MOLECULAR ANALYSES OF GONAD DIFFERENTIATION AND FUNCTION IN ZEBRAFISH MOHAMMAD SOROWAR HOSSAIN (M.S, University of Dhaka, Bangladesh) A THESIS SUBMITTED FOR THE DEGREE OF PHD OF MOLECULAR BIOLOGY DEPARTMENT OF BIOLOGICAL SCIENCES & TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2010 Dedicated to my family i Acknowledgements iii Table of Contents . iv Abstract vii List of Tables . ix List of Figures ix List of Abbreviations and Symbols . xii Gene list xiii ii Acknowledgements Albert Einstein came close to the remark when he wrote: “A hundred times every day I remind myself that my inner and outer life are based on the labors of other man, living and dead, and that I must exert myself in order to give in the same measure as I have received and still receiving.” The World as I See it, Ideas and Opinions (1954) (trans. Sonja Bargmann) My gratitude and debts are owed to a larger and more diverse community than that in which Einstein toiled. I would like to thank my supervisor A/Prof. Laszlo Orban for his advice, guidance, concern and great assistance in the accomplishment of this thesis. I extend my heartfelt gratitude to Ms. Rajini Sreenivasan for her help regarding microarray hybridization and the data analysis. She also meticulously proofread my thesis. I acknowledge Mr. Liew Woei Chang for his suggestions while writing my Thesis. I thank Mr. Alex Chang Kuok Weai who helped me to measure the concentration of 11-KT. I also thank my former colleagues Dr. Richard Bartfai and Dr. Wang Xingang for their valuable suggestions and technical assistance. I also thank current and former colleagues Kwan Hsiao Yuen, Jolly, Leslie Beh Yee Ming and Minnie Cai, Li Yang. I acknowledge my thesis committee members Dr. Naweed Naqvi, Dr. Karuna Sampath and Dr. Sohail Ahmed for their valuable suggestions and guidance. I also thank our collaborator Professor Per-Erik Olsson and his team for their help. I extend my appreciation to Drs. Alexander Emelyanov and Serguei Parinov who provided their transposon-based transgenic technology. I would like to thank all TLL common facilities, such as Sequencing lab, Medium preparing lab,and the Fish keeping facility. Finally, I am grateful to my wife Shameema Ferdous and my daughter Fatima Sorowar for their immense sacrifice and mental support. Without the help from my elder brother Dr. Rabiul Alam, I could not have got the opportunity to Ph.D. My parents and all family members are the real motivators to finish up the long and arduous Ph.D journey. iii Table of Contents Chapter Introduction 1.1 Sex determination and gonad differentiation in vertebrates . Fish Reptiles . Birds Mammals . Sex determination and gonad differentiation in zebrafish 13 1.2.1 Polygenic sex determination proposed in zebrafish 13 1.2.2 The role of primordial germ cells (PGC) in the sexual fate of zebrafish 14 1.2.3 Juvenile hermaphroditism is the mode of sex differentiation in zebrafish 16 1.2.4 Conserved genes in the zebrafish testis differentiation pathway 19 Programmed cell death (apoptosis) . 21 1.3.1 Molecular and cellular events in apoptosis 21 1.3.2 The role of apoptosis in gonad development and maturation . 24 Steroidogenic pathway in vertebrates . 25 1.4.1 Sex steroids . 25 1.4.2 Conserved steroidogenic pathways . 26 The aims of my Thesis 30 1.1.1 1.1.2 1.1.3 1.1.4 1.2 1.3 1.4 1.5. Chapter Materials and Methods 31 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 Origin, rearing and maintaining of fish . 31 Primers 31 Tissue collection, RNA isolation and cDNA synthesis 31 Real-time PCR 32 In situ hybridization 33 Radiation hybrid mapping . 34 Southern blot . 34 Cloning of candidate and apoptosis- related genes . 35 Establishing of zebrafish transgenic lines . 36 Phylogenetic analysis 37 Subcellular localization . 37 Anti-apoptotic drug (QVD) treatment 38 Fadrozole and MT treatment . 39 Flutamide treatment 40 Caspase-3 assay………………………………………………………… .40 DNA ladder assay . 41 Histology . 41 Microarray . 41 2.18.1 Gonad Uniclone Microarray . 41 2.18.2 Microarray target preparation and hybridization . 42 2.18.3 Statistical analysis of microarray data 43 2.18.4 Microarray quality control 43 iv 2.19 Gonadal explants . 44 Chapter Results 45 3.1 The role of apoptosis during testis formation . 45 3.1.1 A broad spectrum caspase inhibitor suppressed apoptosis in zebrafish embryos, juveniles and adults 45 3.1.2 Chemical inhibition of apoptosis in zebrafish juveniles substantially delayed testis formation . 48 3.1.3 Transcriptome analysis of developing gonads exposed to QVD showing differentially expressed genes 54 3.2 The role of steroidogenic pathway in zebrafish reproduction…………………… .60 3.2.1 Disrupting the balance of sex steroids in adult zebrafish by aromatase inhibitor (AI) and methyltestosterone (MT) 60 3.2.2 Incomplete oogenesis due to E2 depletion and overdose of MT . 62 3.2.3 Spermatogenesis-related genes showed upregulated expression in response to E2 depletion…………………………………………………………………… 67 3.2.4 Spermatogenesis- and folliculogenesis -related genes were down-regulated due to MT exposure …………………………………………………………………….70 3.3 Sexually dimorphic expression of steroidogenesis-related genes during gonad differentiation and in adult gonads . 73 3.3.1 Steroidogenesis-related genes favoring testis development . 73 3.3.2 Steroidogenesis-related genes favoring ovary development . 75 3.3.3 Estrogen depletion by aromatase inhibitor caused up-regulation of testicular genes during gonad development 77 3.4 Comparative analyses of candidate genes for the identification of early testicular markers in zebrafish 80 3.4.1 Androgen receptor 83 3.4.1.1 Androgen receptor showed sexual dimorphic expression in developing gonads and adult tissues of zebrafish . 83 3.4.1.2 Sequence homology and phylogenetic analysis of vertebrate androgen receptors . 86 3.4.1.3 Sexually dimorphic expression of the ar gene in zebrafish gonads 88 3.4.1.4 Flutamide treatment to block the androgen receptor during testis formation . 88 3.4.2 A novel gene showing enhanced expression in spermatocytes 92 3.4.2.1 Cloning and characterization of the spermatocyte-expressed gene 92 3.4.2.2 Phylogenetic analysis of scx1 orthologs in vertebrates 94 3.4.2.3 Conserved syntheny of zebrafish scx1 and its human ortholog 95 3.4.2.4 Analysis of scx1expression during gonad development and in adult tissues of zebrafish 98 3.4.3 A novel heat shock transcription factor 98 3.4.3.1 Cloning and characterization of a second novel gene with testis-enhanced expression . 98 v 3.4.3.2 Hsf5 is a new member of heat shock transcription factor family . 100 3.4.3.3 The expression of hsf5 in the embryos, developing and adult gonads of zebrafish 101 3.4.3.4 Dominant-negative approach for the analysis of Hsf5 function in zebrafish ……………………………………………………………………… 108 3.4.4 star is the earliest testicular marker during test differentiation in zebrafish ………………………………………………………………………….108 3.5 Data on gonadal explants ………………………………………………………111 Chapter Discussion 113 4.1 4.2 4.3 4.4 Oocyte apoptosis is required for testis formation in zebrafish . 113 Blocking of zebrafish androgen receptor during gonad development did not influence the sex ratio . 118 Two novel conserved genes, scx1 and hsf5 in the testis pathway 123 Reciprocal expression of steroidogenesis-related genes during gonad differentiation 125 4.5 Hormonal balance is essential for the maintenance and function of adult zebrafish gonads 136 4.5.1 Responses to MT-treatment in the ovary of adult zebrafish . 137 4.5.2 Responses to MT-treatment in the testis of adult zebrafish 140 4.6 4.6.1 4.6.2 Molecular responses to aromatase inhibition in the adult gonads 142 Effects of aromatase inhibition in the adult ovary 142 Effects of aromatase inhibition in the adult testis . 144 4.7 Induction of testicular gene expression during gonad differentiation in response to E2 depletion 147 4.8 Conclusion: estrogen to androgen ratio may hold the key during gonad differentiation in zebrafish 149 vi Abstract Zebrafish is an important vertebrate model organism that has helped researchers to unveil many interesting biological questions, especially those related to early embryogenesis. However, our current knowledge on zebrafish gonad differentiation and function is limited. Juvenile hermaphroditism is the mode of gonad differentiation where both mature ovary and testis are developed from the bipotential ‘juvenile ovary’. Oocyte apoptosis is thought to be involved in the gonadal transformation process. To investigate the role of apoptosis during testis development, we chemically suppressed apoptosis using a broad spectrum anti-apoptotic drug. In our study, when apoptosis was blocked during gonadal transformation, testis development was remarkably delayed. After one week of treatment, prospective individuals destined to be males underwent gonadal transformation, suggesting the necessity of oocyte apoptosis during testis formation in zebrafish. Moreover, expression profiling using Gonad Uniclone microarray also provided evidence for delayed gonadal transformation at the transcriptiome level. We also studied the role of sex steroids during gonad differentiation and in adult gonads. The hormonal balance is essential for the maintenance of spermatogenesis and folliculogenesis in adult zebrafish. Intriguingly, estrogen depletion in the adult testis caused enhanced male function at molecular level. We have also analyzed the expression of a number of steroidogenesis-related genes during gonad differentiation. Our results showed reciprocal expression of these genes during gonad differentiation: foxl2, cyp19a1a, cyp11a, hsd3b and cyp17a1 in ovarian differentiation and star, nr5a1a and cyp11b2 in testicular differentiation. In order to broaden the understanding of zebrafish testis development, we invested our effort to identify early testis markers in zebrafish. As a result, we have identified and characterized zebrafish androgen receptor and two other vii spermatocyte-specific novel genes (scx1 and hsf5). Comparative analyses showed that steroidogenic acute regulatory protein (star) gene is the earliest testis differentiation marker in zebrafish. Our overall findings indicate that the ratio of estrogen to androgen may play an important role in gonad differentiation in zebrafish. viii List of Tables Table 1: Mode of sexuality in teleosts ……………………………………………… . Table 2: Differential expression of selected genes in response to QVD treatment…….59 Table 3: Candidate genes screened by RT-PCR………………………………………. 82 Table 4: Comparative analysis of expression profiles of potential testicular makers….111 List of Figures Fig. 1: Schematic representation of the sequence of events during gonadal development. PGC, primordial germ cell………………………………………… . Fig. 2: Sex determination system is diverse among vertebrates………………………. Fig. 3: Comparative analysis of testis differentiation process between mouse and zebrafish……………………………………………………………………………… 21 Fig. 4: Two major apoptotic pathways in mammals……………………………………25 Fig. 5: Schematic representation of gonadal steroidogenetic pathways in fish……… .28 Fig. 6: Cycloheximide-induced apoptosis in zebrafish embryos in a concentrationdependent manner………………………………………………………………………46 Fig. 7: QVD, a wide range caspase inhibitor, suppressed cycloheximide- (CHX) or camptothecin- (Campt) induced apoptosis in zebrafish embryos………………………47 Fig. 8: Testing the delivery approaches of QVD in adult zebrafish……………………49 Fig. 9: Inhibition of apoptosis during gonad development of zebrafish in response to QVD exposure………………………………………………………………………… 50 Fig. 10: Testis formation was delayed due to QVD exposure as revealed by histological analysis…………………………………………………………………….52 Fig. 11: The sex ratios of adult zebrafish remained the same after QVD treatment… 53 Fig. 12: Histology of adult gonads after QVD treatment………………………………54 Fig. 13: Line graph depicting the expression profiles of genes differentially expressed between QVD-treated, Control-F and Control-M individuals…………… .56 ix Table S5: List of 483 genes that were more than 1.5-fold up-regulated in QVD-treated compared to Control-M individuals. These genes were derived from a 1-Way ANOVA (p-value < 0.01 with Benjamini and Hochberg multiple testing correction) performed on all genes on the Gonad Uniclone Microarray. GenBank ID CO353243 CO353301 EV562968 EV555727 EV557277 CO353830 EV559467 CO350944 CO350467 EV556251 CO351708 EV558567 CO353757 CO352682 CO353884 CO353046 CO353330 EV605649 CO351105 CO353299 CO355792 EW680229 CO356277 CO355042 EV605221 CO351070 EV606155 CO349737 CO354323 CO356260 CO355123 CO354757 CO351534 CO351704 CO355090 CO354400 EV603113 CO354049 EV603611 CO356286 EV563087 CO356336 CO355284 EV557500 CO355630 CO353672 CO355716 EV555872 EV562919 CO349796 EV558046 EV555808 CO353125 EV563620 EV557714 CO356246 EV565115 CO353506 CO354603 CO353303 CO351342 CO353403 CO350558 CO354575 CO356250 CO351042 CO354000 CO355414 CO353335 CO355333 EV558569 CO355373 Control-M Median 1.47 1.47 1.83 1.74 1.58 1.49 1.62 1.29 1.29 1.11 1.74 1.91 1.61 1.91 1.47 2.16 1.33 2.13 1.71 1.71 1.51 1.52 1.50 1.72 1.83 1.85 1.81 1.61 1.43 1.68 1.62 1.73 1.75 2.19 1.71 1.72 1.87 1.77 1.80 1.91 1.23 1.84 1.87 1.84 1.75 1.69 1.78 1.79 2.32 1.94 1.92 2.34 1.74 2.06 1.75 1.88 1.72 1.84 1.92 1.86 1.94 2.54 1.87 2.58 1.75 1.86 2.08 1.89 1.91 1.84 1.10 1.97 Control-F Median 0.90 0.95 0.85 0.90 0.88 0.98 0.88 1.07 1.05 1.09 0.76 0.76 0.96 0.73 1.04 0.61 1.03 0.68 0.95 0.95 1.01 1.06 1.03 0.99 0.93 0.91 0.87 1.02 1.03 0.99 1.00 0.99 0.82 0.84 0.90 0.93 0.98 0.87 0.75 0.95 1.09 0.92 0.98 0.79 0.94 1.02 0.93 0.86 0.79 0.96 0.93 0.77 0.97 0.72 0.87 0.98 0.98 0.86 0.94 0.95 0.76 0.70 1.04 0.83 0.95 0.96 0.93 1.01 0.87 0.74 1.38 0.87 QVD Median 0.98 0.97 1.20 1.14 1.03 0.96 1.01 0.80 0.79 0.69 1.06 1.17 0.98 1.16 0.89 1.28 0.79 1.26 1.01 1.00 0.88 0.88 0.87 0.99 1.06 1.06 1.04 0.92 0.81 0.96 0.92 0.98 0.98 1.21 0.94 0.95 1.03 0.97 0.98 1.05 0.67 1.01 1.02 1.00 0.95 0.91 0.96 0.96 1.24 1.04 1.02 1.24 0.91 1.07 0.91 0.97 0.89 0.95 0.99 0.95 0.99 1.30 0.95 1.31 0.89 0.94 1.04 0.95 0.95 0.91 0.54 0.97 Ratio QVD/Ctrl-F 1.09 1.03 1.43 1.27 1.18 0.98 1.16 0.75 0.76 0.63 1.40 1.53 1.03 1.60 0.86 2.11 0.76 1.86 1.07 1.06 0.88 0.83 0.84 1.00 1.13 1.16 1.19 0.90 0.79 0.97 0.92 0.99 1.19 1.44 1.05 1.03 1.06 1.12 1.30 1.10 0.61 1.09 1.04 1.27 1.01 0.90 1.03 1.11 1.58 1.08 1.10 1.61 0.94 1.50 1.04 0.99 0.91 1.10 1.05 1.01 1.30 1.86 0.91 1.58 0.93 0.98 1.12 0.94 1.10 1.24 0.39 1.11 Ratio QVD/Ctrl-M 0.67 0.66 0.66 0.66 0.65 0.64 0.62 0.62 0.62 0.62 0.61 0.61 0.61 0.61 0.61 0.59 0.59 0.59 0.59 0.59 0.59 0.58 0.58 0.58 0.58 0.57 0.57 0.57 0.57 0.57 0.56 0.56 0.56 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.54 0.54 0.54 0.54 0.54 0.54 0.53 0.53 0.53 0.53 0.52 0.52 0.52 0.52 0.52 0.52 0.51 0.51 0.51 0.51 0.51 0.51 0.51 0.50 0.50 0.50 0.50 0.50 0.50 0.49 BLASTx ribosomal protein S15a [Danio rerio] similar to Rho GTPase activating protein 24 [Danio rerio] unknown unknown fumarate hydratase precursor [Danio rerio] similar to HUS1 checkpoint protein [Canis familiaris] ribosomal protein L24 [Danio rerio] similar to ELAV-like isofor . eukaryotic translation elongation factor beta [Danio rerio] hypothetical protein LOC393742 [Danio rerio] unknown MGC75666 protein [Xenopus tropicalis] unknown small ubiquitin-related protein [Oncorhynchus mykiss] unknown similar to 40S ribosomal protein S27-2 isoform [Danio rerio] unknown GDP dissociation inhibitor [Danio rerio] annexin [Danio rerio] similar to eukaryotic translation initiation factor 3, subunit isoform [Danio rerio] similar to Filamin B (FLN-B) (Beta-filamin) (Actin-binding like protein) [Danio rerio] similar to RAMA1 isoform [Danio rerio] unknown similar to Vacuolar proton translocating ATPase [Danio rerio] H3 histone, family 3A [Homo sapiens] similar to H63 breast cancer expressed gene isoform a [Danio rerio] ubiquinol-cytochrome c reductase core protein II [Danio rerio] glycine dehydrogenase [Danio rerio] unknown similar to F-actin capping protein alpha-1 subunit (CapZ alpha-1) [Danio rerio] citrate synthase [Danio rerio] >gb|AAH45362.1| Citrate synthase [Danio rerio] TIA1 cytotoxic granule-associated RNA bi . hypothetical protein LOC553787 [Danio rerio] similar to CDNA sequence BC027309 [Rattus norvegicus] similar to nascent polypeptide-associated complex alpha polypeptide [Danio rerio] similar to Rsnl2 protein [Danio rerio] similar to splicing factor 3b, subunit isoform isoform [Danio rerio] similar to CG10739-PA [Danio rerio] aminolevulinate, delta-, synthetase [Danio rerio] ubiquinol-cytochrome c reductase core protein II [Danio rerio] similar to High affinity immunoglobulin epsilon receptor alpha-subunit precursor Danio rerio] similar to Ctsd protein isoform [Danio rerio] unknown similar to F-box only protein 10 [Danio rerio] bcl2-associated X protein [Danio rerio] similar to (DAP kinase-related apoptosis-inducing protein kinase 1) [Danio rerio] similar to Kallmann syndrome 1a sequence isoform [Danio rerio] unknown hypothetical protein LOC445124 [Danio rerio] similar to 40S ribosomal protein S27-2 isoform [Danio rerio] unknown unknown myeloid leukemia-associated SET translocation protein [Danio rerio] hypothetical protein LOC402989 [Danio rerio] WD repeat domain 68 [Danio rerio] similar to FLJ00281 protein [Danio rerio] similar to malate dehydrogenase, mitochondrial isoform [Danio rerio] similar to ring finger protein 128 isoform 2, partial [Danio rerio] similar to Mitochondrial import inner membrane translocase subunit [Danio rerio] hypothetical protein LOC449786 [Danio rerio] myosin, light polypeptide 7, regulatory [Danio rerio] cytochrome c oxidase subunit I [Danio rerio] similar to Sorting nexin isoform [Danio rerio] unknown Unknown (protein for IMAGE:5412243) [Danio rerio] similar to Thioredoxin-depend . PRP38 pre-mRNA processing factor 38 (yeast) [Homo sapiens] unknown ribosomal protein L24 [Danio rerio] annexin A11b [Danio rerio] hypothetical protein XP_682942 [Danio rerio] unknown XIV Table S5: continued CO353210 EV562740 EV603076 CO351537 EV556307 CO349919 EV555494 EV605838 CO354068 EV564485 EV560167 CO356135 EV556419 CO350152 EV563917 EV555311 EV555553 EV555923 CO350709 CO351538 EV564137 CO351458 EV557258 CO356153 EV556781 CO355388 EV557666 EV556898 CO355512 CO355349 CO353534 CO354080 CO356053 EV563556 CO353094 CO356232 CO356104 EV557839 CO353236 CO354174 CO353437 CO355335 EV564099 CO355656 CO353372 CO352779 EV605287 EV604282 CO353767 EV564343 CO353339 EV555855 CO354622 EV564341 CO353114 CO355677 EV605281 EV563810 CO353191 CO352675 CO356215 EV605284 CO352974 CO354218 CO354047 EV603914 CO355770 CO355205 EV555413 CO353486 CO353938 CO355498 CO355881 CO354069 2.70 2.66 2.64 2.18 2.75 2.11 2.66 2.85 2.47 2.90 2.97 2.98 2.54 2.18 1.61 2.70 2.19 2.49 2.30 2.71 2.45 3.03 2.54 2.23 2.44 2.33 2.42 2.51 2.80 2.50 3.67 2.97 2.22 2.52 2.49 2.85 2.69 2.00 2.63 3.42 3.89 2.19 1.94 2.76 2.11 2.97 3.10 1.49 2.71 3.08 2.60 3.29 2.71 2.48 2.74 2.59 3.12 2.94 3.84 2.85 2.86 2.81 3.08 5.16 3.26 2.65 2.79 2.72 3.66 3.72 3.25 3.23 2.65 2.60 0.98 0.94 0.70 1.01 0.83 0.95 0.94 0.74 0.97 0.72 0.86 0.86 0.97 1.10 1.02 0.97 0.98 0.99 0.86 0.96 1.06 0.84 0.93 1.13 1.09 0.89 1.00 0.98 0.83 0.97 0.74 0.54 1.00 0.94 0.82 0.98 0.94 0.96 0.96 0.98 0.73 1.03 1.08 0.98 0.96 0.92 0.86 0.95 1.03 0.86 1.09 0.69 0.82 0.88 0.73 0.99 0.97 0.89 0.49 0.90 0.99 0.87 0.87 0.70 0.85 0.92 0.99 1.04 0.74 0.70 0.86 0.86 0.98 0.96 1.04 1.02 1.01 0.83 1.05 0.80 1.01 1.08 0.93 1.09 1.11 1.11 0.95 0.81 0.60 1.00 0.81 0.92 0.84 0.99 0.89 1.10 0.93 0.81 0.89 0.85 0.87 0.90 1.01 0.90 1.31 1.06 0.79 0.89 0.87 1.00 0.94 0.69 0.91 1.18 1.34 0.75 0.66 0.94 0.72 1.01 1.05 0.50 0.92 1.04 0.88 1.10 0.90 0.82 0.91 0.86 1.03 0.97 1.26 0.94 0.94 0.92 1.01 1.68 1.06 0.85 0.89 0.87 1.17 1.19 1.04 1.03 0.84 0.82 1.05 1.09 1.43 0.82 1.27 0.85 1.08 1.46 0.96 1.52 1.29 1.30 0.98 0.73 0.59 1.03 0.83 0.92 0.99 1.03 0.84 1.31 0.99 0.72 0.81 0.95 0.88 0.92 1.21 0.92 1.77 1.96 0.79 0.95 1.07 1.02 1.00 0.72 0.95 1.20 1.84 0.73 0.61 0.96 0.75 1.10 1.23 0.53 0.89 1.20 0.80 1.60 1.10 0.94 1.25 0.87 1.06 1.09 2.57 1.04 0.95 1.06 1.16 2.41 1.24 0.93 0.90 0.84 1.59 1.69 1.21 1.19 0.86 0.86 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.38 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.37 0.36 0.36 0.36 0.36 0.36 0.36 0.36 0.36 0.36 0.36 0.36 0.36 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 similar to Interferon-induced 35 kDa protein homolog (IFP 35) [Danio rerio] hypothetical protein MGC11034-like protein [Danio rerio] hypothetical protein LOC321769 [Danio rerio] unknown unknown similar to WAS protein family, member 3, partial [Danio rerio] Ribosomal protein L36A [Danio rerio] unknown bone morphogenetic protein type II receptor [Rattus norvegicus] similar to Im:6902697 protein [Danio rerio] unknown similar to macrophage expressed gene [Danio rerio] similar to hepatic leukemia factor, partial [Danio rerio] Danio rerio DMbeta1 mRNA, complete cds unknown similar to F-actin capping protein alpha-1 subunit (CapZ alpha-1), partial [Danio rerio] unknown pyruvate dehydrogenase (lipoamide) beta [Danio rerio] unknown putative ISG12-2 protein [Danio rerio] similar to Aspartyl-tRNA synthetase (Aspartate--tRNA ligase) [Danio rerio] hypothetical protein LOC447845 [Danio rerio] similar to KIAA0232 protein [Danio rerio] hypothetical protein LOC393183 [Danio rerio] unknown unknown similar to lysosomal-associated membrane protein precursor [Danio rerio] Similar to Plasmodium falciparum glutamic acid-rich protein [Homo sapiens] similar to SWI/SNF-related,[Danio rerio] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d [Danio rerio] unknown unknown Ba2 globin [Danio rerio] unknown similar to MGC78790 protein [Danio rerio] similar to Serine/threonine-protein kinase Pim-3 [Danio rerio] similar to dystrobrevin binding protein isoform a [Danio rerio] hypothetical protein XP_701410 [Danio rerio] phosphogluconate hydrogenase isoform [Danio rerio] similar to integral membrane protein 2B [Danio rerio] splicing factor, arginine/serine-rich [Danio rerio] unknown similar to pim-1 oncogene [Danio rerio] similar to glutamate dehydrogenase 1a isoform [Danio rerio] unknown similar to Ras association (RalGDS/AF-6) [Danio rerio] unknown unknown unknown unknown ribosomal protein L24 [Danio rerio] similar to complement component C7-2 [Danio rerio] unknown LOC553481 protein [Danio rerio] unknown similar to Exocyst complex component Sec15B [Danio rerio] X-prolyl aminopeptidase (aminopeptidase P) 1, soluble [Danio rerio] similar to CG10011-PA [Danio rerio] cytochrome c oxidase subunit I [Danio rerio] similar to c-Mpl binding protein isoform b, partial [Danio rerio] similar to cAMP response element modulator tau alpha gamma [Danio rerio] unknown E4tf1-60 transcription factor [Danio rerio] NADH dehydrogenase subunit [Danio rerio] similar to Nebulin [Danio rerio] glial fibrillary acidic protein [Danio rerio] similar to 26S proteasome non-ATPase regulatory subunit [Danio rerio] putative ISG12-2 protein [Danio rerio] similar to Moesin (Membrane-organizing extension spike protein), partial [Danio rerio] similar to integral membrane protein 2B [Danio rerio] diazepam binding inhibitor [Danio rerio] hydroxysteroid 11-beta dehydrogenase [Danio rerio] hypothetical protein LOC550245 [Danio rerio] unknown XV Table S6: Amino acid identity of different domains of zebrafish androgen receptor as compared to its orthologs Species TAD DBD LBD Overall Zebrafish 100% 100% 100% 100% Goldfish 77% 97% 96% 84% Fathead minnow 78% 95% 96% 84% Rainbow trout-β 37% 95% 87% 58% Rainbow trout-α 35% 92% 86% 55% European seabass 30% 94% 86% 55% Japaneese eel-β 26% 95% 86% 52% Nile tilapia-β 25% 97% 82% 52% Japeneese eel- α 22% 92% 76% 46% Three-spined stickleback 27% 95% 83% 46% Frog 20% 91% 68% 43% Chicken 16% 91% 68% 43% Nile tilapia-α 13% 77% 66% 40% Mouse 19% 91% 73% 40% Human 17% 95% 73% 39% XVI Table S7: Androgen receptors from different species Family Latin name Common name GenBank ID Ar alpha Ar beta EF42791 AAM09 278 AAF881 38 AAU094 77 AAT764 33 AAO835 72 BAA334 51 AAO616 94 BAA327 85 BAA838 05 BAB200 82 AAL928 78 BAD520 84 ABC686 12 BAC983 01 CAG029 75 AAP558 43 ABD467 46 Cyprinidae Danior rerio Zebrafish - Cyprinidae Carassius auratus Goldfish - Cyprinidae Pimephales promelas Micropogonias undulatus Dicentrarchus labrax Gasterosteus aculeatus Pagrus major Fathead minnow Atlantic croaker European seabass Three spined stickleback Red sea bream - Acanthopagrus schlegelii Oncorhynchus mykiss Anguilla japonica Black sea bream Rainbow trout - Oreochromis niloticus Astatotilapia burtoni Gambusia affinis Nile tilapia Oryziinae Kryptolebias marmoratus Oryzias latipes Tetraodontid ae Squalidae Tetraodon nigroviridis Squalus acanthias Mangrove rivulus Japanese medaka Green spotted pufferfish Spiny dogfish Rajidae Leucoraja erinacea Little skate Sciaenidae Moronidae Gasterosteid ae Sparidae Sparidae Salmonidae Anguillidae Cichlidae Cichlidae Poeciliidae Rivulidae Japanese eel Astatotilapia Mosquitofish - BAA327 84 BAA754 64 BAB200 81 AAD250 74 BAD520 85 - XVII Table S8: Scx1 proteins isolated or predicted from vertebrate species Common name Amino acid identity (%) Danio rerio Zebrafish 100 EF554575 Rutilus rutilus Roach 51 EST: EG537911 (partial Pimephales Fathead 46 EF554576 Salmo salar Salmon 29 ESTs: DW561316, Xenopus levis Frog 21 ENSXETP00000023420 Gallus gallus Chicken 23 XP_423016 Homo sapiens Human 20 AAH40280 Macaca Macaque 21 BAB62932 Mus musculus Mouse 16 AAI20722 Latin name GeneBank or Ensembl ID XVIII Table S9: Amino acid identity of DNA binding domain (DBD) and its mammalian orthologs and HSFL HSFs zHsf5 hHSF5 mHSF5 LW1 HSFY DBD (%) Full-length (%) 100 100 48 28 41 27 38 15 32 15 HSFs DBD (%) hHSF1 100 zHsf1 91 zHsf2 67 zHsf5 39 XIX Table S10: Hsf5 proteins isolated or predicted from vertebrate species Organism Latin name Zebrafish D anio rerio Human Homo sapiens Mouse Mus musculus Chicken Gallus gallus Frog Xenopus levis Rainbow trout Oncorhynchus mykiss Fugu Tetraodon nigricans HSF/HSFL H sf1 H sf2 H sf4 H sf5 HSF1 HSF2 HSF4 HSF5 LW HSF-Y2 HSF1 HSF2 HSF4 HSF5 HSF-Y HSF1 HSF2 HSF3 HSF4 HSF5 H sf1 H sf2 H sf5 H sf1 H sf2 H sf5 H sf1 GenBank ID NP571675 NP571942 NP001013335 FJ969446 NP005517 NP004497 NP001529 XP064190 NP057237 NP714927 NP032322 NP032323 NP036069 CAI35140 NP081937 P38529 P38530 P38531 XP425114 XP415719 JC4199) AAW78930 submitted BAD10989 CAD32483 submitted CAG11654 XX XXI Expression relative to bactin 140 Control Flutamide 120 100 80 in po s pr os a am h a a1 cy p1 dm rt1 b2 cy p1 ar 60 Figure S2: The relative expression of putative Ar-responsive genes in the developing gonad exposed to Flutamide (550 µg/L) from 20-50 dpf shows lack of significant differences in comparison to controls. The developing testes were isolated immediately following the treatment. The transcripts in the real-time PCR were calibrated with internal control, bactin, and normalized (x100%, the value in the control of each gene as 100%) Each value is expressed as mean ± SD (n = 5). No significant differences were found between the control and MT treated males when analyzed by Student t-test. XXII zTs1 NH- hTS1 NH- cTS1 NH- 15 NLS 78 CCD 392 199 168 CCD 150 -COOH 179 217 CCD 40 69 CCD 259 CCD 210 -COOH 256 CCD 412 410 -COOH Figure S3: Structural organization of the zebrafish Scx1 protein and its orthologs. Comparative view of the domains of three vertebrate Scx1 orthologs. Abbreviations for both panels: hs - Homo sapiens; mm - Mus musculus; gg - Gallus gallus; xl - Xenopus laevis; om - Oncorhynchus mykiss; tn - Tetraodon nigroviridis; dr -Danio rerio. XXIII * Expression relative to baction 12.0 Control Heat shock 10.0 8.0 1.0 0.5 0.0 hsf5 hsp70 Figure S4: The relative expression of hsf5 remains the same in the adult zebrafish testis following heat shock treatment. Fish were kept at 37 oC for one hour and RNA isolated from the testis. The transcripts in the real-time PCR were calibrated with internal control, bactin. The expression of hsp70 was used as positive control for this experiment. Each value is expressed as mean ± SD (n = 3). *Significant difference (P[...]... number of randomly picked individuals for staining, which hinders subsequent investigations such as biochemical assays and RNA-based studies In this context, transgenic lines with gonad- specific reporter gene expression (such as EGFP) would be useful to examine the gonad development 17 To explore the dynamic process of gonad differentiation, three transgenic zebrafish lines expressing EGFP in their gonads... (Colombo& Grandi 1996; Nakamura 1984; Takahashi 1983) Unlike in other vertebrates, gonadogenesis in fish is a plastic and relatively complex process Knowledge from other model systems provides little help in the study of gonad differentiation in zebrafish Studying gene function utilizing widely used molecular tools, such as gene knock-out, morpholinos, and cell culture, is also difficult in zebrafish. .. synthesizing cells in some species (Nakamura et al 1989; Strussmann 2002) The induction of sex reversal using exogenous steroids depends mainly on the timing of the onset of treatment, duration of treatment; and the dose and the type of hormone used (Devlin& Nagahama 2002) In general, most investigations in gonochoristic fish suggest that gonadal sex phenotype can only be manipulated around the period of. .. sex-determining gene in bird 8 Knocking down of dmrt1 by RNA interference (RNAi) caused the feminization of the embryonic gonads in genetically male (ZZ) embryos (Smith et al 2009) In the chicken embryo, the expression profile of some mammalian orthologs such as nr5a1 (sf1), sox9, amh, dax1, wint4 is consistent with their conserved roles in gonadal sex differentiation (Smith et al 2007) 1.1.4 Mammals In most... adult organs of zebrafish ………………………………………………………………….105 Fig 47: In situ hybridization of hsf5 onto sections of adult zebrafish gonads……… 106 Fig 48: Subcellular localization of Hsf5…………………………………………… 107 Fig 49: Comparative analyses of early testicular markers in zebrafish ……………109 Fig 50: Hormonal ratio might be critical for gonad differentiation in zebrafish … 151 xi List of Abbreviations and Symbols... steroidogenesis-related genes in adult zebrafish tissues……………………………………………………………… 79 Fig 28: Induction of testicular genes in the juvenile ovary in response to Fadrozole 81 Fig 29: RT-PCR for identifying genes with testis-specific or testis-enhanced expression in zebrafish ……………………………………………………………… 83 Fig 30: The structure of the zebrafish androgen receptor mRNA and protein……… 84 x Fig 31: Single locus of ar in zebrafish. .. containing gene 9a), and ar (androgen receptor)] have been identified and implicated with gonad development in zebrafish In addition, recent large scale transcriptome analyzes also have discovered a substantial number of sexually dimorphically expressed genes (both conserved and novel) in the brain and gonad of zebrafish (Santos et al 2008; Sreenivasan et al 2008a; Villeneuve et al 2009) However, the information... marker) causes the reduction of germ cell number and can also lead to male fate determination (Houwing et al 2007) Transplantation of single PGC in PGC deficient zebrafish results in male fate (Saito et al 2008) Therefore, a threshold number of PGCs might determine the sex in zebrafish 1.2.3 Juvenile hermaphroditism is the mode of sex differentiation in zebrafish Zebrafish is considered to be a juvenile hermaphrodite... Mammals, the testis-determining SRY gene is absent in birds The molecular mechanism of sex determination in birds has been solved very recently (Smith et al 2009) Two hypotheses have been proposed governing avian sex determination Sex in birds might be determined by the dosage of Z-linked gene (two for a male, one for a female) or a dominant ovarydetermining gene(s) located in W chromosome or both may... clustering of 2357 genes that were differentially-expressed……58 Fig 15: The relative expression of vtg1 in the adult liver of both sexes of zebrafish exposed to either Fadrozole or MT…………………………………………………… 61 Fig 16: The gonadosomatic index (GSI) has increased in males following exposure to Fadrozole, and decreased in females following an MT-treatment………………… 63 Fig 17: The relative concentrations of 11-KT in . cyp11a, hsd3b and cyp17a1 in ovarian differentiation and star, nr5a1a and cyp11b2 in testicular differentiation. In order to broaden the understanding of zebrafish testis development, we invested. during gonad differentiation 125 4.5 Hormonal balance is essential for the maintenance and function of adult zebrafish gonads 136 4.5.1 Responses to MT-treatment in the ovary of adult zebrafish. protein (star) gene is the earliest testis differentiation marker in zebrafish. Our overall findings indicate that the ratio of estrogen to androgen may play an important role in gonad differentiation