The methylation patterns of the IGF2 and IGF2R genes in bovine spermatozoa are not affected by flow‐cytometric sex sorting

Abstract

The objectives of this study were to investigate the effect of sexing by flow cytometry on the methylation patterns of the IGF2 and IGF2R genes. Frozen-thawed, unsorted, and sex-sorted sperm samples from four Nellore bulls were used. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). Sperm were isolated from the extender, cryoprotectant, and other cell types by centrifugation on a 40:70% Percoll gradient, and sperm pellets were used for genomic DNA isolation. DNA was used for analyses of the methylation patterns by bisulfite sequencing. Methylation status of the IGF2 and IGF2R genes were evaluated by sequencing 195 and 147 individual clones, respectively. No global differences in DNA methylation were found between NS, SX, and SY groups for the IGF2 (P = 0.09) or IGF2R genes (P = 0.38). Very specific methylation patterns were observed in the 25th and 26th CpG sites in the IGF2R gene. representing higher methylation in NS than in the SX and SY groups compared with the other CpG sites. Further, individual variation in methylation patterns was found among bulls. In conclusion, the sex-sorting procedure by flow cytometry did not affect the overall DNA methylation patterns of the IGF2 and IGF2R genes, although individual variation in their methylation patterns among bulls was observed.

Sex selection of sperm in farm animals: status report and developmental prospects

“…IVF results are also affected by sperm concentration, which needs to be higher than for IVF with unsorted sperm, and by the hydrodynamic pressure used for sorting (BarceloFimbres et al 2011). Although the sorting process has significant effects on sperm viability, and bull effects are obvious, compromised DNA does not affect reduced fertility as most non-viable spermatozoa, and those with aneuploidies are out-gated from sorting by parallel Similarly, bisulphide sequencing of Nellore bull spermatozoa DNA indicated that the sorting process by flow cytometry does not affect the overall DNA methylation patterns of the insulin-like growth factor 2 (IGF2) and IGF2 receptor (IGF2R) genes in sperm, although individual variation in methylation patterns among bulls was observed (Carvalho et al 2012).…”

Aspectos moleculares, estruturais e funcionais de espermatozoides bovinos sexados por citometria de fluxo

“…Durante o processo de sexagem, os espermatozoides podem sofrer danos na sua estrutura, comprometendo sua viabilidade (BLONDIN et al, 2009;CARVALHO et al, 2010). Desta forma, várias características espermáticas, tais como motilidade, integridade de membrana, integridade de acrossoma, integridade de cromatina, capacidade de gerar blastocisto e perfil de mRNA, tem sido avaliadas (BOE-HANSEN et al, 2005;MORTON et al, 2007;BLONDIN et al, 2009;CARVALHO et al, 2010).…”

“…Desta forma, várias características espermáticas, tais como motilidade, integridade de membrana, integridade de acrossoma, integridade de cromatina, capacidade de gerar blastocisto e perfil de mRNA, tem sido avaliadas (BOE-HANSEN et al, 2005;MORTON et al, 2007;BLONDIN et al, 2009;CARVALHO et al, 2010). Entretanto, nenhum destes estudos avaliou se o processo de sexagem poderia causar alguma mudança epigenética no YOUNG et al, 2001;KANG et al, 2001;LONG;CAI, 2007 London, v. 460, p. 473-478, 2009b. HEALY, A.A.; HOUSE, J.K.; THOMSON, P.C.…”

“…Isso porque na PIVE é possível utilizar um menor número de espermatozoides para fecundar um grande número de ovócitos, otimizando o uso de uma dose de sêmen sexado, que além de apresentar menor porcentagem de células viáveis, também apresenta menor concentração de células/dose. Além disto, as melhorias no processo de sexagem (SHARPE; EVANS, 2009) e alterações nos protocolos de PIVE ou preparação espermática (BLONDIN et al, 2009;CARVALHO et al, 2010;VILLAMIL et al, 2012) tem permitido uma melhora nas taxas de blastocisto quando se utiliza sêmen sexado, alcançando taxas semelhantes as do sêmen não sexado (BLONDIN et al, 2009;CARVALHO et al, 2010).…”

“…Esses resultados sugerem que os danos causados nos espermatozoides pelo processo de sexagem (BLONDIN et al, 2009, CARVALHO et al, 2010 Madson, v. 28, p. 312-321, 1983. EMMENS, C.W.…”