PGF2 Research Articles

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.

Ligand-induced activation and G protein coupling of prostaglandin F2α receptor

Prostaglandin F2α (PGF2α), an endogenous arachidonic acid metabolite, regulates diverse physiological functions in many tissues and cell types through binding and activation of a G-protein-coupled receptor (GPCR), the PGF2α receptor (FP), which also is the primary therapeutic target for glaucoma and several other diseases. Here, we report cryo-electron microscopy (cryo-EM) structures of the human FP bound to endogenous ligand PGF2α and anti-glaucoma drugs LTPA and TFPA at global resolutions of 2.67 Å, 2.78 Å, and 3.14 Å. These structures reveal distinct features of FP within the lipid receptor family in terms of ligand binding selectivity, its receptor activation, and G protein coupling mechanisms, including activation in the absence of canonical PIF and ERY motifs and Gq coupling through direct interactions with receptor transmembrane helix 1 and intracellular loop 1. Together with mutagenesis and functional studies, our structures reveal mechanisms of ligand recognition, receptor activation, and G protein coupling by FP, which could facilitate rational design of FP-targeting drugs.

Presynchronization with prostaglandin F2α and gonadotropin-releasing hormone simultaneously improved first-service pregnancy per artificial insemination in lactating Holstein cows compared with Presynch-14 when combined with detection of estrus

This study aimed to determine the effect of 2 simple breeding strategies combining artificial insemination (AI) after detection of estrus (AIED) and timed AI (TAI) on first-service fertility in lactating Holstein cows. Weekly, lactating Holstein cows (n = l,049) between 40 and 46 d in milk (DIM) were randomly assigned to initiate 1 of 2 breeding strategies for first service: Presynch-14 and PG+G. Presynch-14 is a presynchronization strategy with 2 PGF2α treatments 14 d apart with the last PGF2α 14 d before the initiation of the Ovsynch protocol. Cows treated with PG+G receive a simpler presynchronization program that uses PGF2α and GnRH simultaneously 7 d before Ovsynch. In both treatments, cows detected in standing estrus by tail chalk at any time ≥55 DIM were inseminated, and treatment was discontinued (n = 525). Cows completing treatment received TAI from 78 to 84 DIM (n = 526). In a subgroup of cows that received TAI, blood was collected (n = 163) to assess circulating concentrations of progesterone, and ultrasonographic evaluations of ovaries were performed on the day of first GnRH of Ovsynch (n = 162) and PGF2α of Ovsynch (n = 122). The proportion of cows that received TAI was greater for PG+G compared with Presynch-14 (63.5 vs. 31.9%), which increased DIM at first service for cows treated with PG+G compared with Presynch-14 (75.5 ± 0.4 vs. 68.7 ± 0.4). For cows receiving TAI, the ovulatory response to first GnRH of Ovsynch (73.8 vs. 48.8%) and the proportion of cows with functional corpora lutea (92.6 vs. 73.1%) were greater for PG+G than Presynch-14. Cows treated with PG+G had greater overall pregnancy per AI (P/AI) 42 ± 7 d after AI (40.2 vs. 33.6%) and calving per AI (32.1 vs. 25.2%) than Presynch-14. For cows receiving AIED, treatment did not affect P/AI 42 ± 7 d after AI. However, for cows receiving TAI, PG+G increased P/AI compared with Presynch-14 (44.6 vs. 35.2%). Overall, cows receiving TAI had greater P/AI 42 ± 7 d after AI (42.5 vs. 31.5%) and calving per AI (34.1 vs. 23.7%) and decreased pregnancy loss (16.8 vs. 25.2%) than cows receiving AIED. In summary, PG+G increased the proportion of cows receiving TAI and the DIM at first service, P/AI, and calving per AI compared with Presynch-14 when both TAI programs were combined with AIED.

Generation of a novel three-dimensional scaffold-based model of the bovine endometrium

Bovine in vitro endometrial models that resemble tissue function in vivo are needed to study infertility, long-term uterine alterations induced by pathogens and impact of endocrine disruptor chemicals on reproductive function and other reproductive system complications that cause high economic losses in livestock species. The present study aimed to generate an innovative, reproducible, and functional 3D scaffold-based model of the bovine endometrium structurally robust for long term-culture. We developed a multicellular model containing both endometrial epithelial and stromal cells. Epithelial cells organized to form a luminal-like epithelial layer on the surface of the scaffold. Stromal cells produced their own extracellular matrix forming a stable subepithelial compartment that physiologically resembles the normal endometrium. Both cell types released prostaglandin E2 and prostaglandin F2α following a treatment with oxytocin and arachidonic acid. Additionally signal pathways mediating oxytocin and arachidonic acid stimulation of prostaglandin synthesis were analyzed by real time PCR (RT-PCR). Oxytocin receptor (OXTR), prostaglandin E2 receptor 2 (EP2), prostaglandin E2 receptor 4 (EP4), prostaglandin F receptor (PTGFR), prostaglandin E synthase (PTGES), PGF-synthase (PGFS) and prostaglandin-endoperoxide synthase 2 (COX-2) expression was detected in both control and treatment groups, however, only significant changes in abundance of OXTR mRNA transcripts were found. The results obtained by this study are a step forward in bovine in vitro culture technology. This 3D scaffold-based model provides a platform to study regulatory mechanisms involved in endometrial physiology and can set the basis for a broader tool for designing and testing novel therapeutic strategies for recurrent uterine pathologies.

73 Comparison of GnRH Administration day in the 7 & 7 Synch Versus 8 & 6 Synch Protocol for Fixed-Timed Artificial Insemination in Beef Heifers

Abstract The study was conducted to identify effects of a decreased interval of progesterone on the ovulatory follicle before fixed-timed artificial insemination (FTAI) by altering the day of a gonadotropin-releasing hormone agonist (GnRH;00µg gonadorelin; Cystorelin) administration. Evaluation of the 7 & 7 Synch and altered estrous synchronization protocols was done using estrus expression (EE) and FTAI pregnancy rates (PR). Using frozen conventional Angus semen (Select Sires, Inc. Plain City, OH), Angus-cross heifers (n = 68) from the University of Arkansas beef cattle research unit near Fayetteville were stratified into two treatment groups by cyclicity status (CYC) and age. Cyclicity status was determined by using a reproductive tract score (RTS) taken one week prior to beginning synchronization. Scores ranged from 1-5, where RTS 1-3 were considered pre- or peripubertal while scores 4 and 5 were considered cycling. Heifers with a RTS of and 2 were culled prior to synchronization. Each treatment used a controlled internal drug releasing device (CIDR; Eazi-breed; 1.38g progesterone) inserted intravaginally on –14 d simultaneous to the administration of a prostaglandin F2α analogue (PGF; 500 µg cloprostenol sodium; Synchsure). In the first treatment (7 & 7), GnRH was administered on –7 d. In the second treatment (8 & 6), GnRH was administered on –6 d. Both treatments were then administered PGF simultaneous to CIDR removal on 0d, 72 ±h prior to FTAI and the administration of GnRH. Estrus expression was recorded at the time of FTAI using an Estrotect patch (Rockway Inc., Spring Valley, WI). Estrus expression was determined as a patch with 50% or more of the color rubbed off. On the contrary, a patch with less than 50% of the color rubbed off, was not. Pregnancy rates was determined via ultrasound on 58 d. Estrus expression and PR were analyzed by treatment using the GLIMMIX procedure on SAS 9.4 (SAS Institute Inc., Cary, NC) with CYC as a fixed effect for EE; CYC and EE as fixed effects for PR. Statistical differences were determined with α ≤ 0.05 and tendencies were observed between 0.06-0.1. There was an effect of CYC on EE (P = 0.001) and a tendency to affect PR (P = 0.088). There was no treatment effect on EE (P = 0.892) or PR (P = 0.708). In conclusion, the altered time of GnRH administration in the 7 & 7 synch at 72 ± hours did not affect EE or PR; however, research will continue to investigate adjusting length of time from PGF injection to FTAI to evaluate the effects.

55 Investigating the Potential Benefits of an Immunostimulant (Mycobacterium Cell Wall Fraction) Treatment on Increased Pregnancy Success of Embryo Transfer Recipients

Abstract Modulating the vaginal and uterine microbiome through the use of immunostimulants has the potential to improve fertility in cattle. The object of this study was to evaluate the potential benefits of an immunostimulant, mycobacterium cell wall fraction (MCWF), to increase pregnancy success of embryo transfer (ET) recipients. The three main objectives of this study were: determining if injecting ET recipient cows with MCWF increased proportion of cows with a corpus luteum (CL) on ET day, identifying differences in CL volume and progesterone concentration at the time of ET, and determining if ET recipients that received MCWF have greater pregnancy rate. A total of 926 beef cows at eight different locations were enrolled in this study. Primiparous and multiparous cattle were enrolled in a 7-day CO-Synch + CIDR estrous synchronization protocol. During this protocol, cows received an injection of GnRH (100-μg) at CIDR (controlled internal drug release) insertion [d -9], a prostaglandin F2α injection (25 mg), and CIDR removal [d-2] followed by another injection of GnRH (100 μg; d 0) within 60-66 hours after CIDR removal. At each location at (d 0), cows were sorted based on BCS and days post-partum and randomly assigned to one of two treatments: 1) MCWF- a single intramuscular injection (5 mL) of MCWF at (d 0; Amplimune, NovaVive Inc.; n = 452) or 2) Control- a single intramuscular injection (5 mL) of saline (n = 474). Seven days after (d 7), cows were examined by ultrasonography to determine if a CL was present. Recipient cows that had a CL received ET with a frozen or fresh in vivo or in vitro produced embryo in the ipsilateral uterine horn to the CL, with embryo type and grade balanced between treatments. Blood samples were collected in one location (n = 98) for analysis of plasma concentration of progesterone on (d 7) ET day. Pregnancy diagnosis was then performed by ultrasonography between 30 and 90 days of gestation. Data were analyzed using the GLIMMIX and MIXED procedures of SAS. Cow BCS and days postpartum were similar (P > 0.10) between treatments (5.5 ± 0.6 and 103 ± 14 days, respectively). Recipient cow utilization rate was similar between treatments (P = 0.49; 81.2 ± 0.42%). At the time of ET MCWF had larger CL than Control cows (5,768.6 ± 133.7 and 5,408.0 ± 128.5 mm3, respectively, P = 0.03). and increased progesterone concentration (2.68 ± 0.17 and 2.18 ± 0.14 ng/mL, respectively, P = 0.01). However, pregnancy rate to ET was similar (P = 0.922) between treatments (60.9 and 61.3 ± 0.05% for Control and MCWF, respectively). In conclusion, treatment of ET recipient cows with MCWF increased CL volume and concentration of progesterone at the time of ET but failed to generate greater pregnancy rates.

Abstract 460: Catalase Overexpression Modulates Plaque Fibrosis And Vascular Contractile Function In Aged Hyperlipidemic Mice

Background: Plaque fibrosis and re-differentiation of vascular smooth muscle cells (VSMCs) from a contractile to a “synthetic” phenotype are hallmarks of advanced atherosclerosis. While in vitro studies demonstrated a clear role of oxidative stress in the loss of VSMC contractile phenotypes, the roles of specific antioxidant mechanisms and their therapeutic utility remain unclear. Here we test the hypothesis that overexpression of catalase protects against excess plaque fibrosis and losses in contractile phenotypes in vivo . Methods: We used littermate-matched, hypercholesteremic (ldlr -/- /ApoB 100/100 ; LA) mice lacking a transgenic construct (LA-CAT 0/0 ) or harboring a single copy of the human catalase gene (LA-CAT Tg/0 ), which were fed a Western Diet for 3 or 12 months. Isolated organ bath chambers, histopathological analyses, and quantitative RT-PCR were used to assess vasomotor function, structure, and molecular changes in aorta. Results: Oxidative stress increased with disease progression in aortic tissue from LA-CAT 0/0 mice and was modestly reduced in LA-CAT Tg/0 mice. Collagen deposition and fiber thickness increased with disease progression in LA-CAT 0/0 mice, and these increases in fiber thickness were attenuated in LA-CAT Tg/0 mice at 12 months and more pronounced in male mice. In parallel, we observed favorable rightward shifts in the vascular stress-strain relationship in LA-CAT Tg/0 mice that were more pronounced in aged male mice. Interestingly, these structural changes were associated with improved contractile responses to either PGF 2α or serotonin in male LA-CAT Tg/0 mice at 12 months. Consistent with these changes, the contractile marker calponin-1 was profoundly reduced with increasing age in LA-CAT 0/0 mice, with these changes being partially attenuated in aging, male LA-CAT Tg/0 mice. In vitro, treatment of VSMCs with PEG-catalase attenuated BMP2-dependent contractile protein expression. Conclusion: These results demonstrate that moderate overexpression of catalase can attenuate deleterious fibrotic and contractile changes associated with prolonged progression of atherosclerosis. Future mechanistic investigation exploring the context-dependence of these changes (e.g., sex dependence) is warranted.

Effects of the thromboxane receptor antagonist S18886 in the porcine coronary circulation

Thromboxane A2 (TxA 2 ) is a potent coronary vasoconstrictor that has been implicated in promoting decreases in myocardial perfusion in a variety of (patho)-physiologic conditions. S18886 is a promising orally-active TxA 2 receptor antagonist currently approved for investigational clinical use. However, the coronary vascular effects of S18886 are unknown and its specificity and affinity for the thromboxane receptor in the coronary circulation remain unclear. We tested the hypothesis that administration of S18886 dose-dependently attenuates coronary vasoconstriction to the TxA 2 mimetic U46619 without influencing coronary responses to prostaglandin F 2α , acetylcholine, or smooth muscle depolarization (K + ). Experiments to test this hypothesis were performed in male (n = 5) and female (n = 6) domestic swine. Hearts were excised and the left circumflex coronary artery isolated, cleaned of periadventitial fat, and cut into 3 mm rings. Isometric tension of coronary artery rings was measured in response to log order increments of U46619 (1 nM to 1 μM) with and without S18886 (0.1-100 nM). Similar isometric studies were conducted with prostaglandin F 2α (10 nM-10 μM), acetylcholine (0.1-10 μM), and KCl (5-90 mM). U46619 induced concentration dependent increases in tension development of isolated coronary artery rings (average EC50 of 42 ± 19 nM). Incubation of coronary arteries with S18886 (1 nM) significantly attenuated coronary vasoconstriction to U46619 resulting in a rightward shift of the EC50 to 187 ± 38 nM (P < 0.02). Vehicle had no effect on U46619-induced contractions. Higher concentrations of S18886 (10nM and 100nM) dose-dependently reduced U46619-induced contractions by 77% ± 3 and 93% ± 6 respectively. S18886 (1 nM) antagonized coronary vasoconstriction of prostaglandin F2α (10 μM) by 68% ± 5 (P < 0.0001) but had no effect on either acetylcholine or KCl-induced contraction. Data from this investigation indicate that S18886 is an effective antagonist of U46619-induced vasoconstriction in the porcine coronary circulation. While S18886 does not influence coronary smooth muscle response to either acetylcholine or activation of L-type Ca 2+ channels, attenuation of prostaglandin F 2α suggests the antagonists specificity may extend beyond TxA 2 receptor signaling. NIH R01HL158723 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

150 Indomethacin infusion through multiple routes and the resulting Prostaglandin F2α metabolite production during late embryonic development in beef cattle

150 Indomethacin infusion through multiple routes and the resulting Prostaglandin F2α metabolite production during late embryonic development in beef cattle

Review: Maintenance of the ruminant corpus luteum during pregnancy: interferon-tau and beyond.

This manuscript reviews the mechanisms that maintain the corpus luteum (CL) of pregnancy in ruminants. In mammals, ovulation and luteinization of the remaining cells in the CL are due to a surge in Luteinizing Hormone (LH). In cattle, continued secretion of pulses of LH is essential for full development and function of the CL during the estrous cycle (LH pulses), however, the few studies on the CL after d20 of pregnancy do not indicate that LH is essential for maintaining the CL of pregnancy. The first essential step in maintaining the CL of pregnancy in ruminants is overcoming the mechanisms that cause regression of the CL in non-pregnant ruminants (d18-25 in cattle; d13-21 in sheep). These mechanisms have a uterine component involving oxytocin-induced prostaglandin F2α (PGF2A) pulses and a luteal component involving decreased progesterone production and luteal cell death. There is a critical role for embryonic interferon-tau (IFNT) in suppressing the uterine secretion of PGF2A during early pregnancy (d13-21 in sheep; d16-25 in cattle) and preventing luteolysis. There are also effects of IFNT on the expression of interferon-stimulated genes in other tissues including the CL but the physiologic role of these interferon-stimulated genes is not yet clear. After the IFNT period, there is another mechanism that maintains the CL of pregnancy in ruminants since embryonic IFNT is inhibited as attachment occurs and trophoblastic binucleate/giant cells begin secretion of pregnancy-associated glycoproteins. The second mechanism for luteal maintenance has not yet been defined but acts in a local manner (ipsilateral to pregnancy), and remains functional from d25 until just before parturition. The most likely mechanisms mediating later maintenance of the CL of pregnancy are increased uterine blood flow or decreased prostaglandin transporter expression in the utero-ovarian vasculature, preventing PGF2A reaching the CL. Finally, implications of these ideas on pregnancy loss in cattle are explored, highlighting the importance of inappropriate regression of the CL of pregnancy as a mechanism for pregnancy loss in cattle.

Three medicinal plants affecting human ovarian cell viability, hormone release, and response to environmental contaminant toluene

The present study examined the effect of medicinal plants - ginkgo, tribulus (puncture vine), and yucca - on ovarian functions and their response to the toxic influence of toluene. Therefore, we analyzed the effect of toluene with and without these plant extracts on cultured human ovarian granulosa cells. Cell viability and the release of progesterone, insulin-like growth factor I (IGF I), oxytocin, and prostaglandin F (PGF) were analyzed using the trypan blue test, enzyme immunoassay, and enzyme-linked immunosorbent assay, respectively. The ginkgo, tribulus and yucca were able to suppress ovarian cell viability and alter the release of hormones. Toluene suppressed cell viability and the release of PGF, but not of progesterone, IGF-I, or oxytocin. The negative effect of toluene on cell viability was prevented and even reversed by ginkgo and yucca, whereas its effect on PGF was prevented or inverted by all tested plant extracts. These findings (1) demonstrated the direct toxic effect of toluene on ovarian cells, (2) showed the direct effect of some medicinal plants on ovarian cell functions, and (3) demonstrated the ability of these plants to inhibit the effects of toluene and to act as natural protectors against the suppressive effect of toluene on female reproduction.

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.

Addition of equine chorionic gonadotropin in controlled internal drug-release-based synchronization improves reproductive performance of Nili-Ravi buffalo in the sub-tropics

This study evaluated the effect of equine chorionic gonadotropin (eCG) and factors such as body condition score (BCS), cyclicity, and breeding season in a controlled internal drug release (CIDR)-based oestrus synchronization protocol on ovarian follicle dynamics, oestrus expression, ovulation rate, and pregnancy per artificial insemination (AI) (P/AI) in Nili-Ravi buffalo. Adult buffaloes (n = 88), 6.69 ± 1.17 years, parity 3.36 ± 0.97, and BCS 2.76 ± 0.45, were randomly administered eCG or saline, concurrent with prostaglandin F2α (PGF2α) treatment on Day 6 in a CIDR synchronization protocol during the breeding season and low breeding season. Follicular dynamics and oestrus expression were recorded from eCG administration until ovulation. Fixed-time artificial insemination (FTAI) was performed at 48- and 60-hour intervals after CIDR withdrawal. On Day 35 post-AI, pregnancy was diagnosed with ultrasonography. There was no significant difference in the size of the ovulatory follicle between eCG-treated buffaloes (ETB) and saline-treated buffaloes (STB). The growth rate of the dominant follicle, oestrus response and intensity, ovulation, and P/AI were higher (P &lt;0.05) in eCG-treated buffaloes. It was concluded that eCG improved the growth rate of the dominant follicle, oestrus expression, and P/AI in a CIDR-based FTAI programme in Nili-Ravi buffalo. The deleterious effects of poor BCS, acyclicity, and low breeding season could be reduced with a progesterone-based fixed time AI protocol with the addition of eCG.

Estrous synchronization in cattle, sheep and goat

Synchronization of estrous refers to manipulating the estrous cycle or induction of estrous to bring a large number or group of female animals into estrous at a short, determined time. An estrous cycle is a rhythmic event in the female animal from one estrous phase to the next. The normal estrous cycle length is 21 days in cows, 20 days in doe, and 17 days in the ewe. The hormones used in estrous synchronization are estrogen, progesterone, GnRH, and prostaglandin. Estrous synchronization helps shorten the breeding period instead of females being bred over a period of normal length, and this depends on the treatment protocols to be followed. Combining GnRH with prostaglandin F2α (PGF2α) and a progesterone-based synchronization program has shown a novel direction in cattle's estrous synchronization, which causes manipulation in follicular development. Estrous synchronization protocol should be followed based on managemental capabilities and expectations of the farmer. This review article was compiled to summarize the protocols used for estrous synchronization in different species.

Serum concentrations of secretory leukocyte protease inhibitor and IL6 can predict the onset of sepsis in pyometra bitches.

As onset of sepsis adversely affects the prognosis of canine pyometra, finding biomarkers that would distinguish sepsis status would be useful in the clinical management. Accordingly, we hypothesized that differential expression of endometrial transcripts and circulating concentration of certain inflammatory mediators would discriminate pyometra-led sepsis (P-sepsis+) from those of pyometra without sepsis (P-sepsis-). Bitches with pyometra (n = 52) were classified into P-sepsis+ (n = 28) and P-sepsis- (n = 24) based on vital clinical score and total leukocyte count. A group of non-pyometra bitches (n = 12) served as control. The relative fold changes in the transcripts of IL6, IL8, TNFα, IL10, PTGS2, mPGES1 and PGFS, SLPI, S100A8, S100A12 and eNOS were determined by quantitative polymerase chain reaction. Furthermore, the serumconcentrations of IL6, IL8, IL10, SLPI and prostaglandin F2α metabolite (PGFM) were assayed by ELISA. The relative fold changes in S100A12 and SLPI and mean concentrations of IL6 and SLPI were significantly (p < .05) higher in P-sepsis+ than that of P-sepsis- group. Receiver operating characteristic analysis revealed that serum IL6 had a diagnostic sensitivity of 78.6% and a positive likelihood ratio (LR+) of 2.09, at a cut-off value of 15.7 pg/mL to diagnose P-sepsis+ cases. Similarly, serum SLPI had a sensitivity of 84.6% and an LR+ of 2.23, at a cut-off value of 2.0 pg/mL. It was concluded that SLPI and IL6 would serve as putative biomarkers for pyometra-led sepsis in bitches. Monitoring SLPI and IL6would be a useful adjunct to the established haemato-biochemical parameters in customizing the treatment strategies and arriving at the decision for management of pyometra bitcheswith critical illness.

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.

Dibazol-induced relaxation of ophthalmic artery in C57BL/6J mice is correlated with the potency to inhibit voltage-gated Ca2+ channels

We aimed to explore the effect of dibazol on the ophthalmic artery (OA) and ophthalmic artery smooth muscle cells (OASMCs) of C57BL/6J mice as well as the underlying mechanisms. The OA of C57BL/6J mice was isolated under a dissecting microscope for primary OASMCs culture and myogenic tests. OASMCs were identified through morphological and immunofluorescence analyses. Morphology changes in the OASMCs were examined by staining using rhodamine-phalloidin. We performed a collagen gel contraction assay to measure the contractile and relaxant activities of the OASMCs. The molecular probe Fluo-4 AM was used to examine intracellular free Ca2+ levels ([Ca2+]in). The myogenic effects of OA were examined using wire myography. Additionally, the whole-cell patch-clamp technique was used to investigate the mechanisms underlying the relaxant effect of dibazol on L-type voltage-gated Ca2+ channels (LVGC) in isolated cells. 10−5 M dibazol significantly inhibited the contraction of OASMCs and increased the [Ca2+]in response to 30 mM KCl in a concentration-dependent manner. Dizabol had a more significant relaxant effect than 10−5 M isosorbide dinitrate (ISDN). Similarly, dibazol showed a significant dose-dependent relaxant effect on OA contraction induced by 60 mM KCl or 0.3 μM 9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U46619). The current-voltage (I–V) curve revealed that dibazol decreased Ca2+ currents in a concentration-dependent manner. In conclusion, dibazol exerted relaxant effects on the OA and OASMCs, which may involve the inhibition of the Ca2+ influx through LVGC in the cells.

Murine Models and Research Progress on Dysmenorrhea.

Dysmenorrhea is a prevalent gynecological disease among women at reproductive age. It is classified as the primary dysmenorrhea and the secondary dysmenorrhea according to the etiology. The primary dysmenorrhea is caused by uterine hypercontraction without any identifiable pelvic lesions, while the secondary dysmenorrhea is incurred by gynecological disorder with pelvic organic lesions. However, the underlying mechanism of dysmenorrhea is not completely clear. Animal models of dysmenorrhea, especially mouse and rat model, are helpful to explore the pathophysiological mechanism of dysmenorrhea, clarify the therapeutic effect of compounds, and guide clinical treatment. The murine model of primary dysmenorrhea is commonly induced by oxytocin or prostaglandin F2α, while the secondary dysmenorrhea murine model was further created by injecting oxytocin on the basis of the established primary disease model. This review summarizes the current progress of dysmenorrhea models in rodent, including experimental methods, corresponding evaluation indexes, and the advantages and disadvantages of various murine dysmenorrhea models, in order to provide a reference for the selection of murine dysmenorrhea models and the further study of the pathophysiological mechanism of dysmenorrhea.

Successful lactation induction in non-pregnant gilts.

A protocol to induce lactation was applied to non-pregnant gilts. In Experiment I, five gilts with oestrus synchronized through oral supplementation of 20 mg altrenogest for 18 days received: 10 mg oestradiol cypionate (EC) on the last day of oestrous expression (D0); 10 mg EC and 300 mg long-acting progesterone (P4) on D26; and two 0.53 mg doses of a prostaglandin F2α analogue (PGF) 12 h apart on D36. Blood was collected on D12, D19, D26 and D33. Milk secretion started in all gilts 24 h after PGF administration and lasted at least 8 days. Milk samples were collected from D37 to D45. The serum P4 concentration was lower on D12 than subsequently (p < .05), but the oestradiol concentration was unaltered (p > .05). The milk produced during the induced lactation was generally richer in protein and poorer in fat compared to the milk from the lactation of a reference sow. In Experiment II, the same protocol induced lactation in two gilts, which nursed fostered piglets for 22 days. Thus, lactation was induced in all treated gilts and the milk produced was capable to nurture fostered piglets.

Co-exposure to particulate matter and humidity increases blood pressure in hypertensive mice via the TRPV4-cPLA2-COX2 pathway

Epidemiological studies have demonstrated that particulate matter (PM) can induce or exacerbate hypertension. High relative humidity has been associated with elevated blood pressure in certain regions. However, the coupling effect of humidity and PM on elevated blood pressure and the underlying mechanisms remain unknown. Herein, we aimed to explore the effects of exposure to PM and/or high relative humidity on hypertension, as well as elucidate underlying mechanisms. Male C57/BL6 mice were intraperitoneally administered NG-nitro-L-arginine methyl ester (L-NAME) to establish a hypertensive mouse model. The hypertensive mice were exposed to PM (0.15 mg/kg/day) and/or different relative humidities (45/90%) for eight weeks. Histopathological changes, systolic blood pressure (SBP), endothelial-derived contracting factors (thromboxane B2 [TXB2], Prostaglandin F2α [PGF2α], endothelin-1 [ET-1], and angiotensin II [Ang II]), and relaxing factors (prostaglandin I2 [PGI2] and nitric oxide [NO]) were measured to assess the effects of PM exposure and humidity on hypertension in mice. Levels of transient receptor potential vanilloid 4 (TRPV4), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase 2 (COX2) were measured to explore their potential mechanisms. Herein, exposure to 90% relative humidity or PM alone had a slight but insignificant effect on hypertension. However, pathological changes and elevated blood pressure were markedly exacerbated following exposure to PM and 90% relative humidity. Levels of PGF2α, TXB2, and ET-1 were significantly increased, whereas the PGI2 level was substantially decreased. HC-067047-mediated blockade of TRPV4 suppressed TRPV4, cPLA2, and COX2 expression and effectively alleviated the increased blood pressure induced by exposure to PM and 90% relative humidity. These results indicate that 90% relative humidity and PM can activate the TRPV4-cPLA2-COX2 ion channel in the aorta, altering the endothelial-derived contracting and relaxing factors and enhancing blood pressure in hypertensive mice.