Research Article

Gene silencing during development of in vitro-produced female bovine embryos

Published: September 15, 2009
Genet. Mol. Res. 8 (3) : 1116-1127 DOI: 10.4238/vol8-3gmr638

Abstract

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). Dur­ing the course of development, inactivation of the X chromosome equili­brates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the ex­pression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-τ, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fer­tilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-mat­uration. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mM ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocyte-somatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8ºC, in a humidified 5% CO2 atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 106 sperm/mL TALP medium supplemented with BSA and PHE and cultivated un­der the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expres­sion of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to “inacti­vation memory” from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigen­etic memory, leading to frequent failures in nuclear reprogramming.

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). Dur­ing the course of development, inactivation of the X chromosome equili­brates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the ex­pression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-τ, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fer­tilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-mat­uration. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mM ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocyte-somatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8ºC, in a humidified 5% CO2 atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 106 sperm/mL TALP medium supplemented with BSA and PHE and cultivated un­der the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expres­sion of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to “inacti­vation memory” from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigen­etic memory, leading to frequent failures in nuclear reprogramming.