J. Kotwica

Direct Inhibitory Effect of Progesterone on Oxytocin-Induced Secretion of Prostaglandin F2α from Bovine Endometrial Tissue

Abstract The effect of progesterone on oxytocin-induced secretion of prostaglandin (PG) F2α from bovine endometrial tissue explants was examined. Endometrial tissue from the late luteal phase were preincubated for 20 h in control medium. Explants were then treated for 6 h with control medium, oxytocin (10−7 M), progesterone (10−5 M), or both hormones. Oxytocin increased the medium concentration of 13,14-dihydro-15-keto-PGF2α, whereas progesterone completely suppressed the stimulatory effect of oxytocin. In experiment 2, isolated endometrial epithelial cells were incubated with progesterone (10−5 M), oxytocin (10−7 M), and combinations of these hormones with or without actinomycin D (1 ng/ml). Only oxytocin stimulated secretion of PGF2α, and this response was suppressed by progesterone. Oxytocin induced a rapid increase in intracellular concentrations of Ca2+ detected within 1 min of exposure of epithelial cells from the same cows. Progesterone pretreatment diminished this response. In experiment 3, direct effects of progesterone (2 nM–20 μM) on binding of 3H-oxytocin to the membrane preparation from epithelial cells were determined by saturation analysis. Oxytocin binding was suppressed by progesterone at every dosage tested. Progesterone is capable of suppressing the ability of oxytocin to induce endometrial secretion of PGF2α. This effect appears to be mediated through a direct interference in the interaction of oxytocin with its own receptor.

The use of an oxytocin antagonist to study the function of ovarian oxytocin during luteolysis in cattle

Abstract The importance of ovarian oxytocin (OT) in cattle during luteolysis and the mid-luteal phase using a highly specific OT antagonist (CAP-527) was studied. To establish the effective dose of CAP, heifers (n = 4) were infused with saline for 30 min, followed by 50 IU OT into the abdominal aorta on Days 17 and 18 of the estrous cycle. After 5.5 hours later, either 4, 6, 8 or 10 mg of CAP was infused for 30 min, followed by 50 IU OT. Plasma concentrations of 15-keto-13, 14-dihydro-prostagiandin F2α (PGFM) increased after 4 and 6 mg CAP. Therefore, in Experiment 2, 8 mg of CAP was infused and 50 IU OT was given after 3, 4, 6 and 9 h to define how long CAP saturates OT receptors. Concentrations of PGFM increased after 6 and 9 h of OT treatment only. We concluded that 8 mg CAP effectively blocked uterine OT receptors for 4 h in our model; hence in further experiments this dosage of CAP was used. In Experiment 3, CAP was given to 4 heifers every 4 h on Days 15 to 22 of the cycle, and 4 additional heifers received saline and served as the control. The CAP treatment changed neither the duration of the cycle, progesterone, PGFM nor OT plasma concentrations compared with that of the controls. Experiment 4 was designed to study the involvement of OT in noradrenaline (NA)-stimulated progesterone secretion. It was found earlier that ovarian OT stimulates progesterone secretion, and that NA was able to evoke concomitant release of both progesterone and OT. Therefore, in Experiment 4, NA was infused on Days 11 and 12 of the cycle in heifers (n = 4) but it was preceded with 8 mg CAP or with a saline (control) infusion. Concentrations of plasma progesterone concentrations increased after NA treatment in both the experimental and control groups. Thus, we conclude that if ovarian OT and uterine/ovarian OT receptors are involved in luteolysis and steroidogenesis in cattle, they play a more facilitating than mandatory role.

Involvement of ovarian steroids in basal and oxytocin-stimulated prostaglandin (PG) F2α secretion by the bovine endometrium in vitro

It is assumed that exposure of endometrium to spontaneously secreted luteal hormones stimulates PGF2 alpha secretion and modifies oxytocin (OT) influence on the bovine uterus. At first, the time-dependent effect of endogenous luteal products on endometrial PGF2 alpha secretion was examined. Endometrial strips (100 mg) from slaughtered heifers (Days 11 to 17 of the cycle) were incubated alone or with luteal cells (1 x 10(5) cells/mL). The highest PGF2 alpha secretion by the endometrium under influence of hormones secreted from luteal cells was observed after 12 h of incubation compared with the control (P < 0.001). Then, endometrium (Days 11 to 17) was incubated with luteal cells and concomitantly with antagonists of P4 and OT. The P4 antagonist prevented the stimulatory effect of endogenous luteal hormones on PGF2 alpha secretion (P < 0.05), but the OT antagonist did not. Further, direct effects of exogenous P4, OT and estradiol (E2) on endometrial PGF2 alpha secretion (Days 11 to 17) were examined. Both OT and P4 increased PGF2 alpha secretion (P < 0.05); E2 alone had no effect on PGF2 alpha secretion, but it amplified the P4 effect (P < 0.05). Finally, we studied the effect of endogenous luteal products on OT-stimulated PGF2 alpha secretion from endometrium. When endometrium (Days 11 to 17) was incubated without luteal cells, OT stimulated PGF2 alpha secretion (P < 0.001), whereas incubation of endometrium with luteal cells abolished the stimulatory effect of OT on PGF2 alpha secretion (P < 0.001). These treatments did not affect PGF2 alpha secretion from the endometrium collected on Days 1 to 4. In conclusion, P4 stimulates PGF2 alpha secretion by the endometrium and E2 amplifies this effect. As long as the endometrium is under the influence of P4, ovarian OT does not affect PGF2 alpha secretion.

Noradrenaline affects secretory function of corpus luteum independently on prostaglandins in conscious cattle

The aim of this study was to examine whether prostaglandins are involved in the effects of noradrenaline on corpus luteum (CL) function. To establish an effective dose of indomethacin (INDO) to prevent prostaglandin synthesis, different doses (120, 180, 240 and 300 mg) of drug were infused for 30 min on days 17-18 of the estrous cycle in four heifers and followed with 50 IU of OT. Plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) were measured, both to illustrate the concentrations of all prostaglandins and to establish the effective dose of INDO that can effectively block prostaglandins synthesis. In Experiment 2, on days 10-12 of the cycle, heifers (n = 6) were infused in a Latin square design with 4 mg of noradrenaline (NA) and pre-treated with 120 mg of INDO or with saline. In both cases, NA did stimulate progesterone and ovarian oxytocin secretion. We conclude that NA affects secretory function of the CL independently of prostaglandins.

Neural regulation of the bovine corpus luteum.

The ovarian noradrenergic stimulation or noradrenaline (NA) administration directly to the ovary in cow increases ovarian oxytocin (OT) release and post-translational processing of OT synthesis within a few minutes has been established in both in vivo and in vitro studies. Furthermore, NA affects progesterone secretion and its synthesis by an increase of cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase activity. This effect is mediated via luteal cell beta(1)- and beta(2)-receptors. Their total amount correlates with peripheral progesterone concentrations during the luteal phase and this reflects the ability of the ovary to react to beta-stimulation. On the other hand, ovarian denervation causes a decrease of steroidogenic activity in the CL, an increase of beta-receptors on luteal cells, a delay in follicular development and the disruption of cyclicity. Moreover, decrease of progesterone secretion by 20-30% was seen after brief pharmacological blockade of ovarian beta-receptors in the mid-cycle of cattle. We assume that tonic beta-stimulation of the CL ensures the basal secretion of progesterone, whereas acute noradrenergic activation supports the CL during stressful situations which could impair its function. Conversely, long-lasting increase in blood catecholamine concentrations markedly decreases the number of beta-receptors in CL, presumably due to their down-regulation. Concentrations of dopamine (DA) within the CL are highly correlated with those of NA during the estrous cycle, and are higher in the newly-formed than in the developed corpus luteum, the regressed corpus luteum or the corpus luteum of pregnant females. Bovine CL can synthesise de novo NA from DA as a precursor. Concluding, presented data indicate that noradrenergic stimulation can be an important part of mechanism supporting secretory function of CL.

Role of prostaglandin E2 in basal and noradrenaline-induced progesterone secretion by the bovine corpus luteum.

The role of prostaglandin E2 (PGE2) in basal and noradrenaline (NA)-stimulated utilization of high density lipoprotein (HDL) as a source of cholesterol for progesterone synthesis was examined. In Experiment 1, a cannula was inserted into the aorta abdominalis through the coccygeal artery (cranial to the origin of the ovarian artery) in mature heifers, to facilitate infusion of NA (4 mg/30 min; n = 3) on day 10 of the estrous cycle. Three other heifers were similarly cannulated to serve as control. Before, during, and after NA or saline infusion, blood samples from the vena cava were collected every 5-15 min for analysis of PGE2, progesterone, and cholesterol. Each NA infusion stimulated (P < 0.01) secretion of both hormones in heifers. Short-duration increases (P < 0.05) in progesterone were observed due to the infusion of NA while cholesterol was not altered significantly. In addition, increases in PGE2 concentrations (P < 0.05) compared to controls were seen after NA infusion. Therefore, we used an in vitro model to verify the effect of PGE2 on HDL utilization by luteal cells from day 5 to 10 of the estrous cycle. In the preliminary experiment, 10(-6) M of PGE2 out of four different doses examined was selected for further studies, since it evoked the highest release of progesterone. In the next experiment, it was found that HDL increases progesterone secretion by luteal cells and both PGE2 and LH increased (P < 0.05) the response to HDL while NA did not. In the last in vitro experiment, progesterone stimulated PGE2 secretion by luteal cells. In conclusion, PGE2 may be directly involved in the utilization of cholesterol from HDL for progesterone synthesis. Furthermore, PGE2 may influence NA-stimulated progesterone secretion by the corpus luteum (CL). It is concluded that there is a positive feedback loop between progesterone and luteal PGE2 during days 5-10 of the estrous cycle.

Mechanism of action of noradrenaline on secretion of progesterone and oxytocin by the bovine corpus luteum in vitro.

The present studies were conducted: (1) to determine which beta-adrenoceptor subtypes are involved in progesterone and oxytocin (OT) secretion, (2) to examine whether noradrenaline (NA) acts directly on the cytochrome P-450scc and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and (3) to study the effect of prostaglandin F2 alpha (PGF2 alpha) on NA-stimulated steroidogenesis in luteal cells. The effect of NA on progesterone secretion from luteal slices of heifers on days 8-12 of the oestrous cycle was blocked by both atenolol (beta 1-antagonist) and ICI 118.551 hydrochloride (beta 2-antagonist). OT secretion was blocked only after treatment with ICI 118.551 hydrochloride (P < 0.05). Dobutamine (10(-4)-10(-6) M), a selective beta 1 agonist and salbutamol (10(-4)-10(-6) M), a selective beta 2 agonist, both increased progesterone production (P < 0.01) with an efficiency comparable to that produced by NA (P < 0.01). The increase of OT content in luteal slices was observed only after treatment with salbutamol at the dose of 10(-5) M (P < 0.01). Dobutamine had no effect on OT production at any dose. A stimulatory effect of NA on cytochrome P-450scc activity (P < 0.05) was demonstrated using 25-hydroxycholesterol as substrate. 3 beta-HSD activity also increased following NA (P < 0.01) or pregnenolone (P < 0.05) and in tissue treated with pregnenolone together with NA (P < 0.01). PGF decreased progesterone synthesis (P < 0.05) and 3 beta-HSD activity (P < 0.01) in tissue treated with NA. We conclude that NA stimulates progesterone secretion by luteal beta 1- and beta 2-adrenoceptors, while OT secretion is probably mediated only via the beta 2-receptor. NA also increases cytochrome P-450scc and 3 beta-HSD activity. PGF inhibits the luteotropic effect of NA on the luteal tissue.

Changes in ovarian oxytocin secretion as an indicator of corpus luteum response to prostaglandin F2α treatment in cattle

Exogenous prostaglandin F(2alpha) (PGF(2alpha)) rapidly increases ovarian oxytocin (OT) release and decreases progesterone (P4) secretion in cattle. Hence, the measurement of OT secretion (the area under the curve and the height of the peak) after different doses of Oestrophan - PGF(2alpha) analogue (aPGF(2alpha)) on Days 12 and 18 of the estrous cycle (estrus = day 0), could be a suitable indicator of corpus luteum (CL) sensitivity to PGF(2alpha) treatment. Mature heifers (n = 36) were used in this study. Blood samples were collected from the jugular vein for the estimation of OT, P4 and 13, 14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM). In Experiment 1, different doses of aPGF(2alpha) (400, 300, 200 and 100 microg) given on Day 12 of the estrous cycle (n = 8) shortened (P < 0.05) the cycle duration (15.2 +/- 0.6 d) compared with that of the control (21.7 +/- 0.4 d). Successive heifers were also treated on Day 12 with 200 (n = 2), 100 (n = 2), 75 (n = 2) or 50 microg aPGF(2alpha) (n = 2). Only the 50 microg aPGF(2alpha) dose did not cause CL regression, although it increased OT concentrations to levels comparable to those observed during spontaneous luteolysis (50 to 70 pg/ml). In Experiment 2, on Day 18 of the cycle heifers (n = 8) were treated with 50, 40, 30 and 20 microg aPGF(2alpha). There was a dose-dependent effect of aPGF(2alpha) on OT secretion on Day 18 of the estrous cycle (r = 0.77; P < 0.05). In Experiment 3, an injection of 500 microg aPGF(2alpha) on Day 12 (n = 4) and 50 microg aPGF(2alpha) on Day 18 (n = 4) caused a similar (P > 0.05) increase in the OT concentration (288.5 +/- 23.0 and 261.5 +/- 34.7 pg/ml, respectively). Thus the effect of the same dose of aPGF(2alpha) (50 microg) on OT secretion was different on Days 12 and 18 of the cycle. To evoke similar OT secretion on Days 12 and 18 the dose of aPGF(2alpha) on Day 18 could be reduced 10-fold, confirming that CL sensitivity to PGF(2alpha) appears to increase in the late luteal phase.

Influence of dopamine as noradrenaline precursor on the secretory function of the bovine corpus luteum in vitro

1 . Dopamine is assumed to affect the ovary function after its conversion into noradrenaline (NA). 2 . To study this bovine luteal slices from 11–14 days of the oestrous cycle were preincubated for 24 h to recover β‐receptors and next they were incubated for 1, 2 or 4 h with (a) different doses of dopamine; (b) dopamine together with a β‐antagonist (propranolol) or with a dopamine receptor blocker (droperidol); (c) dopamine with a dopamine‐β‐hydroxylase inhibitor. 3 . Dopamine stimulated the luteal content of oxytocin (OT) and progesterone. This effect was inhibited by propanolol but not by droperidol. 4 . Dopamine added to the medium was followed by an increase of noradrenaline there. This rise was dose and time‐dependent. 5 . The dopamine‐β‐hydroxylase inhibitor, inhibited the stimulating effect of dopamine on luteal progesterone and OT content. 6 . Bovine corpus luteum can synthesize de novo NA from dopamine as a precursor.

Influence of noradrenaline on progesterone synthesis and posttranslational processing of oxytocin synthesis in the bovine corpus luteum

Noradrenaline (NA) influences secretory function of the bovine corpus luteum (CL), stimulating secretion of progesterone and ovarian oxytocin (OT). To study whether NA is able to stimulate progesterone synthesis and to affect post-translational OT processing, different doses of NA alone or in combination with different doses of OT were added to bovine CL slices from 8 to 13 d of the estrous cycle. To determine which receptors NA affects, and if dopamine (DA) also affects CL function, we used NA or DA combined with a beta-antagonist (propranolol). The results indicated that NA stimulates both luteal progesterone and OT content; furthermore, it increased the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA), terminal enzymes in synthesis of these 2 hormones. The stimulating effect of NA was inhibited by propranolol and by pre-treatment of CL slices with high OT doses. Post-translational processing of OT synthesis by PGA activation was also stimulated by DA, but this effect was inhibited by beta-receptor blocker. Thus DA acts in CL as a NA precursor. In conclusion, it can be assumed that the noradrenergic system affects CL secretory function on different levels of regulation. Furthermore, a high concentration of OT in CL prevents NA from activating PGA and thus decreases post-translational OT synthesis.