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Recent studies demonstrating a higher incidence of metabolic disorders after calving have challenged the management practice of increasing dietary energy density during the last ~3 wk prepartum. Despite our knowledge at the whole-animal level, the tissue-level mechanisms that are altered in response to feeding management prepartum remain unclear. Our hypothesis was that prepartum body condition score (BCS), in combination with feeding management, plays a central role in the peripartum changes associated with energy balance and inflammatory state. Twenty-eight mid-lactation grazing dairy cows of mixed age and breed were randomly allocated to 1 of 4 treatment groups in a 2 × 2 factorial arrangement: 2 prepartum BCS categories (4.0 and 5.0, based on a 10-point scale; BCS4, BCS5) obtained via differential feeding management during late-lactation, and 2 levels of energy intake during the 3 wk preceding calving (75 and 125% of estimated requirements). Subcutaneous adipose tissue was harvested via biopsy at -1, 1, and 4 wk relative to parturition. Quantitative polymerase chain reaction was used to measure mRNA and microRNA (miRNA) expression of targets related to fatty acid metabolism (lipogenesis, lipolysis), adipokine synthesis, and inflammation. Both prepartum BCS and feeding management had a significant effect on mRNA and miRNA expression throughout the peripartum period. Overfed BCS5 cows had the greatest prepartum expression of fatty acid synthase (FASN) and an overall greater expression of leptin (LEP); BCS5 was also associated with greater overall adiponectin (ADIPOQ) and peroxisome proliferator-activated receptor gamma (PPARG), whereas overfeeding upregulated expression of proadipogenic miRNA. Higher postpartum expression of chemokine ligand 5 (CCL5) and the cytokines interleukin 6 (IL6) and tumor necrosis factor (TNF) was detected in overfed BCS5 cows. Feed-restricted BCS4 cows had the highest overall interleukin 1 (IL1B) expression. Prepartum feed restriction resulted in greater chemokine ligand 2 (CCL2) expression. Overall, changes in mRNA expression were consistent with the expression pattern of inflammation-related miRNA. These data shed light on molecular mechanisms underlying the effect of prepartum BCS and feeding management on metabolic and inflammatory status of adipose tissue during the peripartum period. Data support the use of a controlled feed restriction prepartum in optimally conditioned cows, as well as the use of a higher level of dietary energy in under-conditioned cows.

Although there is significant interest in elucidating the role of placenta-derived exosomes (PdEs) during pregnancy, the exosomal profile in pregnancies complicated by gestational diabetes mellitus (GDM) remains to be established. The aim of this study was to compare the gestational-age profile of PdEs in maternal plasma of GDM with normal pregnancies and to determine the effect of exosomes on cytokine release from human umbilical vein endothelial cells. A prospective cohort of patients was sampled at three time points during pregnancy for each patient (i.e., 11–14, 22–24, and 32–36 weeks' gestation). A retrospective stratified study design was used to quantify exosomes present in maternal plasma of normal (n = 13) and GDM (n = 7) pregnancies. Gestational age and pregnancy status were identified as significant factors contributing to variation in plasma exosome concentration (ANOVA, P < 0.05). Post hoc analyses established that PdE concentration increased during gestation in both normal and GDM pregnancies; however, the increase was significantly greater in GDM (∼2.2-fold, ∼1.5-fold, and ∼1.8-fold greater at each gestational age compared with normal pregnancies). Exosomes isolated from GDM pregnancies significantly increased the release of proinflammatory cytokines from endothelial cells. Although the role of exosomes during GDM remains to be fully elucidated, exosome profiles may be of diagnostic utility for screening asymptomatic populations.

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.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are major obstetric health problems. Higher levels of T-helper (Th) 1 (proinflammatory) cytokines have been observed in pregnancies complicated with PE and IUGR; this is in contrast to the predominant Th2 (anti-inflammatory) cytokine environment found in uncomplicated pregnancies. Myostatin is best known as a negative regulator of muscle development and reportedly has a role in fat deposition, glucose metabolism, and cytokine modulation (outside the placenta). Myostatin concentrations in plasma and protein expression in placental tissue are significantly higher in women with PE. Expression of myostatin in IUGR and PE-IUGR and the effect of this protein on the cytokine production from the placenta is unknown. In the current study, significant differences were identified in the expression of myostatin in pregnancies complicated with IUGR, PE, and PE with IUGR. Furthermore, cytokine production by first-trimester placental tissues was altered following myostatin treatment.

Precalving feeding level alters postcalving energy balance, dry matter intake, the liver and adipose tissue transcriptome, hepatic lipidosis, and the risk of metabolic diseases in both high-production cows consuming total mixed rations and moderate-production cows grazing pasture. We hypothesized that the reported benefits of a controlled restriction before calving are dependent on precalving body condition score (BCS): low BCS animals would not benefit from reduced feeding levels precalving, but high BCS cows would have metabolic and immunomodulatory profiles indicative of an improved health status. One hundred sixty-one days before calving, 150 cows were allocated randomly to 1 of 6 treatment groups (n = 25) in a 2 × 3 factorial arrangement: 2 precalving BCS categories (4.0 and 5.0; based on a 10-point scale: BCS4 and BCS5, respectively) and 3 levels of energy intake during the 3 wk preceding calving (75, 100, and 125% of estimated requirements). Cows in the BCS4 and BCS5 groups were managed through late lactation to ensure that target calving BCS was achieved at dry off. Cows were then fed to maintain this BCS target until 3 wk before expected calving date, at which point they were managed within their allotted precalving energy intake treatments by offering different allowances of fresh pasture/cow per day. Milk production, body weight, and BCS were measured weekly; blood was sampled weekly before and after calving and on d 0, 1, 2, 3, and 4 relative to calving. Aspirated plasma was assayed for nonesterified fatty acids, β-hydroxybutyrate, total protein, albumin, cholesterol, haptoglobin, IL-1β, IL-6, total antioxidant capacity, and reactive oxygen species. Liver was sampled wk 1, 2, and 4 postcalving for triacylglycerol analysis. Results confirm that precalving BCS and precalving feeding level have both independent and interdependent effects on production and health characteristics of transition dairy cows. Irrespective of precalving BCS, a controlled restriction precalving reduced the net release of nonesterified fatty acids from adipose tissue postpartum and increased plasma calcium concentrations, reducing the risk of milk fever. Fatter cows produced more milk but lost more BCS postcalving and had greater blood β-hydroxybutyrate concentrations and increased hepatic lipidosis. In comparison, after calving, indicators of reduced immune competence were accentuated in BCS4 cows subjected to a feed restriction before calving, probably increasing the risk of infectious diseases. It would appear from these results that optimally conditioned cows will benefit from a short-term (2-3 wk) controlled feed restriction (75-90% of requirements), whereas cows in less than optimal condition should be fed to requirements before calving.

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.

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.

In the past few decades, there has been a global decrease in dairy cow reproductive performance. An activated inflammatory system, due to uterine infection, has been associated with decreased cow fertility and as such, there is a need to detect uterine disease earlier. Early detection could be achieved by identifying biomarkers for uterine disease. Exosomes are small nanovesicles known to package and deliver protein, mRNA, and miRNAs to near and distant sites. Therefore, the content of circulating exosomes may have the potential to carry biomarkers for earlier diagnosis of disease. We hypothesized that circulating exosomes from cows with and without uterine infection may contain information representative of endometrial health or disease. We compared the proteomic content of circulating exosomes derived from plasma of dairy cows with (n = 10) or without (n = 10) induced uterine infection, using high-performance liquid chromatography tandem mass spectrometry (HPLC MS/MS). Our results demonstrate that there were a total of 103 bovine and 9 Trueperella pyogenes proteins found in plasma exosomes derived from infected cows (infected exosomes), and 90 bovine and 5 T. pyogenes proteins found in exosomes derived from plasma of non-infected cows (non-infected exosomes). 71 bovine proteins were found to be unique to the infected exosomes while only 4 bovine proteins were found to be unique to the non-infected exosomes. 8 unique T. pyogenes proteins were identified in infected exosomes and 4 were found to be unique to the non-infected exosomes. Pathway analysis showed that infected exosomes had more proteins involved in structural molecule activity and immune system processes than non-infected exosomal protein. Additionally, proteins from infected exosomes were involved in unique pathways: angiogenesis and integrin signaling pathway. Our data provide preliminary evidence of a potential role for exosomes in the early diagnosis of uterine infection in dairy cows.

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.

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.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of decidual-trophoblast interaction, regulation of trophoblast invasion, immune cells at the maternal-fetal interface, and placental inflammation.

Abnormalities in endometrial function contribute to poor fertility and reproductive failure. Exosomes are small lipid vesicles that contain transferable bioactive substances; they participate in intercellular signaling and may have critical roles in reproductive mechanisms, including endometrial remodeling in preparation for pregnancy. In this study, we evaluated the effects of exosomes from heifers with high and low genetic merit for fertility on inflammatory mediator expression by bovine endometrial epithelial and stromal cell lines. Co-incubation of exosomes from low, compared with high, fertility heifers upregulated the gene expression of pro-inflammatory IL1A and IL8 (CXCL8) but downregulated IL4 gene expression in epithelial cells. In contrast, stromal cells co-incubated with exosomes from low, compared with high, fertility heifers downregulated the gene expression of CXCL9, CXCL10, and CX3CL1. Our findings demonstrated that circulating exosomes from high fertility heifers did not alter endometrial inflammatory mediator gene expression. In contrast, circulating exosomes from low fertility heifers enhanced endometrial expression of inflammatory mediators, which may contribute to aberrant inflammation, leading to a reduced fertility in low fertility heifers. However, an in-depth investigation is required to elucidate the role of exosomes in regulating endometrial remodeling events required for enhanced reproductive performance and fertility in dairy cows.

Extraordinary advances are occurring in biomedical science that may revolutionise how we approach health and disease. Many have applications in the dairy industry. We have described one particular area of extracellular vesicles that have already proven to be of interest in diagnostics and prognostics for fertility and assessment of ‘transition’ cows (i.e. evaluation of the problems related to the risk of clinical diseases in dairy cows, such as mastitis and milk fever, during transition period). The addition of measurements of circulating RNA and DNA may prove of value in identifying dairy cows with higher risks of clinical diseases and potentially poor fertility. We describe the exciting opportunity provided by the possibility of generating exosomes to order as therapeutic agents to potentially enhance fertility. The even more radical concept of using exosomes to deliver a CRISPR-linked gene editing function is presented. Undoubtedly, the use of biomedical advances to assist the dairy industry is an obvious and practical approach that has significant merit.

Previous research results have indicated an increase in pregnancy rate in pasture-grazed cows treated with a nonsteroidal antiinflammatory drug (NSAID) 3 to 4. wk postcalving, when a high proportion of nucleated cells from within the uterus were polymorphonucleated; however, no effect on milk production was detected. It was hypothesized that this lack of effect on milk production was because the administration of the NSAID was too late after calving. The aims of the current study were to evaluate the timing of administering a propionic acid-derived NSAID (i.e., carprofen) on milk production, metabolic status, uterine health, and reproductive performance. Six-hundred and thirty-nine cows (134 primiparous and 505 multiparous) calving between July 4 and September 5, 2012, in 2 herds (herd 1: n. = 228; herd 2: n. = 411) were enrolled. Using a randomized block design, cows were allocated to 1 of 3 treatment groups as they calved: (1) no treatment (control; n. = 221), (2) NSAID administered on d 1, 3, and 5 postcalving (early; n. = 214), and (3) NSAID administered on d 19, 21, and 23 postcalving (late; n. = 204). Milk production and composition, and body condition were determined weekly. Blood was sampled at 4 time points (1 precalving and 3 postcalving) to determine the effects of treatment on indicators of metabolic health and energy status. Uterine health was determined by measuring the proportion of nucleated cells that were polymorphonucleated following cytobrush sampling of the uterus between d 13 to 24 and d 30 to 49 postcalving. Irrespective of timing of application, NSAID did not affect milk production, body weight, or body condition during early lactation. Treatment with an NSAID 19 to 23. d postcalving increased the proportion of cows submitted for breeding during the first 3. wk of the seasonal breeding program (control: 85%, early: 83%, and late: 92%), but did not affect conception or pregnancy rates. No detectable effect of treatment on uterine health or circulating metabolites and minerals existed, although cows in the early NSAID treatment group had marginally lower serum β-hydroxybutyrate concentrations (0.1. mmol/L) than the other groups between 2 and 26. d in milk. In conclusion, administration of this particular NSAID at either 1 or 3. wk after calving did not improve milk production, indicators of health, or reproductive performance.

To determine the effect of lipopolysaccharide (LPS) on NF-κB gene expression and proinflammatory cytokine release from trophoblast cell models, JEG-3 and BeWo human choriocarcinoma cells.Serum-starved JEG-3 and BeWo cells were treated with LPS (from Escherichia coli serotype 0111:B4) for 24 or 48h. Cell culture medium was collected and assayed for interleukin (IL)-1β, IL-6, IL-8, and transforming necrosis factor (TNF)-α cytokine release using enzyme-linked immunosorbent assays. RNA was extracted from the cells and real-time PCR was performed to measure NF-κB mRNA expression. All results were analyzed by one-way analysis of variance tests followed by Sidak's post hoc analysis. p0.05 was considered statistically significant.LPS triggered an inflammatory response in JEG-3 cells by inducing a 1.5-fold increase in NF-κB mRNA expression and TNF-α release (0μg/mL: 15.13±2.14, 1μg/mL: 14.94±0.75, 10μg/mL: 23.05±4.50, p0.05) and a 2-fold elevation in IL-6 secretion (0μg/mL: 12.54±5.44, 1μg/mL: 25.54±0.91, 10μg/mL: 24.28±4.43, p0.05). In contrast, BeWo cells were not as sensitive to LPS exposure; NF-κB mRNA expression was unchanged between LPS-treated and control cells, whereas a small but significant 1.3-fold increase in TNF-α release was found (TNF-α: 15.45±1.53pg/mL, control: 12.24±1.00pg/mL, p0.05). The inflammatory pathways in BeWo cells were found to be active given that treatment of these cells with IL-1β and TNF-α induced IL-6 secretion. Interestingly, 1μg/mL LPS appeared to decrease IL-6 and TNF-α release from BeWo cells. IL-1β and IL-8 secretion were not detected from either cell lines.LPS activates the NF-κB pathway in JEG-3 but not BeWo human choriocarcinoma cells and this may be the reason for their differential inflammatory response to LPS exposure.

The objective of this study was to determine if free AA concentrations in uterine luminal fluid (ULF) and plasma differed between dairy cow strains that differ phenotypically for fertility and to evaluate the effect of the presence of a conceptus on ULF AA concentrations. Uterine luminal fluid was obtained postmortem from cows characterized on the basis of genetic ancestry as fertile (n. = 11) or subfertile (n. = 11) strains. At slaughter, cows were at a similar stage of lactation (fertile, 85. ±. 1. d and subfertile, 87. ±. 1. d postpartum, respectively). Cows were slaughtered on either d 17 of the estrous cycle [nonpregnant (n. = 10): fertile n. = 5; subfertile n. = 5] or d 17 of pregnancy [10. d after embryo transfer, which was undertaken 7. d after estrus (n. = 12, pregnant): fertile n. = 6, subfertile n. = 6]. Uterine luminal fluid was collected from each uterine horn of the pregnant (gravid and nongravid horns) and nonpregnant (horn ipsilateral and contralateral to the corpus luteum) cows. Plasma harvested on the day of slaughter and ULF samples were analyzed for AA determination using HPLC. The main effects of genetic strain, reproductive status, and their interactions on ULF and plasma AA content were tested. Additionally, the effect of uterine horn on ULF AA was tested for the pregnant and nonpregnant cows. Reproductive status had the greatest effect on AA concentrations in ULF. The concentrations of Leu, Met, Phe, Val, 1-methyhistidine, Asp, essential, ketogenic, and branched-chain AA, and those AA classified as both glucogenic and ketogenic were greater in the ULF collected from pregnant cows, with taurine being lower. Additionally, we observed effects of uterine horn and genetic strain × uterine horn interaction for ULF AA concentrations. Concentrations of the essential AA plus Met and Phe were greater in the ULF from the gravid horn, irrespective of strain. The ULF from the gravid horn of fertile cows contained the greatest concentrations of nonessential, glucogenic, branched-chain AA, and Leu, Thr, Ala, Ser, and Asp. With the exception of Asp, plasma AA profiles were not different in fertile and subfertile strains. These data support the hypothesis that reproductive status modifies the AA profiles of the ULF and that these profiles differ in fertile and subfertile genetic strains. Successful pregnancy depends on the complex interactions between the developing conceptus and uterine environment. Understanding the mechanisms contributing to maternal-conceptus communication using models with divergent fertility phenotypes could provide information regarding novel mechanisms to improve dairy cow fertility.

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.

A tenet of contemporary obstetrics is that events that compromise placentation increase the risk of complications of pregnancy and contribute to poor pregnancy outcome. In particular, conditions that affect the invasion of placental cells and remodeling of uterine spiral arteries compromise placental function and the subsequent development of the fetus. Extravillous trophoblast cells (EVTs) proliferate and migrate from the cytotrophoblast in the anchoring villi of the placenta and invade the maternal decidua and myometrium. These cells are localised with uterine uterine spiral arteries and are thought to induce vascular remodeling. A newly identified pathway by which EVTs may regulate vascular remodeling within the uterus is via the release of exosomes. Trophoblast cells release exosomes that mediate aspects of cell-to-cell communication. The aim of this brief commentary is to review the putative role of exosomes released from extravillous trophoblast cells in uterine spiral artery remodeling and, in particular, their role in the aetiology of preeclampsia. Placental exosomes may engage in local cell-to-cell communication between the cell constituents of the placenta and contiguous maternal tissues and/or distal interactions, involving the release of placental exosomes into biological fluids and their transport to a remote site of action.

Introduction: Labour and delivery are processes associated with inflammation within intrauterine and cervical tissues. The mechanisms that induce labour-associated changes and, in particular, the role of microRNAs (miRNAs) remain to be elucidated. MiRNAs are small non-coding RNAs that repress gene expression via mRNA degradation and translational repression. Let-7 miRNAs are negatively regulated by RNA-binding protein, Lin28, and both function downstream of NF-kappa B signalling. In non-gestational tissues, let-7 and Lin28 reportedy function as negative and positive regulators of IL-6 expression. We hypothesised that labour-associated inflammation involves the downregulation of let-7 miRNAs and upregulation of Lin28 expression. Aim: To determine the expression of Lin28 protein and let-7 miRNA in human gestational tissue obtained before and after labour. Method: Gestational tissues were collected from women at term by Caesarean section with and without labour and following normal vaginal delivery (n = 6 per group). Protein and RNA was extracted and Lin28 and let-7 miRNA expression was measured by Western blotting and real-time PCR. Results: The data obtained established that let-7 miRNA and Lin28 display tissue-specific expression: Lin28 was strongly expressed in the placenta and choriodecidua, but not measurable in amnion; and let-7b and -7c expression were significantly lower in choriodecidua compare to placenta and amnion, whereas the amnion expressed less let-7d and -7f than other tissues. Conclusion: While the expression of Lin28 protein and let-7 miRNA did not vary significantly with labour onset and delivery, changes in their bioactivity and impact on nuclear signalling pathways in human gestational tissues remain to be established. (C) 2013 Elsevier Ltd. All rights reserved.

Coordinated regulation of endometrial gene expression is essential for successful pregnancy establishment. A nonreceptive uterine environment may be a key contributor to pregnancy loss, as the majority of pregnancy losses occur prior to embryo implantation. DNA methylation has been highlighted as a potential contributor in regulating early pregnancy events in the uterus. It was hypothesized that DNA methylation regulates expression of key genes in the uterus during pregnancy. The correlation between DNA methylation and gene expression was tested. Endometrial samples from fertile and subfertile dairy cow strains were obtained at day 17 of pregnancy or the reproductive cycle. Microarrays were used to characterize genome-wide DNA methylation profiles and data compared with previously published transcription profiles. 39% of DNA methylation probes assayed mapped to RefSeq genes with transcription measurements. Correlations among gene expression and DNA methylation were assessed, and the 1,000 most significant correlations used for subsequent analysis. Of these, 52% percent were negatively correlated with gene expression. When this gene list was compared with previously reported gene expression studies on the same tissues, 42% were differentially expressed when pregnant and cycling animals were compared, and 11% were differentially expressed when pregnant fertile and subfertile animals were compared. DNA methylation status was correlated with gene expression in several pathways implicated in early pregnancy events. Although these data do not provide direct evidence of a causative association between DNA methylation and gene expression, this study provides critical support for an effect of DNA methylation in early pregnancy events and highlights candidate genes for future studies.

To determine whether increased matrix metalloproteinase (MMP) proteolytic activity plays a pathological role in infection/inflammation-induced preterm brain injury, primary cultures of preterm (day 90 of gestation; term 145 days) fetal ovine mixed glia were exposed to 24-96 h of lipopolysaccharide (LPS, 1 μg/ml) or tumour necrosis factor-α (TNF-α, 100 ng/ml). MMP-2 mRNA levels were significantly increased after TNF-α (96 h) and LPS exposure (48 and 96 h), and MMP-9 mRNA levels were significantly increased at 48 and 96 h after TNF-α. On zymography, the active form of secreted MMP-2 was significantly increased 24 h after LPS, but not TNF-α. Both active and latent forms of MMP-9 gelatinolytic activity were significantly increased by TNF-α (96 h) and LPS (72 and 96 h). On reverse zymography, inhibitory activity of TIMP-1 but not TIMP-2 was significantly increased by TNF-α and LPS. SB-3CT-mediated MMP-2 and MMP-9 inhibition transiently reduced LPS-induced oligodendrocyte cell death but had no effect during TNF-α exposure. Collectively, these observations suggest a limited, transient effect of MMPs on immature white matter damage associated with infection but not TNF-α-mediated inflammation.

Despite continuing significant research efforts and an increasing prevalence of preterm labor (PTL) worldwide, a comprehensive understanding of the mechanisms involved remains to be established; such is the complexity of the interactions involved. Closing the knowledge gaps in this field would afford an opportunity to improve mortality and morbidity rates on a global scale. Early identification of pregnancies at risk of PTL would increase the possibility of delaying birth (where appropriate) prior to initiation of labor. This requires identification of appropriate biomarkers that are readily detectable in easily obtained clinical samples. A family of arachidonic acid metabolites, called eicosanoids. Eicosanoids have shown promise as possible diagnostic biomarkers for PTL. The inherent problems of antibody cross-reactivity in immunoassay methods has, however, resulted in possible misinterpretation of past results. The following mini-review outlines the necessity of such diagnostic targets and the problem with previous research methods and also summarizes recent findings in the field.

Preterm delivery is a major obstetric health problem contributing to poor neonatal outcome including low birth weight, respiratory distress syndrome, gastrointestinal, immunologic, central nervous system, hearing, and vision problems. Worldwide, approximately 15 million babies are born prematurely each year. The critical question which remains is how to identify women destined to deliver preterm from those who will achieve a term delivery. Prostaglandins, in all mammals, are important in the parturient process. Increased intrauterine prostaglandin production is associated with labor and in fact prostaglandin E2 (PGE2) or analogs are widely used clinically for cervical ripening and labor induction. Measurements of circulating eicosanoids have been problematic because of the rapid and major clearance by the lungs and then kidneys resulting in very low concentrations in plasma. Moreover, since eicosanoids are produced by all mammalian tissues, the sources of the measured eicosanoids are unknown. Our understanding of how cells communicate has undergone a paradigm shift with the recognition of the role of exosomes in intercellular signaling. Recent publications have identified enzymes and products of arachidonic acid metabolism (eicosanoids) within exosomes. This review will explore the potential roles of exosomes in eicosanoid functions that are critical in preterm labor and delivery.

The greatest risk of metabolic and infectious disease in dairy cows is during the transition from pregnancy to lactating (i.e., the transition period). The objective of this experiment was to determine the effects of extracellular vesicles (microvesicles involved in cell-to-cell signaling) isolated from transition cows on target cell function. We previously identified differences in the protein profiles of exosomes isolated from cows divergent in metabolic health status. Therefore, we hypothesized that these exosomes would affect target tissues differently. To investigate this, 2 groups of cows (n = 5/group) were selected based on the concentration of β-hydroxybutyrate and fatty acids in plasma and triacylglycerol concentration in liver at wk 1 and 2 postcalving. Cows with high concentrations of β-hydroxybutyrate, fatty acids, and triacylglycerol were considered at increased risk of clinical disease during the transition period (high-risk group; n = 5) and were compared with cows that had low concentrations of the selected health indicators (low-risk group; n = 5). At 2 time points during the transition period (postcalving at wk 1 and 4), blood was sampled and plasma exosomes were isolated from the high-risk and low-risk cows. The exosomes were applied at concentrations of 10 and 1 µg/mL to 5 × 103 Madin-Darby bovine kidney cells grown to 50% confluence in 96-well plates. Results indicate a numerical increase in cell proliferation when exosomes from high-risk cows were applied compared with those from low-risk cows. Consistent with an effect on cell proliferation, quantitative reverse transcriptase PCR indicated a trend for upregulation of 3 proinflammatory genes (granulocyte colony-stimulating factor, ciliary neurotrophic factor, and CD27 ligand) with the application of high-risk exosomes, which are involved in cellular growth and survival. Proteomic analysis indicated 2 proteins in the low-risk group that were not identified in the high-risk group (endoplasmin and catalase), which may also be indicative of the metabolic state of origin. It is likely that the metabolic state of the transition cow affects cellular function through exosomal messaging; however, more in-depth research into cross-talk between exosomes and target cells is required to determine whether exosomes influence Madin-Darby bovine kidney cells in this manner.

Cell-to-cell communication in physiological and pathological conditions may be influenced by neighboring cells, distant tissues, or local environmental factors. Exosomes are specific subsets of extracellular vesicles that internalize and deliver their content to near and distant sites. Exosomes may play a role in the maternal-embryo crosstalk vital for the recognition and maintenance of a pregnancy; however, their role in dairy cow reproduction has not been established. This study aimed to characterize the exosome profile in the plasma of 2 strains of dairy cow with divergent fertility phenotypes. Plasma was obtained and characterized on the basis of genetic ancestry as fertile (FERT;23% North American genetics, New Zealand Holstein-Friesian strain, n=8) or subfertile (SUBFERT;92% North American genetics, North American Holstein-Friesian strain, n=8). Exosomes were isolated by differential and buoyant density centrifugation and characterized by size distribution (nanoparticle tracking analysis, NanoSight NS500, NanoSight Ltd., Amesbury, UK), the presence of CD63 (Western blot), and their morphology (electron microscopy). The total number of exosomes was determined by quantifying the immunoreactive CD63 (ExoELISA kit, System Biosciences), and the protein content established by mass spectrometry. Enriched exosome fractions were identified as cup-shape vesicles with diameters around 100 nm and positive for the CD63 marker. The concentration of exosomes was 50% greater in FERT cows. Mass spectrometry identified 104 and 117 proteins in FERT and SUBFERT cows, of which 23 and 36 were unique, respectively. Gene ontology analysis revealed enrichment for proteins involved in immunomodulatory processes and cell-to-cell communication. Although the role of exosomes in dairy cow reproduction remains to be elucidated, their quantification and content in models with divergent fertility phenotypes could provide novel information to support both physiological and genetic approaches to improving dairy cow fertility.

Free to read at publisher's site. Progesterone, estrogen and cyclic adenosine monophosphate (cAMP) together regulate the decidualization of human endometrial stromal cells in a time-dependent manner. The role of DNA methylation and the three active DNA methyltransferases (DNMTs) in the regulation of decidualization is gaining interest but the exact role of this epigenetic mechanism during decidualization is largely unknown. We aimed to understand the effect of the main regulators of decidualization on the expression of the DNMTs and in turn on the expression of steroid hormone receptors during the decidualization. We conducted a time-course analysis from 6 h to 10 days to examine the change in gene expression of the DNMTs and the steroid hormone receptors over time in response to estradiol, medroxy-progesterone acetate (MPA) and dibutyryl-cAMP (db-cAMP) in a human endometrial stromal cells (HESC) cell line. Only the combination treatment with MPA-mix (estradiol MPA db-cAMP) up-regulated ER, PGR, progesterone receptor B (PRB) and androgen receptor at 24 h. Both decidualization pathways of db-cAMP and estradiol/MPA, independently and combined, consistently down-regulated DNMT3B mRNA expression from 6 h till 10 days, whereas DNMT1 and DNMT3A mRNA expression were down-regulated transiently. Forced expression of DNMT3B in HESC for 10 days attenuated IGFBP1 mRNA and protein expression; and forced expression of DNMT3B combined with MPA-mix treatment synergistically increased the expression of PRB at 24 h. The HESC morphology and proliferation remained unchanged in response to forced expression of DNMT3B. In conclusion, mRNA expression of the DNMTs during decidualization is dynamic, so that expression varies according to the cAMP or estradiol/MPA pathway treatments that regulate them in a time-dependent manner. Although forced expression of DNMT3B by itself is insufficient to inhibit decidualization, forced expression of DNMT3B in combination with MPA-mix synergistically up-regulated PRB, as well as attenuated the expression of IGFBP1, the decidualization marker.

Objectives Gangliosides are structural and functional glycosphingolipids, considered to have important roles in neuronal development in fetal and neonatal development and in memory formation. In this report, we have investigated the ability of bovine milk-derived gangliosides GM3 and GD3 to cross the human placenta. Study design We have employed the ex-vivo model of dually-perfused isolated human placental lobules. Results There was significant uptake of both GD3 and GM3 from the maternal perfusate. There was significant increase of GM3 in the fetal side and a non-statistically significant trend for GD3 to increase on the fetal side. Conclusions Hence an apparent preference for GM3 release into fetal circulation. We suggest that gangliosides consumed by the mother enter her circulation, can be transferred across the placenta and may be available to the developing fetus for building neural connections.

A receptive uterine environment is a key component in determining a successful reproductive outcome. We tested the hypothesis that endometrial gene expression patterns differ in fertile and subfertile dairy cow strains. Twelve lactating dairy cattle of strains characterized as having fertile ( n = 6) and subfertile ( n = 6) phenotypes underwent embryo transfer on day 7 of the reproductive cycle. Caruncular and intercaruncular endometrial tissue was obtained at day 17 of pregnancy, and microarrays used to characterize transcriptional profiles. Statistical analysis of microarray data at day 17 of pregnancy revealed 482 and 1,021 differentially expressed transcripts ( P value < 0.05) between fertile and subfertile dairy cow strains in intercaruncular and caruncular tissue, respectively. Functional analysis revealed enrichment for several pathways involved in key reproductive processes, including the immune response to pregnancy, luteolysis, and support of embryo growth and development, and in particular, regulation of histotroph composition. Genes implicated in the process of immune tolerance to the embryo were downregulated in subfertile cows, as were genes involved in preventing luteolysis and genes that promote embryo growth and development. This study provides strong evidence that the endometrial gene expression profile may contribute to the inferior reproductive performance of the subfertile dairy cow strain.

The current study determined whether the pre-implantation conceptus modified endometrial fatty acid concentrations. Oestrus was synchronized in 14 mature lactating cows and embryos were transferred on day 7. Cows were slaughtered 10 d later, with each uterine horn flushed, the preimplantation conceptus located, and inter-caruncular endometrial tissue collected from the gravid horn (containing the pre-implantation conceptus) and non-gravid horn. Endometrial fatty acid concentrations in the gravid and non-gravid horn were compared using linear models in restricted maxiumum likelihood. Investigations of the correlations among selected fatty acids and trophoblast weight or uterine fluid interferon-tau (IFN-tau) concentrations were also undertaken. The presence of the pre-implantation conceptus had relatively minor effects on endometrial fatty acid concentrations, but the omega 6:omega 3 ratio was greater and concentrations of stearic and oleic acid were slightly increased in the gravid horn. In the gravid horn, a negative linear relationship between the concentration of arachidonic acid and conceptus weight and IFN-tau concentration in the uterine luminal fluid were observed. In contrast, there was a positive relationship between concentrations of dihomo-gamma-linolenic acid in the non-gravid horn and conceptus weight. In conclusion, the presence of the preimplantation conceptus appears to modulate endometrial fatty acids, as indicated by the differences in endometria I fatty acid concentrations in the gravid and non-gravid uterine horns. The physiological implication of these local effects of the pre-implantation conceptus, on reproductive success requires further investigation.

Decidualization, the differentiation of endometrial stromal cells is a crucial step for successful implantation of an embryo, development of the placenta and completion of pregnancy to term. Epigenetic mechanisms are thought to be strongly involved in the regulation of processes controlling implantation, placentation, organ formation and foetal growth. Recent studies suggest that decreased DNA methylation facilitates a receptive endometrium. Hence, the aim of this project was to compare the transcriptional profile changes induced by the inhibitor of DNA methylation, 5-Aza-2'-deoxycytidine (AZA) to the transcriptional changes that happen during decidualization. When DNA methylation was inhibited in a human endometrial stromal cell (HESC) line with AZA, it resulted in the fibroblast-like stromal cells being transformed into decidual-like morphology after 9 days. Expression of both prolactin and insulin-like growth factor binding protein-1, the two established decidualization marker genes, were minimally up-regulated by AZA after 10 days of treatment. In a microarray of a three-way experiment between AZA-treated and oestradiol/progestin/cAMP-treated [medroxy-progesterone acetate (MPA)-mix] HESC and the untreated controls, we detected more than 1000 common genes that had a significant difference of expression compared with the controls. AZA-treated cells in the microarray significantly expressed 76 genes in common with the MPA-mix treated cells, and AZA treatment also differentially regulated 148 genes independently to that of MPA-mix treatment. The MPA-mix regulated at least 36 genes in the cell adhesion, extracellular matrix remodelling and RhoGTPase cytoskeletal reorganization pathway; AZA regulated 19 of these genes in common and 15 other RhoGTPase pathway genes. AZA induced some decidualization-like responses of endometrial stromal cells independently of progestins or cAMP, possibly via the cytoskeletal reorganization pathway of the RhoGTPase family.

Objective: To study transplacental transfer and biotransformation of genistein in the human placenta. Study design and outcomes: Human placentae obtained from healthy term singleton pregnancies were utilised in a dual re-circulating model of ex-vivo placental perfusion. Four placentae were perfused for 180 min following addition of genistein (10 ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate integrity of the circuits. Concentrations of genistein and its conjugates were determined by liquid chromatography-mass spectrometry (LC-MS). Results: The transfer percentage for antipyrine and genistein was 25.6 +/- 1.40% and 22.1 +/- 1.61% respectively and the transfer index for genistein was 0.90 +/- 0.04 after 180 min of perfusion. 12.0 +/- 2.40% of genistein in the fetal compartment and 7.36 +/- 4.73% of genistein in the maternal compartment were in the conjugated form. Conclusions: Genistein can transfer across the human placenta at environmentally relevant levels. Placental metabolizing enzymes conjugate a small fraction of genistein into the glucuronide/sulphate form, which is devoid of estrogenic action. (C) 2010 Elsevier Ltd. All rights reserved.

In Table 3 (page 6478), the means and standard errors of the difference (SED) for 9 of the parameters were tabulated as log values, rather than in the original units. All P-values and Tukey comparisons are correct. The corrected table is shown below, with corrected parameters and values in bold. The authors regret the errors.

The endometrium undergoes morphological and functional changes during the menstrual cycle which are essential for uterine receptivity. These changes are driven by estrogen and progesterone and involve the fine control of many different genes-several of which have been identified as being epigenetically regulated. Epigenetic modification may therefore influence the functional changes in the endometrium required for successful implantation. There is, however, only limited information on epigenetic regulation in endometrium. We review the potential role of epigenetic regulation of key processes during the menstrual cycle and present our own findings following a preliminary study into global acetylation levels in the human endometrium. A changing epigenetic state is associated with the differentiation of stem cells into different lineages and thus may be involved in endometrial regeneration. Histone acetylation is implicated in the vascular endothelial growth factor pathway during angiogenesis, and studies using histone deacetylase inhibitors suggest an involvement in endometrial proliferation and differentiation. The processes of decidualization and implantation are also associated with epigenetic change and epigenetic modulators show variable expression across the menstrual cycle. Our own studies found that endometrial global histone acetylation, as determined by western blotting, changed throughout the menstrual cycle and correlated well with expected transcription activity during the different phases. This suggests that epigenetics may be involved in the regulation of endometrial gene expression during the menstrual cycle and that abnormal epigenetic modifications may therefore be associated with implantation failure and early pregnancy loss as well as with other endometrial pathologies.

Here, we provide evidence for a functional role of artemin (ARTN) in progression of endometrial carcinoma (EC). Increased ARTN protein expression was observed in EC compared with normal endometrial tissue, and ARTN protein expression in EC was significantly associated with higher tumor grade and invasiveness. Forced expression of ARTN in EC cells significantly increased total cell number as a result of enhanced cell cycle progression and cell survival. In addition, forced expression of ARTN significantly enhanced anchorage-independent growth and invasiveness of EC cells. Moreover, forced expression of ARTN increased tumor size in xenograft models and produced highly proliferative, poorly differentiated, and invasive tumors. The ARTN-stimulated increases in oncogenicity and invasion were mediated by increased expression and activity of AKT1. Small interfering RNA-mediated depletion or antibody inhibition of ARTN significantly reduced oncogenicity and invasion of EC cells. Thus, inhibition of ARTN may be considered as a potential therapeutic strategy to retard progression of EC.

Intrauterine inflammation plays a major role in the etiology of preterm labor and birth. We established an ex vivo model employing perfused full-thickness term gestational membranes to study membrane transport, function, and inflammatory responses. Exposure of the maternal (decidual) face of the membranes to LPS (5 μg/ml) resulted in increased accumulation of proinflammatory cytokines in the maternal compartment within 4 h, followed by a response in the fetal (amniotic) compartment. Using cytokine arrays, exposure to LPS was found to result in increased secretion of a large number of cytokines and chemokines in both compartments, most notably IL-5, IL-6, IL-7, MDC (macrophage-derived chemokine), MIG (monokine induced by IFN-γ), TARC (thymus and activation-regulated chemokine), TGF-β, and TNF-α. PGE2 accumulation also increased in response to LPS, particularly in the fetal compartment. Cotreatment with sulfasalazine, which inhibited nuclear translocation of NF-κB p65, had a rapid and marked inhibitory effect on the rate of cytokine accumulation in the maternal compartment, with lesser but significant effects observed in the fetal compartment. While membrane integrity was not discernibly impaired with LPS or sulfasalazine exposure, rates of chorionic apoptosis after 20 h were doubled in sulfasalazine-treated tissues. We conclude that the system described provides a means of accurately modeling human gestational membrane functions and inflammatory activation ex vivo. Decidual LPS exposure was shown to elicit a robust inflammatory response in both the maternal and fetal compartments. Sulfasalazine was an effective antiinflammatory agent in this model, but also exerted proapoptotic effects that raise concerns regarding its placental effects when administered in pregnancy.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in brain development and the etiology of adult cerebral injuries. In this study, we determined the MMP-2 and 9 responses following hypoxic ischemia (HI) injury in the developing brain. First, we characterized the developmental changes of MMP activity in the rat brain from embryonic day 18 (E18) to postnatal day 120 (P120). MMP-2 activity was high from E18 to P3 and decreased with age (Por =0.001), while MMP-9 activity was not detectable. MMP-2 immunoreactivity was closely associated with differentiating cortical plate and subplate neurons. Next, we characterized the proteolytic changes after unilateral HI brain injury in 3- (P3) and 21- (P21) day-old rats. Zymography revealed that in the P21 rat brain, MMP-9 activity (150 and 92 kDa forms) was increased at 6 h and remained elevated 24 h post-injury in the ipsilateral injured hemisphere (Por =0.001), whereas there was a gradual increase in MMP-2 (65 kDa) activity, reaching a peak at 5 days (Por =0.001). Similarly, quantitative real time polymerase chain reaction (qRT-PCR) indicated significant elevations in MMP-9 and MMP-2 mRNA expression in the injured cortex (Por =0.05) and hippocampus (Por =0.05) at 1 and 5 days post-injury, respectively in the P21 rat brain. In the P3 rat brain, zymography results revealed that both pro (92 kDa) and cleaved (87 kDa) MMP-9 activities were upregulated in the ipsilateral injured hemisphere from 6 h to 1 day after injury (Por =0.001). In contrast, cleaved MMP-2 (60 kDa) was only moderately upregulated at 6 h (Por =0.01), while pro MMP-2 (65 kDa) levels were unaffected. MMP-9 mRNA expression was also increased at 6 h (Por =0.05) following injury at P3, whereas MMP-2 expression remained unchanged compared with the uninjured contralateral hemisphere. Immunohistochemistry indicated that MMP-9 protein expression was localized predominantly to neurons and peri-vascular astrocytes in the affected regions at early time points, whereas MMP-2 was present on reactive astrocytes surrounding the infarct at later time points. Together, these results indicate that MMP-2 may be primarily associated with the development and differentiation of cortical plate neurons and wound recovery processes. Conversely, MMP-9 appeared to be associated with more acute processes during the period of lesion development.

Key to the success of human reproduction is the capacity of an embryo to attach and implant into the endometrial wall after which a nutrient supply is established through placentation. Herein, we have examined the potential epigenetic regulation of uterine receptivity by use of the receptive RL95-2 and nonreceptive AN3-CA endometrial epithelial carcinoma cell lines. Using an in vitro model of embryo implantation, we demonstrate that inhibition of DNA methylation by 5′-aza-2′-deoxycytidine (AZA), resulted in the nonreceptive AN3-CA cell line becoming receptive to BeWo cell spheroid attachment. Examination of components of the adherens junction complex revealed that AZA specifically increased the expression of E-cadherin and plakoglobin at the mRNA and protein levels in AN3-CA cells, and E-cadherin protein expression was found to localize to sites of intercellular contact. Forced expression of E-cadherin in AN3-CA cells significantly enhanced receptivity. Small interfering RNA (siRNA)-mediated depletion of the individual DNA methyltransferase (DNMT) molecules did not induce E-cadherin expression in AN3-CA cells; however, concomitant siRNA-mediated depletion of both DNMT3A and DNMT3B induced the expression of E-cadherin. Furthermore, E-cadherin expression was significantly increased after the concomitant siRNA-mediated depletion of DNMT-1, -3A, and -3B in AN3-CA cells. Therefore, we have provided evidence that E-cadherin plays an important role in uterine receptivity and that E-cadherin expression is epigenetically regulated in AN3-CA cells, suppressed by the combined actions of DNMT-1, -3A, and -3B. Inhibition of DNA methylation increases both E-cadherin expression and receptivity in an endometrial cell line, suggesting that endometrial receptivity in human pregnancy is epigenetically regulated.

Recent published data have demonstrated elevated levels of human GH (hGH) in endometriosis and endometrial adenocarcinoma. Herein, we demonstrate that autocrine production of hGH can enhance the in vitro and in vivo oncogenic potential of endometrial carcinoma cells. Forced expression of hGH in endometrial carcinoma cell lines RL95-2 and AN3 resulted in an increased total cell number through enhanced cell cycle progression and decreased apoptotic cell death. In addition, autocrine hGH expression in endometrial carcinoma cells promoted anchorage-independent growth and increased cell migration/invasion in vitro. In a xenograft model of human endometrial carcinoma, autocrine hGH enhanced tumor size and progression. Changes in endometrial carcinoma cell gene expression stimulated by autocrine hGH was consistent with the altered in vitro and in vivo behavior. Functional antagonism of hGH in wild-type RL95-2 cells significantly reduced cell proliferation, cell survival, and anchorage-independent cell growth. These studies demonstrate a functional role for autocrine hGH in the development and progression of endometrial carcinoma and indicate potential therapeutic relevance of hGH antagonism in the treatment of endometrial carcinoma.

Trefoil factors (TFFs), in particular TFF1, are classical estrogen-regulated genes and have served as markers of estrogen gene regulation by various environmental estrogens. TFFs are also regulated by several other factors including growth hormone (hGH), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF) and various oncogenic stimuli. TFFs are secreted proteins present in serum and possess the potential to act as growth factors promoting cell survival, anchorage-independent growth and motility. Recent compelling evidence has emerged from experimental and clinical studies to indicate a pivotal role of TFFs in oncogenic transformation, growth and metastatic extension of common human solid tumours. This review will summarize the current evidence for the involvement of TFFs in human cancer.

The appropriate modulation of gene expression in gestational tissues is an essential requirement for a successful pregnancy. Epigenetic control of gene expression through the reversible modifi cation of chromatin has emerged as a fundamental mechanism for the coordination of gene expression in a range of biological systems. Here we summarize recent results in support of our hypothesis that key physiological events and pathological processes occurring throughout human pregnancy are under epigenetic control.

An increase in intrauterine prostaglandin production is critical for the onset and progression of labor in women and indeed all mammalian species studied. Endocannabinoids can act as substrates for enzymes of the prostaglandin biosynthetic pathways and can be utilized to generate other related compounds such as prostamides. The end products are indistinguishable by radioimmunoassay. We have separated such compounds by mass spectrometry. We now show that inflammatory stimuli such as LPS and proinflammatory cytokines act differentially on these pathways in human choriodecidua and preferentially create drive through to prostaglandin end products. These findings create doubt about the interpretation of data on prostaglandin biosynthesis in intrauterine tissues from pregnant women especially in the presence of an infection. The possibility is raised that separation of these products might reduce variability in results and lead to potential uses for their measurement in the diagnosis of preterm labor.

Proteins are key regulators of the biochemistry of cells and, therefore, in the maintenance of good health. It is important to both identify and quantify the proteins that are causative of a particular pathology since this information can inform the clinician as to both the selection of the correct treatment and monitor the success of its use. In the previous chapter, we described the methods used to analyse proteins. Here we turn our attention to the application of proteomics in the clinical context, outline the markers currently in use and describe in more depth several examples of proteins of clinical importance.

Directed by microarray analyses, we report that autocrine human growth hormone (hGH) increased the mRNA and protein expression of DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B in mammary carcinoma cells. Autocrine hGH stimulation of DNMT3A and DNMT3B expression was mediated by JAK2 and Src kinases, and treatment of mammary carcinoma cells with the DNMT inhibitor, 5'-aza-2'-deoxycytidine (AZA), abrogated autocrine hGH-stimulated cellular proliferation, apoptosis and anchorage-independent growth. AZA reversed the epitheliomesenchymal transition of mammary carcinoma cells induced by autocrine hGH, to an epithelioid morphology and abrogated cell migration stimulated by autocrine hGH. Autocrine hGH-stimulated hypermethylation of the first exon of the PLAKOGLOBIN gene and AZA abrogated the ability of autocrine hGH to repress plakoglobin gene transcription. Small interfering RNA (siRNA)-mediated depletion of the individual DNMT molecules did not release autocrine hGH repression of PLAKOGLOBIN promoter activity nor did individual DNMT depletion affect autocrine hGH-stimulated migration. However, concomitant siRNA-mediated depletion of both DNMT3A and DNMT3B abrogated hypermethylation of the PLAKOGLOBIN gene stimulated by autocrine hGH and subsequent repression of plakoglobin gene transcription and increased cell migration. Thus, the autocrine hGH-stimulated increases in DNMT3A and DNMT3B expression mediate repression of plakoglobin gene transcription by direct hypermethylation of its promoter and consequent phenotypic conversion of mammary carcinoma cells. Autocrine hGH, therefore, utilizes DNA methylation as a mechanism to exert its oncogenic effects in mammary carcinoma cells.

Myostatin is a member of the transforming growth factor (TGF)-β superfamily, known for its ability to inhibit muscle growth. It can also regulate metabolism and glucose uptake in a number of tissues. To determine the mechanism of myostatin's effect on glucose uptake, we evaluated its actions using choriocarcinoma cell lines that are widely used as models for placental cells. Protein and mRNA were determined using immunoblotting and RT-PCR/PCR, respectively. Glucose uptake was assessed by uptake of radiolabeled deoxyglucose in vitro. All choriocarcinoma cell lines tested i.e., BeWo, JEG, and Jar, are used as models of placental cells, and all expressed myostatin protein and mRNA. Treatment of BeWo cells with myostatin resulted in inhibition of glucose uptake in a concentration-dependent manner ( P < 0.01). At all concentrations tested, follistatin, a functional inhibitor of myostatin, completely blocked the inhibitory effect of myostatin (40 nM) on glucose uptake by BeWo cells (0.4 nM, P < 0.05). Follistatin treatment alone also increased glucose uptake (0.4 and 4 nM, P < 0.001; 40 nM, P < 0.05). Because BeWo cells proliferated and greater cell densities were achieved, glucose uptake declined irrespective of treatment. Myostatin treatment of BeWo cells did not alter the levels of myostatin receptor, ActRII A/B proteins. The levels of glucose transport proteins also remained unaltered in BeWo cells with myostatin treatment. This study has shown that myostatin specifically inhibits glucose uptake into BeWo cells, suggesting that locally produced myostatin may control glucose metabolism within the placenta.

Following Barker's observations of an association between birth size and later adult diseases, considerable efforts have been made to define the characteristics of low birth weight groups in childhood. In this review, the phenotypic and biochemical characteristics during childhood of three low birth weight groups are summarized: children born following inviter fertilization (IVF), small for gestational age (SGA), or very premature. Each of these groups is likely to have been exposed to an adverse environment at different developmental stages. The triggers and mechanisms leading to programmed changes in growth, development, and metabolism of these groups of children have yet to be identified. Epigenetics has been proposed as a potential mechanism for these programmed changes through environmentally induced changes in gene expression. Data from animal models in which environmental, particularly nutritional, manipulation leads to changes in DNA methylation are presented. The relevance of these animal studies to IVF, SGA, and very premature children are discussed as are potential candidate genes that may have undergone epigenetic modification to alter growth and metabolism.