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Second messenger signalling through cyclic AMP (cAMP) plays an important role in the response of the endometrium to prostaglandin (PG) E(2) during early pregnancy. Arachidonic acid, which is a by-product of the luteolytic cascade in ruminants, is a potential paracrine signal from the epithelium to the stroma. We investigated the effects of arachidonic acid on the response of the stroma to PGE(2). cAMP was measured in bovine endometrial stromal cells treated with agents known to activate or inhibit adenylyl cyclase, protein kinase C (PKC) or phosphodiesterase (PDE). PGE(2) increased the intracellular cAMP concentration within 10 min, and this effect was attenuated by arachidonic acid and the PKC activator, 4beta-phorbol myristate acetate (PMA). The inhibitory effect of arachidonic acid on PGE(2)-induced cAMP accumulation was prevented by the PKC inhibitor, RO318425, and was absent in cells in which PKC had been downregulated by exposure to PMA for 24 h. The effect of arachidonic acid was also prevented by the PDE inhibitor, 3-isobutyl-1-methylxanthine. Arachidonic acid was shown by immunoblotting to prevent induction of cyclooxygenase-2 by PGE(2), forskolin or dibutyryl cAMP. The results indicate that arachidonic acid activates PDE through a mechanism involving PKC, counteracting a rise in intracellular cAMP in response to PGE(2). The data suggest that arachidonic acid antagonizes PGE(2) signalling through cAMP in the bovine endometrium, possibly acting to ensure a rapid return to oestrus in the case of failure of the maternal recognition of pregnancy.

Up-regulation of endometrial oxytocin receptor (OTR) expression followed by an increase in pulsatile endometrial prostaglandin (PG) F(2alpha) secretion causes luteolysis in cattle. Inhibition of luteolysis is essential for the maternal recognition of pregnancy but also occurs in association with endometritis. The factors regulating OTR expression at this time are unclear. The OTR gene promoter region contains binding elements for acute phase proteins but their function has not been established. This study investigated the effects of various cytokines on OTR expression and on PGF(2alpha) and PGE(2) production in explant cultures of bovine endometrium. Endometrium was collected in the late luteal phase (mean day of cycle 15.4+/-0.50) or early luteolysis (mean day of cycle 16.4+/-0.24) as determined by the initial concentration of endometrial OTR. Explants were treated for 48 h with: (i) lipopolysaccharide (LPS) and/or dexamethasone (DEX), (ii) ovine interferon-tau (oIFN-tau), or (iii) human recombinant interleukin (IL)-1alpha, -2 or -6. OTR mRNA was then measured in the explants by in situ hybridisation and the medium was collected for measurement of PGF(2alpha) and PGE(2) by RIA. LPS treatment stimulated production of PGF(2alpha), whereas DEX either alone or in combination with LPS was inhibitory to both PGF(2alpha) and PGE(2). Neither of these treatments altered OTR mRNA expression. oIFN-tau reduced OTR mRNA expression but stimulated production of both PGF(2alpha) and PGE(2). In endometrial samples collected in the late luteal phase, IL-1alpha, -2 and -6 all inhibited OTR mRNA expression, but IL-1alpha and -2 both stimulated PGF(2alpha) production. In contrast, when endometrium was collected in early luteolysis, none of the interleukins altered OTR expression or caused a significant stimulation of PGF(2alpha) production but IL-2 increased PGE(2). Neither IL-1alpha nor -2 altered OTR promoter activity in Chinese hamster ovary cells transfected with a bovine OTR promoter/chloramphenicol acetyl transferase reporter gene construct. In conclusion, the action of interleukins on both OTR mRNA expression and endometrial prostaglandin production alters around luteolysis. Pro-inflammatory interleukins suppress OTR expression in the late luteal phase, while LPS stimulates PGF(2alpha) without altering OTR mRNA expression. IL-I and -2 and LPS are therefore unlikely to initiate luteolysis but may cause raised production of PGF(2alpha) during uterine infection.

A sheep type I interferon receptor (oIFNAR1) cDNA was isolated from a lambda-ZAP library using a reverse transcription (RT)-PCR product probe generated from oestrous endometrial RNA. The oIFNAR1 cDNA was 79, 66 and 95% homologous to human, murine and bovine IFNAR1 cDNAs respectively. The encoded receptor was a 560-amino acid transmembrane protein 80, 66 and 95% similar to human, murine and bovine IFNAR1 respectively. Northern blot analysis of endometrial mRNA revealed the presence of 6.5, 4.3 and 3.7 kb transcripts. Using semi-quantitative RT-PCR the oIFNAR1 mRNA was not found to be down-regulated after 72 h treatment with bovine recombinant IFN-alpha I in in vitro experiments with endometrial explants.

Oxytocin receptor binding activity in explants of caruncular and intercaruncular endometrium collected from luteal phase ewes increased during culture. An initial rise in binding activity occurred during the first 24 h of culture in both tissues; binding activity in intercaruncular endometrium continued to increase until day 6, remained unchanged on day 8 and had decreased by day 10 of culture. The maximum concentration of receptors in caruncular endometrium was significantly lower than that in intercaruncular endometrium (P < 0.001) and did not change significantly between days 4 and 10 of culture. Apparent dissociation constants and maximal binding of oxytocin receptor in cultured caruncular and intercaruncular endometrium were 3.09 and 2.72 nmol l-1 and 249 and 459 fmol [3H]oxytocin bound mg-1 protein, respectively. Concentrations of oxytocin receptor remained constant in myometrium during 96 h of culture. The rise in endometrial oxytocin receptor concentration did not result from exposure to fetal calf serum, phenol red or insulin in the culture medium. Substituting fetal calf serum with sheep serum or BSA did not block the rise in receptor binding activity. Actinomycin D and cycloheximide inhibited the rise in receptor concentration in both tissues. Co-culture of lung or kidney with endometrium had no effect on binding activity, whereas co-culture with luteal tissue effectively reduced the rise in oxytocin receptor concentration. To establish whether synthesis of functional oxytocin receptors occurred during culture, the effect of oxytocin on prostaglandin F2 alpha (PGF2 alpha) production was assessed in fresh tissue and after 48 h in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Slices of caruncular endometrium from steroid-treated ovariectomized sheep were incubated with myo-[2-3H]inositol to label tissue phosphatidylinositol. Effects of oxytocin were determined on the rate of incorporation of radioactivity into phosphatidylinositol and on the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol. Incorporation of radioactivity into phosphatidylinositol was linear during 2 h incubations; 10(-7) M (100 nM)-oxytocin caused a 2.8-fold increase in the rate of incorporation. In the presence of Li+, addition of 10(-7) M-oxytocin to slices in which phosphatidylinositol was pre-labelled caused mean increase of 40-fold in the incorporation of radioactivity into inositol mono-, bis- and tris-phosphates. Inositol 1,3,4-trisphosphate was quantitatively the major trisphosphate formed. The action of oxytocin on phosphoinositide hydrolysis was dose- and time-dependent, occurring at concentrations within the range observed in plasma during episodes of secretion in vivo, and with a time course comparable with that of the action of oxytocin on uterine prostaglandin production. The effect of oxytocin on incorporation of radioactivity into inositol phosphates was not affected by inhibitors of prostaglandin synthesis. Diacylglycerol 1- and 2-lipases in caruncular endometrium converted up to 72% of added 2-[3H]arachidonyldiacylglycerol into [3H]arachidonic acid during 30 min incubations at pH 7.0. Caruncular endometrium contained 1.49 mumol of phosphatidylinositol/g, representing approx. 0.2 mumol/g of phosphatidylinositol arachidonic acid. It is proposed that the stimulation of endometrial prostaglandin synthesis by oxytocin is accounted for by increased hydrolysis of phosphoinositides to diacylglycerol and inositol phosphates with subsequent release of arachidonic acid from diacylglycerol.