The aim of this study was to examine whether active metabolites of phytoestrogens (equol and para-ethyl-phenol) inhibit sensitivity of bovine corpus luteum (CL) to luteinizing hormone (LH) and to auto/paracrine luteotropic factors (prostaglandin E2-PGE2 and prostaglandin F(2alpha)-PGF(2alpha)), and whether they influence pulsatile progesterone (P4) secretion by the bovine CL. In in vivo experiments, high levels of equol and para-ethyl-phenol were found in plasma and in the CL tissue of heifers and cows fed a soy bean diet (2.5 kg/animal/day), along with lower concentrations of P4 (P < 0.05). Both Prostaglandins (PG) and LH strongly stimulated P4 secretion in cultured pieces of CL that were collected from cows fed a standard diet (P < 0.01). There was no effect of PGs and LH on P4 stimulation in CLs obtained from cows fed a diet rich in soy bean. Finally, we examined whether active metabolites of phytoestrogens participated in regulation of pulsatile P4 secretion and LH-stimulated P4 secretion in vitro using a microdialysis system. Equol and para-ethyl-phenol had no effect on basic and pulsatile P4 secretion in CLs during 240 min of perfusion when compared to the control (P < 0.05). However, they inhibited LH-stimulated P4 secretion (P < 0.05). Phytoestrogens and their metabolites may disrupt CL function by inhibiting PG- and LH-stimulated P4 secretion.

Programmed necrosis (necroptosis) is an alternative form of programmed cell death that is regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent, but is a caspase (CASP)-independent pathway. In the present study, to determine if necroptosis participates in bovine structural luteolysis, we investigated RIPK1 and RIPK3 expression throughout the estrous cycle, during prostaglandin F2α (PGF)-induced luteolysis in the bovine corpus luteum (CL), and in cultured luteal steroidogenic cells (LSCs) after treatment with selected luteolytic factors. In addition, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 μM) on cell viability, progesterone secretion, apoptosis related factors and RIPKs expression, were evaluated. Expression of RIPK1 and RIPK3 increased in the CL tissue during both spontaneous and PGF-induced luteolysis (P < 0.05). In cultured LSCs, tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM) up-regulated RIPK1 mRNA and protein expression (P < 0.05). TNF + IFNG also up-regulated RIPK3 mRNA expression (P < 0.05), but not RIPK3 protein. Although Nec-1 prevented TNF + IFNG-induced cell death (P < 0.05), it did not affect CASP3 and CASP8 expression. Nec-1 decreased both RIPK1 and RIPK3 protein expression (P < 0.05). These findings suggest that RIPKs-dependent necroptosis is a potent mechanism responsible for bovine structural luteolysis induced by pro-inflammatory cytokines.

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 PGF2a in the FP and mid-LP. In the mid-LP, E2 and P4+E2 stimulated PGF2a 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.

Interleukin-1 (IL-1) is one of the principal cytokines that participate in local regulation of many reproductive functions. The present study was undertaken to determine whether mRNAs for IL-1α, IL-1β, and IL-1 type I receptor (IL-1R) are expressed in bovine corpora lutea (CL), and whether luteal cells respond to treatment with IL-1α and IL-1β during the luteal phase. Bovine CL were classified into five stages (early, Days 2–3; developing, Days 5–6; mid, Days 8–12; late, Days 15–17; and regressed, Days 19–21). IL-1α, IL-1β, and IL-1R mRNAs were detected by reverse transcription-polymerase chain reaction (PCR) in all luteal stages examined. Densitometric analysis of PCR products revealed increases of the mRNA of IL-1α and IL-1R in the CL of the regressed stage (P < 0.05). There was less mRNA for IL-1β in the regressed stage than in the developing and mid stages (P < 0.05). When developing, mid, and late luteal cells were treated with IL-1α (1–30 ng/ml) or IL-1β (1–30 ng/ml) for 24 h, IL-1α and IL-1β dose-dependently increased prostaglandin (PG) F_2α and PGE₂ production by the luteal cells of all stages (P < 0.05), indicating the presence of functional IL-1R in bovine CL. However, progesterone synthesis was not affected by either IL-1α or IL-1β treatment. Stimulation with IL-1α and IL-1β decreased the PGE₂:PGF_2α ratio in the developing stage (P < 0.05), whereas it increased the ratio in the mid stage (P < 0.05). In the late stage, the ratio of IL-1β-treated cells was greater than that of IL-1α-treated cells (P < 0.05). Overall results indicate that genes for IL-1α and IL-1β are expressed and a functional IL-1R is present in the bovine CL throughout the luteal phase, and suggest that IL-1α and IL-1β have different roles as local modulators to regulate PGF_2α and PGE₂ production during the luteal phase.