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).