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Infection of the uterus with Gram-positive Trueperella pyogenes and Gram-negative Escherichia coli is a common cause of postpartum endometritis in the cattle and buffalo and the condition is treated with antimicrobial drugs. The presence of drug residues in the milk and development of resistant bacteria necessitate the evaluation of alternate therapies for endometritis. Accordingly, we tested the immunomodulatory effect of curcumin in the bubaline endometrial stromal cells after treatment with the lipoteichoic acid (LTA) of Gram-positive Staphylococcus aureus and lipopolysaccharide (LPS) of Gram-negative E. coli that activate toll-like receptors (TLR-2 and TLR-4, respectively). Confluent primary culture of endometrial stromal cells was treated with LTA (1 µg/mL) and/or LPS (0.1 µg/mL), in the presence or absence of curcumin (30 µM for 24 h). PGE 2 was assayed in the supernatant and the relative expression of proinflammatory cytokines (PICs) (IL1B, IL6, IL8 and TNFA) transcripts were quantified using real-time PCR. LTA was not effective in stimulating PGE 2 production or upregulating the PIC expression except IL8. LTA+LPS increased PGE 2 production and upregulated IL6 and IL8 genes. Curcumin inhibited the basal and LTA+LPS induced production of PGE 2 and upregulation of PIC production. It was apparent that LPS, but not LTA, is a potent stimulator of PGE 2 from the bubaline endometrial stromal cells. Curcumin downregulated the expression of LPS and/or LTA induced PICs and PGE 2 and may be an alternate to antimicrobial drugs for the therapeutic management of endometritis.

Developing alternate therapies for bovine endometritis is important in the context of drug residues in the milk and emergence of antimicrobial resistant bacteria. In this regard, we studied the immunomodulatory effect of curcumin 30 µM, on lipopolysaccharide- (LPS) and/or flagellin (100 ng/ml each)-induced prostaglandin E 2 (PGE 2 ) and proinflammatory cytokines (PIC) using primary bubaline endometrial stromal cells. After 24 hr treatment, the supernatant was assayed for PGE 2 while cells were used for relative quantification of cytokines like IL-1β, IL-6, IL-8 and TNF α transcripts using a control group as calibrator. LPS was found to possess potent stimulatory effect on PGE 2 production, whereas the flagellin was not as potent as LPS in stimulating the PGE 2 production either per se or in combination with LPS. LPS markedly up-regulated the transcripts of IL-8 and IL-6 as compared to IL-1β and TNF α in the bubaline endometrial stromal cells. Except for IL-8, flagellin did not up-regulate other PICs. There was no additive effect between LPS and flagellin on the up-regulation of inflammatory cytokines. Curcumin inhibited the LPS-induced up-regulation of PIC with strong down-regulation of IL-8. The inhibitory effects of curcumin on the inflammatory mediators suggest a potential in the treatment of bovine endometritis., (© 2019 Blackwell Verlag GmbH.)

Progesterone (P 4 ) plays a key role in the establishment and maintenance of pregnancy in most mammals. Unravelling the expression of progesterone-regulated genes can expand the understanding of the embryonic mortality. Accordingly, we studied the relative mRNA expression of the P 4 -regulated genes in the buffalo. Uteri were collected from the abattoir and categorized into nonpregnant late luteal phase, stage I (28-38th days of gestation) and stage II (48-56th days of gestation) of pregnancy (n = 6/group). After extraction of total RNA from the endometrial tissues, we carried out qRT-PCR for determining the relative mRNA expression of the P 4 -regulated genes using nonpregnant late luteal phase as calibrator group. The expression of LGALS3BP (essential for maternal recognition of pregnancy) gene was found to be significantly upregulated (p < 0.05), while MUC1 (important for embryo attachment) gene was downregulated in stage I and II of pregnancy. We observed no significant change in the expression of LGALS1, LGALS9 and CTSL genes. The SLC5A11 and SLC2A1 genes (involved in the transport of glucose to endometrium) in early pregnancy were upregulated in the pregnancy stage I (p < 0.05) relative to nonpregnant late luteal phase. The CST3 gene was significantly upregulated in pregnancy stage II (p < 0.01). These results provide molecular insights into the specific pathways involved in foeto-maternal communication during early pregnancy in buffaloes., (© 2018 Blackwell Verlag GmbH.)

Inflammatory markers of endometrial origin are valuable in order to differentiate the pyometra from cystic endometrial hyperplasia in the bitch. In the present study, we hypothesized that histological categorization would distinguish the differential regulation of the proinflammatory genes in the endometrium of bitches with pyometra. Ovariohysterectomy was done on bitches with confirmatory diagnosis of pyometra (n = 18). Using endometrium to myometrium ratio of 0.79 as threshold, the uteri (n = 8/group) were categorized into hyperplastic pyometra (HP) and atrophic pyometra (AP). Two samples were excluded as the diagnosis was inconclusive. In parallel, endometrial tissue was collected for total RNA extraction to study the differential expression of TLR4, IL-6, IL-8, COX-2 and PGFS through real time PCR. Diestrus uterus of non-pyometra bitches (n = 6) served as control. The mean fold change (2 -ΔΔCt ) for the target genes was determined using β-actin as endogenous control and non-pyometra uterus as calibrator group. Except TLR4, other inflammatory genes were upregulated significantly by 1.82 to 3.74 times in the AP as compared to HP with maximum upregulation of COX-2 and PGFS. Further, correlation matrix with Spearman's rho revealed that IL-8 had strong positive correlation with COX-2 and PGFS in the AP group (P < 0.05). It is concluded that histological grading of pyometra into HP and AP revealed differential regulation of inflammatory cytokines and enzymes in the PG synthetic pathway in the canine endometrium that has diagnostic potential under clinical settings., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Decreasing the ratio of n-6 to n-3 polyunsaturated fatty acid (PUFA) through dietary manipulation improves the reproductive functions in the dairy cow. This study was conducted to determine the effect of dietary supplementation of n-3 PUFA rich fish oil that provided different levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to determine the optimum dose with respect to endometrial and ovarian functions in the doe. Normal cyclic goats (n = 20) were divided into four groups (n = 5/group) and the fish oil was supplemented for 55 days such that EPA and DHA content were 0, 72, 156 and 312 mg/Kg body weight for CON, LOW, MED and HIGH groups, respectively. The diet was made isonitrogenous and isocaloric. Estrus was synchronized by two PG regimen at 11 days apart with first PG on day 25 of supplementation. Experimental does were observed from second PG till day 17 post-estrus. Ovarian ultrasound revealed that the follicular attributes were comparable among the groups; however, the diameter of CL on day 5 post-estrus was significantly larger in the MED group. Fish oil supplementation even at the lowest dose significantly decreased the serum estradiol (E 2 ) and basal as well as oxytocin (OXT) induced PGFM during the late luteal phase of the estrous cycle (P < 0.05). Further, the MED group showed a significant decrease in the serum E 2 on day 17 post-estrus as compared to LOW group. A significant increase in the progesterone (P 4 ) on day 14 post-estrus was recorded in the MED group. Neither the effect of treatment nor time was significant either on day 16 or 17 post-estrus with respect to PGEM following OXT challenge (P > 0.05). It is concluded that the dietary fish oil inhibited OXT induced PGF 2α release and decreased E 2 during the late luteal phase of the estrous cycle in the doe, which will have a favourable effect on the maternal recognition of pregnancy. In addition, MED level (156 mg/Kg body weight) increased the CL size and circulating P 4 during the luteal phase., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recently, we showed that dietary supplementation of n-3 PUFA rich fish oil (FO) decreased the metabolites of serum prostaglandin (PG) F 2α and E 2 during the window of pregnancy recognition in the doe. In this study, we investigated its effect on the changes on endometrial PG production in vitro. Cycling does (n = 12) of Rohilkhand region were divided into two equal groups and fed a concentrate diet supplemented with either FO containing 26% n-3 PUFA (TRT; n = 6) or palm oil (CON; n = 6) @ 0.6 mL/kg body weight for 57 days. Estrus was synchronized by two injections of PGF 2α analogue viz, on day 25 and 36 of supplementation and laparo-hysterotomy was performed to obtain endometrial tissue on day 16 of the synchronized estrus. Endometrial explant culture was done using a defined medium.The basal PG production was assayed at 6 and 12 h. Endometrial explant was stimulated with oxytocin (OXT) and/or recombinant ovine interferon tau (roIFN-τ) and PGs were assayed at 3 and 12 h post-treatment. The relative expression of genes related to PG metabolism in the endometrium was done by Quantitative Real Time PCR technique (qRT-PCR). There was a significant (P < 0.05) decline in the basal production of PGF 2α and PGE 2 in the TRT as compared to the CON group. The cultured endometrial tissue produced PGF 2α in a time- dependent fashion in both the groups (P < 0.05). Neither OXT nor roIFN-τ had a significant (P > 0.05) effect on the PGF 2α and PGE 2 production in the TRT group. Similarly, the PG production in the OXT and roIFN-τ was comparable with the control in TRT. Expression of mRNA for cyclooxygenase-2 (COX-2), cytosolic phospholipase A 2 (cPLA 2 ) and PGF synthase (PGFS) was lower (P < 0.05) whereas, PGE synthase and peroxisome proliferator-activated receptors such as PPAR-γ and δ was increased (P < 0.05) in n-3 PUFA fed doe. In conclusion, dietary supplementation of FO decreased the endometrial production of PGF 2α and PGE 2 by downregulating the COX-2, cPLA 2 and PGFS transcripts in the doe. The findings suggest that n-3 PUFA influence embryo survival by modulating the endometrial PG., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Endometritis is one of the leading causes of infertility in the cattle and buffalo and innate immune mechanism plays an important role in clearing the infection. In this regard, endometrial expression and function of Toll Like Receptors (TLR) are focus of investigation in the recent years. In this study, we report the transcriptional profiles of TLR4 and 5 in the buffalo endometrium during the follicular, early, mid and late luteal phases of estrous cycle and 'subclinical and clinical endometritis' and also at true anestrus (n = 10 for each stage) using RT-PCR and qRT-PCR as they are the ligands for the lipopolysaccharide and flagellin components of E.coli, the most common cause of postpartum endometritis. We found a significant positive correlation between TLR4 and 5 in all the groups (r = 0.696-0.803; P < 0.05) except late luteal phase (r = 0.522; P > 0.05). Chi-square analysis showed that the qualitative expression of endometrial TLR4 and 5 transcripts was significantly associated with the phase of estrous cycle and also with uterine infection (P < 0.05). Further, using true anestrus category as a calibrator group, relative quantitation of TLR4 and 5 revealed that the transcriptional expression of TLR4 and 5 genes were highly upregulated (24.6-83.3 folds) during endometritis conditions and moderately upregulated during mid-luteal phase (6.8-16.2) of the estrous cycle (P < 0.05). The results suggested a role of progesterone in the expression of TLR4 and 5.

Prostaglandins (PG) are involved in several female reproductive processes, and their action is regulated at the levels of biosynthesis, catabolism, and signal transduction. Facilitated transport across cell membranes emerges as an additional checkpoint regulating PG action. We have already reported on the influx transporter solute carrier organic anion transporting polypeptide (SLCO2A1) [PG transporter (PGT)] in relation to PG action in the bovine endometrium. In the present study, we report on the functional expression and regulation of multidrug resistance-associated protein 4 (MRP4)/ATP-binding cassette carrier 4, an alternate PG transporter belonging to the ATP-binding cassette carrier (ABC) family. We have found that MRP4 protein was present throughout the estrous cycle and exhibited a pattern of expression similar to that of PGT with maximal expression during early-mid luteal phase in the bovine endometrium. Functional expression and regulation of MRP4 was studied in vitro using the newly developed bovine endometrial epithelial bEEL and stromal CSC cell lines. Oxytocin (OT) stimulated PGF2α production and MRP4 mRNA and protein in a time- and dose-dependent manner but had no effect on PGT. OT induced preferred accumulation of PG outside the cells and secretion toward the basolateral side of polarized bEEL cells grown on membrane inserts. MK-571 and indomethacin, two documented inhibitors of MRP4 activity, blocked preferred accumulation of PG, but interferon-τ and NS-398 had no effect on MRP4 expression or the direction of PG transport. Our results suggest that MRP4 is a functional PG carrier under the regulation of OT in the bovine endometrium.

Oxytocin (OT) triggers the luteolytic pulses of prostaglandin F(2 alpha) (PGF(2 alpha)) from the endometrial epithelial cells in ruminants. We have proposed that the embryonic signal interferon-tau exerts its antiluteolytic effect by disrupting the OT signaling axis. Accordingly, we have attempted to define the signaling pathway of OT-induced PGF(2 alpha) production in the bovine endometrium using our newly characterized epithelial cell line (bEEL). OT receptor was coupled to the classical G alpha(q) pathway as evidenced by calcium release and activation of phospholipase C. Similarly, OT-induced PGF(2 alpha) production was mediated through the canonical ERK1/2 pathway. Because of the importance of receptor and nonreceptor tyrosine kinases in G protein-coupled receptor signaling, we studied the role of epidermal growth factor receptor (EGFR), c-Src, and phosphoinositide 3-kinase (PI3K) on OT-induced PGF(2 alpha) production in association with cyclooxygenase 2 (COX2) expression and ERK1/2 and Akt phosphorylation. The EGFR inhibitor AG1478 (10 microm) nearly abolished basal and OT-induced PGF(2 alpha) production and down-regulated COX2 expression and ERK1/2 phosphorylation. Because the transactivated EGFR can serve as a ligand for the signaling proteins with Src homology 2 (SH2) domain, we hypothesized a role for c-Src and PI3K in OT-induced PGF(2 alpha) production. Inhibitors of c-Src (PP2, 10 microm) and PI3K (LY294002, 25 microm) produced a significant decrease in OT-induced PGF(2 alpha) production and reduced COX2 expression. Also, PP2, but not LY294002, decreased OT-induced ERK1/2 phosphorylation. Because LY294002 did not affect ERK1/2 phosphorylation, but inhibited PGF(2 alpha) production and down-regulated COX2 expression, it is likely that the Akt pathway is also involved in PGF(2 alpha) production. Thus, EGFR may simultaneously activate c-Src and PI3K to amplify the OT signaling to increase the output of PGF(2 alpha) in bEEL cells.

Interferon-tau (IFNtau) is the embryonic signal responsible for pregnancy recognition in ruminants. The primary action of IFNtau is believed to be mediated through inhibition of prostaglandin F(2alpha) (PGF(2alpha)) released from the endometrial epithelial cells in response to oxytocin (OT). Our working hypothesis was that the antiluteolytic effect of IFNtau also involved modulation of PG production downstream of OT receptor (OTR) and/or cyclooxygenase 2 (COX2). There is currently no OT-sensitive endometrial cell line to study the molecular mechanisms underlying our hypotheses. Therefore, we established an immortalized bovine endometrial epithelial cell line (bEEL) exhibiting OT response. These cells were cytokeratin positive, expressed steroid receptors, and exhibited preferential accumulation of PGF(2alpha) over PGE(2). The bEEL cells were highly sensitive to OT, showing time- and concentration-dependent increase in COX2 transcript and protein and PGF(2alpha) accumulation. Interestingly, IFNtau (20 ng/ml) significantly reduced OT-induced PGF(2alpha) accumulation, but surprisingly, the effect was not mediated through down-regulation of either OTR or COX2. Rather, IFNtau up-regulated COX2 in a time- and concentration-dependent manner while decreasing OT-induced PG accumulation. This suggests that COX2 is not a primary target for the antiluteolytic effect of IFNtau. Because IFNtau reduced OT-stimulated PGF(2alpha) accumulation within 3 h, the mechanism likely involves a direct interference at the level of the OT signaling or transcription in addition to the down-regulation of OTR observed in vivo. In summary, bEEL cells offer a unique in vitro model for investigating the cellular and molecular mechanisms underlying OT and IFNtau response in relation with luteolysis and recognition of pregnancy in the bovine.

In ruminants, interferon-tau (IFNtau) is the maternal recognition signal inhibiting prostaglandin (PG) F2alpha production by endometrial epithelial cells and stimulating interferon-stimulated genes in the stroma. Stromal cells mediate the action of progesterone on epithelial cells during pregnancy. Our working hypothesis is that IFNtau acts as a molecular switch that turns on PGE(2) production in endometrial stromal cells while suppressing PGF2alpha production from epithelial cells. In this report we document immortalization and functional characterization of a bovine stromal cell line from the caruncular region of the endometrium [caruncular stromal cell (CSC)]. Primary stromal cells were immortalized by nucleofection with simian virus 40 large T antigen and integrase. The resulting cell line, CSC, expresses stromal cell-specific vimentin, estrogen, and progesterone receptors, and is amenable for transient transfection. Basal and stimulated production of PGE2 is higher than PGF2alpha and associated with cyclooxygenase (COX) 2 expression. Phorbol myristate acetate (PMA) and IFNtau up-regulate COX2 and PG production in a dose-dependent manner. When added together, low concentrations of IFNtau inhibit PMA-induced COX2 expression; whereas this inhibition is lost at high concentrations. Expression of signal transducer and activator of transcription 1 is induced by IFNtau at all concentrations studied but is not modulated by PMA. Because expression of signal transducer and activator of transcription 1 does not exhibit the biphasic response to IFNtau, we investigated the p38 MAPK pathway using the selective inhibitor SB203580. Inhibition of the p38 MAPK pathway abolishes IFNtau action on PG production. In summary, CSC appears as a good stromal cell model for investigating the molecular mechanisms related to IFNtau action and PG production in the bovine.

Cystic endometrial hyperplasia (CEH)-pyometra (CEH-P) is one of the most common reproductive disorders in bitches, posing a risk to both future fertility and life. The aims of the current study were to elucidate the differential expression patterns of inflammatory mediators at transcript and protein levels in the endometrium and to assess the concentrations of key inflammatory mediators in the peripheral circulation of bitches with different graded CEH-P. A total of 25 client-owned intact mixed breed bitches of 3–10 years presented to the outpatient department of RVP-TVCC of the institute were considered for the study. Of which, 22 cases suggestive of pyometra and 3 cases of CEH obtained during routine elective ovariohysterectomy were subjected to histopathological examination. Uteri were categorized into CEH (n = 3), moderate CEH-P (mCEH-P, n = 9), severe CEH-P (sCEH-P, n = 6) and atrophic pyometra (AT-P, n = 7). A group of age matched (n = 12) bitches without pyometra served as control. Endometrial transcripts such as IL6, IL8, PTGS2, PGFS, and SLPI were expressed differentially in the CEH and CEH-P bitch. In addition, a strong immunoreactivity (IR) of IL6, IL8, PTGS2, and mPGES1 was recorded in the sCEH-P uterus, while expression of IL10 was noticed in AT-P. In circulation, serum IL6 was the most relevant marker with high sensitivity of 96.2% and specificity of 84.6% at a cut off concentration 8.5 pg/mL followed by SLPI with 95.2% sensitivity, and 84.6% specificity at cut off concentration of 1.3 ng/mL. Serum IL10, PGFM and SLPI concentration in the peripheral circulation were 1.5–2.23 fold higher in mCEH-P, 0.87–2.5 fold higher in sCEH-P and 2.9–3.5 fold higher in AT-P than that of control. It is concluded that monitoring the serum concentration of IL6, IL10 and SLPI would be useful adjunct to the established hematobiochemical parameters in the management of pyometra in the bitch with critical illness. • Interleukin 6, 8 and prostaglandin synthases expressed differentially in the endometrium of CEH and CEH-P bitch. • Measurement of IL6 and SLPI concentrations in serum serve as potential biomarker in assessing the severity of CEH-P. • Upregulation of IL10 in the endometrium and serum are associated with chronic inflammation in atrophic pyometra in bitch. [ABSTRACT FROM AUTHOR]