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A. P. F. Flint

ABSTRACT

Membrane fractions prepared from the uterine endometrium of untreated ovariectomized sheep contained a 41 × 103 M r protein that was [32P]ADP-ribosylated by pertussis toxin in the presence of [32P]NAD. Progestin and progestin plus oestrogen treatment in vivo increased the concentration of this protein 2·7- and 3·6-fold respectively. Endometrial extracts from untreated or progestin-treated sheep also contained proteins of M r 69 × 103 and 120 × 103 which were ADP-ribosylated in the absence of pertussis toxin; these proteins were not ADP-ribosylated in sheep receiving oestrogen. Incubation of endometrial slices from progestin plus oestrogen-treated sheep with oxytocin in vitro increased phosphoinositide hydrolysis 11-fold. This effect was not altered by prior incubation with pertussis toxin, although toxin treatment reduced by 64% subsequent labelling of the 41 × 103 M r protein when membrane fractions prepared from pretreated slices were incubated with pertussis toxin and [32P]NAD. Thus the endometrium contains a pertussis toxin-sensitive protein which is induced by steroid treatment, but this protein is not involved in the phosphoinositide response to oxytocin.

J. Endocr. (1988) 117, 403–407

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A. P. F. FLINT

SUMMARY

Progesterone in vitro decreases the rates of glucose uptake and of acetate uptake and oxidation, of lipogenesis from acetate and of oxygen consumption, and reduces the intracellular concentrations of ATP and citrate in slices of luteinized rat ovary incubated in a bicarbonate-buffered medium. The effect on glucose uptake was shown to be due to inhibition by progesterone of the membrane transport of glucose. In view of the steroid concentrations used to elicit these effects in vitro and the known actions of steroids such as progesterone on biological membranes, these observations are thought to be due to non-physiological lytic effects.

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A. P. RICKETTS and A. P. F. FLINT

The extra-ovarian contribution to progesterone levels in the circulation has been measured in ovariectomized ewes in which pregnancy was maintained by treatment with medroxy-progesterone acetate. Since adrenal production of progesterone is low this procedure allows the determination of placental secretion. Placental production of progesterone has been shown to rise initially between 50 and 70 days of gestation, with a second phase of increase between 90 and 120 days of gestation. Both periods of increased secretion of progesterone were reflected in rises in progesterone production by placental explants in organ culture and in the activity of 3β-hydroxysteroid dehydrogenase in the tissue. The activity of 3β-hydroxysteroid dehydrogenase exceeded the rate of progesterone production by approximately 30-fold, and it appears unlikely, therefore, that this enzyme is rate-limiting in the synthesis of progesterone. Surgical reduction of the number of cotyledons led to compensation by an increase in weight of individual cotyledons with no significant increase in specific 3β-hydroxysteroid dehydrogenase activity or rate of production of progesterone in organ culture. It was concluded that the rise in placental production of progesterone in the middle period of gestation is unlikely to result either from an increase in 3β-hydroxysteroid dehydrogenase activity or from exposure to factors stimulating placental growth.

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A. P. F. FLINT and P. V. BURROW

SUMMARY

Epitestosterone, a product of the metabolism of androstenedione by the caprine placenta in vitro, is present in the plasma of the pregnant goat. The maternal concentrations of both epitestosterone and unconjugated oestrogens (mostly oestradiol-17α) in the blood increased before parturition and dropped post partum. Measurement of arteriovenous differences at term indicated that epitestosterone was secreted by the uterus; its production was not dependent on the presence of corpora lutea. It is suggested that the concentration of epitestosterone (+ androstenedione + oestrogens) in maternal plasma may be used as an indicator of placental C-17,20 lyase activity; the slight rise in the concentration of these compounds prepartum suggests a relatively small increase in flow through this enzyme.

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E. L. Sheldrick and A. P. F. Flint

ABSTRACT

Peptidyl glycine α-amidating mono-oxygenase (PGA), the terminal enzyme in the pathway of oxytocin synthesis, was measured in extracts of ovine corpora lutea throughout the oestrous cycle. Activity of PGA was low early in the cycle but increased between days 2 and 10 (from 2·3 to 9·0 pmol/mg protein per h) and remained high until day 15. Thereafter, activity declined rapidly at structural luteolysis and was low in corpora albicantia collected 18 and 20 days after ovulation (1·28 and 1·07 pmol/mg protein per h respectively). Luteal concentrations of ascorbic acid, a cofactor for PGA, were high (4·7 μmol/g wet wt tissue) by day 4 after oestrus; concentrations fell rapidly after day 15 (to 2·1 μmol/g on day 16). Concentrations of ascorbic acid were also high in the pituitary gland and in the adrenal medulla and cortex. Concentrations of oxytocin in luteal tissue, which were low (0·3 nmol/g wet wt) on day 2 after oestrus, were highest (2·73 nmol/g) on day 6 and declined thereafter (0·56 nmol/g on day 10, 0·08 nmol/g on day 15 and not detectable on days 18 and 20).

Concentrations of oxytocin, progesterone, PGA and protein were measured in subcellular fractions obtained after density gradient centrifugation of extracts of corpora lutea collected on days 6, 7 and 12 of the oestrous cycle, and on day 7 from an anaesthetized ewe before and after treatment with the prostaglandin F analogue, cloprostenol. PGA co-localized with particle-associated oxytocin in fractions of density 1·049–1·054 g/ml. Exogenous [3H]oxytocin and [3H]progesterone and endogenous progesterone localized in fractions of density 1·035 g/ml. Oxytocin and PGA were depleted from fractions of density 1·049–1·054 g/ml following cloprostenol treatment in vivo.

Fractionation of extracts of ovine corpora lutea by high-performance liquid chromatography (HPLC) followed by radioimmunoassay and radioreceptor assay for oxytocin demonstrated the presence of at least two cross-reacting substances with elution characteristics distinct from oxytocin. Concentrations of these peptides increased as the cycle progressed. These compounds differed from the putative C-terminally extended post-translational processing intermediates, oxytocinyl-glycine, oxytocinyl-glycine-lysine and oxytocinyl-glycine-lysine-arginine, as indicated by their elution positions on HPLC and the specificities of the assays used to detect them, and no conclusions could be drawn on which post-translational processing step was rate-limiting in oxytocin synthesis.

These data are consistent with the suggestion that post-translational processing of oxytocin-neurophysin prohormone takes place in secretory granules in luteal cells. The low activity of PGA early in the cycle may account for the lag previously observed between concentrations of the oxytocin-neurophysin prohormone mRNA and the mature peptide, but post-translational processing intermediates could not be identified. The rate of α-amidation is unlikely to be controlled by availability of ascorbic acid.

Journal of Endocrinology (1989) 122, 313–322

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A. P. F. Flint and R. D. Burton

ABSTRACT

The cytosolic glucocorticoid receptor of ovine placental zona intima has been characterized and measured between day 51 of pregnancy and term, and levels compared with those in fetal lung. By ion-exchange and gel-filtration chromatography the molybdate-stabilized receptor was found to be an acidic molecule with Stokes radius approximately 8 nm; these physicochemical characteristics of the ovine placental receptor are comparable to those of receptors in glucocorticoid target tissues from non-ruminants. Concentrations of cytosolic receptor in placenta (mean, 139 fmol/mg protein) were lower than those in fetal lung (627 fmol/mg) at all stages of gestation investigated. To some extent this difference was accounted for by a twofold higher concentration of protein in placental cytosols compared with those from fetal lung. In both tissues, cytosolic receptor concentrations were maximal between days 91 and 130, when fetal adrenal steroid secretion is low; receptor concentrations decreased before term. Fetal hypophysectomy, which resulted in prolonged gestation, raised receptor concentrations in placenta, but not in fetal lung. In both tissues, apparent dissociation constants for [3H]dexamethasone binding to glucocorticoid receptors were in the range 0·5–7·1 nmol/l; these dissociation constants did not change consistently between day 100 and term. In whole-cell preparations of placenta and fetal lung incubated in vitro there was time-dependent specific binding of [3H]dexamethasone by nuclei, and binding of labelled cytosolic receptor to isolated nuclei occurred at all stages of gestation investigated. Binding of [3H]dexamethasone by cytosolic receptor from placenta and fetal lung was inhibited by progesterone and 17α-hydroxyprogesterone, as well as by cortisol, cortisone, 11-deoxycorticosterone and 11β-hydroxyprogesterone; 20α-hydroxyprogesterone and 17α,20α-dihydroxypregn-4-en-3-one were less effective. In experiments to evaluate the possible antagonistic action of progesterone in whole-cell preparations, uptake of [3H]dexamethasone by nuclei was increased up to twofold in placental minces incubated with aminoglutethimide or epostane, when progesterone synthesis was reduced by 98 and 92 per cent respectively. Nuclear uptake in minces of fetal lung was blocked by concentrations of progesterone found in placenta. The existence of a placental glucocorticoid receptor confirms that fetal cortisol may act directly on the placenta to induce the enzymatic changes controlling the onset of labour. Its availability early in pregnancy is consistent with the ability of administered glucocorticoid to induce labour at any time after day 90 of gestation. Progesterone in the placenta may act as a glucocorticoid antagonist, protecting the fetus against inappropriate induction of preterm labour resulting from high levels of glucocorticoids in the maternal circulation.

J. Endocr. (1984) 103, 31–42

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M. D. MITCHELL and A. P. F. FLINT

SUMMARY

The maternal administration of meclofenamic acid (a prostaglandin synthetase inhibitor) to pregnant sheep prevented the dexamethasone-induced delivery of live lambs and delayed delivery after foetal death in utero. Administration of meclofenamic acid had no effect on the changes in the levels of progesterone and oestrogen in the plasma which occur before lambing in response to foetal glucocorticoid. Despite normal maternal endocrine changes, increased uterine activity did not occur at the expected time, although it could be elicited by vaginal distension or by administration of oxytocin. The rates of cervical ripening and dilatation were reduced by meclofenamic acid and lambing was frequently associated with some degree of cervical dystocia. Withdrawal of meclofenamic acid did not immediately result in an increase in the level of prostaglandin F in the plasma despite the appearance of co-ordinated uterine contractions; the concentration of prostaglandin in the plasma was not raised until vaginal passage of the lambs. It is concluded that the synthesis or release of prostaglandins mediates the effects of changes in the levels of steroids in the maternal plasma on uterine contractility in sheep.

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E. L. Sheldrick and A. P. F. Flint

ABSTRACT

Specific binding of [3H]oxytocin to high affinity sites (hormone receptors) in membrane preparations from uterine tissues of the ewe has been determined at varying stages of the oestrous cycle and in pregnancy. Mean receptor concentrations in caruncular and inter-caruncular endometrium and in myometrium were 14·2, 1·9 and 13·0 fmol/mg protein respectively between days 10 and 13 of the cycle. By the day of oestrus these values had increased to 749, 1085 and 179 fmol/mg protein. These increases in receptor concentrations coincided with luteolysis and falling plasma progesterone levels and followed the preovulatory decline in peripheral oxytocin and rise in ovarian venous oestradiol-17β. Receptor concentrations were low in all uterine tissues from pregnant animals between days 14 and 19 after oestrus. Analysis of binding parameters by Scatchard plot suggested a single population of receptor molecules in each of the tissues studied with apparent dissociation constants in the range 1·9–2·2 nmol/l. A number of naturally occurring neurohypophysial peptides inhibited binding of [3H]oxytocin to the receptor from ewes at oestrus; the cross-reactions of arginine vasopressin and vasotocin exceeded that of oxytocin.

Use of a receptor binding assay to measure oxytocin in extracts of corpora lutea on days 4 and 10 after oestrus gave values similar to those obtained by radioimmunoassay, suggesting the absence of other receptor-active peptides in the corpus luteum.

It is concluded that the oxytocin receptor is present in both components of the endometrium, as well as in the myometrium and that changes in uterine receptor concentrations before oestrus are consistent with receptor activation by steroid hormones.

J. Endocr. (1985) 106, 249–258

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A. P. F. Flint and Marilyn B. Renfree

Oestradiol-17β was measured by radioimmunoassay in cardiac blood from 143 pregnant and post-partum tammar wallabies shot in the wild during reactivation of the diapausing blastocyst, embryonic development, birth and post-partum oestrus. A transient rise in circulating oestradiol on 3 January coincided with or shortly preceded corpus luteum growth and blastocyst expansion; before 5 January mean corpus luteum weight was 14·3± 0·44 mg (n = 65), while thereafter it exceeded 20 mg in two-thirds of the animals. Expanded blastocysts were first noted on 5 January. A second rise in the concentration of oestradiol in plasma, which occurred in late January, preceded parturition and coincided with follicular maturation; the mean (± s.e.m.) oestradiol concentration before 17 January was 27·9 ±1·10 pmol/l (n = 110), whereas on or after this date it was 57·3 ± 4·15 pmol/l (n = 33). Thus oestradiol levels in peripheral plasma increased at parturition and post-partum oestrus, and showed a rise early in gestation which may be related to the termination of diapause.

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M. D. MITCHELL and A. P. F. FLINT

SUMMARY

A technique for the continuous superfusion of small tissue samples in vitro has been applied to the study of prostaglandin production by ovine intra-uterine tissues. Basal and oxytocin-stimulated production of prostaglandins was studied at 120–125 days of pregnancy and after dexamethasone-induced delivery. In general, the relative rate of prostaglandin production by tissues was: foetal cotyledon = maternal cotyledon>myometrium and in quantitative order the prostaglandins produced were prostaglandin E (PGE) > prostaglandin F (PGF) = 13,14-dihydro-15-oxo-prostaglandin F (PGFM). Considerable variation was found between the rates of prostaglandin production in individual sheep. Oxytocin had no effect on the production of prostaglandins by tissues obtained before labour but myometrium and maternal cotyledon obtained at delivery exhibited a significant increase in production of PGE and PGF (though not PGFM) in response to oxytocin. Administration of arachidonic acid increased the production of PGE and PGF by the foetal cotyledon.