Inhibition of FSH augmented adenylate cyclase activity in porcine granulosa cells by ovarian protein.

We sought to evaluate the effects of a fraction of porcine follicular fluid, termed follicle regulatory protein (FRP), on FSH-induced adenylate cyclase activity in porcine granulosa cell membranes using Gpp(NH)p and forskolin as pharmacological probes of adenylate cyclase activity. Without FSH treatment, the addition of 100 micrograms/ml of the FRP fraction induced a significant decrease in Gpp(NH)p stimulated cyclase activity while maximal inhibition of cAMP formation was achieved with 1 mg/ml of FRP. Granulosa cells cultured with FSH reached a maximum in adenylate cyclase activity at 20 min which returned to baseline by 45 minutes. FRP induced a reduction in adenylate cyclase activity during this same interval of time. Adenylate cyclase activity of cells treated with FRP was unchanged in the presence of methyl-isobutal-xanthine. Further, when FRP was heated (56 degrees C, 45 min) or precipitated with 10% TCA, it was unable to inhibit adenylate cyclase. The 50% inhibitory dose (ID50) for FRP inhibition of adenylate cyclase activity in cells stimulated with Gpp(NH)p was 80 micrograms/ml and 500 micrograms/ml when granulosa cells were preincubated with FSH prior to Gpp(NH)p stimulation. The ID50 for the FRP inhibition of forskolin stimulated adenylate cyclase activity was 500 micrograms/ml. Gpp(NH)p stimulated adenylate cyclase activity was more sensitive than forskolin stimulated activity to inhibition by FRP. In conclusion, the data presented here demonstrate that a partially purified fraction of porcine follicular fluid inhibited FSH responsive adenylate cyclase activity in porcine granulosa cells.

Biochemical and physiologic characterization of follicle regulatory protein: a paracrine regulator of folliculogenesis.

Further purification of a porcine 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.

Identification of the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli: recA441 and recA629.

We have identified the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli. The recA441 mutant, which shows constitutive expression of the recA-mediated SOS response at 42 degrees C, contains two amino acid substitutions, glutamic acid to lysine at residue 38 and isoleucine to valine at residue 298. The recA629 mutant is an unusual pseudorevertant of recA441 that is no longer capable of spontaneous expression of SOS functions at 42 degrees C. Purified recA629 protein is cold-labile for several of the wild-type enzymatic activities and is shown here to contain three amino acid substitutions, the two found in the recA441 protein at residues 38 and 298, as well as an aspartic acid-to-glycine change at residue 32. The mutation at residue 32 was verified by restriction digestion of the 5' region of the recA629 structural gene.

Correlation of inhibin and follicle regulatory protein activities with follicular fluid steroid levels in anovulatory patients.

Follicular fluid was obtained from anovulatory patients (n = 12), stimulated with human menopausal gonadotropin, clomiphene, and human chorionic gonadotropin to evaluate the relative responses of inhibin, follicle regulatory protein, and steroid levels in follicles from ovaries requiring exogenous stimulation for follicular development. Follicular fluid concentrations of estradiol, progesterone, androstenedione, testosterone, dihydrotestosterone, and 3 alpha-androstenediol were determined by radioimmunoassay. Follicular fluid inhibin activity was determined by suppression of rat pituicyte follicle-stimulating hormone, and follicle regulatory protein activity was determined by suppression of porcine granulosa cell aromatase. The mean level of steroids were progesterone (7529 +/- 1601 ng/ml), estradiol (1082 +/- 158 ng/ml), androstenedione (15.2 +/- 3.17 ng/ml), 3 alpha-androstenediol (0.90 +/- 0.13 ng/ml), testosterone (2.23 +/- 33 ng/ml), and dihydrotestosterone (0.77 +/- 0.11 ng/ml). Follicle regulatory protein activity was 16.6% +/- 4.3% and mean inhibin level was 62.9 +/- 7.52 U. These results are in contrast to reports of follicular fluid steroid levels from normal ovulatory patients treated with exogenous gonadotropin. Although altered levels of hormones were present within these follicles, they clearly were not atretic, as evidenced by elevated estradiol levels and estradiol/androstenedione ratios. Alterations in the normal follicular response to pharmacologic gonadotropin stimulation in the follicles of anovulatory women suggest the presence of granulosa cell dysynchrony .

Correlation of human follicular fluid inhibin activity with spontaneous and induced follicle maturation.

We sought to correlate the inhibin activity of individual ovarian follicles (greater than 16 mm in diameter) from untreated (7 patients; 7 follicles), clomiphene-stimulated (150 mg/day; menstrual cycle days 5-9; 9 patients, 14 follicles), and human menopausal gonadotropin (hMG)-stimulated (150 IU/day; menstrual cycle days 3-11; 8 patients; 23 follicles) ovarian cycles and to correlate these results with the follicular fluid (FF) steroid concentration. Follicular aspirates were obtained via laparoscopy from 24 regularly menstruating patients when the diameter of the largest follicle reached 20 mm, as determined by serial ultrasonography. FF concentrations of estradiol, progesterone, testosterone, 17-hydroxyprogesterone, and androstenedione were determined by RIA. Inhibin activity was determined using the inhibition of basal 24-h FSH secretion by dispersed rat anterior pituitary cells. Inhibin values were highest among the follicles aspirated from those patients who received hMG [277 +/- 31 (+/- SE) U/ml] compared to untreated subjects (51 +/- 13 U/ml) or those who received clomiphene (96 +/- 14 U/ml). Estradiol was highest in FF from untreated patients (2295 +/- 1155 ng/ml) compared to levels in patients who received hMG (368 +/- 1.76 micrograms/ml) or clomiphene (1049 +/- 174 ng/ml). FF progesterone values were highest in untreated patients (9.4 +/- 2.59 micrograms/ml) compared to those in hMG-treated (5.04 +/- 1.76 micrograms/ml) and clomiphene-treated patients (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values were similarly higher in the untreated (1.55 +/- 0.21 micrograms/ml) and clomiphene-treated (2.54 +/- 0.27 micrograms/ml) patients than in the hMG-treated group (0.73 +/- 0.09 micrograms/ml). FF androstenedione (untreated, 50.7 +/- 30 ng/ml; clomiphene-treated, 73.4 +/- 23.4 ng/ml; hMG-treated, 60.2 +/- 19.8 ng/ml) and testosterone (6.66 +/- 2.45, 5.98 +/- 1.46, and 6.39 +/- 2.16 ng/ml, respectively) concentrations in all three patient groups were similar. In untreated patients, there was a highly significant positive correlation between intrafollicular inhibin activity and FF estradiol, testosterone, and androstenedione concentrations and a statistically significant negative correlation between intrafollicular inhibin activity and FF progesterone concentrations. Patients receiving clomiphene therapy demonstrated at least two different response patterns, one with a positive and one a negative correlation between intrafollicular inhibin activity and FF steroid concentrations. The patients receiving hMG therapy had no statistically significant correlation between intrafollicular inhibin

Inhibition of luteinizing hormone/human chorionic gonadotropin binding to porcine granulosa cells by a follicular fluid protein(s).

Previously, we evaluated the effects of a follicular regulatory protein(s) on granulosa cell follicle-stimulating hormone (FSH) receptors during short-term culture. No demonstrable effect on porcine granulosa cell FSH binding capacity was found. Here, we assessed the effects of follicular regulatory protein(s) on porcine granulosa cell human chorionic gonadotropin (hCG) binding capacity. Follicle regulatory protein was prepared from porcine follicular fluid by saturated ammonium sulfate precipitation, dialysis, lyophilization, and pseudoligand affinity chromatography. Receptor assays were performed with the use of porcine granulosa cells and hCG as the radioligand. After 48 hours in vitro, there was an apparent follicular regulatory protein(s)-induced diminution in overall specific granulosa cell 125I-hCG binding compared to the 24-hour determination. After 72 hours, control cultures and those which received FSH alone had significantly greater specifically bound 125I-hCG compared to cultures which were treated with either follicular regulatory protein(s) or FSH plus follicular regulatory protein(s). This disparity became more apparent by 96 hours of culturing. Granulosa cells which received FSH alone or control cultures had significantly greater specifically bound 125I-hCG compared to granulosa cell cultures treated with either follicular regulatory protein(s) or FSH plus follicular regulatory protein(s). Furthermore, follicular regulatory protein(s) produced a dose-response inhibition of hCG binding. When considered together with previous observations of follicular regulatory protein(s)-associated inhibition of granulosa cell aromatase in both human and porcine granulosa cells, these data suggest that follicular proteins may play a major role in the intraovarian modulation of folliculogenesis.

Activity of a human follicular fluid protein(s) in spontaneous and induced ovarian cycles.

Recently, we identified a human follicular fluid protein(s) (FP) which inhibited human menopausal gonadotropin (hMG)-induced rat ovarian weight gain and FSH-induced aromatase. Here, we assessed FP activity from ovulatory patients who were either untreated (n = 7) or received clomiphene (n = 9; 150 mg/day on cycle days 5-9) or hMG (n = 6; 150 IU/day on cycle day 3). Aspirations were performed when one follicular diameter exceeded 20 mm. FP activity was expressed as the percent inhibition of porcine granulosa cell aromatase activity at three concentrations of extracted follicular fluid (range, 1250-10 micrograms; extrapolated to 50 micrograms). Patients receiving hMG or clomiphene had multiple follicles greater than 16 mm in diameter (3.83; 2.66/patient, respectively), while untreated patients had 1 each. FP activity was 14.1 +/- 5.3% (mean +/- SEM) inhibition for untreated, 18.0 +/- 3.4% inhibition for hMG-treated, and 13.7 +/- 5.3% inhibition for clomiphene-treated patients. Follicular fluid estradiol levels from untreated patients (2590 +/- 1221 ng/ml) were greater than estradiol concentrations from hMG-treated (356 +/- 55 ng/ml; P less than 0.01) or clomiphene-treated (1317 +/- 344 ng/ml; P less than 0.05) patients. Progesterone follicular fluid levels were 9.84 +/- 3.3, 5.18 +/- 61, and 11.3 +/- 2.3 micrograms/ml for untreated, hMG-treated, and clomiphene-treated patients, respectively (P less than 0.05). A similar relationship was present with 17-hydroxyprogesterone (untreated, 1.6 +/- 0.2 micrograms/ml; hMG-treated, 0.76 +/- 0.1 micrograms/ml; clomiphene-treated, 2.16 +/- 0.3 micrograms/ml; P less than 0.05). Androstenedione and testosterone follicular fluid levels were similar in all groups (78.9 +/- 23 and 7.09 +/- 2.14 ng/ml, respectively). Untreated patients had a positive correlation between FP and follicular fluid estradiol (r = 0.689; P less than 0.01) and inhibin activity (r = 0.654; P less than 0.05), and a negative correlation between follicular fluid progesterone levels (r = 0.622; P less than 0.05). Patients treated with hMG had a significant negative correlation between FP activity and follicular fluid progesterone levels (r = 0.756; P less than 0.005) and a biphasic correlation with follicular fluid 17-hydroxyprogesterone (r2 = 0.853; P less than 0.0025). Clomiphene-treated patients had biphasic correlations between follicular fluid estradiol and 17-hydroxyprogesterone levels (r2 = 0.853 and P less than 0.0025, and r2 = 0.637 and P less than 0.025, respectively). These findings indicate that the FP activity of the dominant follicle correlates with its state of differentiation, as described by intrafollicular estradiol, progesterone, 17-hydroxyprogesterone levels and inhibin activity. These relationships are in part dependent upon gonadotropin stimulation.