The methylation patterns of the IGF2 and IGF2R genes in bovine spermatozoa are not affected by flow-cytometric sex sorting

The objectives of this study were to investigate the effect of sexing by flow cytometry on the methylation patterns of the IGF2 and IGF2R genes. Frozen-thawed, unsorted, and sex-sorted sperm samples from four Nellore bulls were used. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). Sperm were isolated from the extender, cryoprotectant, and other cell types by centrifugation on a 40:70% Percoll gradient, and sperm pellets were used for genomic DNA isolation. DNA was used for analyses of the methylation patterns by bisulfite sequencing. Methylation status of the IGF2 and IGF2R genes were evaluated by sequencing 195 and 147 individual clones, respectively. No global differences in DNA methylation were found between NS, SX, and SY groups for the IGF2 (P=0.09) or IGF2R genes (P=0.38). Very specific methylation patterns were observed in the 25th and 26th CpG sites in the IGF2R gene. representing higher methylation in NS than in the SX and SY groups compared with the other CpG sites. Further, individual variation in methylation patterns was found among bulls. In conclusion, the sex-sorting procedure by flow cytometry did not affect the overall DNA methylation patterns of the IGF2 and IGF2R genes, although individual variation in their methylation patterns among bulls was observed. Mol. Reprod. Dev. 79:7784, 2012. (C) 2011 Wiley Periodicals, Inc.

Imprinted gene expression in in vivo- and in vitro-produced bovine embryos and chorio-allantoic membranes

Cloning by nuclear transfer is often associated with poor results due to abnormal nuclear reprogramming of somatic donor cells and altered gene expression patterns. We investigated the expression patterns of imprinted genes IGF2 and IGF2R in 33- to 36-day bovine embryos and chorio-allantoic membranes derived from in vivo- and in vitro-produced embryos by somatic cell nuclear transfer (SCNT), parthenogenetic activation, and in vitro fertilization (IVF). There was a lower IGF2 expression rate in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to in vivo and IVF embryos (2.01; P < 0.05). In the chorio-allantoic membranes, IGF2 showed a baseline expression pattern (P < 0.05) in parthenotes (0.001) when compared to in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was less expressed (P < 0.05) in SCNT chorio-allantoic membranes (0.25) when compared to the in vivo group. The low expression of IGF2 in parthenogenetic embryos and chorio-allantoic membranes confirms its imprinted status in cattle. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in SCNT-derived bovine embryos and chorioallantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes of the low efficiency of SCNT procedures in this species.

Investigation of IGF2/ApaI and H19/RsaI polymorphisms in patients with cutaneous melanoma

Objective: The etiology of cutaneous melanoma is complex, involving both heterogeneous genetic and environmental components. The aim of our study was to verify if single polymorphic sites within IGF2 and H19 genes and their consequent haplotypes influence risk and/or prognosis of familial melanoma. Design: Twenty one patients with clinical criteria of hereditary melanoma (early onset, presence of multiple primary melanoma, and/or one or more affected first- or second-degree relatives) and previously screened for CDKN2A mutations were genotyped by IGF2/ApaI and H19/RsaI PCR-RFLPs. Data were compared between patients and a control group (100 healthy young individuals) using Chi-square and Fisher`s exact tests. We also investigated if these polymorphic sites could be microRNAs potential targets, using RegRNA software. Results: Although the IGF2 and HI9 genotypes/haplotypes were not significantly associated with melanoma, two of the most severe cases (very early onset or multiple melanomas) showed to be heterozygous for both genes. We found an overlap between IGF2/ApaI and miR-615-5p, and between H19/RsaI and miR-574-3p. Conclusions: Some studies have shown H19, and IGF2 genes (or related genes or protein, for example, IGF2R and IMP-3) differential expression in melanoma. However, no study has attempted to examine markers across this cluster in relation to melanoma until now. Since the base change may impair the pairing of microRNA and its binding site, our results suggest a new window for future studies of IGF2 and H19 genetic variability and posttranscriptional regulation. Due to the importance and based on the present results, we suggest that the genotype/haplotype analysis of IGF2 and H19 polymorphisms should be better investigated in large populations with cutaneous melanoma, attempting to tie the association with progression of the disease. (C) 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

Mir211 Is Dysregulated In Conjunctival Melanocytic proliferations

Purpose Downregulation of TRPM1 mRNA, a transient receptor potential cation channel, has been identified in highly metastatic cutaneous melanoma cell lines. TRPM1 mRNA expression is inversely correlated with skin melanoma metastases. Recent evidence has demonstrated that the tumor suppressive activity of TRPM1 is due to miR211 situated in intron 6 of TRPM1. As we have previously identified a downregulation of TRPM1 mRNA expression in conjunctival melanoma, we decided to assess miR211 expression and its potential target gene IGF2R and KCNMA1 in conjunctival melanocytic proliferations. As MITF has been shown to regulate both TRPM1 and mir211 expression, we also assessed MITF expression in our series. Methods Expression of miR211 was assessed by in situ hybridization in 14 conjunctival naevi and 14 conjunctival melanoma. Integrity of miRNA in tissues was evaluated in each sample with the preservation of miR126 expression in endothelial cells. Protein expression of MITF, IGF2R and KCNMA1 was assessed by immunohistochemistry. Statistical analysis was performed with JUMP 8,0 software. In situ hybridization and immunohistochemistry were assessed independently by two observers. Results There were 7 subepithelial nevi and 7 compound nevi. There were 5 female and 9 male. The population mean age was 48.7 ± 6.4 years (SEM). miR211 was found in 11 nevi (79%). MITF was expressed in all the nevi. IGF2R was found in 13 nevi. KCNMA1 was found in 57% of the nevi.The melanoma group was composed of 9 females and 5 males with a mean age of 67 ± 4.8 years (SEM). Using the recent TNM classification, 5 tumors were belonging to the T1, 3 to theT2 and 6 to the T3 categories. miR211 was found in 5 melanoma (36%). There was a significant downregulation of miR211 in the melanoma compared to the nevi (p=0,0219). MITF was found in 13 melanoma (93%). IGF2R and KCNMA1 were respectively found in 71% and 77% of the melanoma. There was no significant differential expression of MITF, KCNMA1 and IGF2R between the nevi and the melanoma as well as no association between miR211 expression and protein expression of two potential target genes Conclusions In vivo miR211 is significantly reduced in conjunctival melanoma compared to conjunctival nevi. No correlation between mir211 expression and two potential target genes KCNMA1 and IGF2R was observed.

Study of imprinted genes in bovine embryos produced by assisted reproductive technologies

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Effects of polymorphic microsatellites in the regulatory region of IGF1 and GHR on growth and carcass traits in beef cattle

Growth hormone (GH), insulin-like growth factors 1 and 2 (IGF1 and IGF2) and their associated binding proteins and transmembrane receptors (GHR, IGF1R and IGF2R) play an important role in the physiology of mammalian growth. The objectives of the present study were to estimate the allele and genotype frequencies of microsatellite markers located in the 5'-regulatory region of the IGF1 and GHR genes in beef cattle belonging to different genetic groups and to determine effects of these markers on growth and carcass traits in these animals under an intensive production system. For this purpose, genotyping was performed on 384 bulls including 79 Nellore, 30 Canchim (5/8 Charolais + 3/8 Zebu) and 275 crossbred animals originating from crosses of Simmental (1/2 Simmental, n = 30) and Angus (1/2 Angus, n = 245) sires with Nellore females. The effects of substituting L allele for S allele of GHR microsatellite across Nellore, Canchim and 1/2 Angus were significant for weight gain and body weight (P < 0.05). The IGF1 microsatellite allele substitutions of 229 for 225 within Nellore group and of 225 for 229 within 1/2 Angus were not significant for any of the traits.

Expressão gênica diferencial em embriões bovinos produzidos in vitro com espermatozóides sexados por gradiente de densidade ou por citometria de fluxo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Epigenética do desenvolvimento em bovinos: DNA metiltransferases e genes imprinted em embriões, fetos e placentas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interação núcleo-citoplasmática em embriões e expressão de genes imprinted em fetos bovinos produzidos in vivo, in vitro e partenogenéticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Imprinted gene expression in in vivo- And in vi/ro-produced bovine embryos and chorio-allantoic membranes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Developmental and Epigenetic Anomalies in Cloned Cattle

Many of the developmental anomalies observed in cloned animals are related to foetal and placental overgrowth, a phenomenon known as the 'large offspring syndrome' (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, that is, genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have sought and identified single nucleotide polymorphisms in Bos indicus DNA useful for the analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. By analysing differentially methylated regions (DMRs) of imprinted genes SNRPN, H19 and the IGF2R in cattle, we demonstrated that there is a generalized hypomethylation of the imprinted allele and the biallelic expression of embryos produced by SCNT when compared to the methylation patterns observed in vivo (artificially inseminated). Together, these results indicate that imprinting marks are erased during the reprogramming of the somatic cell nucleus during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals.

Asynchrone Replikation geprägter und nicht geprägter Chromosomenregionen und Expression relevanter Entwicklungsgene in Präimplantationsembryonen der Maus

Zum besseren Verständnis der epigenetischen Reprogrammierung nach der Befruchtung, wurde in der vorliegenden Studie unter Verwendung eines Interphase-FISH-Assays eine systematische Analyse des Replikationsverhaltens geprägter und nicht geprägter Chromosomenregionen in Präimplantationsembryonen der Maus durchgeführt. Dabei konnte erstmalig gezeigt werden, dass sowohl geprägte als auch nicht geprägte Chromosomen-regionen direkt nach der Befruchtung asynchron replizieren. Vier von fünf nicht geprägten Chromosomenregionen replizierten erst nach dem Zweizell-Embryostadium synchron. Eine asynchrone Replikation geprägter Regionen wurde während der gesamten Präimplantationsentwicklung und in differenzierten Zellen beobachtet. In Morula-Embryonen zeigten der in diesem Stadium nicht exprimierte Dlk1-Gtl2-Locus sowie der biallelisch exprimierte Igf2r-Locus jedoch eine Relaxation der asynchronen Replikation. In einem weiteren Projekt konnte mit Hilfe eines Multiplex-RT-PCR-Ansatzes die sensitive Detektion von multiplen Transkripten in einzelnen Zellen etabliert werden. Anschließend wurden Expressionsmuster von 17 für die epigenetische Reprogrammierung relevanten Entwicklungs-genen in Präimplantationsembryonen sowie in einzelnen Morula-Blastomeren analysiert. Der Transkriptionsfaktor Pou5f1 wurde in allen Präimplantationsembryonen und allen Morula-Blastomeren detektiert, was auf eine uniforme Reaktivierung der Pluripotenz hinweist. Dagegen variierte die mRNA-Expression verschiedener DNA-Cytosin-5-Methyltransferasen, 5-methyl-CpG-Bindeproteine sowie Enzyme der Basenexzisionsreparatur stark zwischen individuellen Zellen des gleichen Embryos und noch stärker zwischen Zellen verschiedener Embryonen. Diese Ergebnisse zeigen, dass sich das für die Reprogrammierungsmaschinerie kodierende Transkriptom zu bestimmten Entwicklungs-zeitpunkten zwischen einzelnen Blastomeren unterscheidet.

Expression of Imprinted Genes Is Aberrant in Deceased Newborn Cloned Calves and Relatively Normal in Surviving Adult Clones

Cattle are the species used most frequently for the development of assisted reproductive technologies, such as nuclear transfer. Cattle cloning can be performed by a large number of laboratories around the world, and the efficiency of nuclear transfer in cattle is the highest among all species in which successful cloning has been achieved. However, an understanding of the expression of imprinted genes in this important species is lacking. In the present study, real time reverse transcription polymerase chain reaction (RT-PCR) was utilized to quantify the expression of the bovine Igf2, Igf2r, and H19 genes in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen, and thymus) of somatic cell cloned calves that died shortly after birth, in three tissues (skin, muscle, and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. We found that, deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs. Large variations in the expression levels of imprinted genes were also seen among these clones, which were produced from the same genetic donor. In surviving adult clones, however, the expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only physically healthy but also relatively normal at the molecular level of those three imprinted genes.

Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P/IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P/IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P/IGF2R-mutated hepatocytes, providing further support for the idea that M6P/IGF2R functions as a liver tumor-suppressor gene.