Effect of maternal intrahepatic cholestasis on fetal steroid metabolism.

Estriol, estriol sulfate, progesterone, and 17 neutral steroid sulfates, including estriol precursors and progesterone metabolites, were determined in 27 cord plasma samples collected after pregnancies complicated by intrahepatic cholestasis of the mother. The levels of these steroids were compared with those in the cord plasma of 42 healthy controls. In the cord plasma, the steroid profile after pregnancies complicated by maternal intrahepatic cholestasis differed greatly from that seen after uncomplicated pregnancy. Two main differences were found. In the disulfate fraction, the concentrations of two pregnanediol isomers, 5alpha-pregnane-3alpha,20alpha-diol and 5beta-pregnane-3alpha,20alpha-diol, were high after cholestasis. Other investigators have shown that, as a result of cholestasis, these pregnanediol sulfates circulate in greatly elevated amounts in the maternal plasma. Our results indicate that in cholestasis these steroids cross the placenta into the fetal compartment, where they circulate in elevated amounts as disulfates. Secondly, the concentrations of several steroid sulfates known to be synthesized by the fetus were significantly lower in the cholestasis group than in the healthy controls. This was especially true of 16alpha-hydroxydehydroepiandrosterone sulfate and 16alpha-hydroxypregnenolone sulfate. These results suggest that, in pregnancies complicated by maternal intrahepatic cholestasis, impairment of fetal steroid synthesis, and especially of 16alpha-hydroxylation, occurs in the fetal compartment.Thus, the changes in maternal steroid metabolism caused by cholestasis are reflected in the steroid profile of the fetoplacental circulation. Furthermore, maternal intrahepatic cholestasis may result in the production of some substance which crosses the placenta and affects fetal steroid metabolism.

Presence of a low molecular weight inhibitor of succinate-supported cholesterol side chain cleavage in rat ovaries.

1. Low molecular weight fractions (mol. wt. 3500-10 000) prepared from cytosols of luteinized rat ovaries inhibited succinate-supported cholesterol side chain cleavage by intact ovarian mitochondria utilizing endogenous or exogenous sterol as substrate. 2. The low molecular weight fractions inhibited steroid secretion by collagenase-dispersed ovarian cells stimulated with lutropin or dibutyryl cyclic AMP. 3. Steroidogenesis by intact mitochondria incubated with NADPH was enhanced by the low molecular weight ovarian fraction, but cholesterol side chain cleavage carried out by sonicated mitochondria incubated with NADPH was unaffected. 4. Succinate-supported mitochondrial respiration was stimulated by the low molecular weight factor, apparently by uncoupling of oxidative phosphorylation. The uncoupling seems to be the mechanism by which steroid synthesis is inhibited. 5. The low molecular weight factor was heat-labile and not extracted by activated charcoal. Similar heat-labile material capable of inhibiting succinate-supported mitochondrial steroid synthesis was not found in low molecular weight fractions prepared from rat kidney, liver, spleen, brain, plasma and bovine corpus luteum. 6. Treatment of rats with cycloheximide 1 h before killing resulted in a reduction of inhibitory activity in ovarian low molecular weight cytosolic fractions. 7. We conclude that ovarian cytosols contain a low molecular weight factor, presumably a protein, which inhibits mitochondrial cholesterol side chain cleavage by uncoupling oxidative phosphorylation. The physiological function of this factor remains to be determined.

The control of steroidogenesis by human fetal adrenal cells in tissue culture. I. Responses to adrenocorticotropin.

A technique of monolayer tissue culture of human fetal adrenal cells was developed in order to study steroidogenic responses to factors such as ACTH. The daily production of 12 steroids [pregnenolone, 17-hydroxy pregnenolone, dehydroepiandrosterone (DHA), DHA sulfate, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, corticosterone, 11-desoxycortisol, cortisol, and aldosterone) was measured by RIA. Initially, fresh fetal adrenal cells produced DHA, DHA sulfate, 17-hydroxypregnenolone, and small amounts of cortisol, but in the absence of ACTH, the production of all steroids declined during culture to low levels. The addition of physiological amounts (1-10(4) pg/ml) of either alpha ACTH-1(1-24) or alpha ACTH-(1-39) or coculture with fetal pituitary cells elicited a progressive rise in steroid production during the first 4-6 days of incubation. The lowest ACTH doses elicited a proportionately greater adrenal androgen response (as reflected in the DHA to cortisol ratio), but with increasing ACTH dosage, there was greater stimulation of cortisol production, which equalled or exceeded that of DHA. The data demonstrate that fetal adrenal cells may be maintained in short term culture and can respond to physiological amounts of ACTH. The progressive increase in the production of cortisol and other delta 4, 3-ketosteroids in vitro suggests that the characteristic fetal pattern of steroidogenesis may result from the interaction of ACTH with some circulating inhibitor of adrenal 3 beta-hydroxysteroid dehydrogenase.

Steroidogenesis in dispersed cells of human fetal adrenal.

Steroidogenesis in dispersed fetal zone cells of midtrimester human fetal adrenal was stimulated acutely by ACTH. Polypeptide hormones such as hCG, alpha MSH, ovine PRL, and LH did not produce a similar stimulation of steroidogenesis. The principal steroid products of ACTH-stimulated fetal adrenal cells were dehydroisoandrosterone sulfate, pregnenolone, pregnenolone sulfate, and 17 alpha-hydroxypregnenolone. Only minimal production of the delta 4-3-ketosteroids, cortisol, corticosterone, and progesterone, was observed. Cyanoketone (2 alpha-cyano-4,4,17 alpha-trimethyl-17 beta-hydroxyandrost-5-en-3-one; an inhibitor of 3 beta-hydroxysteroid dehydrogenase activity) treatment of the cells caused only a minor increase in 3 beta-hydroxysteroid formation, confirming that 3 beta-hydroxysteroid formation is the principal steroidogenic fate of cholesterol in these cells. SU-10603 [7-chloro-3,4-dihydro-2-(3-pyridyl)naphthalen-1-(2H)one; a steroid 17 alpha-hydroxylase inhibitor] treatment of the cells caused a marked accumulation of pregnenolone sulfate, indicating that the C-19 steroids are produced from C-21 steroids in this tissue and possibly that dehydroisoandrosterone sulfate is synthesized directly from pregnenolone sulfate. ACTH-stimulated pregnenolone synthesis was inhibited by AY-9944 [trans-1,4-bis-(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride; an inhibitor of cholesterol biosynthesis]. Thus, cholesterol synthesized de novo was the likely steroidogenic precursor in the acute hormonally stimulated fetal adrenal cells.

Mechanism of action of glucocorticoids in induction of ovine parturition: effect on placental steroid metabolism.

Maternal plasma progesterone levels in sheep may fall dramatically druing the last few days of gestation and following the administration of glucocorticoids to the foetus. To investigate the mechanism of the fall, metabolism of [3H] progesterone in vitro by ovin placental tissue was studied in five ewes before and after intra-foetal administration of dexamethasone in a dosage sufficient to induce parturition, and in one ewe after the spontaneous onset of labour at 143 days of gestation. Manual separation of maternal and foetal placental tissues showed that, in 11 out of 12 cases, the foetal and not the maternal placenta produced progesterone from pregnenolone in vitro. Total activities of cholesterol side-chain cleavage enzyme and 3beta-hydroxysteroid dehydrogenase in the foetal placenta were not influenced by intra-foetal dexamethasone. Befre administration of dexamethasone, homogenates of foetal placenta converted [3H] progesterone to 20alpha-hydroxy [3H]pregn-4-en-3-one in the presence of NADPH. Within 12 h of administration of dexamethasone, and after the natural onset of labour at 143 days, large amounts of 17alpha, 20alpha-dihydroxyI1pregn-4-en-3-one were formed form [3H]progesterone. Intra-foetal dexamethasone treatment also induced the formation of 17alpha, 20alpha-dihydroxy[3H]pregn-4-en-3-one by miced foetal placental tissue incubated with [3H]pregnenolone. This change in steroid metabolism did not occur in foetal placental tissue from a sham-operated animal receiving no dexamethasone. Assay of progesterone in foetal placentae showed that the increased fromation of 17alpha,20alpha-dihydroxypregn-4-en-3-one was unlikely to be caused by a change in the specific activity of added 3H-labelled precursor, although the production of 17alpha, 20alpha-dihydroxypregn-4-en-3-one in vitro increased at a time when both foetal placental and utero-ovarian venous levels of progesterone were decreasing in response to dexamethasone treatment. These observations indicate that intra-foetal dexamethasone treatment induces a placental 17alpha-hydroxylase enzyme, which is also present in foetal placental tissue after the spontaneous onset of labour at term.

Metabolism of pregnenolone by human breast cancer. Evidence for 17 alpha-hydroxylase and 17,20-lyase.

The metabolism of 7-(3)H-pregnenolone was studied in vitro using 16 human breast carcinomas. All mammary tumors transformed pregnenolone to progesterone. All estrogen receptor poor tumors and 4 out of 8 estrogen receptor rich tumors converted pregnenolone to 17-hydroxypregnenolone. Five estrogen receptor poor tumors showed the presence of 17,20-lyase as evidenced by formation of dehydroepiandrosterone and androstenedione. In two estrogen receptor poor tumors, conversions of pregnenolone to progesterone, 17-hydroxy pregnenolone, dehydroepiandrosterone, androstenedione and finally to estradiol was documented, providing a hypothetical pathway for steroid metabolism in human breast cancer. The conversion of pregnenolone to 17-hydroxypregnenolone was significantly less in receptor rich tumors and was totally absent in 4 receptor rich tumors with estrogen receptors of over 45 fmol/mg protein.

[Sequence of conversion of pregnenolone into androgens in virilizing adrenal glands in man]. / Izuchenie posledovatel'nosti prevrashchenii pregnenolona v androgeny v viriliruiushchikh opukholiakh nadpochechnikov cheloveka

Trace amounts of labelled pregnenolone were added to the slices of veriliscent tumor from human adrenal gland in order to study the stepwise conversion of the steroid into androstenedione, 11betaOH-androstenedione and their intermediats--17-alpha-OH pregnenolone, dehydroepiandrosterone, progesterone, and 17-alpha-OH-progesterone. The definite sequence was observed in the maximal incorporation of pregnenolone label into 17-alpha-OH-pregnenolone (2-5 min of incubation) leads to dehydroepiandrosterone (5-15 min) leads to androstenedione (10-20 min). Incorporation of the pregnenolone label into 17-alpha-OH-pregnenolone and dehydroepiandrosterone was distinctly higher than into progesterone and 17-alpha-OH-progesterone in all the experiments within all the studied periods of incubation. The data obtained suggest that in viriliscent tumors of adrenal gland conversion of pregnenolone into androgens proceeded as follows: pregnenolone leads to 17-alpha-OH-pregnenolone leads to dehydroepiandrosterone leads to androstenedione. When formation of androstenedione and 11beta-OH-androstenedione from pregnenolone and progesterone was analyzed during the experiment it was shown that 11-beta-OH-androstenedione was mainly formed via androstenedione and the hormone was the end product of the androgen synthesis.