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 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.     

Contractile effect of PGF2alpha and PGE2 on isolated branches of uterine and ovarian artery in different days of estrous cycle and early pregnancy in pigs

The contractile effects of PGF2alpha (3 x 10(-6) to 10(-4) M) and PGE2 (10(-7) to 10(-5) M) were examined on isolated branches of ovarian artery (OA) and extramyometrial branches of uterine artery (UA) collected from pigs in the luteal (day 10-12) and follicular phase (day 17-20) of the estrous cycle, and during early pregnancy (day 10-12). Strong contraction was demonstrated in both arteries during all investigated periods in response to PGF2alpha, which was significantly higher (P < 0.01) than to PGE2, being negligible in the follicular phase. In UA, the effective dose of PGF2alpha (ED50) amounted 7.9 x 10(-6) M and 6.3 x 10(-6) M in the luteal and follicular phase, and 5.0 x 10(-6) M in early pregnancy. ED50 for PGE2 reached 5.0 x 10(-7) M in the luteal phase, and 4.1 x 10(-7) M in early pregnancy. For both prostaglandins, the contraction was much stronger (P < 0.01) in OA than in UA branches. In OA, the ED50 for PGF2alpha was 1.2 x 10(-5) M in the luteal phase and was significantly higher (P < 0.05) than in the follicular phase (3.1 x 10(-6) M) and early pregnancy (2.7 x 10(-6) M). ED50 for PGE2 amounted 7.3 x 10(-7) M in the luteal phase and 1.7 x 10(-7) M in early pregnancy. Studies showed the influence of the estrous cycle and early pregnancy on OA branches sensitivity to the contractile effect of PGF2alpha and the lack of this effect on UA branches, and the influence of the estrous cycle on UA and OA branch contraction in response to PGE2.     

Regulation of endometrial prostaglandin F(2alpha) synthesis during luteolysis and early pregnancy in cattle

Luteal regression is caused by a pulsatile release of prostaglandin (PG) F(2alpha) from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF(2alpha) secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF(2alpha), the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF(2alpha) secretion. It has been recently demonstrated that tumor necrosis factor-alpha (TNF-alpha) stimulates PGF(2alpha) output from bovine endometrial tissue not only during the follicular phase but also during the late luteal phase, suggesting that TNF-alpha is a factor in the initiation of luteolysis in cattle. Furthermore, our recent study has shown that IFN-tau suppresses the action of TNF-alpha on PGF(2alpha) synthesis by the bovine endometrium in vitro, suggesting that IFN-tau plays a luteoprotective role by inhibiting TNF-alpha-induced PGF(2alpha) production in early pregnancy. On the other hand, factors other than oxytocin or TNF-alpha have also been suggested to be involved in the regulation of PGF(2alpha) synthesis by bovine endometrium. The purpose of this review is to summarize our current understanding of the endocrine mechanisms that regulate the timing and pattern of uterine PGF(2alpha) secretion during the estrous cycle and early pregnancy.     

Release of immunoreactive arachidonate metabolites by equine endometrium in vitro

The ability of equine endometrium to release prostaglandin (PG) F, PGE2, and leukotriene (LT) B4 was studied in vitro, using endometrial tissue from diestrous mares. Because of the high cross-reactivity of the PGF antiserum with PGF1 alpha and with PGF2 alpha, results were quoted as total immunoreactive PGF. Significant concentrations of these arachidonate metabolites were released into tissue culture medium between 1 and 24 hours of incubation. Significantly higher concentrations of PGE, but not of PGE2 or LTB4, were released from endometria of mares with chronic endometritis than from genitally normal mares. Prostaglandin F was released only in low concentrations from the endometrium of a mare with pyometra, but concentrations of PGE2 and LTB4 were similar to those of genitally normal mares.     

Estrous cycle-dependent differences in responsiveness to prostaglandins and contractile agents in sheep (Ovis aries) cervical smooth muscle

We investigated the influence of the phase of the estrous cycle on mechanical responses elicited in sheep cervix by potassium chloride (KCl), acetylcholine chloride (ACh), prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1). The cervix of adult ewes (n = 48) were classified according to the presence or absence of corpora lutea (luteal or follicular phase, respectively). Muscle strips of the circular and longitudinal layers were prepared in an organ bath and coupled to an isometric force transducer. Concentration-response curves were obtained noncumulatively. KCl and ACh produced concentration-dependent contractions in all preparations in both phases of the estrous cycle. However, maximum effect, EC50 and slope values of KCl and ACh were not significantly different between muscle layers, as well as between the phases of the estrous cycle. The prostanoid, PGF2 alpha, produced a significant reduction in the amplitude of spontaneous contractions for all preparations. The depressant effect of PGF2 alpha on spontaneous contractions of circular smooth muscle was significantly greater during the follicular than the luteal phase, whilst the depressant effect of PGF2 alpha on the longitudinal layer did not differ between phases of the estrous cycle. PGE1 significantly reduced the amplitude of spontaneous contractions on circular but not on longitudinal preparations. In conclusion, we have characterized with in vitro preparations of circular and longitudinal muscle layers of ewes during the follicular and luteal phases of the estrous cycle, the parameters of the K- and ACh-induced contractions on cervix and the efficacy of PGF2 alpha and PGE1 on inhibition spontaneous contractile activity.     

Effects of nitric oxide and tumor necrosis factor-alpha on production of prostaglandin F2alpha and E2 in bovine endometrial cells

The purpose of this study was to determine whether nitric oxide (NO) mediates tumor necrosis factor (TNF)alpha influence on the bovine endometrium. TNFalpha influence on the bovine endometrium is limited to the stromal cells. Therefore, it was interesting to find out whether NO production by the stromal cells, stimulated by TNFalpha might influence the endometrial epithelium. Moreover, we investigated the intracellular mechanisms of TNFalpha- and NO-regulated prostaglandin (PG) F(2alpha) and PGE(2) synthesis. Epithelial and stromal cells from the bovine endometrium (Days 2-5 of the oestrous cycle) were separated by means of enzymatic dispersion and cultured for 6-7 days in 48-well plates. The confluent endometrial cells were exposed to a NO donor (S-NAP; 1-1000 microM) for 24 h. S-NAP strongly stimulated PGE(2) production in both bovine endometrial cell types (P<0.001). The effect of SNAP on PGF(2alpha) production was limited only to the stromal cells (P<0.05). To study the intracellular mechanisms of TNFalpha and NO action, stromal cells were incubated for 24 h with TNFalpha or S-NAP and with NO synthase (NOS) inhibitor (L-NAME; 10 microM) or an inhibitor of phosphodiesterase (IBMX; 10 microM). When the cells were exposed to TNFalpha in combination with NOS inhibitor (L-NAME), TNFalpha-stimulated PGs production was reduced (P<0.05). The inhibition of enzymatic degradation of cGMP by IBMX augmented the actions of S-NAP and TNFalpha on PGs production (P<0.05). The overall results suggest that TNFalpha augments PGs production by bovine endometrial stromal cells partially via induction of NOS with subsequent stimulation of NO-cGMP formation. NO also stimulates PGE(2) production in epithelial cells.     

Pig endometrial cells in primary culture: morphology, secretion of prostaglandins and proteins, and effects of pregnancy

Luminal epithelial, glandular epithelial, and stromal cells were isolated from pig endometrium by enzymatic dispersion and sieve filtration. The three cell types, maintained in primary culture, showed distinctly different morphologies when viewed by light and scanning electron microscopy. Immunocytochemical staining indicated that luminal and glandular epithelial cells were positive for both cytokeratin and vimentin. However, stromal cells were positive only for vimentin. Acid phosphatase activity was detected in the culture medium of glandular cells and increased (P less than .05) when progesterone (.1 microM) was included in the culture medium. The secretion of uteroferrin by glandular cells was also indicated by one-dimensional PAGE and Western blot analysis. Stromal cells produced more (P less than .01) prostaglandin E (PGE) than prostaglandin F2 alpha (PGF2 alpha), whereas glandular cells secreted more (P less than .01) PGF2 alpha than PGE. Pregnancy status affected prostaglandin secretion in that stromal cells secreted less (P less than .01) PGE and PGF2 alpha and glandular cells secreted less (P less than .05) PGF2 alpha when they were harvested from pregnant vs cyclic pigs. Furthermore, the PGE:PGF2 alpha ratio in medium from stromal cells was greater (P less than .01) for cells collected from pregnant pigs. This culture system provides an in vitro model for studying the hormonal regulation of the endometrium and potentially may be useful for studying interactions between endometrial cells and embryos in the pig.     

Role of Tumour Necrosis Factor α in Stimulation of Prostaglandins F2α and E2 Release by Cultured Porcine Endometrial Cells

The present studies were undertaken to examine the effect of tumour necrosis factor (TNF) alpha on prostaglandins (PGs) F(2alpha) and E(2) release by cultured porcine endometrial cells harvested on days 13-16 after oestrus in comparison to stimulation with oxytocin (OT) and luteinizing hormone (LH). A time-dependent effect of TNFalpha (10 ng/ml) on PGF(2alpha) release was observed in stromal and luminal epithelial cells. Moreover, TNFalpha increased PGF(2alpha) secretion from both endometrial cell types with effective concentrations of 1 (p < 0.05), 10 and 50 ng/ml (p < 0.01). The effect of TNFalpha (10 ng/ml) on endometrial PGF(2alpha) and PGE(2) release was compared with OT (100 nmol/l) and LH (100 ng/ml). All factors affected PGF(2alpha) secretion from stromal cells, however, the stimulation tended to be more potent after OT and LH (p < 0.01) than after TNFalpha (p < 0.05) treatment. In epithelial cells, only TNFalpha was able to stimulate PGF(2alpha) release (p < 0.001). PGE(2) secretion from stromal cells increased after incubation with TNFalpha and OT (p < 0.05). Only LH stimulated PGE(2) release from epithelium (p < 0.001), and its action was very effective when compared with TNFalpha or OT (p < 0.01). Summarizing, TNFalpha induces both PGs secretion from cultured porcine endometrium, but preferentially stimulates PGF(2alpha) release from luminal epithelial cells. Therefore, similarly to OT and LH, TNFalpha may be considered as a potential modulator of endometrial PGF(2alpha) production during luteolysis in the pig.     

Effects of prostaglandin F(2alpha) and nitric oxide on the secretory function of bovine luteal cells

The objective of the present study was to investigate the influence of prostaglandin F(2alpha) (PGF (2alpha)) and nitric oxide (NO) on production of steroids and PGs by culturing bovine luteal cells obtained from ovaries on days 8-12 of the estrous cycle with a nitric oxide (NO) donor (Spermine NONOate), and a NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester dihydrochloride: L-NAME). When the cells were exposed for 24 h to PGF(2alpha) (10(-7)-10(-5) M), production of progesterone (P(4)) increased significantly at all doses used (P<0.05). Moreover, PGF(2alpha) stimulated PGF(2alpha) production (P<0.01), depressed testosterone (T) production (P<0.05), but did not affect synthesis of prostaglandin E(2) (PGE(2)). Spermine NONOate decreased P(4) production to 66%, 47% and 34% of the control concentration after treatment with 10(-5) M, 10(-4) M and 10(-3) M, respectively, but did not affect T production, and increased PGF(2alpha) synthesis (P<0.05) and PGE(2) (P<0.01) at all doses used. L-NAME increased production of P(4) (P<0.01) but did not affect (P>0.05) secretion of T, PGF(2alpha) and PGE(2). Estradiol-17beta (E(2)) was detectable on the level of sensitivity of assay and was not significantly altered by any treatments. The overall results suggest that PGF(2alpha) and NO produced locally in bovine CL play roles in the regulation of the secretory function of the bovine CL as auto/paracrine factors.     

Effect of dietary fat sources on systemic and intrauterine synthesis of prostaglandins during early pregnancy in gilts

The present experiment was conducted to determine the influence of dietary fatty acids C18:2n-6 and C18:3n-3 on the modulation of intrauterine synthesis of prostaglandin E2 (PGE2) and F2alpha (PGF2alpha) during early pregnancy in pigs. Prostaglandin E2 in uterine fluid has been previously reported to be associated with embryo survival and development. Thirty-two Yorkshire-Landrace nulliparous gilts were randomly allocated to four diets containing 5% supplemental fat. The four dietary treatments were: HT, hydrogenated tallow (26.5% C16:0 and 54.8% C18:0); SO, sunflower oil (61.3% C18:2n-6); LO, linseed oil (50.4% C18:3n-3); and SO(CLA), a mixture of sunflower oil and conjugated linoleic acids to provide 20% CLA. Treatments started 2 d after the first pubertal estrus (d -21) and lasted for 36 d (slaughter), which was 15 d after the second estrus (d 0; insemination). Fatty acids and PGE2 were measured in the peripheral blood plasma on d -19, d -7, d 0, and d 14. Fatty acids in endometrial tissues and PGE2 and PGF2alpha in the uterine fluid collected on d 15 were also measured. Concentrations of fatty acids in the plasma reflected the content of fatty acids in the diet as early as d -7. From d -7, PGE2 concentrations in the plasma were higher in gilts fed SO compared with HT (P < 0.05). Plasma PGE2 concentrations were lower (P < 0.01) on d 14 in gilts fed LO compared with HT. Total PGF2alpha contents in the uterine fluid of gilts fed LO were more than 70% lower (P < 0.05) than for the HT group. A similar trend was observed for total PGE2 content and for the ratio PGF2alpha:PGE2, but the effect (LO vs HT) was less marked (P < 0.07 and P < 0.10, respectively). There was no effect of SO or SO(CLA) on total PGE2 contents in the uterine fluid. Dietary enrichment in C18:2n-6 and/or C18:3n-3 for early pregnant gilts can influence fatty acids in plasma and endometrial tissue and can modulate circulatory and intrauterine prostaglandins.     

In vitro synthesis of prostaglandin E and F2 alpha by pig endometrium in the presence of estradiol, catechol estrogen and ascorbic acid

These experiments were undertaken to determine the potential for estradiol-17 beta (E2), 2-hydroxyestradiol-17 beta (2-OH-E2) and 4-hydroxyestradiol-17 beta (4-OH-E2) to regulate prostaglandin (PG) E and F2 alpha synthesis by pig endometrium. Endometrium was collected from pigs on d 10 of pregnancy and incubated (15 to 20 mg/well) for three 2-h periods in 2 ml of medium in 24-well culture plates. At the end of each period, the medium was removed and frozen. Later media were thawed and assayed for PGE and PGF2 alpha. During Periods 2 and 3, the medium contained 0, 25, 50, 100 or 150 microM 2-OH-E2 (Exp. 1); 0, 25 or 50 microM 4-OH-E2 (Exp. 2); or 0, 25 or 50 microM E2 (Exp. 3). Each experiment was a factorial with 2-OH-E2, 4-OH-E2 or E2 as one main effect and 0 or 1 mM ascorbate as a second main effect. Ascorbate decreased (P less than .01) PGE and PGF2 alpha release in all experiments. Two-hydroxyestradiol-17 beta decreased (P less than .01) PGE and PGF2 alpha release into the medium during Periods 2 and 3 in a dose-dependent manner (Exp. 1). In Exp. 2, 4-OH-E2 decreased (P less than .07) endometrial release of PGE and PGF2 alpha in Periods 2 and 3 and increased (P less than .01) the PGE:PGF2 alpha in Period 3. In Exp. 3, E2 decreased release of PGE during Period 3 and PGF2 alpha release during Period 2. The PGE:PGF2 alpha was not altered by E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of interleukin-1β in the regulation of porcine corpora lutea during the late luteal phase of the cycle and during pregnancy

Interleukin-1β (IL-1β) may regulate ovarian physiology. In this study, the influence of IL-1β on secretory activity within the corpora lutea (CL) of cyclic and gravid pigs was determined in vitro during different stages of the CL lifespan, e.g. on Days 10-11, 12-13 and 15-16 of the oestrous cycle and pregnancy. IL-1β (10 ng/ml) increased prostaglandin E2 (PGE2) secretion from CL of the cyclic and gravid pigs during studied days of the oestrous cycle and pregnancy. Increase (P < 0.05) of prostaglandin F2α (PGF2α) in IL-1β-treated CL was demonstrated only on Days 10-11 of the oestrous cycle. More potent stimulatory effect of IL-1β on PGE2 than PGF2α secretion resulted in the enhancement of the PGE2:PGF2α ratio in cyclic and early pregnant CL. IL-1β increased (P < 0.05) progesterone (P4) secretion only in gravid CL and had no effect on oestradiol-17β (E2) release. Expression of cyclooxygenase-2 (COX-2) mRNA was stimulated (P < 0.05) in IL-1β-treated cyclic and gravid CL. Expression of prostaglandin synthase mRNAs in response to IL-1β did not increase. In conclusion, IL-1β modulates PGE2, PGF2α and P4 secretion from porcine CL, depending on luteal stage and the surrounding hormonal milieu. The cytokine may act locally in porcine CL for luteotrophic support throughout the PGE2-mediated synthesis and secretion.     

Changes in bovine luteal progesterone metabolism in response to exogenous prostaglandin F(2alpha)

An experiment was conducted to determine the effect of prostaglandin F2alpha (PGF2alpha) on luteal synthesis of progesterone (P4) and related progestins. Sixteen beef heifers were assigned in equal numbers to four groups in a 2x2 factorial arrangement of treatments. The experiment consisted of two levels of PGF2alpha analog (0 and 500 microg) and two levels of time (4 and 24 h after injection) of corpus luteum collection. All heifers were injected intravenously with saline (2 ml) or PGF2alpha (cloprostenol) on day 8 of the estrous cycle (estrus=day 0). Jugular blood was collected at 0, 1, 2, 3, 4 and 20, 21, 22, 23, and 24 h after injection. Resulting sera were analyzed for P4 by use of radioimmunoassay. Luteal tissue was analyzed by gas chromatography/mass spectrometry for P4, 20beta-hydroxyprogesterone, pregnenolone, and allopregnanolone (3beta-hydroxy-5alpha-pregnan-20-one). Treatment with PGF2alpha reduced serum concentrations of P4 as early as 1 h after injection (P<0.005) and steroid levels remained low over 24 h. Similarly, administration of PGF2alpha caused a decline in luteal P4 (P<0.005), 20beta-hydroxyprogesterone (P<0.10), and pregnenolone (P<0.05). In contrast, treatment with PGF(2alpha) caused an increase in luteal allopregnanolone over time (time x treatment interaction; P<0.05). These data are interpreted to suggest that PGF2alpha promotes conversion of P4 to the metabolite allopregnanolone.     

Modulation of arachidonic acid metabolism by bovine alveolar macrophages exposed to interferons and lipopolysaccharide

Stimulation of bovine alveolar macrophages with calcium ionophore A23187 resulted in marked production of leukotriene (LT)B4 and a lesser increase in thromboxane (TX)B2, whereas opsonized zymosan (OPZ) resulted in production of TXB2 and relatively small increases in LTB4 and prostaglandin (PG)F2 alpha. Alveolar macrophages incubated with recombinant bovine interferon-gamma or lipopolysaccharide, and subsequently stimulated with A23187 or OPZ, had altered arachidonic acid metabolism, producing markedly increased amounts of TXB2 and PGF2 alpha, and slightly increased LTB4. Incubation of alveolar macrophages with lipopolysaccharide had a more profound effect on the increased amounts of TXB2 and PGF2 alpha, observed in response to stimulation with A23187 or OPZ, than did incubation with interferon-gamma. Alveolar macrophages incubated with recombinant bovine interferon-alpha 1-1 also produced slightly increased amounts of LTB4 when stimulated with A23187 or OPZ. Altered arachidonic acid metabolism by alveolar macrophages exposed to interferons and lipopolysaccharide may contribute to the development of pulmonary inflammation, such as in the early stages of bacterial pneumonia following viral infections that induce interferon production.     

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.     

The effect of progesterone on oxytocin-stimulated intracellular Ca2+ mobilisation and prostaglandin secretion in porcine endometrium

We have studied in the porcine endometrium the expression of oxytocin receptor (OTR) mRNA and the effect of progesterone (P4) on oxytocin/oxytocin receptor (OT/OTR) function concerning intracellular Ca2+ mobilisation ([Ca2+]i), prostaglandin F2alpha (PGF2alpha) and E2 (PGE2; PG) secretion. Tissue was taken from cyclic and early pregnant pigs (days 14-16). A higher expression of OTR mRNA (P < 0.05) was observed in the endometrium of cyclic than pregnant pigs. The stimulatory (P < 0.05) effect of OT (10(-7) M) on [Ca2+]i mobilisation was noticed within 15-60 s and 30-60 s in endometrial stromal cells of cyclic and pregnant pigs, respectively. In the presence of P4 (10(-5) M) basal and OT-stimulated [Ca2+]i concentrations decreased in stromal cells during luteolysis and pregnancy. In stromal cells P4 delayed mobilisation of [Ca2+]i in response to OT by 15 s during luteolysis and had no effect during pregnancy. In cyclic and pregnant epithelial cells OT stimulated mobilisation of [Ca2+]i in 45 s and 60 s, respectively. Oxytocin increased (P < 0.05) PGF2alpha secretion during luteolysis and pregnancy and PGE2 during luteolysis from endometrial slices. Progesterone did not inhibit this stimulatory effect. During luteolysis OT increased (P < 0.05) PGF2alpha in epithelial and stromal cells and PGE2 secretion in epithelial cells. In the presence of P4 this effect of OT was reduced only in stromal cyclic cells (6 h culture). The presence of P4 decreased the effect of OT on [Ca2+]i mobilisation only in stromal cells. We found that, in most conditions, P4 did not inhibit the OT-stimulated secretion of PG in the porcine endometrium.     

Prostaglandin F2alpha (PGF2alpha) independent and dependent regulation of the bovine luteal endothelin system

We have examined the genes of the endothelin system that are targets for regulation by prostaglandin F2alpha (PGF2alpha). The effects of a luteolytic dose of PGF2alpha ) on the mRNA encoding endothelin converting enzyme-1 (ECE-1), pre-pro endothelin-1 (pp ET-1) and the ET receptors ETA, ETB, in bovine corpus luteum (CL) during the early (days 1 and 4), mid (day 10) or late (day 17) luteal phases were examined. The effect of the PGF(2alpha) treatment on ECE-1 protein, Big ET-1 and the biologically active mature ET-1 peptide were also examined. Most importantly, the direct ECE-1 activity was determined. Before day 10 of the cycle, in a PGF2alpha-independent manner, the amounts of mRNA encoding ET-1, ECE-1, ETA, and ETB were increased steadily from day 1. After day 10 of the cycle, expression of mRNA encoding pp ET-1 and ETA acquired responsiveness to exogenous PGF2alpha and both genes were up-regulated by the PGF2alpha treatment. This effect of PGF2alpha was also detected for the proteins corresponding to the mature ET-1. The enzymatic activity of ECE-1 remained unchanged throughout the lifespan of the CL in spite of the detected changes in mRNA and protein. The results suggest that the luteal endothelin system is regulated in a PGF2alpha-independent and -dependent manner. Importantly, an alteration in luteal ET-1 availability is most likely achieved by modulating the expression of mRNA encoding pp ET-1 and not by the amount or activity of ECE-1. This interpretation is supported by the observation that the activity of ECE-1 remained unchanged throughout the ovarian cycle. The combined effects of greater ET-1 availability and gene expression encoding the ETA receptor in the late luteal phase could render the CL, at this developmental stage, more sensitive or responsive to ET-1. If the luteal tissue is responsive to the available ET-1 during the early phase of the ovarian cycle, an additional role for ET-1 should be considered beyond mediating the luteolytic actions of PGF2alpha. Agents blocking the actions of ET-1 might be the best approach to interfere with the luteal ET system and test its physiological role(s) in vivo.     

Effect of PGF2 alpha administration on the release of endogenous PGF2 alpha and oxytocin in indomethacin-treated goats

Repeated intramuscular injection of 1 mg prostaglandin F2 alpha (PGF2 alpha) during the luteal phase of the oestrous cycle of the goat hastened luteolysis and resulted in rapid increases in jugular concentrations of 13,14-dihydro-15-keto PGF2 alpha (PGFM), the primary metabolite of PGF2 alpha, and of oxytocin; similar injections of PGF2 alpha in indomethacin-treated goats had a reduced effect on PGFM and oxytocin concentrations, and failed to induce luteolysis. The same injections of PGF2 alpha were without effect on PGFM and oxytocin concentrations in ovariectomised goats. The results suggest that exogenous PGF2 alpha, or endogenous PGF2 alpha released at luteolysis, may induce the release of ovarian oxytocin in the goat.