Relaxin and progesterone are myometrial inhibitors in the ovariectomized non-pregnant mini-pig

Intravenous bolus injections of pig relaxin which produced short-lived peaks of the hormone equivalent in concentration to those observed at term promptly rendered the uterus almost totally quiescent and the inhibition persisted for about 2.5 h. During this time the uterus remained responsive to oxytocin. The main effect of relaxin was to reduce the frequency of intrauterine pressure cycles rather than the amplitude. In contrast progesterone, which also inhibited myometrial activity, took between 6 and 24 h to exert its maximum effect by reducing both amplitude and frequency of IUP cycles and it also abolished the responsiveness of the myometrium to oxytocin. Its actions were reversible but recovery took between 54 and 140 h. Oestradiol benzoate had no significant effect on myometrial activity in 21 out of 26 treatments. At 24 h after the 5 remaining treatments, however, myometrial activity was virtually zero. No evidence was obtained of a biphasic effect of oestradiol on myometrial activity as reported for the rat and ewe. This work demonstrates that purified pig relaxin is an active myometrial inhibitor in the oestrogen-treated ovariectomized non-pregnant pig in vivo.     

The effect of relaxin on in vivo uterine electromyographic activity in the conscious ovariectomized ewe is oestrogen-dependent

Electromyographic (EMG) activity of the uterus was recorded in vivo in 4 conscious ovariectomized ewes treated with oestrogen (one injection of 100 micrograms of oestradiol benzoate). Doses of 500 micrograms of purified porcine relaxin were injected intravenously 12, 16, 20, 24, 28 and 32 hrs after oestrogen treatment. Relaxin had an inhibitory effect on uterine activity between 16 and 32 hrs after oestrogen treatment. Because the period during which relaxin had a biological effect corresponded to the moment when oestrogen-induced uterine activity was maximal, it is suggested that the ability of relaxin to inhibit uterine activity in the ewe is oestrogen-dependent.     

Blood LH after PGF2alpha in diestrous and ovariectomized cattle.

Two experiments were conducted to determine whether the increased serum LH which occurs within 12 hr after a luteolytic dose of PGF2alpha is dependent upon changes in progesterone or estradiol secretion. In the first experiment, exogenous progesterone abolished the increase in serum LH caused by a subcutaneous injection of 25 mg PGF2alpha in diestrous heifers, but not in ovariectomized heifers. In the second experiment, progesterone pessaries were removed at 6 hr after a subcutaneous injection of 25 mg PGF2alpha. LH remained at pre-PGF2alpha values while the pessaries were in place, but began to increase within 1 hr after they were removed. Blood estradiol also remained at pre-PGF2alpha values until the pessaries were removed, and began to increase at 2 hr after pessary removal. We conclude that the increase in serum LH within 12 hr after PGF2alpha treatment in diestrous cattle is dependent upon withdrawal of progesterone; it is not due to increased serum estradiol.     

Release of PGF(2alpha) during parturition and the postpartum period in the ewe

Peripheral plasma levels of 15-keto-13, 14-dihydro-PGF(2alpha) were determined from one week before parturition until five weeks postpartum in six ewes. Starting at approximately 1 nmol/l one week before parturition, the prostaglandin metabolite levels rose successively during the week preceding parturition and peaked at 70 to 125 nmol/l during parturition. During the first three days postpartum the concentrations of the 15-keto-13, 14-dihydro-PGF(2alpha) decreased to 0.5 to 1 nmol/l. These levels were maintained for three weeks and low levels of 50 to 100 pmol/l were reached around four weeks postpartum. The ewe, as well as other ruminants, has a sustained release of elevated concentrations of PGF(2alpha) postpartum. It seems likely that PGF(2alpha) plays a role in uterine involution.     

An electromyographic study of uterotubal activity in the ewe.

The electrical activity of the oviduct and uterus was automatically registered on a potentiometric pen recorder in ewes fitted with an extracellular multielectrode assembly. The duration and amplitude of local non-propagating activity of the uterus increased and became progressively grouped into phases as natural oestrus progressed. Phases of contractions lasting 5-6 min were initially propagated at a rate of 2/hr. Their frequency increased within 24 hr of the decline in plasma progesterone levels. Grouped activity was then resumed for the next 24 hr. Similar changes were seen in the activity of the oviduct although this region was active earlier in oestrus. The uterotubal activity developed in the same way 24 hr after withdrawal of a progestagen-impregnated sponge but lasted for 3 days. When another oestrus was induced by injection of a prostaglandin analogue, the activity patterns were qualitatively similar although they did not start until 36 hr after injection and lasted for only 2 days.     

Influence of PGF2 alpha on gastrointestinal activity in the conscious piglet.

In 5 conscious piglets with electrodes implanted on the antrum pylori, duodenum, jejunum and ileum, the effect of intravenous infusion of PGF2 alpha, 1 and 10 micrograms/kg/min during 2 h, on gastrointestinal electrical activity was studied. The influence of the PG, 10(-8) to 10(-4) M, on longitudinal tissue strips from the same segments was also examined. The in vitro results demonstrate that PGF2 alpha has only a weak contractile effect on duodenal and jejunal strips. This effect was enhanced in the presence of atropine and indomethacin. In the in vivo part of the study PGF2 alpha induced an inhibition of antral electrical activity as evidenced by a prolongation of the inhibitory phases and a reduction of the frequency of the fast oscillations. In the small intestine only ileal activity was changed significantly. PGF2 alpha provoked an increase in the phase II or irregular spiking activity and an increase in the interval of the migrating myoelectrical complexes in this segment.     

Novel effects of PGF2 alpha on airway function in asthmatic subjects

The effects of inhaled prostaglandin F2 alpha (PGF2 alpha) have been examined in eight subjects with asthma. Incremental PGF2 alpha aerosol concentrations, ranging from 1 to 5,000 micrograms/ml, were administered at 15-min intervals. Plethysmographic specific airway conductance (sGaw), forced expiratory volume at 1 s (FEV1), and maximum expiratory flow at 50% vital capacity breathing air (Vmax50% air) and 80% He-20% O2 (Vmax50% He-O2) were measured after each dose and compared with saline control values. We observed unexpected triphasic dose-response characteristics, i.e., an initial decline in physiological variables at low concentrations (1-100 micrograms/ml), followed by improvement at intermediate concentrations (100-1,000 micrograms/ml) and a subsequent steep decline at high concentrations (1,000-5,000 micrograms/ml). Improvement in FEV1 and Vmax50% air between 100 and 1,000 micrograms/ml was associated with sGaw increases above control levels in six subjects and a significant fall in density-dependent index (Vmax50% He-O2/Vmax50% air) when compared with values before challenge and at low concentrations. Inhaled atropine (5 mg) improved prechallenge lung function but had no effect on PGF2 alpha dose-response characteristics. Intermediate PGF2 alpha concentrations given as a single dose consistently induced greater FEV1 reductions than the same concentration during graded dose challenges. Our findings are consistent with the demonstration of in vivo airway tachyphylaxis and indicate that airway effects of PGF2 alpha are far more complex than previously reported. Moreover, these novel effects suggest that, in addition to its well-known bronchoconstrictor effects, PGF2 alpha directly or indirectly causes airway relaxation, predominantly in large airways.     

Effect of post-mating GnRH analogue (buserelin) treatment on PGF2alpha release in ewes and ewe lambs

The objectives of this study were to determine the effects of buserelin or saline treatment on ovarian function (Experiment 1), plasma PGFM concentrations and oxytocin stimulated prostaglandin F(2alpha) (PGF(2alpha)) release (Experiment 2) in ewe lambs and ewes. Welsh Halfbred ewes (n=26) and ewe lambs (n=24) were mated to vasectomised rams at synchronised oestrus and on Day 12 post-mating each animal was injected intramuscularly either normal saline or 4 microg buserelin. In Experiment 1, plasma progesterone and oestradiol concentrations were determined in samples collected by jugular venepuncture 1h before and at 0, 2, 4, 6, 8, 24, 48 and 72 h after treatment (n=7 per treatment group). Progesterone concentrations increased (P<0.05) from 2 to 8h after buserelin treatment and returned to basal levels after 72 h, whereas oestradiol concentrations were maximal at 2h post-treatment and returned to basal levels after 24h (P<0.05). Oestradiol concentrations were lower (P<0.05) in buserelin-treated animals than controls at 72 h post-treatment. Basal and post-treatment progesterone concentrations were greater (P<0.05) in ewes than in ewe lambs but oestradiol levels were similar for both age groups. Ovulation rate, determined by laparoscopy on Day 14, was similar for both age groups (ewes 1.1; ewe lambs 1.0). Buserelin treatment induced accessory corpora lutea in ewes (4/7; 57%) but not in ewe lambs (0/7; 0%). In the Experiment 2, plasma PGFM concentrations were determined in samples collected at 20-min intervals for 6h on Day 14 and at 20-min intervals for 1h before and at 10-min intervals for 1h and then at 20-min intervals for a further 3h period after an intravenous injection of oxytocin (1IU/kg body weight) on Day 15 post-oestrus. In this experiment there were five ewe lambs and six ewes per treatment group. There was no effect of buserelin treatment or age on basal PGFM concentrations on either Day 14 or 15. Although peak PGFM concentrations tended to be lower in buserelin-treated animals, the difference was not significant (P>0.05). However, peak duration following oxytocin challenge on Day 15 post-mating was shorter (P<0.05) in control ewes compared with control ewe lambs. In conclusion, buserelin treatment given on Day 12 post-oestrus enhances luteal function more in ewes than ewe lambs and after a transitory increase, reduces oestradiol concentrations in both ewes and ewe lambs. However, buserelin treatment does not significantly attenuate the luteolytic signal.     

Effects of diosgenin on myometrial matrix metalloproteinase-2 and -9 activity and expression in ovariectomized rats

Diosgenin, a traditional Yam extraction, has been used in hormone replacement for menopausal women. We aimed to investigate the influences of diosgenin administration upon the MMP-2 and -9 activity and expression and reproductive hormones of ovariectomized (OVX) rats, a model of menopausal status. Seven-week old female Wistar rats with bilateral OVX or sham operation (controls) were divided and administered different dosages of diosgenin (0, 10, 50, or 100 mg/kg/day) for 8 weeks. Serum was then sampled for progesterone (P4) and estradiol (E2) assay and uterine horns harvested. Myometrial MMP-2 and -9 activity and expression were surveyed and myometrial collagen expression was also assayed. The results show higher body weight in OVX rats across the 8 weeks post surgery and no significant differences were noted among OVX or Sham rats with diosgenin supplements. There were lower P4 and E2 concentrations in OVX rats compared to Sham rats, and higher P4 concentration of Sham rats post diosgenin supplement. MMP-2 and -9 mRNA expression and activity was lower in OVX rats, although higher MMP-2 and lower MMP-9 activity/mRNA expression was observed in OVX rats post diosgenin supplementation. Collagen mRNA expression was higher in OVX rats compared to Sham controls, and diosgenin administration decreased collagen mRNA expression in OVX rats. In conclusion, diosgenin is associated with gelatinase expression and collagen metabolism in OVX rats. Diosgenin administration can partially reverse the effects of OVX upon MMP functions and hormone status. Adequate diosgenin supplement might modulate myometrial gelatinase expression and collagen metabolism in menopausal subjects.     

Age-, cycle- and topographic dependency of human myometrial prostaglandin (6-keto-PGF1 alpha, PGF2 alpha)-synthesis in vitro

Specimens of human myometrium (isthmus and fundus) freshly obtained at hysterectomy were immediately transferred in ice cold Tyrode solution and placed in superfusion chambers. Spontaneous contractions were recorded, the effluent of the myometrium was analyzed for PGF2 alpha and 6-keto-PGF1 alpha by use of specific radioimmunoassay systems. Dating of the menstrual cycle was achieved by histological evaluation of the endometrium. The PG release rates expressed as ng/min/g wet weight were correlated to the patients age and to the phase of the menstrual cycle. The production rates of 6-keto-PGF1 alpha were negatively correlated to the age of the patients and declined in fundus specimens from 2.89 +/- 0.35 ng/min/g wet weight in 39-42 years old patients to 0.52 +/- 0.17 ng/min/g wet weight in 48-52 years old women during the secretory phase (p less than 0.001). Similar significant correlations were found in specimens obtained from the isthmus uteri. During the proliferative phase fundus specimens produced on average 1.61 +/- 0.67 ng/min/g wet weight in 39-42 years old patients and 0.49 +/- 0.12 ng/min/g wet weight 6-keto-PGF1 alpha in 48-52 years old women respectively (p les than 0.001). The PGF2 alpha synthesis in myometrial specimens of fundus or isthmus origin was significantly lower than 6-keto-PGF1 alpha and did not correlate to the age of the patients during the proliferative phase. However, PGF2 alpha release rates during the secretory phase were significantly (p less than 0.001) higher in younger women. These results suggest an age-, cycle- and topographic dependency of PGI2 synthesis in human myometrial tissue.     

Luteolytic activity of a synthetic prostaglandin and PGF2alpha in heifers.

Two experiments involving 44 cycling heifers were conducted to evaluate the luteolytic activity of a synthetic prostaglandin, AY 24366, and PGF2alpha. Activity was assessed by the decline in progesterone level of peripheral blood and occurrence of estrus. Progesterone concentrations of jugular blood plasma were quantified by radioimmunoassay. In the first experiment, 36 heifers were treated during diestrus with AY 24366 (A-10mg intrauterine, B-30mg intramuscular and C-60mg im) or with PGF2alpha (D-5mg iu, E-15mg im and F-30mg im). Mean progesterone 0, 24 and 48 hours after treatment were A-6.33, 5.55 and 5.06; B-6.35, 2.79 and 3.92; C-5.23, 2.69 and 3.91; D-5.19, 1.50 and 1.51; E-4.69, 0.85 and 0.61; F-6.66, 0.80 and 0.48 ng/ml. Standing estrus was observed in 1, 1, 1, 4, 5 and 6 females in groups A, B, C, D, E and F respectively within 72 hours of treatment. PGF2alpha resulted in significantly (P less than 0.01) lower progesterone at 24 and 48 hours than AY 24366. However, in administration of the latter did significantly (P less than 0.05) lower progesterone at 24 hours. In the second trial six heifers were treated with either 120 or 180mg of AY 24366 im on day 12 of the cycle. Mean progesterone declined from 3.84 to 2.12 ng/ml (P less than 0.01) by 6 hours and to 1.59 ng/ml by 12 hours. Thereafter the decline was gradual and reached a level of 0.65 ng/ml at 72 hours. All six heifers showed standing estrus at 78 +/-2 hours and were inseminated. Two in each group conceived. Doses of 15mg PGF2alpha and 120mg AY 24366 were effective in causing luteal regression, however, the latter caused respiratory discomfort for 5 to 10 minutes post treatment.     

Effect of PGI-2 on uterine activity in vivo in non-pregnant ovariectomized goats (Capra hircus)

Jugular administration of 200 micrograms PGI-2 salt significantly reduced spontaneous uterine activity in ovariectomized, oestrogen-primed goats; the effect was acute and persisted for about 3 h. Peripheral plasma concentrations of 6-keto-PGF-1 alpha, the stable metabolite of PGI-2, decreased to 50% of initial values after 30 min; but at the start of uterine recovery were in excess of 2 ng.ml-1. Uterine reactivity to both oxytocin and PGF-2 alpha after PGI-2 administration was unaffected.     

A comparison of the contractile activity of PGD2 and PGF2 alpha on human isolated bronchus

Prostaglandins may be implicated in the bronchoconstriction which occurs in asthma. Prostaglandins F2 alpha (PGF2 alpha) and D2 (PGD2) have been reported to produce bronchoconstriction in asthmatic subjects in vivo and PGF2 alpha contracts human isolated airway smooth muscle. We examined the relative efficacy and potency of PGF2 alpha and PGD2 on human bronchial spiral strips taken from 6 patients at thoracotomy. PGF2 alpha had greater efficacy than PGD2. The mean % Tmax (percentage of maximal contractile response) +/- s.e. mean were 84 +/- 7 and 54 +/- 7 respectively (P less than 0.05). PGF2 alpha (mean pD2 +/- s.e. mean = 6.39 +/- 0.6) tended to be more potent than PGD2 (5.68 +/- 0.2). Since, in vivo, PGD2 has greater efficacy and potency than PGF2 alpha, our results suggest that the in vivo effect of these prostaglandins does not result solely from an action on airway muscle.     

Differential effects of probenecid on the levels of endogenous PGF2 alpha and TXB2 in brain cortex

Probenecid in single or repeated doses does not modify levels of PGF2 alpha and TXB2 in rat brain cortex. After administration of subconvulsant dose of pentamethylene tetrazole (PMT) PGF2 alpha increases sharply and rapidly declines subsequently, whereas the elevation of TXB2 is smaller but of longer duration. After probenecid pretreatment PGF2 alpha levels do not decline up to 30 minutes after the initial peak and are still elevated after 60 minutes. Levels of TXB2 tend to be reduced after pretreatment. Differences in transport process or in biosynthetic compartments for these arachidonic acid (AA) metabolites may account for the observed data.     

Characterization of the stimulatory effects of PGF2alpha on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was greater than 90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (greater than 90%) was incorporated into the 2-position. PGF2alpha caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts. The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF2Alpha increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a "trap" for released arachidonic acid. The effect of PGF2Alpha was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed. The effect of PGF2Alpha depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Prostaglandin E2 counteracts the effects of PGF2 alpha in indomethacin treated cycling gilts

Twenty crossbred gilts with at least 2 consecutive estrous cycles of 18 to 21 days in length were used to study the effects of prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) on luteal function in indomethacin (INDO) treated cycling gilts. Intrauterine and jugular vein catheters were surgically placed before day 7 of the treatment estrous cycle and gilts were randomly assigned to 1 of 5 treatment groups (4/group). With exception of the controls (Group I) all gilts received 3.3 mg/kg INDO every 8 h, Groups III, IV and V received 2.5 mg PGF2; 2.5 mg PGF2 alpha + 400 micrograms PGE2 every 4 hr, or 400 micrograms PGE2 every 4 h, respectively. All treatments were initiated on day 7 and continued until estrus or day 23. Jugular blood for progesterone analysis was collected twice daily from day 7 to 30. Estradiol-17 beta (E2-17 beta) concentrations were determined in samples collected twice daily, from 2 d before until 2 d following the day of estrus onset. When compared to pretreatment values, estrous cycle length was unaffected (P greater than 0.05) in Group I, prolonged (P less than 0.05) in Groups II, IV and V; and shortened (P less than 0.05) in Group III. The decline in plasma progesterone concentration that normally occurs around day 15 was unaffected (P greater than .05) in Group I; delayed (P less than 0.05) in Groups II, IV and V; and occurred early (P less than 0.05) in Group III. Mean E2-17 beta remained high (31.2 +/- 4.9 to 49.3 +/- 3.1 pg/ml) in Groups III and IV, while the mean concentrations in Groups III and V varied considerably (17.0 +/- 2.0 to 52.2 +/- 3.5 pg/ml). The results of this study have shown that PGE2 will counteract the effects of PGF2 alpha in INDO treated cycling gilts. The inclusion of PGF2 alpha appeared to either stimulate E2-17 beta secretion or maintain it at a higher level than other treatments.