Differential control of placental lactogen release and progesterone production by ovine placental tissue in vitro.

The hypothesis that placental secretion of progesterone (P4) and ovine placental lactogen (oPL) are controlled through different mechanisms was tested. Placental tissue was obtained at days 133-138 of pregnancy, and explant incubations were established using 200 mg tissue per flask in 5 ml O2-saturated DMEM containing 24 mM HEPES and lacking phenol red (pH 7.4). Following a 30-min preincubation, and a 15-min control period, test substances were added and incubations continued, with periodic gassing, for 4 h at 37 degrees C in a shaking water bath. Dopamine (DA), norepinephrine (NE) and epinephrine significantly stimulated P4 production (P less than 0.05). The enhancement of placental P4 production was mimicked by the addition of 8-bromo-cyclic adenosine monophosphate and forskolin (P less than 0.05). The response to catecholamines was abolished by the addition of propranolol (P less than 0.05) but not by phentolamine (P greater than 0.05). Inclusion of a membrane-permeant substrate for P4 synthesis, 25-hydroxycholesterol, increased basal (P less than 0.05) but did not enhance agonist-induced P4 production (P greater than 0.05). High performance liquid chromatographic analysis of placental tissue demonstrated the presence of DA (80.8 +/- 7.07 pg/mg) and NE (48.8 +/- 5.77 pg/mg), as well as catecholamine metabolites. Addition of 1,2-dioctanoyl-sn-glycerol (DAG) or phorbol 12-myristate-13-acetate (PMA) enhanced oPL secretion (P less than 0.05) without affecting P4 production. The response to DAG and PMA, representing the release of considerably more oPL than can be detected by extracting the tissue, was not influenced by treatment with cycloheximide (P greater than 0.05) indicating that secretion of preformed oPL is regulated by the protein kinase C pathway. These results support the hypothesis that the secretion of oPL and the production of P4 are controlled by different mechanisms.

Support for a physiological role of endogenous catecholamines in the stimulation of bovine luteal progesterone production.

To determine if catecholamines were present in bovine luteal tissue, corpora lutea (CL) were obtained during the mid-luteal phase (Days 10-12) and the concentration of dopamine (DA) and norepinephrine (NE) was determined by high-performance liquid chromatography. Both DA and NE were detected in luteal tissue at mean concentrations of 41.9 +/- 5.73 and 10.2 +/- 2.51 ng/g for DA and NE, respectively. These concentrations represented a luteal content of 306.6 +/- 66.88 ng/CL for DA and 70.5 +/- 16.88 ng/CL for NE. In vitro, DA at concentrations of 1.0 mM to 0.01 mM stimulated the production of progesterone (P4, p less than 0.05). The response to DA was inhibited by propranolol (a beta-adrenergic receptor antagonist, p less than 0.05) but not by phentolamine, phenoxybenzamine (alpha-adrenergic receptor antagonists), or haloperidol (a DA receptor antagonist, p greater than 0.05). Neither L-tyrosine nor L-dopa altered P4 production (p greater than 0.05). Inhibition of DA beta-hydroxylase, the enzyme that catalyzes the conversion of DA to NE by FLA-63 blocked the DA-induced increases in luteal P4 production (p less than 0.05). These results demonstrate the existence of DA and NE in bovine luteal tissue and indicate that exogenous DA can be converted to NE in luteal tissue. The results support a physiological role for catecholamines in the stimulation of bovine luteal function.

The effects of platelet-activating factor and platelet-derived compounds on bovine luteal cell progesterone production.

This study was conducted to characterize bovine platelets with respect to serotonin (5-HT) concentration and platelet-activating factor (PAF)-activation and to examine the in vitro effects of PAF and platelet-derived compounds on bovine luteal progesterone (P4) production. The concentration of 5-HT in platelets, as determined by high-performance liquid chromatography, was 538.8 +/- 40.83 ng/1 x 10(8) platelets. Based on a circulating platelet concentration range of 2.3 x 10(8) 5.8 x 10(8) platelets/ml, the circulating concentration of 5-HT would be approximately 1239-3125 ng/ml of blood. Bovine platelets were found to aggregate in response to PAF (1-40 ng/0.5 ml), with maximal aggregation occurring at 20-40 ng/0.5 ml. Coincubation of luteal cells with platelets (1 x 10(7)-4 x 10(8] enhanced luteal P4 production (p less than 0.05). Addition of the 5-HT receptor antagonist mianserin blocked the platelet-induced increases in P4 (p less than 0.05). Preincubation of platelets with indomethacin did not alter the production of P4 (p greater than 0.05), nor did the addition of propranolol (p greater than 0.05). Platelet-derived growth factor at 8 and 16 ng/ml enhanced basal P4 production (p less than 0.05) but had no effect on the responsiveness of luteal cells to luteinizing hormone (LH) (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Low molecular weight lipid-soluble luteotrophic factor(s) produced by conceptuses in cows.

Peripheral plasma progesterone concentrations are higher (P less than 0.05) in pregnant than in inseminated non-pregnant or cyclic heifers between Days 10 and 18 after insemination. In several experiments, it has been demonstrated that bovine conceptuses produce a low molecular weight (Mr less than 10,000), lipid-soluble, heat-labile, dextran-coated charcoal-adsorbable luteotrophic substance(s) that has the ability to stimulate progesterone synthesis in dispersed bovine luteal cells. This substance does not appear to be PAF, since addition of PAF to dispersed luteal cells at several dose levels failed to affect either basal or LH-stimulated progesterone synthesis. However, results of in-vitro and in-vivo experiments suggest that platelet-derived products are luteotrophic. In in-vitro experiments it was shown that co-incubation of dispersed bovine luteal cells with bovine platelets augments basal progesterone synthesis. Serotonin and platelet-derived growth factor appear to be the major products of platelet activation responsible for the luteotrophic activity of platelets. Products of the arachidonic acid cascade do not appear to be important, since addition of the cyclo-oxygenase blocker indomethacin did not reduce the luteotrophic activity of platelets. In in-vivo experiments, it has been possible to demonstrate a significant thrombocytopenia in pregnant heifers between Days 7 and 16 after insemination. These results are compatible with the concept that release of platelet-derived products under the influence of factors produced by the early embryo play a role in stimulating progesterone synthesis by the corpus luteum during early pregnancy.

Biogenic amine regulation of bovine luteal progesterone production in vivo.

Biogenic amines were administered using osmotic pumps placed subcutaneously in the neck region of regularly cycling, non-lactating dairy cows on Days 9-11 (oestrus = Day 0) of the oestrous cycle. Blood samples were collected using indwelling jugular catheters and the plasma progesterone concentrations were measured. Samples were collected at 4-h intervals for the first 12 h of treatment and thereafter at 12-h intervals for the remainder of the 72-h treatment period. After administration of various doses of noradrenaline, adrenaline and serotonin (0.5-2.0 micrograms/kg/h) significant elevation of plasma progesterone was achieved at a dosage of 2.0 micrograms/kg/h (P less than 0.01). The response to adrenaline was greater than that observed for noradrenaline and serotonin (P less than 0.05). Within-treatment comparison to pretreatment samples showed plasma progesterone concentrations to increase within 4 h after the administration of noradrenaline, adrenaline and serotonin (P less than 0.05) and this enhancement was maintained throughout the treatment period (P less than 0.05). The elevation in plasma progesterone concentrations induced by noradrenaline, adrenaline and serotonin was independent of changes in circulating concentrations of luteinizing hormone. These results support a physiological role for endogenous biogenic amines in the control of bovine luteal progesterone production.

Mechanisms involved in the action of serotonin-induced stimulation of progesterone production by bovine luteal cells in vitro.

Serotonin (5-HT)-induced stimulation or progesterone (P4) production by bovine luteal cells was characterized with respect to the receptor subtype mediating this response, the steroidogenic response to 5-HT metabolites, the role of adenylate cyclase, and the 5-HT concentration of bovine luteal tissue. Addition of 5-HT (10(-5) M) stimulated the production of P4 (P less than 0.05) and this stimulation was inhibited by the 5-HT antagonist mianserin at a concentration of 10(-5) M (P less than 0.05), but not at a mianserin concentration of 10(-7) M. Additionally, the response to 5-HT could not be inhibited by ketanserin (10(-5) M), a 5-HT2 receptor antagonist. Incubation of luteal cells with a specific 5-HT1 agonist, (+/-)-8-hydroxydipropylaminotetralin HBr (DPAT) (10(-4) M), stimulated the production of P4 (P less than 0.05) and this response could not be blocked by mianserin at 10(-7) M or by ketanserin, but was inhibited by mianserin at 10(-5) (P less than 0.05). The addition of the 5-HT metabolite 5-methoxytryptamine (5-MTA) stimulated P4 production (P less than 0.05) and this response could be inhibited by mianserin (10(-5) M, P less than 0.05). Neither, N-acetyl-5-HT nor 5-methoxytryptophan significantly affected P4 production. The addition of the phosphodiesterase inhibitor 3-isobutyl-methylxanthine (IBMX, 0.1 mM) potentiated the effects of 5-HT and DPAT (P less than 0.05), but this effect was additive rather than synergistic. In contrast, the addition of luteinizing hormone (10 ng/ml) plus IBMX resulted in a significant synergistic response (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

A role for alternative pathway catecholamines in the regulation of steroidogenesis in cow luteal cells.

Incubation of bovine luteal cells with the alternative pathway catecholamines octopamine, synephrine and deoxyadrenaline at concentrations of 10(-6) to 10(-3) M enhanced the production of progesterone (P less than 0.05). Tryamine did not alter basal progesterone production (P greater than 0.05). Addition of noradrenaline and adrenaline at concentrations of 10(-4) to 10(-7) M significantly elevated the production of progesterone (P less than 0.05). The steroidogenic response to noradrenaline and adrenaline was greater than that for octopamine, synephrine and deoxyadrenaline (P less than 0.05). Response to both primary (10(-6) M) and alternative (10(-4) M) pathway catecholamines was inhibited by propranolol (10(-5) M, P less than 0.05) but not phentolamine (10(-5) M, P greater than 0.05). These results demonstrate that octopamine, synephrine and deoxyadrenaline can affect steroidogenesis by bovine luteal cells, and their action is mediated by beta-adrenergic receptors.

Serotonin-induced stimulation of progesterone production by cow luteal cells in vitro.

The addition of acetylcholine or histamine (10(-7) to 10(-4) M), gamma-aminobutyric acid, a dopamine agonist, and melatonin (10(-7) to 10(-5) M) did not alter basal or LH-stimulated progesterone production (P greater than 0.05). The addition of the specific beta 2-adrenergic agonist terbutaline and salbutamol did not significantly elevate progesterone production. Treatment of luteal cells with serotonin (5-HT), 10(-6) to 10(-4) M, increased the production of progesterone (P less than 0.05). This stimulated production was inhibited by the addition of mianserin (10(-5) M, a 5-HT antagonist; P less than 0.05). Isoproterenol (10(-7) to 10(-4) M) also resulted in significant increases in progesterone production (P less than 0.05). The combined treatments of 5-HT + LH, isoproterenol + LH, or isoproterenol + 5-HT did not result in a further increase in progesterone above that observed in response to LH or isoproterenol alone (P greater than 0.05). The isoproterenol-induced progesterone production could not be blocked by butoxamine (10(-5) M, a beta 2-antagonist), or practolol (10(-5) M, a beta 1-antagonist), but was inhibited by propranolol (10(-5) M, a general beta-antagonist; P less than 0.05). The response to isoproterenol was unaffected by mianserin (10(-5) M). These results demonstrate a possible role for 5-HT in the regulation of steroidogenesis by the corpus luteum of the cow. Furthermore, these results suggest that serotonin-induced progesterone production is a receptor-mediated event.

Effects of progesterone administered to dairy heifers on sensitivity of corpora lutea to PGF(2)alpha and on plasma LH concentration.

To determine whether progesterone facilitates PGF(2)alpha-induced luteolysis prior to day 5 of the estrous cycle, 48 Holstein-Friestian heifers were assigned at random to four treatments: 1) 4 ml corn oil/day + 5 ml Tris-HCl buffer (control); 2) 25 mg prostaglandin F(2)alpha (PGF(2)alpha); 3) 100 mg progesterone/day (progesterone); 4) 100 mg progesterone/day + 25 mg PGF(2)alpha (combined treatment). Progesterone was injected subcutaneously daily from estrus (day 0) through day 3. The PGF(2)alpha was injected intramuscularly on day 3. Estrous cycle lengths were decreased by progesterone: 20.2 +/- 0.56, 19.2 +/- 0.31 (control and PGF(2)alpha); 13.2 +/- 1.40, and 11.7 +/- 1.27 (progesterone and combined). The combination of progesterone and PGF(2)alpha did not shorten the cycle any more than did progesterone alone (interaction, P>0.05). PGF(2)alpha treatment reduced progesterone concentrations on day 6 (P<0.05) and both progesterone and PGF(2)alpha reduced plasma progesterone on day 8 (P<0.01 and P<0.05, respectively). LH was measured in blood samples collected at 10- min intervals for 4 hr on day 4 from three heifers selected at random from each of the four treatment groups. Mean LH concentration for control heifers ranged from 0.35 to 0.63 ng/ml (overall mean, 0.49 ng/ml) and for progesterone-treated heifers ranged from 0.12 to 0.30 ng/ml (overall mean, 0.23 ng/ml). LH concentrations were greater in control heifers (P<0.01). The mean LH pulse rate for control heifers was 2.7 pulses/heifers/4 hr, while that for the progesterone-treated heifers was 1.7 pulses/heifer/4 hr. The mean pulse amplitude for control and progesterone treatments was 0.47 ng/ml and 0.36 ng/ml, respectively. Neither pulse amplitude nor frequency were different between treatment groups.