Androgen and Oestrogen Affect the Expression of Long Non-Coding RNAs During Phallus Development in a Marsupial

Abstract

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.

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

“…In mice, oestrogen signalling clearly has a regulatory role in phallus development [93,94], as we have found in the tammar [13,15,88]. In the tammar co-expression network, about 50% of estrogen receptor 1 (ESR1) co-expressed coding genes and lncRNAs are also associated with AR [88], suggesting an interaction between androgen receptor (AR) signalling and ESR1 signalling during tammar phallus development [88]. However, those lncRNAs could have other genetic targets because none of them were located within 100 kb upstream or downstream of IGF1, AR, and ESR1.…”

“…Both lnc-BMP5 and lnc-ZBTB16 are downregulated in tammar male phalluses after oestrogen treatment [88]. They are also co-expressed with bone morphogenetic protein (BMP5) and zinc finger and BTB domain containing 16 (ZBTB16), respectively, in our co-expression network [88].…”

“…Lnc-RSPO4 is co-expressed with roof plate-specific spondin-4 (RSPO4) coding gene. Both RSPO4 and lnc-RSPO4 are downregulated in tammar female phalluses after androgen treatment [88], in a similar expression pattern to that of SHH and WNT5A [13]. Interestingly, RSPO4 is a ligand of leucine-rich repeat containing G protein-coupled receptor (LGR) 4-6 receptors that potentiate WNT signalling [95][96][97][98].…”

“…It is also a good way to identify lncRNAs as most of them have extremely low sequence conservation, making them difficult to identify cross species with alignment. In our previous paper, we set up a pipeline by combining WGCNA, DE analysis, and the location of lncRNAs and identified the following three coding gene-neighboring lncRNAs: lnc-RSPO4, lnc-BMP5, and lnc-ZBTB16 [88].…”

“…IGF1 is considered as a hub gene in its co-expression network due to its high correlation with a large number of protein-coding genes and lncRNAs. Within the IGF1 co-expression network, both IGFBP5, an IGF signalling regulator (reviewed in [89]) that inhibits SHH-induced proliferation in cerebellar granule cells in mice [44], and FGF10, a phallus regulating gene in mice and the tammar [15,28,29,77,78], have a high correlation (R ≥ 0.8) with IGF1 [88] (Figure 5). IGF1 is also co-expressed with multiple genes that may have a role in regulating reproductive development ( Figure 5).…”