The molecular control of phallus development in males is still poorly understood, despite the fact that defects of penile development, such as hypospadias, are amongst the commonest developmental defects in newborn boys and the importance of the penis for reproductive function. In this study we investigated the molecular effects of hormonal manipulation of phallus development in the developing tammar wallaby.
We treated male tammar young with oestradiol-17β or castrated them at day 25 pp, to suppress normal penis development. We stimulated penis development in female young using androstanediol treatment from day 25 pp (1, 2). Phalluses of treated and control (untreated) young were sampled at autopsy at day 50 pp when the first macroscopic signs of sexually dimorphic phallus development are seen. Transcriptomes (pooled samples) were generated using RNA-seq. Transcripts were considered to be differentially expressed if there was more than a 2-fold difference in expression between treatments. Of the differentially expressed protein coding genes, most were classified as protein binding or catalytic enzymes. Expression of key regulators of penile development such as SHH, GLI2, β-catenin and EFNB2 were down-regulated in the male phallus after oestradiol-17β treatment and castration and up-regulated in female phallus after androstanediol treatment. Surprisingly, more than 97% of differentially expressed transcripts were predicted to be lncRNAs. Several coding gene-neighbouring long-non coding RNAs were identified. An lncRNA neighbouring MAFB was down-regulated in males by oestradiol-17β treatment and also by castration. Another lncRNA neighbouring EFNB2 was differentially expressed between male and female phalluses, but there was no alteration in expression after any of the experimental treatments, suggesting that this sex difference might be independent of androgen. In summary, both the expression pattern of key regulators and their neighbouring lncRNAs are sexually dimorphic and can be disrupted by a changing endocrine environment during phallus development.