In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum.

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

Sperm activate TLR2/TLR1 heterodimerization to induce a weak proinflammatory response in the bovine uterus.

Toll-like receptor 2 (TLR2) signaling pathway is involved in the sperm-triggered uterine inflammatory response at insemination, but its precise mechanism at molecular-level remains unknown. According to the ligand specificity, TLR2 forms a heterodimer with TLR1 or TLR6 as an initial step to mediate intracellular signaling, leading to a specific type of immune response. Hence, the present study aimed to identify the active TLR2 heterodimer (TLR2/1 or TLR2/6) that is involved in sperm-uterine immune crosstalk in bovine using various models. First, in-vitro (bovine endometrial epithelial cells, BEECs) and ex-vivo (bovine uterine explant) models were employed to test different TLR2 dimerization pathways in endometrial epithelia after exposure to sperm or TLR2 agonists; PAM3 (TLR2/1 agonist), and PAM2 (TLR2/6 agonist). Additionally, in-silico approaches were performed to confirm the dimer stability using de novo protein structure prediction model for bovine TLRs. The in-vitro approach revealed that sperm triggered the mRNA and protein expression of TLR1 and TLR2 but not TLR6 in BEECs. Moreover, this model disclosed that activation of TLR2/6 heterodimer, triggers a much stronger inflammatory response than TLR2/1 and sperm in bovine uterine epithelia. In the ex-vivo model that mimics the intact uterine tissue at insemination, sperm also induced the protein expression of both TLR1 and TLR2, but not TLR6, in bovine endometrium, particularly in uterine glands. Importantly, PAM3 and sperm induced similar and low mRNA expression of pro-inflammatory cytokines and TNFA protein to a lesser extent than PAM2 in endometrial epithelia. This implied that sperm might trigger a weak inflammatory response via TLR2/TLR1 activation which is similar to that of PAM3. Additionally, the in-silico analyses showed that the existence of bridging ligands is essential for heterodimer stability in bovine TLR2 with either TLR1 or TLR6. Altogether, the present findings revealed that sperm utilize TLR2/1, but not TLR2/6, heterodimerization to trigger a weak physiological inflammatory response in the bovine uterus. This might be the way to remove excess dead sperm remaining in the uterine lumen without tissue damage for providing an ideal uterine environment for early embryo reception and implantation.

Hyaluronan regulates sperm-induced inflammatory response by enhancing sperm attachment to bovine endometrial epithelial cells via CD44: in-silico and in-vitro approaches.

Recently, we reported that sperm induce cluster of differentiation 44 (CD44) expression and Toll-like receptor 2 (TLR2)-mediated inflammatory response in bovine uterus. In the present study, we hypothesized that the interaction between CD44 of bovine endometrial epithelial cells (BEECs) and hyaluronan (HA) affects sperm attachment and thereby enhancing TLR2-mediated inflammation. To test our hypothesis, at first, in-silico approaches were employed to define the binding affinity of HA for CD44 and TLR2. Further, an in-vitro experiment using the sperm-BEECs co-culture model was applied to investigate the effect of HA on sperm attachment and inflammatory response. Here, low molecular weight (LMW) HA at different concentrations (0, 0.1, 1, or 10 µg/mL) was incubated with BEECs for 2 h followed by the co-culture without- or with non-capacitated washed sperm (106/ml) for additional 3 h was performed. The present in-silico model clarified that CD44 is a high-affinity receptor for HA. Moreover, TLR2 interactions with HA oligomer (4- and 8-mers) target a different subdomain (h-bonds) compared to TLR2-agonist (PAM3) which targets a central hydrophobic pocket. However, the interaction of LMW HA (32-mers) with TLR2 revealed no stability of HA at any pocket of TLR2. Notably, the immunofluorescence analysis revealed the HA localization in both endometrial stroma and epithelia of ex-vivo endometrial explant. Moreover, ELISA showed significant levels of HA in BEECs culture media. Importantly, BEECs pretreatment with HA prior to sperm exposure increased the number of attached sperm to BEECs, and upregulated the transcriptional levels of pro-inflammatory genes (TNFA, IL-1B, IL-8, and PGES) in BEECs in response to sperm. However, BEECs treated with HA only (no sperm exposure) did not show any significant effect on the transcript abundance of pro-inflammatory genes when compared to the non-treated BEECs. Altogether, our findings strongly suggest a possible cross-talk between sperm and endometrial epithelial cells via HA and HA binding receptors (CD44 and TLR2) to induce a pro-inflammatory response in bovine uterus.

Intrauterine infusion of low levels of interferon-tau on day-8 post-estrus stimulates the bovine endometrium to secrete apolipoprotein-A1: A possible implication for early embryo tolerance.

We previously reported that interferon-tau (IFNT), derived from day-7 blastocyst, generates anti-inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. However, the real in vivo impact of early embryo-derived IFNT on the uterine proteomic profile is mostly unknown. This study aimed to investigate proteomic changes of uterine flush (UF) when infused with a low physiological level of IFNT without embryo on day-8 post-estrus and its possible impact on the uterine immunological microenvironment. First, a fresh medium was infused into the uterine lumen on day-6, from which UF was obtained 24 h later, and this procedure was repeated on day-7 (control UF). On day-8, this procedure was done with a medium containing recombinant bovine IFNT (100 pg/ml) (IFNT-supplemented UF). Control and IFNT-supplemented UF were tested for immune responses in peripheral blood mononuclear cells (PBMCs). Real-time PCR results revealed that IFNT-supplemented UF downregulated pro-inflammatory cytokines (TNFA, IL1B) and upregulated anti-inflammatory cytokine (TGFB1) and PTGES in PBMCs. Through 2-D PAGE, followed by TOF/TOF mass spectrometer, apolipoprotein-A1 (Apo-A1) protein was identified in the IFNT-supplemented UF, which was confirmed by ELISA analysis. Proteomic analysis revealed again that the in vitro stimulation of BEECs by IFNT upregulated Apo-A1 expression. Further, stimulation of PBMCs with recombinant bovine Apo-A1 downregulated TNFA and NFKB and upregulated TGFB1 and PTGES in PBMCs. Altogether, our results suggest that minute amounts of IFNT alone, normally secreted from bovine blastocyst, stimulate Apo-A1 secretion from the endometrial epithelium in the absence of embryo that initiates an anti-inflammatory environment, which could pave the way for the acceptance of early embryo in the uterus.

Progesterone and Inflammatory Response in the Oviduct during Physiological and Pathological Conditions.

Progesterone has been shown to be a potent suppressor of several inflammatory pathways. During pregnancy, progesterone levels increase, allowing for normal pregnancy establishment and maintenance. The dysregulation of progesterone, as well as inflammation, leads to poor pregnancy outcomes. However, it is unclear how progesterone imbalance could impact inflammatory responses in the oviduct and subsequently result in early pregnancy loss. Therefore, in this review, we describe the role of progesterone signaling in regulating the inflammatory response, with a focus on the oviduct and pathological conditions in the Fallopian tubes.

The impact of calcitriol and estradiol on the SARS-CoV-2 biological activity: a molecular modeling approach.

The novel coronavirus disease (COVID-19) is currently a big concern around the world. Recent reports show that the disease severity and mortality of COVID-19 infected patients may vary from gender to gender with a very high risk of death for seniors. In addition, some steroid structures have been reported to affect coronavirus, SARS-CoV-2, function and activity. The entry of SARS-CoV-2 into host cells depends on the binding of coronavirus spike protein to angiotensin converting enzyme-2 (ACE2). Viral main protease is essential for the replication of SARS-CoV-2. It was hypothesized that steroid molecules (e.g., estradiol, progesterone, testosterone, dexamethasone, hydrocortisone, prednisone and calcitriol) could occupy the active site of the protease and could alter the interaction of spike protein with ACE2. Computational data showed that estradiol interacted more strongly with the main protease active site. In the presence of calcitriol, the binding energy of the spike protein to ACE2 was increased, and transferring Apo to Locked S conformer of spike trimer was facilitated. Together, the interaction between spike protein and ACE2 can be disrupted by calcitriol. Potential use of estradiol and calcitriol to reduce virus invasion and replication needs clinical investigation.

Zearalenone interferes with the sperm-triggered inflammation in the bovine uterus in vitro: Negative impact on sperm motility and survival.

Zearalenone (ZEN)-contaminated diets induce detrimental effects on the bovine reproduction. Recently, we reported that active sperm induce pro-inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. This study aimed to investigate the impact of presence of ZEN on the sperm-uterine crosstalk in vitro. BEECs monolayers were stimulated by ZEN (10, 100, and 1000 ng/mL) for 0, 3, 6, 12, or 24 h and gene expressions were analyzed by real-time PCR. Moreover, BEECs were pre-exposed to ZEN (10, 100, and 1000 ng/mL) for 24 h then, co-incubated with sperm for 6 h. Conditioned media (CM) from a sperm-BEECs co-culture, after pre-exposure to ZEN, were harvested and exploited to challenge either polymorphonuclear cells (PMNs) or sperm. Both PMNs phagocytic activity toward sperm and sperm motility parameters were then assessed. Results showed that ZEN alone induced pro-inflammatory responses in BEECs through the induction of mRNA expressions of pro-inflammatory cytokines (TNFA and IL1B) and PGES1 at different time points. Pre-exposure of BEECs to ZEN, amplified the sperm-triggered upregulation of pro-inflammatory cytokines (TNFA and IL1B) and chemokine IL8 mRNA abundance in BEECs. Sperm-BEECs conditioned media, primed by ZEN, stimulated the PMNs phagocytosis for sperm whereas suppressed sperm motility parameters. Taken together, these findings indicate that the presence of ZEN augments the pro-inflammatory cascade triggered by sperm in BEECs, provokes PMNs phagocytosis for sperm, and reduces sperm motility parameters. Such immunological reactions may create a hostile environment for sperm competence and survival in the bovine uterus, thus impair fertility.

The growth hormone and insulin-like growth factor 1 axis in cattle during the peri-ovulatory period activates the synthesis and release of oviductal contraction related substances.

Growth hormone (GH) and insulin-like growth factor 1 (IGF1) are crucial for female reproductive functions. The cyclic regulation of the local GH/IGF1 axis in the oviduct and its involvement in oviductal contraction in cattle has not been investigated. Thus, the messenger RNA (mRNA) expression for GH receptor (GHR), IGF1, IGF1 receptor (IGF1R) in the whole oviducts, as well as in cultured bovine oviductal epithelial cells (BOECs) were evaluated. The GHR, IGF1, and IGF1R mRNA expression was significantly higher during postovulatory phase. The luteinizing hormone (LH), estradiol-17ß (E2), and LH + E2 treatments significantly increased GHR and IGF1 mRNA expression in cultured BOECs. Further, GH and combination of GH with LH and E2 upregulated IGF1 mRNA expression in the BOECs. Moreover, IGF1 + LH and combined IGF1 + LH + E2 treatments significantly increased prostaglandin synthesis cascade enzyme mRNA expression in the BOECs. An ex vivo microdialysis assay revealed that GH and IGF1 induced the release of oviductal contraction related prostaglandins, endothelin-1, and angiotensin II in follicular and postovulatory phases. Together, the findings strongly suggest that the presence of the active GH/IGF1 axis during the peri-ovulatory period, regulating the local system for the release of oviductal contraction related substances, which may provide the optimal oviductal environment for gametes and early embryo.

Sperm interaction with the uterine innate immune system: toll-like receptor 2 (TLR2) is a main sensor in cattle.

During the passage through the female reproductive tract, sperm interact with various compartments and their immune systems. The immune system that protects the female against pathogens also could destroy sperm or prevent them from reaching the site of fertilisation. In particular, the uterine innate immune response is crucial from the perspectives of both the sperm and the uterus. Following insemination, sperm immediately start to trigger inflammation in the uterus by entering uterine glands and activating an innate immune response. In cattle, the activation occurs mainly via TLR2 signalling, if not the only one, between sperm and the uterine epithelium lining the glands. This acute immune response is manifested as the upregulation of mRNA expression of IL8, TNFA, IL1B , and PGES . As a consequence, many sperm are trapped by polymorphonuclear neutrophils, the first and major component of innate immunity. The sperm-induced uterine innate immune responses apparently serve to clear the uterus of excess sperm and, importantly, prepare the endometrium for implantation. Pathophysiological conditions in the uterus seriously disrupt this phenomenon, and thus could directly decrease fertility.

A multilevel analysis identifies the different relationships between amino acids and the competence of oocytes matured individually or in groups.

High-protein diets contribute to an increase in urea follicular concentrations associated with decreased fertility. Urea has been shown to interfere with the epidermal growth factor (EGF)/EGFR system, which has been shown to have a beneficial effect during in vitro maturation (IVM) of oocytes. Of note, the number of cumulus-oocyte complexes (COCs) in the maturation medium can change the maturation and the developmental competence of COCs. Therefore, it was hypothesized that, the presence of urea and EGF may have a differential effect on the depletion/appearance of AAs and competence of COCs matured individually (I-IVM system) or in groups (G-IVM system). In the G-IVM system, COCs increased consumption (depletion) of AAs compared with other groups in the presence of high-level urea (40 mg/dl) + EGF (10 ng/ml). In the I-IVM system, the non-cleaved COCs depleted more AAs than the cleaved COCs, in particular in the presence of urea. The combination of urea and EGF increased the depletion of AAs in the G-IVM system. However, the EGF abrogated the urea-induced depletion of AAs by the I-IVM COCs. The use of N-acetyl-L-cysteine as an EGFR inhibitor canceled urea-induced depletion of AAs. This shows the inhibiting effect of urea over the EGF/EGFR system. In the presence of urea + EGF, COCs had a lower degree of developmental competence than control in both I- and G-IVM systems. Arginine had the best predictive power to identify highly competent COCs in the G-IVM system, while glutamine was the best predictor of the cleavage in the I-IVM system. In conclusion, this multi-level study shows that COCs matured individually or in groups may have different association with AAs metabolism. These findings provide new insights into the relationships between AA metabolism and the subsequent developmental competence of COCs.

Toll-like receptor 2 mediates the immune response of the bovine oviductal ampulla to sperm binding.

We previously reported that sperm binding to cultured bovine oviduct epithelial cells induces an anti-inflammatory immune response. Now we have developed a differentiated explant model to focus on the oviductal ampulla, where fertilization occurs, and to study the effect of sperm capacitation on the immune response. We used heparin to stimulate bovine sperm capacitation. Fluorescence imaging showed that 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide-labeled sperm pretreated with (Hep(+) ) or without (Hep(-) ) heparin rapidly attached to the explant ciliated epithelium in similar numbers. However, only Hep(+) sperm upregulated explant messenger RNA (mRNA) transcription of TLR2, IL8, TGFB1, and PGES, without changes in TNFA and IL-10 expression, while Hep(-) sperm only upregulated PGES. The responses were primarily anti-inflammatory, with a greater response produced by Hep(+) sperm, which also produced a substantial increase in TLR2 protein expression in the epithelium. The addition of TLR1/2 (toll-like receptor 1/2) antagonist to the Hep(+) and (Hep(-) ) sperm-explant coincubations reduced sperm attachment to the epithelium and inhibited TLR2 protein expression and some of the Hep(+) sperm-induced mRNA transcription. Our observations suggest that the ampullar epithelium immunologically reacts more strongly to sperm that have undergone heparin stimulation of capacitation. This anti-inflammatory response could serve to protect capacitated sperm as they approach the oocyte in the ampulla.

Peptidoglycan disrupts early embryo-maternal crosstalk via suppression of ISGs expression induced by interferon-tau in the bovine endometrium.

Uterine infection with bacteria and the release of peptidoglycan (PGN), antigenic cell wall components of both Gram-negative and Gram-positive bacteria, can cause early pregnancy losses in ruminants, but the associated mechanisms remain unsolved. Day 7 blastocyst starts to secrete a minute amount of interferon-tau (IFNT) in the uterine horn which is required for early stage of maternal recognition of pregnancy (MRP) in ruminants, and it induces interferon stimulated genes (ISGs) for driving uterine receptivity in cows. This study investigated if PGN disrupts IFNT response through modulation of endometrial ISGs expressions. Cultured bovine endometrial epithelial cells (BEECs) were treated with embryo culture medium (ECM) or IFNT (1 ng/ml) in the presence or absence of a low level of PGN (10 pg/ml) for 24 h. A real-time PCR analyses revealed that the presence of PGN suppressed IFNT-induced ISGs (OAS1 and ISG15) and STAT1 expressions in BEECs. To visualize the impact of PGN in an ex-vivo model that resembles the in vivo status, endometrial explants were treated by IFNT (1 ng/ml) with or without PGN (10 pg/ml) for 12 h. PGN suppressed IFNT-induced gene expressions of the above factors, but not for IFNA receptor type1 (IFNAR1) or type2 (IFNAR2) in explants. Immunofluorescence analysis illustrated that PGN completely suppressed the IFNT-triggered OAS1 protein expression in the luminal epithelium of explants. Of note, PGN did not stimulate pro-inflammatory cytokines (TNFA and IL1B) or TLR2 mRNA expression in both models. These findings indicate that the presence of low levels of PGN suppresses ISGs expression induced by IFNT secreted from early embryo, at the luminal epithelium of the bovine endometrium. This could severely interfere with early stage of MRP processes in cows, leading to pregnancy failure.

FOXL2 is a Progesterone Target Gene in the Endometrium of Ruminants.

Forkhead Box L2 (FOXL2) is a member of the FOXL class of transcription factors, which are essential for ovarian differentiation and function. In the endometrium, FOXL2 is also thought to be important in cattle; however, it is not clear how its expression is regulated. The maternal recognition of pregnancy signal in cattle, interferon-Tau, does not regulate FOXL2 expression. Therefore, in the present study, we examined whether the ovarian steroid hormones that orchestrate implantation regulate FOXL2 gene expression in ruminants. In sheep, we confirmed that FOXL2 mRNA and protein was expressed in the endometrium across the oestrous cycle (day 4 to day 15 post-oestrus). Similar to the bovine endometrium, ovine FOXL2 endometrial expression was low during the luteal phase of the oestrous cycle (4 to 12 days post-oestrus) and at implantation (15 days post-oestrus) while mRNA and protein expression significantly increased during the luteolytic phase (day 15 post-oestrus in cycle). In pregnant ewes, inhibition of progesterone production by trilostane during the day 5 to 16 period prevented the rise in progesterone concentrations and led to a significant increase of FOXL2 expression in caruncles compared with the control group (1.4-fold, p < 0.05). Ovariectomized ewes or cows that were supplemented with exogenous progesterone for 12 days or 6 days, respectively, had lower endometrial FOXL2 expression compared with control ovariectomized females (sheep, mRNA, 1.8-fold; protein, 2.4-fold; cattle; mRNA, 2.2-fold; p < 0.05). Exogenous oestradiol treatments for 12 days in sheep or 2 days in cattle did not affect FOXL2 endometrial expression compared with control ovariectomized females, except at the protein level in both endometrial areas in the sheep. Moreover, treating bovine endometrial explants with exogenous progesterone for 48h reduced FOXL2 expression. Using in vitro assays with COS7 cells we also demonstrated that progesterone regulates the FOXL2 promoter activity through the progesterone receptor. Collectively, our findings imply that endometrial FOXL2 is, as a direct target of progesterone, involved in early pregnancy and implantation.

Roadmap to pregnancy in the first 7 days post-insemination in the cow: Immune crosstalk in the corpus luteum, oviduct, and uterus.

The first 7 days post-insemination are critical for establishment of pregnancy. The pre-ovulatory luteinizing hormone (LH) surge induces ovulation through disruption of the follicle structure that elucidates pro-inflammatory (Th1) responses. Various types of immune cells are recruited into the corpus luteum (CL) to regulate luteal angiogenesis and progesterone (P4) secretion into the circulation to establish pregnancy. The active sperm-uterine crosstalk also induces Th1 responses, mainly via Toll-like receptor (TLR) 2/4 signaling pathway in vitro. The endometrial glands serve as sensors for sperm signals, which trigger Th1 responses. Conversely, the sperm-oviduct binding generates anti-inflammatory (Th2) responses to support sperm survival until fertilization. It is well-established that embryo-maternal crosstalk starts after the embryo hatches out from the zona pellucida (ZP). However most recently, it was shown that the 16-cell stage bovine embryo starts to secrete interferon-tau (IFNT) that induces Th2 immune responses in the oviduct. Once developing embryos descend into the uterine horn, they induce Th2 responses with interferon-stimulated genes (ISGs) expression in the uterine epithelium and local immune cells mainly via IFNT release. Likewise, multiple embryos in the uterus of superovulated donor cows on D7 post-insemination induce Th2 immune responses with ISGs expressions in circulating immune cells. These findings strongly suggest that the maternal immune system reacts to the embryo during the first 7 days post-insemination to induce fetal tolerance. It became evident that the innate immunity of the developing CL, oviduct, and uterus works together to provide optimal conditions for fertilization and early embryonic development during the first 7 days post-insemination.

Bovine oviduct epithelial cells suppress the phagocytic activity of neutrophils towards sperm but not for bacteria in vitro: Immunofluorescence and electron microscopic observations.

Previously, we reported that polymorphonuclear neutrophils (PMNs) are constantly existent in the bovine oviduct fluid during the pre-ovulatory stage under physiological conditions. Moreover, incubation of PMNs with bovine oviduct epithelial cells-conditioned medium (BOEC-CM) resulted in suppression of their phagocytic activity for sperm. During pathophysiological conditions, cows may be inseminated by infected semen which exposes oviductal PMNs to allogenic sperm simultaneously with pathogens. This study aimed to visually investigate the role of oviduct epithelium in regulating the phagocytic behavior of PMNs toward sperm as a physiological stimulus, with Escherichia coli (E. coli) as a pathological stimulus. In our experiment, PMNs were incubated for 2 h in BOEC-CM. Phagocytosis was then assayed by co-incubation of these PMNs either with sperm, E. coli, or latex beads. BOEC-CM significantly suppressed the direct phagocytosis of PMNs for sperm, but did not affect their phagocytic activity for E. coli or latex beads. Additionally, an investigation with scanning electron microscopy revealed that BOEC-CM suppressed the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. BOEC-CM did not alter NETs formation towards E. coli. A quantification of NETs formation using an immunofluorescence microscopy showed that the areas of NETs formation for E. coli were significantly larger than those formed for sperm. Our data clearly show that the bovine oviduct, through secretions, protects sperm from phagocytosis by PMNs and eliminates bacterial dissemination through maintaining the phagocytic activity of PMNs towards bacteria.

Peptidoglycan Switches Off the TLR2-Mediated Sperm Recognition and Triggers Sperm Localization in the Bovine Endometrium.

In mammals, the uterine mucosal immune system simultaneously recognizes and reacts to most bacteria as well as allogenic sperm mainly through the Toll-like receptors (TLR)2/4 signaling pathway. Here, we characterized the impact of pathogen-derived TLR2/4 ligands (peptidoglycan (PGN)/lipopolysaccharide (LPS)) on the immune crosstalk of sperm with the bovine endometrial epithelium. The real-time PCR analysis showed that the presence of low levels of PGN, but not LPS, blocked the sperm-induced inflammatory responses in bovine endometrial epithelial cells (BEECs) in vitro. Immunoblotting analysis revealed that PGN prevented the sperm-induced phosphorylation of JNK in BEECs. Activation or blockade of the TLR2 system in the endometrial epithelium verified that TLR2 signaling acts as a commonly-shared pathway for PGN and sperm recognition. The impairment of endometrial sperm recognition, induced by PGN, subsequently inhibited sperm phagocytosis by polymorphonuclear neutrophils (PMNs). Moreover, using an ex vivo endometrial explant that more closely resembles those in vivo conditions, showed that sperm provoked a mild and reversible endometrial tissue injury and triggered PMN recruitment into uterine glands, while PGN inhibited these events. Of note, PGN markedly increased the sperm attachment to uterine glands, and relatively so in the surface epithelium. However, addition of the anti-CD44 antibody into a PGN-sperm-explant co-culture completely blocked sperm attachment into glands and surface epithelia, indicating that the CD44 adhesion molecule is involved in the PGN-triggered sperm attachment to the endometrial epithelium. Together, these findings demonstrate that, the presence of PGN residues disrupts sperm immune recognition and prevents the physiological inflammation induced by sperm in the endometrial epithelium via the MyD88-dependent pathway of TLR2 signaling, possibly leading to impairment of uterine clearance and subsequent embryo receptivity.

Ovulatory follicular fluid induces sperm phagocytosis by neutrophils, but oviductal fluid around oestrus suppresses its inflammatory effect in the buffalo oviduct in vitro.

We have recently shown that the conditioned media from bovine oviductal epithelial cell culture suppress sperm phagocytosis by neutrophils, suggesting that the oviduct around oestrus supplies the anti-inflammatory microenvironment. To investigate the immune response of neutrophils toward the sperm at ovulation in the buffalo oviduct, we examined (a) a detailed distribution of neutrophils in the oviduct in buffaloes, (b) the effect of ovulatory follicular fluid (FF) and oviductal fluid (OF) on sperm phagocytosis by neutrophils, and (c) the interaction of the ovulatory FF with OF on sperm phagocytosis by neutrophils in vitro. Buffalo oviducts were collected from healthy reproductive tracts at a local slaughterhouse. A detailed observation by histological examination and transmission electron microscopy revealed that neutrophils exist in the oviduct epithelium and lumen throughout the oestrous cycle in buffaloes. The number of neutrophils at the oestrus stage was higher in ampulla compared with those in isthmus, whereas they remained relatively constant at the dioestrus stage. Two hours of preincubation of neutrophils with FF enhanced sperm phagocytosis through the formation of neutrophil extracellular traps (NETs) together with H2 O2 production, whereas OF around oestrus (eOF) suppressed sperm phagocytosis, NETs formation, and H2 O2 production and relieved the above FF-induced inflammatory response. Our findings show that neutrophils exist in the healthy cyclic oviduct across bovine species, and the OF supplies a strong anti-inflammatory environment that could minimize the inflammatory effect of the FF that flows into the oviduct lumen after ovulation and supports the occurrence of fertilization.

TLR2/4 signaling pathway mediates sperm-induced inflammation in bovine endometrial epithelial cells in vitro.

We have recently shown that sperm attachment to bovine endometrial epithelial cells (BEECs) triggers uterine local innate immunity with induction of a pro-inflammatory response in vitro, however details of the mechanism remain unknown. Here, we investigated the involvement of Toll-like receptor 2/4 (TLR2/4) pathway in mediating sperm-BEECs inflammatory process. Immunohistochemistry of the uterine tissue revealed that TLR2 and TLR4 proteins were present in the luminal and glandular epithelia of bovine endometrium. Moreover, BEECs monolayers were treated with TLR2 agonist (Pam; 0, 10, 100, and 1000 ng/ml) or TLR4 agonist (LPS; 0, 0.1, 1, and 10 ng/ml) for 0, 1, 3, or 6 h, followed by evaluating mRNA expression of the pro-inflammatory genes (TNFA, IL-1B, IL-8, and PGES) in BEECs using a real-time PCR. Both Pam and LPS treatments showed a dose-dependent stimulation of mRNA expression of the pro-inflammatory genes. To elucidate the functional role of TLR2/4 in sperm-BEECs interaction, BEECs monolayers were incubated with either TLR2 antagonist or TLR4 antibody for 2 h prior to the co-culture with sperm for 3 h. Importantly, pre-incubation of BEECs with TLR2 antagonist or TLR4 antibody prevented the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BEECs. Furthermore, sperm increased the phosphorylation levels of TLR2/4 downstream targets (p38MAPK and JNK) in BEECs within 1 h of the co-culture. Treatment of BEECs with TLR2 antagonist prior to sperm addition inhibited JNK phosphorylation, while TLR4 antibody inhibited the phosphorylation of both p38MAPK and JNK. In conclusion, the present in vitro findings strongly suggest that bovine endometrial epithelial cells respond to sperm via TLR2/4 signal transduction.

Understanding the hidden relations between pro- and anti-inflammatory cytokine genes in bovine oviduct epithelium using a multilayer response surface method.

An understanding gene-gene interaction helps users to design the next experiments efficiently and (if applicable) to make a better decision of drugs application based on the different biological conditions of the patients. This study aimed to identify changes in the hidden relationships between pro- and anti-inflammatory cytokine genes in the bovine oviduct epithelial cells (BOECs) under various experimental conditions using a multilayer response surface method. It was noted that under physiological conditions (BOECs with sperm or sex hormones, such as ovarian sex steroids and LH), the mRNA expressions of IL10, IL1B, TNFA, TLR4, and TNFA were associated with IL1B, TNFA, TLR4, IL4, and IL10, respectively. Under pathophysiological + physiological conditions (BOECs with lipopolysaccharide + hormones, alpha-1-acid glycoprotein + hormones, zearalenone + hormones, or urea + hormones), the relationship among genes was changed. For example, the expression of IL10 and TNFA was associated with (IL1B, TNFA, or IL4) and TLR4 expression, respectively. Furthermore, under physiological conditions, the co-expression of IL10 + TNFA, TLR4 + IL4, TNFA + IL4, TNFA + IL4, or IL10 + IL1B and under pathophysiological + physiological conditions, the co-expression of IL10 + IL4, IL4 + IL10, TNFA + IL10, TNFA + TLR4, or IL10 + IL1B were associated with IL1B, TNFA, TLR4, IL10, or IL4 expression, respectively. Collectively, the relationships between pro- and anti-inflammatory cytokine genes can be changed with respect to the presence/absence of toxins, sex hormones, sperm, and co-expression of other gene pairs in BOECs, suggesting that considerable cautions are needed in interpreting the results obtained from such narrowly focused in vitro studies.

Up-regulated mRNA expression of some anti-inflammatory mediators in bovine oviduct epithelial cells by urea in vitro: Cellular pathways by Reactome analysis.

Increased urea concentration is a major cause of low fertility in dairy cows fed high-protein diets. A strong correlation exists between the urea concentration in the blood and oviduct fluid of dairy cows. In this study, bovine oviduct epithelial cells (BOECs) were incubated with varying concentrations of urea (0, 20, 40, and 80 mg/dL) in the absence of ovarian sex steroids (estradiol and progesterone) and luteinizing hormone. The 80 mg/dL urea reduced the cell viability, and thus was excluded in further analysis. Compared to the control (U0), the 20 mg/dL urea (U20) increased the mRNA expression of Toll-like receptor (TLR) 4, interleukin (IL) 10, IL4, and prostaglandin (PG) E synthase (mPGES) but decreased the mRNA expression of tumor necrosis factor α (TNFA). Compared to U0, the 40 mg/dL urea (U40) decreased the mRNA expression of TNFA and increased alpha-1-acid glycoprotein (AGP). U40 also increased TLR2, IL10, and IL4 mRNA expression compared to U0. In addition, compared to U20, the U40 decreased the mRNA expression of TLR4 and IL1B but increased that of AGP and TLR2. Subsequently, the mRNA expression data were then projected into the Reactome database. The Reactome analysis showed that pathways, including cytokine signaling in the immune system (i.e., TNFs bind their physiological receptors) and death receptor signaling (i.e., TNF signaling), were down-regulated in the presence of urea compared to the U0 group. These in vitro data implied that high urea level can alter the balance between pro- and anti-inflammatory responses in BOECs, thus providing a suboptimal environment for the early reproductive events or a weakened innate immune system, predisposing the oviduct to infections.