Alpha-hydroxysteroid Dehydrogenase Activity Research Articles

Regulation of progestin biosynthetic enzymes in cultured rat granulosa cells: effects of prolactin, beta 2-adrenergic agonist, human chorionic gonadotropin and gonadotropin releasing hormone.

Prolactin (Prl), beta 2-adrenergic agents and human chorionic gonadotropin (hCG) are luteotropic in rats, whereas gonadotropin releasing hormone (GnRH) exerts direct inhibitory effects on ovarian steroidogenesis. The present study examined the modulation of the progestin biosynthetic pathway by the luteotropic agents, as well as the actions of GnRH. Rat granulosa cells were primed with follicle-stimulating hormone (FSH) to increase their responsiveness to the luteotropic agents. Subsequent treatment for 2 days with Prl, terbutaline (a beta 2-adrenergic agonist) or hCG stimulated the production of progesterone, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P), pregnenolone and the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). In contrast, treatment with Prl or terbutaline, but not hCG, inhibited 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity by decreasing the apparent maximal velocity of the enzyme with no change in its Km value. Concomitant treatment with GnRH inhibited progesterone, but increased 20 alpha-OH-P production stimulated by Prl or terbutaline. These effects were associated with a stimulation of 20 alpha-HSD activity, while neither 3 beta-HSD activity nor pregnenolone biosynthesis was decreased. In contrast, GnRH inhibited progesterone production in hCG-treated cells without affecting 20 alpha-OH-P production. This was associated with an inhibitory effect of GnRH on pregnenolone biosynthesis with no effect upon 3 beta-HSD activity. Thus, Prl and the beta 2-agonist stimulate progesterone production in granulosa cells by increasing pregnenolone production and 3 beta-HSD activity as well as by decreasing 20 alpha-HSD activity, while hCG stimulates progesterone production by increasing pregnenolone production and 3 beta-HSD activity. The inhibitory effect of GnRH on Prl- or terbutaline-stimulated progesterone production appears to result from a preferential increase in 20 alpha-HSD activity, while the GnRH inhibition of hCG-stimulated progesterone production appears to result from a preferential inhibition of pregnenolone production.

Inactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase by 16-methylene estrone, an affinity alkylator enzymatically generated from 16-methylene estradiol-17 beta.

The substrate 16-methylene estra-1,3,5(10)-triene-3,17 beta-diol (16-methylene estradiol-17 beta) and its enzyme-generated alkylating product, 3-hydroxy-16-methylene estra-1,3,5(10)-triene-17-one (16-methylene estrone), were synthesized to study the 17 beta- and 20 alpha-hydroxysteroid dehydrogenase activities which coexist in homogeneous enzyme purified from human placental cytosol. 16-Methylene estradiol, an excellent substrate (Km = 8.0 microM; Vmax = 2.8 mumol/mg/min) when enzymatically oxidized to 16-methylene estrone in the presence of NAD+ (256 microM), inactivates simultaneously the 17 beta- and 20 alpha-activities in a time-dependent and irreversible manner following pseudo-first order kinetics (t1/2 = 1.0 h, 100 microM, pH 9.2). 16-Methylene estradiol does not inactivate the enzyme in the absence of NAD+. 16-Methylene estrone (Km = 2.7 microM; Vmax = 2.9 mumol/mg/min) is an affinity alkylator (biomolecular rate constant k'3 = 63.3 liters/mol-s, pH 9.2; KI = 261 microM; k3 = 8.0 X 10(-4) S-1, pH 7.0) which also simultaneously inhibits both activities in an irreversible time-dependent manner (at 25 microM; t1/2 = 7.2 min, pH 9.2; t1/2 = 2.7 h, pH 7.0). Substrates (estradiol-17 beta, estrone, and progesterone) protect against inhibition of enzyme activity by 16-methylene estrone and 16-methylene estradiol. Affinity radioalkylation studies using 16-methylene [6,7-3H]estrone demonstrate that 1 mol of alkylator binds per mol of inactivated enzyme dimer. Thus, 16-methylene estradiol functions as a unique substrate for the enzymatic generation of a powerful affinity alkylator of 17 beta,20 alpha-hydroxysteroid dehydrogenase and should be a useful pharmacological tool.

Subcellular distribution of steroid delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the rat epididymis during sexual maturation.

The curve of the specific activity of rat epididymal nuclear delta 4-5 alpha-reductase is bell shaped as a function of age, whereas that of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase does not change significantly with age. The present study examines the subcellular distribution of delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the caput-corpus and cauda epididymidis during development. A 5-step discontinuous sucrose gradient was developed for fractionation of epididymal homogenates. By using enzyme markers specific for different subcellular organelles, the five different subcellular fractions obtained were shown to be of cytoplasmic, microsomal, mitochondrial, nuclear and spermatozoal origin. 3 alpha-Hydroxysteroid dehydrogenase activity was associated only with the cytoplasmic fraction. The activity of the enzyme did not change significantly with age in either the caput-corpus or cauda epididymidis. delta 4-5 alpha-Reductase activity was found in fractions containing microsomal and nuclear markers. delta 4-5 alpha-Reductase activity in the nuclear fraction of the caput-corpus epididymidis was evident in the youngest age group (Day 25), increased 4-fold and peaked in the next age group (Day 35), and declined with each successive age group: Day 45 (60% of maximum), Day 60 (20% of maximum), Day 75 (15% of maximum) and Day 105 (10% of maximum). In contrast, microsomal delta 4-5 alpha-reductase activity increased successively from Day 25 to Day 105; enzyme activity doubled between these two ages. The ratio of nuclear to microsomal delta 4-5 alpha-reductase activity from the caput-corpus epididymidis thus changed markedly with age: Day 25:1.32; Day 35:3.76; Day 45:2.44; Day 60:1.03; Day 75:0.41; and Day 105:0.21. In the cauda epididymidis nuclear delta 4-5 alpha-reductase activity was only evident at Day 35 and Day 45; in microsomal fractions, activity was first found at Day 35 and did not subsequently change with age. These results demonstrate that: 1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity is found only in the cytoplasmic fraction; 2) delta 4-5 alpha-reductase activity is found in nuclear and microsomal fractions; and 3) the subcellular distribution of delta 4-5 alpha-reductase activity changes markedly with age and epididymal section, suggesting differential regulation of nuclear and microsomal delta 4-5 alpha-reductase activities.

Studies on the role of zinc in parturition in the rat.

1. Pregnant rats were fed either low (less than 1 p.p.m.) Zn or control (40 p.p.m. Zn) diets from day 10 of gestation. They were killed at intervals during the last 96 h preceding the normal time for onset of parturition, and differences in plasma progesterone, oestradiol-17 beta and ovarian 20 alpha-hydroxysteroid dehydrogenase were assessed. 2. Gestation was prolonged in Zn-deficient rats. 3. Although the preparturient decline in plasma progesterone began at the same time in all groups, at term, plasma progesterone concentration in Zn-deficient rats remained significantly higher than in normal females. 4. Induction of ovarian 20 alpha-hydroxysteroid dehydrogenase activity was delayed by about 8 h by Zn deficiency. This delay was not observed if prostaglandin F2 alpha was injected previously. 5. The results suggest a Zn-dependent step(s) in uterine synthesis and/or release of prostanoids.

Enhancement of the 7 alpha-dehydroxylase activity of a gram-positive intestinal anaerobe by Bacteroides and its significance in the 7-dehydroxylation of ursodeoxycholic acid

The 7 alpha-dehydroxylation of chenodeoxycholic acid (CDCA) and cholic acid (CA) by a Eubacterium lentum-like intestinal anaerobe was specifically enhanced by the bacteroides present in mixed cultures and also by the addition to the growth medium of cell extracts from the bacteroides. The 7 alpha-dehydroxylating organism also possessed 7 alpha-hydroxysteroid dehydrogenase activity, and, in collaboration with a 7 beta-dehydrogenating organism, converted ursodeoxycholic acid (UDCA) into CDCA. Large quantities of lithocholic acids were produced from UDCA as well as CDCA in in vitro cocultures of these three kinds of microorganisms.

Regulation of ovarian progestin production by epidermal growth factor in cultured rat granulosa cells.

The modulation of ovarian steroidogenesis by epidermal growth factor (EGF) was investigated in cultured rat granulosa cells. Granulosa cells, obtained from ovaries of immature, hypophysectomized, estrogen-treated rats, were incubated for 2 days with EGF, follicle-stimulating hormone (FSH), or EGF plus FSH. Treatment with EGF did not affect estrogen production, but stimulated progestin (i.e. progesterone and 20 alpha-hydroxy-pregn-4-en-3-one) production in a dose-dependent manner. Stimulation of progestin production by EGF appears to be the result of an increase in pregnenolone biosynthesis as well as increases in the activities of 20 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase/isomerase. Treatment with FSH increased both estrogen and progestin production by cultured granulosa cells. When cells were treated concomitantly with EGF, FSH-stimulated estrogen production was inhibited, while progestin production was further enhanced. The EGF enhancement of FSH-stimulated progestin production appears to be the result of synergistic increases in pregnenolone biosynthesis and 20 alpha-hydroxysteroid dehydrogenase activity, resulting in substantial increases in 20 alpha-hydroxypregn-4-en-3-one but not progesterone production. The effects of EGF were shown to be time-dependent. The concept of a direct action of EGF on rat granulosa cells is reinforced by the demonstration of high affinity (Kd approximately 3 X 10(-10) M), low capacity (approximately 5,000 sites/cell) EGF binding sites in these cells. Thus, EGF interacts with specific granulosa cell receptors to stimulate progestin but to inhibit estrogen biosynthesis.

Subcellular distribution of 3 alpha-hydroxysteroid dehydrogenase and antiestrogen action on androgen-metabolizing enzymes in rat pituitary gland.

Gonadectomy of male rats led to a threefold increase of 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH) activity in pituitary homogenates that could be completely reversed by chronic administration of estradiol or 5 alpha-dihydrotestosterone (DHT). 3 alpha-HSDH was found to be distributed mainly between the 10,000 g and 100,000 g sediments from whole homogenates. The microsomal enzyme activity showed a substantial specificity for NADH whereas the cytosolic enzyme (100,000 g supernatant) demonstrated a slight preference for NADPH. The changes in Vmax found in homogenates following gonadectomy and gonadal steroid administration reflected changes in NADH-linked activity of the microsomal, but not the cytosolic enzyme. Estradiol-induced suppression of NADH-linked 3 alpha-HSDH activity in pituitary homogenates from gonadectomized rats of either sex was accompanied by a similar suppression of NADPH-linked 5 alpha-reductase activity and a marked decrease of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release. In the ovariectomized rat chronic administration of nonsteroidal antiestrogens had strong estrogenic effects on 3 alpha-HSDH activity and LH release, but not on 5 alpha-reductase activity and FSH release. In the gonadectomized male rat, which was much less sensitive to intrinsic estrogenicity of the antiestrogens tested, nafoxidine completely blocked estradiol-induced suppression of 5 alpha-reductase activity and FSH release and partially antagonized suppression of LH release. The trans-isomeric, substituted triphenylethylenes, tamoxifen, and enclomiphene, as well as nitromifene (mixture of trans and cis isomers) were able partially to counteract estradiol-induced suppression of 5 alpha-reductase, but not 3 alpha-HSDH activity. It is concluded that estradiol action on pituitary 5 alpha-reductase, but not 3 alpha-HSDH activity, involves an estrogen receptor mechanism.

20 alpha-Hydroxysteroid dehydrogenase and 17 beta-estradiol dehydrogenase localize in cytosol of human term placenta.

The 20 alpha-hydroxysteroid dehydrogenase activity in human term placenta has been localized by different investigators to nuclear, mitochondrial, microsomal, and cytosolic subcellular fractions. Furthermore, in the cytosol, 20 alpha-hydroxysteroid dehydrogenase activity may be a second function of the enzyme that mediates 17 beta-estradiol dehydrogenase activity. To search for a unique 20 alpha-hydroxysteroid dehydrogenase, human placental villous tissue, homogenized in three different buffer systems, was fractionated by differential centrifugation, and the 17 beta- and 20 alpha-activities were measured by radioisotope conversion assay. The enrichment and purity of the subcellular fractions were shown by marker enzyme assays and electron microscopy studies. Under all experimental conditions, 20 alpha-hydroxysteroid dehydrogenase activity was identified only in the 105,000 g placental cytosol: intact, osmotically ruptured, and acetone-extracted mitochondria, nuclei, and microsomes did not convert progesterone to 20 alpha-dihydroprogesterone. Furthermore, because 17 beta-estradiol dehydrogenase activity was in large part soluble in the cytosol, these localization studies are consistent with the hypothesis that the 20 alpha- and 17 beta-oxidoreductase activities in human placenta reside on one soluble protein.

Isolation of a bile salt sulfatase-producing Clostridium strain from rat intestinal microflora

Bile acid sulfates, formed in human and rat livers, are desulfated by the intestinal microflora. In our study we first isolated from conventional rat feces an unnamed bacterium, termed strain S1, which desulfated the 5 beta-bile salt 3 alpha-sulfates in vitro and in vivo after association with gnotobiotic rats. Strain S1 also possessed 12 alpha-hydroxysteroid dehydrogenase and bile salt-deconjugating activities. The strain was a strict anaerobic, CO2-requiring, gram-negative, sporeforming rod and was designated as belonging to the genus Clostridium. Growth was scarce in culture media, unless in the presence of 0.1% taurine, a sulfur-containing amino acid. Addition of this substance raised the number of bacteria in thioglycolate and peptone yeast media from 10(4) per ml to 10(6) to 10(7) per ml and increased the colony diameter on agar medium from 0.2 mm to 0.5 to 0.9 mm. Sulfatase activity was specific for the 5 beta-bile salt sulfates, leaving the 5 alpha-bile salt sulfates unchanged. In addition, the sulfatase activity was cell bound, and its production was dependent on the composition of the culture medium, although no minimal sulfur medium was required for sulfatase activity.

Transformation of bile acids by Clostridium perfringens.

Thirty-five strains of Clostridium perfringens were examined for their ability to transform bile acids, both in growing cultures and by washed whole cells. All of the strains oxidized the 3 alpha-hydroxy group to an oxo group, and all except three converted the same alpha-hydroxy group into a beta-configuration. The oxidative 3 alpha-dehydrogenation was barely detectable under anaerobic cultural conditions but was clearly demonstrated in an aerated system using washed whole cells, with a pH optimum between 7.0 and 9.0. The epimerizing reaction amounting to 10 to 20% conversion was observed in anaerobic cultures and also with resting cells, irrespective of oxygen supply. Both reactions were carried out with seven conventional 3 alpha-hydroxy bile acids, thus producing a series of 3-oxo and 3 beta-hydroxy derivatives that could be examined for gas-liquid chromatographic and mass spectrometric behavior. No evidence for the occurrence of 7 alpha- and 12 alpha-hydroxysteroid dehydrogenase activities among the test strains was found. A highly potent deconjugating hydrolase was elaborated by all of the strains.

Uterine prostaglandin F2 alpha content and 20 alpha-hydroxysteroid dehydrogenase activity of individual ovarian compartments during pseudopregnancy in the rat.

Uterine prostaglandin F2 alpha content and 20 alpha-hydroxysteroid dehydrogenase activity of individual ovarian compartments during pseudopregnancy in the rat.

Direct stimulation of ovarian progesterone-metabolizing enzyme by gonadotropin-releasing hormone in cultured granulosa cells.

The mechanism by which gonadotropin-releasing hormone (GnRH) inhibits ovarian progesterone production was investigated by studying the GnRH modulation of 20 alpha-hydroxysteroid dehydrogenase activity in cultured rat granulosa cells. The granulosa cells obtained from hypophysectomized, estrogen-treated rats were treated with various hormones in vitro and 20 alpha-hydroxysteroid dehydrogenase activity determined by measuring the conversion of radiolabeled 20 alpha-hydroxy-pregn-4-en-3-one to progesterone. Treatment with follicle-stimulating hormone (FSH) increased the 20 alpha-hydroxysteroid dehydrogenase activity of granulosa cells to 1.3-fold of the control value. Concomitant treatment with 10(-8) M GnRH further enhanced the FSH stimulation of enzyme activity to 2.6-fold of the control value. The increases in 20 alpha-hydroxysteroid dehydrogenase activity were associated with increases in maximum velocity, while the Km values remained unchanged. In contrast, prolactin (PRL) treatment of FSH-primed granulosa cells resulted in an 88% decrease in 20 alpha-hydroxysteroid dehydrogenase activity as compared to untreated controls. Concomitant treatment with GnRH partially blocked this inhibitory effect. Furthermore, incubation of granulosa cells for 2 days with GnRH alone resulted in a 2.7-fold increase in 20 alpha-hydroxysteroid dehydrogenase activity, as compared to untreated controls. In all experiments, the effect of GnRH was blocked by concomitant treatment with a GnRH antagonist. These results suggest that one mechanism by which GnRH inhibits FSH- and PRL-stimulated progesterone production in granulosa cells is through the stimulation of the activity of 20 alpha-hydroxysteroid dehydrogenase which converts progesterone to a less active metabolite.

Steroid delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the rat epididymis during development.

delta 4-5 alpha-Reductase activity is apparently regulated by a testicular factor(s) secreted directly into the epididymis, whereas 3 alpha-hydroxysteroid dehydrogenase activity, in this tissue, appears to reflect circulating androgen levels. To test whether the factor(s) regulating delta 4-5 alpha-reductase activity is directly associated with spermatozoa, a developmental study was undertaken to temporally correlate various parameters of the male reproductive tract with enzymatic activities. delta 4-5 alpha-Reductase activity is first detectable at 21 days of age. Activity increases until day 77, after which time enzymatic activity decreases by more than 60%, reaching steady adult values at 105 days. 3 alpha-Hydroxysteroid dehydrogenase activity is detectable as early as 7 days. Levels of this enzyme increase until day 63, after which time constant adult values are maintained until at least 1 yr. Spermatids and/or spermatozoa are first seen in the testes at 42 days, and plateau levels are reached by day 77. Spermatozoa are first seen in the epididymis at 49 days and reach maximal values by 91 days; no significant change occurs thereafter (until 365 days). Increases in seminal vesicle and ventral prostate weights are of a sigmoidal type, paralleling increases in plasma androgens, with the greatest rate of rise between days 35--63. This sigmoidal type of increase in tissue weights and plasma androgens is similar to that seen for epididymal 3 alpha-hydroxysteroid dehydrogenase but markedly different from that found for delta 4-5 alpha-reductase. The importance of delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in the epididymis before the entry of spermatozoa and the decline in delta 4-5 alpha-reductase activity with age is discussed.

Functional luteolysis in the pseudopregnant rat: effects of prostaglandin F2 alpha and 16-aryloxy prostaglandin F2 alpha in vitro.

The present work concerns the luteolytic effects of prostaglandin (PG) F2 alpha and its analogue, 16-aryloxy PGF2 alpha, upon isolated luteal cells. Varying doses of these two prostaglandins were incubated with cells in the presence or absence of an optimum stimulatory dose of LH (1 microgram/ml). The total contents of progesterone and 20 alpha-dihydroprogesterone in flasks were determined after the incubation periods by radioimmunoassay. Both prostaglandins inhibited basal synthesis of progesterone and 20 alpha-dihydroprogesterone, maximum inhibition occurring at concentrations of either PG of between 250 and 500 ng/ml. In this dose range both prostaglandins were found to abolish LH-stimulated progestogen synthesis completely. These effects were discernible within 5 min of incubation. The studies demonstrated that the onset of PG-induced luteolysis in vitro is characterized by an inhibition of the biosynthesis of both progesterone and its weakly progestogenic metabolite, 20 alpha-dihydroprogesterone; induction of 20 alpha-hydroxysteroid dehydrogenase activity by either PG was not found in incubations extending up to 60 min. In contrast to their relative potencies in vivo, PGF2 alpha and 16-aryloxy PGF2 alpha were essentially equipotent in this in-vitro system.

Studies on the mechanism of 3 alpha-androstanediol-induced growth of the dog prostate.

The conditions under which dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol) induce prostatic growth in the castrate dog have been investigated in animals in which the initial size of the prostate was either normal or large. When androgen replacement was commenced immediately after castration of dogs with preexisting prostatic hypertrophy, dihydrotestosterone was as effective as 3 alpha-androstanediol in maintaining prostate weight. Under all other conditions examined (replacement commenced immediately after castration in dogs with small prostates or 2 weeks after castration in dogs with large prostates), 3 alpha-androstanediol but not dihydrotestosterone induced significant prostatic growth. The maximal effect of 3 alpha-androstanediol in inducing prostatic growth was observed in 12 weeks. The administration of 17 beta-estradiol enhanced the growth-promoting action of 3 alpha-androstanediol, but did not affect the action of dihydrotestosterone or influence 3 alpha-hydroxysteroid dehydrogenase activity in prostate. Thus, the synergistic effect of 17 beta-estradiol and 3 alpha-androstanediol seems to be independent of any effect on the interconversion of the two androgens. The activity of the 3 alpha-hydroxysteroid dehydrogenase in dog prostate was high under all conditions of accelerated prostatic growth and appeared to be under androgenic control.

Progesterone metabolism by the ovary of the pregnant rat: discrepancies in the catabolic regulation model.

The intraovarian site of 20 alpha-hydroxysteroid dehydrogenase activity (20 alpha-OH-SDH) was determined biochemically by measuring enzyme activity in homogenates of the whole ovary, or of isolated ovarian compartments, during the last third segment of pregnancy in the rat. In agreement, with previously reported histochemical evidence, an increase in 20 alpha-OH-SDH activity was observed in isolated corpora lutea, but not in the non-luteal compartment of the ovary. Enzyme activity in corpora lutea was low between days 16 and 22 of pregnancy, but increased markedly (4-6 fold) on day 23. Between days 17 and 20 of pregnancy, serum concentrations of progesterone declined from 130 +/- 3 to 80 +/- 3 ng/ml, while 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) concentrations declined from 34 +/- 3 to 18.5 +/- 3 ng/ml. Only later, between days 20 and 22 of pregnancy, was a significant decline in serum progesterone concentrations associated with an increase in serum 20 alpha-OH-P concentrations (50 +/- 15 ng/ml at 0800 h and 128.5 +/- 15 ng/ml at 1400 h on day 22). Thus the decline in progesterone concentration late in pregnancy can be explained only partially by conversion of progesterone to 20 alpha-OH-P. Further, a dissociation between changes in enzyme activity and in serum concentrations of 20 alpha-OH-P was also observed. A marked increase in serum levels of 20 alpha-OH-P on day 22 preceded any increase in enzyme activity by at least 10 h, and continued increases in enzyme activity on day 23 were not associated with any steady increase in peripheral 20 alpha-OH-P levels. We conclude from these observations that luteal regression is a more complex phenomenon than the regulation of a single enzyme, 20 alpha-hydroxysteroid dehydrogenase, and may involve regulation of both the synthesis and degradation of progesterone.

Luteotropic Regulation of Dispersed Rat Luteal Cells in Early Pregnancy

Viable and functional luteal cells were prepared, using a combination of hyaluronidase, collagenase, and a low concentration of trypsin in a Dulbecco's modified Eagle medium containing 0.5% bovine serum albumin and 3.3 mM Ca++, from corpora lutea taken from 2-day pregnant rats. The viability and functional capacity of the dispersed cells were evaluated by electronmicroscopy and by measuring steroidogenic capicity during perifusion. Dispersed luteal cells previously exposed in vivo to biphasic prolactin (PRL) surges were found to respond during perifusion to as little as 0.5 ng/ml LH by increased steroid secretion. The net progesterone synthesis and secretion remained elevated over a time course of 2 1/2 hours perifusion, and the magnitude of the luteotropic stimulation was dose dependent on LH. However, luteotropic stimulation of LH could not be maintained beyond 2 1/2 h without renewed (in vitro) PRL exposure. PRL by itself maintained the low initial secretion rate of progesterone but demonstrated no stimulatory effect. Different steroidogenic responses were noted during the in vitro administration of LH alone and the administration of LH plus PRL. In the former case, the decreasing rate of progesterone secretion was accompanied by an increasing 20 alpha-dihydroprogesterone secretion, suggesting that luteal 20 alpha-hydroxysteroid dehydrogenase activity was not suppressed. In the latter case, progesterone secretion was maintained and 20 alpha-dihydroprogesterone secretion fell suggesting an inhibitory action by PRL against 20 alpha-hydroxysteroid dehydrogenase activity. Dispersed luteal cells, preincubated at 36 C in medium containing only PRL, retained viability and functional capacity in response to LH-PRL stimulation for periods of time up to 48 h. Preincubation with LH alone did not prolong cell viability.