Expression and distribution of oxytocin receptors in the cervix and uterus of the mare

Abstract

Oxytocin is essential in the endocrine regulation of the oestrous cycle, during parturition, milk ejection and for myometrial contractility. It is well described that oxytocin acts on smooth muscles, including the myometrium and myoepithelia of mammary alveoli, stimulating contractions. Myometrial contractility is not only important for expulsion of the foetus during parturition in all species but also for the mechanical drainage of cellular debris and uterine fluid after mating especially in the mare. Failure to clear this intrauterine fluid post-breeding is a major cause of subfertility in the mare. Oxytocin furthermore stimulates the release of equine endometrial prostaglandin F2-alpha (PGF2α) and is thus proposed to be of importance in luteolysis. The important role of oxytocin in both the oestrous cycle and in uterine contractility justifies the need to describe oxytocin receptors (ORs) in the endometrium, myometrium and cervix of mares. Studies have reported that the concentration of ORs in the equine endometrium changes throughout the oestrous cycle with a peak being reached during late dioestrus. There are, however, no reports comparing OR gene expression and distribution in the endometrium, myometrium and cervix of the non-pregnant mare. This study describes the distribution and density of ORs in the mare’s endometrium, myometrium and cervix using immunohistochemistry (IHC) and quantitative reversetranscription polymerase chain reaction (RT-qPCR), respectively. Full-thickness uterine samples were obtained from 27 routinely-slaughtered, cyclic mares of various breeds and ages (range: 2 to 20 years) at three different uterine sites (uterine body, right and left horn) and one sample from the cervix. For IHC, all endometrial, myometrial and cervical samples were immunolabeled using an avidinbiotin- peroxidase complex (ABC) detection system and a polyclonal rabbit antibody against human ORs. Sections were evaluated using an Olympus light microscope and the Olympus cell Sens dimension software. Additional samples obtained from the left uterine horn (endometrium and myometrium) and the cervix (luminal epithelium, propriasubmucosa and muscularis layer) were used for the RT-qPCR assay. Oligonucleotide primers and probe sequences used for detection of OR and β-actin gene expression were obtained from two previous studies evaluating ORs in the equine conceptus, foetal membranes and endometrium from pony mares at parturition. A RT-qPCR was used for detection of the messenger-ribonucleic acid (mRNA). The ΔΔCt method using the StepOnePlus™ software was employed as a descriptive method to quantify different gene expression between tissues and ΔCt values were used for statistical analysis of the data. Immunohistochemistry showed ORs in both the uterus and cervix. Oxytocin receptors were specifically localised to the cytoplasm of the endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and smooth muscle cells of the myometrium. The greatest intensity of labeling occurred consistently in the vascular endothelium. There was a similar pattern of distribution and intensity of OR expression in the uterus and cervix, with the exception of the glandular epithelium which is absent in the cervix. Relative to expression of OR gene in the endometrium, RT-qPCR analysis showed higher expression of the OR gene in the myometrium (4 times) and lower expression in the cervix (1/3). The statistical analysis demonstrated that the myometrium had significantly higher OR gene expression than both cervix (P=0.001) and endometrium (P=0.009). There was no significant difference in the gene expression of ORs when comparing cervix and endometrium (P=1.0).