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.     

Expression of hypoxia-inducible factor-1α and vascular density in mammary adenomas and adenocarcinomas in bitches

Background

The study aimed at examining hypoxia-inducible factor (HIF)1α expression in adenocarcinomas and adenomas in bitches in regard to tumour malignancy grade, proliferation, apoptosis and vascularisation. Therefore, paraffin sections of 15 adenomas and 64 adenocarcinomas sampled from 79 dogs aged 6 to 16 years were analysed.

Results

A significantly higher HIF-1α expression was noted in adenocarcinomas in comparison to adenomas (P < 0.0004). Moreover, HIF-1α expression in adenocarcinomas correlated positively with tumour malignancy grade (r = 0.59, P < 0.05), Ki-67 antigen expression (r = 0.43; P < 0.0005), TUNEL-positive cells (r = 0.62, P < 0001) and tumour vascularity measured by quantification of vessels characterized by the expression of von Willebrand Factor (r = 0.57, P < 0.05).

Conclusion

Results of this study indicate a similar biological role of HIF-1α in dogs and in humans, which may confirm suitability of the animal model in investigations on progression of tumours in humans.     

The role of phosphoinositide-derived second messengers in oxytocin-stimulated prostaglandin F2α release from endometrium of pigs

The mechanism for prostaglandin (PG) F_2α release from pig endometrium after oxytocin (OT) treatment is unknown. OT may rapidly stimulate inositol (1,4,5)-trisphosphate (IP₃) and diacylglycerol (DAG) formation, consistent with the concept of rapid activation of a second-messenger system. In support of this hypothesis, endometrial IP₃ levels were increased (P < 0.05) within 0.5 min after treatment with 0.1 μM OT. In contrast, increased DAG formation was not detected after treatment with OT. However, similar to the stimulation of endometrial PGF_2α secretion observed after OT treatment (P < 0.001), PGF_2α release was increased (P < 0.01) after treatment with phorbol-12myristate-l3-acetate (PMA), which mimics DAG activation of protein kinase C. Further, stimulation of endometrial PGF_2α secretion did not result from cell death induced by PMA or OT because lactate dehydrogenase, a cytosolic marker of cellular integrity, did not leak into the medium after PMA or OT treatment. In contrast, 0.5% saponin (positive control for cell death and concomitant release of lactate dehydrogenase) increased PGF_2α secretion (P < 0.05) and lactate dehydrogenase release (P < 0.001). These results indicate that OT induces endometrial IP₃ production in a rapid manner indicative of a second-messenger system. The finding that increased DAG was not also detected after OT treatment may reflect rapid metabolism or compartmentalized production of DAG involved in the second-messenger stimulation of phospholipase C. The high background of DAG used in the biosynthesis of cellular lipids would obscure the rather small spatially localized changes in DAG levels resulting from the activation of phospholipase C. The finding that DAG was present at approximately 10 to 20-fold higher levels than IP₃ in resting cells was consistent with this conclusion.     

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.     

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.     

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.     

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.     

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.     

Prostaglandin F2α metabolite levels in normal and uterine-infected postpartum cows

The stable metabolite of prostaglandin F₂, 15 keto-13, 14-dihydroprostaglandin F₂ (PGFM), was measured from peripheral blood samples collected at specified intervals postpartum from 7 normal dairy cows and 4 cows with apparent endometritis. Plasma PFGM levels were significantly (P<.05) elevated for the first 5 days postpartum in the cows with endometritis (ranging from 4.0 to 5.0 ng/ml) compared to the controls (approximately 1.0 ng/ml). Beyond 5 days postpartum, plasma PGFM levels were not significantly different and decreased to approximately 0.4 ng/ml by day 13 in both groups. Time to uterine involution was not different between groups (less than 30 days). Therefore, uterine infections in cows during the puerperium was associated with elevated circulating PGFM levels. These findings and the observation that PGF₂ is not uterotonic in the puerperal cow do not suggest a therapeutic use of PGF₂ in order to evacuate the uterus.     

Effect of phthalate esters on the secretion of prostaglandins (F2α and E2) and oxytocin in cultured bovine ovarian and endometrial cells

The influence of phthalate esters di-2-ethylhexyl phthalate (DEHP) and mono-2-ethylhexyl phthalate (MEHP) on uterine prostaglandin (PGF2α and PGE2) and ovarian oxytocin secretion was investigated. Endometrial, granulosa, and luteal cells from cows on days 8–12 of the estrous cycle were treated with DEHP or MEHP (0.1, 1, or 10 ng/mL). We found that DEHP and MEHP stimulated (P < 0.05) secretion of PGF2α and inhibited (P < 0.001) secretion of PGE2 from endometrial cells. The ratio of PGF2α to PGE2 was markedly altered. The endocrine disrupting chemicals also enhanced secretion of oxytocin (P < 0.05) from ovarian cells. Our results indicated that DEHP and its metabolite MEHP could affect the process of the estrous cycle by impairing secretion of prostaglandin from the uterus and oxytocin from the ovary.     

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α

We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8–12 after ovulation) were cultured with PGF (0.01, 0.1, 1 μM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 μM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One μM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.