Donovan Chan

Reproductive and epigenetic outcomes associated with aging mouse oocytes

Female aging entails a decline in fertility in mammals, manifested by reduced oocyte reserves and poor oocyte quality accompanied by chromosomal anomalies and reduced litter size. In addition to compromised genetic integrity, recent studies suggest that epigenetic mechanisms may be altered in aging oocytes, with age affecting the expression of DNA methyltransferases, which catalyze the important epigenetic modification, DNA methylation. Loss of DNA methylation patterns, most notably for imprinted genes, is lethal to mouse embryos. To investigate how maternal age affects embryonic development and underlying DNA methylation patterns, young and aged C57BL/6 females were mated with C57BL/6 or C57BL/6(CAST7) males to allow for the identification of parental alleles; resulting blastocysts and mid-gestation embryos and placentas were evaluated. Although pregnancy, ovulation and implantation rates were similar between age groups, an age-related increase in resorption sites, morphological abnormalities and delayed development was found. Interestingly, placental morphology was also perturbed by aging, with elevated numbers of trophoblast giant cells in aged pregnancies. Normal monoallelic expression of the imprinted genes H19 and Snrpn was unaltered in blastocysts from aged females. We failed to observe any age-related changes in methylation of the differentially methylated regions of imprinted genes Snrpn, Kcnq1ot1, U2af1-rs1, Peg1, Igf2r and H19. Restriction Landmark Genome Scanning showed no significant differences in genome-wide DNA methylation in embryos and placentas, regardless of maternal age. Our findings demonstrate that maternal age affects post-implantation embryo and placental development; however embryos capable of developing to mid-gestation appear to undergo normal acquisition and maintenance of DNA methylation patterning.

Epigenetic Alterations in Sperm DNA Associated with Testicular Cancer Treatment

DNA methylation, a key component of the epigenome involved in regulating gene expression, is initially acquired in the germ line at millions of sites across the genome. Altered sperm methylation patterns are associated with infertility and transgenerational effects in humans and rodents. Testicular cancer is the most common form of cancer among men of reproductive age and has a high cure rate associated with chemotherapy treatment with bleomycin, etoposide, and cis-platinum (BEP). Although these drugs result in improved survival, they also affect the number and quality of germ cells. Our goal was to assess germ cell methylation patterns in a rodent model emulating the BEP treatment regimens used in human testicular cancer treatment. Animals were treated with control, or 0.3× (low) or 0.6× (high) dose of BEP, where a 1× dose is equivalent to human treatment regimens. Both dose-dependent and germ cell-dependent DNA methylation alterations were found at numerous loci throughout the genome. Of about 3000 loci tested, 42 loci were affected by BEP at the round spermatid stage of germ cell development, whereas 101 loci were affected in spermatozoa; 15 loci were consistently altered in spermatozoa of all high dose-treated rats. Both hyper- and hypomethylation were detected, suggesting either an interference with normal methylation patterning or abnormal repair of damaged patterns during spermatogenesis. The results indicate that a combination chemotherapy regimen used for testicular cancer treatment can result in altered DNA methylation patterns in spermatozoa and that some loci are more susceptible to damage than others.

Strain-specific defects in testicular development and sperm epigenetic patterns in 5,10-methylenetetrahydrofolate reductase-deficient mice.

Methylenetetrahydrofolate reductase (MTHFR) is a crucial folate pathway enzyme that contributes to the maintenance of cellular pools of S-adenosylmethionine, the universal methyl donor for several reactions including DNA methylation. Whereas Mthfr(-/-) BALB/c mice show growth retardation, developmental delay, and spermatogenic defects and infertility, C57BL/6 mice appear to have a less severe phenotype. In the present study, we investigated the effects of MTHFR deficiency on early germ cell development in both strains and assessed whether MTHFR deficiency results in DNA methylation abnormalities in sperm. The reproductive phenotype associated with MTHFR deficiency differed strikingly between the two strains, with BALB/c mice showing an early postnatal loss of germ cell number and proliferation that was not evident in the C57BL/6 mice. As a result, the BALB/c MTHFR-deficient mice were infertile, whereas the C57BL/6 mice had decreased sperm numbers and altered testicular histology but showed normal fertility. Imprinted genes and sequences that normally become methylated during spermatogenesis were unaffected by MTHFR deficiency in C57BL/6 mice. In contrast, a genome-wide restriction landmark genomic scanning approach revealed a number of sites of hypo- and hypermethylation in the sperm of this mouse strain. These results showing strain-specific defects in MTHFR-deficient mice may help to explain population differences in infertility among men with common MTHFR polymorphisms.

Impact of the Chemotherapy Cocktail Used to Treat Testicular Cancer on the Gene Expression Profile of Germ Cells from Male Brown-Norway Rats

Abstract Advances in treatment for testicular cancer that include the coadministration of bleomycin, etoposide, and cisplatin (BEP) have brought the cure rate to higher than 90%%. The goal of this study was to elucidate the impact of BEP treatment on gene expression in male germ cells. Brown-Norway rats were treated for 9 wk with vehicle (0×) or BEP at doses equivalent to 0.3× and 0.6× the human dose. At the end of treatment, spermatogenesis was affected, showing altered histology and a decreased sperm count; spermatozoa had a higher number of DNA breaks. After 9 wk of treatment, round spermatids were isolated, and RNA was extracted and probed on Rat230–2.0 Affymetrix arrays. Of the 31 099 probe sets present on the array, 59%% were expressed in control round spermatids. BEP treatment significantly altered the expression of 221 probe sets, with at least a 1.5-fold change compared with controls; 80%% were upregulated. We observed a dose-dependent increase in the expression of oxidative stress response genes and no change in the expression of genes involved in DNA repair. BEP upregulated genes were implicated in pathways related to Jun and Junb protooncogenes. Increased mRNA levels of Jun and Junb were confirmed by quantitative RT-PCR; furthermore, JUN protein was increased in elongating spermatids. Thus, BEP exposure triggers an oxidative stress response in round spermatids and induces many pathways that may lead to the survival of damaged cells and production of abnormal sperm.

Critical period of nonpromoter DNA methylation acquisition during prenatal male germ cell development.

The prenatal period of germ cell development is a key time of epigenetic programming in the male, a window of development that has been shown to be influenced by maternal factors such as dietary methyl donor supply. DNA methylation occurring outside of promoter regions differs significantly between sperm and somatic tissues and has recently been linked with the regulation of gene expression during development as well as successful germline development. We examined DNA methylation at nonpromoter, intergenic sequences in purified prenatal and postnatal germ cells isolated from wildtype mice and mice deficient in the DNA methyltransferase cofactor DNMT3L. Erasure of the parental DNA methylation pattern occurred by 13.5 days post coitum (dpc) with the exception of approximately 8% of loci demonstrating incomplete erasure. For most loci, DNA methylation acquisition occurred between embryonic day 13.5 to 16.5 indicating that the key phase of epigenetic pattern establishment for intergenic sequences in male germ cells occurs prior to birth. In DNMT3L-deficient germ cells at 16.5 dpc, average DNA methylation levels were low, about 30% of wildtype levels; however, by postnatal day 6, about half of the DNMT3L deficiency-specific hypomethylated loci had acquired normal methylation levels. Those loci normally methylated earliest in the prenatal period were the least affected in the DNMT3L-deficient mice, suggesting that some loci may be more susceptible than others to perturbations occurring prenatally. These results indicate that the critical period of DNA methylation programming of nonpromoter, intergenic sequences occurs in male germline progenitor cells in the prenatal period, a time when external perturbations of epigenetic patterns could result in diminished fertility.

Developmental windows of susceptibility for epigenetic inheritance through the male germline

Exposure of developing male germ cells to environmental insults has been linked to adverse effects in the offspring. One mechanism by which germ cell defects may be passed intergenerationally is through perturbations in the epigenome at the level(s) of DNA methylation, histone post-translational modifications and/or small non-coding RNAs. Epigenetic programs are particularly dynamic in germ cells undergoing erasure, re-establishment and maintenance of patterns, events potentially susceptible to prenatal and/or postnatal exposures. In this review, we focus on the epigenetic events occurring at each phase of male germ cell development including the prenatal period covering primordial germ cells and prospermatogonia and the postnatal period covering mitotic spermatogonia, meiotic spermatocytes and post-meiotic haploid spermatids and spermatozoa. Strong barriers to the passage of abnormal epigenetic patterns between generations are erected at two times of genome-wide epigenomic reprogramming, first in the germline in primordial germ cells and second, post-fertilization, during preimplantation development. Evidence from high resolution profiling studies that not all epigenetic marks are erased during germ cell and embryonic reprogramming provides a potential explanation for the intergenerational inheritance of abnormal epigenetic marks that may affect offspring health.

High dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism

Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR.

Transient DNMT1 suppression reveals hidden heritable marks in the genome

Genome-wide demethylation and remethylation of DNA during early embryogenesis is essential for development. Imprinted germline differentially methylated domains (gDMDs) established by sex-specific methylation in either male or female germ cells, must escape these dynamic changes and sustain precise inheritance of both methylated and unmethylated parental alleles. To identify other, gDMD-like sequences with the same epigenetic inheritance properties, we used a modified embryonic stem (ES) cell line that emulates the early embryonic demethylation and remethylation waves. Transient DNMT1 suppression revealed gDMD-like sequences requiring continuous DNMT1 activity to sustain a highly methylated state. Remethylation of these sequences was also compromised in vivo in a mouse model of transient DNMT1 loss in the preimplantation embryo. These novel regions, possessing heritable epigenetic features similar to imprinted-gDMDs are required for normal physiological and developmental processes and when disrupted are associated with disorders such as cancer and autism spectrum disorders. This study presents new perspectives on DNA methylation heritability during early embryo development that extend beyond conventional imprinted-gDMDs.

Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation

STUDY QUESTION Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? SUMMARY ANSWER No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 &mgr;g/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. WHAT IS KNOWN ALREADY Excess dietary folate may be detrimental to health and DNA methylation profiles due to folates role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. STUDY DESIGN, SIZE, DURATION This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996–1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 &mgr;g folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. PARTICIPANTS/MATERIALS, SETTING, METHODS Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. MAIN RESULTS AND THE ROLE OF CHANCE Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. LARGE SCALE DATA Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781. LIMITATIONS, REASONS FOR CAUTION This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated. WIDER IMPLICATIONS OF THE FINDINGS Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 &mgr;g/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome. STUDY FUNDING/COMPETING INTERESTS This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare.

Compromised oocyte quality and assisted reproduction contribute to sex-specific effects on offspring outcomes and epigenetic patterning

Clinical studies have revealed an increased incidence of growth and genomic imprinting disorders in children conceived using assisted reproductive technologies (ARTs), and aberrant DNA methylation has been implicated. We propose that compromised oocyte quality associated with female infertility may make embryos more susceptible to the induction of epigenetic defects by ART. DNA methylation patterns in the preimplantation embryo are dependent on the oocyte-specific DNA methyltransferase 1o (DNMT1o), levels of which are decreased in mature oocytes of aging females. Here, we assessed the effects of maternal deficiency in DNMT1o (Dnmt1Δ1o/+) in combination with superovulation and embryo transfer on offspring DNA methylation and development. We demonstrated a significant increase in the rates of morphological abnormalities in offspring collected from Dnmt1Δ1o/+  females only when combined with ART. Together, maternal oocyte DNMT1o deficiency and ART resulted in an accentuation of placental imprinting defects and the induction of genome-wide DNA methylation alterations, which were exacerbated in the placenta compared to the embryo. Significant sex-specific trends were also apparent, with a preponderance of DNA hypomethylation in females. Among genic regions affected, a significant enrichment for neurodevelopmental pathways was observed. Taken together, our results demonstrate that oocyte DNMT1o-deficiency exacerbates genome-wide DNA methylation abnormalities induced by ART in a sex-specific manner and plays a role in mediating poor embryonic outcome.