Cite this paper:
HU Peng, LIU Bin, MA Qian, LIU Shufang, LIU Xinfu, ZHUANG Zhimeng. Expression profiles of sex-related genes in gonads of genetic male Takifugu rubripes after 17β-estradiol immersion[J]. HaiyangYuHuZhao, 2019, 37(3): 1113-1124

Expression profiles of sex-related genes in gonads of genetic male Takifugu rubripes after 17β-estradiol immersion

HU Peng1,2, LIU Bin1, MA Qian1,2, LIU Shufang1,2, LIU Xinfu1, ZHUANG Zhimeng1,2
1 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
2 Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology & Function Laboratory for Marine Biology and Biotechnology, Qingdao 266000, China
Estradiol treatment during early life stages of tiger puffer Takifugu rubripes induces feminization in genetic males. However, the ovaries in genetic males may revert to testes once estradiol treatment is halted. Therefore studies should investigate molecular mechanisms underlying ovary-to-testis recovery in genetic males after treatment. In the present study, tiger puffer were exposed to 10, and 100 μg/L 17β-estradiol (E2) from 15 to 100 days post-hatching (dph), then gonad phenotypes and expression profiles of six sex-related genes (cyp19a, foxl2, dmrt1, amh, sox9a, and sox9b) were characterized after the exposure. Results showed that both 10 and 100 μg/L E2 induced ovarian development in genetic males at 100 dph. However, all ovaries induced by 10 μg/L E2 first developed into intersexual gonads and subsequently reverted to testes after the exposure. As for treatment of 100 μg/L E2, while the rest of the ovaries maintained morphological stability, percentages of intersexual gonads reached 38%-57%, and none were reverted to testes. Increased mRNA levels of cyp19a, foxl2 and sox9b and decreased mRNA levels of dmrt1, amh, and sox9a were observed during the ovarian development in genetic males. While contrary gene expression profiles were detected during ovary-to-testis transformation. The mRNA levels of all the six genes were increased during the development of intersexual gonads. These results indicated that up-regulation of dmrt1, amh and sox9a is associated with initial ovary-to-intersexual transformation, and suppression of foxl2, cyp19a and sox9b is essential for complete ovary-to-testis recovery in genetic males. This research will help to trace the molecular processes underlying gonadal transformation in teleosts.
Key words:    Takifugu rubripes|ovary-to-testis recovery|sex-related genes|mRNA expression   
Received: 2018-03-19   Revised: 2018-04-24
PDF (1223 KB) Free
Print this page
Add to favorites
Email this article to others
Articles by HU Peng
Articles by LIU Bin
Articles by MA Qian
Articles by LIU Shufang
Articles by LIU Xinfu
Articles by ZHUANG Zhimeng
Aparicio S, Chapman J, Stupka E, Putnam N, Chia J M, Dehal P, Christoffels A, Rash S, Hoon S, Smit A, Gelpke M D, Roach J, Oh T, Ho I Y, Wong M, Detter C, Verhoef F, Predki P, Tay A, Lucas S, Richardson P, Smith S F, Clark M S, Edwards Y J, Doggett N, Zharkikh A, Tavtigian S V, Pruss D, Barnstead M, Evans C, Baden H, Powell J, Glusman G, Rowen L, Hood L, Tan Y H, Elgar G, Hawkins T, Venkatesh B, Rokhsar D, Brenner S. 2002.Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science, 297(5585):1 301-1 310.
Baroiller J F, D'Cotta H. 2001. Environment and sex determination in farmed fish. Comparative Biochemistry and Physiology C:Toxicology & Pharmacology, 130(4):399-409.
Baumann L, Knörr S, Keiter S, Rehberger K, Volz S, Schiller V, Fenske M, Holbech H, Segner H, Braunbeck T. 2014.Reversibility of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the estrogen 17α-ethinylestradiol. Toxicology and Applied Pharmacology, 278(3):230-237.
Bhandari R K, Komuro H, Nakamura S, Higa M, Nakamura M. 2003. Gonadal restructuring and correlative steroid hormone profiles during natural sex change in protogynous honeycomb grouper (Epinephelus merra). Zoological Science, 20(11):1 399-1 404.
Blázquez M, González A, Papadaki M, Mylonas C, Piferrer F. 2008. Sex-related changes in estrogen receptors and aromatase gene expression and enzymatic activity during early development and sex differentiation in the European sea bass (Dicentrarchus labrax). General and Comparative Endocrinology, 158(1):95-101.
Chiang E F L, Pai C I, Wyatt M, Yan Y L, Postlethwait J, Chung B C. 2001. Two Sox9 genes on duplicated zebrafish chromosomes:expression of similar transcription activators in distinct sites. Developmental Biology, 231(1):149-163.
Fleming N I, Knower K C, Lazarus K A, Fuller P J, Simpson E R, Clyne C D. 2010.Aromatase is a direct target of FOXL2:C134W in granulosa cell tumors via a single highly conserved binding site in the ovarian specific promoter. PLoS One, 5(12):e14389.
Georges A, Auguste A, Bessière L, Vanet A, Todeschini A L, Veitia R A. 2013. FOXL2:a central transcription factor of the ovary. Journal of Molecular Endocrinology, 52(1):R17-R33.
Guiguen Y, Baroiller J F, Ricordell M, Iseki K, Mcmeel O M, Martin S A M, Fostier A. 1999. Involvement of estrogens in the process of sex differentiation in two fish species:the rainbow trout (Oncorhynchus mykiss) and a tilapia(Oreochromis niloticus). Molecular Reproduction and Development, 54(2):154-162.
Guiguen Y, Fostier A, Piferrer F, Chang C F. 2010.Ovarian aromatase and estrogens:a pivotal role for gonadal sex differentiation and sex change in fish. General and Comparative Endocrinology, 165(3):352-366.
Horiguchi R, Nozu R, Hirai T, Kobayashi Y, Nakamura M. 2018. Expression patterns of sex differentiation-related genes during gonadal sex change in the protogynous wrasse, Halichoeres trimaculatus. General and Comparative Endocrinology, 257:67-73.
Hu P, Liu B, Meng Z, Liu X F, Jia Y D, Yang Z, Lei J L. 2017.Recovery of gonadal development in tiger puffer Takifugu rubripes after exposure to 17β-estradiol during early life stages. Chinese Journal of Oceanology and Limnology, 35(5):613-623.
Hu Q, Guo W, Gao Y, Tang R, Li D P. 2015. Molecular cloning and characterization of amh and dax1 genes and their expression during sex inversion in rice-field eel Monopterus albus. Scientific Reports, 5:16 667.
Hu Q, Guo W, Gao Y, Tang R, Li D P. 2014. Molecular cloning and analysis of gonadal expression of Foxl2 in the ricefield eel Monopterus albus. Scientific Reports, 4:6 884.
Ijiri S, Kaneko H, Kobayashi T, Wang D S, Sakai F, PaulPrasanth B, Nakamura M, Nagahama Y. 2008. Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus. Biology of Reproduction, 78(2):333-341.
Kamiya T, Kai W, Tasumi S, Oka A, Matsunaga T, Mizuno N, Fujita M, Suetake H, Suzuki S, Hosoya S, Tohari S, Brenner S, Miyadai T, Venkatesh B, Suzuki Y, Kikuchi K. 2012. A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (Fugu). PLoS Genetics, 8(7):e1002798.
Kim Y, Muroki H, Yamamoto K, Deng J M, Behringer R R, Nakamura T, Akiyama H. 2011. Generation of transgenic mice for conditional overexpression of Sox9. Journal of Bone and Mineral Metabolism, 29(1):123-129.
Kobayashi T, Matsuda M, Kajiura-Kobayshi H, Suzuki A, Saito N, Nakamoto M, Shibata N, Nagahama Y. 2004. Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes. Developmental Dynamics, 231(3):518-526.
Kokokiris L, Fostire A, Athanassopoulou F, Petridis D, Kentouri M. 2006. Gonadal changes and blood sex steroids levels during natural sex inversion in the protogynous Mediterranean red porgy, Pagrus pagrus(Teleostei:Sparidae). General and Comparative Endocrinology, 149(1):42-48.
Lambeth L S, Morris K, Ayers K L, Wise T G, O'Neil T, Wilson S, Cao Y, Sinclair A H, Cutting A D, Doran T J, Smith C A. 2016. Overexpression of anti-Müllerian hormone disrupts gonadal sex differentiation, blocks sex hormone synthesis, and supports cell autonomous sex development in the chicken. Endocrinology, 157(3):1 258-1 275.
Lambeth L S, Raymond C S, Roeszler K N, Kuroiwa A, Nakata T, Zarkower D, Smith C A. 2014. Over-expression of DMRT1 induces the male pathway in embryonic chicken gonads. Developmental Biology, 389(2):160-172.
Lau E S, Zhang Z W, Qin M M, Ge W. 2016. Knockout of zebrafish ovarian aromatase gene (cyp19a1a) by TALEN and CRISPR/Cas9 leads to all-male offspring due to failed ovarian differentiation. Scientific Reports, 6:37 357.
Lee K H, Yamaguchi A, Rashid H, Kadomura K, Yasumoto S, Matsuyama M. 2009. Estradiol-17β treatment induces intersexual gonadal development in the pufferfish, Takifugu rubripes. Zoological Science, 26(9):639-645.
Li M H, Sun Y L, Zhao J, Shi H J, Zeng S, Ye K, Jiang D N, Zhou L Y, Sun L N, Tao W J, Nagahama Y, Kocher T D, Wang D S. 2015. A tandem duplicate of anti-Müllerian hormone with a missense SNP on the Y chromosome is essential for male sex determination in Nile tilapia, Oreochromis niloticus. PLoS Genetics, 11(11):e1005678.
Li M H, Yang H H, Li M R, Sun Y L, Jiang X L, Xie Q P, Wang T R, Shi H J, Sun L N, Zhou L Y, Wang D S. 2013.Antagonistic roles of Dmrt1 and Foxl2 in sex differentiation via estrogen production in tilapia as demonstrated by TALENs. Endocrinology, 154(12):4 814-4 825.
Liu H Q, Guan B, Xu J, Hou C C, Tian H, Chen H X. 2013.Genetic manipulation of sex ratio for the large-scale breeding of YY super-male and XY all-male yellow catfish (Pelteobagrus fulvidraco (Richardson)). Marine Biotechnology, 15(3):321-328.
Liu J F, Guiguen Y, Liu S J. 2009. Aromatase (P450arom) and 11β-hydroxylase (P45011β) genes are differentially expressed during the sex change process of the protogynous rice field eel, monopterus albus. Fish Physiology and Biochemistry, 35(3):511-518.
Maack G, Segner H. 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology, 62(4):895-906.
Mair G C, Abucay J S, Abella T A, Beardmore J A, Skibinski D O F. 1997. Genetic manipulation of sex ratio for the large scale production of all-male tilapia Oreochromis niloticus. Canadian Journal of Fisheries and Aquatic Sciences, 54(2):396-404.
Masuyama H, Yamada M, Kamei Y, Fujiwara-Ishikawa T, Todo T, Nagahama Y, Matsuda M. 2012. Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka. Chromosome Research, 20(1):163-176.
Nakamura S, Watakabe I, Nishimura T, Picard J Y, Toyoda A, Taniguchi Y, di Clemente N, Tanaka M. 2012.Hyperproliferation of mitotically active germ cells due to defective anti-Müllerian hormone signaling mediates sex reversal in medaka. Development, 139(13):2 283-2 287.
Nozu R, Horiguchi R, Kobayashi Y, Nakamura M. 2015.Expression profile of doublesex/male abnormal-3-related transcription factor-1 during gonadal sex change in the protogynous wrasse, Halichoeres trimaculatus. Molecular Reproduction and Development, 82(11):859-866.
Pan Q W, Anderson J, Bertho S, Herpin A, Wilson C, Postlethwait J H, Schartl M, Guiguen Y. 2016. Vertebrate sex-determining genes play musical chairs. Comptes Rendus Biologies, 339:258-262.
Pfennig F, Standke A, Gutzeit H O. 2015. The role of Amh signaling in teleost fish-Multiple functions not restricted to the gonads. General and Comparative Endocrinology, 223:87-107.
Piferrer F, Blázquez M. 2005. Aromatase distribution and regulation in fish. Fish Physiology and Biochemistry, 31(2-3):215-226.
Rutaisire J, Levavi-Sivan B, Nyatia N, Booth A. 2008. Juvenile intersexuality in the cyprinid fish Labeo victorianus.Cybium:International Journal of Ichthyology, 32(2):232.
Sekido R, Lovell-Badge R. 2009. Sex determination and SRY:down to a wink and a nudge? Trends in Genetics, 25(1):19-29.
She Z Y, Yang W X. 2017. Sry and SoxE genes:How they participate in mammalian sex determination and gonadal development? Seminars in Cell and Developmental Biology, 63:13-22.
Shen X Y, Cui J Z, Yang G P, Gong Q L, Gu Q Q. 2007.Expression detection of DMRTs and two sox9 genes in Takifugu rubripes (Tetraodontidae, Vertebrata). Journal of Ocean University of China, 6(2):182-186.
Shiraishi E, Yoshinaga N, Miura T, Yokoi H, Wakamatsu Y, Abe S, Kitano T. 2008. Müllerian inhibiting substance is required for germ cell proliferation during early gonadal differentiation in medaka (Oryziaslatipes).Endocrinology, 149(4):1 813-1 819.
Skaar K S, Nóbrega R H, Magaraki A, Olsen L C, Schulz R W, Male R. 2011. Proteolytically activated, recombinant antiMüllerian hormone inhibits androgen secretion, proliferation, and differentiation of Spermatogonia in adult zebrafish testis organ cultures. Endocrinology, 152(9):3 527-3 540.
Strüssmann C A, Nakamura M. 2002. Morphology, endocrinology, and environmental modulation of gonadal sex differentiation in teleost fish. Fish Physiology and Biochemistry, 26(1):13-29.
Sun D, Zhang Y, Wang C, Hua X, Zhang X A, Yan J. 2013.Sox9-related signaling controls zebrafish juvenile ovarytestis transformation. Cell Death and Disease, 4(11):e930.
Suzuki A, Nakamoto M, Kato Y, Shibata N. 2005. Effects of estradiol-17β on germ cell proliferation and DMY expression during early sexual differentiation of the medaka Oryzias latipes. Zoological Science, 22(7):791-796.
Tong S K, Hsu H J, Chung B C. 2010.Zebrafish monosex population reveals female dominance in sex determination and earliest events of gonad differentiation. Developmental Biology, 344(2):849-856.
Uno T, Ishizuka M, Itakura T. 2012. Cytochrome P450 (CYP)in fish. Environmental Toxicology and Pharmacology, 34(1):1-13.
Vizziano-Cantonnet D, Baron D, Mahè S, Cauty C, Fostier A, Guiguen Y. 2008. Estrogen treatment up-regulates female genes but does not suppress all early testicular markers during rainbow trout male-to-female gonadal transdifferentiation. Journal of Molecular Endocrinology, 41(5):277-288.
Vizziano D, Randuineau G, Baron D, Gauty C, Guiguen Y. 2007. Characterization of early molecular sex differentiation in rainbow trout, Oncorhynchus mykiss.Developmental Dynamics, 236(8):2 198-2 206.
Wang D S, Kobayashi T, Zhou L Y, Paul-Prasanth B, Ijiri S, Sakai F, Okubo K, Morohashi K, Nagahama Y. 2007.Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with Ad4 binding protein/steroidogenic factor. Molecular Endocrinology, 21(3):712-725.
Wang D S, Zhao L Y, Kobayashi T, Matsuda M, Shibata Y, Sakai F, Nagahama Y. 2010.Doublesex- and Mab-3-related transcription factor-1 repression of aromatase transcription, a possible mechanism favoring the male pathway in tilapia. Endocrinology, 151(3):1 331-1 340.
Webster K A, Schach U, Ordaz A, Steinfeld J S, Draper B W, Siegfried K R. 2017. Dmrt1 is necessary for male sexual development in zebrafish. Developmental Biology, 422(1):33-46.
Wu G C, Li H W, Luo J W, Chen C, Chang C F. 2015. The potential role of Amh to prevent ectopic female development in testicular tissue of the protandrous black porgy, Acanthopagrus schlegelii. Biology of Reproduction, 92(6):158.
Wu G C, Tomy S, Lee M F, Lee Y H, Yueh W S, Lin C J, Lau E L, Chang C F. 2010.Sex differentiation and sex change in the protandrous black porgy, Acanthopagrus schlegeli.General and Comparative Endocrinology, 167(3):417-421.
Wu G C, Li H W, Tey W G, Lin C J, Chang C F. 2017.Expression profile of amh/Amh during bi-directional sex change in the protogynous orange-spotted grouper Epinephelus coioides. PLoS One, 12(10):e0185864.
Xia W, Zhou L, Yao B, Li C J, Gui J F. 2007. Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers.Molecular and Cellular Endocrinology, 263(1-2):156-172.
Yang Y J, Wang Y, Li Z, Zhou L, Gui J F. 2017. Sequential, divergent and cooperative requirements of foxl2a and foxl2b in ovary development and maintenance of zebrafish. Genetics, 205(4):1 551-1 572.
Yano A, Guyomard R, Nicol B, Jouanno E, Quillet E, Klopp C, Cabau C, Bouchez O, Fostier A, Guiguen Y. 2012. An immune-related gene evolved into the master sexdetermining gene in rainbow trout, Oncorhynchus mykiss.Current Biology, 22(15):1 423-1 428.
Yamaguchi A, Lee K H, Fujimoto H, Kadomura K, Yasumoto S, Matsuyama M. 2006. Expression of the DMRT gene and its roles in early gonadal development of the Japanese pufferfish Takifugu rubripes. Comparative Biochemistry and Physiology Part D:Genomics and Proteomics, 1(1):59-68.
Zhang X B, Li M R, Ma H, Liu X Y, Shi H J, Li M H, Wang D S. 2017. Mutation of foxl2 or cyp19a1a results in female to male sex reversal in XX Nile tilapia. Endocrinology, 158(8):2 634-2 647.
Zhao L, Svingen T, Ng E T, Koopman P. 2015. Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1. Development, 142(6):1 083-1 088.