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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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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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E. L. SHELDRICK, A. P. RICKETTS and A. P. F. FLINT

A major product of progesterone metabolism by the goat placenta in vitro was found to be 5β-pregnane-3α,20α-diol. The concentration of this steroid has been measured by radioimmunoassay in the peripheral circulation during pregnancy. Peripheral plasma concentrations of 5β-pregnane-3α,20α-diol were low (less than 6 nmol/l) in anoestrous and non-pregnant ovariectomized goats, and during the first month of pregnancy but increased progressively after day 45 of pregnancy, reaching 78–94 nmol/l between days 112 and 142. Thereafter levels declined before term. Changes in the plasma concentration of 5β-pregnane-3α,20α-diol during pregnancy in the goat therefore resembled those of progesterone in the sheep. Plasma concentrations of 5β-pregnane-3α,20α-diol between day 70 and term were not influenced by repeated administration of medroxyprogesterone acetate from days 51 to 82 or by lutectomy in goats treated with medroxyprogesterone acetate. Secretion of 5β-pregnane-3α,20α-diol by the uterus and its contents was indicated by a positive venous–arterial difference across the uterus between days 128 and 141 in three ovariectomized pregnant goats receiving medroxyprogesterone acetate. Comparison of the rates of metabolism of progesterone by homogenates of placenta in vitro showed that the placental tissue from goats was three times more active in this respect than was tissue from sheep. The ratio of the plasma concentrations of 5β-pregnane-3α,20α-diol and progesterone in late pregnancy in ovariectomized or lutectomized goats exceeded by a factor of 10 that in sheep at a comparable stage of gestation. It is suggested that reductive metabolism of progesterone before it is secreted may account for the inability of the placenta to maintain pregnancy after ovariectomy in goats.

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E L Sheldrick, H C Flick-Smith, D E Bendall and A P F Flint

Abstract

Oxytocin-induced prostaglandin F (PGF) responses were measured in explants of uterus from ovariectomized ewes on the day of tissue collection or after culture for 72 h in the presence or absence of oestradiol-17β (100 nmol/l). Oxytocin receptor binding activity was 210 ±47 fmol [3H]oxytocin bound per mg protein in fresh tissue and 89 ± 24 and 90 ± 17 fmol/mg in tissue cultured with control medium or with oestradiol respectively (means ± s.e.m.).

PGF production during the hour following oxytocin administration to freshly collected tissue was 272 ± 77 ng/g/h compared with 193 ± 35 ng/g/h in the absence of oxytocin. These rates were 2789 ± 1085 and 353 ± 135 ng/g/h after culture for 72 h in control medium and 2022 ± 496 and 342 ± 134 ng/g/h after culture with oestradiol. Thus oestradiol had no effect on the culture-induced maturation of the PGF response. Short-term exposure to arachidonic acid (66 μmol/l) did not increase PGF production in fresh tissue but significantly increased basal but not oxytocin-induced PGF production after 72 h in culture (P<0·05). There was an absence of oxytocin-induced inositol phosphate turnover in fresh tissue but after culture concentrations of inositol mono-, bis- and trisphosphates were all significandy increased by oxytocin (P<0·005). Antisera directed against G-protein α sub-units αi3, αo, αq, α11 and the common β subunit, and prostaglandin H-synthase-1 detected proteins that were present before and after culture. Oestradiol had no effect on the presence or apparent concentrations of these proteins.

Journal of Endocrinology (1995) 145, 299–305

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J. D. Curlewis, M. B. Renfree, E. L. Sheldrick and A. P. F. Flint

ABSTRACT

Pituitary glands and corpora lutea collected at various stages of the reproductive cycle of the tammar wallaby (Macropus eugenii), were extracted and fractionated by high-performance liquid chromatography, and specific radioimmunoassays were used to measure mesotocin ([Ile8]-oxytocin) and oxytocin. Mesotocin, but not oxytocin, was identified in extracts of pituitary; the mean concentration of mesotocin in this tissue was 0·75 nmol/g wet weight. Neither mesotocin nor oxytocin was detected in extracts of corpus luteum. In female Bennett's wallabies passively immunized against mesotocin during seasonal reproductive quiescence, there was no significant effect on peripheral progesterone concentrations and there were no births, matings or changes in vaginal smears in the 2 months following treatment. Thus mesotocin is unlikely to act as a systemic luteostatic agent during seasonal quiescence.

J. Endocr. (1988) 117, 367–372