87 IS HOXA10 EXPRESSION REGULATED THROUGH PROMOTER DNA METHYLATION IN THE PIG?

Abstract

In utero and early postnatal exposure to exogenous oestrogens may alter the epigenome, possibly influencing health later in life. The reproductive organs are the main target of oestrogens. Uterine HOXA10 expression and promoter DNA methylation have been shown to be sensitive to endocrine disruptors in rodents. In the endometrium of pigs, in vitro exposure to estradiol-17β resulted in an increase of HOXA10 mRNA and protein expression, and a regulation for HOXA10 has also been shown during early pregnancy. However, it is unclear whether modification in promoter DNA methylation was involved herein. In the present study, we investigated the effect of oral estradiol-17β supplementation to pregnant pigs from Day 0 until delivery (0, 0.05, 10, and 1000 µg kg^–1 per day; n = 8–12/group) on HOXA10 expression and promoter DNA methylation in the uterus of prepubertal and the endometrium of postpubertal porcine offspring. In addition, endometrial samples from 2 physiological situations were analyzed, namely the oestrous cycle at Day 0, 3, 6, 12, and 18 (n = 6/day), and early pregnancy at Day 10, 12, and 14 (n = 4/day). Furthermore, different reproductive and nonreproductive tissues from pre- and postpubertal untreated animals were investigated (n = 2/tissue). Gene expression was measured using RT-qPCR. The DNA was first bisulfite converted, amplified by using methylation-sensitive high-resolution melting qPCR, followed by pyrosequencing. Statistical analyses were conducted using one-way ANOVA. In utero estradiol-17β exposure did neither alter HOXA10 expression compared to the control group nor HOXA10 promoter DNA methylation in pre- and postpubertal porcine offspring, respectively. Because the prepubertal group displayed higher HOXA10 mRNA expression than did postpubertal animals, while DNA methylation was lower in the younger animals, a potential role of DNA methylation may be assumed. During the oestrous cycle, HOXA10 expression was significantly regulated (P < 0.001); it was highest at oestrous (Day 0), decreased 2.5-fold by Day 3, and increased again 1.5-fold by Day 6. In early pregnant sows, endometrial HOXA10 transcripts were significantly higher at Day 14 (P = 0.031) compared with nonpregnant controls. In both settings promoter DNA methylation did not change. Regarding HOXA10 expression between different tissues, the DNA methylation at one CpG site was significantly correlated with mRNA expression in prepubertal animals (R² = 0.582) but not in the adult (R² = 0.004). We conclude that HOXA10 expression is regulated through other mechanisms than is promoter DNA methylation in adult sows. Still, in prepubertal pigs promoter DNA methylation may influence HOXA10 expression. In utero estradiol-17β exposure did not affect HOXA10 expression and promoter DNA methylation.