Differential methylation status of IGF2‐H19 locus does not affect the fertility of crossbred bulls but some of the CTCF binding sites could be potentially important

Abstract

Associations between abnormal methylation of spermatozoan DNA with male infertility have been sought in recent years to identify a molecular explanation of differential spermatozoan function. The present work was undertaken to investigate the methylation profile of differentially methylated regions (DMRs) in the IGF2-H19 locus of Bos taurus X Bos indicus crossbred bull spermatozoa. Bulls having more than at least 100 insemination records over a period of 12 years were classified into two groups of five bulls each belonging to low- and high-fertility groups. The IGF2 and H19 DMR sequences in B. indicus cattle were observed to be in absolute homology with B. taurus cattle. The DNA of crossbred bull spermatozoa was isolated, bisulfite treated, and amplified for specific DMR regions using methylation-change-specific primers. The overall degree of methylation at IGF2-H19 DMRs was not found to be significantly different among two groups of bulls. The sixth CTCF binding site (CCCTC) identified in H19 DMR, however, had a significant methylation difference between the high- and low-fertility bulls. It was concluded that alteration of the methylation levels at IGF2-H19 DMRs might not be responsible for the fertility difference of crossbred bulls, although the role played by the specific CTCF binding sites at this locus, which could influence IGF2 expression during spermatogenesis and early embryonic development, deserves further attention.

Sperm DNA Methylation Analysis in Swine Reveals Conserved and Species-Specific Methylation Patterns and Highlights an Altered Methylation at the GNAS Locus in Infertile Boars1

“…This result is not related to poor identification and localization in swine of IGF2 and H19 DMRs, which are highly homologous to the corresponding human gene cluster and well described in the literature [50,51]. Moreover, our results are in accordance with a study on bovine sperm that did not find any significant difference in the methylation level of IGF2/H19 DMRs of high-or low-fertility bulls [52].…”

The Role of CCCTC-Binding Factor (CTCF) in Genomic Imprinting, Development, and Reproduction1

“…Furthermore, DNA methylation alterations are associated with subfertility and/or infertility in humans and animals, and abnormal DNA methylation at the CTCF-binding site 6 of the H19 DMR is closely associated with oligozoospermia [114,115], azoospermia [116], teratozoospermia, and oligo-astheno-teratozoospermia [117] in humans, and altered conception rates in cattle [118]. Interestingly, the same sixth CTCF-binding site was found to be hypomethylated in placentas of pregnancies conceived by IVEP/ICSI [119].…”

DNA methylation in spermatogenesis and male infertility

“…The methylation of constitutive heterochromatic and promoter regions is generally associated with reduced gene transcription (Fig. 1A) (15)(16)(17)(18). Therefore, DNA methylation is a type of epigenetic modification that can effectively promote gene silencing (Fig.…”

“…However, DNMT1 heterozygous mice had a normal reproductive ability, thus suggesting that DNMT1 in the heterozygous state is sufficient to maintain the required DNA methylation pattern (30,75,76). DNMT3a expression in the testis is upregulated prior to birth and during early postnatal life, whereas DNMT3b expression is downregulated during embryonic development and upregulated postnatally (15)(16)(17)(18)(19)(20). Notably, mice lacking DNMT3L have smaller testes, and a negligible number of spermatozoa by adulthood, resulting in sterile animals.…”