Identification of sex differences in zebrafish (Danio rerio) brains during early sexual differentiation and masculinization using 17α-methyltestoterone†

Abstract

Sexual behavior in teleost fish is highly plastic. It can be attributed to the relatively few sex differences found in adult brain transcriptomes. Environmental and hormonal factors can influence sex-specific behavior. Androgen treatment stimulates behavioral masculinization. Sex dimorphic gene expression in developing teleost brains and the molecular basis for androgen-induced behavioral masculinization are poorly understood. In this study, juvenile zebrafish (Danio rerio) were treated with 100 ng/L of 17 alpha-methyltestosterone (MT) during sexual development from 20 days post fertilization to 40 days and 60 days post fertilization. We compared brain gene expression patterns in MT-treated zebrafish with control males and females using RNA-Seq to shed light on the dynamic changes in brain gene expression during sexual development and how androgens affect brain gene expression leading to behavior masculinization. We found modest differences in gene expression between juvenile male and female zebrafish brains. Brain aromatase (cyp19a1b), prostaglandin 3a synthase (ptges3a), and prostaglandin reductase 1 (ptgr1) were among the genes with sexually dimorphic expression patterns. MT treatment significantly altered gene expression relative to both male and female brains. Fewer differences were found among MT-treated brains and male brains compared to female brains, particularly at 60 dpf. MT treatment upregulated the expression of hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), deiodinase, iodothyronine, type II (dio2), and gonadotrophin releasing hormones (GnRH) 2 and 3 (gnrh2 and gnrh3) suggesting local synthesis of 11-ketotestosterone, triiodothyronine, and GnRHs in zebrafish brains which are influenced by androgens. Androgen, estrogen, prostaglandin, thyroid hormone, and GnRH signaling pathways likely interact to modulate teleost sexual behavior.

Analyses of mRNA‐seq and miRNA‐seq of the brain reveal the sex differences of gene expression and regulation before and during gonadal differentiation in 17β‐estradiol or 17α‐methyltestosterone‐induced olive flounder (Paralichthys olivaceus)

“…In this study, egr1 which activates the expression of TGFβ and shisa6 which inhibites the canonical Wnt signaling pathway in mammalians (Tokue et al, ) were identified as DEMs. Consistent with the present result, egr1 was also identified as a female‐biased gene in the zebrafish brain on 40 days postfertilization (Lee et al, ). These results indicate that the TGF‐β and Wnt pathways might also be involved in brain sex differentiation.…”

“…In this study, both at 3 and 8 cm TL, the circadian rhythm core genes were differentially expressed and pathways of circadian rhythm and circadian entrainment were enriched in female brains versus male brains. In juvenile zebrafish brain, genes involved in circadian rhythm regulation were differentially expressed, among which npas2 and cry2 were sexually dimorphic expressed in brain on 40 days postfertilization and per1 was sexually dimorphic expressed in brain on 60 days posthatching (Lee, Horsfield, Black, Rutherford, & Gemmell, ). Consistent with these results, npas2 , cry2 , and per1 were also identified as DEGs in this study.…”

Zebrafish models for personalized psychiatry: Insights from individual, strain and sex differences, and modeling gene x environment interactions

“…Male sex determination begins with the disappearance of oocytes and the decomposition of the ovarian cavity caused by apoptosis (Uchida, Yamashita, Kitano, & Iguchi, ). Several genes, such as brain aromatase ( cyp19a1b ), prostaglandin 3a synthase ( ptges3a ), and prostaglandin reductase 1 ( ptgr1 ) genes, are responsible for fish hormonal regulation and display sexually dimorphic CNS expression patterns (Lee, Horsfield, Black, Rutherford, & Gemmell, ). The Cyp19 gene product enzymatically converts testosterone into 17 beta‐estradiol, and its inhibition leads to male‐to‐female sex reversal (von Hofsten & Olsson, ).…”

“…While androgen treatment stimulates behavioral masculinization, it can be reversed by estrogens (von Hofsten & Olsson, ). Androgen treatment upregulates hydroxysteroid 11‐beta dehydrogenase 2 ( hsd11b2 ), deiodinase iodothyronine type II ( dio2 ), gonadotropin‐releasing hormones 2 and 3 ( gnrh2, gnrh3 ) genes, modulating synthesis of 11‐ketotestosterone, triiodothyronine, and GnRHs in zebrafish brain (Lee et al, ). Furthermore, 42 zebrafish brain genes are differentially expressed between the sexes, including 18 genes over‐expressed in males and 24 genes in females (Santos, Kille, Workman, Paull, & Tyler, ).…”

Latency for facultative expression of male-typical courtship behaviour by female bluehead wrasses depends on social rank: The ‘priming/gating’ hypothesis

“…Gender identity and gender-specific behaviour, however, result from hormonal influences on the sexual differentiation of the brain (Cooke et al, 1998). Genetically male or female individuals can show atypical gender identity if sexual differentiation of the brain is counter to sex chromosome identity; for example, the masculinizing effects of androgens on the fetal brain strongly influence the development of male-specific behaviour (Collaer and Hines, 1995;Lee et al, 2017). Moreover, in many species, all aspects of sex determination are under environmental regulation (Janzen and Phillips, 2006;Kato et al, 2011;Crews et al, 1994).…”