Whole-Genome Resequencing of Worldwide Wild and Domestic Sheep Elucidates Genetic Diversity, Introgression, and Agronomically Important Loci.

Domestic sheep and their wild relatives harbor substantial genetic variants that can form the backbone of molecular breeding, but their genome landscapes remain understudied. Here, we present a comprehensive genome resource for wild ovine species, landraces and improved breeds of domestic sheep, comprising high-coverage (∼16.10×) whole genomes of 810 samples from 7 wild species and 158 diverse domestic populations. We detected, in total, ∼121.2 million single nucleotide polymorphisms, ∼61 million of which are novel. Some display significant (P < 0.001) differences in frequency between wild and domestic species, or are private to continent-wide or individual sheep populations. Retained or introgressed wild gene variants in domestic populations have contributed to local adaptation, such as the variation in the HBB associated with plateau adaptation. We identified novel and previously reported targets of selection on morphological and agronomic traits such as stature, horn, tail configuration, and wool fineness. We explored the genetic basis of wool fineness and unveiled a novel mutation (chr25: T7,068,586C) in the 3'-UTR of IRF2BP2 as plausible causal variant for fleece fiber diameter. We reconstructed prehistorical migrations from the Near Eastern domestication center to South-and-Southeast Asia and found two main waves of migrations across the Eurasian Steppe and the Iranian Plateau in the Early and Late Bronze Ages. Our findings refine our understanding of genome variation as shaped by continental migrations, introgression, adaptation, and selection of sheep.

Gene Expression in Embryos From Norwegian Red Bulls With High or Low Non Return Rate: An RNA-Seq Study of in vivo-Produced Single Embryos.

During the last decade, paternal effects on embryo development have been found to have greater importance than previously believed. In domestic cattle, embryo mortality is an issue of concern, causing huge economical losses for the dairy cattle industry. In attempts to reveal the paternal influence on embryo death, recent approaches have used transcriptome profiling of the embryo to find genes and pathways affected by different phenotypes in the bull. For practical and economic reasons, most such studies have used in vitro produced embryos. The aim of the present study was to investigate the differences in the global transcriptome of in vivo produced embryos, derived from sires with either high or low field fertility measured as the non-return rate (NRR) on day 56 after first AI of the inseminated cows. Superovulated heifers (n = 14) in the age span of 12-15 months were artificially inseminated with semen from either high fertility (n = 6) or low fertility (n = 6) bulls. On day seven after insemination, embryos were retrieved through uterine flushing. Embryos with first grade quality and IETS stage 5 (early blastocyst), 6 (blastocyst) or 7 (expanded blastocyst) were selected for further processing. In total, RNA extracted from 24 embryos was sequenced using Illumina sequencing, followed by differential expression analysis and gene set enrichment analysis. We found 62 genes differentially expressed between the two groups (adj.p-value<0.05), of which several genes and their linked pathways could explain the different developmental capacity. Transcripts highly expressed in the embryos from low fertility bulls were related to sterol metabolism and terpenoid backbone synthesis, while transcripts highly expressed in the high fertility embryos were linked to anti-apoptosis and the regulation of cytokine signaling. The leukocyte transendothelial migration and insulin signaling pathways were associated with enrichments in both groups. We also found some highly expressed transcripts in both groups which can be considered as new candidates in the regulation of embryo development. The present study is an important step in defining the paternal influence in embryonic development. Our results suggest that the sire's genetic contribution affects several important processes linked to pre-and peri implantation regulation in the developing embryo.

Paternal Origins and Migratory Episodes of Domestic Sheep.

The domestication and subsequent global dispersal of livestock are crucial events in human history, but the migratory episodes during the history of livestock remain poorly documented [1-3]. Here, we first developed a set of 493 novel ovine SNPs of the male-specific region of Y chromosome (MSY) by genome mapping. We then conducted a comprehensive genomic analysis of Y chromosome, mitochondrial DNA, and whole-genome sequence variations in a large number of 595 rams representing 118 domestic populations across the world. We detected four different paternal lineages of domestic sheep and resolved, at the global level, their paternal origins and differentiation. In Northern European breeds, several of which have retained primitive traits (e.g., a small body size and short or thin tails), and fat-tailed sheep, we found an overrepresentation of MSY lineages y-HC and y-HB, respectively. Using an approximate Bayesian computation approach, we reconstruct the demographic expansions associated with the segregation of primitive and fat-tailed phenotypes. These results together with archaeological evidence and historical data suggested the first expansion of early domestic hair sheep and the later expansion of fat-tailed sheep occurred ∼11,800-9,000 years BP and ∼5,300-1,700 years BP, respectively. These findings provide important insights into the history of migration and pastoralism of sheep across the Old World, which was associated with different breeding goals during the Neolithic agricultural revolution.

Goats naturally devoid of PrPC are resistant to scrapie.

Prion diseases are progressive and fatal, neurodegenerative disorders described in humans and animals. According to the "protein-only" hypothesis, the normal host-encoded prion protein (PrPC) is converted into a pathological and infectious form (PrPSc) in these diseases. Transgenic knockout models have shown that PrPC is a prerequisite for the development of prion disease. In Norwegian dairy goats, a mutation (Ter) in the prion protein gene (PRNP) effectively blocks PrPC synthesis. We inoculated 12 goats (4 PRNP+/+, 4 PRNP+/Ter, and 4 PRNPTer/Ter) intracerebrally with goat scrapie prions. The mean incubation time until clinical signs of prion disease was 601 days post-inoculation (dpi) in PRNP+/+ goats and 773 dpi in PRNP+/Ter goats. PrPSc and vacuolation were similarly distributed in the central nervous system (CNS) of both groups and observed in all brain regions and segments of the spinal cord. Generally, accumulation of PrPSc was limited in peripheral organs, but all PRNP+/+ goats and 1 of 4 PRNP+/Ter goats were positive in head lymph nodes. The four PRNPTer/Ter goats remained healthy, without clinical signs of prion disease, and were euthanized 1260 dpi. As expected, no accumulation of PrPSc was observed in the CNS or peripheral tissues of this group, as assessed by immunohistochemistry, enzyme immunoassay, and real-time quaking-induced conversion. Our study shows for the first time that animals devoid of PrPC due to a natural mutation do not propagate prions and are resistant to scrapie. Clinical onset of disease is delayed in heterozygous goats expressing about 50% of PrPC levels.

Transcription Profiling of Monocyte-Derived Macrophages Infected In Vitro With Two Strains of Streptococcus agalactiae Reveals Candidate Pathways Affecting Subclinical Mastitis in Cattle.

Macrophages are key cells of innate immune response and serve as the first line of defense against bacteria. Transcription profiling of bacteria-infected macrophages could provide important insights on the pathogenicity and host defense mechanisms during infection. We have examined transcription profiles of bovine monocyte-derived macrophages (bMDMs) isolated from the blood of 12 animals and infected in vitro with two strains of Streptococcus agalactiae. Illumina sequencing of RNA from 36 bMDMs cultures exposed in vitro to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h and unchallenged controls was performed. Analyses of over 1,656 million high-quality paired-end sequence reads revealed 5,936 and 6,443 differentially expressed genes (p < 0.05) in bMDMs infected with ST103 and ST12, respectively, versus unchallenged controls. Moreover, 588 genes differentially expressed between bMDMs infected with ST103 versus ST12 were identified. Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST103 revealed significant enrichment for granulocyte adhesion and diapedesis, while significant enrichment for the phagosome formation pathway was found among down-regulated genes. Moreover, Ingenuity pathway analysis of the differentially up-regulated genes in the bMDMs infected with ST12 showed significant enrichment for type 1/type 2 T helper cell activation, while the complement activation pathway was overrepresented in the down-regulated genes. Our study identified pathogen-induced regulation of key genes and pathways involved in the immune response of macrophages against infection but also likely involved in bacterial evasion of the host immune system. These results may contribute to better understanding of the mechanisms underlying subclinical infection such as bovine streptococcal mastitis.

MicroRNA expression profiles of bovine monocyte-derived macrophages infected in vitro with two strains of Streptococcus agalactiae.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression at the post-transcriptional level and play a key role in the control of innate and adaptive immune responses. For a subclinical infection such as bovine streptococcal mastitis, early detection is a great challenge, and miRNA profiling could potentially assist in the diagnosis and contribute to the understanding of the pathogenicity and defense mechanisms. We have examined the miRNA repertoire and the transcript level of six key immune genes [tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and transforming growth factor beta 1 (TGFß1)] during the early phase response of bovine immature macrophages to in vitro infection with live Streptococcus agalactiae. Next generation sequencing of small RNA libraries from 20 cultures of blood monocyte-derived macrophages exposed to either one of two sequence types of S. agalactiae (ST103 or ST12) for 6 h in vitro and unchallenged controls was performed. RESULTS: Analyzes of over 356 million high quality sequence reads, revealed differential expression of 17 and 44 miRNAs (P < 0.05) in macrophages infected with ST103 and ST12, respectively, versus unchallenged control cultures. We also identified the expression of 31 potentially novel bovine miRNAs. Pathway analysis of the differentially regulated miRNAs and their predicted target genes in the macrophages infected with ST12 revealed significant enrichment for inflammatory response and apoptosis, while significant enrichment for integrin and GABA signaling were found in ST103 infected macrophages. Furthermore, both bacterial strains regulated miRNAs involved in the alternative activation of macrophages. The transcript levels of TNF-α, IL-1ß, IL-6, IL-8 and IL-10 were significantly up-regulated by both bacterial strains, however the expression of TGFß1 was significantly down-regulated only by ST12. CONCLUSIONS: Our study identified pathogen-induced differential regulation of miRNAs controlling inflammation and polarization in bovine macrophages. This implies that miRNAs have potential to serve as biomarkers for early detection of bacterial infection.

Fine mapping of a QTL on bovine chromosome 6 using imputed full sequence data suggests a key role for the group-specific component (GC) gene in clinical mastitis and milk production.

BACKGROUND: Clinical mastitis is an inflammation of the mammary gland and causes significant costs to dairy production. It is unfavourably genetically correlated to milk production, and, thus, knowledge of the mechanisms that underlie these traits would be valuable to improve both of them simultaneously through breeding. A quantitative trait locus (QTL) that affects both clinical mastitis and milk production has recently been fine-mapped to around 89 Mb on bovine chromosome 6 (BTA6), but identification of the gene that underlies this QTL was not possible due to the strong linkage disequilibrium between single nucleotide polymorphisms (SNPs) within this region. Our aim was to identify the gene and, if possible, the causal polymorphism(s) responsible for this QTL through association analysis of high-density SNPs and imputed full sequence data in combination with analyses of transcript and protein levels of the identified candidate gene. RESULTS: Associations between SNPs and the studied traits were strongest for SNPs that were located within and immediately upstream of the group-specific component (GC) gene. This gene encodes the vitamin D-binding protein (DBP) and has multiple roles in immune defense and milk production. A 12-kb duplication that was identified downstream of this gene covered its last exon and segregated with the QTL allele that is associated with increased mastitis susceptibility and milk production. However, analyses of GC mRNA levels on the available samples revealed no differences in expression between animals having or lacking this duplication. Moreover, we detected no differences in the concentrations of DBP and its ligand vitamin D between the animals with different GC genotypes that were available for this study. CONCLUSIONS: Our results suggest GC as the gene that underlies the QTL for clinical mastitis and milk production. However, since only healthy animals were sampled for transcription and expression analyses, we could not draw any final conclusion on the absence of quantitative differences between animals with different genotypes. Future studies should investigate GC RNA expression and protein levels in cows with different genotypes during an infection.

Hematological shift in goat kids naturally devoid of prion protein.

The physiological role of the cellular prion protein (PrP(C)) is incompletely understood. The expression of PrP(C) in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrP(C) knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrP(C). Here we report hematological and immunological analyses of homozygous goat kids lacking PrP(C) (PRNP(Ter/Ter) ) compared to heterozygous (PRNP (+/Ter)) and normal (PRNP (+/+)) kids. Levels of cell surface PrP(C) and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNP (Ter/Ter), intermediate in PRNP (+/Ter) and high in PRNP (+/+) kids. The PRNP (Ter/Ter) animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).

Steroidogenic differential effects in neonatal porcine Leydig cells exposed to persistent organic pollutants derived from cod liver oil.

Seafood products, including fish and fish oils, are major sources of persistent organic pollutants (POPs) which may cause endocrine disruption related to reproductive dysfunction in males. Primary porcine neonatal Leydig cells were exposed to three extracts of POPs obtained from different stages in production of cod liver oil dietary supplement, in the absence and presence of luteinizing hormone (LH). No reduced viability was observed and all POP extracts showed increased testosterone and estradiol levels in unstimulated cells and decreased testosterone and estradiol secretion in LH-stimulated cells. A decrease in central steriodogenic genes including STAR, CYP11A1, HSD3B and CYP17A1 was obtained in both culture conditions with all POP extracts. We implicate both small differences in composition and concentration of compounds as well as "old" POPs to be important for the observed steroidogenic effects.

The early phase transcriptome of bovine monocyte-derived macrophages infected with Staphylococcus aureus in vitro.

BACKGROUND: In the mammary gland, local recruitment and action of macrophages is a key immunological defence mechanism against infection. Macrophages are members of the innate immune system, serve as the first line of the defence against invading pathogens and are critical effectors and regulators of inflammation. We have examined the early phase response of bovine macrophages to infection with live Staphylococcus aureus. Genome-wide transcript profiling of blood monocyte-derived macrophages from six Norwegian Red heifers infected with live S. aureus for 2 and 6 hours in vitro was performed. RESULTS: About 420 of the 17 000 genes on the ARK-Genomics bovine cDNA array were differentially regulated at 6 hours post infection. Approximately 70% of the responding genes had a known identity (Entrez Gene ID) and were used in the identification of overrepresented pathways and biological functions in the dataset.Analysis of a subset of differentially regulated genes (List eQG) obtained by comparison with data from genome-wide association mapping in Norwegian Red cattle identified anti-inflammatory cytokines interleukin 4 and interleukin 13 as putative expression quantitative trait loci, suggesting that S. aureus infection triggers alternative activation of macrophages. Moreover, several classical activation pathways were found, mainly cellular immune response and cytokine signaling pathways, i.e. triggering receptor expressed on myeloid cells 1 (TREM1) and nucleotide-binding and oligomerization domain-like receptor (NLR) pathways. Tumor necrosis factor receptor superfamily member 5 (CD40 ligand) was identified as an upstream regulator which points toward CD40 likely acting as a co-stimulatory receptor during Toll-like receptor 2(TLR2)-mediated inflammatory response of bovine macrophages to S. aureus infection. Furthermore, peptidoglycan was identified as an upstream regulator in the List eQG, which indicates that this bacterial cell-wall component might be pivotal in macrophage intracellular bacterial recognition during early inflammation. CONCLUSIONS: Here we have shown that in vitro infection of bovine macrophages with live S. aureus induced both alternative and classical activation pathways. Alternative activation of macrophages may be a mechanism contributing to intracellular persistence of S. aureus in the course of inflammation such as during mastitis in dairy cattle.

Androstenone and testosterone levels and testicular morphology of Duroc boars related to estimated breeding value for androstenone.

Androstenone and testosterone levels in Duroc boars with an estimated breeding value for androstenone (EBV(androstenone)) were followed in the period from birth to sexual maturity. The breeding value for androstenone had been estimated based on androstenone levels in 1202 Duroc boars at an age of 24 weeks. Testosterone and androstenone levels in plasma were recorded in 19 boars at 1 week of age and in their 15 respective litter-siblings at 3 weeks of age. Between 12 and 27 weeks of age, plasma levels were recorded weekly in a third set of 16 litter-siblings. In the last group, histomorphology was performed at 12, 16, 20, and 27 weeks of age to determine sexual maturity status. The EBV(androstenone) was positively related to plasma androstenone in animals 12 to 27 weeks of age and to plasma testosterone levels in 1- and 3-week-old animals. The EBV(androstenone) was not related to testis morphology. The concentration of fat androstenone was positively correlated to the percentage of immature seminiferous tubules and negatively correlated to the percentage of mature seminiferous tubules at 20 weeks. Testosterone in plasma showed no relationship with testis morphology. Most individuals reached puberty at 20 weeks of age, which indicates that Duroc mature later than crossbred boars. The results indicated that breeding value based on the single trait boar taint parameter EBV(androstenone) was not related to testicular development.

Fetal adrenal development: comparing effects of combined exposures to PCB 118 and PCB 153 in a sheep model.

This study investigated the effects of exposure to the ubiquitous contaminants polychlorinated biphenyls (PCBs) on the fetal adrenal cortex and on plasma cortisol using the domestic sheep (Ovis aries) as a model. Pregnant ewes were intendedly subjected to oral treatment with PCB 153 (98 µg/kg bw/day), PCB 118 (49 µg/kg bw/day) or the vehicle corn oil from mating until euthanasia on gestation day 134 (±0.25 SE). However, because of accidental cross-contamination occurring twice causing a mixed exposure scenario in all three groups, the focus of this paper is to compare three distinct groups of fetuses with different adipose tissue PCB levels (PCB 153high, PCB 118high and low, combined groups) rather than comparing animals exposed to single PCB congeners to those of a control group. When comparing endocrine and anatomical parameters from fetuses in the PCB 153high (n = 13) or PCB 118high (n = 14) groups with the low, combined group (n = 14), there was a significant decrease in fetal body weight (P < 0.05), plasma cortisol concentration (P < 0.001) and adrenal cortex thickness (P < 0.001). Furthermore, adrenal weight was decreased and plasma ACTH was increased only in the PCB 118high group. Expression of several genes encoding enzymes and receptors related to steroid hormone synthesis was also affected and mostly down-regulated in fetuses with high PCB tissue levels. In conclusion, we suggest that mono-and di-ortho PCBs were able to interfere with growth, adrenal development and cortisol production in the fetal sheep model. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.

Healthy goats naturally devoid of prion protein.

Prion diseases such as scrapie in small ruminants, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in man, are fatal neurodegenerative disorders. These diseases result from the accumulation of misfolded conformers of the host-encoded prion protein (PrP) in the central nervous system. To date naturally-occurring PrP free animals have not been reported. Here we describe healthy non-transgenic animals, Norwegian Dairy Goats, lacking prion protein due to a nonsense mutation early in the gene. These animals are predicted to be resistant to prion disease and will be valuable for research and for production of prion-free products.

Salmonella Typhimurium invasion of HEp-2 epithelial cells in vitro is increased by N-acylhomoserine lactone quorum sensing signals.

BACKGROUND: In Gram-negative bacteria, the most commonly studied quorum sensing signals are the N-acylhomoserine lactones (AHLs). In Salmonella, AHLs are recognized by SdiA, which is believed to be a sensor of AHLs produced by other bacteria, since Salmonella does not produce AHLs itself. It has been speculated that AHLs produced by the gastrointestinal flora may influence the regulation of virulence traits in Salmonella. The aim of the present work was to study the effect of AHLs on epithelial cell invasion by Salmonella in vitro. METHODS: Invasion by Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) strain and its isogenc sdiA mutant was studied using a conventional gentamycin invasion assay with HEp-2 cells at 37°C. Gene expression was studied using a semi-quantitative PCR. RESULTS: The S. Typhimurium strain, but not its isogenic sdiA mutant, displayed increased in vitro invasion after addition of both N-hexanoyl-DL-homoserine lactone (C6-AHL) and N-octanoyl-DL-homoserine lactone (C8-AHL). Increased expression of two of the genes in the SdiA regulon (rck and srgE) was observed in the wild type strain, but not in the sdiA mutant. CONCLUSIONS: The results from the present study show that S. Typhimurium can respond to two different AHL quorum sensing signals (C6-AHL and C8-AHL) with increased cell invasion at 37°C in vitro, and that this response most likely is sdiA mediated. These results indicate that if AHLs are present in the intestinal environment, they may increase the invasiveness of Salmonella.

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources.

BACKGROUND: Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. RESULTS: Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1.The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. CONCLUSIONS: This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

In vitro steroidogenic effects of mixtures of persistent organic pollutants (POPs) extracted from burbot (Lota lota) caught in two Norwegian lakes.

This study investigated the effects of two mixtures of persistent organic pollutants (POPs) on steroidogenesis in the H295R cell line. The two mixtures were obtained from the livers of burbot (Lota lota) caught in two Norwegian lakes (Mjøsa and Losna) with different contaminant profiles. Steroid hormone levels in the cell culture medium and mRNA levels of 16 genes involved in steroidogenesis were investigated. The crude Lake Mjøsa extract had to be diluted ten times more than the Lake Losna extract in order to prevent cytotoxicity. The ten times diluted Lake Mjøsa mixture had higher levels of DDT and derivates (∑DDTs, 1.7 times) and brominated flame retardants (∑BDEs and HBCD, 15-25 times) than the Lake Losna mixture, which, on the other hand, had higher concentrations of ∑PCBs (1.5 times higher) and also of HCB, ∑HCH isomers and ∑chlordane isomers (5-20 times higher). In the cell culture media, only cortisol levels were increased at the highest exposure concentration to the Lake Mjøsa mixture, while both cortisol and estradiol levels were increased following exposure to the two highest Lake Losna mixture exposure concentrations. Testosterone levels decreased only at the highest exposure concentration of the Lake Losna mixture. Multivariate models suggested that ∑PCBs, and to a lesser extent ∑DDTs, were responsible for the cortisol responses, while estradiol and testosterone alterations were best explained by HCB and ∑PCBs, respectively. Exposure to the mixtures generally increased mRNA levels, with smaller effects exerted by the Lake Mjøsa mixture than the Lake Losna mixture. It was concluded that both mixtures affected steroidogenesis in the H295R cells. Small differences in mixture composition, rather than the high content of brominated flame retardants in the Lake Mjøsa mixture, were suggested to be the most probable reason for the apparent differences in potencies of the two mixtures.

The effect of valproate and levetiracetam on steroidogenesis in forskolin-stimulated H295R cells.

PURPOSE: Endocrine disruptive effects have been frequently observed in patients using antiepileptic drugs (AEDs). Two different AEDs, valproate (VPA) and levetiracetam (LEV), were tested in forskolin-stimulated human adrenal carcinoma (H295R) cells to explore their effect on steroidogenesis. VPA has a long history as an anticonvulsant and is linked with many of the endocrine disorders associated with AED use. LEV is a newer AED, and no endocrine disruptive effects have been reported in humans to date. METHODS: H295R cells, which are capable of full steroidogenesis, were stimulated with forskolin and exposed to either VPA or LEV for 48 h. Medium was collected and analyzed for hormone production. For the VPA-exposed cells, steroidogenic gene expression analysis was also conducted. RESULTS: VPA exposure resulted in a significant reduction in progesterone and estradiol (E2) production, whereas testosterone (T) levels remained unchanged. There were also significant alterations in expression level for most genes analyzed. LEV exposure resulted in a minor, but statistically significant, reduction in T and E2 production. DISCUSSION: Exposure of forskolin-stimulated H295R cells to VPA led to an increased T/E2 ratio through a significant decrease in estradiol production. Gene analysis suggested that VPA affects NR0B1 expression. NR0B1 inhibits promoters of other genes involved in steroidogenesis, and the altered expression of NR0B1 might explain the observed down-regulation in hormone production. The effects of LEV exposure on hormone secretion were not considered to be biologically significant.

Three structurally different polychlorinated biphenyl congeners (Pcb 118, 153, and 126) affect hormone production and gene expression in the human H295R in vitro model.

Polychlorinated biphenyls (PCB) are ubiquitous environmental pollutants that have been linked to adverse health effects including endocrine disruption. This study compared the mono-ortho-substituted PCB 118 and di-ortho-substituted PCB 153 with the non-ortho-substituted PCB 126, for possible effects on steroid hormone production and on the expression of 10 genes encoding proteins involved in steroidogenesis. The H295R human adenocarcinoma cell line was used as an in vitro model. Cells were exposed for 48 h to solvent control (dimethyl sulfoxide, DMSO) or 6 different concentrations ranging from 40 pM to 4 muM of one of the three test compounds. All three congeners significantly increased the production of estradiol-17beta. PCB 118 produced a rise in progesterone and cortisol in a concentration-dependent manner, similar to PCB 126. Testosterone was significantly reduced in response to PCB 153 but not PCB 118 or PCB 126. All three congeners elevated aldosterone at the highest concentration tested. A significant increase was observed in CYP11B2 mRNA levels in cells exposed to the three congeners. In addition, PCB 126 upregulated CYP19, 3beta-HSD2, StAR, and HMGR mRNA levels at the highest concentration tested, and downregulated CYP21 at 40 nM. In conclusion, all three PCB congeners are capable of modulating steroidogenesis in H295R in a concentration-dependent manner, whereby the hormone profile following PCB 118 exposure resembles that of PCB 126. Where changes in gene expression profile are concerned, exposure to PCB 126 showed the greatest effects.

Revealing the history of sheep domestication using retrovirus integrations.

The domestication of livestock represented a crucial step in human history. By using endogenous retroviruses as genetic markers, we found that sheep differentiated on the basis of their "retrotype" and morphological traits dispersed across Eurasia and Africa via separate migratory episodes. Relicts of the first migrations include the Mouflon, as well as breeds previously recognized as "primitive" on the basis of their morphology, such as the Orkney, Soay, and the Nordic short-tailed sheep now confined to the periphery of northwest Europe. A later migratory episode, involving sheep with improved production traits, shaped the great majority of present-day breeds. The ability to differentiate genetically primitive sheep from more modern breeds provides valuable insights into the history of sheep domestication.

Genetic differentiation between the Old and New types of Serbian Tsigai sheep.

Two Tsigai sheep populations exist in Serbia: the Old type, called Cokan, and the New type. It is assumed that the New type results from upgrading Tsigai sheep with exotic genetic material. We investigated genetic diversity and differentiation of these types by analysing 23 autosomal microsatellites. Tests for Hardy-Weinberg proportions, linkage equilibrium between genotypes across loci and the calculation of inbreeding coefficients were performed and the deficiency in the number of alleles within the Tsigai types was examined using a Wilcoxon sign-rank test. The New type displayed a higher level of genetic variability than the Cokan in terms of allele numbers, but the New Tsigai showed a pattern of heterozygosity deficiency. The positive f value for the Cokan suggests the occurrence of inbreeding in this type. The proportion of linkage disequilibrium was below that expected by chance. Exclusion of two loci in Hardy-Weinberg disequilibrium did not alter our conclusions based on the entire data set i.e. the two Tsigai types are clearly differentiated and the New Tsigai type has been influenced by crossbreeding. Therefore, the Cokan Tsigai should be considered as a distinct endangered breed in the FAO classification.