Raman Spectroscopy Characterization Extracellular Vesicles from Bovine Placenta and Peripheral Blood Mononuclear Cells

Placenta-derived extracellular vesicles (EVs) are involved in communication between the placenta and maternal immune cells possibly leading to a modulation of maternal T-cell signaling components. The ability to identify EVs in maternal blood may lead to the development of diagnostic and treatment tools for pregnancy complications. The objective of this work was to differentiate EVs from bovine placenta (trophoblast) and peripheral blood mononuclear cells (PBMC) by a label-free, non-invasive Raman spectroscopy technique. Extracellular vesicles were isolated by ultracentrifugation. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) were applied to verify the presence and the size distribution of EVs. Raman peaks at 728 cm-1 (collagen) and 1573 cm-1 (protein) were observed only in PBMC-derived EVs, while the peaks 702 cm-1 (cholesterol) and 1553 cm-1 (amide) appeared only in trophoblast-derived EVs. The discrimination of the Raman spectral fingerprints for both types of EVs from different animals was performed by principal component analysis (PCA) and linear discriminant analysis (LDA). The PCA and LDA results clearly segregated the spectral clusters between the two types of EVs. Moreover, the PBMC-derived EVs from different animals were indistinguishable, while the trophoblast-derived EVs from three placental samples of different gestational ages showed separate clusters. This study reports for the first time the Raman characteristic peaks for identification of PBMC and trophoblast-derived EVs. The development of this method also provides a potential tool for further studies investigating the causes and potential treatments for pregnancy complications

Lymphocyte Soluble Factors from Pregnant Cows Modulate mRNA Transcript Abundances Encoding for Proteins Associated with Trophoblast Growth and Development

This study was conducted to determine whether T cell populations are responsible for modulating placental development during gestation in cattle. It was hypothesized that CD4+CD25+ and γ/δ+ T cells modulate gene expression, based on mRNA transcript abundances, and promote proliferation and survival of trophoblast cells. Peripheral blood was collected from cows at 160 to 180 days of gestation and non-pregnant cows, T cell populations CD8+, CD4+, CD4+CD25+, CD24+CD25-, and γ/δ+ T cells were isolated, cultured for 48 h, and supernatant was collected. Placental samples were digested, and trophoblast cells were cultured for 24 h. Trophoblast cells were cultured with 50 μL of T cell-conditioned media and 50 μL of fresh culture media for an additional 48 h. Samples in control wells were treated with unconditioned media. Trophoblast cell proliferation, apoptosis, and mRNA transcript assays were conducted. There was no effect of T cell population on trophoblast apoptosis rate, proliferation, and relative mRNA transcript abundances. The T cell supernatant from pregnant and non-pregnant cows induced greater apoptosis rates in trophoblast cells than unconditioned media. Trophoblast cells proliferated less when treated with T cell supernatant from pregnant compared to unconditioned medium and non-pregnant cows. Treatment with the T cell supernatant from pregnant cows resulted in larger abundances of BMP5, IGF1R, PAG10, FGF2, RSPO3 and TMED2 and also a lesser abundance of FGF2 mRNA transcript than non-pregnant group and unconditioned media treatments. Supernatant from T cell derived from pregnant cows modulates trophoblast mRNA transcript abundances differently from T cell supernatant of non-pregnant cows

Cytokine Gene Expression in the Maternal-Fetal Interface in Somatic Cell Nuclear Transfer Pregnancies in Small Ruminants

The present retrospective study investigates pregnancy rates, incidence of pregnancy losses and large offspring syndrome (LOS), and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesized that significantly higher pregnancy losses observed in sheep SCNT pregnancies compared to goats are due to the increased amounts of T-helper 1 cytokines and pro-inflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days and incidence of LOS were significantly increased in sheep compared with goats. At term, the expression of pro-inflammatory genes in sheep SCNT placentas was increased while the one of goat SCNT was similar to the control animals. Among the genes that had altered expression in sheep SCNT placentas are CTLA4, IL2RA, CD28, IFNG, IL6, IL10, TGFB1, TNF, IL1A and CXCL8. MHC-I protein expression was greater in sheep and goat SCNT placentas at term compared with control pregnancies. An unfavorable immune environment is present at the maternal-fetal interface in sheep SCNT pregnancies

Effects of Pregnancy and Progesterone on Atrial Fibrillation

Atrial Fibrillation (AF) is the most common cardiac arrythmia in adults with likely sex-specific risk factors. Female sex hormones may be important in modulating risk for atrial fibrillation. We hypothesize that pregnancy and progesterone (P4), a hormone found in high levels during pregnancy with abrupt withdrawal immediately after parturition, modulates AF susceptibility in female goats. Cardiac specific TGF-?1 transgenic female goats and age-matched wild-type (WT) female goats were utilized. Pacemakers were implanted in all animals for continuous arrhythmia monitoring and AF inducibility. AF inducibility was evaluated using 5 separate 10 s bursts of atrial pacing (160 - 200 ms). In the first study, all animals were bred (n=12) via natural service and circulating progesterone levels and AF inducibility were monitored throughout gestation. In the second study, seven female goats were treated with high-dose exogenous oral P4 supplementation over a two-week period, and AF inducibility was assessed at baseline, during P4 supplementation and 48-hours following P4 supplementation. Serum P4 was measured using a chemiluminescent assay. Cardiac tissue was collected from both atria from goats in a high P4 state and a P4 withdrawal. RNA was extracted and gene expression analysis was ran on several genes related to AF. Atrial tissue was also During pregnancy, P4 levels increased compared to baseline with a sharp withdrawal during the post-partum period. This decrease in P4 levels post-parturition was associated with an increase in AF inducibility in both TGF-?1 and WT goats. Exogenous P4 supplementation increased circulating P4 compared to baseline with a decrease in P4 levels below baseline at 48-hours post supplementation. Consistent with our hypothesis and observations from the pregnancy studies, abrupt withdrawal of P4 was associated with increased AF inducibility when compared to baseline and P4 supplementation. Several genes were differentially expressed in the P4 withdrawal group compared to the high P4 group. CACN2D, CACNB2, and CAMK2D, all calcium related genes were significantly downregulated in the progesterone withdrawal (P4 WD) group when compared to the high progesterone (HIGH P4) group. These data support the hypothesis that progesterone is an important modulator of AF susceptibility in females. More specifically, abrupt reduction of progesterone significantly increases AF risk. Our gene expression data shows that P4 modulates calcium signaling in the atrium

Expression of MHC-I Proteins by the Placenta of Domestic and Laboratory Animals.

Pregnancy has been described as an immunological paradox because the maternal immune system accepts the fetus even though it expresses paternal antigens. Since placental tissues express maternal and paternal genes the immune system of the mother must tolerate the fetus to avoid its rejection. Placental expression of MHC-I proteins appears to play an important role in the regulation of the maternal immune system and protection of the conceptus from immune-mediated rejection. There are two types of MHC-I proteins. Classical MHC-I proteins, also called MHC-Ia, are highly polymorphic and are expressed by most nucleated cells. The non-classical proteins, also called MHC-Ib, are less polymorphic, are expressed by limited types of cells and are known to have immune modulatory effects in some species. In order to protect the fetus from maternal immune recognition most species down-regulate the expression of placental MHC-Ia proteins. Another commonly used mechanism is the upregulation of MHC-Ib expression by the trophoblast cells. The role of MHC-I proteins in the placenta has been studied in several animal species. This chapter will summarize the current literature on the expression of MHC-I proteins by the placenta of domestic and laboratory species. A lot remains to be learned about the types, function and regulation of expression of placental MHC-I proteins in domestic and laboratory animals

Livestock in biomedical research: history, current status and future prospective

Livestock models have contributed significantly to biomedical and surgical advances. Their contribution is particularly prominent in the areas of physiology and assisted reproductive technologies, including understanding developmental processes and disorders, from ancient to modern times. Over the past 25 years, biomedical research that traditionally embraced a diverse species approach shifted to a small number of model species (e.g. mice and rats). The initial reasons for focusing the main efforts on the mouse were the availability of murine embryonic stem cells (ESCs) and genome sequence data. This powerful combination allowed for precise manipulation of the mouse genome (knockouts, knockins, transcriptional switches etc.) leading to ground-breaking discoveries on gene functions and regulation, and their role in health and disease. Despite the enormous contribution to biomedical research, mouse models have some major limitations. Their substantial differences compared with humans in body and organ size, lifespan and inbreeding result in pronounced metabolic, physiological and behavioural differences. Comparative studies of strategically chosen domestic species can complement mouse research and yield more rigorous findings. Because genome sequence and gene manipulation tools are now available for farm animals (cattle, pigs, sheep and goats), a larger number of livestock genetically engineered (GE) models will be accessible for biomedical research. This paper discusses the use of cattle, goats, sheep and pigs in biomedical research, provides an overview of transgenic technology in farm animals and highlights some of the beneficial characteristics of large animal models of human disease compared with the mouse. In addition, status and origin of current regulation of GE biomedical models is also reviewed

Risk factors for resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows

The objectives of this study were to evaluate factors associated with resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows. Holstein cows, 6396 from four dairy farms were evaluated to determine the relationships among parity, body condition score (BCS) at calving and at AI, season of year when cows calved, and milk yield on resumption of postpartum estrous cycles by 65 days postpartum, and all the previous variables, estrual or anestrus and AI protocol on conception rates and embryonic survival at the first postpartum insemination. Cows had their estrous cycle pre-synchronized with two PGF2α injections given 14 days apart and were inseminated between 69 and 82 days postpartum following either an estrous or ovulation synchronization protocol initiated 12–14 days after the presynchronization. Blood was sampled and analyzed for progesterone twice, 12–14 days apart, to determine whether cows had initiated onset of estrous cycles after calving. Cows were scored for body condition in the week after calving, and again at AI, between 69 and 82 days postpartum. Pregnancy was diagnosed at 30 ± 3 and 58 ± 3 days after AI. Farm influenced all reproductive outcomes evaluated. More (P \u3c 0.0001) multiparous than primiparous cows had initiated estrous cycles. Onset of estrous cycles was also influenced (P \u3c 0.01) by BCS at calving and at AI, BCS change, season, and milk yield. More (P \u3c 0.001) cows that had initiated estrous cycles than anestrous cows were pregnant at 30 and 58 days after AI, but anestrus did not affect pregnancy loss. Conception rates were also influenced (P \u3c 0.01) by parity, BCS at calving and AI, BCS change, and season; however, milk yield and insemination protocol were not associated with conception rates at 30 and 58 days after AI. Factors that reduced conception rate on day 30 after AI also increased pregnancy loss between 30 and 58 days of gestation. Improving BCS at calving and AI, minimizing losses of BCS after calving, and hastening onset of estrous cycles early postpartum are all expected to increase conception because of enhanced embryonic survival

Effect of time and dose of recombinant follicle stimulating hormone agonist on the superovulatory response of sheep

The objective of this study was to determine the superovulatory potential of a single-chain analog of human FSH (Fcα) when administered to ewes either 3 days before, or coincident with, simulated luteolysis (pessary removal [PR]). A total of 40 animals were randomly assigned to receive Fcα at doses of 0.62, 1.25, or 2.5 IU/kg of body weight (bwt) 3 days before PR or 0.31, 0.62, 1.25, or 2.5 IU/kg of bwt at PR. Control ewes received protein without FSH activity. Blood samples were collected during the periovulatory period and ovarian tissue was collected 11 days after PR. Ovulation rate did not differ from the control group in ewes receiving the smallest doses of Fcα (0.31 and 0.62 IU/kg). However, a significant superovulatory response was noted in sheep receiving Fcα at doses of 1.25 and 2.5 IU/kg and this response was comparable in animals receiving the largest dose levels of Fcα at, or 3 days before, PR. The interval between PR and the LH surge was significantly extended and the LH surges were less synchronous in animals receiving Fcα at PR when compared with animals receiving the potent FSH agonist 3 days before PR. Taken together, these data indicate that the human single-chain gonadotropin with FSH activity promotes superovulation in ewe lambs in the breeding season. A single injection of the recombinant gonadotropin 3 days before luteolysis synchronizes the LH surge. The use of the single-chain analog of FSH in assisted reproduction for domestic animals is likely to be of practical significance as an alternative to conventional gonadotropins in superovulation protocols in livestock species