Symposium review: Immunological detection of the bovine conceptus during early pregnancy.

Infertility and subfertility reduce the economic viability of dairy production. Inflammation reduces conception rates in dairy cattle, but surprisingly little information exists about the populations and the functions of immune cells at the conceptus-maternal interface during the periattachment period in dairy cattle. Early pregnancy is accompanied by immune stimulation at insemination and conceptus secretion of IFN-τ, pregnancy-associated glycoproteins, prostaglandins, and other molecules whose effects on immune function during early pregnancy have not been determined. Our working hypothesis is that pregnancy induces changes in immune cell populations and functions that are biased toward immunological tolerance, tissue remodeling, and angiogenesis. This review summarizes current knowledge, starting with insemination and proceeding through early pregnancy, as this is the period of maximal embryo loss. Results indicated that early pregnancy is accompanied by a marked increase in the proportion of endometrial immune cells expressing markers for natural killer (CD335) cells and cytotoxic T cells (CD8) along with an increase in cells expressing major histocompatibility class II antigens (macrophages and dendritic cells). This is accompanied by increased abundance of mRNA for IL-15, a natural killer growth factor, and IL-10 in the endometrium during early pregnancy. Furthermore, expression of indoleamine 2,3 dioxygenase was 15-fold greater in pregnant compared with cyclic heifers at d 17, but then declined by d 20. This enzyme converts tryptophan to kynurenine, which alters immune function by creating a localized tryptophan deficiency and by activation of the aryl hydrocarbon receptor and induction of downstream tolerogenic mediators. Expression of the aryl hydrocarbon receptor is abundant in the bovine uterus, but its temporal and spatial regulation during early pregnancy have not been characterized. Pregnancy is also associated with increased expression of proteins known to inhibit immune activation, including programed cell death ligand-1 (CD274), lymphocyte activation gene-3 (CD223), and cytotoxic T-lymphocyte associated protein-4 (CD152). These molecules interact with receptors on antigen-presenting cells and induce lymphocyte tolerance. Current results support the hypothesis that early pregnancy signaling in dairy heifers involves changes in the proportions of immune cells in the endometrium as well as induction of molecules known to mediate tolerance. These changes are likely essential for uterine wall remodeling, placentation, and successful pregnancy.

Effects of early pregnancy on uterine lymphocytes and endometrial expression of immune-regulatory molecules in dairy heifers.

Natural killer (NK) cells are essential for establishment of human and rodent pregnancies. The function of these and other cytotoxic T cells (CTL) is controlled by stimulatory and inhibitory signaling. A role for cytotoxic cells during early pregnancy in cattle has not been described, but regulation of their function at the fetal-maternal interface is thought to be critical for conceptus survival. The hypothesis that the relative abundance of CTL and expression of inhibitory signaling molecules is increased by the conceptus during early pregnancy was tested. The proportions of lymphoid lineage cells and expression of inhibitory signaling molecules in the endometrium during early pregnancy in dairy heifers were determined using flow cytometry, immunofluorescence, and real-time PCR on days 17 and 20 of pregnancy and day 17 of the estrous cycle. Results revealed an increased percentage of NKp46+ and CD8+ cells in the uterus of pregnant heifers. Furthermore, a large percentage of uterine immune cells coexpressed these proteins. Compared to cyclic heifers, CD45+ uterine cells from pregnant heifers exhibited greater degranulation. Endometrium from pregnant heifers had greater mRNA abundance for the inhibitory molecules, CD274 and lymphocyte activating gene 3 (LAG3), and greater cytotoxic T lymphocyte-associated protein 4 (CTLA4), molecules that can interact with receptors on antigen-presenting cells and induce lymphocyte tolerance. This study demonstrates a dynamic regulation of both cytotoxic immune cells and tolerogenic molecules during the peri-implantation period that may be required to support establishment of pregnancy and placentation.

Discrepancies and Uncertainties in Bottom-up Gridded Inventories of Livestock Methane Emissions for the Contiguous United States.

In this analysis we used a spatially explicit, simplified bottom-up approach, based on animal inventories, feed dry matter intake, and feed intake-based emission factors to estimate county-level enteric methane emissions for cattle and manure methane emissions for cattle, swine, and poultry for the contiguous United States. Overall, this analysis yielded total livestock methane emissions (8916 Gg/yr; lower and upper 95% confidence bounds of ±19.3%) for 2012 (last census of agriculture) that are comparable to the current USEPA estimates for 2012 and to estimates from the global gridded Emission Database for Global Atmospheric Research (EDGAR) inventory. However, the spatial distribution of emissions developed in this analysis differed significantly from that of EDGAR and a recent gridded inventory based on USEPA. Combined enteric and manure methane emissions from livestock in Texas and California (highest contributors to the national total) in this study were 36% lesser and 100% greater, respectively, than estimates by EDGAR. The spatial distribution of emissions in gridded inventories (e.g., EDGAR) likely strongly impacts the conclusions of top-down approaches that use them, especially in the source attribution of resulting (posterior) emissions, and hence conclusions from such studies should be interpreted with caution.

Changes in Myeloid Lineage Cells in the Uterus and Peripheral Blood of Dairy Heifers During Early Pregnancy.

Establishment of pregnancy requires interaction between the developing conceptus and the uterine mucosal immune system. Myeloid lineage cells (macrophages and dendritic cells) are key mediators of pregnancy in rodents and humans but relatively little is known regarding their role and distribution during early pregnancy in ruminants. We tested the hypothesis that myeloid lineage cell number, distribution, and function are altered during early pregnancy in dairy heifers. Dairy heifers were inseminated using sperm from a single bull (Day 0), and uteri and blood were collected at slaughter on Days 17 and 20 of pregnancy to investigate the response of myeloid lineage cells to the presence of a conceptus. Responses were compared to noninseminated heifers on Day 17 of the estrous cycle. Peripheral blood and uterine-derived immune cells were isolated magnetically and examined using flow cytometry. Immunohistochemical analysis was used to evaluate the spatial distribution of myeloid lineage cells in the endometrium and quantitative polymerase chain reaction was conducted to quantify abundance of mRNA transcripts associated with myeloid lineage cell function. Transcripts for major histocompatibility complex (MHC) II, cluster of differentiation (CD) 80, CD86, CD163, and indoleamine 2,3-dioxygenase (IDO) 1 were greater in endometrium of pregnant compared to cyclic heifers. Immunofluorescence analysis revealed increased labeling for MHCII and SIRPA in pregnant compared to cyclic heifers. There were approximately 50% more CD14+CD11c+ cells in the peripheral circulation of pregnant compared to cyclic heifers. A greater number of myeloid lineage cells were observed during early pregnancy, and this increase was most pronounced in and around the shallow glands. Furthermore, expression of molecules associated with a tolerogenic or alternatively activated phenotype of these cells also increased in pregnant heifers. The results support the hypothesis that myeloid lineage cells with a tolerogenic phenotype are involved in establishment of pregnancy in dairy heifers.

Embryo Mortality Around the Period of Maintenance of the Corpus Luteum Causes Alterations to the Ovarian Function of Lactating Dairy Cows.

Objectives were to identify cows with embryo mortality (EM) around the period of corpus luteum maintenance by interferon tau (IFNT) and to characterize ovarian function in cows that underwent EM. Lactating Holstein cows received artificial insemination (AI) (Day = 0) with semen or extender only. From Day 14 to 42 transrectal ultrasonography was performed daily to monitor ovarian dynamics and uterine contents whereas blood was collected every 48 h to determine ISG15 and MX2 mRNA abundance in blood mononuclear cells (Day 14 to 22 only) and determination of hormone concentrations. Cows were classified in the following reproductive status groups: cyclic (inseminated with extender; n = 15), pregnant (embryo present on Day 42; n = 23), no embryo (n = 23), and EM (n = 14). EM was defined as the presence of an embryo based on interferon-stimulated genes (ISG) mRNA abundance and concentrations of pregnancy-specific protein B (PSPB) above specific cutoff points but no embryo visualized by ultrasonography. Within the EM group, early EM (up to Day 22) was when ISG fold changes were above specific cutoff points from Day 18 to 22 and PSPB below 0.7 ng/ml on and after Day 24, whereas late EM (after Day 22) was when PSPB was above 0.7 ng/ml on or after Day 24 regardless of ISG expression. This experiment provided evidence that the combination of ISG expression patterns and PSPB concentrations is a reasonable method to determine EM around the period of corpus luteum maintenance by IFNT because cows with evidence of EM had patterns of ISG expression more similar to pregnant than cyclic cows or cows with no embryo. Within the EM group, only cows with late EM had delayed luteal regression and longer interovulatory intervals. No major alterations in follicular function were observed after the onset of luteolysis. Our results suggest that embryo development needs to continue beyond 22 days after AI to effectively prevent luteolysis and extend the luteal phase.

Low doses of bovine somatotropin enhance conceptus development and fertility in lactating dairy cows.

Objectives were to evaluate the effects of administering either one or two low doses of slow-release recombinant bovine somatotropin (bST) on hormone concentrations, conceptus development, and fertility in dairy cows. Cows from two farms were detected in estrus on or after 50 days postpartum (n = 1483), inseminated, and enrolled in the study (Day 0). Within farm, cows were blocked by parity and assigned randomly to receive a single placebo injection at insemination (control), a single injection with 325 mg of bST at insemination (S-bST), or two injections with 325 mg of bST administered on Days 0 and 14 (T-bST). From a subset of cows, blood was collected twice weekly from Day 0 to 42 for determination of hormone concentrations and on Day 19 for isolation of leucocytes and analysis of transcript abundance of selected interferon-stimulated genes. Pregnancy was diagnosed on Days 31 and 66, and ultrasonographic morphometry of the conceptus was performed on Days 34 and 48 in a subset of cows. Cows that received T-bST had increased plasma concentrations of GH and IGF1 for 4 wk, increased mRNA expression of ISG15 and RTP4 in leukocytes, earlier rise in the pregnancy-specific protein B in plasma of pregnant cows, increased conceptus size, and enhanced fertility. Cows that received S-bST had increased concentrations of GH and IGF1 for only 2 wk and it was insufficient to alter conceptus development and fertility. In conclusion, supplementation with low doses of bST during the pre- and peri-implantation periods enhanced conceptus development, reduced embryonic losses, and improved fertility in dairy cows.

Effects of conceptus proteins on endometrium and blood leukocytes of dairy cattle using transcriptome and meta-analysis.

This study investigates the short and long-term effects of IFNT and PAG on the transcriptome of endometrium and blood leukocytes. Holstein heifers received intrauterine infusions of one of the following treatments: 20 mL of a 200 µg/mL bovine serum albumin solution (BSA; vehicle) from day 14 to 16 of the estrous cycle (BSA), vehicle + 10 µg/mL of IFNT from day 14 to 16 (IFNT3), vehicle + 10 µg/mL of IFNT from day 14 to 19 (IFNT6), and vehicle + 10 µg/mL of IFNT from day 14 to 16 followed by vehicle + 10 µg/mL of IFNT + 5 µg/mL of PAG from day 17 to 19 (IFNT+PAG). RNA-seq analysis was performed in endometrial biopsies and blood leukocytes collected after treatments. Acute IFNT signaling in the endometrium (IFNT3 vs BSA), induced differentially expressed genes (DEG) associated with interferon activation, immune response, inflammation, cell death, and inhibited vesicle transport and extracellular matrix remodeling. Prolonged IFNT signaling (IFNT6 vs IFNT3) altered gene expression related to cell invasion, retinoic acid signaling, and embryo implantation. In contrast, PAG induced numerous DEG in blood leukocytes but only 4 DEG in the endometrium. In blood leukocytes, PAG stimulated genes involved in development and TGFB signaling while inhibiting interferon signaling and cell migration. Overall, IFNT is a primary regulator of endometrial gene expression, while PAG predominantly affected the transcriptome of circulating immune cells during early pregnancy. Further research is essential to fully grasp the roles of identified DEG in both the endometrium and blood leukocytes.

Pregnancy Establishment and Diagnosis in Livestock.

This comprehensive review explores the complex processes of reproduction, pregnancy establishment, and pregnancy diagnostic methods in cattle, sheep, goats, swine, horses, and camelids. It provides an overview of the history of pregnancy detection and an in-depth exploration of the physiology of pregnancy in livestock. The detection of conceptus tissue and fluids, conceptus-produced hormones, and maternal responses to conceptus signals, crucial for pregnancy diagnosis, are also discussed in detail, as are emerging methods for pregnancy diagnosis in livestock species. Overall, this review emphasizes the direct impact of pregnancy diagnosis and efficient pregnancy management for profitability of livestock enterprises.

Immunological detection of pregnancy: Evidence for systemic immune modulation during early pregnancy in ruminants.

Mammalian pregnancy creates unique challenges for immune systems highly evolved to detect and eliminate invading pathogens. Recognition of the challenges created by gestating a semi-allogeneic fetus evolved from the discipline of transplantation biology and were informed by studies on the unique natural parabiosis that occurs when female calves are gestated with twin male fetuses. These pregnancies typically result in an intersex female termed a freemartin, which revealed insights into development of the male and female reproductive tracts. However, they also uncovered important clues on immune tolerance with wide-ranging implications to reproductive biology, transplantation biology and autoimmune disease. Many studies focused on identifying mechanisms through which the fetus evades maternal immune detection and elimination. These included studies characterizing immune interactions between the fetus and mother at the nourishing interface of the placenta and uterine endometrium. This immunological forbearance only occurs under high concentrations of circulating progesterone. Beyond the requirement for progesterone, there has been considerable progress towards understanding the effects of conceptus signals on maternal immune function. One common theme is that pregnancy induces a T helper 2 immune bias as shown in several mammalian species, including domestic ruminants. However, a growing body of evidence shows that the fetus not only evades, but also provokes immune responses locally in the uterus and in peripheral tissues. This is perhaps most dramatically illustrated by domestic ruminants where the conceptus secretes a unique interferon in the opening salvo of hormonal communication with the maternal immune system. The role of interferon tau in regulating expression of genes of the innate immune system in the uterus has been extensively studied. More recently, it was determined that these same genes are also induced in peripheral immune cells and other tissues throughout the body. In addition to interferon tau and progesterone, pregnancy associate glycoproteins and chaperonin 10 (aka Early Pregnancy Factor) are implicated in altering immune function both locally and systemically during pregnancy. While it is tempting to speculate that this activation of innate immunity is designed to counteract selective immunosuppression, knowledge of the importance of local and systemic immune activation to the success of pregnancy remains incomplete. This area remains fertile ground for developing better approaches to diagnose and treat infertility in domestic farm species and humans alike.

Author Correction: Evolution of placental invasion and cancer metastasis are causally linked.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanical shear controls bacterial penetration in mucus.

Mucus plays crucial roles in higher organisms, from aiding fertilization to protecting the female reproductive tract. Here, we investigate how anisotropic organization of mucus affects bacterial motility. We demonstrate by cryo electron micrographs and elongated tracer particles imaging, that mucus anisotropy and heterogeneity depend on how mechanical stress is applied. In shallow mucus films, we observe bacteria reversing their swimming direction without U-turns. During the forward motion, bacteria burrowed tunnels that last for several seconds and enable them to swim back faster, following the same track. We elucidate the physical mechanism of direction reversal by fluorescent visualization of the flagella: when the bacterial body is suddenly stopped by the mucus structure, the compression on the flagellar bundle causes buckling, disassembly and reorganization on the other side of the bacterium. Our results shed light into motility of bacteria in complex visco-elastic fluids and can provide clues in the propagation of bacteria-born diseases in mucus.

Evolution of placental invasion and cancer metastasis are causally linked.

Among mammals, placental invasion is correlated with vulnerability to malignancy. Animals with more invasive placentation (for example, humans) are more vulnerable to malignancy. To explain this correlation, we propose the hypothesis of 'Evolved Levels of Invasibility' proposing that the evolution of invasibility of stromal tissue affects both placental and cancer invasion. We provide evidence for this using an in vitro model. We find that bovine endometrial and skin fibroblasts are more resistant to invasion than are their human counterparts. Gene expression profiling identified genes with high expression in human but not in bovine fibroblasts. Knocking down a subset of them in human fibroblasts leads to stronger resistance to cancer cell invasion. Identifying the evolutionary determinants of stromal invasibility can provide important insights to develop rational antimetastatic therapeutics.

Ovine endometrial expression of fibroblast growth factor (FGF) 2 and conceptus expression of FGF receptors during early pregnancy.

In ruminants, conceptus development beyond the blastocyst state requires input from uterine-derived factors. Fibroblast growth factor 2 (FGF2) is expressed by the bovine endometrium throughout the estrus cycle and early pregnancy and stimulates trophectoderm expression of interferon-tau, the maternal recognition of pregnancy factor in ruminants. The objective of this study was to examine the expression of FGF2 in ovine endometrium and peri-attachment conceptuses and FGF receptors (FGFR) in conceptuses. FGF2 mRNA was present in the ovine endometrium with specific localization within the luminal and glandular epithelium. No pregnancy-dependent changes in endometrial FGF2 mRNA abundance were detected until placental attachment was well underway. FGF2 protein was detected in the uterine lumen throughout the estrous cycle and early pregnancy. Concentrations of luminal FGF2 protein did not differ based on pregnancy status. However, uterine luminal FGF2 protein levels increased at days 12-13 after estrus in both cyclic and pregnant ewes. Ovine conceptuses collected at days 14-19 after mating contained transcripts for FGF2 and FGFR types 1, 2 and 3. In summary, FGF2 is expressed by the ovine endometrium and conceptus during early pregnancy, and peri-attachment conceptuses possess several FGFR types. Concentrations of FGF2 protein in the uterine lumen increase coincident with the initiation of pregnancy recognition in ewes. These observations support the concept that FGF2 and potentially other FGFs may affect conceptus development and/or gene expression during early pregnancy in ruminants.

Differential sensitivity of viruses to the antiviral activity of Shiga toxin 1 A subunit.

The non-toxic enzymic A subunit of Shiga toxin 1 (StxA1) reduces expression and replication of the bovine retroviruses, bovine leukemia virus and bovine immunodeficiency virus (BIV). Here, the impact of StxA1 on representative positive and negative stranded RNA viruses was compared. BIV and equine infectious anemia virus were sensitive to picomolar concentrations of StxA1 while poliovirus, rhinovirus, and vesicular stomatitis virus were only marginally sensitive to nanomolar concentrations of toxin. Thus, the length of the reproductive cycle and/or other factors, but not viral encapsulation may play a role in determining sensitivity to StxA1. The effects of StxA1 at concentrations from 0.01 to 10 microg/ml on the most sensitive virus (BIV-infected cultures of fetal bovine lung cells) were analyzed by electron microscopy 48 h post challenge. Cells treated with 0.1 microg StxA1/ml or higher toxin concentrations were similar in appearance and showed progressively fewer viral factories with increasing toxin concentration. However, cells treated with 0.01 microg/ml StxA1 had a radically different appearance, exhibiting smooth cell membranes and high vacuolization. These results showed that complex retroviruses were more sensitive to StxA1 than single-stranded RNA viruses and that StxA1 interfered with retroviral replication in a concentration-dependent manner.

The Transcriptomic Evolution of Mammalian Pregnancy: Gene Expression Innovations in Endometrial Stromal Fibroblasts.

The endometrial stromal fibroblast (ESF) is a cell type present in the uterine lining of therian mammals. In the stem lineage of eutherian mammals, ESF acquired the ability to differentiate into decidual cells in order to allow embryo implantation. We call the latter cell type "neo-ESF" in contrast to "paleo-ESF" which is homologous to eutherian ESF but is not able to decidualize. In this study, we compare the transcriptomes of ESF from six therian species: Opossum (Monodelphis domestica; paleo-ESF), mink, rat, rabbit, human (all neo-ESF), and cow (secondarily nondecidualizing neo-ESF). We find evidence for strong stabilizing selection on transcriptome composition suggesting that the expression of approximately 5,600 genes is maintained by natural selection. The evolution of neo-ESF from paleo-ESF involved the following gene expression changes: Loss of expression of genes related to inflammation and immune response, lower expression of genes opposing tissue invasion, increased markers for proliferation as well as the recruitment of FOXM1, a key gene transiently expressed during decidualization. Signaling pathways also evolve rapidly and continue to evolve within eutherian lineages. In the bovine lineage, where invasiveness and decidualization were secondarily lost, we see a re-expression of genes found in opossum, most prominently WISP2, and a loss of gene expression related to angiogenesis. The data from this and previous studies support a scenario, where the proinflammatory paleo-ESF was reprogrammed to express anti-inflammatory genes in response to the inflammatory stimulus coming from the implanting conceptus and thus paving the way for extended, trans-cyclic gestation.

The myxovirus-resistance protein, MX1, is a component of exosomes secreted by uterine epithelial cells.

PROBLEM: Dairy cattle suffer from high percentages of early embryonic loss, and therefore, it is critical to study the function of the uterus at this time. We hypothesize that the antiviral protein, myxovirus resistance (MX)1, regulates secretion in uterine glandular cells during early pregnancy. METHOD OF STUDY: Uterine epithelial cells were used to study uterine function, in vitro. Sucrose gradients, Western blotting, and transmission electron microscopy were used to isolate and identify exosomes. Immunofluorescence and ceramide inhibitors were used for the characterization of exosomes. RESULTS: Myxovirus resistance1 was associated with exosomes and protected from proteases, indicating it was inside exosomes. MX1 partially colocalized with exosomal protein CD63, and a ceramide inhibitor reduced numbers of MX1-associated exosomes. CONCLUSION: This study is the first to characterize MX1-associated exosomes, and we postulate that MX1 regulates secretion in epithelial cells by playing a role in exosome formation or trafficking.

Estrone and estrone sulfate concentrations in milk and milk fractions.

Dairy products naturally contain estrogens, and some consumer groups contend these estrogens cause adverse health effects. The objectives of this research were to characterize estrone (E(1)) and estrone sulfate (E(1)S) concentrations in milk from a large number of individual cows, in skim and fat fractions of milk, and in retail milk to provide food and nutrition practitioners with information to estimate potential consumption. Milk was from Holstein cows. Data are presented as means and standard deviations. Analysis of variance was used to determine differences in E(1) and E(1)S content of whole milk and its skim and fat fractions. Mean E(1) and E(1)S concentrations (n=173 cows) were 7.0±12.7 and 46.7±62.1 pg/mL (25.89±46.96 and 172.74±229.71 pmol/L), respectively. Analysis of milk fractions (n=50 samples) demonstrated that 55% of E(1) and 14% of E(1)S were associated with the fat fraction with the remainder associated with the skim fraction. Concentrations of E(1) and E(1)S in pasteurized-homogenized whole milk (n=8) averaged 10.3±0.6 and 85.9±7.3 pg/mL (38.09±2.22 and 317.74±27.00 pmol/L), respectively. Production rates of E(1) plus estradiol in human beings range from 54,000 to 630,000 ng/day. US Food and Drug administration guidelines state that no physiologic effects occur when consumption is ≤1% of the endogenous quantities produced by the segment of the population with the lowest daily production. This threshold value for intake would be 540 ng/day. Estimated total E(1) intake from three servings of whole milk was 68 ng/day, which represents 0.01% to 0.1% of daily production rates in human beings. These findings support levels below the current guidelines for safe consumption.