Prostaglandins F2α and E2 Secretion by Porcine Epithelial and Stromal Endometrial Cells on Different Days of the Oestrous Cycle

The endometrial tissue of the uterus plays a key role in reproduction and is a source of hormones and factors responsible for the proper physiological function of reproductive tract during the oestrous cycle and pregnancy. In this study, we investigated the pattern of PGF(2alpha) and PGE(2) secretion from cultured porcine endometrial cells at different days of the oestrous cycle. Epithelial and stromal cells were isolated by differential enzymatic digestion on days 6-8, 10-12 and 14-16. After attachment cells were incubated for 3 and 24 h to estimate PGF(2alpha) and PGE(2) output. The purity of culture was 85-90% for epithelial and 95-98% for stromal cells as determined by immunofluorescent staining. Release of PGF(2alpha) and PGE(2) was affected by cell type, days of the oestrous cycle and the time of incubation. After 3 h of incubation epithelial cells secreted more PGF(2alpha) than PGE(2) during all studied periods of the oestrous cycle (p < 0.01 and p < 0.001, respectively), whereas stromal cells released more PGE(2) (p < 0.01) on days 10-12 and 14-16. Longer incubation of stromal cells revealed that PGF(2alpha) output tended to overcome PGE(2) on days 10-16. The lowest secretion of prostaglandins was observed on days 6-8 in both cell types. The highest secretion of PGF(2alpha) from epithelium was measured on days 10-12 after 24 h of incubation when compared with other days studied (p < 0.001). In stromal cells, PGE(2) output increased on consecutive days studied (p < 0.001) after 3 h of incubation. The differential properties of endometrial cell types seem to play an important role in the profile of PGF(2alpha) and PGE(2) release before and during luteolysis. Described endometrial cells culture might serve as the model for further studies on the hormonal regulation of prostaglandin production in the pig.     

Effect of flunixin meglumine on prostaglandin F2α synthesis and metabolism in the pig

The effect of flunixin meglumine on prostaglandin synthesis and metabolism was evaluated in the pig in vivo. It was found that the prostaglandin metabolite, 15-ketodihydro-PGF2 alpha, was decreased in the peripheral circulation within 20 min of injection of the drug. In therapeutic doses in the pig the drug had no effect on the metabolism of PGF2 alpha. Flunixin was compared with some other non-steroidal anti-inflammatory drugs in an in vitro test system utilizing sheep vesicular gland microsomes. It was concluded that this drug is a potent inhibitor of prostaglandin synthesis.     

Luteolytic Effect of Prostaglandin F2α on Bovine Corpus Luteum Depends on Cell Composition and Contact

Prostaglandin F_2α (PGF_2α) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF_2α action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF_2α action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF_2α) for 0.5 h. Dinoprost caused a short‐time increase in progesterone (P4), whose concentration decreased thereafter (at 6‐, 10‐, 12‐ and 24‐h after treatment). In experiment 2, the direct effect of PGF_2α on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF_2α after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF_2α in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C₄ were determined. Although PGF_2α treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC₄ increased after the treatment of PGF_2α both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF_2α action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF_2α role in vitro.     

Timing of Ovulation and Fertility of Heifers After Synchronization of Oestrus with GnRH and Prostaglandin F2α

Synchronization of oestrus and/or ovulation can reduce workload in heifer reproductive management. The objective of this study was to compare two protocols to synchronize oestrus and/or ovulation using GnRH and prostaglandin F_2α (PGF_2α) in dairy heifers concerning their effect on follicular dynamics and reproductive performance. Four trials were carried out. In trial 1, 282 heifers were treated with GnRH and PGF_2α 7 days apart (GP protocol). One group was inseminated on detection of oestrus (IDO 1), and the other group received two timed artificial inseminations (AI) 48 and 72 h after PGF_2α administration (TAI 1). In trial 2, 98 heifers were synchronized with the same GP protocol. Heifers in IDO 2 were treated as in IDO 1, heifers in TAI 2 received two TAI 48 and 78 h after PGF_2α administration. In trial 3, heifers in IDO 3 (n = 71) were again treated as in IDO 1. Heifers in TAI 3 (n = 166) received a second dose of GnRH 48 h after PGF_2α (GPG protocol) and TAI together with this treatment and 24 h later. Trial 4 compared the timing of ovulation after the GP and the GPG protocol, using a subgroup of the heifers from trials 1 to 3. The ovaries of the heifers were scanned via ultrasound at 48, 56, 72, 80, 96 and 104 h after PGF_2α administration. Timing of ovulation and size of the ovulatory follicles were compared between the two groups. In trials 1 to 3, conception rates to first service were between 49 and 66%. They did not differ significantly between IDO and TAI groups within or between trials. Pregnancy rates per synchronization were numerically higher in the TAI groups, but the difference was not significant. Conception rates to breeding on spontaneous oestrus in heifers returning to oestrus were higher than that after synchronized oestrus. In trial 4, more heifers ovulated before the end of the observation period in GPG than in GP (96.5% vs 74.7%; p < 0.001). Overall, ovulatory follicles were smaller in GPG (13.1 ± 1.9 mm vs 14.3 ± 1.9 mm; p < 0.001).     

Specific binding of dl-cloprostenol and d-cloprostenol to PG F2α receptors in bovine corpus luteum and myometrial cell membranes

Prostaglandin F_2α receptors (PGF_2α Rs) were measured in bovine corpus luteum and myometrial cell membranes using a radiometric method. The inhibition of labelled PGF_2α binding exerted by d-cloprostenol, dl-cloprostenol, PGF_2α and PGE₁ (l0–¹¹ M to 10^–4 M) was evaluated in vitro. Results strongly suggest that cloprostenol binding to PGF_2αRs is stereospecific. d-Cloprostenol and PGF_2α were equipotent, about 150 times more potent than d-cloprostenol (P < 0.05) and approximately 280 times more potent than PGE, (P < 0.05) in inhibiting [3H]PGF_2α binding to corpus luteum cell membranes. Such differences were less evident in myometrial cell membranes, where d-cloprostenol and PGF_2α were about 10 times more potent than d-cloprostenol (P < 0.05) and approximately 95 times more potent than PGE_l (P < 0.05).     

Pharmacokinetics of flunixin and its effect on prostaglandin F2α metabolite concentrations after oral and intravenous administration in heifers

Flunixin meglumine (FM) was administered either orally as granules or intravenously to six heifers in a two period crossover study. Single doses of 2.2 mg/kg body weight were used. Pharmacokinetic variables were calculated using statistical moment methods. The effect exerted by flunixin was measured as changes in the basal plasma concentration of the main metabolite of prostaglandin (PG) F_2α. After oral FM the arithmetic means of pharmacokinetic variables were: MRT = 12.7 h; MAT = 6.3 h; C _max= 0.9 μg/mL; t _max= 3.5 h. The bioavailability was 60% and the mean half-life (harmonic mean) was 6.2 h. Oral administration of FM inhibited as effectively as intravenous administration the prostaglandin biosynthesis. The concentration of the PG metabolite decreased almost as rapidly as after intravenous administration. The duration of the effect was prolonged and the PG metabolite concentration was significantly lower between 10 and 30 h after oral than after intravenous administration. The results indicate that oral dosing of flunixin, in the form of granules, can be an alternative to intravenous administration for therapeutic use in cattle.     

Bovine Endothelial Cells Interact with Fully-luteinized, but Not Luteinizing, Granulosa Cells in the mRNA Expression of Endothelin-1 System in Response to Prostaglandin F2α

The corpus luteum (CL) undergoes regression by prostaglandin (PG)F(2alpha) from uterus and endothelin-1 (ET-1) plays an important role during luteolysis as a local mediator of PGF(2alpha) in the cow. Endothelial cells (EC) and luteal cells are main cell types making up the CL and their interactions are vital for CL function. We aimed to examine the relevance of interactions between EC and luteal cells on stimulation of genes which involved ET-1 synthesis by PGF(2alpha). We further focused the impact of maturity of luteal cells on the stimulation of the genes. To make a microenvironment which resembles the CL, we used bovine aortic endothelial cells (BAEC) and luteinizing or fully-luteinized granulosa cells (GC) and evaluated the effect of PGF(2alpha) on the expression for mRNA of ET-1 system by using real-time RT-PCR. PGF(2alpha) stimulated the expression of preproET-1 and endothelin converting enzyme-1 mRNA only in the co-cultures of BAEC with fully-luteinized GC, but not with luteinizing GC. The data suggest that interactions between BAEC and fully-luteinized GC enhance the capability of BAEC to produce ET-1 in response to PGF(2alpha). This mechanism may contribute to the local induction of luteolytic action of PGF(2alpha) which is dependent on the age/maturation of the CL.     

Utilization of F1 information in estimating QTL effects in F2 crosses between outbred lines

Quantitative trait loci (QTL) analysis in designed experiments is investigated using a mixed model framework through the modification of segment mapping techniques. Allele effects are modelled in the F₁ generation allowing the estimation of additive substitution effects while accounting for QTL segregation within lines and differences in mean QTL effects between lines. The resulting approach is called F₁ segment mapping. Simulation is used to illustrate the method and its properties. F₁ segment mapping has advantages over F₂ segment mapping in the derivation of exact additive genetic covariances and in the computation time for variance component estimation.     

Stimulation of Libido in Pubertal and Mature Boars with Prostaglandin F2α Analogs: Clinical Observations.

Contents Observations on 156 pubertal and 120 mature boars trained and used for semen collection at a.i. studs indicated that treatment with 25 mg PGF₂α (Enzaprost) 30 min. before collection, overcame to a large extent lack or loss of libido. It improved markedly the success of the first training session of pubertal boars: more than 90% accepted a dummy and allowed for semen collection. Treatment reduced reaction time in older boars with fatigue considerably to normal values. Repeated treatments, if required, showed similar favorable responses.     

Effects of Ram Introduction After the Second Prostaglandin F2α Injection on Day 11 on the LH Surge Characteristics in Fat-Tailed Ewes

The aim of this study was to evaluate the effects of ram introduction after the second prostaglandin F_2α (PG F_2α) injection on day 11 on the secretion characteristics of pre‐ovulatory LH surge of fat‐tailed ewes. Multiparous Morkaraman ewes (n=12) were divided into three groups by balancing the groups for liveweight (BW) and body condition score (BCS). On the day of second PGF_2α injection (0 h), performance tested rams (n=2) were either introduced to the ewes at 0 h (ram 0 group, n=4) or at 18 h (ram 18 group, n=4) or were not introduced (control group, n=4). Blood samples were collected at 6, 18, 42, 48, 56, 62, 66, 70, 74, 78 and 90 h for the determination of pre‐ovulatory LH surge. BCS and BW during the experimental period were 2.2 ± 0.2 units and 50.9 ± 2.3 kg, 2.4 ± 0.4 units and 49.2 ± 6.2 kg, 2.1 ± 0.3 units and 45.9 ± 4.4 kg, respectively for the ram 0, ram 18 and control groups (p > 0.05). No significant difference was observed in LH surge characteristics for the experimental groups. Peak LH concentrations were also not different between groups (p > 0.05) and they were 12.2 ± 8.3, 29.1 ± 9.9 and 15.8 ± 9.5 μg/l for the ram 0, ram 18 and control groups, respectively. There was, however, a significant correlation between peak LH concentrations and BCS (p < 0.05, R²=0.373). In conclusion, it appears that, compared with ram introduction, variability in body condition of the ewe has much pronounced effect on the amount of LH secreted after the usage of two PGF_2α injections (11 days apart) as a tool for oestrus synchronization.     

Assessment of histamine, bradykinin, prostaglandins E1 and E2 and carrageenin as vascular permeability agents in the horse

The vascular leakage induced by histamine, bradykinin, serotonin and prostaglandin E1 and E2 was assessed. The test agents were injected intradermally into the shaved thoracic skin of horses and the vascular leakage estimated either semi-quantitatively by recording the diameter of the lesions or by measuring the actual volume of extravasated plasma in microliters using iodine-125-labelled human serum albumin (125I-HSA) as a marker in the blood plasma. Using the latter method, the vascular leakage induced by carrageenin and the effect of coadministered prostaglandins E1 and E2 upon the vascular leakage of both histamine and bradykinin were also investigated. No obvious lesions resulted when serotonin (10(-2) mol/l) was injected but histamine and bradykinin produced circular lesions which increased in diameter for approximately 30 min. The size of the lesions and volume of extravasated plasma was dose dependent. On a molar basis, bradykinin (10(-6) mol/l, 10(-5) mol/l) was more potent than histamine but they were equipotent at 10(-4) mol/l. The size of the lesions induced by carrageenin were independent of their anatomical location on the thorax. Except for the second hour, the hourly volume of vascular leakage increased until the fifth hour when the experiment was concluded. The maximum vascular leakage resulting from the injection of prostaglandin E1 or E2 (1, 10, 100 or 1000 ng) was 7 microliters but when co-administered with bradykinin (10(-6) mol/l), the volume of leaked plasma increased from 29 to 78 microliters. No synergy was observed when either prostaglandin was co-administered with histamine (10(-5) mol/l).     

Fate of etiproston, a synthetic analogue of PGF2α, in cows

The pharmacokinetics and metabolic fate of labelled compounds were investigated after intramuscular administration of ³H-radiolabelled etiproston to nine cows. Elimination was rapid ( t _1/2β= 2.8 h). Forty-eight h after administration 92.6% of the radioactivity had been eliminated, mainly via the urinary (66% at 48 h) and faecal routes (26% at 48 h). In comparison, little elimination in milk occurred (less than 0.034% dose/l by 24 h). Radioactivity at the injection site 48 h after administration was seen in one cow (< 4.68 × 10^-5% dose/g). No radioactivity was detected in the tissues. Urinary metabolites were purified and isolated using XAD-2 extraction and preparative HPLC in reverse and normal phases. The main urinary metabolite, identified by mass spectrometry, was the tetranor acid derivative in equilibrium with its lactone form.     

The effects of pentoxifylline infusion on plasma 6-keto-prostaglandin F1α and ex vivo endotoxin-induced tumour necrosis factor activity in horses

Pentoxifylline (7.5 mg/kg) was bolused intravenously to eight healthy horses and was immediately followed by infusion (1.5 mg/kg/h) for 3 h. Clinical parameters were recorded and blood samples were collected for 24 h. Plasma was separated and concentrations of pentoxifylline, its reduced metabolite I, and 6-keto-prostaglandin F_1α were determined. Heparinized whole blood was also incubated ex vivo with 1 ng Escherichi coli endotoxin/mL blood for 6 h before determination of plasma tumour necrosis factor activity. The peak plasma concentrations of pentoxifylline and metabolite I occurred at 15 min after bolus injection and were 9.2± 1.4 and 7.8± 4.3 μg/mL, respectively. The half-life of elimination ( t _½β) of pentoxifylline was 1.44 h and volume of distribution ( V d_area) was 0.94 L/kg. The mean plasma concentration of 6-keto-prostaglandin F_1α increased over time, with a significant increase occurring 30 min after the bolus administration. Ex vivo plasma endotoxin-induced tumour necrosis factor activity was significantly decreased at 1.5 and 3 h of infusion. These results indicate that infusion of pentoxifylline will increase 6-keto-prostaglandin F_1α and significantly suppress endotoxin-induced tumour necrosis factor activity in horses during the period of infusion.