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Biomedical subjects

W Gibb

Publications and source records attributed to W Gibb.

At least 55 records · Page 3Linked to original sources
PubMed1986

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Primary culture of cells from human chorion laeve: steroid metabolism and properties of cells grown in defined media supplemented with 0.1% or 10% fetal calf serum.

The purpose of this study was to develop primary cultures of human chorion laeve cells and examine certain aspects of steroid metabolism during culture. Tissues obtained by elective cesarean section at term (38-40 weeks) were dispersed with collagenase. Cells were isolated on Percoll gradients at the interface between 20% and 40% Percoll and examined in primary culture for up to 1 week. Cultures were carried out in chemically defined media supplemented with 10% or 0.1% fetal calf serum (FCS). The morphological and biochemical properties of the cells were different in the two systems. In 0.1% FCS, cells formed clumps of tissue within 16 h of plating, and there was no cell replication. In contrast, in 10% FCS, the cells formed a carpet of tissue and reached confluence after 5 days in culture, resulting in increased DNA and protein content and thymidine incorporation in the dishes. Three steroidogenic enzymes were studied during culture: alkyl steroid sulfatase, estrogen sulfatase and 3 beta-hydroxysteroid dehydrogenase. The sulfatases had higher activities in 0.1% than in 10% FCS, and their activities decreased markedly during the culture period. In contrast, 3 beta-hydroxysteroid dehydrogenase activity was higher in 10% FCS than in 0.1% FCS. Activity remained constant during the culture period in 0.1% FCS and increased in 10% FCS. In the latter system this increase resulted in the enzyme maintaining a constant specific activity during culture. These studies describe two viable systems of chorion laeve cells in primary culture, which may be valuable for studying long term and/or subtle effects on various metabolic aspects of this tissue.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Isolation, purification and culture of Sertoli cells from immature piglet testes.

Several studies suggest a role of Sertoli cells in the control of Leydig cell steroidogenesis. In order to verify this hypothesis, we have developed a system for the purification of pig Sertoli cells. These cells were then characterized by their morphological appearance in light and electron microscopy, their ability to bind [125I]follicle stimulating hormone (FSH) and their functional capacity as evaluated by adenosine 3',5' monophosphate (cAMP) accumulation and lactate production when in primary culture under basal and FSH-stimulated conditions. Crude Sertoli cell suspensions from immature porcine testes were fractionated on discontinuous Percoll gradients (densities 1.025, 1.039, 1.055, 1.080 g/ml). Highly purified Sertoli cells were contained in the second band (d: 1.039) generated on the gradient. These cells demonstrated morphological and functional integrity as evidenced by binding specifically [125I] FSH and by responding to FSH stimulation (by an increased production of cAMP and lactate after 3 days in primary culture), but not to human chorionic gonadotrophin (hCG). This preparation represents a useful model for the study of Sertoli cell functions and their interation with Leydig cells in the regulation of testicular steroidogenesis.

Animals↗
PubMed1986

Nocardiosis mimicking Wegener's granulomatosis.

A 45-year-old man presented with persistent nasal discharge and later developed arthropathy, cutaneous vasculitis, a macular rash and radiographic lung opacities. Early relapse after starting immunosuppressive therapy consisted of new cavitating lung opacities, as seen in Wegener's granulomatosis, and subcutaneous nodules. Lymph node biopsy showed Nocardia asteroides. One month after high dose cotrimoxazole the chest X-ray was normal.

Diagnosis, Differential↗
PubMed1985

Steroid metabolism by human chorion laeve from dichorionic twin pregnancies.

Certain steroid metabolic properties of chorion laeve from dichorionic twin pregnancies were examined to determine whether they were present in chorion not contaminated by decidua or serum. In the chorion situated between the two amniotic sacs and not in contact with decidua, aryl sulfatase, 3 beta-hydroxysteroid dehydrogenase, and aromatase activities were found. This indicates that these reactions are present in chorion laeve and were not previously ascribed to this tissue because of decidual contamination. Specific cortisol binding was also present in this area of chorion laeve, which excludes serum contamination. It is suggested that the specific steroid-binding protein in the membranes may be derived from the transcortin-like protein present in amniotic fluid.

3-Hydroxysteroid Dehydrogenases↗
PubMed1985

Kinetic comparison of the 3 beta-hydroxysteroid dehydrogenase activity in human placenta, chorion laeve, and ovary.

Recent studies from our laboratory and others have shown that Km values for steroid substrates of the 3 beta-hydroxysteroid dehydrogenase in the human placenta were in the nanomolar range compared with micromolar values previously described. The purpose of the present study was to measure the kinetic parameters of the 3 beta-hydroxysteroid dehydrogenase in other human tissues, namely the ovary and chorion laeve, and to determine whether they were similar to those of the placental enzyme. In chorion laeve microsomes the 3 beta-hydroxysteroid dehydrogenase had Km values for dehydroepiandrosterone and pregnenolone similar to those found in placenta. Microsomes from human ovaries, on the other hand, had Km values for both substrates 10- to 20-fold higher. However, the ability of various steroids to inhibit the ovarian enzyme was similar to that previously described from the placenta and the chorion laeve.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Role of catecholamines in the inhibitory effect of immobilization stress on testosterone secretion in rats.

Immobilization stress applied for 6 h induced, in adult male rats, a rise of epinephrine (E) and norepinephrine (NE) plasma levels and a decrease of baseline plasma testosterone (T) values and of human chorionic gonadotropin (hCG)-induced T response. Treatment of the animals for 5 weeks with guanethidine (G), a sympathetic neuron toxic agent, significantly decreased E and NE responses to stress and partly antagonized the inhibitory effects exerted by immobilization on T biosynthesis. Adrenalectomy totally suppressed circulating E and reduced the stress-induced NE increase while partly antagonizing the inhibitory effects exerted on T biosynthesis. Combined G and adrenalectomy treatments totally suppressed plasma E and NE, and completely blocked the effects of immobilization on T levels. Treatment of the animals with the alpha 1-adrenergic blocker, prazosin, and the beta 1-adrenergic blocker, metoprolol, did not modify the effects of stress on T biosynthesis. Treatment with propranolol or with butoxamine, a nonspecific beta- and a specific beta 2-adrenergic receptor blocker, respectively, antagonized the testicular hyposensitivity to hCG induced by stress. Stress- or treatment-induced changes of plasma luteinizing hormone (LH) and hCG levels were not consistently correlated with plasma T modifications. These findings suggest that at least part of the inhibitory effects of immobilization stress on T biosynthesis is exerted by catecholamines through a beta 2-adrenergic receptor.

Adrenal Glands↗
PubMed1984

Effect of glucocorticoids on testosterone production by porcine Leydig cells in primary culture.

For this study, purified immature porcine Leydig cells in primary culture were used. After 2 days of culture, the cells were incubated with dexamethasone (5 X 10(-9), 1 X 10(-7) M) for various periods of time (3-45 h). The media were discarded and treatment was repeated with or without the addition of human chorionic gonadotropin (HCG, 10 mIU/mL) for 3 h. Dexamethasone (10(-7) M) decreased testosterone production of HCG-treated cells (up to 40%) in a time-dependent fashion while the lower dose was ineffective. The effect of varying doses (10(-8) and 10(-6) M) of natural glucocorticoids (corticosterone, cortisol) or synthetic glucocorticoids (triamcinolone, triamcinolone acetonide, betamethasone, dexamethasone) and that of a synthetic progestin (R-5020) on cultured Leydig cells was also studied. After 18 h of preincubation, the various synthetic but not the natural steroids nor R-5020, were able to decrease testosterone production of control and HCG-treated cells by 20-40%. Of a number of other hormonal and nonhormonal substances studied at concentrations of 10(-9)-10(-5) M, only lysine8-vasopressin at a concentration of 10(-6) M was able to inhibit testosterone production by these cells. These results indicate that dexamethasone and other synthetic glucocorticoids, and to a lesser degree lysine8-vasopressin, may exert a direct inhibitory effect on testosterone production by purified porcine immature Leydig cells in vitro.

Animals↗
PubMed1984

The 3 beta-hydroxysteroid dehydrogenase activity of cultured porcine Leydig cells in primary culture.

Previous studies have shown that there is a spontaneous progressive increase in human chorionic gonadotropin (HCG)-stimulated testosterone (T) production by immature porcine Leydig cells during primary culture for 3 days, whereas basal T production is unaltered. Preliminary data indicated that during this time, there was an increase in the activity of the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The purpose of the present study was to further characterize this increase. 3 beta-HSD was assayed by measuring the conversion of tritiated dehydroepiandrosterone (DHEA) to androstenedione in the presence of NAD+. A ninefold increase in activity occurred between days 1 and 2 and only twofold between days 2 and 3. A 5- to 20-fold increase of the apparent maximum velocity of the enzyme was observed after 3 days of culture while the apparent Km remained unchanged. Cycloheximide treatment (150 ng/mL) completely abolished this spontaneous increase in enzyme activity. In parallel experiments, the stimulatory effect of HCG on T and DHEA production was inhibited by cycloheximide treatment by 90% and 60%, respectively, whereas basal T production was not affected. These results suggest that the spontaneous increase of 3 beta-HSD activity in primary cultures of porcine Leydig cells is secondary to de novo protein synthesis and that this phenomenon may be partially responsible for the increased responsiveness of these cells to HCG during the culture period.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Kinetic studies on the formation of estrogens from dehydroepiandrosterone sulfate by human placental microsomes.

Using microsomes isolated from term human placentae kinetic analyses of each of the enzymes involved in estrogen synthesis from dehydroepiandrosterone sulfate have been carried out and the following parameters were found: sulfatase, Michaelis-Menten constant (Km) = 16,000 +/- 5,000 nM, maximum velocity (Vm) = 2.0 +/- 0.5 nmol X min-1 X mg protein-1; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), Km = 15 +/- 3 nM, Vm = 1.8 +/- 0.4 nmol X min-1 X mg protein-1; aromatase, Km = 14 +/- 4 nM, Vm = 0.12 +/- 0.02 nmol X min-1 X mg protein-1. From these values one can predict that, theoretically, the rate-limiting enzyme in estrogen synthesis from dehydroepiandrosterone sulfate (DS) should change from the sulfatase at low concentrations of substrate to the aromatase at higher concentrations. In order to test this hypothesis we developed a system which allowed the formation of estrogens from DS, dehydroepiandrosterone, and androstenedione to be measured and the appropriate intermediates to be isolated. The sulfatase was found to be rate limiting at concentrations of DS below 2 microM and the aromatase was found to be rate limiting at higher concentrations. These data may explain why previous perfusion studies of human placentae indicated the sulfatase was the rate-limiting enzyme in estrogen synthesis yet in vitro studies found that it was the aromatase. Steroids previously shown to inhibit the 3 beta-HSD were examined for their ability to inhibit the formation of estrogens from DS. Although 3 beta-HSD activity was markedly inhibited this had little effect on the overall conversion of DS to estrogens, until high concentrations of inhibitors were used. The data also underline the importance of studying enzyme systems rather than single enzymes when studying steroid synthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Role of arginine-vasopressin (AVP) in stress-induced inhibition of testicular steroidogenesis in normal and in AVP-deficient rats.

It has been recently demonstrated that immobilization stress induces in rats a state of testicular desensitization to gonadotropins as well as a post-cAMP blockade of testosterone (T) biosynthesis. Since arginine-vasopressin (AVP) has recently been found to antagonize in rats the in vitro T-releasing effect of human CG, with this work we have verified whether AVP might be involved in stress-induced inhibition of T biosynthesis. In Sprague-Dawley and Long-Evans adult male rats chronically cannulated in the jugular vein, a small but statistically significant rise of plasma AVP levels was observed after 2 h of immobilization stress. The iv infusion of AVP (1 micrograms/kg/h) to chronically cannulated rats induced a fall of plasma T levels. A dose-dependent inhibition of plasma T values was also observed 3 h after ip administration of AVP (1, 5, 25 micrograms/kg) in animals killed by decapitation. An antagonist of AVP pressor activity [1-(beta-mercapto-beta 1 beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine] AVP, antagonized, when injected ip at a dose of 30 micrograms/kg, the T-inhibitory effects of 3 h of immobilization stress. No consistent changes in plasma LH levels were observed in these experiments. To further evaluate the role of AVP in stress-induced T inhibition, AVP-deficient Brattleboro rats were submitted to 2 or 3 h of immobilization stress concomitantly with rats of the original Long-Evans strain. After 2 h and even more after 3 h of stress, plasma T levels fell in Long-Evans rats together with basal and human CG- or cAMP-stimulated T production by Percoll purified Leydig cells. In Brattleboro rats, 2 h of stress had no effects on plasma T levels nor in vitro basal or stimulated T production, whereas 3 h of immobilization were as effective as in Long-Evans animals. These results suggest, therefore, that at least part of T inhibitory effects of immobilization, those occurring during the first 2 h of stress, are due to an AVP-induced, post-cAMP blockade of T biosynthesis. Since plasma corticosterone, during 2 h of stress, rose to similar, albeit smaller, levels in Brattleboro rats as compared to those in Long-Evans animals, this glucocorticoid does not seem to be involved in the testicular effects of stress.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Maturation of the hypothalamic--pituitary--gonadal axis in the male lamb: a review.

We have studied the activity of the hypothalamic--pituitary--gonadal (HPG) axis in the male ovine fetus and newborn lamb. Circulating levels of gonadotropins, prolactin, cortisol (F), testosterone (T), dehydroepiandrosterone sulfate and delta 4androstenedione (delta 4A) were measured in fetal plasma in the third trimester of gestation and the testicular response to hCG was studied in ovine fetuses at 95 - 141 days. Ultradian variations of LH, FSH and testosterone and the testicular response to hCG also were assessed from 1 to 28 days postnatally. The data indicate that (1) fetal plasma LH, FSH, delta 4A and T levels are low from 95 days of gestation to term, while F levels increase in the last 10 days. Postnatally, F decreases rapidly. Spontaneous T peaks may occur as early as 36 hr of life. (2) Spontaneous LH and concomitant or subsequent T secretory peaks are observed by 3 days of age. (3) hCG can induce an increase in T production by interstitial cells in vitro and can increase the T testicular content and its release in the plasma from 95 days of intrauterine life. Desensitization to hCG also can be observed throughout the last trimester of gestation. We conclude that the HPG axis of the male lamb is active ty 36 - 72 hr of postnatal life and that the steroidogenic capacity of the ovine testis is developed several weeks prenatally. Consequently, the relative quiescence of the axis prenatally and in the first 24 hr of life seems to result from relatively low LH secretion and release, related to an undefined endogenous control, together with decreased Leydig cell sensitivity and relatively low enzyme activity limiting T release.

Animals↗
PubMed1983

Studies with purified immature porcine Leydig cells in primary culture.

The steroidogenic capacity of purified immature porcine Leydig cells in culture was studied over several days. The cells were obtained by fractionating crude testicular interstitial cell suspensions on a discontinuous Percoll gradient (d = 1.037, 1.042, 1.052, 1.098 g/ml), and characterized by specific binding of 125I-human chorionic gonadotropin (hCG), testosterone (T) and cyclic adenosine 3':5'-monophosphate (cAMP) production in response to hCG, and the enzymatic determination of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The Leydig cells were recovered in a density band between 1.052-1.068 g/ml and grown in a chemically defined medium (Mather et al., 1981). In the absence of hCG, T production was low throughout the 6 days of culture. However, in response to hCG (10 mIU/ml), the cultured Leydig cells showed a progressive increase in T synthesis, which reached a maximum at Days 3-4. 8-Br-cAMP (1 mM) induced a comparable rise in T production to that obtained with hCG throughout the culture period. In contrast, 8-Br-cAMP induced a near maximal increase in dehydroepiandrosterone (DHEA) production from Day 1. This paper demonstrates that purified immature porcine Leydig cells in primary culture are a valuable model to study the ontogeny of Leydig cell function.

3-Hydroxysteroid Dehydrogenases↗
PubMed1982

Characterization of specific steroid binding in human amnion at term.

In human fetal membranes (amnion and chorion) the appearance, during the last few weeks of pregnancy, of a protein which binds progesterone has been suggested as playing an important role in the inset of labor. The purpose of the present study was to quantitatively characterize specific binding in fetal membranes. Amnion, rather than chorion, was studied because it could be obtained free from contamination by other tissues. The cytosol (105,000 X g supernatant) was used after being extracted for 1 h with dextran-coated charcoal. The specific binding of cortisol in the cytosol, determined with a charcoal assay, was stable after storage at -20 degrees C or -60 degrees C and after heating for 1 h at 37 degrees C. It was reversible, and showed high-affinity, KD = 1 +/- 0.5 nM (mean +/- SEM, n = 6) and a large number of sites were found, 1497 +/- 666 fmoles/mg protein (mean +/- SEM, n = 6). In contrast, progesterone binding in the cytosol had a 10-fold lower affinity, KD = 12 +/- 5 nM (mean +/- SEM, n = 5) with 802 +/- 246 fmol bound/mg protein (mean +/- SEM, n = 5). A number of steroids were examined for their ability to compete with cortisol binding. The following were found to express affinities relative to cortisol which was considered to be 100: corticosterone (100), progesterone (10), cortisone (8), 5 alpha-pregnane-3, 20-dione (2), and 20 alpha-hydroxy-4-pregnen-3-one (less than 1). No competition was found for dexamethasone, betamethasone, triamcinolone, triamcinolone acetonide, R5020, medroxyprogesterone acetate, estrone or estradiol. These studies indicate that the steroid binding protein associated with human amnion at term exhibits a higher affinity for a cortisol and corticosterone than progesterone. Thus it is perhaps involved in the metabolism of glucocorticoids by the membranes, rather than in the local withdrawal of progesterone, as proposed by previous investigators.

Amnion↗