Placental Transcriptome Analysis in Connection with Low Litter Birth Weight Phenotype (LBWP) Sows.

It is possible to identify sub-populations of sows in every pig herd that consistently give birth to low birth weight (BW) piglets, irrespective of the litter size. A previous study from our group demonstrated that placental development is a main factor affecting the litter birth weight phenotype (LBWP) in sows, thereby impacting the BW of entire litters, but the biological and molecular pathways behind this phenomenon are largely unknown. The aim of this study was to investigate the differential gene expression in placental tissues at day 30 of gestation between low LBWP (LLBWP) vs. high LBWP (HLBWP) sows from a purebred Large White maternal line. Using mRNA sequencing, we found 45 differentially expressed genes (DEGs) in placental tissues of LLBWP and HLBWP sows. Furthermore, (GO) enrichment of upregulated DEGs predicted that there were two biological processes significantly related to cornification and regulation of cell population proliferation. To better understand the molecular interaction between cell proliferation and cornification, we conducted transcriptional factor binding site (TFBS) prediction analysis. The results indicated that a highly significant TFBS was located at the 5' upstream of all four upregulated genes (CDSN, DSG3, KLK14, KRT17), recognized by transcription factors EGR4 and FOSL1. Our findings provide novel insight into how transcriptional regulation of two different biological processes interact in placental tissues of LLBWP sows.

Postnatal development of skeletal muscle in pigs with intrauterine growth restriction: morphofunctional phenotype and molecular mechanisms.

Intrauterine growth restriction (IUGR) is a serious condition which impairs the achievement of the fetus' full growth potential and occurs in a natural and severe manner in pigs as a result of placental insufficiency. Reduced skeletal muscle mass in the fetus with IUGR persists into adulthood and may contribute to increased metabolic disease risk. To investigate skeletal muscle postnatal development, histomorphometrical patterns of the semitendinosus muscle, myosin heavy chain (MyHC; embryonic I, IIA, IIB and IIX isoforms) fiber composition and the relative expression of genes related to myogenesis, adipogenesis and growth during three specific periods: postnatal myogenesis (newborn to 100 days old), hypertrophy (100-150 days old), and postnatal development (newborn to 150 days old) were evaluated in female pigs with IUGR and normal birth weight (NW) female littermates. NW females presented higher body weights compared to their IUGR counterparts at all ages evaluated (P < 0.05). Moreover, growth restriction in utero affected the semitendinosus muscle weight, muscle fiber diameter, and muscle cross-sectional area, which were smaller in IUGR pigs at birth (P < 0.05). Notwithstanding the effects on muscle morphology, IUGR also affected muscle fiber composition, as the percentage of MyHC-I myofibers was higher at birth (P < 0.05), and, in 150-day-old gilts, a lower percentage of MyHC-IIX isoform (P < 0.05) and the presence of embryonic MyHC isoform were also observed. Regarding the pattern of gene expression in both the postnatal myogenesis and postnatal development periods, IUGR led to the downregulation of myogenic factors, which delayed skeletal muscle myogenesis (PAX7, MYOD, MYOG, MYF5 and DES). Altogether, growth restriction in utero affects muscle fiber number and size at birth and muscle fiber composition through the downregulation of myogenic factors, which determines the individual´s postnatal growth rate. This fact, associated with delayed myofiber development in growth-restricted animals, may affect meat quality characteristics in animal production. Hence, knowledge of the morphofunctional phenotype of the skeletal muscle throughout postnatal development in individuals with IUGR, and the mechanism that governs it, may provide a better understanding of the mechanisms that limit postnatal muscle growth, and help the establishment of potential strategies to improve muscle development and prevent the onset of later-life metabolic diseases.

Gene ontology analysis of expanded porcine blastocysts from gilts fed organic or inorganic selenium combined with pyridoxine.

BACKGROUND: Gene ontology analysis using the microarray database generated in a previous study by this laboratory was used to further evaluate how maternal dietary supplementation with pyridoxine combined with different sources of selenium (Se) affected global gene expression of expanded porcine blastocysts. Data were generated from 18 gilts randomly assigned to one of three experimental diets (n = 6 per treatment): i) basal diet without supplemental Se or pyridoxine (CONT); ii) CONT + 0.3 mg/kg of Na-selenite and 10 mg/kg of HCl-pyridoxine (MSeB610); and iii) CONT + 0.3 mg/kg of Se-enriched yeast and 10 mg/kg of HCl-pyridoxine (OSeB610). All gilts were inseminated at their fifth post-pubertal estrus and euthanized 5 days later for embryo harvesting. Differential gene expression between MSeB610 vs CONT, OSeB610 vs CONT and OSeB610 vs MSeB610 was performed using a porcine embryo-specific microarray. RESULTS: There were 559, 2458, and 1547 differentially expressed genes for MSeB610 vs CONT, OSeB610 vs CONT and OSeB610 vs MSeB610, respectively. MSeB610 vs CONT stimulated 13 biological processes with a strict effect on RNA binding and translation initiation. OSeB610 vs CONT and OSeB610 vs MSeB610 impacted 188 and 66 biological processes, respectively, with very similar effects on genome stability, ceramide biosynthesis, protein trafficking and epigenetic events. The stimulation of genes related with these processes was confirmed by quantitative real-time RT-PCR. CONCLUSIONS: Gene expression of embryos from OSeB610 supplemented gilts was more impacted than those from MSeB610 supplemented gilts. Whereas maternal OSeB610 supplementation influenced crucial aspects of embryo development, maternal MSeB610 supplementation was restricted to binding activity.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Morphologic and transcriptomic assessment of bovine embryos exposed to dietary long-chain fatty acids.

The main objectives of this study were to determine the influence of diets enriched in α-linolenic, linoleic or oleic acid on the development and transcriptomic profile of embryos collected from dairy cattle. Non-lactating Holstein cows received one of the three diets supplemented with 8% rolled oilseeds: flax (FLX, n = 8), sunflower (SUN, n = 7) or canola (CAN, n = 8). After a minimum 35-day diet adaptation, cows were superovulated, artificially inseminated and ova/embryos recovered non-surgically after 7.5 days. Cows fed FLX had less degenerated embryos and more viable embryos than those fed CAN or SUN. In total, 175 genes were differentially expressed in blastocysts from cows fed FLX than in cows fed CAN or SUN. These differentially expressed genes were mainly involved in cellular growth and proliferation, cellular development, and cell survival and viability. In conclusion, dietary n-3 polyunsaturated fatty acids reduced early embryonic degeneration possibly through improving embryonic cell survival and viability.

Identification of differentially expressed genes in sexed pig embryos during post-hatching development in primiparous sows exposed to differing intermittent suckling and breeding strategies.

The aim of commercial pig breeding programs is to maximize the number of pigs produced per sow per year. Given that sows exhibit an estrus during lactation is a potential means of increasing productivity of a pig breeding herd without reducing in lactation length, conventionally, weaning of piglets at a relatively young age is often related to post-weaning piglet performance which compromises piglet welfare. Therefore, intermittent suckling (IS) is a management technique in which lactating sows are separated from their piglets for a fixed period of the days and allowing sows to continue nursing piglets while exhibiting estrus and being breed during lactation, thereby promoting both piglet well-being and sow reproductive performance [1]. For this study, primiparous sows (PP) were exposed to 28 day (D28) lactation with intermittent suckling (IS) during the final week prior to weaning. The sows detected to be in estrus during lactation were either bred at this first estrus (FE) during lactation (IS21FE), or were "skipped" and bred at their second estrus which occurred after final weaning at D28 (IS21SE). Despite the benefits of IS, the effects of the maternal physiology related to breeding during lactation on embryonic transcriptome are largely unknown. Recent advances in the ability to assess embryonic gene expression in both sexes have made these analyses possible. Here, we describe the experimental procedures of two color microarray analyses and annotation of differentially expressed (DE) genes in detail corresponding to data deposited at NCBI in the Gene Expression Omnibus under accession number GSE53576 and GSE73020 for day 9 embryos (D9E) and day 30 embryos (D30E) respectively. Although only a few DE genes were discovered between IS21FE and IS21SE in both sexes from D9E or D30E, the raw data are still valuable for future use to understand the gene expression profiling from two different developmental stages.

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR.

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo's sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

RNA amplification for pseudogene detection using RNA-seq.

Transcriptomic research using microarrays and RNA-Sequencing (RNA-seq) is now possible starting from minute biological samples, such as clinical specimens or embryos, due to the development of highly sensitive and reproducible cDNA synthesis methods. Here, we describe a quick method of RNA amplification and double-stranded cDNA synthesis starting with 10 ng of high-quality total RNA extracted from porcine embryos. The final product (double-stranded DNA) is adequate for the detection by RNA-seq of protein-coding transcripts, as well as of all the other classes of noncoding RNAs, including pseudogenes.

Characterization of the altered gene expression profile in early porcine embryos generated from parthenogenesis and somatic cell chromatin transfer.

The in vitro production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from in vitro Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than in vivo-derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of in vivo (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The "eIF2 signalling", "mitochondrial dysfunction", "regulation of eIF4 and p70S6K signalling", "protein ubiquitination", and "mTOR signalling" pathways were down-regulated in PA HB. Dysregulation of notch signalling-associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.

The effects of glial cell line-derived neurotrophic factor on the in vitro matured porcine oocyte transcriptome.

It is well documented that oocytes from small antral follicles are less competent than those derived from large follicles, and we have previously shown that glial cell line-derived neurotrophic factor (GDNF) enhances developmental competence in oocytes from antral follicles. Exactly how GDNF effects this change and if it depends on the stage of oocyte development is currently unknown. The objective of this study was to examine the transcriptomic effects of follicle size and GDNF on the in vitro maturation of porcine oocytes. Microarray analysis uncovered differentially expressed transcripts among in vitro-matured porcine oocytes from different-size antral follicles, in the absence or presence of GDNF. Oocytes isolated from small follicles showed a lower state of maturation than those from large follicles, with several transcripts associated with meiotic arrest. Addition of GDNF to the culture media had effects that depended on the stage of the follicle from which the oocyte was isolated, with those from small follicles showing decreased expression of genes associated with acetyltransferase activity while those from large follicles showed decreased metabolic activity. In summary, our results revealed considerable differences between the transcriptomes of small- and large-follicle-derived oocytes. Furthermore, GDNF affects the developmental competence of oocytes in follicle-stage dependent manner. Thus, improving our understanding of the requirements for successful in vitro maturation of porcine oocytes will inform current reproductive technologies, with implications for the future of animal and human health.

Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation.

BACKGROUND: The domestic pig is an important livestock species and there is strong interest in the factors that affect the development of viable embryos and offspring in this species. A limited understanding of the molecular mechanisms involved in early embryonic development has inhibited our ability to fully elucidate these factors. Next generation deep sequencing and microarray technologies are powerful tools for delineation of molecular pathways involved in the developing embryo. RESULTS: Here we present the development of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using 454 Titanium pyrosequencing technology. Over one million high-quality EST sequences were obtained and used to develop the EMbryogene Porcine Version 1 (EMPV1) microarray composed of 43,795 probes. Based on an initial probe sequence annotation, the EMPV1 features 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA, 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control on the EMPV1 was performed and revealed an even distribution of ten clusters of spiked-in control spots and array to array (dye-swap) correlation was 0.97. CONCLUSIONS: Using next-generation deep sequencing we have produced a large EST dataset to allow for the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo-specific array was confirmed with a high-level of reproducibility using current Agilent microarray technology. With more than an estimated 20,000 unique genes represented on the EMPV1, this platform will provide the foundation for future research into the in vivo and in vitro factors that affect the viability of porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos.

Differential gene expression profiling in HELLP syndrome placentas.

The aim of this study was to identify differentially expressed genes by suppression subtractive hybridization (SSH) in HELLP placentas. Two cDNA libraries were constructed; HSI (HELLP subtracted induced or upregulated) and HSS (HELLP subtracted suppressed or downregulated). Two hundred eighty-eight cDNA clones were sequenced; 37 were matched to GenBank entries and included genes in cell communication and organization, cellular processes, genetic information processing, and metabolic processes. A subgroup of 11 genes of interest was further selected for real-time quantitative polymerase chain reaction confirmation. Results showed no differences in expression of chosen upregulated genes between HELLP and non-HELLP placentas; 6 HELLP downregulated genes were significantly suppressed. Two genes related to production of secreted proteins, CTHRC1 and SERPINE2. SERPINE2 (PAI-1) is a soluble protease inhibitor and is a potential biomarker by Western blot analysis, and the protein is significantly decreased in HELLP placentas. SERPINE2 might be tested clinically in patients for early diagnosis of HELLP syndrome.

Gene expression in diapause-destined embryos of the crustacean, Artemia franciscana.

Diapause-destined embryos of the crustacean Artemia franciscana cease development as gastrulae, encyst, and enter a resting stage characterized by greatly reduced metabolic activity and extreme stress resistance. To better understand diapause induction and maintenance in Artemia embryos gene expression was analyzed by subtractive hybridization at two days post-fertilization, a time early in this developmental process. Eighty-five of 264 cDNA clones sequenced matched GenBank entries and they fell into categories designated as environmental information processing, cellular processes, genetic information processing and metabolism. Semi-quantitative RT-PCR of cDNAs populating the subtractive library identified seventeen up-regulated and four down-regulated transcripts, the former including those encoding a human transcription cofactor homologue, three small heat shock proteins, putative cell growth suppressor proteins and several enzymes. As examples, p8 may modulate gene expression during diapause in Artemia embryos. BRCA1 associated protein-1 (BAP1) and other functionally related proteins may influence cell growth and division during transition into diapause, a time when these processes are inhibited, whereas small heat shock proteins protect embryos from stress. This study represents the first systematic molecular characterization of diapause in crustaceans. Several differentially expressed genes were identified, expanding the repertoire of proteins potentially modified during diapause and suggesting mechanistic pathways indigenous to the initiation and maintenance of this physiological state.

Markers of oxidative stress in placental villi exposed to ethanol.

OBJECTIVE: Ethanol exposure during pregnancy may result in fetal alcohol syndrome (FAS). The mechanism by which this occurs is unknown. Recent studies in several organ systems, including the placenta, suggest that oxidative stress is involved. In this study we investigated the presence and levels of three oxidative stress markers in placental villous tissue exposed to ethanol. METHODS: Villous tissues from normal placentas were perfused with Dulbeco's modified Eagle's medium (DMEM) with HEPES buffer, sodium bicarbonate, and glucose at pH 7.4. After stabilization, 100 mM ethanol was added to the perfusate. After 2 hours of perfusion, the tissue was removed, fixed and stained for nitrotyrosine, 4-hydroxy-2-nonenal (4HNE) and 8-hydroxyguanosine (8-OHDG). Staining within the trophoblasts was quantified with densitometry. RESULTS: Nitrotyrosine and 4HNE immunostaining was seen in the trophoblasts. 4HNE was also seen in the stroma. In contrast, 8-OHDG was seen only in the stroma and endothelial cells in the fetal circulation. Ethanol exposure significantly increased nitrotyrosine levels in the trophoblasts beyond levels in the control tissue. Nitrotyrosine and 8-OHDG levels were also increased in stroma. CONCLUSION: Within the placental villi, markers of oxidative stress are present in the trophoblasts and stroma after a short period of ethanol exposure. There is an increase in oxidative stress, primarily involving the nitric oxide pathway, in the trophoblasts as well as DNA damage in the stroma. Lipid peroxidation is not acutely changed in our 2-hour exposure window.

Identification of a transcript encoding a soluble form of toll-like receptor 5 (TLR5) in Atlantic salmon during Aeromonas salmonicida infection.

Toll-like receptors (TLRs) are involved in the innate immune response against microbial pathogens in vertebrates and insects. The extracellular region of a TLR recognizes pathogen-associated molecules, while the intracellular region initiates the signaling pathway leading to immune response. Membrane-bound TLRs have been found in most vertebrates, but few soluble forms have been reported. A novel transcript corresponding to a portion of a soluble TLR was identified in liver of infected Atlantic salmon. The complete coding sequence of this TLR was obtained and BLASTN analysis showed the highest sequence identity to a recently described full-length cDNA sequence of a soluble TLR5 from rainbow trout (GenBank Accession No.: ). The deduced protein is 40% identical to the mammalian counterpart of the leucine-rich repeat (LRR)/LRR-like motifs of TLR5. Based on the structure of human TLRs, it contains 21 LRRs with conserved LxxLxLxxNx*xx*xxxxFxxL pattern. Since TLR5 is essential for the recognition of bacterial flagellins, we hypothesize that flagellin and perhaps some other pathogen-derived factors from Aeromonas salmonicida bind to this soluble TLR through an unknown binding domain within the LRR.

Dietary rapeseed oil affects the expression of genes involved in hepatic lipid metabolism in Atlantic salmon (Salmo salar L.).

Supplies of marine fish oils (FO) are limited, and sustainable production in aquaculture dictates that alternatives that do not compromise fish health and product quality, such as vegetable oils, must be found. Nutrigenomics will increase our understanding of how nutrition influences metabolic pathways and homeostatic control, and may be used to measure and validate subtle changes in organ-specific, metabolic gene expression signatures. We compared 2 groups of Atlantic salmon fed diets containing 100% FO or 75% rapeseed oil (RO) for 42 wk. A small-scale cDNA microarray was constructed to screen for changes in the expression of lipid metabolism genes in the liver resulting from this partial substitution of RO for FO. Delta5 fatty acid desaturase gene expression was significantly greater in fish fed 75% RO than in fish fed the control diet; this was confirmed by quantitative real time PCR analysis. In addition, several genes, among these mitochondrial proteins, peroxisome proliferator-activated receptor gamma, as well as other transcription factors, coactivators, and signal transducers, showed significant differential regulation. This partially validated microarray may be used for further gene expression profiling using other dietary comparisons, and for further characterization of selected genes.

Identification of genes differentially expressed in Atlantic salmon (Salmo salar) in response to infection by Aeromonas salmonicida using cDNA microarray technology.

The response of Atlantic salmon, Salmo salar, to infection by the bacterial pathogen Aeromonas salmonicida (the causative agent of furunculosis), was investigated using a cohabitation model and a custom Atlantic salmon cDNA microarray consisting of over 4000 different amplicons. Pooled samples of each of three immune-relevant tissues (spleen, head kidney and liver) were obtained from fish exposed to infected salmon for 13 days. Reverse transcription-PCR assays were used to verify the differential expression of 12 candidate genes uncovered by microarray analysis. Among the differentially expressed genes were several previously revealed by suppression subtractive hybridization and EST surveys and that are recognized to encode humoral components of the innate immune system. Other genes identified in this study were not previously associated with infection. In addition, a number of genes with no known homologs were uncovered. Determination of their specific roles during infection may lead to a better understanding of innate immunity.

Molecular cloning of ovine endothelial nitric oxide synthase and expression in COS-7 cells.

While studies of human and bovine endothelial nitric oxide synthase (eNOS) demonstrate activation by Ca(2+)/calmodulin, recent progress demonstrates that eNOS phosphorylation can alter sensitivity to intracellular free calcium ([Ca(2+)](i)). The sheep, however, is widely used as a model for cardiovascular adaptation to pregnancy and ovine uterine artery endothelial cell (UAEC) eNOS undergoes pregnancy-specific (P) enhancement of activity associated with increased Ca(2+) and protein kinase signaling in response to a number of agonists, including adenosine triphosphate (ATP). The degree of homology between the ovine and human full-length cDNAs was not previously known and yet is necessary to determine the validity in using an ovine model to study human physiology. The objectives of this study were to isolate and validate the clone of ovine eNOS cDNA and investigate ovine eNOS activation when expressed in COS-7 cells. The ovine eNOS cDNA has high homology to published human and bovine sequences and shares identity with the bovine amino acid sequence. When ovine eNOS was transiently expressed in COS-7 cells (COS-7/oeNOS), A23187 increased specific catalytic activity in a dose- and time-dependent manner. A23187-stimulated activation of eNOS was, however, also accompanied by phosphorylation of eNOS S1179 and dephosphorylation of T497, demonstrating that an increase in [Ca(2+)](i) may not be the sole mechanism of activation. The physiologic relevance of this was further underscored by the finding that ATP dose-dependently increased peak [Ca(2+)](i) and eNOS activity in COS-7/oeNOS, but also increased eNOS p-S1179 and decreased p-T497. This finding was similar to those in ovine P-UAEC treated with the Ca(2+)-mobilizing agonist ATP, wherein activation of eNOS was again concomitant with a rise p-S1179 as well as a slight decrease in p-T497. In conclusion, we describe the full-length ovine eNOS cDNA sequence and show that both physiologic and nonphysiologic calcium-mobilizing agents, which activate ovine eNOS in COS-7 and P-UAEC, do so in association with changes in eNOS phosphorylation. Given this information we can now begin to dissect the relationship between Ca(2+) elevation and specific phosphorylation events in eNOS activation in the ovine model, and thereby gain insight into the possible basis for pregnancy-related dysfunction.

Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, kidney and spleen tissues following vaccination against Vibrio anguillarum and Aeromonas salmonicida.

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.

cDNA microarray analysis of gene expression profiles in human placenta: up-regulation of the transcript encoding muscle subunit of glycogen phosphorylase in preeclampsia.

OBJECTIVE: Third-trimester human placentas from normal and preeclamptic pregnancies were evaluated for possible changes in gene expression patterns by microarray analysis. METHODS: Placentas from four normal pregnancies and four pregnancies complicated by preeclampsia were studied. In a preliminary effort to identify possible differences between the two groups, complementary DNA (cDNA) probes were prepared from pooled total RNA by reverse transcription in the presence of (33)P-dCTP. After hybridization to human GeneFilter cDNA microarrays (GF211; Research Genetics, Huntsville, AL), 319 positive signals were detected above background out of a possible 4131 human cDNAs spotted on the filters. RESULTS: Ten most highly expressed mRNA species, ten most up-regulated, and ten most down-regulated genes in placentas from both groups of patients were identified for future studies. Of the 319 positive hybridizations, one transcript was clearly elevated in preeclamptic pregnancy. This cDNA encodes the muscle subtype of glycogen phosphorylase (GP-M) and was increased more than 2.8-fold (P <.05) in the preeclamptic placentas. In contrast, cDNA for glycogen synthase (muscle and liver isoforms) was not significantly increased, being near the limits of detection. The preeclampsia-induced increase of placental GP-M mRNA expression (approximately 3.5-fold) was confirmed by northern blot analysis. CONCLUSION: We conclude that microarray analysis can detect trends in mRNA and gene expression in placentas from normal and preeclamptic pregnancies that may be further studied in a more targeted fashion. We found that placental GP-M mRNA level is up-regulated in preeclampsia, which is consistent with previous reports of increased glycogen phosphorylase activity, and we suggest that it may be largely regulated at the level of transcription. Further studies may determine whether such up-regulation might be a response to hypoxia.