Publication Database

Influence of cycle stage, age and biopsy score on oxytocin receptor gene expression and distribution in the cervix and uterus of non-pregnant mares (2018)

Art

Hochschulschrift

Autor

Ströhle, Ruth (WE 17)

Quelle

Berlin: Mensch und Buch Verlag Berlin, 2018 — x, 127 Seiten

ISBN: 978-3-86387-923-5

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/22999

Kontakt

Pferdeklinik

Oertzenweg 19 b
14163 Berlin
+49 30 838 62299 / 62300
pferdeklinik@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Each year numerous mares fail to fall pregnant despite optimised breeding management (no obvious reason of infertility in mare, good semen quality of stallion, correct timing of breeding, good insemination technique and postbreeding management; Da Silva 2008). These mares are considered subfertile (Da Silva 2008). Various reproductive pathologies may cause a mare to not conceive or to lose a pregnancy at an early stage, with uterine pathologies including post-breeding endometritis being one of the most important reasons (Carnevale 2008). Uterine clearance is one of the most important components of the uterine defence mechanism against infection (Troedsson 1999). Mares incapable of efficient uterine clearance and consequently incapable of resolving the transient physiological endometritis are likely to develop a persistent post-breeding endometritis leading to prolonged inflammation of the endometrium and consequently embryonic loss and subfertility (Troedsson 1999; Troedsson et al. 2001; Watson 2000). Oxytocin, a neuropeptide hormone produced by the hypothalamus and endometrium of the mare (Behrendt–Adam et al. 1999, Handler 2001, Gimpl and Fahrenholz 2001, Bae and Watson 2003) is the main hormone responsible for contractions of smooth muscles in the oviduct, uterus and the myoepithelial cells of the mammary gland and is thus of importance for uterine clearance (Handler 2001). Oxytocin is additionally considered to be important in luteolysis (Betteridge et al. 1985; Goff et al. 1987; Goff et al. 1993). Oxytocin acts on target tissues by binding to its receptors. Oxytocin receptors (ORs) have been described in various tissues including tissues outside the genital tract (Gimpl and Fahrenholz 2001). The important role of oxytocin in the equine oestrous cycle as well as in uterine clearance supports the need to investigate the location of ORs in the mare’s reproductive tract (endometrium, myometrium and cervix). Previous studies have shown changes in the concentration of ORs in the equine endometrium throughout the oestrous cycle, with a reported peak during late dioestrus. There are, however, no reports comparing OR density and distribution in the myometrium and cervix of the non-pregnant mare during the different oestrous cycle stages. Despite considerable support for oxytocin playing an important role in the uterine defence mechanism and hence fertility, its exact significance is poorly defined. This study described the distribution and density of ORs in the mare’s endometrium, myometrium and cervix during the luteal and non-luteal phases and additionally within different mare categories based on age and endometrial biopsy score using both immunohistochemistry (IHC) and quantitative reversetranscription polymerase chain reaction (RT-qPCR). Full-thickness uterine samples and endometrial biopsy samples were obtained from 27 routinely-slaughtered, cyclic mares of various breeds and ages (10 mares > 10 years old, 17 mares < 10 years old) at three different uterine sites (uterine body, right and left horn) and one sample from the cervix. Endometrial biopsy sample sections were stained with haematoxylin and eosin by standard techniques and sections were graded I, IIa, IIb, or III according to the Kenney and Doig categorisation scheme. For IHC, all endometrial, myometrial and cervical samples were immunolabeled using an avidin-biotinperoxidase complex (ABC) detection system and a polyclonal 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, propria-submucosa and muscularis layer) were used for the RT-PCR assay. Oligonucleotide primers and probe sequences used for detection of OR and β-actin gene expression were obtained from three previous studies evaluating OR gene expression and distribution in the equine conceptus, fetal membranes, endometrium of pony mares at parturition and in endometrium, myometrium and cervix of non-pregnant mares. A RT-qPCR was used for detection of the OR 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 analyse of the data. Five (18.52 %; one old mare and four young) mares showed a grade I biopsy score, sixteen (59.26 %, six old and 10 young) mares showed a grade IIA biopsy score and six (22.22 %) mares showed a grade IIB biopsy score. None of the mares in the study population showed a grade III biopsy score. All mares with biopsy score I were in the luteal phase and all mares with biopsy score IIB were in the non-luteal phase of their cycles. There was no significant difference in biopsy scores between old and young mares (P = 0.386). Mares in the non-luteal phase had significantly lower biopsy scores (I = highest score, IIB = lowest score) compared to mares in the luteal phase (P = 0.003). By means of immunolabeling, ORs could be identified as fine brown granules in the cytoplasm of selected cells in both negative- and positive-tissue control tissues and in the test sections. No difference in OR distribution nor intensity of staining could be detected between mares in luteal and nonluteal phases, between old and young mares and between mares with different biopsy scores. Looking at fold differences, relative to expression of OR gene RT-qPCR analysis showed higher expression of the OR gene during the non-luteal phase in myometrium (26 times), endometrium (2 times) and cervix (3 times). Statistically the myometrium was found to have significantly higher OR gene expression during non-luteal than during luteal phases (P = 0.004). There was no significant difference in the gene expression of ORs when comparing luteal and non lutealphases in endometrium (P = 0.574) and the cervix (P = 0.339). Statistical analysis of the RT-qPCR data did not demonstrate a significant difference between OR gene expression in young and old mares or between mares with different endometrial biopsy scores (P = 0.601).