The Effects of Synthetic Estrogen Exposure on the Sexually Dimorphic Liver Transcriptome of the Sex-Role-Reversed Gulf Pipefish

Abstract

Species exhibiting sex-role reversal provide an unusual perspective on the evolution of sex roles and sex differences. However, the proximate effects of sex-role reversal are largely unknown. Endocrine disruptors provide an experimental mechanism to address hormonal regulation of sexually dimorphic gene expression in sex-role-reversed taxa. Here, we investigate gene expression patterns in the liver of the sex-role-reversed Gulf pipefish, because the liver is known to be sexually dimorphic and estrogen-regulated in species with conventional sex roles. Using next-generation RNA-sequencing technology (RNA-seq), we detected sexually dimorphic hepatic gene expression patterns, with a total of 482 differentially expressed genes between the sexes in Gulf pipefish. Two-thirds of these genes were over-expressed in females, and the sex-specific transcriptomes of this sex-role-reversed pipefish's liver were superficially similar to those of fishes with conventional sex-roles. We exposed females, pregnant males, and non-pregnant males to 17α-ethinylestradiol (EE2) at ecologically relevant concentrations of 5ng/L and compared gene expression patterns in the livers of exposed fish to control fish. Several genes that were up-regulated in EE2-exposed males relative to control males were also found to be female-biased in control animals. These genes included several of the classic estrogen biomarkers, such as vitellogenin, choriogenin, and zona pellucida. Thus, estrogen exposure induced feminization of the male liver transcriptome in a sex-role-reversed pipefish. These results suggest that the ancestral state of estrogen-regulated female reproductive physiology has been retained in all sex-role-reversed vertebrates thus far studied, despite substantial evolution of the hormonal regulation of ornamentation and mating behavior in these interesting taxa.

Fig 1. Sexually Dimorphic Gene Expression Patterns.

Heat map showing hierarchical clustering of mean expression values for the six treatments including control females, pregnant males, and non-pregnant males, as well as EE2 exposed females, pregnant males, and non-pregnant males, for all genes which showed sexually dimorphic expression patterns in control fish. The colors of the bars represent either up-regulated (red) or down-regulated (blue) genes. The male treatments cluster together with the exception of the EE2 exposed pregnant males which cluster with the both control and EE2 females.

Fig 2. The Overlap of Female Biased and EE2 Responsive Genes.

Venn diagram of control females (CF), EE2 exposed pregnant males (EP), and non-pregnant males (ENP) to show overlap of female biased genes and EE2 responsive genes. Gene expression levels for control females are either over or under expressed compared to the levels of expression in control males, and EE2 exposed male genes were differentially expressed from their control counterparts. A list of genes up-regulated between all three groups can be found in Table 3.

Fig 3. Pipefish and Zebrafish Comparisons of Expression Patterns.

Venn diagrams of sexually dimorphic and EE2 responsive genes for pipefish and zebrafish showing EE2 up-regulated genes in 3a and EE2 down-regulated genes in 3b. Numbers of female biased genes in control females are presented for control pipefish females in this study (PF) and for control zebrafish from a study by [23]. (ZF). Male EE2 responsive genes are presented for both pregnant and non-pregnant, EE2 exposed pipefish males (PM) and for EE2 exposed males from [23](ZM).