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B F Mitchell

Publications and source records attributed to B F Mitchell.

At least 37 records · Page 2Linked to original sources
PubMed1996

Relationships among sex steroids, oxytocin, and their receptors in the rat uterus during late gestation and at parturition.

Sex steroids and oxytocin (OT) produced within intrauterine tissues have been implicated in the regulation of parturition. The purpose of these studies was 1) to determine the relationships among estradiol (E2), progesterone (P4), OT, and their receptors in uterine tissues during late gestation and parturition in the rat; 2) to observe the effects of the estrogen antagonist tamoxifen (TAM) on these factors; and 3) to evaluate the rat as a potential model for events at human parturition. Concentrations of E2, P4, PGE2, and OT were measured by RIA. E2 receptor (ER) was measured by enzyme immunoassay, and P4 receptor (PR) and OT receptor (OTR) were measured by binding assays. OT messenger RNA (mRNA) was measured by ribonuclease protection assay. Groups (n = 5) of pregnant rats (normal gestation = 22 days) were treated with TAM (200 mg/day) or vehicle and killed on gestation day 19, 21, 21.5, or 22 or after delivery of the first pup. Serum E2 increased throughout late gestation accompanied by an increase in uterine OT mRNA and ER. Serum P4 declined after day 19, and uterine PR did not change significantly. Uterine PGE2 increased progressively, reaching peak levels the evening before delivery. Uterine OTR did not increase until the morning of delivery, and uterine OT peptide concentrations increased only during parturition. Parturition was significantly delayed by 24 h in the TAM-treated group. TAM inhibited the increase in serum E2, uterine ER, and OT mRNA and peptide, but had no effect on serum P4 or uterine PR levels. With TAM, the responses of uterine OTR and PGE2 were significantly delayed, but still underwent a significant increase before the delayed parturition. These results support the hypothesis that E2 stimulates the synthesis of ER, OT, and OTR within the rat uterus and is essential for normal parturition. P4 withdrawal may be more important to the increases in OTR and PGE2, but these are delayed in the absence of estrogen. These data also suggest that the rat may be a relevant model for human parturition.

Animals↗
PubMed1995

Estrogen stimulates oxytocin gene expression in human chorio-decidua.

We have recently shown that oxytocin (OT) is synthesized within human amnion, chorion, and decidua during late gestation. The levels of OT messenger ribonucleic acid (mRNA) increased around the time of parturition, suggesting that locally produced OT may play a role in this poorly understood process. In this report, we present results from investigations into the effects of estrogen and progesterone on the synthesis of OT by human chorio-decidua. Using an in vitro incubation system, estradiol at physiological concentrations more than doubled the concentration of OT mRNA. This was reflected by an increase in the amount of OT peptide secreted into the medium. The increase in OT mRNA was antagonized by tamoxifen, suggesting that the effects were estrogen receptor mediated. Progesterone had no effect on OT mRNA synthesis. Using ribonuclease protection assays, mRNAs for estrogen receptor (ER) and progesterone receptor (PR) were detected in all tissues examined. The highest levels were found in decidua, with lower amounts in chorion and very small amounts in amnion and placenta. This is the same relative tissue distribution that we previously demonstrated for OT mRNA. A single transcript was present for ER, and two transcripts were protected for PR. The concentrations of ER mRNA in chorio-decidua were 3-fold higher in tissues obtained after spontaneous labor onset than in tissues obtained from cesarean section at a similar gestational age but before labor onset. Levels of PR did not change significantly. We conclude that synthesis of OT in human chorio-decidua may be regulated in part by estrogen, and that regulation of ER levels may be an important factor modulating this effect. These data support the hypothesis of a paracrine network within human fetal membranes and decidua that may participate in regulating the timing of human birth.

Chorion↗
PubMed1995

Metabolism of oxytocin in human decidua, chorion, and placenta.

Oxytocin (OT) synthesized within human decidua may influence the timing of human parturition. Metabolism of OT within intrauterine tissues may regulate local concentrations. We hypothesized that a decrease in OT metabolism may contribute to an increase in local tissue concentrations around the time of parturition. Thus, we compared OT degradation in human decidua with that in chorion and placenta obtained before or after labor onset at term. We measured kinetic parameters for OT metabolism and determined pathways of degradation. Both cytosol and microsomal fractions contained aminopeptidase and postproline endopeptidase activities. Metabolism in the microsomal fractions was predominantly by an aminopeptidase enzyme that cleaves the ring structure of OT and removes amino acid residues from the N-terminal end. Metabolism in the cytosol fractions was predominantly via postproline endopeptidase activity, which cleaves the C-terminal Leu8-Gly9NH2. The resultant OT-(1-7) also is a substrate for aminopeptidase activity. The apparent maximum velocities of OT metabolism in the cytosol subcellular fractions of decidua (0.87 +/- 0.30 nmol/mg protein.min) and chorion (1.04 +/- 0.47) were significantly (P < or = 0.05) higher than those in corresponding microsomal fractions (0.17 +/- 0.05 and 0.29 +/- 0.10, respectively). Placental cytosols (1.08 +/- 0.34) were similar to decidua and chorion, but the microsomal fractions had significantly greater activity (0.82 +/- 0.22). The Km values for all tissues were in the range of 8-20 mumol/L. There were no significant changes in the kinetic parameters for OT metabolism around the time of labor onset. We conclude that human decidua and chorion as well as placenta actively metabolize OT, but changes in metabolism do not occur around parturition. If increasing decidual concentrations of OT play a role in the timing of human labor onset, mechanisms that increase production or secretion are of primary importance.

Aminopeptidases↗
PubMed1995

Synthesis and metabolism of oxytocin in late gestation in human decidua.

Several studies in the past few years have supported the hypothesis that oxytocin (OT) is synthesized in a paracrine system within the pregnant human uterus and that this paracrine system may be an important regulator of the timing of human parturition. Using ribonuclease protection assays, we have demonstrated a three-fold increase in the rate of synthesis of OT mRNA in human decidua around the time of parturition. We also have shown that a similar increase in OT mRNA and peptide synthesis can be stimulated in vitro by physiological concentrations of estradiol. This increase is inhibited by concomitant use of the estrogen receptor (ER) blocker tamoxifen or by transcription inhibitors. Progesterone had little, if any effect. We also detected mRNAs for ER and progesterone receptor (PR) in amnion, chorion and decidua with the same relative tissue concentrations as OT mRNA. The concentrations of ER but not PR increased significantly around the time of labour onset. To determine if local OT concentrations may be regulated by changes in OT metabolism, we determined kinetic parameters for OT metabolism in decidua, chorion and placenta. [3H]tyrosyl-OT was used as substrate. Metabolites were separated using HPLC and identified using amino acid analysis and mass spectrometry. Metabolism in decidua and chorion occurred predominantly via a cytosolic post-proline endopeptidase and the activity was comparable to placenta. In microsomal fractions, cystine aminopeptidase activity predominated and placenta had significantly more activity than decidua and chorion. There were no changes in any Km or apparent vmax values around the time of parturition. These findings support the existence of a paracrine system within human decidua that involves sex steroids regulating synthesis of OT and that undergoes significant changes around the time of parturition. Changes in local OT concentrations are controlled by rates of synthesis rather than rates of metabolism.

Amino Acid Sequence↗
PubMed1993

Changes in 17 beta,20 alpha-hydroxysteroid dehydrogenase activity supporting an increase in the estrogen/progesterone ratio of human fetal membranes at parturition.

OBJECTIVE: Our purpose was to measure the activity of the reversible enzyme 17 beta,20 alpha-hydroxysteroid dehydrogenase around parturition with estrogen and progestogen substrates. STUDY DESIGN: Classic kinetic studies and explant cultures were used to determine kinetic parameters and net enzyme activities in both oxidative and reductive directions for both sets of substrates. RESULTS: Affinity constant values for estrone, estradiol, and 20 alpha-dihydroprogesterone were 1 to 8 mumol/L. Affinity constant for progesterone was 9 to 25 mumol/L. Maximal velocities for all substrates in the chorion were 20- to 70-fold higher than in amnion and severalfold higher for estrogen substrates compared with the progestins. Around parturition there was a significant change toward net formation of the stronger estrogen (estradiol) and the weaker progestin (20 alpha-dihydroprogesterone), suggesting an increase in the local estrogen/progesterone ratio. CONCLUSION: The enzyme 17 beta,20 alpha-hydroxysteroid dehydrogenase may be an important regulator of the local estrogen/progesterone ratio in fetal membranes around the time of parturition.

20-Hydroxysteroid Dehydrogenases↗
PubMed1993

Synthesis of oxytocin in amnion, chorion, and decidua may influence the timing of human parturition.

Despite the widespread clinical use of oxytocin (OT) as a potent and specific stimulant of labor, previous research data have not supported a role for OT in the physiology of normal human parturition. We have demonstrated synthesis of OT mRNA in amnion, chorion, and decidua using Northern blot analysis, ribonuclease protection assays, and in situ hybridization. Probes directed towards both the 3' and 5' ends of the gene have been used. Levels were highest in decidua with considerably less in chorion and amnion and very low levels in placenta. The transcript size in decidua appears to be 60-80 nucleotides smaller than the transcripts in amnion and chorion. OT gene expression in chorio-decidual tissues increased three- to fourfold around the time of labor onset. Estradiol stimulated synthesis of OT mRNA during in vitro incubation. These results support the hypothesis of a paracrine system involving OT and sex steroids within intrauterine tissues wherein significant changes could occur without being reflected in the maternal circulation. Such a paracrine system could rationalize a long-sought role for oxytocin in the physiology of human labor. These data may lead to novel approaches towards prevention or treatment or preterm labor.

Amnion↗
PubMed1993

The dynamics of prostaglandin metabolism in human fetal membranes and decidua around the time of parturition.

To address whether prostaglandins (PGs) produced in the amnion can gain access to the myometrium, we used three in vitro systems to determine the characteristics of PG secretion, metabolism, and transfer in amnion, chorion, and decidua around the time of parturition. PG metabolism, measured in explant cultures or cytosol preparations, occurs predominantly by the enzyme PG dehydrogenase, which was highest in chorion and was 2- to 3-fold more active for PGE2 than PGF2 alpha. The activity increased significantly around the time of labor onset. The activity of PG-9-ketoreductase, which interconverts the E and F series of PGs, was 2-3 orders of magnitude less than that of PG dehydrogenase. Using a dual chamber perfusion apparatus, we demonstrated that similar amounts of PGE2 were secreted from the fetal (amnion) and maternal (chorio-decidua) surfaces, and this ratio did not change with labor. Using radiolabeled PGE2, radioactivity traversed full thickness membranes at the rate of 4%/h. Only approximately 12% of the transferred radioactivity remained as intact PGE2, and very little conversion to PGF2 alpha was detected. No changes in transfer were detected around the onset of labor. We conclude that it is unlikely that PGE2 produced in the amnion acts directly on the myometrium.

Amnion↗
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↗