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B F Mitchell

Publications and source records attributed to B F Mitchell.

15 recordsLinked to original sources
PubMed1990

Steroid sulfohydrolase in human chorion and decidua: studies using pregnenolone sulfate and dehydroepiandrosterone sulfate as substrate.

Human chorion and decidua use pregnenolone sulfate (P5S) and dehydroepiandrosterone sulfate (DHAS) as substrates for local estrogen and progesterone synthesis. We hypothesized that the local estrogen/progesterone ratio may influence contractility of the adjacent myometrium and hence effect the timing of parturition. Thus, we studied steroid sulfohydrolase activity for P5S in these tissues and investigated the potential interaction of other steroids on the rates of hydrolysis of P5S and DHAS. The enzyme was present in both tissues, predominantly in the microsomal fraction. With P5S as substrate, the Michaelis-Menten constant (Km) was similar in chorion (1.3 +/- 0.2 mumol/L, mean +/- SEM) and decidua (0.9 +/- 0.1 mumol/L) but the maximum velocity (Vmax) was significantly greater in chorion (2.6 +/- 0.4 vs. 1.1 +/- 0.3 nmol/mg protein/15 min, P less than 0.05). In both tissues there was a tendency towards greater activity in tissues obtained before labor compared to tissues obtained after spontaneous labor onset. Using either DHAS or P5S as substrate, there was significant inhibition of sulfohydrolase activity by other steroids at concentrations similar to those in late pregnancy fetal and maternal plasma. In microsomal preparations using DHAS as substrate, activity was inhibited by equimolar concentrations of estrone sulfate (E1S, by 38 +/- 2%), P5S (by 74 +/- 2%), and cholesterol sulfate (C27S, by 38 +/- 3%). With P5S as substrate, equimolar concentrations of E1S, DHAS, and C27S caused inhibition of sulfohydrolase activity by 19 +/- 5%, 16 +/- 4%, and 18 +/- 2%, respectively. These inhibitory effects also were observed using a tissue explant system with intact cells. In kinetic inhibition studies using DHAS as substrate, E1S and P5S were competitive inhibitors with inhibition constants (Ki) of 4.8 +/- 1.3 and 0.7 +/- 0.1 mumol/L, respectively. Using P5S as substrate, E1S and DHAS also were competitive inhibitors with Ki values of 8.2 +/- 2.1 and 9.6 +/- 1.2 mumol/L, respectively. For both substrates, the pattern of inhibition by C27S was complex. Preliminary experiments to distinguish, on the basis of differing physical-chemical properties, separate enzymes for different substrates were inconclusive. We conclude that human chorion and decidua can hydrolyze several steroid sulfoconjugates and this activity may regulate local estrogen and progesterone synthesis. There are significant interactions among steroid sulfoconjugates in regulating this activity. These activities may be important components of a paracrine system that determines myometrial contractility and the timing of parturition.

Arylsulfatases↗
PubMed1990

Regulation of neural oxytocin gene expression by gonadal steroids in pubertal rats.

We have previously demonstrated that neuronal oxytocin mRNA increases during the pubertal development of female rats. In this paper we have examined the factors that regulate this developmental increase in both male and female rats. Northern blot analysis demonstrated that neural oxytocin mRNA increased 5- to 10-fold from postnatal day 20 (P20) to P60 in animals of both sexes, coincident with puberty. Mature male rats and females at all stages of the estrous cycle expressed similar levels of neural oxytocin mRNA. Pubertal up-regulation of oxytocin mRNA was largely, but not completely, inhibited by prepubescent gonadectomy, indicating a requirement for intact gonads as well as some other as yet undefined factor(s). Pubertal treatment of gonadectomized animals with estradiol or testosterone abolished the effects of gonadectomy; treated animals expressed levels of neural oxytocin mRNA similar to those in controls. However, treatment of prepubertal animals with estradiol or testosterone from P10 to P20 had no effect on oxytocin mRNA levels, suggesting that neural maturation or other factors are necessary requisites for steroid sensitivity. To determine whether neural activin played any role in regulating oxytocin mRNA during puberty, we examined levels of inhibin/activin beta A-chain mRNA. This mRNA was expressed at similar levels in all brain regions and did not vary as a function of gonadectomy or steroid treatment, making it unlikely that activin mediates the observed changes. Together, these data indicate that neural oxytocin mRNA is induced by gonadal steroids during puberty, and suggest a mechanism for coordinating development of reproductive functions with other pubertal changes.

Activins↗
PubMed1988

Steroid sulfohydrolase activity in human chorion. I. Interactions of other steroids with estrone sulfate as substrate.

Human chorion contains steroid sulfohydrolase activity and synthesizes free estrogens from estrone sulfate (E1S). We hypothesized that the free estrogen thus formed may influence the contractility of the adjacent myometrium in late pregnancy. In this study we measured the abilities of various steroids and steroid conjugates to influence the hydrolysis of E1S by the 105,000 x g pellet of chorion tissue obtained from women after spontaneous onset of labor and vaginal delivery and from women delivered by cesarean section before labor onset. No differences were found in tissues obtained before or after the onset of labor. None of the steroids increased the rate of hydrolysis. Several unconjugated steroids caused significant inhibition, but only at concentrations well beyond physiological ranges in maternal or fetal blood. However, conjugated steroids had marked inhibitory effects at circulating concentrations. At equimolar concentrations with the E1S substrate, the sulfoconjugates of dehydroepiandrosterone, pregnenolone, and cholesterol caused 27 +/- 1% (+/- SE), 64 +/- 1%, and 40 +/- 1% inhibition, respectively. These results were confirmed using a tissue explant system. Using enzyme inhibition kinetic analysis, we determined that the inhibition by dehydroepiandrosterone sulfate was non-competitive, with Ki = 8.7 +/- 1.7 mumol/L. The inhibition by pregnenolone sulfate was competitive, with Ki = 1.6 +/- 0.4 mumol/L, and that by cholesterol sulfate was primarily noncompetitive, with Ki = 7.4 +/- 1.2 mumol/L. We conclude that there is significant interaction among sulfurylated steroids that may influence local free estrogen synthesis within human chorion. This interaction may affect the contractility of the late pregnancy myometrium.

Chorion↗
PubMed1987

Progesterone synthesis by human amnion, chorion, and decidua at term.

We investigated the ability of human fetal membranes to produce progesterone from a variety of substrates. Chorion is more active than decidua, and amnion produces little progesterone. Cholesterol or low-density lipoprotein cholesterol was not used as a substrate. Chorion used pregnenolone, pregnenolone sulfate, and 20 alpha-dihydroprogesterone for progesterone synthesis. Decidua also used these three substrates but produced significantly less progesterone than chorion. Amnion used only 20 alpha-dihydroprogesterone as a substrate. Exogenous human chorionic gonadotropin or gonadotropin-releasing hormone or its analogues did not influence progesterone production by any of the tissues. There were several significant changes in substrate usage for progesterone synthesis by the tissues around the time of the onset of labor. Tissue concentrations of progesterone are approximately 4 ng/mg protein, and it appears that local production rates could completely account for this. These data are compatible with the hypothesis that local regulatory mechanisms may determine progesterone concentrations in fetal membrane tissues and that important changes may occur around the time of the onset of parturition.

20-alpha-Dihydroprogesterone↗
PubMed1986

Human chorion cells respond to growth factors but lose steroidogenic capacity in primary monolayer cell culture.

This study has defined a method for preparation and monolayer culture of cells from chorion laeve. Cell number and cell protein content are stable over 7 d in culture. The cells will divide in response to epidermal growth factor in the presence of a supplemented, enriched medium and a collagen matrix, but they lose steroidogenic activity over time in culture. This culture system can be used as the starting point for the development of a chemically defined hormone-supplemented, serum-free culture system for studies of chorion cell differentiation and fetal membrane cell interactions.

Cell Differentiation↗
PubMed1986

Substrate utilization for estrogen synthesis by human fetal membranes and decidua.

We have investigated the ability of tissue explants of human amnion, chorion, and decidua to produce estrone when incubated alone or in the presence of estrone sulfate, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, or testosterone. Amnion produced very little estrone from any substrate. Chorion utilized all substrates and decidua utilized estrone sulfate and dehydroepiandrosterone sulfate for estrone production. For both chorion and decidua, estrone sulfate was quantitatively the most important substrate. Chorionic tissues obtained after spontaneous labor produced greater levels of estrone than tissues obtained before labor (p less than 0.05). We could demonstrate no effect of cortisol, estriol, progesterone, prostaglandins, oxytocin, or dibutyryl cyclic adenosine monophosphate on estrogen production. We also measured endogenous concentrations of estrone and estradiol in fetal membranes. We found no significant difference in tissue concentrations between the two methods of delivery. There was no significant correlation between estrone and estradiol concentrations and distance from the placenta. We conclude that human chorion and decidua can produce estrogen, which may have some role in determining the timing of parturition.

Androstenedione↗
PubMed1986

Changes in prostaglandin transfer across human fetal membranes obtained after spontaneous labor.

Increased prostaglandin E2 production from amnion is thought to be a critical step in the initiation of human parturition. However, it is not known whether amniotic prostaglandin E2 can reach the decidua and/or myometrium. We examined whether prostaglandin E2 could cross the amnion and full-thickness membranes and whether this capacity changed with active labor. Using an in vitro system we found that there was a time-dependent cumulative transfer of total radioactivity and of radioactivity corresponding chromatographically to prostaglandin E2 across amnion and full-thickness membranes. The rate of transfer across the amnion was faster than across full-thickness membranes and varied according to the site of tissue sampling within the uterus. The permeability constant for prostaglandin E2 transfer across full-thickness membranes was significantly higher in tissue collected after the spontaneous onset of labor than in tissue collected at elective cesarean section at term. We conclude that prostaglandin E2 produced in human amnion at term may escape metabolism in the chorion and reach the decidua and/or myometrium.

Decidua↗
PubMed1986

Effects of steroids on progesterone output by explants of human chorion.

Human chorion can synthesize and metabolize progesterone, and changes in progesterone synthesis by chorion at term might be important in the processes leading to parturition. We examined whether other steroids present within the maternal compartment and amniotic fluid during late pregnancy influence progesterone output by explants of chorion. We also sought differences in steroid effects on progesterone output in association with labor. Explants were prepared from chorion collected after the spontaneous onset of labor and vaginal delivery and chorion collected after cesarean section without active labor. To study the short-term effects of steroids on progesterone output by chorion, explants were incubated for 4 h with 3 microM pregnenolone and 3 microM of a potential interacting steroid. Other explants were preincubated for 24 h with steroid, then rinsed and incubated for 4 h with 3 microM pregnenolone and 3 microM of the same steroid as during preincubation. Under these conditions, dehydroepiandrosterone and androstenedione inhibited progesterone output by explants of chorion obtained at spontaneous labor and at cesarean section. Testosterone also inhibited progesterone output, but only in cesarean section chorion. If explants were preincubated for 24 h with steroid and then rinsed and incubated for 4 h with pregnenolone only, progesterone synthesis returned to control values. This finding indicates that the mechanism of action of these inhibitory steroids is likely through an effect on 3 beta-HSD activity and not due to a change in the rate of enzyme synthesis. We also noted apparent stimulatory effects of steroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1986

Androstenedione metabolism in the late gestation sheep fetus.

We have determined metabolic parameters for androstenedione (A) in chronically catheterized late gestation (day 130) sheep fetuses. The MCR (MCRA) was 3210 +/- 229 (SEM, n = 12) ml/min, the fetal arterial whole blood concentration of A [A] was 65 +/- 5 pg/ml, and the blood production rate (PRA) was 204 +/- 20 ng/min. Pulsatile administration of ACTH in amounts that raised fetal arterial plasma cortisol concentrations by 5- to 7-fold increased [A] to 154 +/- 20 pg/ml and PRA to 471 +/- 31 ng/min with no change in MCRA. In the presence of metopirone to block fetal adrenal cortisol output, ACTH treatment still provoked elevations in [A] (to 198 +/- 23 pg/ml) and PRA (539 +/- 158 ng/min), without altering MCRA. The major radiolabeled product in blood of infused [3H]A was [3H]testosterone; smaller amounts of phenolic steroids were formed. Extensive metabolism of [3H]A occurred in whole blood in vitro. The major product was [3H]testosterone; the 17-oxidoreductase activity was associated with the red blood cells. Umbilical vein [A] was greater than umbilical artery [A]; ACTH treatment increased [A] in both vessels. Concomitant metopirone abolished the arteriovenous difference by eliminating the ACTH-induced increase in venous [A], although arterial [A] rose significantly. The venous [A] and the arteriovenous gradient were restored with exogenous glucocorticoid treatment to the fetus. Collagenase-dispersed fetal adrenal cells secreted A. Adrenal cells from fetuses pretreated with ACTH in vivo had higher basal and ACTH-induced output of A in vitro than cells from fetuses pretreated with saline in vivo. We conclude that the MCRA in fetal sheep is extremely high, in part due to conversion of A to testosterone in fetal blood. The elevated PRA after ACTH plus metopirone and the lack of an umbilical arteriovenous gradient of [A] in this, but not other groups of fetuses, suggests a source of A production independent of the cortisol-induced changes in the placenta. Direct evidence is provided for fetal adrenal secretion of A which is enhanced by ACTH pretreatment of the fetus in vivo and for the utilization of circulating A in the fetus as a precursor for estrogen in both fetal and maternal compartments.

Adrenal Glands↗
PubMed1986

Sulfohydrolase activity for estrone sulfate and dehydroepiandrosterone sulfate in human fetal membranes and decidua around the time of parturition.

We examined the distribution and kinetic parameters of sulfohydrolase activity in human amnion, chorion, and decidua using estrone sulfate (E1S) and dehydroepiandrosterone sulfate as substrates. Amnion contained low levels of sulfatase activity. Chorion had active sulfohydrolase activity for both substrates, but a significantly greater maximum velocity (Vmax) for E1S. The Km was not different between the two substrates. However, there was a slight but statistically significant decrease in Km and increase in Vmax for sulfohydrolase activity using E1S in chorion from patients delivering vaginally after the spontaneous onset of labor compared to those delivering by elective cesarean section before the onset of labor but at a similar gestational age. Decidua possessed sulfohydrolase for E1S with similar Km and Vmax as chorion. There were no changes occurring around the onset of labor. Using dehydroepiandrosterone sulfate as substrate, the decidua had a similar Km as the chorion, but its Vmax was significantly less. In both tissues for both substrates, the enzyme had highest specific activity in the 105,000 X g pellet, with almost no activity in the soluble fraction. The greatest total sulfohydrolase activity was contained in the 800 X g pellet despite several methods of homogenization and washing of the 800 X g pellet. We conclude that the sulfohydrolase activity of human chorion and decidua may be an important factor in regulating free steroid levels within the pregnant uterus. The significant change in the kinetic parameters of E1S sulfatase may partially explain the increased ability of chorion to hydrolyze E1S which occurs in association with the spontaneous onset of labor.

Carbon Radioisotopes↗
PubMed1985

Relation between cyclic adenosine monophosphate and prostaglandin output by dispersed cells from human amnion and decidua.

We have examined the ability of activators of adenylate cyclase and cyclic adenosine monophosphate to affect the output of prostaglandins E and F by dispersed cells of amnion and decidua collected from women following spontaneous labor. Cyclic adenosine monophosphate production by amnion and decidua cells was stimulated in a dose-dependent fashion by cholera toxin and by forskolin in the absence or presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine. Forskolin and cholera toxin also stimulated prostaglandin E and F output from amnion and decidua cells. Similar effects were seen with cells incubated with dibutyryl cyclic adenosine monophosphate +/- 3-isobutyl-1-methyl xanthine. The beta-adrenergic receptor agonists salbutamol, isoproterenol, and epinephrine all stimulated prostaglandin E and F output from dispersed cells of both tissues. The stimulatory effect of 3-isobutyl-1-methyl xanthine was partially additive with the Ca2+ ionophore A23187. Basal outputs of prostaglandin and outputs stimulated by A23187 and by N6, O2'-dibutyryl adenosine 3':5'-cyclic monophosphate were attenuated by the calmodulin antagonist trifluoperazine in a dose-dependent fashion. We conclude that mechanisms exist for stimulation of adenylate cyclase in human amnion and decidua resulting in enhanced prostaglandin output. This pathway requires basal interaction with Ca2+-calmodulin and may be additive with cyclic adenosine monophosphate-independent mechanisms for prostaglandin stimulation.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Modulation by cortisol of adrenocorticotropin-induced activation of adrenal function in fetal sheep.

We examined the hypothesis that cortisol (F) modulates the activation of adrenal function induced by treating fetal sheep in vivo with pulsatile ACTH (P-ACTH). Chronically catheterized sheep fetuses were infused in utero for 100 h between day 127 and day 131 of pregnancy with P-ACTH; P-ACTH plus metopirone; P-ACTH plus metopirone plus F; P-ACTH plus metopirone plus dexamethasone, or saline (controls). After 100 h, basal and ACTH-stimulated output of 11-desoxycortisol (S), F, and progesterone from collagenase-dispersed fetal adrenal cells was measured. Adrenal cells from fetuses treated with P-ACTH in vivo had significantly greater basal and stimulated (delta) outputs of F and S in vitro than controls. These effects were attenuated in fetuses pretreated with P-ACTH plus metopirone. Concurrent in vivo treatment with ACTH plus metopirone plus F restored basal and delta outputs of F and S to values that were not significantly different from those after P-ACTH alone. In vivo treatment with dexamethasone in addition to P-ACTH plus metopirone significantly raised basal outputs of F and S, but the cells were unresponsive to ACTH in vitro. Basal output of progesterone was significantly greater after in vivo P-ACTH plus metopirone plus dexamethasone, but no treatment raised delta progesterone output over controls. These results support a role for glucocorticoids in modulating ACTH-induced activation of adrenal function in late gestation fetal sheep.

Adrenal Glands↗