Aminoglutethimide Phosphate Research Articles

Testosterone Propionate Induced Changes in Testicular Phosphatases of Musk Shrew (Suncus murinus L.)/Testosteronpropionatinduzierte Veränderungen der Hoden-Phosphatasen von Bisammaus (Suncus murinus L.)

In order to investigate the role of testosterone (TP) on the physiology of the testis of the musk shrew, activities of acid phosphatase, alkaline phosphatase and adenosine triphosphatase, and DNA, protein and phospholipid contents ot the testis were compared under high and suppressed testosterone conditions. The result indicates that testosterone propionate administration to intact shrews resulted in a significant increase in the activities of testicular enzymes. Following TP the testis phospholipid: DNA ratio increased, whilst no change was observed in the enzyme activities in response to aminoglutethimide phosphate. The role of testosterone in regulating testis function of this non-scrotal primitive eutherian mammal is discussed.

Effect of dexamethasone and cytochrome P 450 inhibitors on the formation of 7α-hydroxydehydroepiandrosterone by human adipose stromal cells

7α-Hydroxydehydroepiandrosterone (7α-OHDHA) is a major metabolite of dehydroepiandrosterone (DHA) using adipose stromal cells. To gain a better understanding of the factors regulating DHA metabolism, we examined the effect of dexamethasone and cytochrome P 450 inhibitors on the formation of 7α-OHDHA. Dexamethasone (10 −9 to 10 −7 M) stimulated 7α-OHDHA formation in a dose-dependent manner with a 2- to 5-fold stimulation at 10 −7 M. The dexamethasone stimulated 7α-OHDHA formation was inhibited by RU486 in a dose-dependent manner with suppression to basal levels at 10 −6 M. Progesterone (10 −7 M) had no effect on 7α-OHDHA formation suggesting that the dexamethasone stimulation was acting through the glucocorticoid receptor. Conversion of DHA to 7α-OHDHA was inhibited by ketoconazole and metyrapone. An inhibition of 70–80% was obtained with ketoconazole and 25–60% with metyrapone at concentrations of 10 −5 M. Aminoglutethimide phosphate was less effective than either ketoconazole or metyrapone in inhibiting 7α-OHDHA formation with <30% inhibition at 10 −5 M. These studies indicate that 7-hydroxylation provides an alternative pathway for the metabolism of DHA in peripheral tissues. This pathway, which is regulated by glucocorticoids, may influence the amount of DHA available for conversion to androstenedione and its subsequent aromatization to estrone. The biological role of the 7-oxygenated metabolites and their effects on other steroidogenic pathways have not been established.

Effects of aminoglutethimide phosphate on litter size and birth weight in superovulated rats

In this study, litter sizes, birth weights and survival rates of neonates in superovulated rats treated with aminoglutethimide phosphate (AGP) were studied. Young (8- to 9-week old) and adult (14- to 16-week old) rats were treated with 20 or 40 IU PMSG between 1100 and 1300 hours on the day of early diestrus, followed by identical doses of hCG 52 to 54 hours later. These rats received 10 mg AGP on Days 0 to 2 of pregnancy (AGP rats). The duration of pregnancy in the AGP-treated and control rats was about the same. Mean live litter sizes in young and in adult AGP rats treated with 20 IU PMSG and 20 IU hCG were significantly higher (15.5 and 15.4; P<0.01) than in the control rats (11.5 and 12.6). When AGP was given to young and adult rats pretreated with 40 IU PMSG and 40 IU hCG, they produced means of 17.7 and 17.3 live neonates, respectively. The birth weights for young (5.55 to 5.88 g) and adult (5.89 to 5.96 g) AGP rats were smaller than those of respective control rats (6.44 and 6.40 g; P < 0.05−0.01). When the number of neonates was adjusted to 8 per dam for nursing and rearing, all of them were alive at 5 weeks after birth and showed normal growth. However, body weight gains of the progeny of AGP dams were lower than those of controls. These results indicate that rats treated with large doses of gonadotropins and AGP produce larger litter sizes than nontreated controls.

Effects of aminoglutethimide phosphate administration on the incidence of pregnancy and number of live fetuses in superovulated rats

This study examined the effects of aminoglutethimide phosphate (AGP) administration on the incidence of pregnancy and number of live fetuses in superovulated rats. Immature (31- to 33-day-old) young (8- to 9-week-old) and (14- to 16-week-old) rats were treated with 20 or 40 IU of PMSG and the same dosses of hCG at 52 to 54 hour-intervals (GTH rats). One half of the GTH rats were injected with 10 mg AGP twice a day for the first 3 days of pregnancy (AGP rats). Pregnancy rates of all AGP rats (95 to 100%) were much higher than the GTH rats (12.7). Young and adult AGP rats treated with 20 IU of GTH had 14.7 and 15.3 live fetuses per dam, respectively. These numbers of live fetuses increased to 17.8 and 18.4 after treatment with 40 IU of GTH. These values were significantly greater than those of the control rats (12.3 to 12.8; P<0.01), but the fetal weights were lower. The results show that AGP administration improves pregnancy rates and the number of live fetuses in young and adult superovulated rats.

Effects of aminoglutethimide phosphate administration on embryonic development and implantation and on steroid hormone levels in superovulated rats

The present study was undertaken to determine the relationship between preimplantation development of embryos and hormone levels in superovulated immature rats treated with aminoglutethimide phosphate (AGP), an inhibitor of steroid synthesis. In addition, the effects of AGP on incidence of pregnancy and the number of implantation sites in superovulated rats were studied. Immature or adult rats were pretreated with 20 or 40 IU of pregnant mare's serum gonadotropin (PMSG) at 11.00–13.00 h followed by 20 or 40 IU of human chorionic gonadotropin (hCG) to the respective groups 52 to 54 h later. Ten or 20 mg AGP/day was administered once on day 0, 1 or 2; twice during days 0 to 2; or three times on days 0 to 2 of pregnancy. Animals were killed 24 to 120 h after hCG injection (estimated 12 or 108 h after ovulation) or on day 8 of pregnancy. Pregnancy rates in superovulated immature and adult rats given 10 or 20 mg AGP for the first 3 days were 73.3–80.0 or 93.3%, respectively. Injection of 10 or 20 mg AGP to these animals resulted in 10.9–16.2 or 20.2–21.6 implantation sites per mated rat, respectively. Transport of ova in superovulated rats receiving no AGP treatment was faster than in AGP-treated rats by 72 h after hCG injection, and most embryos were lost from the uterus at 120 h in the former group, but not in the latter. Serum progesterone concentrations at 48 and 72 h and estradiol-17β levels at 48 h in AGP-treated rats were significantly lower than those in controls ( P < 0.05-0.01). These findings suggest that the diminished incidence of implantations is caused by hypersecretion of estrogen, and this was counteracted by AGP treatment.

Increased activity of ovarian thecal cytochrome P45017α in the hamster induced by endogenous LH

The in vivo regulatory action of LH on the ovarian androgen synthesizing enzyme cytochrome P450(17 alpha) (17 alpha-hydroxylase/C17,20-lyase) was studied in cyclic golden hamsters. Immunohistochemical studies using anti-porcine testicular cytochrome P450(17 alpha) antibody indicated that the enzyme was limited to thecal cells. Transient removal of the negative feedback action of endogenous steroids by use of aminoglutethimide phosphate produced an increase in endogenous gonadotropins. A single dose (30 mg) of aminoglutethimide on days 1, 2 or 3 of the estrous cycle (day 1 = estrus), increased serum levels of LH, but only for about 12 h. Increases in enzyme activities, which persisted for at least 24 h, followed. Two doses of aminoglutethimide, 12 h-apart, maintained elevated levels of serum LH for about 36 h, and enzyme activities for at least 48 h. However, when the drug was given after the pre-ovulatory surge of LH on proestrus, and again on the morning of estrus, neither serum LH nor enzyme activity levels increased within 24 h. Induced increases in enzyme activity had no effect upon ovulation rate, as determined by the number of oviductal eggs present on the morning of estrus. The results demonstrate clearly a close correlation between serum concentrations of LH and ovarian androgen synthesizing enzyme activity.

Effects of follicle‐stimulating hormone and ovarian steroids during vitro meiotic maturation on fertilization of rat oocytes

The present study examined the effects of gonadotropins and ovarian steroids during in vitro meiotic maturation of rat oocytes on their ability to undergo in vitro fertilization. Fully grown oocytes were isolated from antral follicles of immature rats and cultured as oocyte-cumulus cell complexes (OCC) under conditions in which completion of meiotic maturation occurs spontaneously. They were then exposed to spermatozoa under conditions in which oocytes matured in vivo exhibit high fertilization rates. Compared with oocytes from pregnant mare's serum gonadotropin (PMSG) or follicle-stimulating hormone (FSH)-treated rats, a similar proportion of the oocytes (greater than 80%) from untreated rats underwent germinal vesicle breakdown, but such oocytes had a lower rate of fertilization (70% vs. 20%). The presence of FSH during in vitro maturation restored the fertilization rate for oocytes from untreated rats, while a cytochrome P450 inhibitor, aminoglutethimide phosphate abolished this beneficial effect of FSH. The addition of progesterone during the in vitro maturation period duplicated the beneficial effect of FSH on fertilization rate of oocytes from untreated rats; oestradiol-17 beta was less effective in this regard, and 5 alpha-dihydrotestosterone was ineffective. These findings indicate that FSH and progesterone, although having no apparent effect on nuclear maturation of the oocyte, play an important role during oocyte maturation in enabling normal fertilization to occur.

Formation of 4-oestrene-3,17-dione (19-norandrostenedione) by porcine granulosa cells in vitro is inhibited by the aromatase inhibitor 4-hydroxyandrostenedione and the cytochrome P-450 inhibitors aminoglutethimide phosphate and ketoconazole.

The origin and biosynthesis of 4-oestrene-3,17-dione (19-norandrostenedione), a major steroid in porcine ovarian follicular fluid, was investigated by culturing granulosa cells from 4-6 mm follicles of prepubertal gilts with radiolabelled androstenedione and 19-hydroxyandrostenedione. Steroid metabolites were purified by solvent extraction and lipophilic column chromatography, and analysed by C18 reverse-phase high-performance liquid chromatography. 19-Hydroxyandrostenedione, 19-norandrostenedione and oestradiol-17 beta were obtained as major metabolites from androstenedione, while 19-norandrostenedione and oestradiol-17 beta were the major products from 19-hydroxyandrostenedione. Serum alone or serum plus FSH significantly enhanced formation of 19-norandrostenedione and oestradiol-17 beta from each substrate, compared with controls. Micromolar concentrations (1 mumol/l) of 4-hydroxyandrostenedione, an aromatase inhibitor, significantly reduced formation of 19-norandrostenedione and oestradiol-17 beta by granulosa cells cultured with serum and FSH. Formation of 19-norandrostenedione and oestradiol-17 beta from androstenedione and 19-hydroxyandrostenedione was also significantly inhibited by aminoglutethimide phosphate, a cytochrome P-450 inhibitor known to block the conversion of androstenedione to oestrogens. Ketoconazole, an inhibitor of the cytochrome P-450 dependent 17,20-lysase, blocked formation of 19-norandrostenedione and oestradiol-17 beta only at millimolar concentrations. These results suggest that 19-norsteroid and oestrogen formation from C19 aromatizable androgens may share a common or overlapping pathway, and imply that 19-norsteroid and oestrogen synthesis is mediated by cytochrome P-450 dependent enzymes.

Formation and metabolism of 5(10)-estrene-3 β,17 β-diol, a novel 19-norandrogen produced by porcine granulosa cells from C 19 aromatizable androgens

The biosynthesis of non-aromatic 19-norsteroids has been studied using primary cultures of porcine granulosa cells. Formation of 5(10)-estrene-3 beta,17 beta-diol, a novel 19-norsteroid, from androstenedione and 19-hydroxyandrostenedione by porcine granulosa cells is reported for the first time. The structure was deduced from (i) comparison of its elution times on C18 reverse phase HPLC with authentic 5(10)-estrene-3 beta,17 beta-diol (ii) identification with 5(10)-estrene-3 beta,17 beta-diol-diacetate after acetylation (iii) oxidation/acid catalysed isomerization to 19-norandrostenedione. Serum or serum plus FSH significantly stimulated (seven fold increase) formation of 5(10)-estrene-3 beta,17 beta-diol from androstenedione and 19-hydroxyandrostenedione. Formation of 5(10)-estrene-3 beta,17 beta-diol from both substrates was significantly (p less than 0.01) reduced by the aromatase inhibitors 4-hydroxyandrostenedione (15 microM) and aminoglutethimide phosphate (10(-4)M). These results suggest that 5(10)-estrene-3 beta,17 beta-diol (and 19-norandrostenedione) may be formed by enzymes similar to the aromatase complex required for estradiol-17 beta biosynthesis. 5(10)-Estrene-3 beta,17 beta-diol is converted by granulosa cells to four metabolites. 19-Norandrostenedione was identified by crystallization to constant specific activity; 19-nortestosterone is a minor product. Production of 19-norandrostenedione and 19-nortestosterone indicates that granulosa cells possess the enzymes necessary for the transformation of 5(10)-estrene-3 beta,17 beta-diol and other 3-hydroxy-5(10)-estrenes to 19-nor-4-ene-3-ketosteroids. The formation of 5(10)-estrene-3 beta,17 beta-diol and 19-norandrostenedione as substantial metabolites of androstenedione suggest a physiological role for these 19-norsteroids in ovarian follicular development.

Effect of calcium ionophore A23187 on pregnenolone metabolism to progesterone in rat granulosa cells.

The effect of calcium ionophore A23187 on the metabolism of pregnenolone to progesterone was examined in rat granulosa cells during a 24-h culture period. Granulosa cells harvested from pregnant mare's serum gonadotropin treated immature rats were incubated in the presence and absence of the divalent cation ionophore A23187. The ionophore induced progesterone synthesis from both endogenous sterol substrate and exogenous pregnenolone in a time- and concentration-dependent manner. Pregnenolone metabolism was examined in the presence of aminoglutethimide phosphate, an inhibitor of endogenous pregnenolone production. Steroid secretion resulting from metabolism of endogenous substrate was more sensitive to A23187 in that a lower concentration of the ionophore was required to induce a significant increase than that noted for exogenous pregnenolone metabolism. In addition, progesterone production from endogenous sterol occurred 6 h earlier than the observed increase in the conversion of pregnenolone to progesterone. These results indicate that A23187 and therefore possibly enhanced calcium influx may play a significant role in the regulation of pregnenolone metabolism in granulosa cells depending on the duration of incubation. The earlier steroidogenic response from endogenous substrate may be a reflection of an acute effect of A23187 on certain steroidogenic steps proximal to pregnenolone production.

The effect of ovarian steroidogenesis on ovulation and fertilizability in the in vitro perfused rabbit ovary.

The effects of aminoglutethimide phosphate (AGP) on ovulation, ovum maturation, fertilizability, and steroid production were studied with the use of an isolated perfused rabbit ovary preparation. AGP (10(-3) or 10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused in medium alone. Thirty minutes later human chorionic gonadotropin (hCG) (50 IU) was added to the perfusate of all ovaries. No difference was observed in time of ovulation or ovulatory efficiency between controls and AGP-treated ovaries. The degree of ovum maturity and degeneration was also comparable in the two groups. Progesterone and estradiol production were significantly reduced by AGP treatment. A second experiment examined fertilizability of ova ovulated in vitro after perfusion with 10(-3) M AGP. AGP significantly reduced the rate of normal fertilization as observed 12 h after insemination. The percentage of inseminated ova with evidence of degeneration was greater in ova from AGP-treated ovaries than in those from controls, however, this difference was not significant. The study indicates that AGP affects neither hCG-induced ovulation nor meiotic resumption; however, fertilizability of ova from ovaries treated with AGP is impaired. These data suggest that the intrafollicular steroid environment may participate in cytoplasmic maturation of ovulated ova.

Evidence for a positive feedback interaction between progesterone and luteinizing hormone in the induction of ovulation in the hen, Gallus domesticus

Two experiments were conducted to test the hypothesis that progesterone (P 4) acts to induce a preovulatory rise of luteinizing hormone (LH) by initiating a true positive feedback interaction between P 4 and LH. In each experiment, ten hens were stereotaxically implanted with a third ventricle cannula. Aminoglutethimide phosphate (AGP), an inhibitor of steroidogenesis, was administered (200 mg iv) to hens at 10 and 6 hr (Experiment 1) and 10 and 7 hr (Experiment 2) before an expected C 1 ovulation. A 20-μg injection of P 4 ( n = 5) or the vehicle ( n = 5) was made intraventricularly at 6 hr (Experiment 1) or 7 hr (Experiment 2) before the expected ovulation. Blood samples were taken via branchial vein cannula at regular intervals after the injection. In Experiment 1, four of five P 4-treated hens ovulated, and no atretic follicles were found in any of the five P 4-treated hens, including the one which did not ovulate. Ovulations were always accompanied by preovulatory LH peaks. By comparison, none of the vehicle-injected animals ovulated and in four of five hens the largest follicle was determined to be atretic. No ovulations occurred in Experiment 2 for either the P 4- or vehicle-injected hens. Atresia of the largest follicle occurred in all five hens in the P 4-treated group and four of five hens of the vehicle-treated group. The difference in results between Experiment 1 and Experiment 2 can be explained assuming that the steroidogenesis-inhibiting action of the initial AGP injection in Experiment 1 (at 10 hr before ovulation) had diminished prior to the second AGP treatment (at 6 h before ovulation). In both Experiments 1 and 2, intraventricular P 4 injection caused a significant ( P < 0.001 for Experiment 1; P < 0.03 for Experiment 2) rise in plasma P 4 within 10 min after injection due to leakage of P 4 from the third ventricle into the systemic circulation. Nevertheless, in the absence of any steroid feedback from the ovary (Experiment 2), the small rise in LH induced by the P 4 injection was not potentiated, and no preovulatory surge of LH occurred. These results support the hypothesis that a positive feedback interaction between P 4 and LH, in contrast to a stimulatory effect of P 4, is necessary for the preovulatory LH surge and ovulation.

Effect of aminoglutethimide on murine fetal hepatic erythroid colony formation.

Pretreatment of pregnant mice with aminoglutethimide phosphate, an inhibitor of glucocorticoid synthesis, increases the content of fetal liver erythroid colony-forming cells (CFU-E), as assessed by the formation of erythroid colonies in vitro by fetal liver cells in plasma clots containing exogenous erythropoietin. In addition, the inability of aminoglutethimide to influence erythroid colony formation in vitro suggests that endogenous glucocorticoids exert a suppressive effect on the number of functional CFU-E in the fetal liver.

Follicular plasminogen activator: involvement in ovulation.

Production of plasminogen activator (PA) by granulosa cells (GC) and its stimulation by gonadotropins led to the suggestion that PA is involved in ovulation. However, whereas only LH may be regarded as the ovulation-inducing hormone in the rat, FSH was found to be much more potent than LH in enhancing PA production by GC. Assuming that the entire follicular wall, rather than isolated GC, is involved in follicular rupture, we have examined activity of PA in intact follicles. LH (NIH-LH-S23) was 5-fold more potent than FSH (NIH-FSH-S14), and purified ovine LH and FSH were equally potent in enhancing follicular PA activity. Furthermore, injection into the ovarian bursa of proestrous rats of epsilon-amino-caproic acid and benzamidine (0.05-0.25 mmol), inhibitors of serine proteases, including PA and plasmin, resulted in a dose-dependent inhibition of ovulation without causing changes discernible by histological examinations of the ovaries. Whereas steroids did not change basal follicular PA production in culture, addition of estradiol-17 beta [(E2) 1 microgram/ml] but not progesterone or testosterone, further enhanced LH-stimulated PA. Aminoglutethimide phosphate (10(-3) M) and 17 beta-formamidoandrost-4-en-3-one inhibited LH-induced increase in follicular PA and this inhibition was reversed by addition of E2. Intrabursal injection of indomethacin, an inhibitor of cyclooxygenase, and of nordihydroguaiaretic acid, an inhibitor of lipoxygenase pathway of arachidonic acid metabolism at doses which effectively blocked ovulation (0.3 mg/bursa) had no effect on PA content of the follicles. Likewise, indomethacin (10 microM) and nordihydroguaiaretic acid (100 microM) did not affect LH-stimulated PA in vitro. In conclusion, LH, the physiological trigger of ovulation is, at least, as potent as FSH in stimulating follicular PA activity. The role of serine proteases, most probably of PA and plasmin, in ovulation is further corroborated by a pharmacological approach. LH stimulation of follicular PA appears to be enhanced by E2 but is not mediated by arachidonic acid metabolites.

Follicle-stimulating hormone induction of luteinizing hormone receptor in cultured rat granulosa cells: an examination of the need for steroids in the induction process.

The induction of LH receptors by FSH in cultured rat granulosa cells and the effects of ovarian steroids on this process were examined. Granulosa cells were isolated from the ovaries of untreated immature rats (25 days old) and cultured with highly purified FSH (Sairam; 250 ng/ml). After culture (48-96 h in chemically defined media), both the binding of [125I]hCG and the responsiveness (cAMP and progesterone production) to an acute LH stimulus (100 ng/ml; NIH B8) were measured. The appearance of LH/hCG-binding sites and LH responsiveness indicates the presence of functional LH receptors. The induction of LH receptors by FSH requires a lag period of 24-48 h. After 48 h, the concentration of LH receptors in cultured granulosa cells continues to increase with time in culture with FSH; the continuous presence of FSH is required to maintain the induction process. If granulosa cells are cultured without hormone for 24-48 h before FSH is added, no induction of LH receptors occurs. However, if 17 beta-estradiol (5 X 10(-7) M) is added during this initial period, then the cells are responsive to the later addition of FSH. This maintenance of FSH responsiveness is not observed when dihydrotestosterone or progesterone is substituted for 17 beta-estradiol in the initial culture period. The FSH-dependent induction of LH receptors in cultured rat granulosa cells can be blocked if an inhibitor of steroidogenesis, such as aminoglutethimide phosphate (AGP; 1 mM), is added along with FSH at the initiation of the culture. The inclusion of progesterone, dihydrotestosterone, or 17 beta-estradiol (but not 20 alpha-dihydroprogesterone) in the culture together with FSH and AGP will overcome the inhibition by AGP and restore the induction of LH receptors. The results suggest that steroids produced by the developing follicle can modulate the FSH-dependent induction of LH receptors, and this may play a role in the development of follicular responsiveness to LH.

Effects of steroid blockers on LH-induced ovulation in the domestic fowl, Gallus domesticus.

The spontaneous ovulation of hens was suppressed by daily injection of PMSG. The LH injected to overcome the block was accompanied by one of 4 compounds known to inhibit steroidogenesis at different sites in the biosynthetic pathway. A dose of 300 mg aminoglutethimide phosphate, which inhibits the conversion of cholesterol to 20 alpha-hydroxycholesterol, blocked the LH-induced ovulation and prevented the normal rise in plasma progesterone. Metyrapone, an inhibitor of 11 beta-hydroxylase, and SC 12937 and AY 9944, inhibitors of cholesterol synthesis, did not prevent ovulation or the progesterone rise induced by exogenous LH. Administration of progesterone overcame the inhibitory effect of aminoglutethimide, and it is suggested that progesterone is involved in the ovulatory process of the fowl.

Blastocyst implantation in ovariectomized, adrenalectomized hamsters treated with inhibitors of steroidogenesis during the pre-implantation period

The possible involvement of blastocyst estrogen production in the initiation of implantation, as indicated by the presence of areas of increased endometrial vascular permeability on Day 5 of pregnancy, was examined in hamsters. The animals were ovariectomized and adrenalectomized on Day 3 to remove maternal sources of estrogen, and pregnancy was maintained with medroxyprogesterone acetate. Aminoglutethimide phosphate (AGP) and cyano-ketone, inhibitors of steroidogenesis, administered from Days 3 to 5 of pregnancy, did not affect the proportion of hamsters in which implantation was initiated. However, the AGP treatment was associated with a lower proportion of embryos, recovered on Day 4, which were blastocysts, fewer implantation sites on Day 5, and smaller implantation swellings on Day 9. AGP treatment had no significant effect on the uterine concentrations of prostaglandins (PGs) of the E series, which were higher in the implantation sites than elsewhere in the uterus on Day 5. These results suggest that neither maternal nor blastocyst estrogen production is essential for the initiation of implantation in the hamster. In addition, the data suggest that the localized elevated PGE concentrations at implantation sites are induced by a blastocyst signal which is independent of blastocyst steroidogenesis.

Prevention of cystic ovary induction in rabbits by aminoglutethimide.

Polycystic ovaries were induced in thyroidectomized rabbits by the administration of either 25 IU human chorionic gonadotrophin or 10 IU pregnant mare serum daily for a period of 20 days. Cystic ovaries in rabbits exhibited subnormal (P < 0.01) ovarian delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) concentration and specific activity. Since weight of cystic ovaries was markedly elevated, however, total capacity for delta 4-3-ketosteroid production was enhanced. Concomitant administration of chorionic gonadotrophin and aminoglutethimide phosphate to thyroidectomized rabbits prevented the formation of cystic ovaries indicating that a steroid, or steroids formed beyond cholesterol in the biosynthesis pathway is necessary for the experimental induction of cystic ovaries in this laboratory animal.

Effect of adrenocorticotrophin on intracellular cholesterol transport.

The rate of side-chain cleavage of cholesterol by mitochondria derived from mouse adrenal tumour cells was measured. Incubation of the cells in the presence of adrenocorticotrophin (ACTH) for periods of up to 1 h was without effect on the subsequent side-chain cleavage by mitochondria. However, if the cells were incubated in the presence of aminoglutethimide phosphate (0·76 mmol/l), addition of ACTH (final concentration 86 u./l) to the medium containing the cells increased the subsequent rate of side-chain cleavage by the isolated mitochondria. This response reached a maximum after incubation of cells with ACTH for 2 h and decayed when the isolated mitochondria were left at 0 °C, although a significant difference was still apparent after 120 min. Similar stimulation of mitochondrial side-chain cleavage by ACTH was observed when the reaction was inhibited by anaerobiosis instead of aminoglutethimide phosphate. Addition of dibutyryl cyclic AMP, at final concentrations greater than 10−5 mol/l, to cells during incubation with aminoglutethimide phosphate (0·76 mmol/l) also stimulated the conversion of cholesterol to pregnenolone by the mitochondria. Provided the adrenal tumour cells were incubated with aminoglutethimide, or anaerobically, the mean cholesterol content of the inner mitochondrial membrane was significantly higher (P &lt; 0·01) when ACTH was included in the incubation medium than when it was not. It is concluded that ACTH increases the movement of cholesterol to the mitochondrial membrane which contains the side-chain cleavage enzyme system and that part of this cholesterol is used for the enhanced conversion of cholesterol to pregnenolone brought about by ACTH.

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.