Effects of androgen and oestrogen on IGF pathways controlling phallus growth

in Reproduction
Correspondence should be addressed to M B Renfree; Email: m.renfree@unimelb.edu.au
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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.

Downloadable materials

  • Table 1: list of qPCR primers used
  • Table 2: list of primers used for section in situ hybridization probes
  • Supplementary table 3: list of the antibodies used
  • Supplementary Figure 1

 

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    mRNA distribution of FGF10, FGFR2IIIb, EFNB2, MAFB and DLX5, in day 20 pp male and female phalluses irrespective of sex, mRNA of FGF10, FGFR2IIIb, EFNB2, MAFB and DLX5 were all detected in the urethral epithelial cells and phallus epithelial cells in the phalluses at day 20 pp. EFNB2, MAFB and DLX5 were also expressed in the mesenchyme in male and female phalluses. Section in situ hybridisation staining of FGF10 (A1–A4), FGFR2IIIb (B1–B4), EFNB2 (C1–C4), MAFB (D1–D4) and DLX5 (E1–E4). E, epithelium; M, mesenchyme; UE, urethral epithelium; Dist, distal; Prox, proximal. Scale bar: 200 μm.

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    mRNA distribution of FGF10, FGFR2IIIb, EFNB2, MAFB and DLX5, in day 90 pp male and female phalluses. Irrespective of sex, mRNA of FGF10, FGFR2IIIb, EFNB2, MAFB and DLX5 were all detected in the urethral epithelial cells and phallus epithelial cells in the phalluses. FGF10 and DLX5 were expressed in the mesenchyme in male phalluses at day 90 pp. The urethral closure is complete at the base of male phalluses at this stage. There is a slight variation in sectioning depth shown in the proximal sections of phalluses, so apparent differences in the degree of urethral closure are due to the level of sectioning used. Section in situ hybridisation staining of FGF10 (A1–A4), FGFR2IIIb (B1–B4), EFNB2 (C1–C4), MAFB (D1–D4) and DLX5 (E1–E4). D, diverticulum; E, epithelium; M, mesenchyme; U, urethra; UE, urethral epithelium; Dist, distal; Prox, proximal. Scale bar: 200 μm.

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    Transcriptomic expression of FGF10, EFNB2 and MAFB in normal and hormone treated phalluses at day 50 pp. FGF10 was higher in male phalluses than female phalluses at day 50 pp, upregulated in female phalluses after androgen treatment, and downregulated in males after castration. EFNB2 expression was higher in male phalluses than female phalluses at day 50 pp, upregulated in female phalluses after androgen treatment and downregulated in males after oestrogen treatment or castration. MAFB expression was higher in female phalluses at day 50 pp than that in male phalluses, upregulated after castration in males, but not affected by androgen treatment nor oestrogen treatment. A, androgen; E2, oestrogen; FPKM, Fragments Per Kilobase of transcript per Million mapped reads. *P < 0.05, **P < 0.05. Error bar: s.e.m.

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    qPCR analysis on the expression of FGF10, FGFR2IIIb, EFNB2, MAFB and DLX5 during postnatal phallus development. The expression level of FGF10, FGFR2IIIb, EFNB2, MAFB and DLX5 was significantly higher in male phalluses than in female phalluses at day 90 pp. The expression of FGF10 and EFNB2 were similar from day 20 pp to day 90 pp. There was no differences in expression level between male phalluses and female phalluses until after day 50 pp. MABF was significantly higher in female phalluses at day 50 pp. DLX5 was significantly reduced in the female phalluses at day 90 pp. The expression of all genes was relative to 18S, GAPDH and HMBS. *P < 0.05. Error bar: s.e.m.

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    RNA-Seq analysis on the expression pattern of AP-1 genes in phalluses at day 50 pp. The expression of C-FOS, FOSB, ATF3, C-JUN, JUNB and JUND were increased after androgen treatment. The expression level of those genes was significantly higher in male phalluses at day 50 pp than that in female phalluses. A, androgen; E2, oestrogen; FPKM, Fragments Per Kilobase of transcript per Million mapped reads. *P < 0.05, **P < 0.05, ***P < 0.001. Error bar: s.e.m.

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    IGF1 distribution in male and female phalluses at day 90 pp. In both male and female phalluses, IGF1 was primarily found in the extracellular matrix and cytoplasm of epithelial cells and in the diverticulum. Some IGF1 was detected in the corpora cavernosa. In the URS, IGF1 was found only in male phalluses (see insets). CC, corpus cavernosum; D, diverticulum; E, epithelium; U, urethra; UE, urethral epithelium; UG, urethra groove; URS, urorectal septum. Red staining: IGF1. Blue staining: DAPI. Scale bar: 1.0 mm.

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    PCNA localisation in male and female phalluses at day 90 pp. In both male and female phalluses, PCNA staining was found in the nuclear of basal urethral epithelial cells, phallus epithelial cells and diverticulum. The nuclear PCNA was also found in mesenchymal cells in the corpora cavernosa. In the URS, nuclear PCNA was found only in male phalluses (see insets). Cytoplasmic PCNA was detected in the urethral epithelium, phallus epithelium and diverticulum in both sexes, and there was some mesenchymal cytoplasmic and extracellular matrix staining. A, anus; CC, corpus cavernosum; D, diverticulum; E, epithelium; M, mesenchyme; U, urethra; UE, urethral epithelium; UG, urethra groove; URS, urorectal septum. Red staining: PCNA. Blue staining: DAPI. Scale bar: 1.0 mm.

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    Expression pattern of IGF1, IGFBP3 and IGFBP6 in the phalluses. In RNASeq data (left), IGF1 was higher in male phalluses than female phalluses at day 50 pp. It was upregulated after androgen treatment in females and oestrogen treatment in males but was downregulated after castration in males. IGFBP3 was higher in female phalluses than male phalluses at day 50 pp. It was downregulated after androgen treatment in females. IGFBP6 was both upregulated in males after oestrogen treatment and in females after androgen treatment. In qPCR (right), IGF1 was higher in male phalluses than female phalluses at day 50 pp, day 90 pp and day 150 pp. It was upregulated after androgen treatment in females but was not affected by oestrogen treatment or castration at day 50 pp. IGFBP3 was higher in female phalluses than male phalluses at day 50 pp, day 90 pp and day 150 pp. A, androgen; E2, oestrogen; FPKM, Fragments Per Kilobase of transcript per Million mapped reads. *P < 0.05, **P < 0.05, ***P < 0.001. Error bar: s.e.m.

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    Summary of androgen and oestrogen effects on gene expression of IGF1, IGFBPs, FGF10, EFNB2, MAFB and AP-1 in phalluses (top) and diagram of how IGF1 regulates urethral closure (bottom). Adiol treatment upregulated gene expression of IGF1, IGFBP6, FGF10, EFNB2 and AP-1, but downregulated IGFBP3 expression. Oestrogen increased IGF1 and IGFBP6 expression but decreased EFNB2 and C-FOS expression. Castrating males induced an upregulation of MAFB and a downregulation of IGF1, FGF10 and EFNB2 in phalluses. IGF1 was higher in male phalluses from day 50 pp to day 90 pp, and induced URS proliferation and urethral closure. In contrast, IGFBP3 was higher in female phalluses and inhibited urethral closure. Adiol treatment and oestrogen treatment both increased IGF1 expression and induced urethral closure of day 150 pp phalluses. Castrating males decreased IGF1 expression in phalluses and prevented urethral closure. A, anus; D, diverticulum; E, epithelium; E2, oestrogen; M, mesenchyme; U, urethra; UG, urethral groove. Red indicates decreased levels; green indicates increased levels.

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