RESUMO
Although it is well known that mRNA is present in mammalian spermatozoa, the relevance of mRNA to capacitation and early embryo development in the pig remains unclear. In the present study, we investigated differences in the abundance of selected mRNAs coding for MYC, CYP19, ADAM2, PRM1 and PRM2 in purified porcine spermatozoa depending on embryo cleavage rate and capacitation (n=20 semen samples). Semen samples were used in IVF procedures, with subsequent embryo development classified into one of two groups based on cleavage rate (i.e. high (>75%) and low (<75%) cleavage groups) and mRNA abundance in purified spermatozoa compared between these two groups. In addition, mRNA abundance was compared between capacitated and non-capacitated spermatozoa. Comparison of mRNA levels between porcine spermatozoa revealed that the abundance of MYC, CYP19, ADAM2, PRM1 and PRM2 mRNA was significantly greater in the high cleavage group (n=10 high cleavage group semen samples) than in the low cleavage group (n=10; P<0.05). Significant downregulation of MYC mRNA was observed in capacitated spermatozoa (n=12; P<0.05). The results of the present study suggest that the amount of specific mRNAs could be used for estimating the quality of spermatozoa in the pig.
Assuntos
Regulação da Expressão Gênica/fisiologia , RNA Mensageiro/metabolismo , Capacitação Espermática/fisiologia , Espermatozoides/citologia , Suínos/embriologia , Suínos/genética , Proteínas ADAM/genética , Animais , Aromatase/genética , Primers do DNA/genética , Fertilinas , Regulação da Expressão Gênica/genética , Genes myc/genética , Modelos Lineares , Masculino , Glicoproteínas de Membrana/genética , Protaminas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Espermatozoides/metabolismoRESUMO
In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos.
