The functions of testosterone in sex differentiation and reproduction of zebrafish
Gang Zhai#,1, Tingting Shu#,1,2, Yuguo Xia#,3, Guohui Shang1,2, Xia Jin1, Jiangyan He1 and Zhan Yin 1*
1. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China, 430072.
2. University of the Chinese Academy of Sciences, Beijing, China.
3. Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China.
#These authors contributed equally to this work.
The sex is determined by sex chromosomes in mammals. However, unlike mammals, the mechanism in teleost fish shows greater complexity and plasticity. We knockout cyp17a1, which has both 17alpha-hydroxylase and 17, 20-lyase activities, to block the steroidogenic pathway that produces androgens in zebrafish. Interestingly, all the cyp17a1-deficient fish were fertile males with normal testis and sperm but had impaired male secondary sexual characteristics (SSCs), including breeding tubercles (BTs), pigmentations of the body and anal fin, and mating behaviors. Further analyses showed that the differentiation of the juvenile ovary into the mature ovary failed due to increased apoptosis during the sex differentiation stage. More importantly, the all-male phenotype and compromised SSCs in the cyp17a1-deficient fish was rescued by testosterone or estradiol treatment, and the optimum concentration for sex reversal of testosterone and estradiol is 7.5 μg/L and 0.1 μg/L, respectively. Our results support that the ovarian differentiation is estrogen dependent, whereas the SSCs are closely correlated with testosterone. Moreover, the upregulated expressions of endocrine (pituitary gonadotropin) and paracrine (testicular growth factor) parameters may be involved in spermatogenesis for compensation in the cyp17a1-deficient fish.
It has been suggested that androgen signaling regulates critical genes for the differentiation and development of the testis. To elucidate the exact regulatory mechanisms involved in Anti-Müllerian hormone (amh) transcription mediated by androgen signaling, androgen signaling was studied in cyp17a1-deficient fish and Flutamide treatment. In cyp17a1-deficient and Flutamide-treated testes, up-regulated sry-box 9a (sox9a) and down-regulated amh were observed. Moreover, a physical association of the zebrafish androgen receptor (AR) and SOX9A was found. The interaction between AR and SOX9A was mediated via the DNA binding domain (DBD) of AR and the transactivation domain (TA) of SOX9A, and was further enhanced by 5-alpha dihydrotestosterone (DHT), one of the most potent androgens. Intriguingly, together with SOX9A, androgen signaling synergistically promoted amh transcription, mainly through the proximal 1 kb of the amh promoter region. Taken together, our data demonstrate a critical mechanism underlying the direct synergy of androgen signaling and SOX9A in the regulation of amh transcription.
Gang Zhai#,1, Tingting Shu#,1,2, Yuguo Xia#,3, Guohui Shang1,2, Xia Jin1, Jiangyan He1 and Zhan Yin 1*
1. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China, 430072.
2. University of the Chinese Academy of Sciences, Beijing, China.
3. Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China.
#These authors contributed equally to this work.
The sex is determined by sex chromosomes in mammals. However, unlike mammals, the mechanism in teleost fish shows greater complexity and plasticity. We knockout cyp17a1, which has both 17alpha-hydroxylase and 17, 20-lyase activities, to block the steroidogenic pathway that produces androgens in zebrafish. Interestingly, all the cyp17a1-deficient fish were fertile males with normal testis and sperm but had impaired male secondary sexual characteristics (SSCs), including breeding tubercles (BTs), pigmentations of the body and anal fin, and mating behaviors. Further analyses showed that the differentiation of the juvenile ovary into the mature ovary failed due to increased apoptosis during the sex differentiation stage. More importantly, the all-male phenotype and compromised SSCs in the cyp17a1-deficient fish was rescued by testosterone or estradiol treatment, and the optimum concentration for sex reversal of testosterone and estradiol is 7.5 μg/L and 0.1 μg/L, respectively. Our results support that the ovarian differentiation is estrogen dependent, whereas the SSCs are closely correlated with testosterone. Moreover, the upregulated expressions of endocrine (pituitary gonadotropin) and paracrine (testicular growth factor) parameters may be involved in spermatogenesis for compensation in the cyp17a1-deficient fish.
It has been suggested that androgen signaling regulates critical genes for the differentiation and development of the testis. To elucidate the exact regulatory mechanisms involved in Anti-Müllerian hormone (amh) transcription mediated by androgen signaling, androgen signaling was studied in cyp17a1-deficient fish and Flutamide treatment. In cyp17a1-deficient and Flutamide-treated testes, up-regulated sry-box 9a (sox9a) and down-regulated amh were observed. Moreover, a physical association of the zebrafish androgen receptor (AR) and SOX9A was found. The interaction between AR and SOX9A was mediated via the DNA binding domain (DBD) of AR and the transactivation domain (TA) of SOX9A, and was further enhanced by 5-alpha dihydrotestosterone (DHT), one of the most potent androgens. Intriguingly, together with SOX9A, androgen signaling synergistically promoted amh transcription, mainly through the proximal 1 kb of the amh promoter region. Taken together, our data demonstrate a critical mechanism underlying the direct synergy of androgen signaling and SOX9A in the regulation of amh transcription.