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ABSTRACT

Pertussis toxin catalysed the ADP-ribosylation of a protein of M _r 40 000 in ovine luteal tissue. Ribosylation of 45% of this protein in whole cell incubations (as judged by subsequent ribosylation of cell-free preparations in the presence of [³²P]NAD) attenuated the prostaglandin (PG)F_2α-stimulated hydrolysis of [³H]inositol-labelled phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate by 60%, but did not affect the inhibition by PGF_2α of LH-stimulated accumulation of cyclic AMP. It is concluded that activation of phospholipase C by PGF_2α involves a pertussis toxin-sensitive protein, probably a G protein, and that the inhibitory effect of PGF_2α on LH-stimulated adenylate cyclase is unlikely to be directly mediated by such a protein.

ABSTRACT

The polymerase chain reaction (PCR) was used to generate a 131 bp cDNA encoding part of the sheep endometrial oxytocin receptor. The nucleotide sequence of this cDNA was 93.8% identical to the human oxytocin receptor sequence in this region. When used to probe Northern blots of sheep endometrial RNA the PCR product identified a 6.7 kb mRNA which appeared and disappeared during the oestrous cycle in parallel with the oxytocin receptor molecule as measured by ligand binding. The sheep endometrial receptor mRNA was significantly larger than the human myometrial mRNA (4.7 kb). It is suggested that the cloned cDNA described here is an appropriate probe for use where it is required to measure sheep oxytocin receptor mRNA.

ABSTRACT

A sheep type I interferon receptor (oIFNAR1) cDNA was isolated from a λ-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-α_I in in vitro experiments with endometrial explants.

ABSTRACT

A bovine trophoblast interferon (IFN-τ) gene promoter sequence (− 450 to +26 bp relative to the transcription start site) led to expression of reporter gene (CAT) constructs transfected into L929 (murine fibroblast) or JAR (human choriocarcinoma) cells. Expression depended on the presence of an exogenous (SV40) enhancer. Poly(I)(C) activated endogenous IFN production in L929 and JAR cells but had no consistent effect on CAT expression. Similar results were obtained in L929 cells with inactivated Newcastle disease virus. There was no 'priming' effect of exogenous Type I IFN. Deletion mutants revealed sites exerting negative control on expression between −338 and −247 bp, and between −150 and −71 bp; these regions contained sequences resembling previously identified negative regulatory domains. In the absence of viral inducibility it is proposed that negative regulation contributes towards the stringent control of expression characteristic of IFN-τ genes.

ABSTRACT

A sheep endometrial oxytocin receptor (OTR) cDNA (1·5 kb) was isolated from a λ-ZAP library using a reverse transcription-PCR product probe generated from oestrous endometrial mRNA. The sheep OTR cDNA shared an overall similarity of 82% with human OTR cDNA, 85% with pig OTR cDNA and 76% with rat OTR cDNA. The encoded receptor was a 391 amino acid polypeptide 94% similar to human OTR, 94% similar to pig OTR and 93% similar to rat OTR. The sheep OTR contained two additional amino acids compared with human OTR which were located in the highly GC-rich third intracytoplasmic loop. This region is thought to be associated with G protein coupling and signal transduction. Expression of the cDNA in Cos-7 cells and measurement of oxytocin-induced phosphoinositide turnover confirmed that it coded for a functional product. The affinity of the expressed receptor was comparable with that observed for the in vivo receptor.

ABSTRACT

Genomic and cDNA clones for bovine trophoblast interferon (IFN) have been isolated by probing a bovine genomic library and a bovine embryonic (day-18 post coitus) cDNA library respectively with the ovine trophoblast IFN cDNA. The two DNA sequences were identical; sequence analysis demonstrated 80% identity between the amino acid sequence of bovine trophoblast IFN and ovine trophoblast IFN, and 70% identity with a previously identified bovine IFN-α2. Southern blotting of bovine genomic DNA indicated the presence of a minimum of three trophoblast IFN genes. Primer extension analysis identified the transcription start site in the 5′ flanking region of the bovine IFN gene. Computer-aided analysis of the 5′ flanking sequence demonstrated a similarity with that of bovine IFN-α2 and the existence of a possible viral induction sequence.

ABSTRACT

A synthetic 45-mer oligonucleotide corresponding to part of the ovine endometrial oxytocin receptor cDNA was hybridized to sections of ovine uterus collected from 40 ewes at different stages during the oestrous cycle, the first 3 weeks of pregnancy and seasonal anoestrus. The quantity of oxytocin receptor mRNA was measured as the optical density (OD) value on autoradiographs using image analysis. Message first appeared in the luminal epithelium on days 14–15 of the cycle, increasing to a peak OD of 0·48 at oestrus and decreasing again between days 2 and 5. Oxytocin receptor mRNA in the superficial glands, deep glands and caruncular stroma increased between day 15 and oestrus to peak OD values of 0·17, 0·11 and 0·11 respectively, declining again by day 2 and reaching basal values (OD<0·015) by day 5. Hybridization to the myometrium tended to rise from a mean OD value of 0·01 on days 2–15 to a peak of 0·03±0·01 (mean±s.e.m.) on days 0–1, but the change was not significant. In pregnant ewes there was no detectable oxytocin receptor mRNA on days 14–15 in any region, but hybridization to the luminal epithelium was present in two of three ewes on day 21. In anoestrous ewes oxytocin receptor mRNA concentrations in all areas of the endometrium were approximately half those measured at oestrus.

Optical density readings for oxytocin receptor mRNA in the various uterine compartments were compared with measurements of oxytocin receptors in the same regions as assessed by binding studies using the ¹²⁵I-labelled oxytocin antagonist d(CH₂)₅[Tyr(Me)²,Thr⁴,Tyr-NH₂ ⁹]-vasotocin (¹²⁵I-labelled OTA). In the endometrium, receptor mRNA and ¹²⁵I-labelled OTA binding patterns changed in parallel, and both sets of measurements were significantly correlated (P<0·01). In the myometrium, a significant increase in ¹²⁵I-labelled OTA binding occurred at oestrus; this was not accompanied by a similar increase in oxytocin receptor mRNA hybridization.

This study helps to confirm that the previously identified cDNA clone is derived from the ovine oxytocin receptor, as patterns of oxytocin receptor mRNA expression in the endometrium closely resembled those of oxytocin binding. Maximum expression and binding both occurred at oestrus, suggesting that regulation of the oxytocin receptor gene in the uterus occurs principally at the transcriptional, rather than at the translational, level. Failure to detect a significant increase in myometrial mRNA expression at oestrus may indicate that the endometrial and myometrial oxytocin receptors are of different isoforms.

ABSTRACT

A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-α family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)⁺ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.