Sperm induce proinflammatory responses in the uterus and peripheral blood immune cells of artificially inseminated cows.

This in vivo study aimed to investigate local and systemic immune responses induced by sperm in cows after artificial insemination (AI). Initially, 12 multiparous Japanese Black cows were subjected to intrauterine AI (AI group, n = 6) or saline infusion (control group, n = 6). The uterine body and horn ipsilateral to the ovulatory follicle were mini-flushed with 2 ml of RPMI-1640 medium at different time points (0, 1, 6, 10, 24, 48 h, and 7 days after AI), centrifuged, and the sediments were examined under a light microscope. Vaginal smears were prepared at 0, 1, 6, and 10 h after AI to investigate the sperm backflow. Subsequently, another experiment was conducted by assigning cows to three groups: intrauterine AI (AI group, n = 5), heat-inactivated AI (Heat-AI group, n = 5), or saline infusion (control group, n = 5). Blood samples were collected, and polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were separated and analyzed for gene expression using real-time PCR. The results showed that most sperm were rapidly transported either forward into the uterine horn or backward into the vagina within 1 h after AI. The PMNs migrated into the uterine lumen 6 hours after AI. Only active sperm-induced proinflammatory responses in PMNs and PBMCs via upregulation of TNFa, IL8, IL1B, and PGES and downregulation of IL10 at 6 h after AI. These data provide evidence that sperm generate transient proinflammatory responses locally in the uterus and systemically in the peripheral immune cells, which may be prerequisites for uterine clearance, embryo receptivity, and implantation in cows.

Energy balance and hippo effector activity in endometrium and corpus luteum of early pregnant ewes.

CONTEXT: The establishment of pregnancy in cows requires uterine activity regulation of the main Hippo signalling effector yes-associated protein 1 (YAP). It remains unknown (1) how YAP activity at the corpus luteum (CL) correlates with early pregnancy-related events in ruminants; and (2) if YAP activity in the uterus and CL can be affected by metabolic disorders that may lead to pregnancy failure in ruminants. AIMS AND METHODS: To determine the effect of early pregnancy on total and phospho-YAP expression and its transcriptional activity in the CL, we compared non-pregnant vs pregnant ewes. To understand the YAP activity dysregulation with disorders that may result in pregnancy loss, we induced negative energy balance in pregnant ewes. KEY RESULTS AND CONCLUSIONS: Our main results indicate that early pregnancy alters the expression and activity patterns of YAP in the ovine CL but not in the endometrium. In addition, while our NEB-induced model fails to alter YAP activity at the endometrium level, we found that fasting during the first but not second week of pregnancy affects YAP activity in the CL of pregnant ewes. IMPLICATIONS: The data presented herein provide considerable insight into the activity of a signalling pathway that may be a key player in pregnancy recognition and establishment in ewes.

Increased ß-hydroxybutyrate (BHBA) concentration affect follicular growth in cattle.

Metabolic stress conditions caused by negative energy balance (NEB) have been associated with reduced fertility in cows. ß-hydroxybutyrate (BHBA) is the main circulating ketone body, which accumulates within follicular fluid. The aim of this study was to evaluate the effects of BHBA on follicle growth and on ovulatory mechanisms in cattle. At 72 h after intrafollicular injection, there was a decrease in follicular diameter in BHBA group compared to control (P = 0.02). Furthermore, follicle growth rate was reduced post-treatment with BHBA in comparison to the control group (P < 0.03). The BHBA intrafollicular injection in follicles ≥ 12 mm, however, did not affect E2 and P4 concentrations in the follicular fluid. In addition, the relative abundance of genes involved in the ovulatory cascade (ADAM 17, AREG, EREG, PTGS2), steroidogenesis (CYP19A1, 3BHSD, STAR), cellular stress (SOD1, CAT, GPX1, HSPA5, XBP1s, XBP1u, ATF4, ATF6), monocarboxylic acid transporters (SLC16A1, SLC16A7) and apoptosis (XIAP) was similar between groups. In conclusion, the results of this study indicate that the increase in intrafollicular concentrations of BHBA affects follicular growth, but it does not compromise the ovulatory cascade and cellular homeostasis in bovine granulosa cells.

Possible impact of neutrophils on immune responses during early pregnancy in ruminants.

The interaction between early embryo and maternal immune system for the establishment of pregnancy is the focus of several studies; however, it remains unclear. The maternal immune response needs to keep a balance between avoiding any damage to the conceptus and maintaining its function in combating microbes as well. When conceptus-maternal crosstalk cannot achieve this balance, pregnancy losses might occur. Intercommunication between mother and conceptus is fundamental during early pregnancy to dictate the outcome of pregnancy. In ruminants, the embryo reacts with the maternal system mainly via interferon tau (IFNT) release. IFNT can act locally on the embryo and endometrial cells and systemically in several tissues and cells to regulate their response via the expression of interferon-stimulated genes (ISGs). Also, IFNT can induce the expression of inflammatory-related genes in immune cells. Day 7 embryo induces a shift in the maternal immune response towards anti-inflammatory (Th2) immune responses. During maternal recognition of pregnancy, peripheral mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) express markers that configure an anti-inflammatory response. However, PMNs response is more sensitive to the effects of IFNT. PMNs are more likely to express interferon-stimulated genes (ISGs), transforming growth factor-beta (TGFB), interleukin 10 (IL10), and arginase-1 (ARG1), configuring one of the most rapid immune responses to early pregnancy. This review focus on the local and peripheral immune responses during early pregnancy in ruminants, mainly the PMNs function in the immune system.

Heat stress modulates polymorphonuclear cell response in early pregnancy cows: I. interferon pathway and oxidative stress.

One of the major causes of early pregnancy loss is heat stress. In ruminants, interferon tau (IFNT) is the embryo signal to the mother. Once the interferon signaling pathway is activated, it drives gene expression for interferon-stimulated genes (ISGs) and alters neutrophils responses. The aim of the present study was to evaluate interferon (IFN) pathway, ISGs and gene expression in polymorphonuclear leukocytes (PMN) and oxidative stress in dairy cows under heat stress. Pregnant cows had their estrous cycle synchronized and randomly assigned to a comfort or heat stress group. Blood samples were collected at artificial insemination (AI) and on Days 10, 14 and 18 following AI. Pregnant cows were pregnancy checked by ultrasound on Day 30 and confirmed on Day 60 post-AI. Results are presented as mean ± SEM. The corpus luteum (CL) diameter was not different between groups of pregnant cows; concentration of progesterone of pregnant cows on Day 18 following AI was greater in comfort group compared to heat stressed group. Comfort pregnant cows had higher expression of all analyzed genes from interferon pathway, except for IFNAR1, on both Days 14 and 18. Conversely, heat stressed cows did not show altered expression of IFNT pathway genes and ISGs between Days 10, 14, and 18 after AI. The oxidative stress, determined as malondialdehyde (MDA) levels, was greater in heat stress group on Days 10, 14 and 18, independent of pregnancy status. Heat stress negatively influences expression of ISGs, IFN pathway gene expression in neutrophils, and oxidative stress. Our data suggest that lower conception rates in cows under heat stress are multifactorial, with the association of interferon pathway activation and the unbalanced oxidative stress being main contributing factors.

Neutrophils recognize and amplify IFNT signals derived from day 7 bovine embryo for stimulation of ISGs expression in vitro: A possible implication for the early maternal recognition of pregnancy.

Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs' immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs' immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle.

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.

Heat stress on oocyte or zygote compromises embryo development, impairs interferon tau production and increases reactive oxygen species and oxidative stress in bovine embryos produced in vitro.

Interferon tau (IFNT) is the cytokine responsible for the maternal recognition of pregnancy in ruminants and plays a role modulating embryo-maternal communication in the oviduct inducing a local response from immune cells. We aimed to investigate IFNT production, reactive oxygen species, and oxidative stress under the influence of heat stress (HS) during different stages of bovine in vitro embryo production. HS was established when the temperature was gradually raised from 38.5°C to 40.5°C in laboratory incubator, sustained for 6 hr, and decreased back to 38.5°C. To address the HS effects on IFNT production, reactive oxygen species, and oxidative stress, ovaries from a slaughterhouse were used according to treatments: control group (38.5°C); oocytes matured under HS; oocytes fertilized under HS; zygotes cultured in the first day under HS; and cells submitted to HS at oocyte maturation, fertilization, and the first day of zygote culture. The HS negatively affected cleavage and blastocyst rates, in all HS groups. On Day 7, all HS-treated embryos showed decrease IFNT gene and protein expressions, whereas reactive oxygen species were increased in comparison to the control. In conclusion, the compromised early embryo development due to higher temperatures during in vitro oocyte maturation, fertilization, and/or zygote stage have diminished IFNT expression and increased reactive oxygen species in bovine.

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.

Bacteriological, cytological and histopathological evaluation of the reproductive tract of slaughtered cows / Avaliação bacteriológica, citológica e histopatológica do trato reprodutivo de vacas abatidas

Reproductive diseases, mainly endometritis, are important hurdles in cattle raising, In the current study we evaluated gross, bacteriological, cytological, and histological findings from selected sites of the genital from 23 slaughtered cows and tested whether there is an association between these findings and the probability of reaching a reliable diagnosis. The results from the examinations of macroscopic aspects of uterine secretions, the cytological, bacteriological, and histopathological findings were then correlated. There was no significant correlation (P>0.05) of the statistical data from different parts of the genital tract. Trueperella pyogenes and Escherichia coli were isolated from the vagina in 3/23 cases. In only 2/23 samples Enterococcus faecalis and a gram-negative, oxidase-positive bacteria were isolated from the cervix uteri. Only Staphylococcus epidermidis, in 1/23 case, was isolated from the uterus. Histopathological findings in uterus from samples of Groups II (moderate lesions) and III (severe lesions) did not translated in grossly visible changes. Samples from reproductive tracts with secretion in the vagina and cervix uteri had no detectable changes in the other parameters analyzed from this portion. Uterus with positive bacterial culture had evidence of ascendant inflammation judging by the high granulocyte count in the three analyzed portions. This study reinforces that vaginitis and cervicitis in the cow diagnosed only by clinical examination does not reflect the real status of the uterine health. For this reason, treatment of uterine disorders should be conducted based on reliable tests to determine the appropriate therapy for each situation.(AU)

Doenças reprodutivas causam altas perdas econômicas nos rebanhos bovinos, sendo a endometrite uma das alterações mais relevantes. Os objetivos desta pesquisa foram avaliar a associação dos achados fisiopatológicos em exames macroscópicos, bacteriológicos, citológicos e histopatológicos nas porções do trato genital de 23 vacas abatidas, bem como avaliar a necessidade de associação destes exames para efetuar diagnóstico fidedigno. A avaliação macroscópica da secreção, os exames histopatológico, citológico e as bactérias identificadas foram correlacionados. Não houve associação (P>0,05) dos resultados nas diferentes porções do trato genital. Na vagina foram isoladas as bactérias Trueperella pyogenes e Escherichia coli. Na cérvix, em apenas 2/23 (8,6%) amostras isolou-se Enterococcus faecalis e gram negativo oxidase positiva. No útero houve isolamento apenas da bactéria Staphylococcus epidermidis. As amostras histopatológicas classificadas em grupo II e III não apresentaram alterações detectadas no exame macroscópico. As amostras com secreção não fisiológica na vagina e cérvix não apresentaram alterações nos outros exames na porção uterina. A amostra com cultura bacteriológica positiva no útero demonstrou uma infecção ascendente através da alta contagem de granulócitos nas três porções analisadas. O presente estudo reforça o conceito de que a vaginite e cervicite diagnosticadas pelo exame clinico na vaca não retratam o real status da saúde uterina e por esta razão, o tratamento do útero deve ser conduzido com critério, alicerçado nos exames complementares para definir a terapia adequada para cada situação.(AU)