Hormone-responsive genes in the SHH and WNT/β-catenin signaling pathways influence urethral closure and phallus growth†

Abstract

Environmental endocrine disruptors (EEDs) that affect androgen or estrogen activity may disrupt gene regulation during phallus development to cause hypospadias or a masculinized clitoris. We treated developing male tammar wallabies with estrogen and females with androgen from day 20-40 postpartum (pp) during the androgen imprinting window of sensitivity. Estrogen inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment in females did not promote phallus elongation but initiated urethral closure. Phalluses were collected for transcriptome sequencing at day 50 pp when they first become sexually dimorphic to examine changes in two signaling pathways, sonic hedgehog (SHH) and wingless-type MMTV integration site family (WNT)/β-catenin. SHH mRNA and β-catenin were predominantly expressed in the urethral epithelium in the tammar phallus, as in eutherian mammals. Estrogen treatment and castration of males induced an upregulation of SHH, while androgen treatment downregulated SHH. These effects appear to be direct since we detected putative estrogen receptor α (ERα) and androgen receptor (AR) binding sites near SHH. WNT5A, like SHH, was downregulated by androgen, while WNT4 was upregulated in female phalluses after androgen treatment. After estrogen treatment, WIF1 and WNT7A were both downregulated in male phalluses. After castration, WNT9A was upregulated. These results suggest that SHH and WNT pathways are regulated by both estrogen and androgen to direct the proliferation and elongation of the phallus during differentiation. Their response to exogenous hormones makes these genes potential targets of EEDs in the etiology of abnormal phallus development including hypospadias.

Hormonal and Molecular Regulation of Phallus Differentiation in a Marsupial Tammar Wallaby

“…In the tammar, SHH expression remains low in males when testicular testosterone is high, but increases after the content of testosterone (ng/mg protein) in the testes drops [7,9]. Similarly, in phallus transcriptome data ( Figure 2), SHH expression increases after removing the testes, but decreases in female phalluses when given androgen [13]. The negative association between SHH expression and androgen is also seen in a lymph node carcinoma of the prostate (LNCaP) cell line [14].…”

“…Through steroid treatment and RNA-sequencing (RNA-seq) data analysis in the tammar, a number of genes are shown to have a similar expression pattern to that of SHH. SHH, Wnt family member 5A (WNT5A), and MAF BZIP transcription factor B (MAFB) are all downregulated in female phalluses by androgen treatment at day 50 pp, but are upregulated after castration in males [13,15], while fibroblast growth factor 10 (FGF10) is upregulated by androgen treatment, but downregulated after castration in males [15]. The development of male tammar phallus is androgen dependent [5,6], like that of eutherian mammals.…”

“…Like SHH, WNT5A is also downregulated by androgen treatment in females, but increases in males after castration in the tammar [13]. The interaction between androgen and activation of SHH and WNT5A can be critical to maintain masculinization of tammar phalluses, as seen in mice [17,18].…”

“…WNT inhibitory factor 1 (Wif1) negatively regulates WNT/β-catenin signalling to balance cell apoptosis in mice [20][21][22]. In the tammar, WNT Inhibitory Factor 1(WIF1) is higher in male phalluses and is downregulated by oestrogen treatment, which is opposite to that of SHH [13]. This opposite expression pattern suggests that maintaining phallus development requires a balanced SHH signalling and WIF/WNT signalling in the tammar.…”

“…In the tammar, FGF10 expression is upregulated by androgen [15], unlike SHH, WNT5A, and MAFB that are downregulated [13,15]. In mice, high levels of SHH inhibits FGF10 transcription in the endoderm during lung morphogenesis [26].…”