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CONGRESS OF THE ESPU
15:38–15:41
S3-2 (PP)
THE EFFECT OF XENOESTROGENS ON FORESKIN
FIBROBLASTS AND THE ETIOLOGY OF HYPOSPADIAS
Karen AITKEN
1
, Jia-Xin JIANG
2
, Shreya BATRA
2
, Sevan HOPYAN
2
and
Darius BAGLI
2
1) PGCRL room 159420TUV, DSCB, Sickkids Research Institute, Toronto, CANADA - 2) Hospital for Sick Children,
Developmental and Stem Cell Biology, Research Institute, Toronto, CANADA
PURPOSE
Hypospadias is a developmental defect in males where the development of the urethra foreskin and
ventricle surface of the penis is altered. Although the etiology of hypospadias is still debated, it has
been suggested that hypospadias is linked to environmentally altered gene expression. Previous
studies have found that in utero exposure to xenoestrogens (XE) underlying many congenital anom-
alies also alters the development of the genital tubercle (GT) through developmentally important
genes wnt5a and hoxa13. We hypothesize that expression of hoxa13 and wnt5a are rescued in the
presence of a methylase inhibitors indicating that XE mediation of GT is under epigenetic regulation.
MATERIAL AND METHODS
Human foreskin fibroblast cell line (BJ cells) were maintained in EMEM, 10 % heat-treated charcoal-
stripped (hormone-free) fetal calf serum in 37°C, 5 % CO2 incubator. For exposure to XE, cells
were starved of serum for 24–48 hours, and exposed to Diethylstilbestrol for 0–120 hours. EZH
(methylase) inhibitor (UNC 1999) or DNAmethylation inhibitor (decitibine) were introduced to the BJ
cells for 48–120 hours. Cells were harvested and real time PCR was performed using SYBR-Green
Master Mix. Relative levels of hoxa13 and wnt5a was determined with GAPDH used to normalize
the findings. p<0.05 was considered to be significant.
RESULTS
Exposure to XE decreased hoxa13 and wnt5a expression in human foreskin fibroblasts. After the
introduction of UNC1999, the expression of hoxa13 at day 6 was rescued, p<0.05. Wnt5a expres-
sion was partially rescued by DNA methylation inhibition, p<0.05.
CONCLUSIONS
XE have the potential to affect genital tubercle development through epigenetic mechanisms,
though the mechanisms may differ depending on the genes. Further work is underway to uncover
the regulation of wnt5 and hoxa13 by histone and DNA methylation profiles using ChIP-PCR and
pyrosequencing.