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

Abstract

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-t, 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 oocytesomatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8°C, in a humidified 5% CO 2 atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 10 6 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.

Chemically Assisted Enucleation Results in HigherG6PDExpression in Early Bovine Female Embryos Obtained by Somatic Cell Nuclear Transfer

“…Thus, a disruption of normal XIST expression likely occurs in IVF embryos, evidenced by the high percentage of embryos expressing the XIST gene, as shown in previous studies (Merighe et al, 2009;Wrenzycki et al, 2002).…”

Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos

“…The X-chromosome dosage compensation mechanism by random inactivation of one X-chromosome is an essential process during the early phases of embryonic development. Failure to achieve complete inactivation during in vitro production would initiate epigenetic differences between female and male bovine BLs [33]. Moreover, a higher abundance of DNMTs transcript was detected in the early embryonic stage of X-embryos, while expression was decreased at the BL stage relative to the Y counterpart group (Figure 6C,D).…”

HDAC inhibition decreases XIST expression on female IVP bovine blastocysts

“…Controversial results were found regarding G6PD expression, which was shown to be increased in some studies (Gutiérrez-Adán et al 2000, Peippo et al 2002, Wrenzycki et al 2002, Lopes et al 2007, Morton et al 2007, Bermejo-Alvarez et al 2011 and decreased in others (Merighe et al 2009), when comparing female and male IVP blastocysts. Considering that G6PD varies its expression according to respiration and metabolism (Lopes et al 2007), their different mRNA levels among systems might be related to stressful culture conditions.…”