De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering.

To better understand molecular aspects of equine endometrial function, there is a need for advanced in vitro culture systems that more closely imitate the intricate 3-dimensional (3D) in vivo endometrial structure than current techniques. However, development of a 3D in vitro model of this complex tissue is challenging. This study aimed to develop an in vitro 3D endometrial tissue (3D-ET) with an epithelial cell phenotype optimized by treatment with a Rho-associated protein kinase (ROCK) inhibitor. Equine endometrial epithelial (eECs) and mesenchymal stromal (eMSCs) cells were isolated separately, and eECs cultured in various concentrations of Rock inhibitor (0, 5, 10 µmol) in epithelial medium (EC-medium) containing 10% knock-out serum replacement (KSR). The optimal concentration of Rock inhibitor for enhancing eEC proliferation and viability was 10 µM. However, 10 µM Rock inhibitor in the 10% KSR EC-medium was able to maintain mucin1 (Muc1) gene expression for only a short period. In contrast, fetal bovine serum (FBS) was able to maintain Muc1 gene expression for longer culture durations. An in vitro 3D-ET was successfully constructed using a collagen-based scaffold to support the eECs and eMSCs. The 3D-ET closely mimicked in vivo endometrium by displaying gland-like eEC-derived structures positive for the endometrial gland marker, Fork headbox A2 (FOXA2), and by mimicking the 3D morphology of the stromal compartment. In addition, the 3D-ET expressed the secretory protein MUC1 on its glandular epithelial surface and responded to LPS challenge by upregulating the expression of the interleukin-6 (IL6) and prostaglandin F synthase (PGFS) genes (P < 0.01), along with an increase in their secretory products, IL-6 (P < 0.01) and prostaglandin F2alpha (PGF2α) (P < 0.001) respectively. In the future, this culture system can be used to study both normal physiology and pathological processes of the equine endometrium.

Prostaglandin F2α Affects the Cycle of Clock Gene Expression and Mouse Behavior.

Prostaglandins are bioactive compounds, and the activation of their receptors affects the expression of clock genes. However, the prostaglandin F receptor (Ptgfr) has no known relationship with biological rhythms. Here, we first measured the locomotor period lengths of Ptgfr-KO (B6.129-Ptgfrtm1Sna) mice and found that they were longer under constant dark conditions (DD) than those of wild-type (C57BL/6J) mice. We then investigated the clock gene patterns within the suprachiasmatic nucleus in Ptgfr-KO mice under DD and observed a decrease in the expression of the clock gene cryptochrome 1 (Cry1), which is related to the circadian cycle. Moreover, the expression of Cry1, Cry2, and Period2 (Per2) mRNA were significantly altered in the mouse liver in Ptgfr-KO mice under DD. In the wild-type mouse, the plasma prostaglandin F2α (PGF2α) levels showed a circadian rhythm under a 12 h cycle of light-dark conditions. In addition, in vitro experiments showed that the addition of PTGFR agonists altered the amplitude of Per2::luc activity, and this alteration differed with the timing of the agonist addition. These results lead us to hypothesize that the plasma rhythm of PGF2α is important for driving clock genes, thus suggesting the involvement of PGF2α- and Ptgfr-targeting drugs in the biological clock cycle.

Effect of photobiomodulation on alleviating primary dysmenorrhea caused by PGF2α.

Photobiomodulation (PBM) has attracted widespread attention in suppressing various pain and inflammation. Primary dysmenorrhea (PD) primarily occurs in adolescents and adult females, and the limited effectiveness and side effects of conventional treatments have highlighted the urgent need to develop and identify new adjunct therapeutic strategies. In this work, the results of pain and PGs demonstrated that 850 nm, 630 nm, and 460 nm all exhibited pain inhibition, decreased PGF2α and upregulated PGE2, while 630 nm PBM has better effectiveness. Then to explore the underlying biological mechanisms of red light PBM on PD, we irradiated prostaglandin-F2α induced HUSM cells and found that low-level irradiance can restore intracellular calcium ion, ROS, ATP, and MMP levels to normal levels. And, red light enhanced cell viability and promoted cell proliferation for normal HUSM cells. Therefore, this study proposes that red light PBM may be a promising approach for the future clinical treatment of PD.

A century of research on the uteroovarian pathway for uterine-induced luteolysis in mammals.

Year 2023 is the 100-year anniversary of the discovery in guinea pigs that the lifespan of the corpus luteum (CL) is controlled by the uterus. The CL is the gatekeeper between two fundamental reproductive events - the estrous cycle and pregnancy. Uteroluteal research for the initial 33 years was productive but limited to laboratory species until the inclusion of farm animals in 1956. In the early 1960s, it was found that uterine luteolysin in sows travels unilaterally from a uterine horn to the adjacent CL which likely accounted for the heyday of uteroluteal research in the 1960s-70s. The luteolytic properties of PGF2α were demonstrated in rats in 1969. In 1971, (1) surgical separation of the lengthwise adherence between the uteroovarian vein and ovarian artery interfered with luteolysis in ewes, (2) species with primarily unilateral vs systemic uterine-induced luteolysis have a strong vs an absent or weak unilateral venoarterial transfer pathway, and (3) vascular infusions identified PGF2α as a uterine luteolysin. Vascular and PGF2α studies were beginning to merge. In 1973, a venoarterial pathway was firmly demonstrated in ewes and later in heifers by surgical anastomosis of a uterine vein or ovarian artery from a uterine-intact side to the corresponding vessel on the unilaterally hysterectomized side. More recent studies described how prostaglandins likely transfer through the walls of uterine and ovarian vessels using concentration gradients in sows and a prostaglandins transporter system in cows.

Myeloid Bmal1 deletion suppresses the house dust mite-induced chronic lung allergy.

Asthma is the chronic pulmonary inflammatory response that could lead to respiratory failure when allergic reactions exacerbate. It is featured by type 2 immunity with eosinophilic inflammation, mucus, and IgE production, and Th2 cytokine secretion upon repeated challenge of allergens. The symptom severity of asthma displays an apparent circadian rhythm with aggravated airway resistance in the early morning in patients. Bmal1 is the core regulator of the circadian clock, while the regulatory role of Bmal1 in asthma remains unclear. Here, we investigate whether the myeloid Bmal1 is involved in the pathogenesis of house dust mite (HDM)-induced lung allergy. We found that knockdown of Bmal1 in macrophages suppressed the time-of-day variance of the eosinophil infiltration in the alveolar spaces in chronic asthmatic mice. This was accompanied by decreased bronchial mucus production, collagen deposition, and HDM-specific IgE production. However, the suppression effects of myeloid Bmal1 deletion did not alter the allergic responses in short-term exposure to HDM. The transcriptome profile of alveolar macrophages (AMs) showed that Bmal1-deficient AMs have enhanced phagocytosis and reduced production of allergy-mediating prostanoids thromboxane A2 and prostaglandin F2α synthesis. The attenuated thromboxane A2 and prostaglandin F2α may lead to less induction of the eosinophil chemokine Ccl11 expression in bronchial epithelial cells. In summary, our study demonstrates that Bmal1 ablation in macrophages attenuates eosinophilic inflammation in HDM-induced chronic lung allergy, which involves enhanced phagocytosis and reduced prostanoid secretion.

8-Iso-prostaglandin F2α as a potential biomarker in patients with unipolar and bipolar depression.

OBJECTIVE: Previous studies have shown that the disturbance of redox homeostasis plays a role in the pathogenesis of mood disorders. It is currently unclear whether oxidative stress parameters can be used as biomarkers (state vs. trait). The aim of the present study was to investigate oxidative stress markers in patients with major depressive disorder (MDD) and bipolar disorder (BP) in acute depressive episodes and remission, and healthy individuals. PATIENTS AND METHODS: Thirty-two patients with a diagnosis of MDD, 32 patients with a diagnosis of BP and 32 matched healthy controls were included in the study. We measured the serum levels of markers of oxidative damage, including 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-Iso-prostaglandin F2α (8-iso-PGF2α; 8-isoprostane), and malondialdehyde (MDA), and also serum activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR) in both acute and remission phase, and in control group. RESULTS: After controlling for the effects of age, sex, body mass index, and smoking status, serum 8-iso-PGF2α levels were significantly higher in both patient groups compared to controls, regardless of disease phase. The activities of GPX and GR were significantly lower in the acute phase in MDD patients compared to controls. Serum GR activity was lower in both acute and remission phase in MDD compared to BP. CONCLUSIONS: Our results suggest that both MDD and BP are associated with a disturbed redox balance with a particularly pronounced increase in serum 8-iso-PGF2α levels in both groups and the presence of glutathione metabolism disorders in MDD patients. Further research is needed to confirm the importance of oxidative stress parameters as potential biomarkers of MDD and BP.

Peony Pollen Protects against Primary Dysmenorrhea in Mice by Inhibiting Inflammatory Response and Regulating the COX2/PGE2 Pathway.

Peony pollen contains multiple nutrients and components and has been used as a traditional Chinese medicine with a long history, but the effect of the treatment of primary dysmenorrhea remains to be clarified. The aim of this study is to investigate the therapeutic effect of peony pollen on primary dysmenorrhea mice and the potential mechanism. A uterus contraction model in vitro and primary dysmenorrhea mice were used to evaluate the treatment effect of peony pollen on primary dysmenorrhea. The primary dysmenorrhea mice were treated with 62.5 mg/kg, 125 mg/kg, or 250 mg/kg of peony pollen, and the writhing response, latency period, histopathological changes in the uterus, prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) levels, and infiltration of neutrophils and macrophages were investigated. Protein expression of interleukin 1 ß (IL-1ß), interleukin 6 (IL-6), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cyclooxygenase-2 (COX-2), microsomal prostaglandin-E synthase 1 (mPGEs-1), BCL2-Associated X (Bax), B-cell lymphoma-2 (BCL-2), caspase-3, and cleaved caspase-3 were detected by Western blot, and the oxidative stress related marker malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and reactive oxygen species (ROS) were evaluated. Peony pollen could attenuate spontaneous or oxytocin-induced uterus contractions in vitro. Moreover, peony pollen decreased the writhing times, prolonged the writhing latency, and reduced the pathological damage of uterine tissues. Furthermore, the inflammatory cell infiltration and the protein expression of IL-1ß, IL-6, and NLRP3 were decreased. The COX-2/PGE2 pathway was inhibited; oxidative stress and apoptosis in the uterus also improved in the uterus of primary dysmenorrhea mice. Peony pollen exerts a positive effect on primary dysmenorrhea by inhibiting the inflammatory response and modulating oxidative stress and apoptosis by regulating the COX-2/PGE2 pathway.

PGF2α signaling drives fibrotic remodeling and fibroblast population dynamics in mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptgfr) are implicated as a TGF-ß1-independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER-SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen-treated IER-SftpcI73T mice developed an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr-null (FPr-/-) line showed attenuated weight loss and gene dosage-dependent rescue of mortality compared with FPr+/+ cohorts. IER-SftpcI73T/FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single-cell RNA-Seq, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts, which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2α /FPr-dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

Prostaglandin F2α analogue, bimatoprost ameliorates colistin-induced nephrotoxicity.

Colistin (polymyxin E) is an antibiotic that is effective against multidrug-resistant gram-negative bacteria. However, the high incidence of nephrotoxicity caused by colistin limits its clinical use. To identify compounds that might ameliorate colistin-induced nephrotoxicity, we obtained 1707 compounds from the Korea Chemical Bank and used a high-content screening (HCS) imaging-based assay. In this way, we found that bimatoprost (one of prostaglandin F2α analogue) ameliorated colistin-induced nephrotoxicity. To further assess the effects of bimatoprost on colistin-induced nephrotoxicity, we used in vitro and in vivo models. In cultured human proximal tubular cells (HK-2), colistin induced dose-dependent cytotoxicity. The number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, indicative of apoptosis, was higher in colistin-treated cells, but this effect of colistin was ameliorated by cotreatment with bimatoprost. The generation of reactive oxygen species, assessed using 2,7-dichlorodihydrofluorescein diacetate, was less marked in cells treated with both colistin and bimatoprost than in those treated with colistin alone. Female C57BL/6 mice (n = 10 per group) that were intraperitoneally injected with colistin (10 mg/kg/12 hr) for 14 days showed high blood urea nitrogen and serum creatinine concentrations that were reduced by the coadministration of bimatoprost (0.5 mg/kg/12 hr). In addition, kidney injury molecule-1 (KIM1) and Neutrophil gelatinase-associated lipocalin (NGAL) expression also reduced by bimatoprost administration. Further investigation in tubuloid and kidney organoids also showed that bimatoprost attenuated the nephrotoxicity by colistin, showing dose-dependent reducing effect of KIM1 expression. In this study, we have identified bimatoprost, prostaglandin F2α analogue as a drug that ameliorates colistin-induced nephrotoxicity.

Possible roles of bone morphogenetic protein 4 in regulating endometrial function in cows.

This study investigated the expression dynamics of bone morphogenetic protein 4 (BMP4) and its receptors (BMPR1A, BMPR1B, and BMPR2) in bovine endometrium and examined the physiological function and regulatory mechanism of BMP4 expression. The messenger RNA (mRNA) expression of BMP4 and its receptors was detected in bovine endometrium of both ipsilateral (corpus luteum [CL]-side) and contralateral (non-CL-side) uterine horns during the estrous cycle and early pregnancy. BMP4 protein levels were higher in the endometrial tissues obtained from those cows in early pregnancy than in the estrous cycle. Immunohistochemical analysis showed that BMP4 and its receptors were localized in endometrial epithelial cells. The addition of BMP4 to cultured endometrial epithelial cells did not affect caspase-3/-8 mRNA expression, whereas it significantly inhibited cell proliferation. Both prostaglandin (PG) E2 and PGF2α concentrations in the culture supernatant were decreased when stimulated by BMP4. Furthermore, BMP4 mRNA expression was increased by stimulation with tumor necrosis factor-α (TNF) and interferon-γ (IFNG). In conclusion, BMP4 is produced in bovine endometrial epithelial cells and may contribute to the regulation of cell proliferation and suppression of PG secretion through autocrine or paracrine mechanisms. BMP4 expression in the bovine endometrium may be regulated by TNF and IFNG.

Prostaglandin F2α requires activation of calcium-dependent signalling to trigger inflammation in human myometrium.

Introduction: Preterm birth is one of the major causes of neonatal morbidity and mortality across the world. Both term and preterm labour are preceded by inflammatory activation in uterine tissues. This includes increased leukocyte infiltration, and subsequent increase in chemokine and cytokine levels, activation of pro-inflammatory transcription factors as NF-κB and increased prostaglandin synthesis. Prostaglandin F2α (PGF2α) is one of the myometrial activators and stimulators. Methods: Here we investigated the role of PGF2α in pro-inflammatory signalling pathways in human myometrial cells isolated from term non-labouring uterine tissue. Primary myometrial cells were treated with G protein inhibitors, calcium chelators and/or PGF2α. Nuclear extracts were analysed by TranSignal cAMP/Calcium Protein/DNA Array. Whole cell protein lysates were analysed by Western blotting. mRNA levels of target genes were analysed by RT-PCR. Results: The results show that PGF2α increases inflammation in myometrial cells through increased activation of NF-κB and MAP kinases and increased expression of COX-2. PGF2α was found to activate several calcium/cAMP-dependent transcription factors, such as CREB and C/EBP-ß. mRNA levels of NF-κB-regulated cytokines and chemokines were also elevated with PGF2α stimulation. We have shown that the increase in PGF2α-mediated COX-2 expression in myometrial cells requires coupling of the FP receptor to both Gαq and Gαi proteins. Additionally, PGF2α-induced calcium response was also mediated through Gαq and Gαi coupling. Discussion: In summary, our findings suggest that PGF2α-induced inflammation in myometrial cells involves activation of several transcription factors - NF-κB, MAP kinases, CREB and C/EBP-ß. Our results indicate that the FP receptor signals via Gαq and Gαi coupling in myometrium. This work provides insight into PGF2α pro-inflammatory signalling in term myometrium prior to the onset of labour and suggests that PGF2α signalling pathways could be a potential target for management of preterm labour.

Basic fibroblast growth factor induces proliferation and collagen production by fibroblasts derived from the bovine corpus luteum†.

Cyclic regression of the ovarian corpus luteum, the endocrine gland responsible for progesterone production, involves rapid matrix remodeling. Despite fibroblasts in other systems being known for producing and maintaining extracellular matrix, little is known about fibroblasts in the functional or regressing corpus luteum. Vast transcriptomic changes occur in the regressing corpus luteum, among which are reduced levels of vascular endothelial growth factor A (VEGFA) and increased expression of fibroblast growth factor 2 (FGF2) after 4 and 12 h of induced regression, when progesterone is declining and the microvasculature is destabilizing. We hypothesized that FGF2 activates luteal fibroblasts. Analysis of transcriptomic changes during induced luteal regression revealed elevations in markers of fibroblast activation and fibrosis, including fibroblast activation protein (FAP), serpin family E member 1 (SERPINE1), and secreted phosphoprotein 1 (SPP1). To test our hypothesis, we treated bovine luteal fibroblasts with FGF2 to measure downstream signaling, type 1 collagen production, and proliferation. We observed rapid and robust phosphorylation of various signaling pathways involved in proliferation, such as ERK, AKT, and STAT1. From our longer-term treatments, we determined that FGF2 has a concentration-dependent collagen-inducing effect, and that FGF2 acts as a mitogen for luteal fibroblasts. FGF2-induced proliferation was greatly blunted by inhibition of AKT or STAT1 signaling. Our results suggest that luteal fibroblasts are responsive to factors that are released by the regressing bovine corpus luteum, an insight into the contribution of fibroblasts to the microenvironment in the regressing corpus luteum.

Prostaglandin F2α regulates mitochondrial dynamics and mitophagy in the bovine corpus luteum.

Prostaglandins are arachidonic acid-derived lipid mediators involved in numerous physiological and pathological processes. PGF2α analogues are therapeutically used for regulating mammalian reproductive cycles and blood pressure, inducing term labor, and treating ocular disorders. PGF2α exerts effects via activation of calcium and PKC signaling, however, little is known about the cellular events imposed by PGF2α signaling. Here, we explored the early effects of PGF2α on mitochondrial dynamics and mitophagy in the bovine corpus luteum employing relevant and well characterized in vivo and in vitro approaches. We identified PKC/ERK and AMPK as critical protein kinases essential for activation of mitochondrial fission proteins, DRP1 and MFF. Furthermore, we report that PGF2α elicits increased intracellular reactive oxygen species and promotes receptor-mediated activation of PINK-Parkin mitophagy. These findings place the mitochondrium as a novel target in response to luteolytic mediator, PGF2α. Understanding intracellular processes occurring during early luteolysis may serve as a target for improving fertility.

Conjugated linoleic acid supplementation changes prostaglandin concentration ratio and alters the expression of genes involved in maternal-fetal recognition from bovine trophoblast cells in vitro.

Early embryonic mortality caused by maternal-fetal recognition failure in the three weeks after fertilization represents a major cause of reproductive inefficiency in the cattle industry. Modifying the amounts and ratios of prostaglandin (PG) F2α and PGE2 can benefit the establishment of pregnancy in cattle. Adding conjugated linoleic acid (CLA) to endometrial and fetal cells culture affects PG synthesis, but its effect on bovine trophoblast cells (CT-1) is unknown. The aim of this study was to determine the effects of CLA (a mixture of cis- and trans-9, 11- and -10,12-octadecadienoic acids) on PGE2 and PGF2α synthesis and the expression of transcripts involved with maternal-fetal recognition of bovine trophectoderm. Cultures of CT-1 were exposed to CLA for 24, 48 and 72 h. Transcript abundance was determined by qRT-PCR and hormone profiles were quantified by ELISA. The PGE2 and PGF2α concentrations were reduced in the culture medium of CLA-exposed CT-1 compared to that of unexposed cells. Furthermore, CLA supplementation increased the PGE2:PGF2α ratio in CT-1 and had a quadratic effect (P < 0.05) on the relative expression of MMP9, PTGES2, and PTGER4. The relative expression levels of PTGER4 were reduced (P < 0.05) in CT-1 cultured with 100 µM CLA than in the unsupplemented and 10 µM-CLA groups. Treatment of CT-1 with CLA decreased PGE2 and PGF2α synthesis but a biphasic effect of CLA was observed on the PGE2:PGF2α ratio and relative abundance of transcripts with 10 µM CLA providing maximal improvements in each endpoint. Our data suggest that CLA may influence eicosanoid metabolic process and extracellular matrix remodeling.

Raftlin and 8-iso-prostaglandin F2α levels and gene network analysis in patients with Modic changes.

PURPOSE: Oxidative stress in the vertebral endplates of patients with low back pain and Modic changes (MCs) (types I, II, and III) endplate changes on magnetic resonance imaging. 8-iso-prostaglandin F2α (8-iso-PGF2α) has been proposed as new indicator of oxidative stress. Raftlin, as an inflammatory biomarker, has been previously reported in inflammatory diseases. Oxidative stress plays an important role in various human diseases. This study was aimed to assess Raftlin and 8-iso-PGF2α levels in patients with MCs. METHODS: Patients with MCI, II, and III (n = 45) and age- and sex matched controls subjects (n = 45) were enrolled in this study. 8-iso-PGF2α and Raftlin levels in the serum samples of both groups were measured with enzyme-linked immunosorbent assay. RESULTS: In our study results, raftlin levels changed in parallel with prostaglandin levels (p < 0.05). Raftlin levels changed in parallel with prostaglandin levels (p < 0.05). The levels of 8-iso-PGF2α and Raftlin levels showed increase in patients with MCs and the control group (p < 0.05). In addition, a significant positive correlation was found between MC-I, MC-II, MC-III and Raftlin (r = 0.756, 0.733, 0.701 p < 0.001, respectively). A significant positive correlation was found between ISO (Respectively; r = 0.782, 0.712, 0.716 p < 0.001). In our evaluation between Raftlin and Iso, a significant positive relationship was determined. (r = 0.731, p < 0.001). CONCLUSION: Our findings indicated that oxidative stress in patients with MC-I may be aggravated and it may cause an inflammation formation of the lesion areas in these patients. Also, the increased 8-iso-PGF2α and Raftlin levels in patients with MC-II and MC-III may be an adaptive response to against oxidative stress.

Prostaglandin F2α Regulates Adipogenesis by Modulating Extracellular Signal-Regulated Kinase Signaling in Graves' Ophthalmopathy.

Prostaglandin F2α (PGF2α), the first-line anti-glaucoma medication, can cause the deepening of the upper eyelid sulcus due to orbital lipoatrophy. However, the pathogenesis of Graves' ophthalmopathy (GO) involves the excessive adipogenesis of the orbital tissues. The present study aimed to determine the therapeutic effects and underlying mechanisms of PGF2α on adipocyte differentiation. In this study primary cultures of orbital fibroblasts (OFs) from six patients with GO were established. Immunohistochemistry, immunofluorescence, and Western blotting (WB) were used to evaluated the expression of the F-prostanoid receptor (FPR) in the orbital adipose tissues and the OFs of GO patients. The OFs were induced to differentiate into adipocytes and treated with different incubation times and concentrations of PGF2α. The results of Oil red O staining showed that the number and size of the lipid droplets decreased with increasing concentrations of PGF2α and the reverse transcription-polymerase chain reaction (RT-PCR) and WB of the peroxisome proliferator-activated receptor γ (PPARγ) and fatty-acid-binding protein 4 (FABP4), both adipogenic markers, were significantly downregulated via PGF2α treatment. Additionally, we found the adipogenesis induction of OFs promoted ERK phosphorylation, whereas PGF2α further induced ERK phosphorylation. We used Ebopiprant (FPR antagonist) to interfere with PGF2α binding to the FPR and U0126, an Extracellular Signal-Regulated Kinase (ERK) inhibitor, to inhibit ERK phosphorylation. The results of Oil red O staining and expression of adipogenic markers showed that blocking the receptor binding or decreasing the phosphorylation state of the ERK both alleviate the inhibitory effect of PGF2a on the OFs adipogenesis. Overall, PGF2α mediated the inhibitory effect of the OFs adipogenesis through the hyperactivation of ERK phosphorylation via coupling with the FPR. Our study provides a further theoretical reference for the potential application of PGF2α in patients with GO.

Role of intraluteal and intrauterine prostaglandin signaling in LH-induced luteolysis in pregnant rats.

Luteal dysfunctions lead to fertility disorders and pregnancy complications. Normal luteal function is regulated by many factors, including luteinizing hormone (LH). The luteotropic roles of LH have been widely investigated but its role in the process of luteolysis has received little attention. LH has been shown to have luteolytic effects during pregnancy in rats and the role of intraluteal prostaglandins (PGs) in LH-mediated luteolysis has been demonstrated by others. However, the status of PG signaling in the uterus during LH-mediated luteolysis remains unexplored. In this study, we utilized the repeated LH administration (4×LH) model for luteolysis induction. We have examined the effect of LH-mediated luteolysis on the expression of genes involved in luteal/uterine PG synthesis, luteal PGF2α signaling, and uterine activation during different stages (mid and late) of pregnancy. Further, we analyzed the effect of overall PG synthesis machinery blockage on LH-mediated luteolysis during late pregnancy. Unlike the midstage of pregnancy, the expression of genes involved in PG synthesis, PGF2α signaling, and uterine activation in late-stage pregnant rats' luteal and uterine tissue increase 4×LH. Since the cAMP/PKA pathway mediates LH-mediated luteolysis, we analyzed the effect of inhibition of endogenous PG synthesis on the cAMP/PKA/CREB pathway, followed by the analysis of the expression of markers of luteolysis. Inhibition of endogenous PG synthesis did not affect the cAMP/PKA/CREB pathway. However, in the absence of endogenous PGs, luteolysis could not be activated to the full extent. Our results suggest that endogenous PGs may contribute to LH-mediated luteolysis, but this dependency on endogenous PGs is pregnancy-stage dependent. These findings advance our understanding of the molecular pathways that regulate luteolysis.

Expression and regulation of visfatin/NAMPT in the porcine corpus luteum during the estrous cycle and early pregnancy.

Visfatin/NAMPT creates a hormonal link between energy metabolism and female reproduction. A recent study documented visfatin expression in the ovary and its action on follicular cells; however, the expression of visfatin in luteal cells is still unknown. The aim of this study, therefore, was to investigate the transcript and protein expression of visfatin as well as its immunolocalization in the corpus luteum (CL) and to examine the involvement of extracellular signal-regulated kinases (ERK1/2) in the regulation of visfatin level in response to LH, insulin, progesterone (P4), prostaglandin E2 (PGE2) and F2α (PGF2α). Corpora lutea were harvested from gilts on days 2-3, 10-12 and 14-16 of the estrous cycle and on days 10-11, 12-13, 15-16 and 27-28 of pregnancy. The current study demonstrated that visfatin expression depends on hormonal status related to the phase of the estrous cycle or early pregnancy. Visfatin was immunolocalized to the cytoplasm of small and large luteal cells. Moreover, visfatin protein abundance was increased by P4, and decreased by both prostaglandins, while LH and insulin have modulatory effects, depending on the phase of the cycle. Interestingly, LH, P4 and PGE2 effects were abolished in response to the inhibition of ERK1/2 kinase. Thus, this study demonstrated that expression of visfatin in the porcine CL is determined by the endocrine status related to the estrous cycle and early pregnancy and by the action of LH, insulin, P4 and prostaglandins via activation of the ERK1/2 pathway.

Phloroglucinol inhibits oxytocin-induced contraction in rat gastric circular muscle and uterine smooth muscle.

Phloroglucinol is commonly used to alleviate dysmenorrhoea and stomach cramps. However, there is little evidence of phloroglucinol in the mechanism of primary dysmenorrhoea (PD) development. In this study, a PD rat model was established. The effects of phloroglucinol on the contraction of rat gastric circular muscle and uterine smooth muscle induced by oxytocin (OT) were investigated. The writhing response, and levels of oestradiol (E2), prostaglandin e2 (PGE2), and prostaglandin f2α (PGF2α) were determined. The protein and mRNA levels of OT receptor (OTR) were detected. OT showed a significant promoting effect on gastric circular muscle and uterine smooth muscle contraction. However, phloroglucinol strongly inhibited the contraction induced by 10-6 mol/L of OT. We also found that phloroglucinol reduced writhing response and attenuated uterine damage. Compared to the blank group, E2 and PGF2α were significantly increased, but PGE2 was significantly decreased in the PD model group. Phloroglucinol was found to reverse the changes of E2, PGF2α and PGE2. Moreover, phloroglucinol reduced the protein and mRNA levels of OTR. In conclusion, phloroglucinol could attenuate PD and inhibit the contraction of rat gastric circular muscle and uterine smooth muscle induced by OT. The mechanism might be related with the regulation of OTR expression.IMPACT STATEMENTWhat is already known on this subject? Phloroglucinol is commonly used to alleviate dysmenorrhoea and stomach cramps. However, there is little evidence of phloroglucinol in the mechanism of primary dysmenorrhoea (PD) development.What do the results of this study add? Phloroglucinol could attenuate PD and inhibit the contraction of rat gastric circular muscle and uterine smooth muscle induced by OT. The underlying mechanisms of phloroglucinol for PD treatment may be associated with OTR.What are the implications of these findings for clinical practice and/or further research? These findings provide novel ideas for the role of phloroglucinol in PD development.

Current knowledge about the processes of luteolysis and maternal recognition of pregnancy in camelids.

Camelids have many unique reproductive features that considerably differ from those of other domestic species. Females are induced ovulators with subsequent development of a corpus luteum (CL) with a short lifespan. Plasma progesterone concentration starts to increase on day 4, peaks on day 8-9 and, in non-pregnant animals, basal concentration is reached around day 10-11 post-induction of ovulation. Luteolytic pulses of prostaglandin F2α (PGF2α ) are firstly detected on day 7 or 8 (approximately on day 5-6 after ovulation), with maximal luteolytic peaks observed between days 9 and 11 post-mating, in coincidence with a high endometrial expression of cyclooxygenase 2, a limiting enzyme in prostaglandins synthesis. Unlike other species, oxytocin seems not to be involved in the luteolytic process in these species. The CL is the main source of progesterone secretion, and its function is required to support pregnancy. Despite constant research efforts, aspects of reproduction and maternal recognition of pregnancy in camelids remain not fully understood. A transient decrease and subsequent recovery in plasma progesterone concentration are observed after day 9 post-mating in pregnant animals in association with a pulsatile release of PGF2α and a transitory decrease in CL vascularization. Thus, embryo recognition should occur between days 8 and 12 post-mating. In camels, conceptus tissues exhibit aromatizing activity with the capacity to synthesize large amounts of oestradiol. Similarly, llama blastocysts secrete oestradiol-17ß during the preimplantation stage, with a higher production during the elongation period. An increase in the endometrial expression of oestrogen receptor α is also observed on day 12 post-mating. All these evidences suggest that oestrogen could be the signal released by the embryo at the time of its recognition in camelids. Besides, nearly 98% of pregnancies are carried out in the left horn. A decrease in the endometrial expression of mucin 1 and 16 genes has been reported, suggesting that these changes are crucial for successful embryo implantation; however, no differences have been observed between horns. Thus, maternal recognition of pregnancy in camelids is a particularly complex process that must occur in a concise time to allow the rescue of the CL and embryo survival.