Interactions between oocytes of different maturational stages in the carp Cyprinus carpio: effects on maturational and steroidogenic activity in vitro.

Ovarian fragments from carp which had received injections of saline (control) or carp hypophysial homogenate (primed) were incubated both alone and a cocultures of primed and control tissue in the presence of carp pituitary extract. The results showed that the cocultures were not simply the sums of separate incubations. Oocyte maturation was retarded in primed tissue but advanced in control tissue and concentrations of 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P) in the medium were significantly lower than that expected from separate incubations. The results indicate that local factors may act to synchronize oocyte maturation throughout the ovary to enable spawning of the maximum number of eggs.

In vitro effects of cycloheximide and luteinizing hormone on the estradiol-to-progesterone shift in follicular steroidogenesis.

The objectives of this study were to establish a completely in vitro system that would simulate the in vivo effects of cycloheximide (cyclo) on preovulatory serum levels of estradiol (E2) (prolonged) and progesterone (P4) (reduced). Graafian follicles were removed from proestrous hamsters at 0900 h and incubated for a basal hour (Hour 1) with various doses of cyclo before the medium was replaced; in Hour 2, 100 ng luteinizing hormone (LH) was added with cyclo added every hour for 5 or 6 h. The endpoints were steroid levels/follicle/h per ml medium of P4, 17 alpha-hydroxyprogesterone (170HP), androstenedione (A), and E2. The goal was best accomplished with hourly addition of 400 ng cyclo, which reduced follicular protein synthesis by 76%. Cyclo suppressed P4 and 170HP and prolonged the accumulation of A and E2, in Hour 5 and Hour 6, correlated with sustained thecal C-17,20-lyase/17 alpha-hydroxylase as determined by enzyme assays. Cyclo therefore prevented the early demise of the enzyme complex after LH stimulation and hence prolonged the ability of the theca to provide androgens for conversion to E2 by the granulosa cells. Our earlier work established that one of the major effects of LH is to recruit the granulosa compartment as a source of C-21 steroids, and cyclo interferes with the availability of cholesterol to mitochondrial side-chain cleavage (Greenwald and Limback, 1984). Thus, cyclo affects follicular steroidogenesis through different mechanisms in theca and granulosa.

Maintenance of testosterone production by purified adult rat Leydig cells for 3 days in vitro.

Using a preparation of highly purified, adult rat Leydig cells and conditions of culture which we found to optimize testosterone production during 24 h, we sought to maintain optimal testosterone production for 3 d. Leydig cells cultured on Cytodex 3 beads at 19% O2 in Dulbecco's modified Eagle's medium-Ham's nutrient mixture F12 (1:1; vol/vol) containing 0.5 mg/ml total bovine lipoproteins (less than 1.222 g/ml) with maximal luteinizing hormone (LH) stimulation failed to maintain a constant amount of testosterone for 3 d. These cells did however secrete a similar amount of total delta 4-3-ketosteroids on each of the 3 culture d, indicating that their viability was preserved. The predominance of progesterone and 170H-progesterone relative to the amount of androstenedione found on Days 2 and 3 suggested that the activity of the cytochrome P450 C17-hydroxylase-C17,20-lyase enzyme in the smooth endoplasmic reticulum was diminished when Leydig cells were maintained in our primary culture for longer than 24 h. Decreasing the oxygen tension of the cultures from 19 to 5%, and decreasing the concentration of LH used to stimulate the Leydig cells from 100 to 0.1 ng/ml, were necessary to achieve maintenance of testosterone secretion without accumulation of other delta 4-3-ketosteroids during a 3-d period. Cells cultured in this fashion were still able to respond to maximal LH stimulation during Day 3, producing as much testosterone as if cultured for 24 h on Day 1 at 19% O2 with 100 ng/ml LH stimulation.

Steroidogenesis expression depends on negative control(s): analysis in Leydig X adrenal intraspecific cell hybrids.

Hybrids constructed by fusing mouse Leydig cells with mouse adrenal Y1 cells were able to randomly express all the parental specific traits but for the response to gonadotropin (hCG) and corticotropin (ACTH): three of them, YDYL 14, 17 and 19, metabolized both progesterone and dehydroepiandrosterone into testosterone accounting for 17 alpha-hydroxylase, 17-20-lyase, 17-ketoreductase and 3 beta-hydroxysteroid dehydrogenase activities. Under basal conditions, 17 alpha-hydroxylase and 17-20-lyase activities were high in the three clones as compared to parental Leydig cells, and were no longer stimulated by cAMP in YDYL 17 and 19. The hybrids responded to various hormones such as prostaglandin E2 (PGE2), vasoactive intestinal peptide (VIP) and prolactin (PRL) which are not directly implicated in the expression of steroidogenesis; they generally retained the Y1 morphological response to 8-bromo cAMP. On extended culture, reexpression of ACTH sensitivity occurred in one clone, YDYL 9. This reexpression was correlated with a Robertsonian translocation between mouse chromosomes 2 and 11, while extinction required the presence of an intact mouse chromosome 11.

Experimental control of the differentiation of Leydig cells in the rat fetal testis.

In the developing fetal testis, in vitro as well as in vivo, two kinds of endocrine cells differentiate successively: Sertoli cells, which produce the Müllerian inhibitor (or anti-Müllerian hormone) and aggregate with germ cells into seminiferous cords; and Leydig cells, which release androgens. Serum added to the synthetic culture medium prevents the morphogenesis of the seminiferous cords but not the cytodifferentiation of the endocrine cells. L-Azetidine 2-carboxylic acid (LACA), a proline competitor, introduced into the medium also prevents differentiation of seminiferous cords. In the present experiments, the effects of LACA on the endocrine cells were studied. It did not suppress production of the Müllerian inhibitor, but it opposed differentiation of Leydig cells. Histochemically detectable 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was virtually absent and the release of testosterone, delta 4-androstenedione, 17-hydroxyprogesterone, or progesterone into the medium became undetectable. Moreover, dibutyryl cAMP added to the medium during the final day in vitro had very little effect on the parameters of steroidogenesis. An excess of proline added to the LACA-containing medium permitted normal morphogenesis of seminiferous cords, normal steroidogenesis, and normal response to cAMP. LACA did not prevent the appearance of 3 beta-HSD activity in the adrenals, nor did it reduce the expression of laminin and fibronectin (data not shown) in the mesonephric structures as much as in the testes. The differentiation of the testis and especially of the Leydig cells appears to have special requirements for proline.

Involvement of 3',5'-cyclic adenosine monophosphate in the control of follicular steroidogenesis of amago salmon (Oncorhynchus rhodurus).

The role of cAMP in the control of follicular steroidogenesis of amago salmon (Oncorhynchus rhodurus) was studied using in vitro incubation of isolated thecal and granulosa layers. Particular attention was paid to the role of cAMP in the shift in the steroidogenic responses of follicle layers to gonadotropin (partially purified chum salmon gonadotropin, SGA) during oogenesis. First, the effects of SGA and forskolin, an activator of adenylate cyclase, on intratissue accumulation of cAMP were determined using isolated thecal and granulosa layers from various stages of development. Regardless of the stage of development, SGA and forskolin stimulated cAMP formation in both layers within 1 hr of incubation. Second, the effects of SGA, forskolin, dibutyryl cAMP, and inhibitors of phosphodiesterase on testosterone and 17 alpha-hydroxyprogesterone production by thecal layers and on the activities of aromatase and 20 beta-hydroxysteroid dehydrogenase in granulosa layers from follicles of various developmental stages were investigated. All steroidogenic actions of SGA were mimicked by those agents known to raise the cellular level of cAMP. These results provide evidence that the steroidogenic actions of gonadotropin in both thecal and granulosa layers depend on increased intracellular cAMP, and they further suggest that a change in cellular events at a step(s) subsequent to cAMP production is involved in regulating the shift in the steroidogenic responses of follicle layers to gonadotropin.

Effect of a luteinizing hormone-releasing hormone agonist (Buserelin) on steroidogenesis of cultured human preovulatory granulosa cells.

In order to ascertain whether there is a direct effect of luteinizing hormone-releasing hormone (LH-RH) agonists on ovarian steroidogenesis, human preovulatory granulosa cells were cultured in the presence of an LH-RH agonist (Buserelin, Hoechst Roussel Pharmaceuticals, Paris, France). Cultures were performed without serum but with precursors of estrogens and progestins. In basal conditions, Buserelin, at 1 ng/ml, increased estradiol and progesterone (P) secretion, while at concentrations of 10 and 100 ng/ml, no effect was observed. In the presence of follicle-stimulating hormone (FSH) or LH, Buserelin at 1 and 10 ng/ml did not modify the cell's steroidogenesis. However, at a concentration of 100 ng/ml, Buserelin decreased LH stimulation of P secretion. These results suggest that LH-RH agonists, at concentrations in the range of those obtained in clinical use, can modulate ovarian steroidogenesis by direct action.

Developmental changes in steroidogenic responses of ovarian follicles of amago salmon (Oncorhynchus rhodurus) to chum salmon gonadotropin during oogenesis.

Changes in amago salmon (Oncorhynchus rhodurus) ovarian thecal and granulosa layer function in association with the production of two biologically important ovarian mediators of oocyte growth and maturation in salmonids, estradiol-17 beta and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog), were investigated using isolated follicular preparations in vitro. A distinct shift of steroidogenic responses of intact follicles from estradiol-17 beta to 17 alpha,20 beta-diOHprog in response to partially purified chum salmon gonadotropin (SGA) occurred immediately prior to oocyte maturation. Aromatase activity in granulosa layers increased during vitellogenesis and decreased rapidly prior to oocyte maturation. This decrease in aromatase activity was coincident with the decreased ability of intact follicles to produce estradiol-17 beta in response to SGA. Since testosterone production in thecal layers did not decline during this time, the reduced production of estradiol-17 beta by postvitellogenic follicles is due, in part, to decreased aromatase activity in granulosa layers. Immediately prior to oocyte maturation, intact follicles acquire an increased ability to produce 17 alpha,20 beta-diOHprog in response to SGA. Although granulosa layers first acquired the ability to convert exogenous 17 alpha-hydroxyprogesterone (17 alpha-OHprog) to 17 alpha,20 beta-diOHprog (20 beta-hydroxysteroid dehydrogenase, 20 beta-HSD, activity) in response to SGA about 2 months prior to oocyte maturation, thecal layers did not develop the ability to produce 17 alpha-OHprog in response to SGA until immediately prior to oocyte maturation. Thus, changes in thecal cell function are critical for intact follicles to acquire the ability to produce 17 alpha,20 beta-diOHprog in response to gonadotropin.

Steroidogenic activities of two distinct salmon gonadotropins.

The effects of salmon gonadotropins, GTH I and GTH II, on production of two major steroid hormones in female salmonid reproduction, estradiol-17 beta and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) were compared using amago salmon (Oncorhynchus rhodurus) intact ovarian follicles in vitro. In addition, the production of 17 alpha-hydroxyprogesterone (17 alpha-OHprog) by thecal layers and 17 alpha,20 beta-diOHprog by granulosa layers in response to GTH I and II was examined during oocyte maturation. Both GTHs enhanced estradiol-17 beta production by midvitellogenic ovarian follicles in a dose-dependent manner; there was no significant difference in potency between GTH I and II. In postvitellogenic follicles, GTH II appeared to be more effective in stimulating 17 alpha,20 beta-diOHprog production than GTH I. GTH II was also found to be more potent than GTH I in stimulating 17 alpha-OHprog production by thecal layers and 17 alpha,20 beta-diOHprog production by granulosa layers in the presence of 17 alpha-OHprog. Thus, GTH II appears to differ from GTH I showing a reproductively high specificity for 17 alpha,20 beta-diOHprog production during oocyte maturation.

Influence of follicular development on steroid production in the medaka (Oryzias latipes) ovarian follicle in response to exogenous substrates.

Changes in the capacity of medaka, Oryzias latipes, ovarian follicles to convert exogenous 17 alpha-hydroxyprogesterone (17 alpha-OHprog) or testosterone to testosterone, estradiol-17 beta, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) were examined using 18-hr incubations. Under a constant long photoperiod (14 hr light-10 hr dark) at 26 degrees medaka spawn daily within 1 hr of the onset of light. Under these conditions, vitellogenesis and oocyte maturation of individual follicles occur within 72 hr, allowing accurate determination of the time of oocyte maturation and ovulation. In the absence of substrates, vitellogenic follicles isolated between 28 and 16 hr before spawning produced increased amounts of estradiol-17 beta, while postvitellogenic follicles between 14 and 8 hr produced a large amount of 17 alpha,20 beta-diOHprog. However, under the same conditions, testosterone levels were very low in follicles from all stages of development. The capacity of follicles to produce estradiol-17 beta in response to 17 alpha-OHprog or testosterone increased as follicles developed from the early to late vitellogenic stage, but declined during oocyte maturation. Maximum estradiol-17 beta production was observed in follicles at 20 hr before spawning. In contrast, the conversion of 17 alpha-OHprog to 17 alpha,20 beta-diOHprog was low in vitellogenic follicles and increased in follicles isolated immediately prior to or during oocyte maturation, with maximum 17 alpha,20 beta-diOHprog production occurring in follicles isolated at 14 hr before spawning. These results demonstrate a distinct shift in the activities of steroidogenic enzymes from C17-20 lyase, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), and aromatase to 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) occurring in the medaka ovarian follicles immediately prior to oocyte maturation.

Interleukin-1 stimulates steroidogenesis in cultured rat Leydig cells.

We have studied the effects of human interleukin-1 beta on steroidogenesis in cultured immature rat Leydig cells. In the presence of low concentrations of LH or in its absence interleukin-1 beta markedly stimulates the production of C19-steroids (testosterone and androstenedione) and C21-steroids (progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxypregn-4-en-3-one). In the presence of maximally effective concentrations of LH, on the contrary, interleukin-1 beta inhibits C19-steroid production by provoking a block at the level of the 17,20-desmolase. These actions were observed with similar low doses of interleukin-1 beta (ED50 = 1 U/ml), but the stimulatory effects are evident within the first 2 h of incubation whereas the inhibitory actions appeared after a latent period of 6 h. None of the effects of interleukin-1 beta is accompanied by measurable changes in cAMP output, and the effects are much less pronounced in freshly isolated Leydig cells than in cultured cells. At maximally effective doses the effects of interleukin-1 beta are additive with those of a number of other Leydig cell agonists: LHRH, epidermal growth factor, arginine vasopressin and Sertoli cell-derived factor(s), suggesting that these agonists act by mechanisms different from that of interleukin-1 beta. The possibility is considered that Leydig cells may act as target cells for interleukin-1 beta derived from testicular macrophages or for interleukin-1-like factors derived from testicular tubules.

In vitro biosynthesis of 17 alpha,20 alpha,20 beta-dihydroxy-4-pegnen-3-one by the ovaries, testes, and head kidneys of the Atlantic salmon Salmo salar.

Ovaries, testes, and head kidneys of sexually mature Atlantic salmon, Salmo salar, biosynthesized 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHP) from equimolar amounts of [3H]pregnenolone plus [4-14C]progesterone in vitro. The 3H:14C isotope ratios of steroid metabolites indicated that the biosynthetic pathways to 17 alpha,20 beta-diOHP in the testes differed from those observed in the ovaries and head kidneys. [4-14C]Progesterone appeared to be the principal precursor of 17 alpha,20 beta-diOHP in the testes, whereas both precursors were efficiently biotransformed to 17 alpha,20 beta-diOPH in the ovaries and head kidneys. 17 alpha-Hydroxy-4-pregnen-3-one (17 alpha-OHP) was the immediate precursor to 17 alpha,20 beta-diOHP in all tissues. However, appreciable amounts of 17 alpha,20 beta-diOHP accumulated in vitro in the testes only in the presence of exogenous [14C]progesterone. Incubation of the testes, ovaries, and head kidneys with [14C]pregnenolone resulted in high yields of 17 alpha,20 beta-diOHP in the ovaries and head kidneys but no detectable amounts of the steroid in the testes. The results confirm that progesterone is the favored precursor to 17 alpha,20 beta-diOHP in the testes. The results also suggest that the head kidneys may be an excellent cellular source of 17 alpha,20 beta-diOHP in both male and female. Atlantic salmon and may play an important role in the sexual maturation process in this fish. It is suggested that biosynthetic control mechanism affecting 17 alpha,20 beta-diOHP synthesis and/or spermiation and ovulation may differ in male and female Atlantic salmon.

The cytoskeleton and rat granulosa cell steroidogenesis: possible involvement of microtubules and microfilaments.

The participation of both microtubules and microfilaments in granulosa cell steroidogenesis was assessed by monitoring the effects of colchicine (0-250 microM) and/or cytochalasin B (0-10 micrograms/ml) or dihydrocytochalasin B (0-2.0 micrograms/ml) on cellular morphology and production of progestins during 24 h of culture. Both colchicine and the cytochalasins increased granulosa cell production of progesterone and of 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) in a dose-dependent manner. The largest increase in steroidogenesis (about 2- to 3-fold) was observed at 4-250 microM colchicine and at 2-10 micrograms/ml cytochalasin. Those concentrations of the inhibitors of microtubule or microfilament polymerization that stimulated basal progestin production also markedly influenced cell spreading. Whereas cells cultured for 24 h in medium alone became very flattened with numerous cytoplasmic extensions, those cultured with colchicine (0.2-250 microM) or cytochalasin (0.4-2 micrograms/ml) were much less spread and progressively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions in mean contour index values from 5.7 +/- 0.1 (control) to 3.9 +/- 0.1 (250 microM colchicine), 4.2 +/- 0.1 (2 micrograms/ml cytochalasin B), or 4.1 +/- 0.1 (2 micrograms/ml dihydrocytochalasin B). Cultures containing both colchicine and cytochalasin B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For example, granulosa cell progesterone production was stimulated almost 2-fold by 4 microM colchicine or 2 microM/ml cytochalasin B, but 5.5-fold by 4 microM colchicine plus 2 micrograms/ml cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid synthesis inhibition by ketoconazole: sites of action.

Ketoconazole is an antifungal agent that, in high doses, inhibits testicular and adrenal steroid synthesis. The ability of ketoconazole to block steroid synthesis has prompted us to use it in the treatment of advanced prostatic carcinoma. This study was designed to determine the site of steroid synthetic blockade that was induced by ketoconazole. Twelve patients with metastatic prostate carcinoma on long term high dose ketoconazole therapy were compared with 12 control volunteers. Values of serum progesterone, 17-hydroxyprogesterone, androstenedione, dehydroepiandrosterone sulphate, testosterone, and cortisol were measured in a baseline state and after Cosyntropin and human chorionic gonadotropin stimulation. Baseline data showed that serum levels of testosterone, androstenedione, and dehydroepiandrosterone sulphate were lower and that plasma progesterone, luteinizing hormone, and adrenocorticotropin were higher in the ketoconazole group. With Cosyntropin, plasma cortisol, androstenedione, and dehydroepiandrosterone sulphate increased only in the control group. With human chorionic gonadotropin, testosterone increased only in the control group. Basal 17-hydroxyprogesterone and progesterone rose after Cosyntropin only in the ketoconazole group. Following human chorionic gonadotropin, progesterone rose in the ketoconazole group but not in the control group. These results suggest that ketoconazole is a potent inhibitor of steroid synthesis. The major site of action appears to be in the inhibition of 17-20 desmolase. A moderate blockade of 17-hydroxylase may be present. There is a marked inhibition of 21- and/or 11-hydroxylase. The ability of ketoconazole to inhibit steroid synthesis should have therapeutic potential in the treatment of steroid dependent disease. Frequent high dose ketoconazole therapy can inhibit adrenal steroid synthesis, which can be important for patients undergoing stressful situations.

Reciprocal inhibition of the long-acting luteinizing hormone releasing hormone agonist Buserelin and human chorionic gonadotropin in stimulating Leydig cell steroidogenesis.

The interference between human chorionic gonadotropin (hCG, Pregnyl, Organon 1500 IU i.m.) and the long acting LHRH agonist Buserelin (500 micrograms s.c.) in stimulating Leydig cell steroidogenesis was studied in 6 eugonadal men. Simultaneous administration of a single injection of the agonist (LHRH alpha) and hCG blunted the plasma testosterone response observed 24 h after LHRH alpha alone, enhanced the secretion of oestradiol without affecting 17-hydroxy-progesterone and aggravated the late steroidogenic block at the 17,20-lyase locus. As compared to hCG alone, combined LHRH alpha and hCG administration also decreased the maximum and 48 h testosterone increments and the testosterone production reflected by the area under the curve, enhanced the production of oestradiol and again aggravated the 17,20-lyase lesion. The data show that the long acting agonist LHRH alpha and hCG reciprocally inhibit their stimulatory effect on Leydig cell testosterone secretion probably by a suppressive effect of oestrogens on the conversion of C21- to C19-steroids. The mechanism underlying this reciprocal inhibition remains to be elucidated.

The temporal sequence of changes in oocyte maturation and ovarian steroid hormone production during induced ovulation in the common carp, Cyprinus carpio.

Oocytes taken from carp sacrificed 3-24 hr after injection with either carp hypophysial homogenate (chh) (primed) or saline (control) were incubated with or without chh for 24 hr at 20 degrees. In oocytes from the primed fish the percentage of peripheral germinal vesicles at sacrifice progressively increased with time from injection. No such change occurred in control fish. After incubation with chh, tissue from primed fish showed a progressive increase in oocyte maturity with increasing time between priming and sacrifice, with germinal vesicle breakdown (GVBD) in 50% of the oocytes in incubations begun 24 hr after priming. In contrast, incubated tissue from control fish showed no variation with time between saline injection and sacrifice, and oocyte maturity did not exceed 50% with peripheral germinal vesicles. GVBD was not significant in these fish. 17-Hydroxyprogesterone, testosterone, and testosterone glucuronide production in vitro showed no major changes with the interval between injection and sacrifice, nor between control and primed fish. In vitro production of 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P), however, increased significantly between tissue taken from animals at 3 and at 6 hr after injection of chh, and thereafter remained unchanged. 17,20 beta P production preceded the increase in GVBD in primed fish but neither this hormone nor GVBD was found in incubations of control fish, supporting the suggestion that 17,20 beta P is the maturation-inducing hormone in carp. Steroid production was not detectable in the absence of chh in the incubation medium and oocyte maturation showed little advance on that at sacrifice.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect.

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.

Progesterone derivatives that bind to the digitalis receptor: synthesis of 14 beta-hydroxyprogesterone. A novel steroid with positive inotropic activity.

The synthesis of 14-hydroxy-14 beta-pregn-4-ene-3,20-dione (14 beta-hydroxyprogesterone) is described. This novel steroid is about 10 times more potent than progesterone and one-tenth as potent as ouabagenin in an [3H]ouabain radioligand binding assay and is the first in a series of progesterone congeners that interact at the cardiac glycoside receptor both to possess the C/D cis ring junction and to enhance contractility of isolated cardiac tissue.

[Studies on the significance of 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17 alpha,20 alpha-P) in pregnancy induced hypertension (PIH) and its site of production].

We have measured 17 alpha,20 alpha-P, 17 alpha-OHP and aldosterone in maternal peripheral blood, umbilical arterial and venous blood in normal pregnancy and PIH. The PIH cases studies had a systolic blood pressure above 140 mmHg and a diastolic blood pressure above 90 mmHg for more than two weeks. The 800 G supernatant of the ovary and placenta from human was used as an enzyme source for in vitro studies. Incubation was carried out in a Dubnoff-type incubator at 37 degrees C for 2, 4, 8 and 12 hours. As substrates, [4-14C]-P5,[4-14C]-P and [4-14C]-17 alpha-OHP were used. Concentrations of aldosterone and 17 alpha-OHP of PIH cases were not significantly different from those of control. Concentrations of 17 alpha,20 alpha-P in normal pregnancy and PIH were as follows. Peripheral vein blood, 0.39 +/- 0.08 and 0.64 +/- 0.19 ng/ml, umbilical arterial blood, 0.33 +/- 0.09 and 0.65 +/- 0.19 ng/ml, umbilical vein blood 0.50 +/- 0.12 and 0.84 +/- 0.17 ng/ml, respectively. The 17 alpha,20 alpha-P levels in the maternal peripheral venous, umbilical arterial and venous blood of hypertensive cases were all higher than those of the normal pregnancy. The synthesis of 17 alpha,20 alpha-P in vitro occurred from all the substrates used in the ovary and only from [4-14C]17 alpha-OHP in the placenta. These results suggest that 17 alpha,20 alpha-P is somehow related to the increase in blood pressure and the amount of this steroid is significantly greater in PIH cases than controls.(ABSTRACT TRUNCATED AT 250 WORDS)