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ABSTRACT

Luteal oxytocin-containing secretory granules have been isolated and characterized in terms of their physicochemical parameters. The isopynic sedimentation density (1·03 ±0·003 g/ml) and sedimentation coefficient (1670 S, 0·32 mol sucrose/l, 4 °C) of these granules have been estimated. Based upon these estimates, the average vesicle diameter (258 ± 17 nm) and vesicle weight (9·92 ±0·67 fg/vesicle) were calculated. The exchangeable water content (58·2%) of these granules was determined using density gradients prepared with deuterium oxide. Luteal oxytocin-containing granules displayed similar physicochemical characteristics to those reported for neurohypophysial peptide-containing granules, with the exception of particle size. Luteal granules were 1·3 times greater in diameter than neurohypophysial granules.

J. Endocr. (1988) 116, 267–272

ABSTRACT

The subcellular distribution and compartmentalization of choriomammotrophin (CM) and progesterone within ovine placentomes was investigated using differential and density gradient centrifugation techniques. Approximately 67% of placental CM and 45% of progesterone was associated with subcellular particles. The 10 000 g particulate fraction contained the highest specific activity of both CM and progesterone (19·1 ±3·8 (s.e.m.) μg/mg and 71·5 ± 9·2 pmol/mg protein respectively). This fraction was also shown to contain electron-dense granules with morphology similar to that of hormone-containing secretory granules isolated from other endocrine tissues. Particle-associated CM sedimented to a density of 1·051–1·054 g/ml in colloidal silica gradients and displayed physicochemical characteristics consistent with its storage in secretory granules. During in-vitro incubations, particle-associated CM was stable for up to 90 min, but dissociated when incubated in hypoosmotic medium. Particulate progesterone, which was also present in the CM-rich fraction and was stable for up to 90 min of incubation, was not affected by decreasing the osmolality of the incubation medium. These data suggest that ovine CM (but not progesterone) is stored within a population of secretory granules located within placentomes.

J. Endocr. (1986) 111, 217–223

Abstract

Although it is well established that the formation of eicosanoids by ovine intrauterine tissues increases during pregnancy and at the time of labour, the biochemical mechanisms involved remain to be clearly established. In this study, we tested the hypothesis that the gestational and labour-associated increases in eicosanoid formation are associated with a reduction in the activity of the reacylating enzyme, acyl Coenzyme A lysophosphatide acyltransferase (LAT). To evaluate this proposal, in vitro LAT activity was quantified in ovine placenta (cotyledons) obtained during pregnancy (85–147 days of gestation and at the time of labour). Ovine placental LAT increased from 1·81 ± 0·06 nmol/min per mg protein at 85 days of gestation to 2·34 ± 0·10 nmol/min per mg protein at 142 days of gestation (P<0·005, n=15). The apparent K _m did not vary significantly between the 85- and 142-day groups. V_max, however, was significantly greater in the late-gestation group (2·98 ± 0·02 nmol/min per mg protein) than in the mid-gestation group (2·38 ± 0·13 nmol/min per mg protein, P<0·05). In association with labour, placental LAT activity decreased by 16% (1·96 ± 0·13 nmol/min per mg protein) when compared with that observed in tissue obtained from the non-labouring ewe (P<0·01). The data obtained are consistent with the hypothesis that changes in LAT activity in ovine placenta do not contribute to the gestational increase in prostaglandin formation, but a contribution to the labour-associated increase in non-esterified arachidonic acid availability and eicosanoid formation cannot be negated.

Journal of Endocrinology (1996) 148, 241–247

ABSTRACT

The subcellular distribution of progesterone and oxytocin within the ovine corpus luteum was investigated using differential and density gradient centrifugation. Progesterone and oxytocin were associated with particles which sedimented to a density of 1·049–1·054 g/ml and 1·054–1·061 g/ml respectively. Particle-associated progesterone did not, however, display physical or biochemical characteristics consistent with its storage within secretory granules. When particle-associated progesterone was incubated in HEPES buffer at 37 °C, 70% of the total progesterone was recovered in the incubation medium. The remaining stable particle-associated progesterone was not affected by treatments which stimulated oxytocin release and which have been shown to cause the release of peptides and biogenic amines from secretory granules. These results suggest that particle-associated progesterone represents the intercalation of progesterone into cell membranes and they do not support the hypothesis that progesterone is stored, in a protein-bound form, in luteal secretory granules.

J. Endocr. (1986) 108, 109–116

ABSTRACT

The capacity of cotyledonary microsomes, prepared from pregnant ewes (20–145 days of gestation), to metabolize exogenous arachidonic acid was quantified using a radiolabel technique. During gestation, the capacity of microsomes to metabolize arachidonic acid increased 25-fold, from 0·36±0·06μmol arachidonic acid/incubation (n = 8) at <100 days of gestation to 9·06±1 ·02μmol arachidonic acid/incubation at 130–145 days of gestation (n = 5; P<0·05). Arachidonic acid was metabolized to prostaglandin E₂ and F_2α, as determined by thin-layer chromatography and reverse-phase high performance liquid chromatography. The profile of prostaglandins synthesized by cotyledonary microsomes did not change throughout gestation. These data suggest that the increase in cotyledonary prostaglandin synthesis that occurs during late gestation and at term may reflect an increase in the tissue content of prostaglandin H₂ synthase.

J. Endocr. (1988) 118, 265–270

Abstract

The aims of this study were to investigate the concentration and release of interleukin-1α (IL-1α) at the time of human term labour, and to study the regulation of IL-1α release from human gestational tissue explants by bacterial endotoxin. Immunoreactive IL-1α concentrations in maternal plasma, amniotic fluid and conditioned media from human amniotic, choriodecidual and placental explants were quantified before and after spontaneous term labour-onset and delivery. Furthermore, the effects of a bacterial endotoxin, lipopolysaccharide (LPS), on the release of IL-1α from human gestational tissue explants over a time course of 24 h (n=3) and LPS concentrations ranging from 10–10⁷ pg/ml (n=3) were investigated. IL-1α concentrations in maternal plasma and amniotic fluid did not change significantly with spontaneous term labour-onset. In contrast, IL-1α was released in detectable amounts from human amniotic and choriodecidual explants only in association with term labour-onset and delivery. Similarly, placental release of IL-1α was increased significantly in explant cultures in association with term labour-onset and delivery. LPS increased IL-1α release significantly only from human placental explants from both term not-in-labour and term after-labour tissues. The data demonstrate differential regulation of IL-1α release from human gestational tissues in association with labour and LPS treatment and the observations support the hypothesis that the labour-associated increase in IL-1α release from the fetal membranes is independent of exposure to bacterial endotoxin.

Journal of Endocrinology (1996) 150, 515–522

Abstract

In this study, we quantified interleukin-6 (IL-6) concentrations in amniotic fluid at term and preterm labour, and determined the gestational tissue source of IL-6. In addition, aspects of the regulatory mechanisms involved in IL-6 release at the time of term labour and in response to bacterial endotoxin, lipopolysaccharide (LPS), have been established. IL-6 concentrations were 2-fold higher in amniotic fluid collected at term compared with preterm gestation, with an additional 2-fold increase in association with term labour. IL-6 was released from all choriodecidual and placental explants but was detected in only 33% of amniotic explant cultures of tissues obtained before labour onset. In contrast, IL-6 was detected in all amniotic, choriodecidual and placental cultures of tissues obtained after term labour onset and delivery, and the mean IL-6 release was significantly higher than that measured in explant cultures of both amniotic (80-fold increase, P<0·0001) and choriodecidual (3-fold increase, P<0·02) but not placental explants taken at the time of elective Caesarean section at term before labour onset. LPS significantly (P<0·05) increased the release of IL-6 from human choriodecidual and placental explants but not amniotic explants, in a time- and dose-dependent manner. IL-6 is a physiological constituent of amniotic fluid and its production by gestational tissues is differentially regulated by LPS and spontaneous labour onset and delivery.

Journal of Endocrinology (1996) 149, 431–439

ABSTRACT

The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234·4 ± 32·8 pmol/g per h (n = 24) during 60-min incubations.Activators of protein kinase C: phorbol 12,13-dibutyrate (n = 8), phorbol 12-myristate,13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0·2 μmol/l). Phospholipase C (PLC; 50–250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4).

These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum.

Journal of Endocrinology (1990) 124, 225–232

ABSTRACT

Inhibition of microsomal prostaglandin (PG) biosynthesis by allantoic fluid, obtained from ewes at 80–120 days of gestation, was examined. Inhibition of cotyledonary microsomal PGE₂ and PGF_2α biosynthesis by lyophilized allantoic fluid occurred in a dosedependent manner. The concentration of allantoic fluid required to inhibit PGE₂ and PGF_2α production by 50% averaged 17·9 ± 3·2 (s.e.m.) mg dry weight/ml (n = 5). Microsomal PG biosynthesis was markedly enhanced by the addition of arachidonic acid (30 μmol/l). Synthesis of PGE₂ and PGF_2α was increased to 245 ± 65% and 184±14% of control (P<0·05, n = 5) respectively. Treatment of cotyledonary microsomes with porcine phospholipase A₂ (PLA₂; 0·125 units/ml) also stimulated PG synthesis, PGE₂ increasing to 216 ± 27% and PGF_2α to 172 ± 14% of control (P<0·05, n=5) respectively. Allantoic fluid (20 mg dry weight/ml) inhibited arachidonic acid-stimulated PG synthesis (PGE₂ by 48·6 ± 13·8% and PGF_2α by 44·2 ± 7·7%) and PLA₂-stimulated PG synthesis (PGE₂ by 60·6±11·6% and PGF_2α by 74·8 ± 8·5%). Allantoic fluid, however, did not affect PLA₂-stimulated release of arachidonic acid from microsomes, thus negating the possibility that allantoic fluid suppresses PG synthesis by inhibiting PLA₂ activity. These data indicate that allantoic fluid inhibits PG production at the level of PG synthase enzymes. Prostaglandin inhibitor(s) in allantoic fluid may play a role in maintaining uterine quiescence throughout gestation and its withdrawal, at term, may be involved in the initiation of labour.

J. Endocr. (1987) 114, 295–300

Abstract

Parturition in the sheep is preceded by an increase in the synthesis of prostaglandins by intrauterine tissues. Prostaglandin G/H synthase (PGHS) is the central enzyme involved in prostanoid production. Its expression is enhanced during late gestation in the ewe. Recent studies have identified two PGHS isozymes, termed PGHS-1 and PGHS-2. The labour-associated expression of the two isozymes of PGHS in the sheep has not been characterized.

This study investigated the changes in expression of immunoreactive PGHS-1 and PGHS-2 in ovine amnion and placenta following glucocorticoid-induced labour. Ewes underwent surgery to implant fetal and maternal vascular cannulae and uterine electromyogram electrodes between 118 and 125 days of gestation. Fetal sheep were administered either the glucocorticoid betamethasone (n=5) or saline (control n=6) by direct transabdominal intrafetal injection. Ewes from the betamethasone-injected group were killed in the first stage of labour as indicated by uterine electromyographic activity. Ewes from the saline-injected group were killed at the same time to obtain age-matched control tissue. The time taken to euthanasia following induced-labour onset in the glucocorticoid-injected animals was 56·6 ± 0·8 h post-injection.

Plasma endocrine profiles in the maternal and fetal circulation following glucocorticoid injection were comparable to those observed following normal spontaneous delivery. At post-mortem, amnion and cotyledons were collected in liquid N₂ and stored at −70 °C. Solubilized tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PGHS-1 and PGHS-2 isozymes. Fetal amnion contained PGHS-1 isozyme at day 133 of gestation, as demonstrated in the saline-injected animals. Slightly higher PGHS-1 immunoreactivity was observed following induced-labour onset, although this did not reach statistical significance (P>0·05). PGHS-2 enzyme was not detectable in amnion. PGHS-2 expression was also not induced following labour onset.

In contrast, PGHS-2 demonstrated enhanced expression following glucocorticoid-induced labour in ovine cotyledon. This tissue contained PGHS-1 enzyme, but immunoreactive levels were minimal and demonstrated limited regulation at labour.

These data suggest that the previously reported rise in placental PG production at term in the sheep is predominantly due to increased expression of the PGHS-2 isozyme. This suggests that PGHS-2 contributes to PG production at term labour in sheep or is induced by the mechanisms controlling ovine parturition. PGHS-1 isozyme is produced constitutively in ovine amnion and may contribute to the gestational increase in PG formation by intrauterine tissues.

Journal of Endocrinology (1996) 151, 125–135