SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows.

The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin; thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal; n = 30) and Aged (Primiparous; n = 20) dairy cows (Bos taurus) of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV; p value = 3.302e-07) and 0.95 (plasma; p value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (*p = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (***p = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle.

A complete proteomic profile of human and bovine milk exosomes by liquid chromatography mass spectrometry.

BACKGROUND: The present study investigates the proteomic content of milk-derived exosomes. A detailed description of the content of milk exosomes is essential to improve our understanding of the various components of milk and their role in nutrition. METHODS: The exosomes used in this study were isolated as previously described and characterized by their morphology, particle concentration, and the presence of exosomal markers. Human and bovine milk exosomes were evaluated using Information-Dependent Acquisition (IDA) Mass Spectrometry. A direct comparison is made between their proteomic profiles. RESULTS: IDA analyses revealed similarities and differences in protein content. About 229 and 239 proteins were identified in the human and bovine milk exosome proteome, respectively, of which 176 and 186 were unique to each species. Fifty-three proteins were common in both groups. These included proteins associated with specific biological processes and molecular functions. Most notably, the 4 abundant milk proteins lactadherin, butyrophilin, perilipin-2, and xanthine dehydrogenase/oxidase were present in the top 20 list for both human and bovine milk exosomes. CONCLUSION: The milk exosome protein profiles we have provided are crucial new information for the field of infant nutrition. They provide new insight into the components of milk from both humans and bovines.

Exosomal Cargo May Hold the Key to Improving Reproductive Outcomes in Dairy Cows.

The reproductive status of dairy cows remains a challenge for dairy farmers worldwide, with impaired fertility linked to a significant reduction in herd profitability, due in part to impaired immunity, increased metabolic pressure, and longer postpartum anestrous interval (PPAI). Exosomes are nanovesicles released from a variety of cell types and end up in circulation, and carry proteins, bioactive peptides, lipids, and nucleic acids specific to the place of origin. As such, their role in health and disease has been investigated in humans and animals. This review discusses research into exosomes in the context of reproduction in dairy herds and introduces recent advances in mass-spectrometry (MS) based proteomics that have a potential to advance quantitative profiling of exosomal protein cargo in a search for early biomarkers of cattle fertility.

The physiology of intrapartum fetal compromise at term.

Uterine contractions in labor result in a 60% reduction in uteroplacental perfusion, causing transient fetal and placental hypoxia. A healthy term fetus with a normally developed placenta is able to accommodate this transient hypoxia by activation of the peripheral chemoreflex, resulting in a reduction in oxygen consumption and a centralization of oxygenated blood to critical organs, namely the heart, brain, and adrenals. Providing there is adequate time for placental and fetal reperfusion between contractions, these fetuses will be able to withstand prolonged periods of intermittent hypoxia and avoid severe hypoxic injury. However, there exists a cohort of fetuses in whom abnormal placental development in the first half of pregnancy results in failure of endovascular invasion of the spiral arteries by the cytotrophoblastic cells and inadequate placental angiogenesis. This produces a high-resistance, low-flow circulation predisposing to hypoperfusion, hypoxia, reperfusion injury, and oxidative stress within the placenta. Furthermore, this renders the placenta susceptible to fluctuations and reduction in uteroplacental perfusion in response to external compression and stimuli (as occurs in labor), further reducing fetal capillary perfusion, placing the fetus at risk of inadequate gas/nutrient exchange. This placental dysfunction predisposes the fetus to intrapartum fetal compromise. In the absence of a rare catastrophic event, intrapartum fetal compromise occurs as a gradual process when there is an inability of the fetal heart to respond to the peripheral chemoreflex to maintain cardiac output. This may arise as a consequence of placental dysfunction reducing pre-labor myocardial glycogen stores necessary for anaerobic metabolism or due to an inadequate placental perfusion between contractions to restore fetal oxygen and nutrient exchange. If the hypoxic insult is severe enough and long enough, profound multiorgan injury and even death may occur. This review provides a detailed synopsis of the events that can result in placental dysfunction, how this may predispose to intrapartum fetal hypoxia, and what protective mechanisms are in place to avoid hypoxic injury.

The Role of Exosomal Epigenetic Modifiers in Cell Communication and Fertility of Dairy Cows.

Abnormal uterine function affects conception rate and embryo development, thereby leading to poor fertility and reproduction failure. Exosomes are a nanosized subclass of extracellular vesicles (EV) that have important functions as intercellular communicators. They contain and carry transferable bioactive substances including micro RNA (miRNA) for target cells. Elements of the cargo can provide epigenetic modifications of the recipient cells and may have crucial roles in mechanisms of reproduction. The dairy industry accounts for a substantial portion of the economy of many agricultural countries. Exosomes can enhance the expression of inflammatory mediators in the endometrium, which contribute to various inflammatory diseases in transition dairy cows. This results in reduced fertility which leads to reduced milk production and increased cow maintenance costs. Thus, gaining a clear knowledge of exosomal epigenetic modifiers is critical to improving the breeding success and profitability of dairy farms. This review provides a brief overview of how exosomal miRNA contributes to inflammatory diseases and hence to poor fertility, particularly in dairy cows.

Genes upregulated in the amnion at labour are bivalently marked by activating and repressive histone modifications.

Inflammatory genes are expressed increasingly in the foetal membranes at late gestation triggering birth. Here we have examined whether epigenetic histone modifications contribute to the upregulation of proinflammatory genes in the amnion in late pregnancy and at labour. Amnion samples were collected from early pregnancy, at term in the absence of labour and after spontaneous birth. The expression of the labour-associated proinflammatory genes PTGS2, BMP2 and NAMPT was determined by reverse transcription-coupled quantitative real-time PCR (qRT-PCR). Chromatin immunoprecipitation (ChIP) and sequential double ChIP were performed to determine the levels and co-occurrence of activating histone-3, lysine-4 trimethylation (H3K4me3) and repressive histone-3, lysine-27 trimethylation (H3K27me3) at the gene promoters. H3K4 methyltransferase, H3K27me3 demethylase and H3K27 methyltransferase expression was determined by qRT-PCR and immunofluorescence confocal microscopy. PTGS2, BMP2 and NAMPT expression was upregulated robustly between early pregnancy and term (P < 0.05). The promoters were marked bivalently by both the H3K4me3 and H3K27me3 modifications. Bivalence was reduced at term by the decrease of the H3K27me3-modified fraction of promoter copies marked by H3K4me3 indicating epigenetic activation. Messenger RNAs encoding the H3K4-specific methyl transferases MLL1,-2,-3,-4, SETD1A,-B and the H3K27me3-specific demethylases KDM6A,-B were expressed increasingly while the H3K27 methyl transferase EZH2 was expressed decreasingly at term. Histone modifying enzyme proteins were detected in amnion epithelial and mesenchymal cells. These results with prototypical proinflammatory genes suggest that nucleosomes at labour-promoting genes are marked bivalently in the amnion, which is shifted towards monovalent H3K4me3 modification at term when the genes are upregulated. Bivalent epigenetic regulation by histone modifying enzymes may control the timing of labour.

The Interplay between the Endocannabinoid System, Epilepsy and Cannabinoids.

Epilepsy is a neurological disorder that affects approximately 50 million people worldwide. There is currently no definitive epilepsy cure. However, in recent years, medicinal cannabis has been successfully trialed as an effective treatment for managing epileptic symptoms, but whose mechanisms of action are largely unknown. Lately, there has been a focus on neuroinflammation as an important factor in the pathology of many epileptic disorders. In this literature review, we consider the links that have been identified between epilepsy, neuroinflammation, the endocannabinoid system (ECS), and how cannabinoids may be potent alternatives to more conventional pharmacological therapies. We review the research that demonstrates how the ECS can contribute to neuroinflammation, and could therefore be modulated by cannabinoids to potentially reduce the incidence and severity of seizures. In particular, the cannabinoid cannabidiol has been reported to have anti-convulsant and anti-inflammatory properties, and it shows promise for epilepsy treatment. There are a multitude of signaling pathways that involve endocannabinoids, eicosanoids, and associated receptors by which cannabinoids could potentially exert their therapeutic effects. Further research is needed to better characterize these pathways, and consequently improve the application and regulation of medicinal cannabis.

Proteome profiling of exosomes derived from plasma of heifers with divergent genetic merit for fertility.

The current study evaluated exosomes isolated from plasma of heifers bred to have high or low fertility through developing extreme diversity in fertility breeding values, however, key animal traits (e.g., body weight, milk production, and percentage of North American genetics) remained similar between the 2 groups. The exosomes were isolated by a combined ultracentrifugation and size exclusion chromatography approach and characterized by their size distribution (nanoparticle tracking analysis), morphology (transmission electron microscopy), and presence of exosomal markers (immunoblotting). In addition, a targeted mass spectrometry approach was used to confirm the presence of 2 exosomal markers, tumor susceptibility gene 101 and flotillin 1. The number of exosomes from plasma of high fertility heifers was greater compared with low fertility heifers. Interestingly, the exosomal proteomic profile, evaluated using mass spectrometry, identified 89 and 116 proteins in the high and low fertility heifers respectively, of which 4 and 31 were unique, respectively. These include proteins associated with specific biological processes and molecular functions of fertility. Most notably, the tetratricopeptide repeat protein 41-related, glycodelin, and kelch-like protein 8 were identified in plasma exosomes unique to the low fertility heifers. These proteins are suggested to play a role in reproduction; however, the role of these proteins in dairy cow reproduction remains to be elucidated. Their identification underscores the potential for proteins within exosomes to provide information on the fertility status and physiological condition of the cow. This may potentially lead to the development of prognostic tools and interventions to improving dairy cow fertility.

Effect of exosomes from plasma of dairy cows with or without an infected uterus on prostaglandin production by endometrial cell lines.

A contributing factor to declining fertility in dairy cows is an activated inflammatory system associated with uterine infection. Detecting uterine disease using biomarkers may allow earlier diagnosis and intervention with resultant improvements in fertility. Exosomes are known to participate in intercellular communication, paracrine, and endocrine signaling. Exosomes carry a cargo of proteins, lipids, and nucleic acids that represent specific cellular sources. Prostaglandins are lipids that are critical determinants of bovine fertility. In this study exosomes were isolated from the plasma of cows before (d 0) and during (d 10) the study in healthy animals or those with an induced uterine infection in a 2 × 2 factorial design. Exosomes were characterized for size and number (nanoparticle tracking analysis), exosomal marker expression (Western blot), and morphology (transmission electron microscopy). No significant differences were observed in exosome size or number. The abundance of exosome-enriched markers was confirmed in noninfected and infected animals. Transmission electron microscopy confirmed the morphology of the exosomes. These exosomes were co-incubated with bovine endometrial epithelial and stromal cells. Exosomes from d-10-infected animal plasma decreased PGF2α production in endometrial epithelial but not stromal cells. For future research, the identification of effectors in the cargo may provide a useful basis for early diagnosis of uterine infection using an exosomal characterization approach.

Characterization of exosomal release in bovine endometrial intercaruncular stromal cells.

BACKGROUND: Cell-to-cell communication between the blastocyst and endometrium is critical for implantation. In recent years, evidence has emerged from studies in humans and several other animal species that exosomes are secreted from the endometrium and trophoblast cells and may play an important role in cell-to-cell communication maternal-fetal interface during early pregnancy. Exosomes are stable extracellular lipid bilayer vesicles that encapsulate proteins, miRNAs, and mRNAs, with the ability to deliver their cargo to near and distant sites, altering cellular function(s). Furthermore, the exosomal cargo can be altered in response to environmental cues (e.g. hypoxia). The current study aims to develop an in vitro system to evaluate maternal-embryo interactions via exosomes (and exosomal cargo) produced by bovine endometrial stromal cells (ICAR) using hypoxia as a known stimulus associated with the release of exosomes and alterations to biological responses (e.g. cell proliferation). METHODS: ICAR cells cultured under 8 % O2 or 1 % O2 for 48 h and changes in cell function (i.e. migration, proliferation and apoptosis) were evaluated. Exosome release was determined following the isolation (via differential centrifugation) and characterization of exosomes from ICAR cell-conditioned media. Exosomal proteomic content was evaluated by mass spectrometry. RESULTS: Under hypoxic conditions (i.e. 1 % O2), ICAR cell migration and proliferation was decreased (~20 and ~32 %, respectively) and apoptotic protein caspase-3 activation was increased (∼1.6 fold). Hypoxia increased exosome number by ~3.6 fold compared with culture at 8 % O2. Mass spectrometry analysis identified 128 proteins unique to exosomes of ICAR cultured at 1 % O2 compared with only 46 proteins unique to those of ICAR cultured at 8 % O2. Differential production of proteins associated with specific biological processes and molecular functions were identified, most notably ADAM10, pantetheinase and kininogen 2. CONCLUSIONS: In summary, we have shown that a stimulus such as hypoxia can alter both the cellular function and exosome release of ICAR cells. Alterations to exosome release and exosomal content in response to stimuli may play a crucial role in maternal-fetal crosstalk and could also affect placental development.

Prepartum feeding level and body condition score affect immunological performance in grazing dairy cows during the transition period.

Precalving feeding level affects dry matter intake, postcalving energy balance, the risk of hepatic lipidosis and metabolic disease, and gene expression in liver and adipose tissue. These coincide with a higher risk of disease postpartum and, very likely, a failure to reach optimum production as well as reproductive targets. Current interpretation of the available evidence suggest that metabolic stressors affect the immune system of transition dairy cows and lead to reduced immunocompetence. The objective of the current study was to investigate the effect of precalving body condition score (BCS) and level of feeding on immunocompetence during the peripartum period. Twenty-three weeks before calving, 78 cows were allocated randomly to 1 of 6 treatment groups (n=13) in a 2 × 3 factorial arrangement: 2 precalving BCS categories (4.0 and 5.0, based on a 10-point scale) and 3 levels of energy intake during the 3 wk preceding calving (75, 100, and 125% of estimated requirements). Blood was sampled precalving and at 1, 2 and 4 wk after calving. Cells were analyzed by flow cytometry and quantitative real-time PCR. The numbers of T helper lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), natural killer cells (CD335+), and γδ T lymphocytes (WC1+) as well as their activation status [IL-2 receptor (CD25)+ cells] were highly variable between animals, but there was no evident effect of BCS, feeding level, or time. All groups presented with an increase in expression of cytokines in unstimulated blood cells in the week after calving, although this was significant only for IFNG in the BCS 4.0 group. Analysis of in vitro-stimulated cells allowed 2 general observations: (1) cows with high energy intake precalving (125%) had increased cytokine expression precalving, and (2) all cows had increased cytokine expression in the week after calving. The present study provides evidence that prepartum feed management can affect immunocompetence during the transition period. Considering the current results, optimally conditioned animals might benefit from a restricted precalving diet, whereas underconditioned cows can be fed to requirements.

Modulation of the immune system during postpartum uterine inflammation.

Postpartum uterine inflammation (endometritis) in the dairy cow is associated with lower fertility at both the time of infection and after the inflammation has resolved. We hypothesized that aberrant DNA methylation may be involved in the subfertility associated with uterine inflammation. The objective of this study was to characterize genome-wide DNA methylation and gene expression in the endometrium of dairy cows with subclinical endometritis (SCE). Endometrial tissues were obtained at 29 days postpartum (n = 12), and microarrays were used to characterize transcription and DNA methylation. Analyses revealed 1,856 probes differentially expressed in animals with SCE (n = 6) compared with controls (CON, n = 6, P < 0.05, Storey Multiple testing correction) and 2,976 probes with significant correlation between gene expression and bacteriology score. No significant associations among DNA methylation and gene expression were detected. Analysis of transcription data using the Database for Annotation, Visualization, and Integrated Discovery and Gene Set Enrichment Analysis identified several pathways and processes enriched in SCE cows, with the majority related to the immune response. Furthermore, the top ontology terms enriched in genes that had expression data correlated to bacteriology score were: Defense response, inflammatory response, and innate immune response. Gene expression profiles in cows with subclinical endometritis in this study indicate that the immune response is activated, potentially resulting in a local proinflammatory environment in the uterus. If this period of inflammation is prolonged it could result in tissue damage or failure to complete involution of the uterus, which may create a suboptimal environment for future pregnancy.

Applying SWATH Mass Spectrometry to Investigate Human Cervicovaginal Fluid During the Menstrual Cycle.

Inherent interindividual and intraindividual variation in the length of the menstrual cycle limits the accuracy of predicting days of peak fertility. To improve detection of days of peak fertility, a more detailed understanding of longitudinal changes in cervicovaginal fluid (CVF) biomarkers during the normal menstrual cycle is needed. The aim of this study, therefore, was to characterize longitudinal changes in CVF proteins during the menstrual cycle using a quantitative, data-independent acquisition mass spectrometry approach. Six serial samples were collected from women (n = 10) during the menstrual cycle. Samples were obtained at two time points for each phase of the cycle: early and late preovulatory, ovulatory, and postovulatory. Information-dependent acquisition (IDA) of mass spectra from all individual CVF samples was initially performed and identified 278 total proteins. Samples were then pooled by time of collection (n = 6 pools) and analyzed using IDA and information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]). The IDA library generated contained 176 statistically significant protein identifications (P < 0.000158). The variation in the relative abundance of CVF proteins across the menstrual cycle was established by comparison with the SWATH profile against the IDA library. Using time-series, pooled samples obtained from 10 women, quantitative data were obtained by SWATH analysis for 43 CVF proteins. Of these proteins, 28 displayed significant variation in relative abundance during the menstrual cycle (assessed by ANOVA). Statistical significant changes in the relative expression of CVF proteins during preovulatory, ovulatory, and postovulatory phases of menstrual cycle were identified. The data obtained may be of utility not only in elucidating underlying physiological mechanisms but also as clinically useful biomarkers of fertility status.

Regulation of TIMP-1 in Human Placenta and Fetal Membranes by lipopolysaccharide and demethylating agent 5-aza-2'-deoxycytidine.

BACKGROUND: An appropriate transcriptional profile in the placenta and fetal membranes is required for successful pregnancy; any variations may lead to inappropriate timing of birth. Epigenetic regulation through reversible modification of chromatin has emerged as a fundamental mechanism for the control of gene expression in a range of biological systems and can be modified by pharmacological intervention, thus providing novel therapeutic avenues. TIMP-1 is an endogenous inhibitor of MMPs, and hence is intimately involved in maintaining the integrity of the fetal membranes until labor. OBJECTIVE AND METHODS: To determine if TIMP-1 is regulated by DNA methylation in gestational tissues we employed an in vitro model in which gestational tissue explants were treated with demethylating agent 5-aza-2'-deoxycytidine (AZA) and lipopolysaccharide (LPS). RESULTS: Quantitative Real-Time PCR (qRT-PCR) revealed that TIMP-1 transcription was significantly increased by combined treatment of AZA and LPS, but not LPS alone, in villous, amnion and choriodecidua explants after 24 and 48 hrs, whilst western blotting showed protein production was stimulated after 24 hrs only. Upon interrogation of the TIMP-1 promoter using Sequenom EpiTyper MassARRAY, we discovered sex-specific differential methylation, in part explained by x-linked methylation in females. Increased TIMP-1 in the presence of LPS was potentiated by AZA treatment, signifying that a change in chromatin structure, but not in DNA methylation at the promoter region, is required for transcriptional activators to access the promoter region of TIMP-1. CONCLUSIONS: Collectively, these observations support a potential role for pharmacological agents that modify chromatin structure to be utilized in the therapeutic targeting of TIMP-1 to prevent premature rupture of the fetal membranes in an infectious setting.

Decidualisation of human endometrial stromal cells is associated with increased expression and secretion of prorenin.

BACKGROUND: In pregnancy, the decidualised endometrium expresses high levels of prorenin and other genes of the renin-angiotensin system (RAS) pathway. In this study we aimed to determined if the RAS was present in endometrial stromal cells and if decidualisation upregulated the expression of prorenin, the prorenin receptor ((P)RR) and associated RAS pathways. Immortalised human endometrial stromal cells (HESCs) can be stimulated to decidualise by combined treatment with medroxyprogesterone acetate (MPA), 17ß-estradiol (E2) and cAMP (MPA-mix) or with 5-aza-2'-deoxycytidine (AZA), a global demethylating agent. METHODS: HESCs were incubated for 10 days with one of the following treatments: vehicle, MPA-mix, a combination of medroxyprogesterone acetate (MPA) and estradiol-17ß alone, or AZA. Messenger RNA abundance and protein levels of prorenin (REN), the (P)RR (ATP6AP2), angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1), vascular endothelial growth factor (VEGF), and plasminogen activator inhibitor-1 (PAI-1) were measured by real-time PCR and ELISA's, respectively. Promyelocytic zinc finger (PLZF) and phospho-inositol-3 kinase (PIK3R1) mRNA abundances were also measured. RESULTS: HESCs expressed the prorenin receptor (ATP6AP2), REN, AGT, ACE and low levels of AGTR1. MPA-mix and AZA stimulated expression of REN. Prorenin protein secretion was increased in MPA-mix treated HESCs. E2 + MPA had no effect on any RAS genes. MPA-mix treatment was associated with increased VEGF (VEGFA) and PAI-1 (SERPINE1) mRNA and VEGF protein. CONCLUSIONS: An endometrial prorenin receptor/renin angiotensin system is activated by decidualisation. Since (P)RR is abundant, the increase in prorenin secretion could have stimulated VEGF A and SERPINE1 expression via Ang II, as both ACE and AGTR1 are present, or by Ang II independent pathways. Activation of the RAS in human endometrium with decidualisation, through stimulation of VEGF expression and secretion, could be critical in establishing an adequate blood supply to the developing maternal placental vascular bed.

The rat placental renin-angiotensin system - a gestational gene expression study.

BACKGROUND: The placenta is an essential organ that provides nutrients and oxygen to the developing fetus and removes toxic waste products from the fetal circulation. Maintaining placental blood osmotic pressure and blood flow is crucial for viable offspring. The renin-angiotensin system (RAS) in the placenta is a key player in the regulation of maternal-fetal blood flow during pregnancy. Therefore, the aim of this study was to determine if RAS genes are differentially expressed in mid to late gestation in rat placenta. METHODS: Whole placental tissue samples from pregnant Sprague Dawley rats at embryonic (E) days 14.25, 15.25, 17.25 and 20 (n = 6 for each gestational age) were used for genome-wide gene expression by microarray. RAS genes with expression differences of >2 fold were further analyzed. Quantitative Real-Time PCR (qPCR) was performed on independent samples to confirm and validate microarray data. Immunohistochemisty and Western blotting were performed on a differentially expressed novel RAS pathway gene (ANPEP). RESULTS: Six out of 17 genes of the RAS pathway were differentially expressed at different gestational ages. Gene expression of four genes (Angiotensin converting enzyme (Ace), angiotensin converting enzyme 2 (Ace2), membrane metalloendopeptidase (Mme) and angiotensin II receptor 1A (Agtr1a)) were significantly upregulated at E20 whereas two others (Thimet oligopeptidase 1 (Thop1) and Alanyl aminopeptidase (Anpep)) were downregulated at E20 prior to the onset of labour. These changes were confirmed by qPCR. Western blots revealed no overall differences in ANPEP protein expression in the placentae. Immunohistochemical studies, however, indicated that the localization of ANPEP differed at E17.25 and E20 as ANPEP localization in the giant trophoblast cell of the junctional zone was no longer detectable at E20. CONCLUSIONS: The current study investigated the expression of members of the RAS pathway in rat placentae and observed significantly altered expression of 6 RAS genes at 4 gestational ages. These findings present the need for further comprehensive investigation of RAS genes in normal and complicated pregnancies.

Placental exosomes in normal and complicated pregnancy.

While there is considerable contemporary interest in elucidating the role of placenta-derived extracellular vesicles in normal and complicated pregnancies and their utility as biomarkers and therapeutic interventions, progress in the field is hindered by a lack of standardized extracellular vesicle taxonomy and isolation protocols. The term "extracellular vesicle" is nonspecific and refers to all membrane-bound vesicles from nanometer to micrometer diameters and of different biogenic origins. To meaningfully ascribe biological function and/or diagnostic and therapeutic utility to extracellular vesicles, and in particular exosomes, greater specificity and vesicle characterization is required. The current literature relating to exosome biology must be interpreted in this context. Exosomes are a subtype of extracellular vesicle that are specifically defined by an endosomal biogenesis and particle size (40-120 nm) and density (1.13-1.19 g/mL(-1)). Exosomes are specifically package with signaling molecules (including protein, messenger RNA, microRNA, and noncoding RNA) and are released by exocytosis into biofluid compartments. Exosomes regulate the activity of both proximal and distal target cells, including translational activity, angiogenesis, proliferation, metabolism, and apoptosis. As such, exosomal signaling represents an integral pathway mediating intercellular communication. During pregnancy, the placenta releases exosomes into the maternal circulation from as early as 6 weeks of gestation. Release is regulated by factors that include both oxygen tension and glucose concentration and correlates with placental mass and perfusion. The concentration of placenta-derived exosomes in maternal plasma increases progressively during gestation. Exosomes isolated from maternal plasma are bioactive in vitro and are incorporated into target cells by endocytosis. While the functional significance of placental exosomes in pregnancy remains to be fully elucidated, available data support a role in normal placental development and maternal immunotolerance. Similarly, the role of exosomes in the etiology and progression of complications of pregnancy remains in a formative stage. Changes in the release of placenta- and nonplacenta-derived exosomes, their concentration in maternal plasma, composition, and bioactivity have been reported in association with pregnancies complicated by gestational diabetes and preeclampsia. The data, however, are confounded by the use of different isolation methodologies and vesicle subpopulations. The application of specific and well-characterized isolation methodologies is requisite to resolving the precise role of exosomes in complications of pregnancies and their ultimate clinical utility.

Gestation Related Gene Expression of the Endocannabinoid Pathway in Rat Placenta.

Mammalian placentation is a vital facet of the development of a healthy and viable offspring. Throughout gestation the placenta changes to accommodate, provide for, and meet the demands of a growing fetus. Gestational gene expression is a crucial part of placenta development. The endocannabinoid pathway is activated in the placenta and decidual tissues throughout pregnancy and aberrant endocannabinoid signaling during the period of placental development has been associated with pregnancy disorders. In this study, the gene expression of eight endocannabinoid system enzymes was investigated throughout gestation. Rat placentae were obtained at E14.25, E15.25, E17.25, and E20, RNA was extracted, and microarray was performed. Gene expression of enzymes Faah, Mgll, Plcd4, Pld1, Nat1, Daglα, and Ptgs2 was studied (cohort 1, microarray). Biological replication of the results was performed by qPCR (cohort 2). Four genes showed differential expression (Mgll, Plcd4, Ptgs2, and Pld1), from mid to late gestation. Genes positively associated with gestational age were Ptgs2, Mgll, and Pld1, while Plcd4 was downregulated. This is the first comprehensive study that has investigated endocannabinoid pathway gene expression during rat pregnancy. This study provides the framework for future studies that investigate the role of endocannabinoid system during pregnancy.

Grazing dairy cows had decreased interferon-γ, tumor necrosis factor, and interleukin-17, and increased expression of interleukin-10 during the first week after calving.

Peripartum, and especially during the transition period, dairy cows undergo dramatic physiological changes. These coincide with an increased risk of disease during the first 2 wk after calving and have been linked to dairy cows failing to achieve production as well as reproductive targets. Previous evidence suggests that these physiological changes affect the immune system and that transition dairy cows experience some form of reduced immunocompetence. However, almost all of these studies were undertaken in high-production, housed dairy cows. Grazing cows have much lower levels of production and this study aimed to provide clarity whether or not the dysfunctional attributes of the peripartum immune system reported in high production housed cows are evident in these animals. Therefore, cell culture techniques, flow cytometry, and quantitative PCR were applied to analyze the cellular composition of peripheral blood mononuclear cells from transition dairy cows as well as the performance of these cells in an in vitro assay. First, a combination of in vitro stimulation and quantitative PCR for cytokines was validated as a quantifiable immunocompetence assay in 29 cattle and a correlation of quantitative PCR and ELISA demonstrated. Second, the relative number of T helper cells, cytotoxic T cells, B cells, γδ T cells, natural killer cells, and monocytes in peripheral blood was measured, of which B cells and natural killer cells increased in number postcalving (n=29) compared with precalving. Third, following in vitro stimulation cytokine profiles indicated decreased expression of IFNγ, tumor necrosis factor, and IL-17 and increased expression of IL-10 wk 1 after calving, which later all returned to precalving values (n=39). Additionally, treatment of transition cows with a nonsteroidal anti-inflammatory drug (i.e., carprofen) administered on d 1, 3, and 5 postcalving (n=19; untreated control n=20) did not affect the cytokine expression at any time point. In conclusion, an immunocompetence assay has been developed that highlights a characteristic expression pattern for IFNγ, tumor necrosis factor, IL-17, and IL-10 that reflects a state of reduced immunocompetence in moderate-yielding pasture-based transition cows after calving, which is similar to that described for higher-yielding housed cows.