Enhanced Response to Oxytocin in Myometrium of Proven Breeder Compared to Virgin Rats Does Not Depend on Augmented Expression of Oxytocin Receptors

Abstract

ObjectiveOur overall hypothesis is that pregnancy's mechanical and hormonal stimuli induce permanent changes in the properties of uterine tissue. We have shown previously that the response to oxytocin of the non‐pregnant (NP) myometrium is more pronounced in proven breeder than in virgin animals. Our objective here was to investigate some of the possible mechanisms that could produce such difference: 1) Altered number of oxytocin receptors (OXTR); 2) Altered production of intracellular prostaglandins; 3) Altered availability of voltage gated calcium channels (VGCC).MethodVirgin (V) and proven breeder (PB) NP female 18 week old rats were staged and euthanized at proestrus. One uterine horn was dissected in strips suspended in a tissue bath containing Krebs solution (in mM, NaCl 117, KCl 4.7, NaHCO3 25, MgCl2 1.2, KH2PO4 1.2, CaCl2 2.5, glucose 11, pH= 7.4) at 37°C and bubbled with 95% O2 and 5% CO2. Dose response curves to oxytocin (10E‐10 to 10E‐5 M) were recorded in the presence of indomethacin or after exposure to a 0 CaCl2 modified Krebs. The other uterine horn was flash frozen in liquid NO2 and later used for ELISA (kit from Wuhan Fine Biotech, Wuhan China) and qRT‐PCR experiments (Taqman® gene expression assays (Life Technologies) and QuantStudio™ 5 real‐time PCR system (Applied Biosystems, Grand Island, NY) were used to examine the gene of interest mRNA expression using the comparative CT method (Schmittgen and Livak, 2008).ResultsIn the ELISA experiments, NP samples showed a barely significant (32±14%, P=0.040) elevation of OXTR in V (n = 8) vs PB myometrium. This is in open contradiction to our previous motility data. To investigate whether this inversion trend in protein level is matched at the mRNA level, we evaluated OXTR gene expression in NP strips (n=5, in quadruplicates). No significant difference was detected between V and PB expression of β‐actin (P= 0.099). Instead, a significant difference was found in the OXTR gene expression (P<0.01) and the 2−ΔΔCt (RQ) was 0.18 (Min RQ=0.12, Max RQ=0.27, indicative of an 8.3 to 3.7 folds decrease in the OXTR mRNA level in PB vs V samples. On the other hand, in the presence of 10 μM indomethacin (COX inhibitor) or in the absence of extracellular calcium the differences in the responses to oxytocin in V and PB myometrial strips were attenuated (−98% and −96% respectively). This suggest important roles of second messengers activated pathways in the more robust effects of oxytocin in NP PB strips.ConclusionsWhile our previous functional experiments suggested that PB myometrium might contain a higher number of OXTR, ELISA and PCR experiments imply that the opposite might be true. The gene expression of the OXTR is frankly stronger in V, while the actual protein expression is not significantly different between V and PB. A better explanation for our pharmacological observations involves differences in the activity of channels and intracellular mechanisms activated by oxytocin, some of which we presented here and are currently under investigation.Support or Funding InformationThis study was funded by an MWU intramural research grant to MP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.