DNA methylation and gene expression changes in mouse pre- and post-implantation embryos generated by intracytoplasmic sperm injection with artificial oocyte activation

Background The application of artificial oocyte activation (AOA) after intracytoplasmic sperm injection (ICSI) is successful in mitigating fertilization failure problems in assisted reproductive technology (ART). Nevertheless, there is no relevant study to investigate whether AOA procedures increase developmental risk by disturbing subsequent gene expression at different embryonic development stages. Methods We used a mouse model to explore the influence of AOA treatment on pre- and post-implantation events. Firstly, the developmental potential of embryos with or without AOA treatment were assessed by the rates of fertilization and blastocyst formation. Secondly, transcriptome high-throughput sequencing was performed among the three groups (ICSI, ICSI-AOA and dICSI-AOA groups). The hierarchical clustering and Principal Component Analysis (PCA) analysis were used. Subsequently, Igf2r/Airn methylation analysis were detected using methylation-specific PCR sequencing following bisulfite treatment. Finally, birth rate and birth weight were examined following mouse embryo transfer. Results The rates of fertilization and blastocyst formation were significantly lower in oocyte activation-deficient sperm injection group (dICSI group) when compared with the ICSI group (30.8 % vs. 84.4 %, 10.0 % vs. 41.5 %). There were 133 differentially expressed genes (DEGs) between the ICSI-AOA group and ICSI group, and 266 DEGs between the dICSI-AOA group and ICSI group. In addition, the imprinted gene, Igf2r is up regulated in AOA treatment group compared to control group. The Igf2r/Airn imprinted expression model demonstrates that AOA treatment stimulates maternal allele-specific mehtylation spreads at differentially methylated region 2, followed by the initiation of paternal imprinted Airn long non-coding (lnc) RNA, resulting in the up regulated expression of Igf2r. Furthermore, the birth weight of newborn mice originating from AOA group was significantly lower compared to that of ICSI group. The pups born following AOA treatment did not show any other abnormalities during early development. All offspring mated successfully with fertile controls. Conclusions AOA treatment affects imprinted gene Igf2r expression and mehtylation states in mouse pre- and post-implantation embryo, which is regulated by the imprinted Airn. Nevertheless, no significant differences were found in post-natal growth of the pups in the present study. It is hoped that this study could provide valuable insights of AOA technology in assisted reproduction biology.

Double paternal uniparental isodisomy 7 and 15 presenting with Beckwith-Wiedemann spectrum features

Here we describe for the first time double paternal uniparental isodisomy (iUPD) 7 and 15 in a baby boy with features in the Beckwith-Wiedemann syndrome spectrum (BWSp) (placentomegaly, hyperinsulinism, enlarged viscera, hemangiomas, and earlobe creases) in addition to conjugated hyperbilirubinemia. His phenotype was also reminiscent of genome-wide paternal uniparental isodisomy. We discuss the most likely origin of the UPDs; a maternal double monosomy 7 and 15 rescued by duplication of the paternal chromosomes after fertilization. So far, paternal UPD7 is not associated with an abnormal phenotype, while paternal UPD15 causes Angelman syndrome. Methylation analysis for other clinically relevant imprinting disorders, including BWSp, was normal. Therefore, we hypothesized that the double UPD affected other imprinted genes. To look for such effects, patient fibroblast RNA was isolated and analyzed for differential expression compared to six controls. We did not find apparent transcription differences in imprinted genes outside chromosomes 7 and 15 in patient fibroblast. PEG10 (7q21.3) was the only paternally imprinted gene on these chromosomes upregulated beyond double-dose expectation (6-fold). We speculate that a high PEG10 level could have a growth-promoting effect as his phenotype was not related to aberrations in BWS-locus on 11p15.5 after DNA, RNA, and methylation testing. However, many genes in gene sets associated with growth were upregulated. This case broadens the phenotypic spectrum of UPDs but did not show evidence of involvement of an imprinted gene network.

Association of allele-specific methylation of the ASNS gene with asparaginase sensitivity and prognosis in T-ALL

Asparaginase therapy is a key component of chemotherapy for T-cell acute lymphoblastic leukemia (T-ALL) patients. Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, while leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Since the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite PCR products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from childhood T-ALL patients in Japanese cohorts (n = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In childhood T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively showed ASNS hypomethylation status. These observations demonstrate that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL.

Zfp57 inactivation illustrates the role of ICR methylation in imprinted gene expression during neural differentiation of mouse ESCs

ZFP57 is required to maintain the germline-marked differential methylation at imprinting control regions (ICRs) in mouse embryonic stem cells (ESCs). Although DNA methylation has a key role in genomic imprinting, several imprinted genes are controlled by different mechanisms, and a comprehensive study of the relationship between DMR methylation and imprinted gene expression is lacking. To address the latter issue, we differentiated wild-type and Zfp57-/- hybrid mouse ESCs into neural precursor cells (NPCs) and evaluated allelic expression of imprinted genes. In mutant NPCs, we observed a reduction of allelic bias of all the 32 genes that were imprinted in wild-type cells, demonstrating that ZFP57-dependent methylation is required for maintaining or acquiring imprinted gene expression during differentiation. Analysis of expression levels showed that imprinted genes expressed from the non-methylated chromosome were generally up-regulated, and those expressed from the methylated chromosome were down-regulated in mutant cells. However, expression levels of several imprinted genes acquiring biallelic expression were not affected, suggesting the existence of compensatory mechanisms that control their RNA level. Since neural differentiation was partially impaired in Zfp57-mutant cells, this study also indicates that imprinted genes and/or non-imprinted ZFP57-target genes are required for proper neurogenesis in cultured ESCs.

Imprinted Gene Expression and Function of the Dopa Decarboxylase Gene in the Developing Heart

Dopa decarboxylase (DDC) synthesizes serotonin in the developing mouse heart where it is encoded by Ddc_exon1a, a tissue-specific paternally expressed imprinted gene. Ddc_exon1a shares an imprinting control region (ICR) with the imprinted, maternally expressed (outside of the central nervous system) Grb10 gene on mouse chromosome 11, but little else is known about the tissue-specific imprinted expression of Ddc_exon1a. Fluorescent immunostaining localizes DDC to the developing myocardium in the pre-natal mouse heart, in a region susceptible to abnormal development and implicated in congenital heart defects in human. Ddc_exon1a and Grb10 are not co-expressed in heart nor in brain where Grb10 is also paternally expressed, despite sharing an ICR, indicating they are mechanistically linked by their shared ICR but not by Grb10 gene expression. Evidence from a Ddc_exon1a gene knockout mouse model suggests that it mediates the growth of the developing myocardium and a thinning of the myocardium is observed in a small number of mutant mice examined, with changes in gene expression detected by microarray analysis. Comparative studies in the human developing heart reveal a paternal expression bias with polymorphic imprinting patterns between individual human hearts at DDC_EXON1a, a finding consistent with other imprinted genes in human.

Effects of Plasma Choline Concentrations on Placental Development and Fetal Growth, With Potential Mechanistic Roles of Imprinted Genes

Objectives Imprinted genes are epigenetically regulated and play critical roles in placental development and fetal growth. We aimed to examine (1) the impact of maternal one-carbon (methyl donor) nutrition on placental imprinted gene expression, placental development, and fetal growth; (2) whether imprinted gene expression alterations mediate effects of one-carbon nutrition on placental development and fetal growth; (3) interaction effects between one-carbon nutrients and imprinted genes in placental development and fetal growth. Methods Histopathology and expression of 109 imprinted genes (Nanostring) were assessed in placentas from 101 women recruited at initiation of antenatal care in a prospective cohort study examining developmental effects of prenatal alcohol exposure in South Africa. Women were interviewed prenatally about demographics, alcohol, smoking, and drug use, and erythrocyte folate, serum vitamin B12, and plasma choline concentrations were assayed at recruitment. Infant weight and height were assessed at age 2 wk. Results In limma tests, women with plasma choline concentrations below the median had lower placental expression of EPS15, IGF2R, LINC00657, SGCE, ZC3H12C, and ZNF264 than women above the median (p < .05, FDR < .10). In regression models adjusted for potential confounders (maternal age, gravidity, education, alcohol and drug use), plasma choline (μM) was associated with larger placental weight (g) (B = 14.0(1.9, 26.2)) and reduced maternal vascular underperfusion (MVU) prevalence (B = −.07(−.12, −.02). In causal inference analyses, there were trends for mediation of the relation between choline and MVU by decreased LINC00657, ZC3H12C, and ZNF264 expression. In regression models examining plasma choline X imprinted gene expression interaction effects, choline modified relations of EPS15, ZC3H12C, and ZNF264 to placental weight and fetal growth. Conclusions Maternal plasma choline was associated with decreased placental expression of 6 imprinted genes, 3 of which may mediate effects of choline on placental development. Choline modified effects of 3 genes on placental and fetal growth. These findings suggest maternal choline status may impact placental and fetal development, with imprinted genes playing mechanistic roles. Funding Sources NIH/NIAAA; Lycaki-Young Fund.

A Novel Imprinted Gene (Sp-Pol) with Sex-specific SNP Locus and Sex-biased Expression Pattern Provides Insights into the Gonad Development of Mud Crab (Scylla Paramamosain)

Background: We previously discovered a potential sex-specific single nucleotide polymorphism (SNP) locus named SNP1888 in mud crab (Scylla paramamosain). Methods: In this study, we first verified SNP1888 to be truly a female-specific locus and then we identified a novel imprinted gene (designated as Sp-Pol) at the upstream of SNP1888 (SNP1888 is located at the 3’-UTR of Sp-Pol). Moreover, SNP1888 together with Sp-Pol were mapped on LG32 of a high-density genetic map. Results: Phylogenetic analysis showed that Sp-Pol may need to be classified as a new gene family due to the very low sequence identity with other known genes. Sp-Pol was expressed at a higher level in gonads compared to other tissues and its expression level in the testis was much higher than in the ovary. Coincidentally, mono-allelic expression was observed in the ovary. Moreover, Sp-Pol exhibited sex-biased expression with approximately 3- to 4-fold higher in males than in females at fifth (C5) and sixth (C6) crablet stages. During the zoeae development, Sp-Pol had the highest expression at the zoea I stage. After unilateral eyestalk ablation, the expression level of Sp-Pol significantly increased in testis and hepatopancreas in males, while it was downregulated in the hepatopancreas of females. Fluorescence in situ hybridization (FISH) assay revealed that Sp-Pol transcripts were strongly localized in the epithelia of seminiferous tubules of the testis and in the ovary it was detected in the oogonium cells.Conclusion: These results demonstrated that Sp-Pol may play important roles in the sexual development of S. paramamosain.