School of BioSciences - Theses
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ItemHormone-responsive networks influence urethral closure and phallus growth( 2018)Phallus development in mammals is androgen-dependent but can be affected by oestrogen. Many genes that are important in regulating phallus development in mice are also androgen and/or oestrogen responsive. The balance between androgen receptor (AR) and oestrogen receptor α (ERα) activity is important to maintain normal penile development. Interfering this balance either by treating with sex steroids or AR inhibitors, such as flutamide, induces abnormal phallus phenotype in mice and rat. However, very few studies have looked into the global regulation of gene networks on phallus development under the direct effects of sex steroids. Different from eutherian mammals, tammar wallaby has a unique phallus developmental process, where the phallus differentiation occurs postnatally. Such unique postnatal development allows us to observe the direct effects of sex hormones on phallus development without considering maternal physiology and placental transfer. In this thesis, male tammar wallabies were treated with oestrogen and females were treated with the adiol from day 20-40 postpartum (pp) during the established androgen imprinting window (day 25-30 pp). Another group of male tammars were castrated at day 25 pp. Phalluses were collected for morphological comparison at day 150 pp, when urethral closure was almost completed, and for transcriptomic analysis at day 50 pp when they first become sexually dimorphic to examine changes in multiple signalling pathways, including SHH/WNT signalling pathways, IGF1 signalling pathway and AP-1 signalling pathway, as well as to identify novel hormonal responsive lncRNAs, such as, lnc-BMP5, lnc- ZBTB16 and lnc-RSPO4, during phallus development in the tammar. Administration of oestrogen to males inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment during the imprinting window in females did not promote phallus elongation but did initiate urethral closure. Along with multiple stage hormonal treatments conducted by Leihy, et al., (2004) in the tammar phallus, the data suggests that short-term exposure of androgen during the window of sensitivity is necessary for urethral closure, but a constant level of androgen is required to maintain later phallus elongation. Hormonal manipulation altered gene expression in multiple signalling pathways during tammar phallus development. Adiol treatment suppressed SHH expression and its associated genes, including MAFB, WNT5A and RSPO4, but upregulated IGF1 and AP 1 genes in male phalluses at day 50 pp. After day 50 pp, SHH was transiently increased in male phalluses, which might be regulated by the increase level of IGF2 in the liver. The androgen-SHH switch might be the signalling constraint for the timing of phallus differentiation. While, the later activation of both endocrine IGF2-SHH and androgen- IGF1 signalling may be required to trigger and maintain male urethral closure. More interestingly, the activation of androgen dependent window (day 40-120 pp) altered IGF1 signalling, which may not only maintain phallus elongation by regulating cell proliferation, but also contribute to urethral closure by increasing the proliferation in urorectal septum. During the androgen dependent window, IGFBP3 expression was negatively correlated with that of IGF1 in phalluses, suggesting that IGFBP3 may negatively regulate IGF1 signalling in the tammar phalluses. Hormonal manipulation also altered non-coding gene expression profile of phalluses. I identified several predicted trans-regulatory lncRNAs that appear to be co-expressed with the hormone-responsive coding genes IGF1, a candidate gene regulating urethral closure and phallus growth, AR and ESR1. Interestingly, more than 50% of ARcorrelated coding genes and lncRNAs were also co-expressed with ESR1. In addition, I identified and validated three novel cis-regulatory and hormone-responsive lncRNAs: lnc-BMP5, lnc-ZBTB16 and lncRSPO4. Lnc-BMP5 was detected in the urethral epithelium in day 20 and day 90 post-partum (pp) male phalluses, and was downregulated by oestrogen in males. Lnc-ZBTB16 was downregulated by oestrogen treatment in male phalluses at day 50 pp. LncRSPO4 was downregulated by adiol treatment in female phalluses but increased in male phalluses after castration. In addition, another lncRNA, LEAT1, which has a short but highly conserved region in mammals, was also co-expressed with its neighbouring regulating gene, EFNB2, in the tammar phalluses at day 30 and day 50 pp. Thus, exogenous steroid hormones appear to have significant effects on the complex network of both coding genes and lncRNAs controlling phallus development. Phallus development is under strictly programmed molecular regulation of SHH and IGF1 signalling as well as lncRNAs, which are susceptible to exogenous sex steroids. This study offered new insights to understand the complex gene networks between coding genes and lncRNAs during phallus development under the regulation of a balanced endocrine environment.