Change of steroidogenic pathways in the ovary of a tropical catfish, Clarias macrocephalus, Gunther, after hCG treatment.

Adult female catfish received an im injection of 454 IU hCG in 0.2 ml saline. Sixteen hours later, the ovarian tissue from the hCG-treated or control fish was aerobically incubated in vitro with 4-[14C]progesterone or 17 alpha-hydroxyprogesterone at 30 degrees for 60 min. When progesterone was employed as the substrate, significant production of androstenedione and testosterone was observed in the control group. However, after the hCG injection, a markedly higher amount of 20 beta-hydroxy-4-pregnen-3-one was produced. Furthermore, the androgen production was diminished, and the production of 5 beta-reduced C21 metabolites such as 5 beta-pregnane-3,20-dione and 3 alpha-hydroxy-5 beta-pregnan-20-one was also reduced in the hCG-treated group. From 17 alpha-hydroxyprogesterone as a substrate, considerable amounts of androstenedione and testosterone were obtained as the metabolites in the control group. However, after the hCG treatment, production of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-diOHprog) and its 5 beta-reduced metabolite was markedly stimulated, while the androgen production was reduced drastically. By evaluating the yield of each product, it was suggested that the tentatively calculated activity of 17 alpha-hydroxylase and C-17-C-20 lyase was diminished by the hCG treatment and that 20 beta-hydroxysteroid dehydrogenase was activated. It indicates that hCG changed the ovarian steroidogenic pathway from androgen production to formation of 17 alpha, 20 beta-diOHprog, an inducer of germinal vesicle breakdown.

Purification and characterization of rat adrenal 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene-isomerase.

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.

Changes in enzyme activities related to steroidogenesis in human ovaries during the menstrual cycle.

Follicles and corpora lutea were isolated from the ovaries of 25 patients at several stages of the normal menstrual cycle. To measure the steroidogenic enzyme activities, cell-free homogenates of the ovaries were incubated aerobically with 4-14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, and testosterone in the presence of appropriate cofactor(s). The delta 5-3 beta-hydroxysteroid dehydrogenase plus delta 4-delta 5 isomerase activity in the follicle was low during early follicular phase (greater than 11 days before ovulation), but gradually increased toward ovulation and showed maximal values in the corpus luteum at the midluteal phase (6-10 days after ovulation). The activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the follicle reached the highest value at the late follicular phase (within 5 days of ovulation), but markedly decreased after luteinization. 17 alpha-Hydroxylase activity in the corpus luteum increased again during the midluteal phase, whereas the activity of C-17-C-20 lyase remained at a low level throughout the luteal phase. The aromatase activity was low during the follicular phase, but increased markedly with luteinization. These findings are discussed in light of the known plasma steroid concentrations and their changes during the menstrual cycle.

Postovulatory decrease in estrogen production is caused by the diminished supply of aromatizable androgen to ovarian aromatase.

After administration of testosterone to immature rats which had ovulated after treatment with PMSG and hCG, a significant increase in ovarian content of estradiol-17 beta was observed. Together with our previous result on the immediate decrease in ovarian 17 alpha-hydroxylase and C-17-C-20 lyase after PMSG and hCG treatment, it is concluded that the drastic decrease in serum estradiol-17 beta after ovulation is caused by a shortage of supply of aromatizable androgen(s) to the ovarian aromatase, due to diminished activities of the above two enzymes.

Effect of pregnant mare's serum gonadotrophin on the activities of delta 4-5 alpha-reductase, aromatase, and other enzymes in the ovaries of immature rats.

After incubation of progesterone, 17 alpha-hydroxyprogesterone, androstenedione, and testostrone with an ovarian preparation (supernatant fluid at 10,000 x g) of immature rats (21-23 days of age) in the presence of NADPH, 3 alpha- and 3 beta-hydroxy-5 alpha-reduced steroids were obtained as the major metabolites. Among the enzyme activities relevant to the metabolism, delta 4-5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase were intracellularly localized to the microsomal fraction (10,000--105,000 x g), and 3 alpha-hydroxysteroid dehydrogenase was detected exclusively in the cytosol fraction (supernatant fluid at 105,000 x g). Within 2 days after a single injection of pregnant mare's serum gonadotrophin (10 IU/rat) to 21-day-old female rats, the following occurred: 1) an enhancement of 17 alpha-hydroxylase and C-17-C-20 lyase activities; 2) a suppression of delta 4-5 alpha-reductase activity; and 3) an increase in aromatizing activity. From the above-mentioned results, it was concluded that the increased secretion of estrogen from ovaries of immature rats stimulated by pregnant mare's serum gonadotrophin administration was caused by a modification of the ovarian enzyme activities relevant to estrogen production.

Steroid metabolism in normal mammary gland and in the dimethylbenzanthracene-induced mammary tumor of rats.

After incubation of [4-14C]progesterone with cell-free homogenates of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced mammary tumor of rats, 20 alpha-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione, 20 alpha-hydroxy-5 alpha-pregnan-3-one, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol were identified as the metabolites. In normal mammary tissue, however, 4-pregnene-3 alpha-diol was isolated in addition to 5 alpha-reduced, and 3 alpha- and 20 alpha-hydroxy metabolites. When radioactive testosterone was employed as a substrate, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were obtained as the metabolites of the mammary tumor. In the normal mammary gland, only 4-andorstene 3 alpha, 17 beta-diol was formed as its metabolite. Although the enzyme activities relevant to the metabolism varied among the tumor examined, the activity of 20 alpha-hydroxysteroid dehydrogenase in the mammary tumor was significantly lower than that in the normal mammary gland, whereas the activity of 5 alpha-reductase was higher in some of the mammary tumors than in the normal gland. The 5 alpha-reductase activity in the normal mammary gland was mostly localized in the crude microsomal fraction, whereas the same enzyme activity in the tumor was detected in all the organelle fractions. The activities of 20 alpha-hydroxysteroid dehydrogenase and NADPH-linked 3 alpha-hydroxysteroid dehydrogenase were found mainly in the cytosol fractions of the tumor and the normal tissue. The NADH-linked 3 alpha-hydroxysteroid dehydrogenase activity was detected only in the cytosol fraction of the normal mammary gland, but in the tumor studied, the activity of this enzyme was detected in all the subcellular fractions examined.

Endocrine studies in Sertoli-cell-only syndrome.

Pituitary-gonadal endocrine functions were studied in 15 patients with Sertoli-cell-only syndrome and a comparison was made with other testicular diseases, such as Klinefelter's syndrome. Elevated levels of serum luteinizing hormone as well as follicle stimulating hormone and lowered levels of serum testosterone suggested the existence of Leydig cell failure in addition to germ cell failure. However, the degree of these endocrinological abnormalities in patients with Sertoli-cell-only syndrome was mild compared to that in patients with Klinefelter's syndrome. Reserve capacity of Leydig cells in patients with Sertoli-cell-only syndrome was considered to be preserved as suggested by the human chorionic gonadotropin stimulation test.

Direct evidence on incorporation of the receptor into the nuclei of rat ventral prostate in the form of the complex with dihydrotestosterone.

Cytosol 9S receptor was prepared from the supernatant fluid at 105,000 X g of rat prostate homogenates by a Sephadex G-100 gel chromatography, and was labeled with 131I. The 131I-labeled 9S receptor retained the activity of forming a complex with 3H-dihydrotestosterone, similarly to the intact cytosol receptor, when examined by a sucrose density gradient centrifugation. When the 131I-labeled cytosol 9S receptor was incubated with isolated prostatic nuclei in the presence of 3H-dihydrotestosterone, it was found that the 131I-labeled receptor was directly incorporated into the nuclei in a form of the complex bound to 3H-dihydrotestosterone. 131I-Labeled receptor-3H-dihydrotestosterone complex which was incorporated into the nuclei was extracted with 0.5 M KCl solution, and the nuclear complex sedimented with a velocity of 5S. The incorporation of 131I-labeled receptor-3H-dihydrotestosterone complex into the nuclei increased along with the raised temperature of incubation, whereas association of the complex with the chromatin reached maximum at 35 degrees C and then decreased gradually beyond this temperature. In the time course study, either the incorporation of the complex into the nuclei or the association with the chromatin reached the maximal levels within 10 min and leveled off up to 60 min. On the other hand, 131I-labeled serum protein was far less efficiently incorporated into the nuclei than the radioiodinated 9S receptor, and association of the serum protein with the chromatin was limited to a very little extent. The 131I-labeled 9S receptor-dihydrotestosterone complex associated with the chromatin was found to be preferably distributed into the non-histone protein as well as the DNA itself of the chromatin. The radioactivity lost by dehistonization of the chromatin was almost negligible. Furthermore, the cytosol 9S receptor fraction bound to 3H-dihydrotestosterone was purified about 100 fold by the two consecutive column chromatographies. The partially purified receptor fraction which was labeled with radioiodine incorporated mostly into non-histone protein and DNA fractions.