Environmental endocrine disruptors (EEDs) that affect androgen or estrogen activity may disrupt gene regulation during normal 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 established androgen imprinting window. Administration of estrogen 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. Phalluses were collected for transcriptome sequencing at day 50 pp when they first become sexually dimorphic to examine changes in two key signaling pathways, SHH and 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 ERα and AR binding sites upstream of SHH. WNT5A, like SHH, was downregulated by androge, 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.

Sex determination and sexual differentiation pathways are highly conserved between marsupials and eutherians. There are 2 different pathways of prostaglandin D2 (PGD2) synthesis: prostaglandin D synthase (PTGDS) and haematopoietic prostaglandin D synthase (HPGDS). PGD2 regulates the subcellular localization of SOX9 during gonadal sexual differentiation. To investigate the function of PGD2 in the tammar gonad, we cultured undifferentiated male gonads in the presence of the HPGDS inhibitor HQL-79 and female gonads with exogenous PGD2 to mimic activation of the PTGDS-PGD2 pathway. Tammar PTGDS and HPGDS have only 50% similarity with mouse and human orthologues, but functional domains are conserved. The expression of SOX9 was unchanged by the treatments in cultured gonads, but its subcellular localization was markedly affected. SOX9 remained cytoplasmic in the Sertoli cells of testes treated with HQL-79. Treated testes developed a thickened ovary-like surface epithelium. In contrast, SOX9 became nuclear in the granulosa cells of developing ovaries treated with PGD2 and the surface epithelium was thin, as in testes. These results demonstrate that PGD2 regulates the subcellular localization of SOX9 and subsequent gonadal development in the developing marsupial gonads, as it does in mice, and that it must have been an ancestral mechanism.

Marsupials are experiencing devastating population declines across Australia. Exposure to environmental endocrine disruptors, through ingestion of contaminated resources in the environment, could be contributing to this decline. Atrazine (ATZ), a widely used herbicide in Australia, is an endocrine disruptor with the ability to cause reproductive abnormalities in a diverse range of vertebrates. We exposed adult female wallabies (Macropus eugenii) to drinking water containing ATZ (450 p.p.m) throughout pregnancy, parturition and lactation. We assessed the outcome of this exposure to the reproductive development of their young by assessing gonad and phallus development. Both these organs are especially sensitive to perturbations in the hormonal environment during development. Although no gross abnormalities were seen in gonad structure, exposure to ATZ did alter the expression of genes required for normal testis function. Furthermore, long-term exposure to ATZ resulted in a significant reduction in penis length. These results demonstrate that ATZ exposure during gestation and lactation can significantly affect the development of male young by affecting virilisation. Given the known vulnerability of macropodid marsupials to endocrine disruption, as well as their overlapping distribution with agricultural areas, these data raise major concerns for the use of pesticides in areas with fragile marsupial populations.

There is increasing evidence that long non-coding RNAs (lncRNAs) are important for normal reproductive development, yet very few lncRNAs have been identified in phalluses so far. Unlike eutherians, phallus development in the marsupial tammar wallaby occurs post-natally, enabling manipulation not possible in eutherians in which differentiation occurs in utero. We treated with sex steroids to determine the effects of androgen and oestrogen on lncRNA expression during phallus development. Hormonal manipulations altered the coding and non-coding gene expression profile of phalluses. We identified several predicted co-regulatory lncRNAs that appear to be co-expressed with the hormone-responsive candidate genes regulating urethral closure and phallus growth, namely IGF1, AR and ESR1. Interestingly, more than 50% of AR-associated coding genes and lncRNAs were also associated with ESR1. In addition, we identified and validated three novel co-regulatory and hormone-responsive lncRNAs: lnc-BMP5, lnc-ZBTB16 and lncRSPO4. Lnc-BMP5 was detected in the urethral epithelium of male phalluses and was downregulated by oestrogen in males. Lnc-ZBTB16 was downregulated by oestrogen treatment in male phalluses at day 50 post-partum (pp). LncRSPO4 was downregulated by adiol treatment in female phalluses but increased in male phalluses after castration. Thus, the expression pattern and hormone responsiveness of these lncRNAs suggests a physiological role in the development of the phallus.

The development of the mammalian phallus involves hormone-dependent mesenchymal–epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.

Background: RSPO1 and FOXL2 are female sex‐determining genes involved in the differentiation and organization of the ovary in some eutherian mammals. Mutations or loss of function of these genes are associated with partial to full sex reversal in mice, humans, and goats. RUNX1 may also play a role in ovarian development, but its expression in marsupials has not been examined as yet. We studied the conservation and protein localization of RSPO1, FOXL2, and RUNX1 orthologs in the marsupial tammar wallaby (Notamacropus eugenii) compared to other vertebrates. Results: RSPO1, FOXL2, and RUNX1 were highly conserved in their sequences across all vertebrates examined. The localization of these proteins in the tammar ovary was studied from Day 18 postpartum to adulthood. RSPO1, FOXL2, and RUNX1 were expressed in the granulosa cells of the early ovary and granulosa cells of the mature ovary, while RSPO1 expression was also found in the intra‐ovarian rete and cell membrane of germ cells during the period of germ cell meiosis and meiotic arrest. Conclusions: RSPO1, FOXL2, and RUNX1 are highly conserved in the vertebrate ovary‐determining pathway and were expressed in the tammar wallaby in a manner consistent with their role in the ovarian differentiation of eutherian mammals.