Maria Rosa Rebordão

Effect of cytokines and ovarian steroids on equine endometrial function: an in vitro study

Regulation of immune-endocrine interactions in the equine endometrium is not fully understood. The aims of the present study were to: (1) investigate the presence of tumour necrosis factor alpha (TNF), interferon gamma (IFNG), Fas ligand (FASLG) and their receptors in the mare endometrium throughout the oestrous cycle; and (2) assess endometrial secretory function (prostaglandins), angiogenic activity and cell viability in response to TNF, oestradiol (E2), progesterone (P4) and oxytocin (OXT). Transcription of TNF and FASLG mRNA increased during the early and late luteal phase (LP), whereas IFNG mRNA increased in late LP. Transcription of the mRNA of both TNF receptors was highest in the mid-LP. All cytokines and receptors were expressed in surface and glandular epithelium, as well as in the stroma. Expression of TNF and its receptor TNFRSF1A increased during the follicular phase (FP) and mid-LP. IFNG was expressed in the mid-LP, whereas its receptor IFNR1 was expressed in the in mid- and late LP. The highest expression of FASLG and FAS occurred during the late LP. OXT increased the secretion of prostaglandin (PG) E2 and PGF2α in the FP and mid-LP. In the mid-LP, E2 and P4+E2 stimulated PGF2α secretion, whereas TNF and P4 increased cell viability. All treatments, with the exception of P4, increased nitric oxide and angiogenic activity in both phases. The coordinated action of cytokines and ovarian hormones may regulate secretory, angiogenic and proliferative functions in the equine endometrium.

Opposing Roles of Leptin and Ghrelin in the Equine Corpus Luteum Regulation: An In Vitro Study

Metabolic hormones have been associated with reproductive function modulation. Thus, the aim of this study was: (i) to characterize the immunolocalization, mRNA and protein levels of leptin (LEP), Ghrelin (GHR) and respective receptors LEPR and Ghr-R1A, throughout luteal phase; and (ii) to evaluate the role of LEP and GHR on progesterone (P4), prostaglandin (PG) E2 and PGF2α, nitric oxide (nitrite), tumor necrosis factor-α (TNF); macrophage migration inhibitory factor (MIF) secretion, and on angiogenic activity (BAEC proliferation), in equine corpus luteum (CL) from early and mid-luteal stages. LEPR expression was decreased in late CL, while GHR/Ghr-R1A system was increased in the same stage. Regarding secretory activity, GHR decreased P4 in early CL, but increased PGF2α, nitrite and TNF in mid CL. Conversely, LEP increased P4, PGE2, angiogenic activity, MIF, TNF and nitrite during early CL, in a dose-dependent manner. The in vitro effect of LEP on secretory activity was reverted by GHR, when both factors acted together. The present results evidence the presence of LEP and GHR systems in the equine CL. Moreover, we suggest that LEP and GHR play opposing roles in equine CL regulation, with LEP supporting luteal establishment and GHR promoting luteal regression. Finally, a dose-dependent luteotrophic effect of LEP was demonstrated.

The Effect of Coumestrol on Progesterone and Prostaglandin Production in the Mare: In Vitro and In Vivo Studies

ABSTRACT Coumestrol (Cou) is a plant-derived phytoestrogen that induces various pathologies in the female reproductive tract. Although effects of phytoestrogens on reproductive function in other species are well documented, their influence on progesterone (P4) and prostaglandin (PG) secretion in the mare is unknown. The aim of this study was to determine if Cou directly affects P4 and PG concentrations (in vivo) and endometrial PG secretion (in vitro) in the mare. In experiment 1, the mares (n = 4) were fed for 14 days on a diet containing increasing proportions of alfalfa pellets (250 g–1 kg/day). An additional 4 mares were fed a standard diet (control group). Sequential blood samples were obtained for 8 h after feeding on Days 13 and 14 (1 kg/day alfalfa pellets). Feeding the mares alfalfa pellets up-regulated PGE2 and 13,14-dihydro-15-ketoprostaglandin F2alpha (PGFM) and down-regulated P4 in the blood plasma compared to those in the control group (P < 0.05). In experiment 2, epithelial and stromal cells were exposed to E2 (10−9 M) or Cou (10−8 M) for 24 h. In the in vitro study, Cou increased PG secretion in epithelial and stromal cells (P < 0.05). In both types of endometrial cells, Cou up-regulated PTGS-2 protein expression (P < 0.05). Moreover, PGES and PGFS proteins were up-regulated by Cou in epithelial cells (P < 0.01). These results indicate that Cou can disturb reproductive function by affecting reproductive hormone secretion and altering the endometrial milieu through PG stimulation. Coumestrol therefore may impair physiologic regulation of the estrous cycle and early pregnancy.

TGFB1 modulates in vitro secretory activity and viability of equine luteal cells

HighlightsTGF beta‐1 (TGF) and its receptor ALK5 expression rises in later stages of cyclic corpus luteum in the mare.TGF decreases progesterone (P4) and prostaglandin (PG) E2 secretion from luteal cells through downregulation of StAR, CYP11A1, cPGES and mPGES1.TGF supports PGF2alpha secretion from luteal cells through PTGS2 and PGFS activity.TGF decreases luteal cells viability and through BAX and FASL expression and caspase 3 activity.ALK5, ALK4 and ALK7 are important mediators of PGF2alpha luteolytic actions. &NA; In the present report we describe the involvement of transforming growth factor B1 (TGF) in functional regression and structural luteolysis in the mare. Firstly, TGF and its receptors activin‐like kinase (ALK) 5 and TGF receptor 2 were identified in corpus luteum (CL) steroidogenic, endothelial and fibroblast‐like cells. Also, TGF and ALK5 protein expression were shown to be increased in Mid‐, and Late‐CL (p < 0.05). Subsequently, using an in vitro model with Mid‐CL cells, we studied the role of TGF on secretory activity and cell viability. Cell treatment with TGF decreased progesterone (P4) and prostaglandin (PG) E2 concentrations in culture media (p < 0.05), and downregulated mRNA and protein of StAR, CYP11A1, cPGES and mPGES1 (p < 0.05). Conversely, TGF augmented PGF2a concentration in culture media, through PTGS2 and PGFS gene expression activation (p < 0.05). When cells were incubated with PGF2a, both TGF and ALK5 were upregulated (p < 0.05). Additionally, treatment with the pharmacological inhibitor of ALK5, ALK4 and ALK7 ‐ SB431542 (SB) attenuated PGF2a functional and structural luteolytic actions. Indeed, SB blocked: (i) PGF2a inhibitory effect on StAR, CYP11A1, 3BHSD and mPGES1; (ii) PGF2a auto‐amplification signal via PTGS2 and PGFS expression (p < 0.05); (iii) the PGF2a‐induced BAX and FASL expression (p < 0.05). Finally, TGF decreased cell viability (p < 0.05) and promoted caspase 3 activity (p = 0.08) and the expression of pro‐apoptotic FASL and BAX (p < 0.05). Our results suggest that TGF supports functional regression and structural luteolysis, and also confirm the importance of ALK5, ALK4 and ALK7 activation during PGF2a mediated luteolysis in mares.

Bacteria causing pyometra in bitch and queen induce neutrophil extracellular traps

Neutrophils are capable of releasing their DNA in response to infectious agents to form neutrophil extracellular traps (NETs) to destroy pathogens. Even though pyometra in queens and bitches is a common disease, its pathogenesis is not fully understood. The aim of this study was to assess the presence of NETs in the endometrium of queens and bitches suffering from pyometra. Pyometra and normal uteri were obtained after ovariohysterectomy from adult queens and bitches in diestrus. Uterine contents were evaluated for bacterial isolation and identification and for NETs presence. Escherichia coli were isolated in 5/5 queens and 4/5 bitches, and Streptococcus spp in one bitch. Sterile glass coverslips were placed on the endometrium surface to obtain material for NETs that were evaluated by immunocytochemistry (histone, neutrophil elastase or myeloperoxidase), fluorescence microscopy or scanning electron microscopy. NETs in endometrium content were positively stained by DNA histone DAPI, myeloperoxidase and by neutrophil elastase. NETs were spread in all observed queen and bitch endometria of pyometra cases. Ultrastructure images of NETs depicted clusters of globular material with fine filaments deposited on or around thick filaments and trapped bacteria. To the best of our knowledge these are the first findings confirming NETs endometrial presence in queen and bitch pyometra. Nevertheless, the precise role of NETs in pyometra in the bitch and queen, either to contribute to the defeat of infection or to its persistence remains to be unraveled.