Production of prostaglandin F2α and estrogen by embryonal membranes and endometrium and metabolism of prostaglandin F2α by embryonal membranes,endometrium and lung from gilts

A 3 hr incubation of endometrium and embryonal membranes was used to assess potential contributions of these tissues to the prostaglandin F₂α (PGF₂α) and unconjugated estrogen (UE) present in the uteri of pregnant gilts. Metabolism of [³H]PGF₂α was determined during a 6 hr incubation of endometrium, lung and embryonal membranes to assess the contribution of these tissues in conversion of PGF₂α to less active forms during the estrous cycle and early pregnancy. Tissue was collected from 12 cyclic gilts on days 13, 16 or 19 and from 17 pregnant gilts on days 13, 16, 19 and 25 after the onset of estrus (day 0). Concentration of PGF₂α (ng/g of tissue) in incubation medium after incubation of endometrium at 37 C was 4- to 6-fold greater (P<.001) on days 16 and 19 for cyclic gilts than for pregnant gilts. Concentration of PGF₂α in medium after incubation of embryonal membranes recovered on days 13 and 16 was similar to that found after incubation of endometrium from cyclic gilts on days 16 and 19. Percentage of [³H]PGF₂α converted by endometrium and lung tissue to other metabolites (61.8 and 79.5%, respectively) did not differ significantly among days of the cycle or pregnancy. The percentage of [³H]PGF₂α metabolites recovered as [³H]13,14-dihydro-15-keto-PGF₂α (PGFM) for endometrium (50.3%) and for lung (64.6%) was not affected significantly by pregnancy status.Embryonal membranes recovered on days 13 and 16 converted more (P<.05) [³H]PGF₂α to other metabolites (%/μg of DNA) than embryonal membranes recovered on days 19 and 25, lung or endometrium. The % of [³H]PGF₂α metabolites recovered as [³H]PGFM for embryonal membranes increased (P<.05) from 37.4 on day 13 to 68.6 on day 25. Concentration of UE (ng/g of tissue) in medium after incubation of embryonal membranes from day 13 was about 100-fold greater than for endometrium. Concentration of UE and estrone sulfate (E₁SO₄) (ng/g of tissue) in medium after incubation was greater (P<.05) for endometrium from pregnant gilts on day 25 than that for all days of the cycle or pregnancy. Concentration of UE in medium after incubation of endometrium or embryonal membranes was not significantly affected by incubation treatment, but more E₁SO₄ accumulated in the presence of indomethacin (P<.01). These results indicate that endometrium from pregnant gilts produces less PGF₂α than that of cyclic gilts in vivo and this may contribute to the maintenance of corpora lutea. The high concentrations of PGF₂α and estradiol in uteri of pregnant gilts may originate from embryonal membranes and be converted to biologically less active forms before leaving the uterus.     

The influence of clenbuterol on prostaglandin F2α-induced dyspnoea in calves

Pulmonary function tests were performed in six healthy calves. Prostaglandin F₂ causes severe narrowing of both upper and lower airways (total lung resistance increased, dynamic compliance decreased). Clenbuterol administered intravenously fifteen minutes prior to prostaglandin F₂ aerosol, and in increasing doses (0, 0.4, 0.8, 1.2 g/kg), on days 1, 2, 4 and 6 of the experiment, effectively but not entirely suppressed these responses.These data indicate that -adrenergic receptors are present in the bovine airways and that the use of clenbuterol (0.8 g/kg) may be effective in treating clinical respiratory disease such as bronchopneumonia in calves.     

Cellular mechanisms by which oxytocin mediates uterine prostaglandin F2α synthesis in bovine endometrium: role of calcium

The objective of these experiments was to determine the role of Ca²⁺ during oxytocin-stimulated prostaglandin (PG) F_2α release from bovine endometrial tissue in vitro. Uteri were collected from dairy cows on the day after spontaneous luteal regression. Caruncular endometrial explants were dissected and incubated in vitro to determine phospholipase C activity or PGF_2α release. A23187 (a calcium ionophore) and maitotoxin (an activator of voltage-gated L-type calcium channels) stimulated release of PGF_2α in a concentration-dependent manner (P < 0.05). Thapsigargin (induces accumulation of Ca²⁺ in the cytoplasm by inhibiting endoplasmic reticulum Ca²⁺/ATPase pumps) stimulated release of PGF_2α in a concentration-dependent manner as well (P < 0.13). Oxytocin (10⁻⁶ M), AlF₄⁻ (a nonspecific activator of G-proteins; 10⁻⁵ M), A23187 (10⁻⁵ M), and melittin (a stimulator of phospholipase A₂; 10⁻⁴ M) stimulated PGF_2α release when explants were incubated in Ca²⁺-free medium (P < 0.10); however, oxytocin, A23187, or melittin were unable to stimulate PGF_2α release when explants were incubated in Ca²⁺-free medium containing the calcium chelator EGTA (P < 0.10). This treatment did not prevent oxytocin or AlF₄⁻ from stimulating phospholipase C activity (P < 0.08). CoCl₂ (a nonspecific Ca²⁺ channel blocker) and methoxyverapamil (a specific voltage-gated L-type Ca²⁺ channel blocker) prevented oxytocin from stimulating PGF_2α release (P < 0.05). Our results suggest that both extracellular and intracellular Ca²⁺ may be required for oxytocin to stimulate PGF_2α secretion in bovine endometrial tissue.     

Influence of prostaglandin F2α analogues on the secretory function of bovine luteal cells and ovarian arterial contractility in vitro

Although prostaglandin (PG) F_2α analogues are routinely used for oestrus synchronisation in cattle, their effects on the function of the bovine corpus luteum (CL), and on ovarian arterial contractility, may not reflect the physiological effects of endogenous PGF_2α. In the first of two related experiments, the effects of different analogues of PGF_2α (aPGF_2α) on the secretory function and apoptosis of cultured bovine cells of the CL were assessed. Enzymatically-isolated bovine luteal cells (from between days 8 and 12 of the oestrous cycle), were stimulated for 24 h with naturally-occurring PGF_2α or aPGF_2α (dinoprost, cloprostenol or luprostiol). Secretion of progesterone (P4) was determined and cellular [Ca²⁺]_i mobilisation, as well as cell viability and apoptosis were measured.Naturally-occurring PGF_2α and dinoprost stimulated P4 secretion (P < 0.05), whereas cloprostenol and luprostiol did not influence P4 synthesis. The greatest cytotoxic and pro-apoptotic effects were observed in the luprostiol-treated cells, at 37.3% and 202%, respectively (P < 0.001). The greatest effect on [Ca²⁺]_i mobilisation in luteal cells was observed post-luprostiol treatment (200%; P < 0.001).In a second experiment, the influence of naturally-occurring PGF_2α and aPGF_2α on ovarian arterial contraction in vitro, were examined. No differences in the effects of dinoprost or naturally-occurring PGF_2α were found across the studied parameters. The effects of cloprostenol and luprostiol on luteal cell death, in addition to their effects on ovarian arterial contractility, were much greater than those produced by treatment with naturally-occurring PGF_2α.     

Calcium potentiates the effect of estradiol on PGF2α production in the bovine endometrium

Background: Estradiol(E2) is required for luteolysis in cows and its injection stimulates prostaglandin F2α(PGF2α)release. The main goal of our study was to investigate the ability of endometrial explants and cells treated with E2 and the calcium ionophore(CI) A23187 to synthesize PGF2α.Results: Treatment with E2 in vivo resulted in a 48.4% increase of PGF2α production by endometrial explants treated in vitro with A23187. Production of PGF2α was better stimulated with A23187 at concentrations of 10-6and10-5mol/L compared with other concentrations used. The concentration of PGF2α for untreated bovine endometrial cell cultures was 33.1 pg/m L, while for cultures treated with E2, A23187, or a combination of E2 and A23187, the PGF2α concentration was 32.5, 92.4 and 145.6 pg/m L, respectively.Conclusions: Treatment with A23187 tended to stimulate PGF2α production. In the presence of E2, A23187 significantly stimulated PGF2α synthesis. It appears that A23187 potentiates the effects of E2 with respect to synthesis of endometrial PGF2α in cattle.     

Calcium potentiates the effect of estradiol on PGF2α production in the bovine endometrium

Background

Estradiol (E₂) is required for luteolysis in cows and its injection stimulates prostaglandin F2α (PGF2α) release. The main goal of our study was to investigate the ability of endometrial explants and cells treated with E₂ and the calcium ionophore (CI) A23187 to synthesize PGF2α.

Results

Treatment with E₂in vivo resulted in a 48.4% increase of PGF2α production by endometrial explants treated in vitro with A23187. Production of PGF2α was better stimulated with A23187 at concentrations of 10^-6 and 10^-5 mol/L compared with other concentrations used. The concentration of PGF2α for untreated bovine endometrial cell cultures was 33.1 pg/mL, while for cultures treated with E₂, A23187, or a combination of E₂ and A23187, the PGF2α concentration was 32.5, 92.4 and 145.6 pg/mL, respectively.

Conclusions

Treatment with A23187 tended to stimulate PGF2α production. In the presence of E2, A23187 significantly stimulated PGF2α synthesis. It appears that A23187 potentiates the effects of E₂ with respect to synthesis of endometrial PGF2α in cattle.     

Control of in vitro prostaglandin F2α and E2 synthesis by caruncular and allantochorionic tissues from cows that calved normally and those with retained fetal membranes

High concentrations of PGF_2α and PGE₂ are produced by the uterus during the early postpartum period in cows and may play an important role in both placental separation and uterine involution. In the present study, we have examined the hormonal and intracellular control mechanisms involved in PGF_2α and PGE₂ secretion by caruncular and allantochorionic tissue in vitro. Tissue explants, obtained about 6 hr postpartum from cows that delivered normally (NFM, n = 10) or cows with retained fetal membranes (RFM, n = 4), were incubated for 6 hr and PGF_2α and PGE₂ concentrations in the medium were determined by radioimmunoassay. Addition of oxytocin (100 μU/ml), platelet activating factor (PAF, 100 ng/ml) and epidermal growth factor (EGF, 100 ng/ml) had no effect on secretion of PGF_2α from the caruncle, but oxytocin and PAF did stimulate PGE₂. There was no difference between groups of cows. All three substances stimulated PGF_2α from the allantochorion of NFM, but not RFM, cows and stimulated PGE₂ secretion from the allantochorion of both groups of cows. Incubation of the tissues with cholera toxin (100 ng/ml), dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl cAMP, 1mM), calcium ionophore A23187 (5 μM) or phorbol ester 12-myristate-13 acetate (PMA, 100 nM) showed that PGF_2α secretion is essentially via the calcium-protein kinase C effector pathway. However, calcium-protein kinase C and cAMP second messenger systems appear to be involved in the secretion of PGE₂. Prostaglandin secretion was sensitive to cycloheximide in both caruncular and allantochorionic tissues, suggesting that protein synthesis may be involved. In conclusion, these data show that in vitro PGF_2α secretion can be modulated by the agonists used only in allantochorion and is essentially via the calcium-protein kinase C effector pathway. PGE₂ secretion can be modifified in both caruncular and allantochorion tissues and involves both inositol triphosphate-diacylglycerol and cAMP second messenger systems.     

Acute changes in the concentrations of prostaglandin F2α (PGF) and cortisol in uterine and ovarian venous blood during PGF-induced luteolysis in cows

Prostaglandin F2α (PGF) is considered to be the main luteolysin in cattle. We have previously demonstrated that cortisol (Cr) suppresses PGF production in non-pregnant bovine endometrium. This study was carried out to test whether exogenous PGF increases ovarian and/or uterine PGF production and to determine the temporal relationship between PGF and Cr in ovarian and uterine circulations during PGF-induced luteolysis in cows. Catheters were inserted into the ovarian vein (OV), uterine vein (UV) and jugular vein (JV) of 10 cows on Day 9 of the oestrous cycle (Ovulation = Day 0) for frequent blood collection. On Day 10, the cows were divided randomly into two groups and treated with a luteolytic dose of a PGF analogue (cloprostenol) or saline solution. Blood samples were collected at -0.25, 0, 0.25, 0.5, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). The basal concentrations of PGF and Cr in OV and UV plasma were not significantly different. Injection of a PGF analogue induced more than twofold increases in the levels of PGF between 0.25 and 1 h in UV plasma, but not in OV plasma. PGF increased (p < 0.05) the concentrations of Cr in OV, UV and JV plasma between 0.5 and 1 h. The Cr levels in OV, UV and JV plasma were similar. The PGF levels in UV plasma decreased after Cr reached its highest levels. The overall results suggest that the uterus rather than the ovary increases PGF production in response to PGF injection. Based on the temporal changes of PGF and Cr in the ovarian and uterine circulations, Cr may act to reduce uterine PGF production in non-pregnant cows in vivo.     

The influence of progesterone and prostaglandin F2α on decorin and the adhesion of caruncular epithelial cells of bovine placenta at early-mid pregnancy—Part II

One of the most important processes determining the proper course of gestation and its physiological termination in cows is the adhesion of epithelial cells allowing for direct contact of maternal and foetal parts of the placenta. Throughout pregnancy, placental cells are under strict hormonal control, which among others regulates the concentration and activity of specific proteins participating in the extracellular matrix remodelling of foetal membranes. The aim of the study was to evaluate the influence of progesterone and prostaglandin F_2α on the adhesion of epithelial cells at early-mid pregnancy in cows. Additionally, the impact of selected hormones on anti-adhesive properties of decorin was evaluated. Caruncular epithelial cells were isolated from healthy cows during pregnancy, immediately after slaughter. Primary cell cultures derived from the 2nd and 4th month of gestation were used in the experiments. The viability of cells was assessed by MTT assay. The adhesion of cells to fibronectin was measured spectrophotometrically. The activity of metalloproteinases was confirmed by the metalloproteinase assay. Progesterone (10^–5 and 10^–7 mol/L) and prostaglandin F_2α (10^–4_, 10^–5 and 10^–7 mol/L) increased the viability of bovine caruncular epithelial cells in the 2nd month of pregnancy. The treatment with prostaglandin F_2α significantly reduced the number of adherent cells from the 2nd month of gestation at the doses of 10^–4 and 10^–5 mol/L. Both progesterone and prostaglandin F_2α were shown to have an effect of decorin resulting in both a decrease in metalloproteinase activity and an increase in adhesion of cells to fibronectin.     

The effect of two different routes of administration of oxytocin on peripheral plasma prostaglandin F(2α) metabolite levels in early post-partum dairy cows

Various parenteral treatment forms of oxytocin, as often used under praxis circumstances, may act differently on contractility of the uterus during the first days of the puerperium. Various patterns of such induced uterotonic responses may lead to alterations in the emptying characteristics of the uterine lumen, thus influencing, as a late consequence, the process of involution. Therefore, this study was designed to test whether two different parenteral administration forms of oxytocin induce changes in peripheral plasma concentrations of 15-ketodihydro-prostaglandin F(2α) (PGF(2α) metabolite) in early post-partum cows. Between 13 and 15 h after uncomplicated calving, healthy dairy cows without retained foetal membranes were treated with 50 IU oxytocin, either intramuscularly (OT-IM group; n = 15) or intravenously (OT-IV group; n = 16). Saline solution was administered intramuscularly as controls (CON group; n = 15). Jugular blood samples were taken at 10-min intervals from 1 h before to 2 h after treatment. Plasma PGF(2α) metabolite levels were measured by radioimmunoassay. No significant differences in peripheral plasma PGF(2α) metabolite concentrations occurred in the OT-IM and CON groups, but mean values significantly increased in the OT-IV group, peaking at 20 min after treatment and reaching pre-treatment baseline values again at 120 min. Although the source of prostaglandins was not investigated in this study, our results suggest that exogenous oxytocin may enhance secretion of prostaglandins by the uterus during the first day after normal calving. These prostaglandins might contribute, by an endocrine or paracrine route, to the stimulation of myometrial contractility when exogenous oxytocin is given during this early post-partum stage.     

The role of chicken IL-1β, IL-6 and TNF-α in the occurrence of amyloid arthropathy

In this study, the roles of IL-1β, IL-6 and TNF-α in amyloid arthropathic chickens with variable amounts (severe, moderate and mild) of amyloid accumulation were investigated. The presence and the levels of cytokines were evaluated in serum and in joint tissues by using ELISA and immunohistochemistry, respectively. One hundred brown layer chicks were allocated into four groups and intra-articular injections of Freund’s adjuvant were used to induce amyloid arthropathy in Groups II, III and IV. Vitamin A in group II, and methylprednisolone in Group IV were added to enhance and to reduce the severity of amyloidosis, respectively. At the end of the study, a positive correlation was observed among the incidence and severity of amyloidosis, the serum amyloid A levels and the IL-1β values both in the serum and tissues. Elevation in the tissue TNF-α levels in parallel with the severity of amyloidosis has also been noted. As a conclusion, IL-1β appears to play an important role in avian AA amyloidosis either alone or in combination with TNF-α. Further investigation is needed for understanding the role of the pro-inflammatory cytokines in avian AA amyloidosis.