Sharon A. Tonetta

Thecal cell 3-beta hydroxysteroid dehydrogenase activity: Modulation by human chorionic gonadotropin, progesterone, estradiol-17 beta and dihydrotestosterone

Thecal cell steroidogenesis plays a major role in folliculogenesis within the porcine ovary. Accordingly, the effects of physiological concentrations of steroids on 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD) were determined. Theca was excised from large porcine follicles and prepared in a monolayer culture in 1 ml of serum-free media. Cells were treated 24 h after culture as follows: (1) control, (2) hCG (5 IU); (3) progesterone (P, 3 micrograms); estradiol-17 beta (E, 4 micrograms); 5 beta-dihydrotestosterone (DHT, 1 microgram); (4) hCG + P or E or DHT. At 3, 6, 12, 24 and 48 h after treatment, media were assessed for P levels. For 3 beta-HSD activity, P formation by microsomal fractions incubated with 1 microM pregnenolone + 5 microM NAD+ for 1 h (37 degrees C) was monitored. Thecal cell P secretion increased from 27 to 72 h. hCG significantly (P less than 0.05) increased P levels after 36 h compared to controls. E or E + hCG decreased P levels at 36, 48, and 72 h and DHT prevented the hCG-induced increase in P secretion. 3 beta-HSD activity in thecal microsomes increased significantly from 27 to 72 h. hCG had little effect on 3 beta-HSD activity compared with controls from 27 to 36 h, but significantly (P less than 0.05) decreased 3 beta-HSD activity at 48 and 72 h. However, P or P + hCG significantly (P less than 0.05) decreased 3 beta-HSD activity at all times. In addition, E or E + hCG significantly (P less than 0.05) decreased 3 beta-HSD activity at 48 and 72 h. DHT prevented the hCG-induced decrease in 3 beta-HSD activity. In conclusion, porcine thecal secretion of P and microsomal 3 beta-HSD activity increased during 72 h of culture. Paradoxically, the addition of hCG to cultures enhanced media P concentrations but inhibited 3 beta-HSD activity. Further, the addition of E to cultures decreased media concentrations of P while P or E decreased 3 beta-HSD activity. Therefore, paracrine/autocrine effects of locally produced steroids may play a role in modulating thecal cell steroidogenesis.

7 Paracrine regulation of follicular maturation in primates

Summary Taken together, the studies reviewed here suggest that although gonadotropins are necessary for follicular growth, they are insufficient by themselves to explain the dynamics of folliculogenesis. Indeed, the role of gonadotropins in follicular maturation must necessarily be permissive: that is LH and FSH initiate a synchronized cascade of follicular events directly mediated by paracrine and autocrine factors.

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.

Follicle regulatory protein: a novel marker for granulosa cell cancer patients.

Follicle regulatory protein (FRP) is secreted by the granulosa cell of the ovary and plays a role in modulating follicle development. A dual epitope immunoassay using two murine monoclonal antibodies, isotype IgG1 (raised against porcine FRP), in tandem was developed to measure FRP in serum. The levels of FRP in the serum of women with granulosa cell tumors, normal, menstruating women, and postmenopausal women were determined. The levels of FRP were elevated in the serum of 79% of the women with granulosa cell tumors compared to the normal controls. FRP levels in serial samples from women with granulosa cell tumors generally correlated with the clinical course of the disease. Thus, FRP may provide a useful marker for granulosa cell tumors.

A postulated role for naturally occurring aromatase inhibitors in follicle selection

The studies reviewed here indicate that follicle regulatory protein (FRP) alters aromatase and 3B-hydroxysteroid dehydrogenase activity in porcine, human, and rat granulosa cells. The inhibitory effect of FRP on granulosal aromatase activity depend upon the response of the cell to FSH: large amounts of FSH can partially overcome FRP inhibition while relatively small amounts of FSH sensitize the granulosal aromatase system to FRP. Although androgens potentiate FSH-mediated granulosal functions, they also sensitize granulosa cell steroidogenic enzymes to inhibition by FRP. The demonstration that FRP acts primarily on granulosa cells of less mature antral follicles to inhibit aromatase supports the hypothesis that FRP may facilitate follicle selection and suggests a role for FRP in atresia. Most of the effects of FRP on granulosal activities reflect an interplay between the systemic endocrine and local paracrine systems. That FRP functions, at least in part, by modulating follicular response to FSH is consistent with the hypothesis that paracrine effectors are important mediators of folliculogenesis in the presence of gonadotropins.

Plasma iodothyronines in the domestic fowl: newly hatched to early adult stages, with special reference to reverse triiodothyronine (rT3)

Circulating concentrations of thyroxine (T4), triiodothyronine (T3) and reverse triiodothyronine (rT3) were measured in chicks before, during, and after hatching, up to 9 weeks of age. T4 decreased prior to hatching, rose after emergence, and was variable in the immature domestic fowl. T3 increased prior to emergence, decreased until 5 days after hatching, and increased again by 1 week of age, after which the levels declined. Plasma rT3 declined prior to hatching, remained low until 5 days after emergence, and then increased, again, to 0.14-0.19 ng/ml between 1-9 weeks of age.

Analysis of Binding Sites for IGF-I on Membranes from Granulosa Cells of Small, Medium, and Large Porcine Follicles

We examined binding of IGF-I to granulosal membranes from small ( 8 mm) porcine follicles. Granulosa cells were aspirated and membranes prepared by dounce homogenization. Membranes from each group were incubated for 16 h at 4°C (optimal conditions) with I-IGF-I in the absence or presence of unlabeled IGF-I (0.01–1 μg/ml), IGF-II (0.5–5 μg/ml), or insulin (0.1–20 μg/ml). Scatchard analysis of the data demonstrated a curvilinear plot for all three groups. The high-affinity sites had the following Ka values (mean ± SEM; nM): small = 6.50 ± 0.09; medium = 6.45 ± 0.44; large = 6.48 ± 0.44. The binding capacities of the high-affinity sites for each group were (pmol/mg protein): small = 1.51 ± 0.17; medium = 1.15 ± 0.14; large = 1.40 ± 0.15. The low-affinity sites had the following Ka values (nM): small = 0.053 ± 0.008; medium = 0.077 ± 0.019; large = 0.077 ± 0.007. The low-affinity sites had the following binding capacities (pmol/mg protein): small = 20.40 ± 3.10; medium = 13.76 ± 0.78; large = 12.99 ± 0.55. Preferential binding for the high-affinity sites was IGF-I > IGF-II > insulin. These data show that although IGF-I plays a role in granulosal differentiation, the affinity and number of high-affinity binding sites do not change during follicular maturation. However, the number of low-affinity binding sites appears to be greater in granulosa cells from small follicles, suggesting a possible role for IGF-II and/or insulin in early follicular maturation.

Comparison of norethindrone and medroxyprogesterone acetate with natural progesterone and estradiol in stimulating prolactin production from cultured endometrial stromal cells

Progestins stimulate prolactin production from endometrial stromal cells in culture. We compared the potencies of the synthetic progestins norethindrone and medroxyprogesterone acetate to natural progesterone in inducing stromal prolactin production. Modifications of the culture system provided an increase in stromal cell yield, thus permitting multiple comparisons from the treatment of cells from a common endometrial sample. The effects of high-dose estradiol also were evaluated in this system. The findings suggest relative potencies of 50:1 for medroxyprogesterone acetate and progesterone. Norethindrone gave intermediate and more variable responses. Estradiol potentiated prolactin production from only submaximal progestins doses. The differences between the progestin effects, in large measure, were due to differential culture growth as reflected by culture mass. Compared to controls and estradiol alone, all the progestins induced much greater prolactin production. Thus during decidualization, progestins probably promote both stromal growth and intracellular prolactin production. High-dose estradiol may not interfere with these events.

Binding Sites for IGF-I Identified on Theca Cells from Large Porcine Follicles

Theca from large porcine follicles (>8mm) were examined for specific IGF-I binding sites. Thecal membranes were prepared and then incubated overnight at 4°C with I-IGF-I with or without increasing concentrations of IGF-I, IGF-II and insulin. Specific high-affinity IGF-I binding sites were demonstrated. Scatchard analysis gave a curvilinear plot with a Ka for the high affinity sites of 1.97 ± 0.35 nM (binding capacity, 305.7±37.2 fmol/mg protein). Preferential binding was IGF-I>IGF-II>insulin. Interestingly, IGFs modulate steroidogenesis in porcine theca cells with a similar order of potency. These data demonstrate high affinity, low capacity binding of IGF-I to porcine thecal membranes and suggest that IGF-I and related peptides act on thecal steroidogenesis through IGF-I receptors.

Modulation of 17α-hydroxylase/C17,20-lyase activity in porcine theca cells

The major source of ovarian androgen is the theca cells. Androgens are produced by the conversion of progestins by the 17 alpha-hydroxylase/C17,20 lyase enzymatic system (lyase). The 3 beta-hydroxysteroid dehydrogenase and aromatase enzymes in the theca cells are modulated by gonadotropins as well as by steroids produced locally. Therefore, the combined effects of hCG plus progesterone, estradiol, or dihydrotestosterone (DHT) on microsomal lyase activity in theca cells from large and medium-sized follicles were determined. Theca cells (3 x 10(6) cells/6 ml/well) were cultured in Medium 199 (M199) containing only insulin (10 micrograms/ml) and transferrin (5 micrograms/ml). At 24 h, theca cells were treated with M199, hCG (15 ng/ml), progesterone, estradiol, or DHT (100 ng/ml) or a combination of hCG + one of the three steroids. Media were removed at various times of culture (27-72 h) and levels of androgen determined by RIA. Microsomes were incubated with 1 microCi [3H]progesterone +0.5 mM NADPH and radioactive conversion products were measured after purification by thin layer chromatography. Administration of progesterone, estradiol, or DHT alone had little effect on lyase activity in theca cells from medium-sized follicles whereas the addition of hCG alone significantly increased lyase activity in these cells. However, concomitant addition of any steroid with hCG inhibited the increase in lyase activity after the addition of hCG alone. Theca cells from large porcine follicles had a higher basal level of lyase activity compared to theca cells from the smaller follicles. Lyase activity in theca cells from large follicles was enhanced by progesterone; estradiol was inhibitory. DHT initially stimulated lyase activity in theca cells from large follicles, but was inhibitory later in culture. In contrast to its marked effect on theca cells from medium follicles, hCG had only a small effect on lyase activity in theca cells from large follicles. Thus, thecal lyase activity increased as the follicle matured, providing more androgen substrate for the production of estrogen. Lyase activity in theca cells of medium follicles appears to be regulated predominantly by gonadotropin from the pituitary while intraovarian regulation of lyase activity by steroids may be more important in larger follicles.