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Recent studies in which [7α-3H]dehydroepiandrosterone sulphate (DHA-S) was shown to be converted to oestrogen by minced corpora lutea (Fahmy, Griffiths & Turnbull, 1968a), also showed the formation of 19-hydroxyandrostenedione (19-OH-A) but not 19-hydroxytestosterone (19-OH-T). Little is known concerning the role of testosterone as a direct precursor of oestradiol-17β synthesis in the ovary, and of 19-OH-T as an obligatory intermediate. Even in the placenta the role of 19-OH-T remains equivocal. Perfusion studies of Bolté, Mancuso, Dray, Baulieu & Diczfalusy (1964) suggested that testosterone was directly converted to oestradiol-17β, whereas incubation studies have produced contradictory results (Baulieu, Wallace & Lieberman, 1963; Menini & Engel, 1967). It was therefore decided to investigate further the precursor role of 19-OH-A and 19-OH-T in human luteal tissue.
Radioactive 19-OH-A and 19-OH-T were prepared by incubating either [4-14C]- or [7α-3H]androstenedione with golden hamster adrenal homogenates (Griffiths & Giles, 1965). Incubations
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SUMMARY
Microsomal fractions isolated from post-partum ovine placentae catalysed the synthesis of [3H]oestrone and [3H]oestradiol from [3H]17α-hydroxyprogesterone and NADPH; oestrone and oestradiol were formed in a ratio of approximately 50:1. The expected intermediate, [3H]androstenedione, did not accumulate during these incubations but was shown by trapping experiments to be the intermediate involved. Mean ( ± s.d.) yields of [3H]oestrone (% conversion of substrate) during incubation for 1 h of placentae from five animals in late pregnancy before the onset of labour, from five animals which delivered spontaneously at term and from four animals in which labour was induced by administration of dexamethasone to the foetus were: in tissue obtained before labour, 3·2 ± 0·44; in tissue obtained after the spontaneous onset of labour, 20·6 ± 10·2 (P < 0·01) and in tissue obtained after dexamethasone-induced labour, 24·4 ± 2·13 (P < 0·001). This increase in oestrone synthesis suggests activation of steroid C-17,20 lyase, since this is the step limiting the rate of synthesis of oestrone in vitro. The enzyme is probably activated by foetal glucocorticoid. The findings are discussed in relation to the site of synthesis of oestrogens which in the sheep increase in concentration in the peripheral circulation at term, and with reference to a possible mechanism by which foetal glucocorticoid may control the onset of labour in this species.
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Cortisol: cortisone interconversion was studied in human decidua obtained from three groups of patients at term (37-42 weeks): before the onset of labour (at elective Caesarean section), after labour of spontaneous onset, and after labour of induced onset. When intact tissue was incubated with [3H]cortisol or [3H]cortisone in phosphate buffer without added substrate or cofactors, cortisone to cortisol was the dominant conversion. However, when damaged cells or tissue homogenates were used in the same conditions, the dominant direction of the reaction was reversed, with a large increase in oxidative (cortisol to cortisone) activity. Cortisol: cortisone interconversion was similar in the three groups of samples using either intact tissue or homogenates, as was the total 11β-hydroxysteroid dehydrogenase (11β-HSD) activity measured in tissue homogenates in the presence of added substrate (cortisol or cortisone) and cofactors (NADP+ or NADPH). Endogenous cortisol concentrations in decidua were higher than those of cortisone, and the ratio of cortisol to cortisone was similar in the three groups. These findings suggest that there are no changes in human decidual 11β-HSD activity in relation to parturition. Specific activity of 11β-HSD decreased at high protein concentrations, suggesting the presence of some enzyme inhibitor(s) in homogenized decidual tissue.
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SUMMARY
Using specific radioimmunoassays, 17α,20α-dihydroxypregn-4-en-3-one and progesterone were measured in maternal utero-ovarian venous plasma from four sheep during parturition induced with dexamethasone and two sheep at spontaneous delivery. 17α,20α-Dinydroxypregn-4-en-3-one was also measured in maternal jugular venous and foetal posterior vena caval plasma from one dexamethasone-induced animal over the same period. Basal utero-ovarian venous levels of 17α,20α-dihydroxypregn-4-en-3-one were in the range 3– 7 ng/ml; at the time of the pre-labour fall in maternal progesterone, the concentration of 17α,20α-dihydroxypregn-4-en-3-one increased, reaching 17–76 ng/ml at delivery. Maternal levels fell rapidly after delivery. In contrast to foetal progesterone concentrations, which are low, foetal 17α,20α-dihydroxypregn-4-en-3-one levels were approximately the same as those in the maternal utero-ovarian vein. Maternal jugular venous levels in one animal were 50–80% of those in the utero-ovarian vein. These findings confirm earlier results obtained in vitro which indicated that exposure to glucocorticoid results in increased activity of a steroid 17α-hydroxylase in the ovine foetal placenta.
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Placental tissue was removed from a ewe at 127 days' gestation and, without separating foetal from maternal contributions, incubated as a mince (4 g) with equimolar amounts of [4-14C]androstenedione (2 μCi, 56 mCi/mmol) and [1,2-3H]epitestosterone (15 μCi, 420 mCi/mmol) at 39·5 °C. The reactions were stopped after 2 h by adding acetone and freezing. The non-radioactive carrier steroids, androstenedione, testosterone, epitestosterone, oestrone, oestradiol-17β, oestradiol-17α, testosterone sulphate and epitestosterone sulphate were added in 500 μg amounts.
After mixing, steroids and their conjugates were extracted from the incubation medium with 2 × 100 ml acetone followed by 2 × 100 ml ether: ethanol (3:1, v/v). Nonpolar lipid material was removed by partitioning the pooled, dried extract between 70% (v/v) aqueous methanol and light petroleum (b.p. 40–60 °C). The methanol was evaporated and unconjugated steroids extracted from the aqueous residue with 3 × 30 ml ether before saturating with ammonium
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ABSTRACT
The effects of an inhibitor of 3β-hydroxysteroid dehydrogenase (epostane) on uterine activity and cervical softening have been studied in eight sheep during late pregnancy. Treatment with epostane led to a rapid decline in the concentration of progesterone measured in utero-ovarian venous plasma, to less than 10% of the pretreatment value within 30 min of bolus injection. This was followed by a significant (P<0·02) increase in the concentrations of metabolites of prostaglandins E and F in utero-ovarian venous plasma and uterine activity similar to that seen in the final stages of normal labour. Measurements of cervical tissue extensibility made ex vivo showed the cervix to have softened considerably. These changes occurred without any significant change in the concentration of oestradiol-17β in utero-ovarian venous plasma. Infusion of mefenamic acid, an inhibitor of prostaglandin synthesis, prevented the changes in uterine activity and cervical softening that occurred after injection of epostane alone. Mefenamic acid also reduced the increase in concentrations of metabolites of prostaglandins E and F in plasma, although the concentration of progesterone in these animals showed the same abrupt fall which occurred in sheep after injection of epostane alone. These results suggest that progesterone withdrawal, in the absence of any subsequent rise in circulating oestrogen concentrations, is sufficient stimulus to induce cervical softening in the ewe. Cervical softening following progesterone withdrawal can be prevented by inhibition of prostaglandin synthesis.
J. Endocr. (1985) 105, 227–233
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ABSTRACT
Relaxin, prolactin and prostaglandin synthase were localized by the avidin-biotin immunoglucose oxidase method in human amnion, chorion and decidua. Specimens from ten normal spontaneous deliveries and four elective Caesarean section deliveries with no labour were compared. Relaxin was found more consistently in the cells of the chorionic cytotrophoblast than in the cells of the parietal decidua adherent to the fetal membranes. Only half the tissues after spontaneous delivery contained positive relaxin-stained cells, whereas all the tissues from elective Caesarean sections contained cells positively stained with antiserum to relaxin. In both series of tissues prolactin was localized predominantly in the parietal decidual cells and was very infrequently found in the chorionic cytotrophoblast. Polyclonal antiserum to prostaglandin synthase was used to identify those cells producing prostaglandin in amnion, chorion and decidua. The cells of the amnion and chorion showed positive immunolocalization with no differences between tissues collected before or after labour. Double immunostaining using avidin-biotin immunoperoxidase for prolactin, followed by avidin-biotin immunoglucose oxidase for prostaglandin synthase, produced identical results in the same series of tissues examined with the single-staining method.
J. Endocr. (1987) 114, 491–496
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This paper reports the metabolism of labelled androstenedione and pregnenolone by the liver of foetal sheep. Isolation of reduction products was a major feature of this study.
The livers were removed from sheep foetuses at (a) 129 days', and (b) 143 days' gestation immediately after Caesarean section under spinal analgesia. The tissue was maintained at 0 °C until incubated 1 h later. Chopped tissue (2 g) was incubated in 24 ml Krebs—Ringer bicarbonate glucose solution at 39–5 °C in 95% O2:5% CO2 with the following: incubation (a), 57·4 nmol each of [4-14C]androstenedione (34·8 mCi/mmol) and [7α-3H]pregnenolone (14·7 Ci/mmol); incubation (b), 57·4 nmol of [4-14C]androstenedione (34·8 mCi/mmol). Reactions were stopped after 2 h with acetone containing 500 μg each of the listed carrier steroids (Table 1). The following were unobtainable as sulphates and were added as free steroids to the conjugate fraction before
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ABSTRACT
Human term decidua produces prostaglandins (PGs) which have been implicated in the initiation of human parturition. Using flow cytometry to isolate pure cell populations, we have investigated the cell types responsible for decidual PG production. Cell dispersions were prepared enzymatically from decidua vera isolated from term placentae, and were incubated in Dulbecco's Modified Eagle's Medium containing 0·25% bovine serum albumin at 37 °C. PGF2α and PGE2 output were measured by radioimmunoassay of the conditioned medium. Production of PGF2α (fmol/106 cells per 3 h) exceeded that of PGE2 at 273 (108–322) 322) versus 97 (38–127) respectively (median (range)). The decidual cell dispersions were then incubated with monoclonal antibodies (anti-CD45 which labels the leukocyte common antigen or anti-human leukocyte antigen class II (HLA-DR) which is specific for macrophages in this tissue) and sorted by flow cytometry. The resultant antibody-positive and -negative cell populations were incubated and PG production was measured. Controls showed that antibody labelling and sorting did not alter PG production. PGF2α and PGE2 output by bone marrow-derived (CD45-positive) cell populations exceeded that of non-bone marrow-derived (CD45-negative) cells. Furthermore, we were able to demonstrate that the HLA-DR-positive macrophage population had the highest PGF2α and PGE2 production rates in human term decidua in vitro.
Journal of Endocrinology (1991) 131, 327–334
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SUMMARY
Maternal plasma progesterone levels in sheep may fall dramatically during the last few days of gestation and following the administration of glucocorticoids to the foetus. To investigate the mechanism of the fall, metabolism of [3H]pregnenolone and/or [3H]progesterone in vitro by ovine placental tissue was studied in five ewes before and after intra-foetal administration of dexamethasone in a dosage sufficient to induce parturition, and in one ewe after the spontaneous onset of labour at 143 days of gestation. Manual separation of maternal and foetal placental tissues showed that, in 11 out of 12 cases, the foetal and not the maternal placenta produced progesterone from pregnenolone in vitro. Total activities of cholesterol side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase in the foetal placenta were not influenced by intra-foetal dexamethasone. Before administration of dexamethasone, homogenates of foetal placenta converted [3H]progesterone to 20α-hydroxy [3H]pregn-4-en3-one in the presence of NADPH. Within 12 h of administration of dexamethasone, and after the natural onset of labour at 143 days, large amounts of 17α,20α-dihydroxy[3H]pregn4-en-3-one were formed from [3H]progesterone. Intra-foetal dexamethasone treatment also induced the formation of 17α,20α-dihydroxy[3H]pregn-4-en-3-one by minced foetal placental tissue incubated with [3H]pregnenolone. This change in steroid metabolism did not occur in foetal placental tissue from a sham-operated animal receiving no dexamethasone. Assay of progesterone in foetal placentae showed that the increased formation of 17α,20αdihydroxypregn-4-en-3-one was unlikely to be caused by a change in the specific activity of added 3H-labelled precursor, although the production of 17α,20α-dihydroxypregn-4-en3-one in vitro increased at a time when both foetal placental and utero-ovarian venous levels of progesterone were decreasing in response to dexamethasone treatment. These observations indicate that intra-foetal dexamethasone treatment induces a placental 17α-hydroxylase enzyme, which is also present in foetal placental tissue after the spontaneous onset of labour at term.
