange column. Samples obtained from Mono P chromatography were injected onto preparative and analytical scale gel exclusion columns. Eluent fractions in the apparent molecular weight of 16,000 daltons were tested for aromatase inhibition. Throughout each step, parallelism of an aromatase inhibitor was apparent in both a cell-free microsomal assay and a granulosa cell assay. Follicle regulatory protein, purified about 6666-fold from the orange A-bound fraction of porcine follicular fluid, had a 50% inhibitory concentration of 25 ng/ml for granulosa cell aromatase activity.

We studied 15 anovulatory women undergoing ovulation induction with purified human urinary FSH or purified human urinary FSH and LH [human menopausal gonadotropins (hMG)]. All patients had either sporadic or no vaginal bleeding after progesterone therapy and failed to ovulate after receiving clomiphene (250 mg for 5 days) plus hCG. Other causes of infertility were ruled out. Sixteen cycles of FSH and 12 cycles of hMG were administered according to a standard protocol. Estradiol, progesterone, androstenedione, testosterone, LH, and FSH concentrations were quantitated by RIA. Follicular diameter was determined using ultrasound. There was no significant difference in the amount of FSH or hMG used per patient, in the duration of therapy before hCG administration, or in the length of the luteal phase in any patient. There was a difference in the number of follicles greater than 1000 mm3 per cycle in those patients receiving FSH compared to the number in those receiving hMG [2.8 +/- 1.3 (+/- SEM) vs. 4.4 +/- 1.5 follicles; P = 0.026). The maximum follicular phase serum estradiol (18.3 vs. 34.8 ng/ml) and maximum luteal phase progesterone concentrations (1289 vs. 2808 pg/ml; P = 0.026) were also different between the FSH and hMG groups. Linear regression analysis revealed a significant correlation between the peripheral serum estradiol levels and the total follicular volume of follicles in the hMG-treated group which was not apparent in the FSH-treated group. These findings suggest that exogenous LH may not be required to induce folliculogenesis in anovulatory patients.