Research ArticleORGANISMAL BIOLOGY

Stress, novel sex genes, and epigenetic reprogramming orchestrate socially controlled sex change

See allHide authors and affiliations

Science Advances  10 Jul 2019:
Vol. 5, no. 7, eaaw7006
DOI: 10.1126/sciadv.aaw7006
  • Fig. 1 Sex change in the bluehead wrasse.

    Schematic of sex change summarizing changes in external coloration, behavior, serum steroid levels, and gonadal histology across time. Within hours of removing TP males, the largest female displays aggression and male courtship behaviors, and adopts darker spawning coloration, but still has healthy ovaries (stage 1). Transitioning females establish dominance within 1 to 2 days, after which serum estrogen [17β-estradiol (E2)] levels collapse and ovarian atresia is observable (stage 2). Ovarian atresia is advanced by 3 to 4 days (stage 3). Testicular tissues are observed by days 4 to 5, and serum 11-ketotestosterone (11-KT) begins to rise (stage 4) before spermatogenesis begins by days 6 to 7 (stage 5). Within 8 to 10 days, transitioning fish are producing mature sperm and successfully reproducing with females (stage 6). Full TP male coloration develops within ~20 days. Gonadal stages are classified according to (17). Hormonal changes are predicted on the basis of patterns in the congener Thalassoma duperrey (17). For detailed descriptions of behavioral and morphological changes, see (5, 18). Photo credit: J. Godwin, North Carolina State University (fish images); H. Liu, University of Otago (histology images).

  • Fig. 2 Global gene expression changes during sex change.

    (A) PCA showing close clustering of brain samples but separation of gonad samples by sex change stage. (B) PCA (10,000 most variable transcripts) of gonad samples. The transition from ovary to testis is captured along PC1 (58% variance), whereas PC2 (17% variance) delineates fully differentiated gonads of control females (bottom left) and TP males (bottom right) from those of transitioning fish. (C) Inset: Component loadings were used to identify transcripts contributing most to PC1 and PC2. Shaded sections define 5th and 95th percentiles defining four spatial regions: “Female” (left), “Male” (right), “Differentiated” (bottom), and “Transitionary” (top). The Euler diagram shows numbers of transcripts uniquely assigned to each region. (D) Expression patterns across sex change of transcripts uniquely assigned to each of the four spatial regions, showing four distinct patterns of expression: Female (declining), Male (increasing), Transitionary (highest mid-sex change), and Differentiated (lowest mid-sex change) and (E) representative GO terms for these transcripts. LC-FACS bios. process, long-chain fatty-acyl–coenzyme A biosynthetic process; TGF β receptor binding, transforming growth factor–β receptor binding; CF, control female; S1 to S6, stages 1 to 6; TP, TP male; ATP, adenosine 5′-triphosphate.

  • Fig. 3 Sex change involves transition from female- to male-specific expression and gene neofunctionalization.

    (A) Mammalian model of sex determination and development. In males, SRY (sex-determining region Y) activates SOX9 (SRY-related HMG box 9) to initiate male development while blocking expression of feminization genes that would, in turn, antagonize masculinizing expression (9). (B) Teleost model of sexual development. Diverse factors determine sex in fishes, yet conserved downstream effectors act in feminizing and masculinizing pathways to promote female or male development, respectively, while antagonizing the opposing sexual pathway. Testosterone is a prohormone and is converted to estrogen (E2) by gonadal aromatase (encoded by cyp19a1a) to promote ovarian function or to 11-KT by the products of hsd11b2 and cyp11c1 to promote testicular function. (C) Normalized expression of classical sex-pathway genes across sex change in bluehead wrasse gonads. At stage 2, cyp19a1a is sharply down-regulated, after which feminizing expression collapses as female fish transition to males. Up-regulation of masculinizing gene expression begins with amh and its receptor amhr2. Classically feminizing genes within the R-spondin/Wnt/β-catenin signaling pathway (wnt4a/b, rspo1, and two transcripts annotated as fstl4 are shown as examples), and also foxl2, are duplicated in the bluehead wrasse with orthologs showing testis-specific expression.

  • Fig. 4 Dynamic sex-specific expression of androgenic and glucocorticoid factors.

    (A) Normalized gonadal expression of androgen synthesis and cortisol pathway genes across sex change. Up-regulation of cyp11c1 at stage 2 implies increased local cortisol production. Then, changes in expression of 11β-HSD enzymes at stage 3 suggest a shift from cortisone-cortisol regeneration (hsd11b1a down-regulated) to cortisol-cortisone inactivation (hsd11b2 up-regulated). Concurrent up-regulation of hsd11b2 would also promote 11-KT synthesis, the most potent teleost androgen. Genes encoding the glucocorticoid (nr3c1) and mineralocorticoid (nr3c2) receptors show opposing sex-specific expression patterns that also imply highest cortisol activity at early sex change. These expression patterns suggest a window of high cortisol activity at stage 2 that is concurrent with arrested aromatase expression and ovarian atresia, followed by the stimulation of 11-KT production by stage 3. (B) Schematic showing cross-talk between the androgen synthesis and glucocorticoid pathways. The 11β-hydroxylase enzyme (Cyp11c1, homologous to mammalian CYP11B) and two 11β-hydroxysteroid dehydrogenases (HSD11b1 and HSD11b2) are together responsible for 11-KT production and the interconversion of cortisol. Cortisol is produced via the action of Cyp11c1, whereas 11β-HSD enzymes control its interconversion with inactive cortisone, thus mediating the stress response. Cortisol itself stimulates 11β-HSD expression, resulting in the production of both 11-KT and inactive cortisone.

  • Fig. 5 Epigenetic factors orchestrate sex change.

    (A) Schematic summary of sex-specific expression patterns for epigenetic factors across sex change. (B) Normalized gonadal expression of DNA methyltransferase genes, showing a turnover in sex-specific expression across sex change. The maintenance methyltransferase ortholog dnmt1 is female biased and has lowest expression at mid-sex change, whereas most methyltransferase dnmt3 orthologs, responsible for de novo methylation, show increasing expression toward maleness. DNMTs, DNA methyltransferases. (C) Ten-eleven translocation (TET) methylcytosine dioxygenases, which demethylate 5-methylcytosines, show the highest expression during late sex change. (D) Genes encoding core proteins (eed, ezh2, and suz12b) and cofactors (jarid2) of the chromatin remodeling PRC2 are suppressed at mid-sex change. The variant histone H2A.Z gene (h2afz) shows a similar expression pattern.

  • Fig. 6 Global methylation changes and relationship between methylation and gene expression during sex change.

    (A) Global CG methylation levels during sex change examined by low-coverage sequencing. The horizontal bar indicates the mean. Gray dots, brain samples; colored dots, gonadal samples. (B) Comparison of methylation (2-kb running windows) between female and TP male gonadal methylomes. Only probes with >100 calls were included for the analysis. (C) Violin plot showing distribution of methylation at transcription start sites (TSS) of genes classified into quartiles according to expression level (highest, 4). Each violin is scaled to the same maximum width (total area is not constant between violins) to demonstrate distributions for each quartile. Black dots denote the median. (D) Relationship between CpG methylation and RNA expression for cyp19a1a and dmrt1. CG methylation track shows methylation levels for dinucleotides with >10 calls; CGIs were predicted according to the Gardiner-Garden and Frommer criteria. Changes in TSS methylation containing CGIs (dashed box) are negatively correlated with gene expression for both genes (bottom).

  • Fig. 7 Mechanistic hypothesis of socially cued sex change.

    Perception of a social cue (absence of a dominant male) promotes sex change in the largest female of a social group via raised cortisol. In the brain, cortisol increases isotocin expression to promote male-typical behaviors that rapidly establish social dominance. Other neuroendocrine factors may play a role [e.g., arginine vasotocin (AVT), dopamine (DA), and norepinephrine (NE)], although expression changes were not seen for their encoding genes. In the gonad, cortisol promotes transition from ovary (red) to testis (blue) via three pathways: (i) down-regulates aromatase (cyp19a1a) expression causing estrogen (E2) production to cease and feminizing expression to decline, causing ovarian atresia; (ii) up-regulates amh expression, which, as a transcription factor (TF) and a germ cell regulator, can suppress feminizing genes and promote oocyte apoptosis while promoting masculinizing expression and spermatogonial recruitment; and (iii) up-regulates androgenic genes cyp11c1 and hsd11b2 to increase 11-KT production to support testicular development. Epigenetic reprogramming, via changes in sexually dimorphic DNA methylation (represented by lollipops; open, unmethylated and filled, methylated), rewrites cellular memory of sexual fate and canalizes sex-specific expression. Photo credit: E. D’Alessandro, Oregon State University (male fish); R. Fenner, wetwebmedia.com (female fish).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw7006/DC1

    Supplementary Materials and Methods

    Fig. S1. Lack of sex-biased variation in global gene expression in the bluehead wrasse forebrain.

    Fig. S2. Intermediate gonads are molecularly distinct from ovary and testis.

    Fig. S3. Differential transcript expression in forebrain and gonad of bluehead wrasses across sex change.

    Fig. S4. Writers and erasers of histone acetylation are dynamically expressed across sex change.

    Table S1. Genes enriched in the JAK-STAT signaling pathway are up-regulated across sex change.

    Data S1. GO enrichment detailed results.

    Data S2. Differential expression statistical results.

    Data S3. RNA-seq metadata for bluehead wrasse brain and gonad samples.

    Data S4. WGBS metadata for bluehead wrasse gonads.

    References (6476)

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Lack of sex-biased variation in global gene expression in the bluehead wrasse forebrain.
    • Fig. S2. Intermediate gonads are molecularly distinct from ovary and testis.
    • Fig. S3. Differential transcript expression in forebrain and gonad of bluehead wrasses across sex change.
    • Fig. S4. Writers and erasers of histone acetylation are dynamically expressed across sex change.
    • Table S1. Genes enriched in the JAK-STAT signaling pathway are up-regulated across sex change.
    • References (6476)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Data S1 (Microsoft Excel format). GO enrichment detailed results.
    • Data S2 (.zip format). Differential expression statistical results.
    • Data S3 (Microsoft Excel format). RNA-seq metadata for bluehead wrasse brain and gonad samples.
    • Data S4 (Microsoft Excel format). WGBS metadata for bluehead wrasse gonads.

    Files in this Data Supplement:

Navigate This Article