11β-HSD1 Research Articles

The heterogeneity of isolated adult rat Leydig cells separated on Percoll density gradients: an immunological, cytochemical, and functional analysis.

Intertubular cells, isolated from adult rat testes by collagenase dispersal under conditions designed to minimize cell damage, were fractionated on Percoll density gradients. In the gradient fractions, there was a close cellular correlation between the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), determined by cytochemistry, and other Leydig cell markers (nonspecific esterase, autofluorescence, and an antigen defined by monoclonal antibody LC-1C6). As the reagents for 3 beta HSD cytochemistry are excluded by intact membranes, Leydig cells with damaged plasma membranes were identified by 3 beta HSD reactivity in suspended cell preparations, and the total number of 3 beta HSD-positive (3 beta HSD+) cells in the same preparations was determined after lysis of the cell membrane. Whole cells were differentiated from cytoplasmic fragments by counterstaining with the nuclear dye propidium iodide, and the number of intact Leydig cells in each preparation was determined subsequently by subtracting the number of damaged nucleated 3 beta HSD+ cells from the total number of nucleated 3 beta HSD+ cells. The majority of intact isolated Leydig cells were found in gradient fractions of 1.054-1.096 g/ml density. Acute (3-H) basal and hCG-stimulated testosterone production per intact Leydig cell were dependent upon the concentration of Leydig cells per assay well, indicating that there is cooperativity among Leydig cells in vitro. There was no difference in steroidogenic function among intact Leydig cells from different fractions of the above density gradient range at assay concentrations greater than 10,000 Leydig cells/well. At lower cell concentrations, Leydig cells from gradient fractions of lower density (1.054-1.064 g/ml) produced slightly less testosterone in response to hCG stimulation than Leydig cells from more dense fractions (1.070-1.096 g/ml). Prolonging the exposure of isolated cells to the dispersal conditions caused declines in the apparent buoyant density and basal testosterone and hCG-stimulated cAMP and testosterone production of all Leydig cells, without detectable changes in cell integrity. The data indicate that both the absolute steroidogenic function and the functional heterogeneity of isolated intact Leydig cells are, at least in part, dependent upon the procedures used for their isolation.

Delta (5)3 beta hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: a continuous monitoring microdensitometric study with unfixed ovarian tissue sections.

delta (5)3 beta hydroxysteroid dehydrogenase activity transforms biologically inactive delta (5)3 beta hydroxy steroids into the active delta (4)3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for delta (5)3 beta HSD we have analysed the initial velocity rates (Vo) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20 alpha hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 micron) of the dioestrous and proestrous rat ovary. The results are mean +/- S.E.M. The relationship between DHEA concentration (0 to 50 microM) and delta (5)3 beta HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/Vo versus 1/S plot, the x intercept being positive. Using a 1/Vo versus 1/S2 plot the Vmax was determined to be 1.0 +/- 0.08 mumol min-1 mg-1 CL (n = 6). The Hill constant was 2.7 +/- 0.02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 +/- 1 mumoles (n = 6). In the corpora lutea cells of the proestrous ovary, the Vmax for DHEA transformation was unchanged (0.95 +/- 0.04 mumol min-1 mg-1, n = 3) whilst the S0.5 was significantly increased to 27 +/- 0.1 (p less than 0.01, n = 3). The Hill constant remained positive being 2.9 +/- 0.2 (n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that the same structural gene encodes testicular and adrenal 3 beta-hydroxysteroid dehydrogenase-isomerase.

Thermostability of 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) activity was examined in testes and adrenal glands from several inbred lines and feral mice. A thermolabile variant of 3 beta HSD was detected in the feral Brno mice. The thermostability (t1/2) of 3 beta HSD was approximately 7 min for both testes and adrenal glands from C57BL/6J mice, compared with 4 min for both tissues from Brno mice. Comparison of testicular and adrenal 3 beta HSD thermostability in six kinds of mice indicated that the t1/2 of 3 beta HSD was correlated in the two tissues and could be classified into two distinct types, thermolabile and thermostable. In contrast, quantitative variants in 3 beta HSD activity were not correlated in the two tissues. These data are consistent with the hypothesis that testicular and adrenal 3 beta HSD is encoded by the same structural gene but that expression of 3 beta HSD activity is independently controlled in testes and adrenal glands.

Size distribution and hormonal responsiveness of dispersed rabbit luteal cells during pseudopregnancy.

Due to the evidence for two distinct steroidogenic cell types in corpora lutea of large domestic animals, cells of the rabbit corpus luteum were characterized with respect to cell diameters, relative abundance, steroidogenic capacity and responsiveness to hormones. Pseudopregnancy was induced in New Zealand rabbits by injection of 30-160 IU pregnant mare's serum gonadotropin (PMSG) followed in 2-4 days by an i.m. injection of 20-35 micrograms gonadotropin-releasing hormone (GnRH). Corpora lutea were obtained 2, 5 and 9 days after injection of GnRH and dissociated into single cell suspensions. Suspended steroidogenic cells were incubated (2 h, 37 degrees C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 microM). Media were collected and assayed for progesterone content. Secretion of progesterone (means +/- SE, n = 4) was stimulated (p less than 0.05) by oLH on each day: Day 2 = 1.7 +/- 0.2-fold; Day 5 = 3.5 +/- 0.4-fold; and Day 9 = 3.1 +/- 0.6-fold stimulation above controls. Isoproterenol also stimulated (p less than 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity provided identification of cells with steroidogenic capacity. The diameters (means +/- SE) for steroidogenic cells increased (p less than 0.05) from Days 2 to 9 (Day 2 = 15.2 +/- 0.2 micron; Day 5 = 22.4 +/- 0.4 micron; Day 9 = 28.3 +/- 1.6 micron). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9.(ABSTRACT TRUNCATED AT 250 WORDS)

Short- and long-term effects of ACTH on the adrenal zona glomerulosa of the rat. A coupled stereological and enzymological study.

Short-term ACTH treatment provoked a decrease in volume of the lipid-droplet compartment in rat zona glomerulosa cells, and a rise in plasma and intracellular concentrations of corticosterone and aldosterone. It enhanced activities of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 11 beta-hydroxylase (11 beta OH) and 18-hydroxylase (18OH). Long-term ACTH administration produced a hypertrophy of the zona glomerulosa and its parenchymal cells, a result of the increase in volume of the smooth endoplasmic reticulum and the mitochondrial compartment. The surface area per cell of mitochondrial inner membranes increased; the tubular cristae were transformed into a homogeneous population of vesicles. The plasma and intracellular concentrations of corticosterone further increased, whereas those of aldosterone fell below basal levels (the "aldosterone-escape" phenomenon). The activities of 3 beta HSD and 11 beta OH were enhanced, that of 180H decreased. Therefore, ACTH stimulates zona glomerulosa growth and transforms parenchymal elements into zona fasciculata cell-types. Cyanoketone nullified acute ACTH effects on plasma and intracellular concentrations of corticosterone and aldosterone, but did not affect the activities of 11 beta OH and 18OH. Chronic ACTH treatment produced similar results, although 18OH activity was not suppressed. The mechanism underlying the "aldosterone-escape" phenomenon may thus involve a rise in the intracellular concentration of corticosterone, caused by the enhanced synthesis and activation of 3 beta HSD and 11 beta OH.

Steroid metabolism by cells from human chorion laeve isolated on Percoll gradients.

Collagenase-dispersed cells from human chorion laeve were examined on Percoll gradients. The 3 beta-hydroxysteroid dehydrogenase (a trophoblast marker) and steroid sulfatase activities of the cells were measured and a system was developed to isolate enriched preparations of the trophoblast cells. No cells were found to sediment at Percoll concentrations greater than 50%, and using continuous gradients of Percoll there appeared to be cells with different 3 beta-hydroxysteroid dehydrogenase (3 beta HSD): steroid sulfatase ratios sedimenting in different regions of the gradient. Cells with a high ratio were found in the denser region of the gradient. Continuous gradients provided inadequate separations of distinct populations of cells, thus to obtain a more reproducible system to isolate cells, discontinuous gradients of Percoll were studied. A discontinuous gradient composed of 5, 20, 40, and 60% Percoll was developed and three bands of cells were found sedimenting at the 20, 40 and 60% interfaces, respectively. The number and appearance of cells at the 20 and 60% interfaces varied from tissue to tissue. In contrast, the cells sedimenting at the 40% interface were less variable, a substantial number was found to be present in every tissue studied, they were similar in appearance to the trophoblast cells and had high 3 beta HSD:sulfatase ratios.

Progesterone production by human fetal membranes: An in vitro incubation system for studying hormone production and metabolism

We have established an in vitro tissue explant incubation system to study endocrine functions of human amnion, chorion, and decidua. By means of this technique, tissues remain histologically similar for at least 72 hours, actively use glucose for at least 48 hours, and demonstrate no evidence of release of lactate dehydrogenase into the medium by 24 hours. All three tissues produced progesterone, measured by specific radioimmunoassay, in a dose-dependent fashion from added pregnenolone. However, chorion was many times more active in this respect than were the other tissues. These results were corroborated by demonstrating conversion of 3H-pregnenolone to radiochemically pure 3H-progesterone. This activity was inhibited by a 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) enzyme inhibitor, trilostane. Histochemical staining identified the site of 3 beta HSD activity as being located predominantly in the trophoblast layer of the chorion. We conclude that: (1) this in vitro system is a simple and reliable method by means of which to study endocrine function of amnion, chorion, and decidua; and (2) human fetal membranes, particularly the trophoblast layer of the chorion, can produce progesterone, and hence may be an important regulator of local progesterone levels, which subsequently may affect myometrial contractility.

The relationship between 17 beta-hydroxysteroid dehydrogenase activity and oestrogen concentrations in human breast tumours and in normal breast tissue.

The activity of 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) was measured in human breast tumours and in normal breast tissue from premenopausal, perimenopausal and postmenopausal women. Enzyme activity was higher in tumour tissue than in normal tissue from the same breast and under the conditions of the assay the oxidation of oestradiol was higher than the reduction of oestrone. The physiological status of the women in the study did not relate to the activity of the enzyme in either normal or tumour tissue although fibroadenomas had less activity than adenocarcinomas. In postmenopausal women tumour tissue oestrogens were 2-3 fold higher than in normal tissue from the same breast. Furthermore, tumour tissue concentrations of oestradiol tended to be higher than those of oestrone although in normal tissue the two oestrogens were present in similar concentrations. In plasma from the same women oestrone was the predominant oestrogen. There appears to be no direct relationship between 17 beta HSD activity and oestrogen concentrations but the enzyme may play a part in determining the balance between oestrone and oestradiol according to substrate and cofactor availability.

Ovarian steroidogenic enzyme activities during the rat estrous cycle.

We have correlated the concentrations of serum LH, estradiol and progesterone with the activities of 2 ovarian steroid biosynthetic enzymes during the rat estrous cycle. Ovarian 3 beta-hydroxysteroid dehydrogenase isomerase (3-beta HSD) activity decreased from 29 +/- 6 nmol/mg protein/min (mean +/- SEM) in diestrus, to 7 +/- 0.4 nmol/mg protein/min in late proestrus (p less than 0.005), and subsequently increased to 36 +/- 9 nmol/mg protein/min in metestrus (p less than 0.01). Ovarian 17-hydroxylase (17-OH) activity decreased from early to late proestrus (3.3 +/- 0.2 vs 2.2 +/- 0.2 nmol/mg protein/min, p less than 0.0025), and subsequently increased to 3.9 +/- 0.2 in metestrus (p less than 0.001). Serum LH, estradiol and progesterone peaked during proestrus, and reached a nadir during estrus. We conclude that the activities of 3-beta HSD and 17-OH in the rat ovary vary markedly during the estrous cycle. These changes may underlie the pattern of steroid secretion characteristic of this process.

On the Structural Changes of Granulosa Cells Cultured in vitro

Granulosa cells from porcine ovarian follicles were cultured in vitro in standard medium and in medium supplemented with HCG. After culture the cellular 3 beta HSD enzymatic activity was evaluated and the cells were studied by TEM and SEM. A stereological evaluation of the smooth and rough endoplasmic reticulum was carried out. The results indicate that the cultured cells undergo a luteinization process which is more evident in the presence of HCG. In fact, in comparison with the controls, in these cells the enzymatic activity is higher, the rough endoplasmic reticulum decreases and the smooth endoplasmic reticulum increases, the cytoplasm shows lipid droplets and vesicular mitochondria. The cell surface develops a number of microvillus-like evaginations.

FOLLICULAR GROWTH DURING THE NORMAL CYCLE AND AFTER TREATMENT WITH PROGESTAGENS IN THE EWE

Follicular development in the ewe was studied during the normal estrous cycle and during and after treatment for 13 days with 6alpha-methyl-17alpha-acetoxy-progesterone (MAP). Estradiol-17beta concentrations in the liquor folliculi were also studied. During the estrous cycle growth waves were observed from Days 1 to 10 and from Days 6 to 17. Estradiol concentrations were low through Day 14 and reached maximum values on Day 16. The presence of 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta HSD 17beta HSD) activities in the theca interna cells of follicles from Days 1 to 16 may explain the variations in the synthesis of estrogen. The administration of MPA prolonged follicular life. 3beta-HSD activity which was not observed in the membrane granulosa of follicles was detectable for at least 6 days in the membrane granulosa after treatment. MAP did not inhibit the growth of new follicles and only slightly inhibited follicular steroid synthesis. Rapid new follicular growth was evident after termination of MAP treatment. Some of these net follicles showed 3beta-HSD activity in the membrane granulosa and 17beta-HSD activity in the membrane granulosa and 17beta-HSD activity in the theca interna. This indicates that ovarian secretion of estrogens can occur in the 1st estrous cycle a fter discontinuation of treatment with progestogens. It is hypothesized that subfertility in the 1st estrus after treatment with progestagens may be due to insufficient suppression of follicular growth and steroid synthesis in the ovaries during treatment.