An N-ethyl-N-nitrosourea random mutation screen was used to identify recessive modifiers of gene silencing in the mouse using an epigenetically sensitive reporter transgene. One of the mutant lines, MommeR1, was identified as a suppressor of variegation and it showed female-specific age-associated infertility in homozygotes. Linkage analysis identified a region on chromosome 10, containing the Foxo3a gene, previously shown to play a critical role in female gametogenesis. Foxo3a is a transcription factor with roles in cell cycle control, apoptosis, neural and hematopoietic cell differentiation, and DNA repair. Sequencing of the Foxo3a gene in MommeR1 mice revealed a point mutation that causes an amino acid substitution in the highly conserved Forkhead DNA-binding domain. In vitro transcription assays showed that the point mutation causes loss of FOXO3a transactivation activity. Compound heterozygotes made with Foxo3a-null mice (carrying the targeted deletion of exon 2) displayed complementation with respect to both the activation of the reporter transgene and defects in folliculogenesis similar to those seen in MommeR1 homozygotes, supporting the conclusion that this is the causative mutation. Approximately one in six female MommeR1 homozygotes develop teratomas, a phenotype not reported in Foxo3a-null mice. Ovulated oocytes from MommeR1 homozygotes display a number of abnormalities. The MommeR1 mice provide a novel platform to investigate teratocarcinogenesis and link Foxo3a with parthenogenesis and ovarian cancer. The finding of Foxo3a as a modifier of epigenetic reprogramming is discussed.

Biomarker discovery applied to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a disabling disease of inconclusive aetiology, has identified several cytokines to potentially fulfil a role as a quantitative blood/serum marker for laboratory diagnosis, with activin B a recent addition. We explored further the potential of serum activin B as a ME/CFS biomarker, alone and in combination with a range of routine test results obtained from pathology laboratories. Previous pilot study results showed that activin B was significantly elevated for the ME/CFS participants compared to healthy (control) participants. All the participants were recruited via CFS Discovery and assessed via the Canadian/International Consensus Criteria. A significant difference for serum activin B was also detected for ME/CFS and control cohorts recruited for this study, but median levels were significantly lower for the ME/CFS cohort. Random Forest (RF) modelling identified five routine pathology blood test markers that collectively predicted ME/CFS at ≥62% when compared via weighted standing time (WST) severity classes. A closer analysis revealed that the inclusion of activin B to the panel of pathology markers improved the prediction of mild to moderate ME/CFS cases. Applying correct WST class prediction from RFA modelling, new reference intervals were calculated for activin B and associated pathology markers, where 24-h urinary creatinine clearance, serum urea and serum activin B showed the best potential as diagnostic markers. While the serum activin B results remained statistically significant for the new participant cohorts, activin B was found to also have utility in enhancing the prediction of symptom severity, as represented by WST class.

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Previous studies on the epigenetic regulator DNA methyltransferase 3-Like (DNMT3L), have demonstrated it is an essential regulator of paternal imprinting and early male meiosis. Dnmt3L is also a paternal effect gene, i.e., wild type offspring of heterozygous mutant sires display abnormal phenotypes suggesting the inheritance of aberrant epigenetic marks on the paternal chromosomes. In order to reveal the mechanisms underlying these paternal effects, we have assessed X chromosome meiotic compaction, XY chromosome aneuploidy rates and global transcription in meiotic and haploid germ cells from male mice heterozygous for Dnmt3L. XY bodies from Dnmt3L heterozygous males were significantly longer than those from wild types, and were associated with a three-fold increase in XY bearing sperm. Loss of a Dnmt3L allele resulted in deregulated expression of a large number of both X-linked and autosomal genes within meiotic cells, but more prominently in haploid germ cells. Data demonstrate that similar to embryonic stem cells, DNMT3L is involved in an auto-regulatory loop in germ cells wherein the loss of a Dnmt3L allele resulted in increased transcription from the remaining wild type allele. In contrast, however, within round spermatids, this auto-regulatory loop incorporated the alternative non-coding alternative transcripts. Consistent with the mRNA data, we have localized DNMT3L within spermatids and sperm and shown that the loss of a Dnmt3L allele results in a decreased DNMT3L content within sperm. These data demonstrate previously unrecognised roles for DNMT3L in late meiosis and in the transcriptional regulation of meiotic and post-meiotic germ cells. These data provide a potential mechanism for some cases of human Klinefelter's and Turner's syndromes.