Activities of cyclooxygenases, and levels of prostaglandins E2 and F2alpha, in fetopathy associated with experimental diabetic gestation.

AIM: The present study investigated the cyclooxygenase (COX) pathway to elucidate any changes that may be involved in the mechanism(s) underlying diabetic fetopathy. METHODS: Diabetes was induced in female rats (n=12) by two successive daily injections of 55 mg/kg streptozotocin, while control animals (n=10) were injected with a buffer solution; hyperglycaemia was confirmed by blood glucose levels greater than 11 mmol/L. The study female rats were made pregnant and, on day 15 of gestation, the rats were sacrificed, and the fetuses, placentas and membranes dissected out of the uterine horns. Following morphological examination, the fetuses, placentas and membranes were homogenized, and used to measure COX activities and prostaglandin (PG) E(2) and PGF(2alpha) levels. RESULTS: Fetuses from diabetic mothers exhibited significantly (P<0.05) shorter crown-to-rump lengths, lower body weights and heavier placental weights. The activity of COX-1 in the fetuses, placentas and membranes from diabetic mothers represented a small percentage of total COX activity compared with that of COX-2. The presence of a COX-1 inhibitor in the control and diabetic rats was investigated and found to be negative. The activity of COX-2 in malformed fetuses from diabetic mothers was significantly lower (P<0.05) compared with non-malformed fetuses from control and diabetic mothers. The mean level of PGE(2) in fetuses from diabetic mothers was significantly (P<0.05) lower than that in controls. In contrast, the biggest increases in PGF(2alpha) were observed in the malformed diabetic fetuses, placentas and membranes. CONCLUSION: The increased production of PGF(2alpha) probably proceeds, at least in part, independently of the COX pathway and via the isoprostane route. However, it is unclear whether the relatively high levels of PGF(2alpha) are causally related to, or simply coincidental with, fetal malformation.

Expression and localization of the contractile prostaglandin F receptor in pregnant rat myometrium in late gestation, labor, and postpartum.

A polyclonal antibody was raised against amino acids 7-18 in the first extracellular loop of rat prostaglandin F (FP) receptor to monitor expression and localization in pregnant rat myometrium at Gestational Days 16, 18, 20, 21, 21.5, 22 (delivery), and 23 (1-day postpartum; n = 5 per group). The antibody recognized a protein of approximately 43 kDa on Western blot analysis in both membrane (soluble and nonsoluble) and cytosolic fractions of myometrium on each day of gestation. Expression of FP protein increased significantly (P < 0.05) during late gestation in both soluble membrane and cytosolic fractions, being significantly greater at Day 21.5 than at Day 20 of gestation in the soluble membrane fraction and in the cytosolic fraction of tissues collected during labor compared with those obtained before labor. The total concentration of FP receptor in the membrane (soluble plus nonsoluble) remained high throughout late gestation and fell significantly (P < 0.05) in the postpartum period. The FP receptor in the soluble membrane fraction (compared to the total membrane FP receptor) was significantly (P < 0.05) higher in late gestation than earlier, whereas the ratio of FP protein in cytosolic to that in the total membrane was significantly (P < 0.05) higher on Day 23 than earlier in gestation, suggesting a dynamic movement of FP with advancing gestational age. Immunoreactive FP receptor localized to circular and longitudinal smooth muscle at all gestational ages, but changes in intracellular localization were observed in late gestation with a staining pattern similar to alpha-actin, suggesting an association with myofibrils. Our study suggests an increase in FP-receptor protein in myometrium with advancing gestation and a marked elevation at term. This supports a role for uterine FP receptors in mediation of uterine contractility at term.

Effect of finadyne on oestradiol-induced ovarian oxytocin and uterine PGF2alpha secretory systems on day 15 after oestrus in ovarian autotransplanted ewes.

This study was undertaken to determine whether induction of ovarian oxytocin after oestradiol treatment on day 15 after oestrus is mediated through prostaglandin secretion by blocking prostaglandin synthesis using finadyne, an inhibitor of the cyclo-oxygenase pathway. Nine ewes with ovarian autotransplants were assigned randomly to receive an i.m. injection of either oestradiol benzoate (50 microg) in peanut oil ( n= 5) or oestradiol benzoate plus finadyne (2.2 mg kg (-1)) ( n= 4) at 3 h intervals starting at the time of oestradiol injection. Blood samples were collected from the ovarian and contralateral jugular veins at 30 min intervals for 6 h before and at 15 min intervals for up to 9 h after the oestradiol and finadyne injections. The secretion rate of ovarian progesterone remained high in all ewes, thus indicating the presence of a functional corpus luteum. Peripheral oestradiol concentrations were significantly (P < 0.001) higher during the 9 h after oestradiol injection in both groups. None of the oestradiol-finadyne-treated ewes showed significant pulses in either ovarian oxytocin secretion or release of the prostaglandin F(2alpha) metabolite 13,14-dihydro-15-keto PGF(2alpha) (PGFM) after injections. In ewes treated with oestradiol only, at least one detectable pulse of ovarian oxytocin and jugular PGFM was observed with mean +/- SEM amplitude of 17.7 +/- 7.29 ng min (-1) and 237.18 +/- 43.13 pg ml (-1), respectively. The areas under the curve for ovarian oxytocin and jugular PGFM pulses were significantly increased after oestradiol treatment. These findings demonstrate that initiation of the arachidonic acid cascade is important for the secretion of oxytocin after oestrogen treatment.

Co-localization of Rab3B and oxytocin to electron dense granules of the sheep corpus luteum during the estrous cycle.

Oxytocin and its carrier protein, neurophysin, are both associated with luteal secretory granules which migrate from the paranuclear region to the cell membrane where exocytosis takes place. Rab3 proteins are thought to be associated with membrane vesicles or granules undergoing exocytotic fusion with the plasma membrane. The objective of this study was to determine whether Rab3B is co-localized with oxytocin within the same secretory granules of large luteal cells obtained from corpora lutea of 16 Merino cross ewes at day 3, 7, 12 or 15 of the estrous cycle using immunocytochemistry. The mean granule density (granules/microm3) was not significantly different (P > 0.05) between the days examined. Electron microscopic immunocytochemistry showed that oxytocin and Rab3B were co-localized to the secretory granules on all days evaluated. Rab3B immunostaining was primarily located within secretory granules scattered throughout the cytoplasm. The mean intensity of labelling (number of gold particles) for oxytocin per microm2 cytoplasmic luteal tissue was significantly decreased on day 15 compared to those observed on days 3, 7 and 12 of estrous cycle. No significant changes were observed in the mean intensity of the Rab3B label at the different times of the cycle. The present study provides evidence that a member of the subfamily of Rab proteins, Rab3B, is present and co-localized with oxytocin in the same secretory granules of the ovine corpus luteum. These results implicate Rab3B protein directly or indirectly in the hormone secretory pathway of ovarian tissue.

Effects of progesterone on expression of messenger RNA encoding oxytocin-neurophysin, oxytocin receptor and prostaglandin G/H synthase-1 and -2 during the early oestrous cycle in the ovine corpus luteum.

This study was conducted to determine whether early progesterone treatment plays a role in the regulation of messenger RNA (mRNA) expression for oxytocin-neurophysin, oxytocin receptor, prostaglandin G/H synthase (PGHS)-1 and PGHS-2 in the ovine corpus luteum. The expression of ovarian oxytocin, oxytocin receptor, PGHS-1 and PGHS-2 mRNA was investigated in control, progesterone- or RU486-treated ewes. Fifteen ewes were randomly assigned to three groups to receive intramuscular injections of progesterone (12.5 mg; n = 5), RU486, (2.5 mg kg(-1) bodyweight; n = 4) or corn oil (1 mL; n = 6) twice daily from Day 1 to Day 3 post oestrus. On the morning of Day 4 post oestrus, the corpora lutea were collected and analysed for oxytocin-neurophysin mRNA by Northern blot using a labelled cDNA probe, and for the expressions of the oxytocin receptor, PGHS-1 and PGHS-2 mRNA using the reverse transcription polymerase chain reaction. Administration of progesterone or suppression of progesterone activity with RU486 did not affect expression of oxytocin-neurophysin mRNA (P>0.05). Pretreatment of the ewes with progesterone resulted in the enhancement of luteal oxytocin receptor mRNA expression and suppression of PGHS-1 and PGHS-2 mRNA (P<0.001). These results indicate that early progesterone treatment does not control the expression of oxytocin-neurophysin mRNA in the ovine ovary but may be involved in the regulation of ovarian oxytocin receptor and PGHS expression. It is proposed, on the basis of these results, that progesterone may play a role in premature corpus luteum regression through an intra-ovarian mechanism involving the induction of ovarian oxytocin receptor mRNA expression.

Oestrogenic effects of ICI 182,780, a putative anti-oestrogen, on the secretion of oxytocin and prostaglandin F2 alpha during oestrous cycle in the intact ewe.

The effect of ICI 182,780, oestrogen antagonist, on the concentration of oxytocin and uterine PGF2 alpha was investigated in intact Border Leicester Merino cross ewes during the late oestrous cycle. Twelve cyclic ewes (n = 6 per group) were randomly assigned to receive, at 6 h intervals, intra-muscular injection of either peanut oil or ICI 182,780 (1.5 mg kg-1 day-1) in oil for 2 days, starting at 1900 h on day 13 until 1300 h on day 15 post-oestrus. Hourly blood samples were collected via a jugular catheter from 0800 h on day 14 for 37 h and then daily over days 16, 17 and 18 post-oestrus. Peripheral plasma concentrations of oxytocin, the metabolite of prostaglandin F2 alpha, 15-keto-13,14-dihydro-prostaglandin F2 alpha, (PGFM) and progesterone were measured by radioimmunoassay. All ewes treated with ICI 182,780 exhibited functional luteal regression as indicated by a marked reduction in plasma progesterone concentrations to less than 1000 pg/ml over the period of 18-36 h during sampling period on days 14 and 15 of the oestrous cycle. In five of six vehicle-treated ewes, progesterone concentrations declined between day 16 and day 18 post-oestrus. In the remaining control ewe, progesterone concentrations reach less than 1000 pg/ml within 36 h of the commencement of the sampling period. During the frequent sampling period, the number of oxytocin pulses in the ICI 182,780 treated ewes was significantly higher compared to control ewes (2.7 +/- 0.3 vs. 0.8 +/- 0.3). The mean amplitude of oxytocin pulses observed was also greater (70.4 +/- 19.5 pg/ml) in ewes treated with ICI 182,780, but was not significantly different from the control ewes (33.5 +/- 12.9 pg/ml). Oxytocin pulses may however have occurred following the initial two ICI 182,780 injections but before commencing blood sampling. The oxytocin pulses were detected at a mean of 3.2 +/- 0.2 h following each injection with ICI 182,780 during blood sampling. In the ICI 182,780-treated ewes, the pulsatile pattern of plasma PGFM in jugular blood samples over the 37 h sampling period on days 14 and 15 post-oestrus had a higher amplitude (512.9 +/- 158.9 vs 121.7 +/- 78.7 pg/ml) and pulse area (618.1 +/- 183.3 vs 151.5 +/- 102.9 (ph/ml)tau) compared to the vehicle-treated ewes (P < 0.05) respectively.. The average number of PGFM pulses observed per ewe was 3.0 +/- 0.7 in the ICI 182,780-treated group and was significantly (P < 0.02) higher than the number of pulses (0.5 +/- 0.3) observed in ewes treated with vehicle alone. The PGFM pulses were detected at 4.2 +/- 0.6 h following each injection with ICI 182,780 during blood sampling. The percentage of PGFM pulses that occurred coincidently with significant elevation of oxytocin concentrations was 44.4% in ICI 182,780-treated compared to 66.6% in control ewes. We conclude that administration of oestrogen antagonist ICI 182,780 accelerated development of the luteolytic mechanism by enhancing pulsatile secretion of oxytocin and PGFM which suggests that ICI 182,780 acts as an agonist for oxytocin and prostaglandin f2 alpha release in intact ewes when administered at 1.5 mg/kg/day over day 13 to 15 post-oestrus.

Stimulation of ovarian oxytocin secretion and uterine prostaglandin release by exogenous progesterone early in the cycle of the ovarian auto-transplanted ewe.

The present study was undertaken to determine whether the administration of progesterone, early in the oestrous cycle, had an influence on ovarian oxytocin secretion and on peripheral concentrations of the prostaglandin F2 alpha metabolite 13,14-dihydro-15-keto PGF2 alpha (PGFM) in the ovarian auto-transplanted ewe. Twelve ewes with ovarian auto-transplants (n = 6 per group) were randomly assigned to receive an i.m. injection of progesterone (12.5 mg) or vehicle, twice a day, on days 1, 2 and 3 of the oestrous cycle. Beginning on day 7, blood samples were collected at intervals of 1 h from the ovarian and contralateral jugular veins for up to 70 h. Ovarian oxytocin secretion rate and jugular concentrations of PGFM and progesterone were determined by radioimmunoassay. The number of ewes that showed pulses of both ovarian oxytocin and PGFM was significantly (P < 0.05) greater in progesterone-treated ewes than in control ewes. In progesterone-treated ewes, the average number of ovarian oxytocin pulses per ewe was 9.66 +/- 5.5 (mean +/- SD) and the interval between pulses was 7.18 +/- 5.8 h. The mean amplitude and amount of oxytocin released, as calculated by the area under the curve of ovarian oxytocin pulses, were 6.27 +/- 1.98 ng min-1 and (10.05 +/- 8.91 ng min-1)tau, respectively (where tau is the number of hours between the last time point before and the first time point after a significant increase in hormone concentration was detected by the Pulsar program). The mean amplitude and area under the curve of PGFM pulses were 317.22 +/- 5.65 pg ml-1 and (383.36 +/- 1.77 pg ml-1)tau, respectively. The average number of pulses of plasma PGFM observed per ewe was 5.8 +/- 1.9 and interpulse interval for plasma PGFM pulses was 10.32 +/- 8.7 h between day 7 and day 9 after oestrus. These data indicate that administration of progesterone during the first 3 days of the oestrous cycle results in the premature release of ovarian oxytocin and uterine prostaglandin F2 alpha.

Effect of oestradiol on ovarian oxytocin secretion rate and luteolysis in the ewe after ovarian auto-transplantation.

The release of ovarian oxytocin and uterine prostaglandin (PG)F2alpha in response to an oestradiol stimulus was investigated. On Day 15 post-oestrus, ten ewes with ovarian auto-transplants (n=5 per group) received an intra-muscular injection of either oestradiol benzoate (50 microg) or vehicle. Blood samples were collected from the ovarian and jugular veins at 30 and 0 min before, and at 15-min intervals up to 540 min after, injection. The secretion rate of ovarian progesterone remained elevated in four of five treated ewes and in all control ewes, indicating the presence of a functional corpus luteum. Peripheral oestradiol concentrations were significantly (P < 0.001) higher in treated than in control ewes. The number of ewes that released pulses of ovarian oxytocin > or =240 min following oestradiol benzoate injection was significantly (P < 0 05) greater than that in control ewes. Mean amplitude and area under both ovarian-vein oxytocin and jugular-vein 15 keto-13,14 dihydro prostaglandin F2alpha (PGFM) pulses were significantly increased in the treated ewes. These findings demonstrate that the administration of exogenous oestrogen provides a positive stimulus for the release of ovarian oxytocin and uterine PGF2alpha in the ovarian auto-transplanted ewe.