Quantitative allele-specific expression and DNA methylation analysis of H19, IGF2 and IGF2R in the human placenta across gestation reveals H19 imprinting plasticity

Extent: 11p.Imprinted genes play important roles in placental differentiation, growth and function, with profound effects on fetal development. In humans, H19 and IGF2 are imprinted, but imprinting of IGF2R remains controversial. The H19 non-coding RNA is a negative regulator of placental growth and altered placental imprinting of H19-IGF2 has been associated with pregnancy complications such as preeclampsia, which have been attributed to abnormal first trimester placentation. This suggests that changes in imprinting during the first trimester may precede aberrant placental morphogenesis. To better understand imprinting in the human placenta during early gestation, we quantified allele-specific expression for H19, IGF2 and IGF2R in first trimester (6–12 weeks gestation) and term placentae (37–42 weeks gestation) using pyrosequencing. Expression of IGF2R was biallelic, with a mean expression ratio of 49:51 (SD = 0.07), making transient imprinting unlikely. Expression from the repressed H19 alleles ranged from 1–25% and was higher (P<0.001) in first trimester (13.5±8.2%) compared to term (3.4±2.1%) placentae. Surprisingly, despite the known co-regulation of H19 and IGF2, little variation in expression of the repressed IGF2 alleles was observed (2.7±2.0%). To identify regulatory regions that may be responsible for variation in H19 allelic expression, we quantified DNA methylation in the H19-IGF2 imprinting control region and H19 transcription start site (TSS). Unexpectedly, we found positive correlations (P<0.01) between DNA methylation levels and expression of the repressed H19 allele at 5 CpG’s 2000 bp upstream of the H19 TSS. Additionally, DNA methylation was significantly higher (P<0.05) in first trimester compared with term placentae at 5 CpG’s 39–523 bp upstream of the TSS, but was not correlated with H19 repressed allele expression. Our data suggest that variation in H19 imprinting may contribute to early programming of placental phenotype and illustrate the need for quantitative and robust methodologies to further elucidate the role of imprinted genes in normal and pathological placental development.Sam Buckberry, Tina Bianco-Miotto, Stefan Hiendleder and Claire T. Robert

Pronounced inter-individual variation in plasma cortisol response to fluoxetine hydrochloride in the pig

Published: 18 March 2020Animal welfare assessment requires measures for positive affective state. Pharmacological agents that manipulate affective state can be used to evaluate novel biomarkers for affective state assessment. However, to validate that an agent has modified brain function, a reliable indicator is required. Circulating cortisol has been used as a reporter for effective delivery of the antidepressant selective serotonin reuptake inhibitor (SSRI) fluoxetine hydrochloride to the brain in humans and sheep. Here, we tested cortisol as a reporter for effective delivery of fluoxetine hydrochloride to the pig brain. We hypothesized that following administration of SSRI, innervation of the serotonergic reward pathway would result in activation of the hypothalamic-pituitary-adrenal (HPA) axis, leading to increased circulating cortisol levels. Large White-Landrace gilts received either a single intravenous dose of 100 mg fluoxetine hydrochloride suspended in 10 mL saline (n = 4), or 10 mL saline alone (n = 4). Blood samples were collected every 15 min for one hour prior to, and six hours post-treatment. The interaction of treatment x time on mean plasma cortisol levels between 15–165 min post-treatment was significant (p = 0.048) with highest cortisol concentrations of SSRI treated pigs versus controls (+ 98%) at 135 min post-treatment. However, individual cortisol profiles after SSRI treatment revealed high inter-individual variation in response. We conclude that, while combined data imply that plasma cortisol may be a readout for SSRI efficacy, inter-individual variation in SSRI response may preclude application of this approach in the pig. Given the current limited sample size, further research to confirm these findings is needed.Laura E. Marsh, Robyn Terry, Alexandra L. Whittaker, Stefan Hiendleder and Cameron R. Ralp

Energy efficient plasma processing of industrial wastes

The paper presents the results of thermodynamic modeling of the process of joint plasma treatment of non-combustible and combustible industrial wastes. The compositions of water-salt-organic compositions based on these wastes and regimes providing their energy-efficient joint treatment in air plasma have been determined

Dual-locus DNA metabarcoding reveals southern hairy-nosed wombats (Lasiorhinus latifrons Owen) have a summer diet dominated by toxic invasive plants

Habitat degradation and summer droughts severely restrict feeding options for the endangered southern hairy-nosed wombat (SHNW; Lasiorhinus latifrons). We reconstructed SHNW summer diets by DNA metabarcoding from feces. We initially validated rbcL and ndhJ diet reconstructions using autopsied and captive animals. Subsequent diet reconstructions of wild wombats broadly reflected vegetative ground cover, implying local rather than long-range foraging. Diets were all dominated by alien invasives. Chemical analysis of alien food revealed Carrichtera annua contains high levels of glucosinolates. Clinical examination (7 animals) and autopsy (12 animals) revealed that the most degraded site also contained most individuals showing signs of glucosinolate poisoning. We infer that dietary poisoning through the ingestion of alien invasives may have contributed to the recent population crashes in the region. In floristically diverse sites, individuals appear to be able to manage glucosinolate intake by avoidance or episodic feeding but this strategy is less tractable in the most degraded sites. We conclude that recovery of the most affected populations may require effective Carrichtera management and interim supplementary feeding. More generally, we argue that protection against population decline by poisoning in territorial herbivores requires knowledge of their diet and of those food plants containing toxic principles

INSL3 in the Ruminant: A Powerful Indicator of Gender- and Genetic-Specific Feto-Maternal Dialogue

The hormone Insulin-like peptide 3 (INSL3) is a major secretory product of the Leydig cells from both fetal and adult testes. Consequently, it is a major gender-specific circulating hormone in the male fetus, where it is responsible for the first phase of testicular descent, and in the adult male. In most female mammals, circulating levels are very low, corresponding to only a small production of INSL3 by the mature ovaries. Female ruminants are exceptional in exhibiting high INSL3 gene expression by the thecal cells of antral follicles and by the corpora lutea. We have developed a specific and sensitive immunoassay to measure ruminant INSL3 and show that, corresponding to the high ovarian gene expression, non-pregnant adult female sheep and cows have up to four times the levels observed in other female mammals. Significantly, this level declines during mid-pregnancy in cows carrying a female fetus, in which INSL3 is undetectable. However, in cows carrying a male fetus, circulating maternal INSL3 becomes elevated further, presumably due to the transplacental transfer of fetal INSL3 into the maternal circulation. Within male fetal blood, INSL3 is high in mid-pregnancy (day 153) corresponding to the first transabdominal phase of testicular descent, and shows a marked dependence on paternal genetics, with pure bred or hybrid male fetuses of Bos taurus (Angus) paternal genome having 30% higher INSL3 levels than those of Bos indicus (Brahman) paternity. Thus INSL3 provides the first example of a gender-specific fetal hormone with the potential to influence both placental and maternal physiology

Gaur genome reveals expansion of sperm odorant receptors in domesticated cattle

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Phylogenetic Position of a Copper Age Sheep (Ovis aries) Mitochondrial DNA

BACKGROUND: Sheep (Ovis aries) were domesticated in the Fertile Crescent region about 9,000-8,000 years ago. Currently, few mitochondrial (mt) DNA studies are available on archaeological sheep. In particular, no data on archaeological European sheep are available. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the first portion of mtDNA sequence of a Copper Age European sheep. DNA was extracted from hair shafts which were part of the clothes of the so-called Tyrolean Iceman or Ötzi (5,350-5,100 years before present). Mitochondrial DNA (a total of 2,429 base pairs, encompassing a portion of the control region, tRNA(Phe), a portion of the 12S rRNA gene, and the whole cytochrome B gene) was sequenced using a mixed sequencing procedure based on PCR amplification and 454 sequencing of pooled amplification products. We have compared the sequence with the corresponding sequence of 334 extant lineages. CONCLUSIONS/SIGNIFICANCE: A phylogenetic network based on a new cladistic notation for the mitochondrial diversity of domestic sheep shows that the Ötzi's sheep falls within haplogroup B, thus demonstrating that sheep belonging to this haplogroup were already present in the Alps more than 5,000 years ago. On the other hand, the lineage of the Ötzi's sheep is defined by two transitions (16147, and 16440) which, assembled together, define a motif that has not yet been identified in modern sheep populations

Trans-Species Polymorphism and Selection in the MHC Class II DRA Genes of Domestic Sheep

Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the induction of protective immunity

Association between MUC1 gene polymorphism and expected progeny differences in Nelore cattle (Bos primigenius indicus)

MUC1 is a heavily glycosylated mammalian transmembrane protein expressed by mucosal secretory tissues for both protection against microbial infection and lubrication. An important characteristic of MUC1 is its variable number of tandem repeats (VNTR) containing several sites for O-glycosylation. VNTR length has been associated with many human diseases and with certain economically important traits in domestic ruminants. The aim of the present study was to correlate the length of MUC1 gene VNTR with expected progeny differences (EPDs) obtained for growth, fertility and carcass traits. Five alleles were identified, with alleles containing short VNTRs being more frequent than those with long, thereby demonstrating that Brazilian Nelore cattle are characterized by high frequencies in short MUC1 VNTRs. Statistical analyses revealed there to be no significant association between VNTR length and EPDs for weight at 120 days (W120 ), scrotal circumference at 365 (SC 365 ) and 450 (SC 450 ) days, age at first calving (AFC), and rib eye area (REA)