Effects of inhibitors of steroid biosynthesis on prostaglandin formation in rabbit ovarian follicles

Rabbit ovarian follicles were incubated without stimulation, with LH and with LH + an inhibitor of steroid biosynthesis. Formation of prostaglandins PGE and PGF and of progesterone and estradiol was measured in these incubates. It was found that aminoglutethimide phosphate (AGP) inhibited the LH stimulated biosynthesis of both prostaglandins and steroids. However U 30870 and Metyrapone, while completely inhibiting the LH stimulated biosynthesis of progesterone and estradiol respectively, had no effect on the formation of prostaglandins. Further, the inhibition of prostaglandin formation by AGP could not be reversed by exogenous steroids. It, therefore, appears that the effect of AGP on prostaglandin biosynthesis may not be related to its effect on steroid biosynthesis. However, the response of rabbit follicles to AGP is contrary to that reported for rat follicles and indicates different control mechanisms for prostaglandin formation in the follicles of the two species.     

Cycloheximide inhibition of ovulation, prostaglandin biosynthesis and steroidogenesis in rabbit ovarian follicles

Cycloheximide (5 mg/kg, i.v.) significantly inhibited ovulation in the rabbit when it was administered as early as 20 h before the ovulation process was initiated by hCG, and as late as 1 h after hCG. The ovulation rate was significantly reduced, but follicular biosynthesis of prostaglandins E and F was only partly inhibited. The biosynthesis of progesterone and oestradiol in follicles during the early stages of the ovulation process was also inhibited. Cycloheximide may therefore inhibit ovulation by a mechanism which is different from the action of indomethacin, and this mechanism may involve the suppression of ovarian steroidogenesis.     

Stimulation of prostaglandin F synthesis by luteinizing hormone in rabbit ovarian follicles grown in organ culture

Pre-ovulatory follicies of rabbits were cultivated in organ culture dishes for 3 days in media with or without luteinizing hormone (LH). Prostaglandin F (PGF) levels were measured in the media harvested after various incubation periods. PGF levels found in media were very low (between 0.55 ± 0.19 ng and 0.91 ± 0.15 ng per follicle) throughout incubation periods in the absence of LH. In the LH-treated group, PGF levels were significantly higher than those in corresponding controls, at every time interval. Within the treated group, PGF concentration rose sharply, reaching maximal levels between 12 and 36 hours (11.25 ± 3.08 ng and 11.33 ± 5.04 ng per follicle), then fell but still remained above basal level (1.63 ± 0.55 ng per follicle) by 72 hours.     

Selective activation of rabbit ovarian protein kinase isozymes in rabbit ovarian follicles and corpora lutea

The magnitude of activation of the type I and type II forms of cAMP-dependent protein kinase was investigated in estrous follicles and corpora lutea (CL) obtained from ovaries of control rabbits and rabbits injected acutely with human chorionic gonadotropin (hCG). To this end, a chromatographic technique which permitted quantitative evaluation of the in vivo activational state of the two forms of cAmP-dependent protein kinase was developed and verified. Results revealed that in follicles obtained from ovaries of untreated estrous rabbits, 15% of the soluble cAMP-dependent protein kinase, all of which exists as the type II isozyme, is activated. Intravenous administration of a single bolus of hCG promoted a concentration-dependent activation (in 10 min) of this protein kinase isozyme. In CL obtained from ovaries of control, 4-day pseudopregnant rabbits, 32% of the total soluble cAMP-dependent protein kinase exists as the type I form and 68% exists as the type II form. Both types of protein kinase are approximately 10% dissociated in CL from ovaries of untreated rabbits. Upon intravenous administration of hCG, only the type I form of cAMP-dependent protein kinase is further activated (in 10 min). Dissociation of this protein kinase is dependent upon the time and concentration of hCG. Preferential activation of the type I form of cAMP-dependent protein kinase in CL is also demonstrable in in vitro studies using exogenous cAMP. These data suggest that the physiological intracellular mediator of acute cAMP-regulated, hCG-triggered functions in rabbit ovarian follicles is the type II isozyme of cAMP-dependent protein kinase while in CL of 4-day pseudopregnant rabbits, it is the type I enzyme form.     

Steroid hormone formation in bovine ovarian follicles.

In an attempt to assess histophysiological implication of the follicular compartment of the bovine ovary in steroid hormone formation and the effect of human chorionic gonadotropin (hCG) in vitro on follicular steroidogenesis, minces of follicular tissues from non-gravid bovine ovaries were incubated with radioactive testosterone or acetate in the presence and absence of hCG. Significant amounts of estrone and estradiol-17beta were formed on incubation with testosterone-4-14C; hCG decreased the conversion approximately by 30%. The major radioactive products formed from acetate-l-14C were androstenedione and testosterone with lesser amounts of dehydroepiandrosterone and 17-hydroxyprogesterone. In addition, small amounts of progesterone, 17-hydroxypregnenolone, estrone and estradiol-17beta were formed. Histology of the dissected follicle specimens was characterized by dominant theca cells undergoing luteinization with small amounts of granulosa cells, which showed neither proliferation nor luteinization. The pattern of distribution of radioactivity among the steroids formed from acetate-14C was considered to represent steroidogenic profile of bovine atretic follicles. The addition of hCG in vitro increased the overall incorporation of radioactive acetate into the steroids approximately by 50%, although the range of increase was not uniform in the individual steroids under the exprimental conditions.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.     

Prostaglandins and steroidogenesis by isolated rabbit ovarian follicles

Prostaglandins of the F and E series at concentrations from 1 to 100 microgram/ml had no effect on steroidogenesis by isolated rabbit follicles. Indomethacin and 7-oxa-13-prostynoic acid at doses lower than 100 microgram/ml failed to prevent the LH-induced increase in testosterone accumulation by follicles. At 1 mg/ml these inhibitors prevented the LH effects. Prostaglandin E2 and F2alpha had no effect on testosterone accumulation. However, prostaglandin E2 seemed to enhance the LH-induced accumulation of androstenedione and progesterone by the follicles. These data suggest that prostaglandins play a minor role in steroidogenesis by isolated rabbit ovarian follicles.     

Therapy of Cushing''s syndrome with steroid biosynthesis inhibitors

Several substances with different inhibitory effects on adrenal steroid biosynthesis were investigated in patients with Cushing's syndrome. It has been shown that trilostane, a 3β-hydroxysteroid-dehydrogenase inhibitor, is not potent enough to block cortisol biosynthesis in patients with hypercortisolism. Aminoglutethimide inhibits side chain cleavage of cortisol synthesis, but it has been demonstrated that the blocking effect on cortisol secretion is not strong enough to normalize urinary cortisol excretion in patients with Cushing's disease. For metyrapone, an inhibitor of adrenal 11β-hydroxylase, promising results were reported for the treatment of Cushing's syndrome. However, the drug has several side effects and depending on the definition of the desired reduction of cortisol secretion a true remission was only found in a minority of patients. The antifungal drug ketoconazole in vitro predominantly blocks 17,20-desmolase (IC₅₀ 1 μM) and to a lesser extent 17-hydroxylase (IC₅₀ 10 μM) and 11β-hydroxylase (IC₅₀ 15–40 μM). Therefore, ketoconazole in vivo most potently suppresses androgen secretion and only to a lesser extent cortisol biosynthesis. Several therapeutic trials with ketoconazole treatment in patients with pituitary Cushing's disease showed various remission rates between 30 and 90%. In contrast, in almost all patients with benign, primary adrenal Cushing's syndrome cortisol levels were normalized. In patients with ectopic ACTH syndrome ketoconazole was effective in about 50% of all reported cases, while cortisol hypersecretion due to adrenocortical carcinoma was only rarely inhibited by ketoconazole. The main side effect of ketoconazole treatment was liver toxicity which occurred in 12% of all treated patients. In contrast to ketoconazole, the narcotic drug etomidate shows a strong inhibitory effect on 11β-hydroxylase (IC₅₀ 0.03–0.15 μM) but only a weak inhibition of 17,20 desmolase (IC₅₀ 380 μM). This correlates with in vivo studies where even low, non-hypnotic doses of etomidate induced a pronounced fall in serum cortisol levels in normals and in patients with Cushing's syndrome. However, its clinical use is limited by its mandatory intravenous application and its sedative effects. In conclusion, ketoconazole remains the only available steroid-inhibitory drug for a therapeutic trial in patients with Cushing's syndrome who cannot be treated definitively by surgery.     

Steroid hormone formation in human ovarian follicles in vitro.

Ovarian follicles of 5 to 15 mm in diameter were isolated from 45 ovaries of 34 patients in the follicular and luteal phases of the cycle. Three experiments were done. In the first, follicles were minced and incubated in Krebs-Ringer bicarbonate buffer containing 1 to 2muCi of testosterone-4-14C in the presence or absence of 100 IU human chorionic gonadotropan (hCG). In the second, minced follicles were incubated with 100 muCi of sodium acetate-I-14C under identical conditions. In the third, ten follicles from a single patient in the late proliferative stage of endometrial dating were cut in halves and incubated with 100 muCi of acetate-I-14C under identical conditions. The minced follicle preparation was capable of aromatizing testosterone-4-14C into radioactive estrone and estradiol in significant amounts. Incorporation of radioactive acetate into pregenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, estradiol and estrone was assessed by reverse dilution analysis with recrystallization to constant specific activity. The major radioactive products formed were androstenedione and 17-hydroxyprogesterone in the latter two experiments. Dehydroepiandrosterone was one of the major steroids in the second experiment. The minor products were testosterone, progesterone and pregnenolone. Smaller, but definite incorporations of radioactive acetate into estradiol and estrone occurred in the second experiment. On histological examination, the follicles were characterized by atretic changes. This distribution pattern of radioactive acetate among the steroids was considered to represent the steroidogenic profile of unstimulated or atretic follicles.     

Effects of bivalent cations on prostaglandin biosynthesis and phospholipase A2 activation in rabbit kidney medulla slices.

The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3--5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.