I-50: Embryo Loss Due to Epigenetic Anomaliesin the Male Germ Line: Role of Estrogen

Background: To investigate if aberrant methylation and expression of imprinted genes of the Igf2-H19 locus in the spermatozoa and embryos could be a paternal epigenetic factor involved in early embryo loss To elucidate the role of estrogen in acquisition of the imprinting at the Igf2-H19 locus during spermatogenesis Materials and Methods: Adult male rats of Holtzman strain were administered tamoxifen citrate at a dose of 0.4mg/kg/day for 60 days. Control males rats were administered water only. Control and tamoxifen treated males were mated with normal cycling females a week before sacrifice. Caudal spermatozoa and normal and resorbed embryos sired by these males were processed for DNA methylation status at the Igf2-H19 imprinting control region (ICR) by Bisulfite sequencing, Methyl Specific PCR and Combined Bisulfite Restriction Analysis. The effect of tamoxifen treatment of global DNA methylation in spermatozoa was studied by methylation mapping of Line 1 elements and 5-methyl cytosine content by flow cytometry. Expression of Igf2 protein and H19 transcript was studied in normal and resorbed embryos. To elucidate the role of estrogen, estrogen response elements (ERE) were identified in the Igf2-H19 ICR (H19 DMR) and its functionality determined in the male germ cells by Chromatin-immunoprecipitation with DNAβ antibody. In addition, interaction of ER β with Estrogen receptor methyltransferase, Dnmt1 and PCNA in the male germ cells was studied by immunoprecipitation and confocal studies.Results: Methylation analysis by bisulfite sequencing of CpG island at H19 DMR in control and tamoxifen treated male rats showed association between methylation at H19 DMR in the spermatozoa and embryo loss. This observation suggests potential application of H19 DMR methylation in spermatozoa as a predictive factor to assess embryonic development. The study also showed hypomethylation at H19 DMR in spermatozoa upon tamoxifen treatment indicating an effect of tamoxifen in the establishment and/ or maintenance of methyl imprint during spermatogenesis. Since tamoxifen is a selective estrogen receptor modulator, the effects could be mediated through estrogen associated signaling pathways. An indicator of genome wide methylation, Line1 and 5-methyl cytosine content did not show any change with tamoxifen treatment confirming Igf2-H19 locus specific effect of tamoxifen treatment. Combined bisulfite restriction analysis and bisulfite sequencing ofH19 DMR in embryo revealed loss of methylation on the paternal allele in resorbed embryo. Resorbed embryo from control and tamoxifen group showed decrease in Igf2 protein expression and no change in H19 gene expression. This observation indicated deregulation of imprinting of Igf2 in embryo leading to aberrant expression of this gene. Deregulation of imprinting leading to aberrant expression of Igf2 could be one of the factors leading to resorption of embryo. Methylation error on the paternal allele could be responsible for loss of imprinting bringing about aberrant expression of Igf2 leading to embryo resorption. In rat testicular germ at H19 DMR was β cells, a functional estrogen response element binding ER was β detected. Also, a colocalization and interaction between Dnmt1 and ER observed in rat testis. These observations of protein–protein and protein– DNA interaction prompted us to propose a model for DMR methylation. Dnmt1 complexed , bound to ERE on Igf2/H19 locus could catalyze methylation of β with PCNA and ER-ERE β CpG island on H19 DMR. With tamoxifen treatment, reduction in ER interaction was observed which could be attributed to antiestrogenic action of tamoxifen leading to inhibited targeting of PCNADnmt1 complex. This could result into methylation error in spermatozoa either by interfering with maintenance or spreading activity of Dnmt1.Conclusion: The study demonstrates significant hypomethylation at the H19 DMR in the spermatozoa of tamoxifen treated males, which is transmitted to its progeny affecting their developmental potential. The study indicates that methylation at the H19 DMR in the spermatozoa could be used as a predictive factor for pregnancy outcome. The study suggests involvement of estrogen in the acquisition of imprint in the male germ cells and explains the possible impact of environmental estrogens on male reproductive health.