Cholesterol side chain cleavage and aromatase activities in the corpus luteum of the pregnant rhesus monkey.

Cholesterol side-chain cleavage (CSCC) and aromatase activities were measured in luteal mitochondria and tissue pieces, respectively, from rhesus monkeys on days 22, 49, 128 and 160 of gestation. CSCC activity did not vary significantly during gestation and thus probably does not respond to chorionic gonadotropin which is elevated on day 22 of pregnancy. It is not known, however, whether CSCC can be stimulated prior to day 22 when the corpus luteum is steroidogenically more active. Both 3H-pregnenolone and 3H-progesterone were synthesized from [1,2-3/]cholesterol. Aromatase activity declined from high levels on days 22 and 49 to a nadir on day 128 of pregnancy. Utilizing either [1beta-3H]androstenedione or [1beta-3H]testosterone as substrate yielded comparable results throughout gestation.     

Progestin metabolism in the rhesus monkey corpus luteum

For the purpose of describing the pathway by which estrogens are synthesized in the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) corpus luteum (CL), CL were obtained during the midluteal phase of the menstrual cycle and fragments incubated with equimolar amounts of [7-³H]pregnenolone plus [4-¹⁴C]progesterone. Metabolites including ³H-progesterone, ³H, ¹⁴C-20α-dihydroprogesterone, ³H, ¹⁴C-17-hydroxyprogesterone, ³H-estrone and ³H-estradiol-17β appeared in the medium during the first 20 minutes of incubation, ³H, ¹⁴C-Androstenedione was not consistently noted until after 60 minutes. Despite the fact that the ¹⁴C/³H-17-hydroxyprogesterone ratio quickly approached a constant value in the medium, ¹⁴C-estrogens were not detected in the medium or tissue fragments suggesting that progesterone was not a principal precursor for estrogen synthesis. As evidenced by the observation that the ¹⁴C/³H-progesterone ratio was significantly higher in luteal fragments than the 17-hydroxyprogesterone ratio, 17-hydroxyprogesterone appeared to be synthesized from pregnenolone both by way of progesterone and by another route which did not include progesterone. C₂₁- and C₁₈-Steroids were more concentrated in tissue fragments after 120 minutes of incubation than in the medium indicating that these steroids were sequestered by luteal tissue.     

The in vitro synthesis of 7-hydroxy dehydroepiandrosterone by human mammary tissues

Normal and tumorous human mammary tissues were incubated in vitro with [7α-³H] dehydroepiandrosterone and [7α-³H] dehydroepiandrosterone sulphate. Tritium-labelled 7α-hydroxy dehydroepiandrosterone was identified as a principal metabolite from four of the five studies with normal tissue and all seven studies with tumorous tissue.     

A difference in the in vitro accumulation and metabolism of testosterone -1,2-3H by the rat prostate gland following incubation with ovine or bovine prolactin

The $\text{in vitro}$ actions of ovine or bovine prolactin on the accumulation and metabolism of testosterone-1,2-³H by various lobes of the rat prostate gland were examined. Ovine prolactin at a concentration of 2 IU/ml of incubation media significantly increased the accumulation of total radiosteroid by the anterior and dorsal lobes of the rat prostate gland. Although the levels of both testosterone-1,2-³H and its principle metabolite 5α-dihydrotestosterone-1,2-³H were increased by this concentration of ovine prolactin, the ratio of the two steroids was not altered. Ovine prolactin did not alter either the accumulation or metabolism of testosterone-1,2-³H by the ventral or lateral lobes of the rat prostate gland.In contrast to ovine prolactin, bovine prolactin in a wide range of concentrations (0.02–8 IU/ml) significantly reduced the accumulation of total labeled radiosteroid by the dorsal lobe of the rat prostate gland. The reduction in total radiosteroid was reflected in concomitent decreases in both testosterone-1,2-³H and 5α-dihydrotestosterone-1, 2-³H. The anterior lobe responded with similar reductions but only at relatively high concentrations of bovine prolactin (4 and 8 IU/ml). No significant alterations were observed in either the ventral or lateral lobes as a result of incubation with bovine prolactin.     

Factors affecting the adrenocorticotropic hormone stimulation of rabbit adrenal 17α-hydroxylase activity

Adrenocorticotropic hormone (ACTH)-stimulated 17α-hydroxylase activity of rabbit adrenal tissue has been shown to be associated with the subcellular fractions sedimented from 0.25 M sucrose at 33 000 × g for 60 min and at 105 000 × g for 60 min. The fraction sedimenting at 9000 × g for 20 min (mitochondria) contained the majority of the 11β-hydroxylase activity but also had a significant amount of 17α-hydroxylase activity. All subcellular 17α-hydroxylase activity showed an apparent preference for pregnenolone over progesterone. A 1 : 1 mixture of wholehomogenates of adrenal tissue from control and ACTH-stimulated rabbits incubated with[4-¹⁴C]pregnenolone synthesized as much 17α-hydroxylated corticosteroids as homogenate from the ACTH-stimulated tissue alone. However, the mixed homogenate synthesized only 1/4th–1/5th as much 17-deoxycorticosteroids as control, non-stimulated tissue, suggesting that the control tissue contained no inhibitor of 17α-hydroxylation, whereas ACTH-stimulated tissue may contain an inhibitor of 17-deoxycorticoid formation. 24-h dialysis of whole homogenates and subcellular fractions of adrenal tissue from control and ACTH-stimulated animals showed that 17α-hydroxylation was not activated in control tissue and somewhat inactivated in ACTH-stimulated tissue by this treatment. On the other hand, dialysis activated 17-deoxycorticoid formation by whole homogenates, but not in subcellular fractions, of both ACTH-stimulated and control adrenal tissue. Injection of 5 mg/kg cycloheximide prior to the first of 2 daily ACTH injections caused an average of 270 g body weight loss while not affecting the increase in adrenal weight effected by the ACTH. Adrenal tissue homogenates from cycloheximide injected animals produced only 50% as much 17α-hydroxycorticosteroids as homogenates of tissue from animals injected with ACTH alone and produced an amount of17-deoxycorticoids intermediate between homogenates of control and ACTH-stimulated tissue, suggesting the requirement of protein synthesis for 17α-hydroxylation stimulating activity of ACTH.     

Steroid specificity of human placental 5-ene-3β-hydroxysteroid oxidoreductase

The properties of 5-ene-3β-hydroxysteroid oxidoreductase (3β-HSD) from human placental homogenates were studied invitro. The apparent Michaelis constants for 3β-HSD with the substrates pregnenolone (Δ⁵P) and dehydroepiandrosterone (DHA) were 170 nM and 40 nM respectively. The optimal pH for both these substrates was between 10 and 12. With NAD as the substrate, the Km for pregnenolone was 20 μM and for DHA, 17 μM. The activity of 3β-HSD was inhibited by various steroids. Competitive inhibitors (pregnenolone substrate) included: ethynylestradiol (inhibition constant Ki=7.3 nM), DHA (Ki=46 nM), estradiol-17β (Ki=46 nM), cholesterol (Ki=0.68 μM) and 16α-hydroxydehydroepiandrosterone (16αOHDHA) (Ki=2.2 μM). When the substrate was DHA, competitive inhibition occurred with the following steroids: ethynylestradiol (Ki=6.4 nM), estradiol-17β (Ki=69 nM), pregnenolone (Ki=91 μM), cholesterol (Ki=1.3 μM) and 16αOHDHA (Ki=1.9 μM). 4-Ene-3-ketosteroids such as androstenedione, progesterone (Δ⁴P), norethindrone and chlormadinone acetate acted as noncompetitive inhibitors towards both substrates.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Testicular steroidogenesis in the baboon Papio anubis.

We recently reported that the baboon testis converts pregnenolone to testosterone through the delta-4 pathway. The present studies were to determine the metabolism of intermediates of the delta-4 and delta-5 pathway by the baboon testis. Fragments (50 mg) were incubated for 3 hr with 10 muCi of the following tritium-labelled substrates: pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, or testosterone. Pregnenolone was converted to testosterone primarily through the delta-4 pathway, with accumulation of progesterone, 17-hydroxyprogesterone and 20alpha-dihydroprogesterone as predominant intermediates. Similar results were obtained in progesterone incubations. 17-hydroxyprogesterone was not efficiently metabolized by the fragments, while 17-hydroxypregnenolone and dehydroepiandrosterone were efficiently converted into testosterone and androstenedione. Androstenedione was metabolized primarily to testosterone, while testosterone was not a suitable substrate. Some 5alpha-androstanediol was identified in each incubate. These results suggest that although testosterone is formed from pregnenolone through the delta-4 pathway, the delta-5 intermediates are more suitable substrates for testosterone synthesis in the baboon testis.     

Failure to detect delta 5-3 beta-hydroxysteroid oxidoreductase activity in the preimplantation rabbit embryo

Preimplantation rabbit embryos were incubated with pregnenolone and dehydroisoandrosterone under conditions which gave formazan precipitation by the histochemical technique. The metabolic fate of the labeled steroids was assessed simultaneously. There was no concomitant transformation of pregnenolone to progesterone and dehydroisoandrosterone was not transformed to androstenedione. It is concluded that the formazan precipitation is coupled with an "activity" other than delta 5-3 beta-hydroxysteroid oxidoreductase.     

Intracellular distributian and substrate specificity of the 16 alpha-hydroxylase in the adrenal of human fetus

When [7α-³H] dehydroepiandrosterone was incubated with the adrenal homogenates of human fetus at 22 to 26 weeks gestational age, 16α-hydroxydehydroepiandrosterone and/or its sulfate was formed as the only detectable metabolite. The 16α-hydroxylase activity was concentrated in the microsomal fraction of the adrenal homogenate.[1,2-³H]Androstenedione, [4-¹⁴C] pregnenolone and [7α-³H] progesterone were also 16α-hydroxylated by incubation with the microsomal fraction. Amoung these substrates, progesterone gave the highest yield of 16α-hydroxylated products. By incubation with the microsomal fraction, formation of following steroids were also established: 6β-hydroxyandrostenedione from androstenedione; 17-hydroxypregnenolone, 17,21-dihydroxypregnenolone and dehydroepiandrosterone from pregnenolone; 17-hydroxy-progesterone, deoxycorticosterone, 11-deoxycortisol and androstenedione from progesterone.     

Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

Effect of estradiol-17β on the conversion of pregnenolone to progesterone in human term placenta incubated in vitro

The effect of different doses of estradiol-17β (E₂) on the netabolic pregnenolone to progesterone pathway in fragments of human term placenta incubated $\text{in vitro}$ was studied. Doses considered as being physiological of 0.09 and 0.9 μM had a stimulatory effect on the conversion (p < 0.008 to 0.0l6). However, a supraphysiological dose of 45 μM showed an inhibitory activity related to the maximal stimulation (p < 0.03). A dose of 0.9 μM E₂ favoured the accumulation of (³H)-progesterone in the tissue (p < 0.05). These results suggest that E₂ may regulate the synthesis of progesterone in human term placenta.     

Reduction of 4-androstene-3,17-dione by growing cucumber plants

4-[4-¹⁴C]Androstene-3,17-dione was applied to the leaves of growing cucumber plants, Cucumis sativus, twice a week. Four weeks after the first     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

Biotransformation of pregnenolone - 7alpha-3H, progesterone - 7alpha-3H and dehydroepiandrosterone - 7alpha-3H by porcine fetal and maternal adrenal homogenate preparations.

The biotransformation of pregnenolone-7alpha-3H and of progesterone-7alpha-3H by porcine fetal and maternal adrenal homogenates at 56 and 112 days of pregnancy and of dehydroepiandrosterone-7alpha-3H by fetal adrenal homogenates has been investigated in vitro. Both pregnenolone-7alpha-3H and progesterone-7alpha-3H were metabolized extensively by maternal adrenal preparations, the principal radioactive metabolites isolated being cortisol, corticosterone, 11-deoxycortisol, deoxycorticosterone, 11beta-hydroxyprogesterone and androstenedione. In addition, 17alpha-hydroxyprogesterone, 20alpha-dihydroprogesterone and cortisone were formed from both substrates and 17alpha-hydroxypregnenolone and progesterone were formed from pregnenolone. Although essentially the same radioactive metabolites were isolated after incubation of fetal adrenal glands with pregnenolone-7alpha-3H or progesterone-7alpha-3H, a greater proportion of the radioactivity was associated with corticosteroids at 112 days of pregnancy than at 56 days. 11beta-Hydroxyandrostenedione and androstenedione were isolated and identified together with an unknown polar metabolite, after incubation of fetal adrenal tissue with dehydroepiandrosterone-7alpha-3H. These results are discussed in relation to feto-placental steroid biosynthesis and metabolism and the role of the fetal adrenal in the initiation of parturition in the pig.     

Placental and luteal steroidogenesis in the pregnant rhesus monkey.

Progesterone, 20alpha-dihydroprogesterone, estrone and estradiol-17beta concentrations were estimated by radioimmunoassay in blood plasma from uterine, uteroovarian and femoral veins of rhesus monkeys (Macaca mulatta) on days 22, 49, 128 and 160 of gestation. Steroids were consistently more concentrated in uterine and uteroovarian that in femoral venous plasma and in many cases levels in the uteroovarian vein were also higher than those in the uterine vein indicating luteal secretion of both progestins and estrogens thoughout gestation. In some animals, however, the corpus luteum appeared quiescent. As reflected in the decline in the uterine venous progesterone/estradiol-17beta concentration ratio, a shift in steroid contribution from the uterus and its contents occurred between days 22 and 49 of gestation with progesterone declining more rapidly than estradiol-17beta. Progesterone/20alpha-dihydroprogesterone was higher in both uterine and uteroovarian than in femoral venous plasma suggesting peripheral metabolism of progesterone to 20alpha-dihydroprogesterone.     

Modification of steroidogenesis in a mouse adrenal cell line (Y-1) transformed by simian adenovirus SA-7

Transformation of a steroidogenic mouse adrenal cell line (Y-1) by simian adenovirus SA7 produced a cell line with low apparent steroidogenic activity. The effect of ACTH and cholera toxin on cyclic AMP production was similar in both not transformed and virus-transformed cells and activity of cyclic AMP-dependent protein kinase was also similar in both cells. In transformed cells, cholesterol was metabolized to delta 5-3 beta-hydroxysteroids, mainly 20 alpha-dihydropregnenolone while in not transformed cells, the major metabolites were delta 4-3 ketosteroids (20 alpha-dihydro- and 11 beta-hydroxy-20 alpha-dihydroprogesterone). In both cell lines ACTH increased the metabolism of cholesterol. Further studies with labelled pregnenolone and progesterone revealed a loss of delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase and 11 beta-hydroxylase activity in the transformed cells.     

Metabolism of 4-cholesten-3-one to 5α-cholestan-3-one by leaf homogenates

Young leaf homogenates of Digitalis purpurea, Cheiranthus cheiri and Strophanthus kombe' were incubated at 30° for 2 hr with 4-cholesten-3-one-[4-¹⁴C] in a buffered medium containing an NADPH generating system. A single identical metabolite was observed with each tissue. The metabolite was identified by co-chromatography and co-crystallization to constant specific activity as 5α-cholestan-3-one. Leaf homogenates of D. purpurea exhibited the greatest metabolic activity. Addition of non-radioactive progesterone effectively competed with the radioactive substrate, markedly reducing the yield of 5α-cholestan-3-one.     

Steroid delta 4-5 beta-reductase in human mammary tumors.

Steroid delta 4-5 alpha- and delta 4-5 beta-reductase activity was determined in 16 human mammary tumors and 8 DMBA-induced rat mammary tumors using a spectrophotometric assay. Steroid delta 4-5 alpha-reductase was present in all tumors investigated while delta 4-5 beta-reductase was detected in only 6 estrogen receptor negative human breast tumors and absent in all estrogen receptor positive human breast tumors as well as in all rat mammary tumors. Further support for the presence of delta 4-5 beta-reductase was established by using a dual-labelling technique consisting of incubating tumor slices with [14C] testosterone and adding [3H] etiocholanolone, [3H] testosterone and [3H]-5 alpha-dihydrotestosterone at the end of the reaction. Following extraction and chromic acid oxidation, 4-androstenedione, 5 beta-androstanedione and 5 alpha-androstanedione were isolated and purified, and the constancy of the 14C/3H ratio was used as proof of 5 alpha-reductase and 5 beta-reductase. These results were shown to be consistent with the data obtained using the spectrophotometric assay.