Characterization of serum and tissue oxytocinase and tissue oxytocin in the pregnant and nonpregnant mare

Leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase) is a key regulator of oxytocin. Our objectives were to measure serum concentrations of LNPEP and determine the presence of LNPEP and oxytocin (OXT) in equine tissue. LNPEP was measured by ELISA in serum obtained: every other day from ovulation (D0) until D21-22 in nonpregnant and pregnant mares; and at around 320 days of gestation, at 24 hours before foaling, and 20 min and 2 hrs post-foaling. Placenta from the pregnant horn (PH), nonpregnant horn (NPH) and body (B) and from tissues of 6 nonpregnant mares were homogenized and extracts obtained for protein determination and LNPEP ELISA assay. Identification of LNPEP and OXT protein in tissues was also performed via western blot (WB), immunohistochemistry (IHC) and liquid chromatography-mass spectrometry (LC-MS/MS). Furthermore, in situ hybridization was performed for LNPEP and OXT on endometrium, myometrium, pituitary and corpus luteum (CL). No difference in serum LNPEP concentration was identified between any of the groups. The NPH had the lowest quantities of LNPEP (P<0.05). The highest to lowest LNPEP U/mg protein by tissue were: myometrium> follicle wall> endometrium> kidney>CL> liver. Oxytocin was identified in the equine pituitary, CL and placenta. LNPEP may regulate the availability of luteal OXT locally.