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.
Epigenetics in spermatogenesis Trasler, Jacquetta M.
Molecular and cellular endocrinology,
07/2009, Letnik:
306, Številka:
1
Journal Article
Recenzirano
DNA methylation is a heritable epigenetic modification of cytosine residues within CpG dinucleotides associated with the modulation of gene expression and found at 20–30 million sites throughout the ...mammalian genome. The methylation of DNA is catalyzed by DNA (cytosine-5)-methyltransferases (DNMTs), regulates genes during development and plays a role in genomic imprinting and X inactivation. Abnormalities in DNA methylation lead to growth and behavioral abnormalities as well as cancer. Genomic methylation patterns are acquired in the germline and then further modified during embryogenesis. Methylation in the male germline is unique in comparison to that in somatic tissues, begins in the fetal gonad before birth, and is completed during postnatal spermatogenesis. In rodents, altered expression of the DNMTs through gene-targeting or drug treatment is associated with abnormal DNA methylation patterns in germ cells and perturbations in spermatogenesis. In man altered sperm DNA methylation patterns have been reported in individuals with oligospermia.
The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. ...Dnmt1o(mat-/-) mouse embryos born to Dnmt1(Δ1o/Δ1o) female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1(Δ1o/Δ1o) mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.
Imprinted genes play important roles in embryonic growth and development as well as in placental function. Many imprinted genes acquire their epigenetic marks during oocyte growth, and this period ...may be susceptible to epigenetic disruption following hormonal stimulation. Superovulation has been shown to affect growth and development of the embryo, but an effect on imprinted genes has not been shown in postimplantation embryos. In the present study, we examined the effect of superovulation/in vivo development or superovulation/3.5dpc (days post-coitum) embryo transfer on the allelic expression of Snrpn, Kcnq1ot1 and H19 in embryos and placentas at 9.5 days of gestation. Superovulation followed by in vivo development resulted in biallelic expression of Snrpn and H19 in 9.5dpc placentas while Kcnq1ot1 was not affected; in the embryos, there was normal monoallelic expression of the three imprinted genes. We did not observe significant DNA methylation perturbations in the differentially methylated regions of Snrpn or H19. Superovulation followed by embryo transfer at 3.5dpc resulted in biallelic expression of H19 in the placenta. The expression of an important growth factor closely linked to H19, Insulin-like growth factor-II, was increased in the placenta following superovulation with or without embryo transfer. These results show that both maternally and paternally methylated imprinted genes were affected, suggesting that superovulation compromises oocyte quality and interferes with the maintenance of imprinting during preimplantation development. Our findings contribute to the evidence that mechanisms for maintaining imprinting are less robust in trophectoderm-derived tissues, and have clinical implications for the screening of patients following assisted reproduction.
Abstract
STUDY QUESTION
Could clinically-relevant moderate and/or high dose maternal folic acid supplementation prevent aberrant developmental and epigenetic outcomes associated with assisted ...reproductive technologies (ART)?
SUMMARY ANSWER
Our results demonstrate dose-dependent and sex-specific effects of folic acid supplementation in ART and provide evidence that moderate dose supplements may be optimal for both sexes.
WHAT IS KNOWN ALREADY
Children conceived using ART are at an increased risk for growth and genomic imprinting disorders, often associated with DNA methylation defects. Folic acid supplementation is recommended during pregnancy to prevent adverse offspring outcomes; however, the effects of folic acid supplementation in ART remain unclear.
STUDY DESIGN, SIZE, DURATION
Outbred female mice were fed three folic acid-supplemented diets, control (rodent daily recommended intake or DRI; CD), moderate (4-fold DRI; 4FASD) or high (10-fold DRI; 10FASD) dose, for six weeks prior to ART and throughout gestation. Mouse ART involved a combination of superovulation, in vitro fertilisation, embryo culture and embryo transfer.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Midgestation embryos and placentas (n = 74-99/group) were collected; embryos were assessed for developmental delay and gross morphological abnormalities and embryos and placentas were examined for epigenetic defects. We assessed methylation at four imprinted genes (Snrpn, Kcnq1ot1, Peg1 and H19) in matched midgestation embryos and placentas (n = 31-32/group) using bisulfite pyrosequencing. In addition, we examined genome-wide DNA methylation patterns in placentas (n = 6 normal placentas per sex/group) and embryos (n = 6 normal female embryos/group; n = 3 delayed female embryos/group) using reduced representation bisulfite sequencing (RRBS).
MAIN RESULTS AND THE ROLE OF CHANCE
Moderate, but not high dose supplementation, was associated with a decrease in the proportion of developmentally delayed embryos. Although moderate dose folic acid supplementation reduced DNA methylation variance at certain imprinted genes in embryonic and placental tissues, high dose supplementation exacerbated the negative effects of ART at imprinted loci. Furthermore, folic acid supplements resolved female-biased aberrant imprinted gene methylation. Supplementation was more effective at correcting ART-induced genome-wide methylation defects in male versus female placentas; however, folic acid supplementation also led to additional methylation perturbations which were more pronounced in males.
LARGE-SCALE DATA
The RRBS data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE123143.
LIMITATIONS REASONS FOR CAUTION
Although the combination of mouse ART utilised in this study consisted of techniques commonly used in human fertility clinics, there may be species differences. Therefore, human studies, designed to determine the optimal levels of folic acid supplementation for ART pregnancies, and taking into account foetal sex, are warranted.
WIDER IMPLICATIONS OF THE FINDINGS
Taken together, our findings support moderation in the dose of folic acid supplements taken during ART.
STUDY FUNDING/COMPETING INTEREST(S)
This work was funded by the Canadian Institutes of Health Research (FDN-148425). The authors declare no conflict of interest.
Imprinted genes are differentially marked during germ cell development to allow for their eventual parent-of-origin specific expression. A subset of imprinted genes becomes methylated during oocyte ...growth in both mouse and human. However the timing and mechanisms of methylation acquisition are unknown. Here, we examined the methylation of the Snrpn, Igf2r, Peg1 and Peg3 differentially methylated regions in postnatal growing mouse oocytes. Our findings indicate that methylation was acquired asynchronously at these different genes. Further analysis of Snrpn DMR1 revealed that parental alleles retain an epigenetic memory of their origin as the two alleles were recognized in a parental-specific manner in the absence of DNA methylation. In addition, we show that methylation acquisition was probably related to oocyte diameter and coincided with the accumulation of Dnmt3a, Dnmt3b and Dnmt3L transcripts. Methylation of the repetitive retroviral-like intracisternal A particle also occurred during this same window of oocyte growth. These findings contribute to our understanding of the epigenetic mechanisms underlying imprint acquisition during female germ cell development and have implications for the practice of assisted reproductive technologies.
In the male germ line, sequence-specific methylation patterns are initially acquired prenatally in diploid gonocytes and are further consolidated after birth during spermatogenesis. It is still ...unclear how DNA methyltransferases are involved in establishing and/or maintaining these patterns in germ cells, or how their activity is regulated. We compared the temporal expression patterns of the postulated de novo DNA methyltransferases DNMT3a and DNMT3b in murine male germ cells. Mitotic, meiotic and post-meiotic male germ cells were isolated, and expression of various transcript variants and isoforms of
Dnmt3a and
Dnmt3b was examined using Quantitative RT-PCR and Western blotting. We found that proliferating and differentiating male germ cells were marked by distinctive expression profiles. Dnmt3a2 and Dnmt3b transcripts were at their highest levels in type A spermatogonia, decreased dramatically in type B spermatogonia and preleptotene spermatocytes and rose again in leptotene/zygotene spermatocytes, while Dnmt3a expression was mostly constant, except in type B spermatogonia where it increased. In all cases, expression declined as pachynema progressed. At the protein level, DNMT3a was the predominant isoform in type B spermatogonia, while DNMT3a2, DNMT3b2, and DNMT3b3 were expressed throughout most of spermatogenesis, except in pachytene spermatocytes. We also detected DNMT3a2 and DNMT3b2 in round spermatids. Taken together, these data highlight the tightly regulated expression of these genes during spermatogenesis and provide evidence that DNMTs may be contributing differentially to the establishment and/or maintenance of methylation patterns in male germ cells.
The acquisition of genomic DNA methylation patterns, including those important for development, begins in the germ line. In particular, imprinted genes are differentially marked in the developing ...male and female germ cells to ensure parent-of-origin-specific expression in the offspring. Abnormalities in imprints are associated with perturbations in growth, placental function, neurobehavioural processes and carcinogenesis. Based, for the most part, on data from the well-characterised mouse model, the present review will describe recent studies on the timing and mechanisms underlying the acquisition and maintenance of DNA methylation patterns in gametes and early embryos, as well as the consequences of altering these patterns.
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.
The organochlorine dichlorodiphenyltrichloroethane (DDT) is banned worldwide owing to its negative health effects. It is exceptionally used as an insecticide for malaria control. Exposure occurs in ...regions where DDT is applied, as well as in the Arctic, where its endocrine disrupting metabolite,
dichlorodiphenyldichloroethylene (
DDE) accumulates in marine mammals and fish. DDT and
DDE exposures are linked to birth defects, infertility, cancer, and neurodevelopmental delays. Of particular concern is the potential of DDT use to impact the health of generations to come via the heritable sperm epigenome.
The objective of this study was to assess the sperm epigenome in relation to
DDE serum levels between geographically diverse populations.
In the Limpopo Province of South Africa, we recruited 247 VhaVenda South African men and selected 50 paired blood serum and semen samples, and 47 Greenlandic Inuit blood and semen paired samples were selected from a total of 193 samples from the biobank of the INUENDO cohort, an EU Fifth Framework Programme Research and Development project. Sample selection was based on obtaining a range of
-DDE serum levels (
). We assessed the sperm epigenome in relation to serum
-DDE levels using MethylC-Capture-sequencing (MCC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq). We identified genomic regions with altered DNA methylation (DNAme) and differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3) in sperm.
Differences in DNAme and H3K4me3 enrichment were identified at transposable elements and regulatory regions involved in fertility, disease, development, and neurofunction. A subset of regions with sperm DNAme and H3K4me3 that differed between exposure groups was predicted to persist in the preimplantation embryo and to be associated with embryonic gene expression.
These findings suggest that DDT and
DDE exposure impacts the sperm epigenome in a dose-response-like manner and may negatively impact the health of future generations through epigenetic mechanisms. Confounding factors, such as other environmental exposures, genetic diversity, and selection bias, cannot be ruled out. https://doi.org/10.1289/EHP12013.
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