The Endometrium Contributes to the Differential Contractile Response to Oxytocin of Uterine Tissue from Virgin and Proven Breeder Non‐pregnant Rats

Abstract

Introduction We hypothesized that the hormonal and physical stimuli experienced by the uterus during pregnancy induce epigenetic modifications that affect the physiological performance and health status of this organ. We have previously shown that the contractile response of the uterus from virgin (V) and proven breeder (PB) rats to oxytocin is different. Here we investigated the impact of the endometrium in this response. Methods Virgin (V) and proven breeder (PB) NP female 18 week-old rats were staged and euthanized at proestrus (IACUC #2758). Uterine horns were dissected in strips, suspended in a tissue bath containing Krebs solution at 37°C, and bubbled with 95% O2 and 5% CO2. Contractility of tissue in response to increasing levels of oxytocin (10-10 to 10-5 M) were recorded. Oxytocin receptor (OXTR) mRNA expression was evaluated withTaqman® gene expression assays (Life Technologies) and QuantStudio™ 5 real-time PCR system (Applied Biosystems, Grand Island, NY). Distribution of OXTR in the myometrium and endometrium was detected via immunohistochemistry(Primaries: OXTR antibody (BIOSS #bs-1314R) and primary β-actin antibody (Santa Cruz Biotechnology INC #sc-47778). Secondaries: Sheep anti-rabbit FITC, Sigma Aldrich #F7512 and donkey anti-mouse, Alexa Fluor 594 –(Invitrogen, #A21203) Dilution 1:200). Samples were mounted with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories #H1800) and observed with a Nikon A1R Laser Scanning Confocal Microscope. Results Immunohistochemical imaging of the uterine samples we used in our motility experiments revealed a significant presence of endometrium. Thus, we hypothesized that previously observed differences in the response of V and PB tissue to oxytocin might be in part caused by the endometrium. Dose response to oxytocin performed on tissue fully stripped of the endometrium showed that V samples had stronger responses to oxytocin than PB samples (P<0.01). In particular, PB samples showed a significantly lower range of response (max-min) than their unstripped counterparts. In V samples, both maximal response and response range were more elevated than in unstripped counterparts (P<0.01). qRT-PCR did not showed any significant difference in the expression of OXTR in the endometrium of V and PB. Hence, the differences in the endometrial control of uterine contractility observed in the two groups are likely determined by other factors. Conclusions This study showed that the complete removal of the endometrium significantly alters the differences previously observed in the contractile response to oxytocin. Although no parity-related difference in the expression of the OXTR was detected in the endometrium, we speculate that other aspects of the endometrial regulation of uterine smooth muscles might be altered by pregnancy.