Oxytocin (OXT) has been used to prolong the luteal phase in mares, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of chronic exogenous OXT administration to mid-luteal phase mares on luteal maintenance. Also, endometrial expression of prostaglandin endoperoxide synthase 2 (PTGS2), prostaglandin F2α, E2 and I2 synthases (AKR1C3, PTGES, and PTGIS), oxytocin receptor (OXTR), progesterone receptor (PGR), and estrogen receptors 1 (ESR1) and 2 (ESR2) were assessed in mares experiencing luteal maintenance 2 weeks after chronic exogenous OXT administration. Control mares (n = 5; C group) received 6 mL of saline im, whereas OXT (60 units/mare) was administered im (n = 6; OXT group), every 12 hours, on days 7 to 14 postovulation. After endometrial biopsy in groups C (Day 10) and OXT (Day 24), luteolysis occurred within 3 or 6 days, respectively. Luteal maintenance took place in 4 of 6 (67%) of OXT-treated mares. Progesterone in C group was the highest on biopsy day (P < 0.05). In OXT mares, PTGS2, ESR1 (P < 0.05), PTGES, PTGIS, PGR, and ESR2 (P < 0.01) gene transcription decreased, whereas OXTR increased (P < 0.05) in comparison with the C group. In OXT-treated mares, endometrial ESR2 protein expression decreased (P < 0.05), but OXTR increased (P < 0.05) compared with control animals. In both experimental groups, PTGS2 was mainly immunolocalized in surface epithelium, whereas AKR1C3, PTGES, PTGIS, and PGR were in surface and glandular epithelia. ESR1 and ESR2 were found in glandular epithelium and OXTR in stromal cells. High immunolabeling for PTGES, PTGIS, PGR, and OXTR and low for ESR2 was detected in endometrium of OXT-group mares with extended diestrus. Prolonged luteal function associated with chronic OXT treatment may be related to different spatial expression of OXTR and PGR in the endometrium. The observed reduction of endometrial ESR2 may be responsible for the maintenance of PGR in luminal and glandular epithelium. Also, ESR2 may attenuate the transcriptional activity of ESR1 in mare endometrium. This study offers new knowledge on the endometrial expression of ovarian steroids and OXT receptors in OXT pharmacologically induced luteal maintenance in the mare.

Endometrosis, a fibrotic disease of mare endometrium, impairs uterine function. Prostaglandins (PG), despite modulating reproductive physiological functions, may also cause local pathological collagen deposition (fibrogenesis). We have previously shown that neutrophil extracellular traps (NETs) may also favor mare endometrosis. The aim of this study was to investigate the effect of enzymes present in NETs on PGF2α-pathway activation. Kenney and Doig's type I/IIA and IIB/III mare endometria, from follicular phase (FLP) and mid-luteal (MLP) phase, were cultured in vitro in the presence of NETs enzymes (elastase, cathepsin-G or myeloperoxidase). Production of PGF2α (EIA) and transcription (qPCR) of its synthases (PTGS2, AKR1C3) and receptor (PTGFR) genes were evaluated. PGF2α and PTGFR were influenced by endometrial category and estrous cycle phase. In FLP endometrium, NETs enzymes induced both high PGF2α production and/or PTGFR transcription. In MLP type I/IIA tissues, down-regulation of PTGFR transcripts occurred. However, in MLP type IIB/III endometrium, high levels of PTGFR transcripts were induced by NETs enzymes. As PGF2α-pathway activation facilitates fibrogenesis in other tissues, PGF2α may be involved in endometrosis pathogenesis. In the mare, the endocrine microenvironment of healthy and pathological endometrium might modulate the PGF2α pathway, as well as fibrosis outcome on endometrium challenged by NETs enzymes.

Neutrophil extracellular traps (NETs) are DNA complexes carrying nuclear and cytoplasmic proteins, such as elastase (ELA), cathepsin-G (CAT) and myeloperoxidase (MPO). Mare endometrosis is a chronic degenerative process characterized by excessive collagen in endometrium. While NETs fight bacteria that cause endometritis, they may trigger endometrial fibrogenesis. The aim was to evaluate the in vitro effect of some NETs components on mare endometrial fibrogenesis and determine its relationship with histopathology or estrous cycle. Endometrial explants were incubated with NETs components (ELA, CAT, MPO or oxytocin). Collagen type I (COL1) protein and type I and III (COL3) gene transcription were evaluated in follicular and mid-luteal phases endometria (Kenney and Doig type I/IIA and IIB/III). Increased COL1 occurred with all NETs proteins, although endometrial response to each NETs protease depended on estrous cycle and/or endometrial category. Since ELA enhanced COL1 production, NETs persistence might be linked to endometrosis. Estrous cycle influenced COL1 protein concentration and COL3 transcripts, suggesting that follicular phase may favor endometrial collagen production. However, luteal phase endometria with moderate or severe lesions may be also susceptible to fibrotic effects of NETs constituents. These data propose that NETs involvement in chronic endometritis in mares may act as putative endometrial fibrogenic mediators.

In the present work, we investigated the role of Nodal, an embryonic morphogen from the TGFβ superfamily in corpus luteum (CL) secretory activity using cells isolated from equine CL as a model. Expression pattern of Nodal and its receptors activin receptor A type IIB (ACVR2B), activin receptor-like kinase (Alk)-7, and Alk4, as well as the Nodal physiological role, demonstrate the involvement of this pathway in functional luteolysis. Nodal and its receptors were immune localized in small and large luteal cells and endothelial cells, except ACVR2B, which was not detected in the endothelium. Nodal mRNA in situ hybridization confirmed its transcription in steroidogenic and endothelial cells. Expression analysis of the aforementioned factors evidenced that Nodal and Alk7 proteins peaked at the mid-CL (P < .01), the time of luteolysis initiation, whereas Alk4 and ACVR2B proteins increased from mid- to late CL (P < .05). The Nodal treatment of luteal cells decreased progesterone and prostaglandin (PG) E2 concentrations in culture media (P < .05) as well as mRNA and protein of secretory enzymes steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, cytosolic PGE2 synthase, and microsomal PGE2 synthase-1 (P < .05). Conversely, PGF2α secretion and gene expression of PG-endoperoxidase synthase 2 and PGF2α synthase were increased after Nodal treatment (P < .05). Mid-CL cells cultured with PGF2α had increased Nodal protein expression (P < .05) and phosphorylated mothers against decapentaplegic-3 phosphorylation (P < .05). Finally, the supportive interaction between Nodal and PGF2α on luteolysis was shown to its greatest extent because both factors together more significantly inhibited progesterone (P < .05) and promoted PGF2α (P < .05) synthesis than Nodal or PGF2α alone. Our results neatly pinpoint the sites of action of the Nodal signaling pathway toward functional luteolysis in the mare.

The oviduct presents the ideal conditions for fertilization and early embryonic development. In this study, (i) vascularization pattern; (ii) microvascular density; (iii) transcripts of angiogenic factors (FGF1, FGF2, VEGF) and their receptors-FGFR1, FGFR2, KDR, respectively, and (iv) the relative protein abundance of those receptors were assessed in cyclic mares' oviducts. The oviductal artery, arterioles and their ramifications, viewed by means of vascular injection-corrosion, differed in the infundibulum, ampulla and isthmus. The isthmus, immunostained with CD31, presented the largest vascular area and the highest number of vascular structures in the follicular phase. Transcripts (qPCR) and relative protein abundance (Western blot) of angiogenic factors fibroblast growth factor 1 (FGF1) and 2 (FGF2) and vascular endothelial growth factor (VEGF), and their respective receptors (FGFR1, FGFR2, VEGFR2 = KDR), were present in all oviduct portions throughout the estrous cycle. Upregulation of the transcripts of angiogenic receptors FGF1 and FGFR1 in the ampulla and isthmus and of FGF2 and KDR in the isthmus were noted. Furthermore, in the isthmus, the relative protein abundance of FGFR1 and KDR was the highest. This study shows that the equine oviduct presents differences in microvascular density in its three portions. The angiogenic factors VEGF, FGF1, FGF2 and their respective receptors are expressed in all studied regions of the mare oviduct, in agreement with microvascular patterns.

The regulation of corpus luteus (CL) luteolysis is a complex process involving a myriad of factors. Previously, we have shown the involvement of Nodal in functional luteolysis in mares. Presently, we ask the extent of which Nodal mediation of luteolysis is done through regulation of angioregression. We demonstrated the interaction between Nodal and hypoxia-inducible factor 1 α (HIF1α) and thrombospondin 1/thrombospondin receptor (TSP1/CD36) systems, could mediate angioregression during luteolysis. First, we demonstrated the inhibitory effect of Nodal on the vascular marker platelet/endothelial cell adhesion molecule 1 (CD31). Also, treatment of mid CL explants with vascular endothelial growth factor A (VEGFA) showed a trend on activin-like kinase 7 (Alk7) protein inhibition. Next, Nodal was also shown to activate HIF1α and in vitro culture of mid CL explants under decreased oxygen level promoted Nodal expression and SMAD family member 3 (Smad3) phosphorylation. In another experiment, the crosstalk between Nodal and TSP1/CD36 was investigated. Indeed, Nodal increased the expression of the anti-angiogenic TSP1 and its receptor CD36 in mid CL explants. Finally, the supportive effect of prostaglandin F2α (PGF2α) on TSP1/CD36 was blocked by SB431542 (SB), a pharmacological inhibitor of Nodal signaling. Thus, we evidenced for the first time the in vitro interaction between Nodal and both HIF1α and TSP1 systems, two conserved pathways previously shown to be involved in vascular regression during luteolysis. Considering the given increased expression of Nodal in mid CL and its role on functional luteolysis, the current results suggest the additional involvement of Nodal in angioregression during luteolysis in the mare, particularly in the activation of HIF1α and TSP1/CD36.