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.

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.

Oviduct epithelium induces interferon-tau in bovine Day-4 embryos, which generates an anti-inflammatory response in immune cells.

Recent studies indicate that communication between the bovine embryo and the mother begins in the oviduct. Here, we aimed to investigate the effect of embryos on bovine oviducts for their immune responses using an in vitro model. First, zygotes were cultured with or without bovine oviduct epithelial cells (BOECs) for 4 days, when embryos had reached the 16-cell stage. At that time, we detected interferon-tau (IFNT) in embryos co-cultured with BOECs, but not in embryos cultured alone. Next, peripheral blood mononuclear cells (PBMCs) were incubated either in media from embryo alone cultures or from co-cultures of embryos with BOECs. The medium from embryo alone cultures did not modulate PBMCs gene expression; whereas the embryo-BOEC co-culture medium increased interferon-stimulated genes (ISGs: ISG15, OAS1, MX2), STAT1, PTGES and TGFB1 but suppressed IL17 expression in PBMCs. Both IFNT-treated BOEC culture medium and IFNT-supplemented fresh medium alone without BOEC, modulated PBMCs gene expressions similar to those by the embryo-BOEC co-culture medium. Further, specific antibody to IFNT neutralized the effect of embryo-BOEC co-culture medium on PBMCs gene expression. Our results indicate that BOECs stimulate embryos to produce IFNT, which then acts on immune cells to promote an anti-inflammatory response in the oviduct.

Evidence that interferon-tau secreted from Day-7 embryo in vivo generates anti-inflammatory immune response in the bovine uterus.

Recent studies suggest that Day-7 bovine embryo starts to communicate with the uterine epithelium through interferon-tau (IFNT) signaling. However, immune modulatory role of IFNT in the uterus just after the embryo moves from the oviduct is unclear. We aimed to examine the hypothesis that Day-7 bovine embryo secretes IFNT in the uterus, which induces anti-inflammatory response in immune cells. The uterine flush (UF) with multiple embryos was collected from Day-7 donor pregnant cows and peripheral blood mononuclear cells (PBMCs) were then cultured in UF. Transcripts detected in PBMCs revealed that UF from pregnant cows down-regulated pro-inflammatory cytokines (TNFA, IL1B) and up-regulated anti-inflammatory cytokine (IL10) expression, with activation of interferon-stimulated genes (ISGs; ISG15, OAS1) as compared with UF from non-pregnant cows. An addition of specific anti-IFNT antibody to the UF inhibited the effect on PBMCs, indicating that IFNT is a major factor for such immune modulation. The observation that conditioned media from bovine uterine epithelial cells both stimulated with IFNT in vitro and supplemented with fresh IFNT induced similar PBMCs gene expression, confirming that IFNT directly acts on this immune crosstalk. This study shows that IFNT secreted from Day-7 embryo in vivo generates anti-inflammatory response in immune cells, which may provide immunological tolerance to accept the embryo.

A proinflammatory response of bovine endometrial epithelial cells to active sperm in vitro.

In the cow, cryopreserved semen is inseminated into the uterus, and most of sperm are removed by backflow and phagocytes. Nevertheless, the mechanism responsible for sperm phagocytosis is unclear. Here, we used cultured bovine uterine epithelial cells (BUECs) to investigate the uterine response to sperm and the mechanism that activates polymorphonuclear neutrophils (PMNs). BUEC monolayers were co-cultured with different numbers of washed sperm obtained from cryopreserved semen (104 , 105 , and 106 sperm/ml) for 3 hr. Sperm dose-dependently up-regulated IL8 (Interleukin 8). Sperm at 106 /ml increased mRNA expression of TNFA (Tumor necrosis factor alpha), IL1B (Interleukin 1B), NFKB2 (Nuclear factor kappa B2), and C3 (Complement factor 3), as well as PGES (Prostaglandin E synthase) expression and PGE2 release. Live sperm, but not dead sperm, attached to BUECs, and dead sperm did not induce an acute inflammatory response. Time-dependent effects were evaluated by co-culture of 106 /ml washed sperm with BUECs for 0, 1, 3, and 6 hr. The number of detached sperm increased gradually toward 6 hr. Maximum mRNA expression of IL8, TNFA, IL1B, and NFKB2 was induced at 3 hr, while C3 continued to increase toward 6 hr. Sperm did not stimulate mRNA expression of anti-inflammatory cytokines TGFB1 (Transforming growth factor beta 1) or IL10 (Interleukin 10). Medium conditioned by sperm co-incubated with BUECs stimulated PMNs phagocytosis of sperm in vitro. Fresh media supplemented with low levels of IL1B, TNFA, and PGE2 up-regulated sperm phagocytosis by PMNs as well. In conclusion, our findings strongly suggest that the active sperm attach to BUECs and trigger uterine local innate immunity with induction of a pro-inflammatory response that enhances sperm phagocytosis by PMNs.

Zearalenone (ZEN) disrupts the anti-inflammatory response of bovine oviductal epithelial cells to sperm in vitro.

Dietary contamination by Zearalenone (ZEN) has a detrimental effect on bovine fertility. Recently, we showed a novel anti-inflammatory response of bovine oviductal epithelial cells (BOEC) to active sperm cells in vitro. The aim of the present study was to investigate the effect of ZEN exposure of BOEC on the immune-related cytokine expression in response to bovine sperm. At concentrations of 100 and 1000ng/mL, ZEN induced the expression of TNF and IL1B (pro-inflammatory cytokines) as well as IL8 (chemokine) in BOEC in a dose-dependent manner. Furthermore, ZEN induced PTGES expression and PGE2 secretion in BOEC. Sperm co-culture induced an anti-inflammatory response in BOEC with upregulation of TGFB, secretion of PGE2 and downregulation of TNF. Most importantly, ZEN at 1-1000ng/mL eliminated the response of BOEC to sperm. Estradiol-17ß (5ng/mL) treatment did not produce the same effects as ZEN, suggesting that the response of BOEC to ZEN is, at least in part, not mediated by estrogen receptors. Taken together, ZEN can produce inflammatory effects on BOEC by stimulating the expressions of pro-inflammatory cytokines and disrupt the normal interaction between sperm and BOEC at the level of cytokine expressions and PGE2 production. Thus, exposure of the bovine oviduct to ZEN may negatively affect sperm survival and reduce fertility.

Bovine embryo induces an anti-inflammatory response in uterine epithelial cells and immune cells in vitro: possible involvement of interferon tau as an intermediator.

Recent observations suggest that the bovine uterus starts to react to the early embryo immediately after its arrival from the oviduct. The present study aimed to investigate the effect of the early developing embryo on the immune-related gene profile in bovine uterine epithelial cells (BUECs) in vitro, and to further examine the impact of conditioned media (CM), either from embryo-BUEC co-culture or embryo culture alone, on gene expression in peripheral blood mononuclear cells (PBMCs). First, BUECs were co-cultured with morulae (n = 10) for D5-D9 (D0 = IVF), and gene expression in BUECs was analyzed. Subsequently, PBMCs were cultured in CM from embryo-BUEC co-culture or D5-D9 embryo culture, and gene expression was evaluated. In BUECs, the embryo induced interferon (IFN)-stimulated genes (ISGs: ISG15, OAS1, and MX2), a key factor for IFN-signaling (STAT1), and type-1 IFN receptors (IFNAR1 and IFNAR2), with suppression of NFkB2, NFkBIA and pro-inflammatory cytokines (TNFA and IL1B). The embryo also stimulated PTGES and PGE2 secretion in BUECs. In PBMCs, both CM from embryo-BUEC co-culture and embryo culture alone induced ISGs, STAT1 and TGFB1, while suppressing TNFA and IL17. Similarly, interferon tau (IFNT) at 100 pg/ml suppressed NFkB2, TNFA and IL1B in BUECs, and also stimulated TGFB1 and suppressed TNFA in PBMCs. Our findings suggest that the bovine embryo, in the first four days in the uterus (D5-D9), starts to induce an anti-inflammatory response in epithelial cells and in immune cells. IFNT is likely to act as one of the intermediators for induction of the anti-inflammatory response in the bovine uterus.