Evaluation of Xist expression in preattachment equine embryos.

The research investigated the expression of the Xist gene, which is the marker for Barr-body or X-chromosomes inactivation in female equine embryos and assessed its potential use for determining gender in equine embryos.

Research Background and Purpose

• The aim of the research was to explore the role and expression of the X-inactivated-specific transcript (Xist) gene in equine embryos.
• The Xist gene marks the point of X-chromosomes inactivation in female mammals and its product, RNA, coats and deactivates one of the X chromosomes.
• The researchers wanted to observe if the Xist gene could be used alongside Y-chromosome-specific sequences as a more accurate tool for determining the sex of equine embryos earlier in their development.

Methodology

• Preattachment embryos at varying times after ovulation were analyzed for Xist RNA expression using quantitative and qualitative reverse transcription-polymerase chain reaction (RT-PCR).
• Primary equine dermal cell cultures from females and males were used as positive and negative controls, respectively.
• Embryos that tested Xist-negative were further evaluated for the male-specific eSRY gene expression using a qualitative PCR at the DNA level.
• The researchers also examined the counter transcript Tsix in the horse.

Findings

• The researchers found that 50% of the embryos analyzed via qualitative RT-PCR showed positive Xist expression. Also, 55% of the embryos tested by quantitative RT-PCR showed positive Xist expression.
• All Xist-negative equine embryos were positive for eSRY gene, which is male-specific, using quantitative PCR.
• Unlike in many other species, neither Xist nor Tsix was expressed in male equine testicular tissue.
• The results indicate that Xist expression is exclusive to female embryos in horses.
• The study thus concludes that in addition to Y-chromosome markers, Xist gene can be used as an X-inactivation-specific marker for sex determination in equine.