Sex-dependent insulin like growth factor-1 expression in preattachment equine embryos.

An adjustment of sex ratio of offspring to the conditions present at conception is seen in many mammals including horses. This depends on preferential survival of male embryos under conditions of high energy intake. In several species, growth factors including insulin like growth factor (IGF)-1 have been shown to promote embryonic development by decreasing apoptosis and increasing cell proliferation. We hypothesized that sex-related differences in IGF-1 expression in equine embryos during the phase of maternal recognition of pregnancy might exist and thus contribute to preferential survival of embryos from either of both sexes under specific environmental conditions. Insulin like growth factor-1 mRNA expression of in vivo-produced equine embryos on different days of pregnancy (Day 8, N = 6; Day 10, N = 8; Day 12, N = 14) was analyzed. Insulin like growth factor-1 mRNA expression was evaluated by reverse transcription quantitative polymerase chain reaction. The sex of the embryo was determined by detection of X-inactivation specific transcript (Xist) RNA and equine sex determining region of the Y chromosome DNA. Embryos positive for Xist expression were classified as female, and Xist negative and equine sex determining region of the Y chromosome positive embryos were classified as male. From 28 embryos tested, 15 (54%) showed positive Xist expression and were thus classified as female. Insulin like growth factor-1 mRNA expression was influenced by sex (P = 0.01) but not by day of pregnancy (relative expression of IGF-1 in relation to ß-actin, Day 8: male 5.1 ± 2.1, female 11.4; Day 10: male 5.2 ± 1.6, female 17.4 ± 6.7; Day 12: male 2.6 ± 0.3, female 11.6 ± 2.4). Results demonstrate an increased expression of IGF-1 in female equine embryos. Sex-related influences on expression of the IGF system are probably related to a gradual X chromosome inactivation.

Evaluation of Xist expression in preattachment equine embryos.

Until now, sex determination in equine embryos has been performed by detection of Y-chromosome-specific sequences only. In the present study, expression of a Barr-body-specific marker, the X-inactivated-specific transcript (Xist) gene, whose gene product consists of RNA which coats and thereby inactivates one of the X chromosomes, was investigated in equine embryos produced in vivo. Preattachment embryos at different times after ovulation (Day 8: n = 9; Day 10: n = 12; Day 12: n = 15) were analyzed for Xist RNA expression using quantitative and qualitative reverse transcription-polymerase chain reaction (RT-PCR). Female and male primary equine dermal cell cultures were used as positive and negative controls, respectively. Embryos tested negative for Xist were evaluated for expression of the male-specific eSRY gene by qualitative PCR at the DNA level. From 36 embryos assessed by qualitative RT-PCR, 18 showed positive Xist expression (50%). From 29 embryos tested by quantitative RT-PCR, 16 showed positive Xist expression (55%). All of the Xist-negative equine embryos tested by quantitative PCR were positive for eSRY. We also demonstrated by strand-specific RT-PCR that in the horse, as in humans, the counter transcript Tsix seems to be truncated not reaching Exon 1. In contrast to many other species, neither Xist nor Tsix was expressed in equine male testicular tissue. The results demonstrate that expression of Xist is restricted to female equine embryos. Xist can thus be considered an X-inactivation-specific marker which can be used in concert with Y-specific markers for sex determination.