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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

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.