Loss-of-function of sox3 causes follicle development retardation and reduces fecundity in zebrafish

Abstract

Folliculogenesis is essential for production of female gametes in vertebrates. However, the molecular mechanisms underlying follicle development, particularly apoptosis regulation in ovary, remain elusive. Here, we generated sox3 knockout zebrafish lines using CRISPR/Cas9. sox3 knockout led to follicle development retardation and a reduced fecundity in females. Comparative analysis of transcriptome between sox3^−/− and wild-type ovaries revealed that Sox3 was involved in pathways of ovarian steroidogenesis and apoptosis. Knockout of sox3 promoted follicle apoptosis and obvious apoptosis signals were detected in somatic cells of stages III and IV follicles of sox3^−/− ovaries. Moreover, Sox3 can bind to and activate the promoter of cyp19a1a. Up-regulation of Cyp19a1a expression promoted 17β-estradiol synthesis, which inhibited apoptosis in follicle development. Thus, Sox3 functions as a regulator of Cyp19a1a expression, via 17β-E2 linking apoptosis suppression, which is implicated in improving female fecundity.

Key words： Sox3; follicle development; apoptosis; Cyp19a1a; zebrafish

References

1 Baker KE, Parker R (2004) Nonsense-mediated mRNA decay: terminating erroneous gene expression. Curr Opin Cell Biol 16:293–299

2 Bauters M, Frints SG, Van Esch H, Spruijt L, Baldewijns MM, de Die-Smulders CEM, Fryns JP, Marynen P, Froyen G (2014) Evidence for increased SOX3 dosage as a risk factor for X-linked hypopituitarism and neural tube defects. Am J Med Genet Part A 164:1947–1952

3 Brion F, Tyler CR, Palazzi X, Laillet B, Porcher JM, Garric J, Flammarion P (2004) Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio). Aquat Toxicol 68:193–217

4 Bylund M, Andersson E, Novitch BG, Muhr J (2003) Vertebrate neurogenesis is counteracted by Sox1-3 activity. Nat Neurosci 6:1162–1168

5 Chen S, Zhang H, Wang F, Zhang W, Peng G (2016) nr0b1 (DAX1) mutation in zebrafish causes female-to-male sex reversal through abnormal gonadal proliferation and differentiation. Mol Cell Endocrinol 433:105–116

6 Crowder CM, Lassiter CS, Gorelick DA (2018) Nuclear androgen receptor regulates testes organization and oocyte maturation in zebrafish. Endocrinology 159:980–993

7 Dee CT, Hirst CS, Shih YH, Tripathi VB, Patient RK, Scotting PJ (2008) Sox3 regulates both neural fate and differentiation in the zebrafish ectoderm. Dev Biol 320:289–301

8 Dranow DB, Hu K, Bird AM, Lawry ST, Adams MT, Sanchez A, Amatruda JF, Draper BW (2016) Bmp15 is an oocyte-produced signal required for maintenance of the adult female sexual phenotype in zebrafish. PLoS Genet 12:e1006323

9 Eppig JJ (2001) Oocyte control of ovarian follicular development and function in mammals. Reproduction 122:829–838

10 Facchinello N, Skobo T, Meneghetti G, Colletti E, Dinarello A, Tiso N, Costa R, Gioacchini G, Carnevali O, Argenton F (2017) nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor. Sci Rep 7:4371

11 Foster JW, Graves JAM (1994) An Sry-related sequence on the marsupial X-chromosome- implications for the evolution of the mammalian testis determining gene. Proc Natl Acad Sci USA 91:1927–1931

12 Fu XZ, Cheng YB, Yuan J, Huang CH, Cheng HH, Zhou RJ (2015) Loss-of-function mutation in the X-linked TBX22 promoter disrupts an ETS-1 binding site and leads to cleft palate. Hum Genet 134:147–158

13 Gou Y, Guo J, Maulding K, Riley BB (2018a) Sox2 and Sox3 cooperate to regulate otic/epibranchial placode induction in zebrafish. Dev Biol 435:84–95

14 Gou Y, Vemaraju S, Sweet EM, Kwon HJ, Riley BB (2018b) Sox2 and Sox3 play unique roles in development of hair cells and neurons in the zebrafish inner ear. Dev Biol 435:73–83

15 Guo YQ, Cheng HH, Huang X, Gao S, Yu HS, Zhou RJ (2005) Gene structure, multiple alternative splicing, and expression in gonads of zebrafish Dmrt1. Biochem Bioph Res Co 330:950–957

16 Haines B, Hughes J, Corbett M, Shaw M, Innes J, Patel L, Gecz J, Clayton-Smith J, Thomas P (2015) Interchromosomal insertional translocation at Xq26.3 alters SOX3 expression in an individual with XX male sex reversal. J Clin Endocr Metab 100:E815–E820

17 Hou Y, Yuan J, Zhou X, Fu XZ, Cheng HH, Zhou RJ (2012) DNA demethylation and USF regulate the meiosis-specific expression of the mouse miwi. PLoS Genet 8:e1002716

18 Hsu SY, Lai RJ, Finegold M, Hsueh AJ (1996) Targeted overexpression of Bcl-2 in ovaries of transgenic mice leads to decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis. Endocrinology 137:4837–4843

19 Hutt KJ (2015) The role of BH3-only proteins in apoptosis within the ovary. Reproduction 149:R81–R89

20 Jelsig AM, Diness BR, Kreiborg S, Main KM, Larsen VA, Hove H (2018) A complex phenotype in a family with a pathogenic SOX3 missense variant. Eur J Med Genet 61:168–172

21 Jeng SR, Wu GC, Yueh WS, Kuo SF, Dufour S, Chang CF (2018) Gonadal development and expression of sex-specific genes during sex differentiation in the Japanese eel. Gen Comp Endocrinol 257:74–85

22 Jones RL, Pepling ME (2013) Role of the antiapoptotic proteins BCL2 and MCL1 in the neonatal mouse ovary. Biol Reprod 88:1–8

23 Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T (2008) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36:D480–D484

24 Kim D, Landmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360

25 Kitano T, Takamune K, Nagahama Y, Abe SI (2000) Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus). Mol Reprod Dev 56:1–5

26 Kobayashi T, Kajiura-Kobayashi H, Nagahama Y (2003) Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia. Cytogenet Genome Res 101:289–294

27 Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R (1991) Male development of chromosomally female mice transgenic for Sry. Nature 351:117–121

28 Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25

29 Lau ESW, Zhang ZW, Qin MM, 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. Sci Rep 6:37357

30 Laumonnier F, Ronce N, Hamel BCJ, Thomas P, Lespinasse J, Raynaud M, Paringaux C, van Bokhoven H, Kalscheuer V, Fryns JP (2002) Transcription factor SOX3 is involved in X-linked mental retardation with growth hormone deficiency. Am J Hum Genet 71:1450–1455

31 Leerberg DM, Sano K, Draper BW (2017) Fibroblast growth factor signaling is required for early somatic gonad development in zebrafish. PLoS Genet 13:e1006993

32 Li B, Dewey CN (2011) RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform 12:323

33 Li EB, Truong D, Hallett SA, Mukherjee K, Schutte BC, Liao EC (2017) Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model. PLoS Genet 13:e1007009

34 Liew WC, Bartfai R, Lim Z, Sreenivasan R, Siegfried KR, Orban L (2012) Polygenic sex determination system in zebrafish. PLoS ONE 7:e34397

35 Lin Q, Mei J, Li Z, Zhang X, Zhou L, Gui JF (2017) Distinct and cooperative roles of amh and dmrt1 in self-renewal and differentiation of male germ cells in zebrafish. Genetics 207:1007–1022

36 Liu D, Wang Z, Xiao A, Zhang Y, Li W, Zu Y, Yao S, Lin S, Zhang B (2014a) Efficient gene targeting in zebrafish mediated by a zebrafish-codon-optimized Cas9 and evaluation of off-targeting effect. J Genet Genom 41:43–46

37 Liu J, Yao W, Yao Y, Du X, Zhou J, Ma B, Liu H, Li Q, Pan Z (2014b) MiR-92a inhibits porcine ovarian granulosa cell apoptosis by targeting Smad7 gene. FEBS Lett 588:4497–4503

38 Matsuda F, Inoue N, Manabe N, Ohkura S (2012) Follicular growth and atresia in mammalian ovaries: regulation by survival and death of granulosa cells. J Reprod Dev 58:44–50

39 Michael MD, Kilgore MW, Morohashi K, Simpson ER (1995) Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. J Biol Chem 270:13561–13566

40 Miller WL (2017) Steroidogenesis: unanswered questions. Trends Endocrinol Metab 28:771–793

41 Miura C, Higashino T, Miura T (2007) A progestin and an estrogen regulate early stages of oogenesis in fish. Biol Reprod 77:822–828

42 Moalem S, Babul-Hirji R, Stavropolous DJ, Wherrett D, Bagli DJ, Thomas P, Chitayat D (2012) XX male sex reversal with genital abnormalities associated with a de novo SOX3 gene duplication. Am J Med Genet Part A 158A:1759–1764

43 Nagahama Y (1997) 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: mechanisms of synthesis and action. Steroids 62:190–196

44 Okuda Y, Ogura E, Kondoh H, Kamachi Y (2010) B1 SOX coordinate cell specification with patterning and morphogenesis in the early zebrafish embryo. PLoS Genet 6:e1000936

45 Orban L, Sreenivasan R, Olsson PE (2009) Long and winding roads: testis differentiation in zebrafish. Mol Cell Endocrinol 312:35–41

46 Oshima Y, Naruse K, Nakamura Y, Nakamura M (2009) Sox3: a transcription factor for Cyp19 expression in the frog Rana rugosa. Gene 445:38–48

47 Pannetier M, Fabre S, Batista F, Kocer A, Renault L, Jolivet G, Mandon-Pepin B, Cotinot C, Veitia R, Pailhoux E (2006) FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development. J Mol Endocrinol 36:399–413

48 Pennisi D, Bowles J, Nagy A, Muscat G, Koopman P (2000a) Mice null for sox18 are viable and display a mild coat defect. Mol Cell Biol 20:9331–9336

49 Pennisi D, Gardner J, Chambers D, Hosking B, Peters J, Muscat G, Abbott C, Koopman P (2000b) Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice. Nat Genet 24:434–437

50 Perez GI, Robles R, Knudson CM, Flaws JA, Korsmeyer SJ, Tilly JL (1999) Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency. Nat Genet 21:200–203

51 Peters MJ, Parker SK, Grim J, Allard CAH, Levin J, Detrich HW3rd (2018) Divergent Hemogen genes of teleosts and mammals share conserved roles in erythropoiesis: analysis using transgenic and mutant zebrafish. Biol Open 7:bio035576

52 Popp MW, Maquat LE (2016) Leveraging rules of nonsensemediated mRNA decay for genome engineering and personalized medicine. Cell 165:1319–1322

53 Qiu M, Liu J, Han C, Wu B, Yang Z, Su F, Quan F, Zhang Y (2014) The influence of ovarian stromal/theca cells during in vitro culture on steroidogenesis, proliferation and apoptosis of granulosa cells derived from the goat ovary. Reprod Domest Anim 49:170–176

54 Quirk SM, Cowan RG, Harman RM (2006) The susceptibility of granulosa cells to apoptosis is influenced by oestradiol and the cell cycle. J Endocrinol 189:441–453

55 Ratts VS, Flaws JA, Kolp R, Sorenson CM, Tilly JL (1995) Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology 136:3665–3668

56 Regan SLP, Knight PG, Yovich JL, Leung Y, Arfuso F, Dharmarajan A (2018) Granulosa cell apoptosis in the ovarian follicle-a changing view. Front Endocrinol 9:Article 61

57 Rizzoti K, Brunelli S, Carmignac D, Thomas PQ, Robinson IC, Lovell-Badge R (2004) SOX3 is required during the formation of the hypothalamo-pituitary axis. Nat Genet 36:247–255

58 Sadraie SH, Saito H, Kaneko T, Saito T, Hiroi M (2000) Effects of aging on ovarian fecundity in terms of the incidence of apoptotic granulosa cells. J Assist Reprod Genet 17:168–173

59 Santos JM, Menezes VG, Barberino RS, Macedo TJ, Lins TL, Gouveia BB, Barros VR, Santos LP, Goncalves RJ, Matos MH (2014) Immunohistochemical localization of fibroblast growth factor-2 in the sheep ovary and its effects on pre-antral follicle apoptosis and development in vitro. Reprod Domest Anim 49:522–528

60 Shen M, Liu Z, Li B, Teng Y, Zhang J, Tang Y, Sun SC, Liu H (2014) Involvement of FoxO1 in the effects of follicle-stimulating hormone on inhibition of apoptosis in mouse granulosa cells. Cell Death Dis 5:e1475

61 Sifer C, Benifla JL, Bringuier AF, Porcher R, Blanc-Layrac G, Madelenat P, Feldmann G (2002) Could induced apoptosis of human granulosa cells predict in vitro fertilization-embryo transfer outcome? A preliminary study of 25 women. Eur J Obstet Gyn Reprod Biol 103:150–153

62 Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN (1990) A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346:240–244

63 Su YQ, Wu XM, O’Brien MJ, Pendola FL, Denegre JN, Matzuk MM, Eppig JJ (2004) Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Dev Biol 276:64–73

64 Su YQ, Sugiura K, Eppig JJ (2009) Mouse oocyte control of granulosa cell development and function: paracrine regulation of cumulus cell metabolism. Semin Reprod Med 27:32–42

65 Sun D, Zhang Y, Wang C, Hua X, Zhang XA, Yan J (2013) Sox9-related signaling controls zebrafish juvenile ovary-testis transformation. Cell Death Dis 4:e930

66 Sutton E, Hughes J, White S, Sekido R, Tan J, Arboleda V, Rogers N, Knower K, Rowley L, Eyre H (2011) Identification of SOX3 as an XX male sex reversal gene in mice and humans. J Clin Investig 121:328–341

67 Takahashi H (1977) Juvenile hermaphroditism in the zebrafish, Brachydanio rerio. Bull Fac Fish Hokkaido Univ 28:57–65

68 Takehana Y, Matsuda M, Myosho T, Suster ML, Kawakami K, Shin IT, Kohara Y, Kuroki Y, Toyoda A, Fujiyama A (2014) Cooption of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena. Nat Commun 5:4157

69 Thome RG, Domingos FF, Santos HB, Martinelli PM, Sato Y, Rizzo E, Bazzoli N (2012) Apoptosis, cell proliferation and vitellogenesis during the folliculogenesis and follicular growth in teleost fish. Tissue Cell 44:54–62

70 Uchida D, Yamashita M, Kitano T, Iguchi T (2002) Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. J Exp Biol 205:711–718

71 Uchida D, Yamashita M, Kitano T, Iguchi T (2004) An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal. Comp Biochem Phys Part A 137:11–20

72 Wang DS, Kobayashi T, Zhou LY, Paul-Prasanth B, Ijiri S, Sakai F, Okubo K, Morohashi K, Nagahama Y (2007a) 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 1. Mol Endocrinol 21:712–725

73 Wang XG, Bartfai R, Sleptsova-Freidrich I, Orban L (2007b) The timing and extent of ‘juvenile ovary’ phase are highly variable during zebrafish testis differentiation. J Fish Biol 70:33–44

74 Wang DS, Zhou LY, 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 151:1331–1340

75 Watanabe M, Tanaka M, Kobayashi D, Yoshiura Y, Oba Y, Nagahama Y (1999) Medaka (Oryzias latipes) FTZ-F1 potentially regulates the transcription of P-450 aromatase in ovarian follicles: cDNA cloning and functional characterization. Mol Cell Endocrinol 149:221–228

76 Webster KA, Schach U, Ordaz A, Steinfeld JS, Draper BW, Siegfried KR (2017) Dmrt1 is necessary for male sexual development in zebrafish. Dev Biol 422:33–46

77 Weiss J, Meeks JJ, Hurley L, Raverot G, Frassetto A, Jameson JL (2003) Sox3 is required for gonadal function, but not sex determination, in males and females. Mol Cell Biol 23:8084–8091

78 Wilson CA, High SK, McCluskey BM, Amores A, Yan YL, Titus TA, Anderson JL, Batzel P, Carvan MJ, Schartl M (2014) Wild sex in zebrafish: loss of the natural sex determinant in domesticated strains. Genetics 198:1291–1308

79 Xia Y, Papalopulu N, Vogt PK, Li J (2000) The oncogenic potential of the high mobility group box protein Sox3. Cancer Res 60:6303–6306

80 Xia H, Zhong CR, Wu XX, Chen J, Tao BB, Xia XQ, Shi MJ, Zhu ZY, Trudeau VL, Hu W (2018) Mettl3 mutation disrupts gamete maturation and reduces fertility in zebrafish. Genetics 208:729–743

81 Yan Q, Wang F, Miao Y, Wu X, Bai M, Xi X, Feng Y (2016) Sexdetermining region Y-box3 (SOX3) functions as an oncogene in promoting epithelial ovarian cancer by targeting src kinase. Tumor Biol 37:12263–12271

82 Yang YJ, Wang Y, Li Z, Zhou L, Gui JF (2017) Sequential, divergent, and cooperative requirements of Foxl2a and Foxl2b in ovary development and maintenance of zebrafish. Genetics 205:1551–1572

83 Yao B, Zhou L, Wang Y, Xia W, Gui JF (2007) Differential expression and dynamic changes of SOX3 during gametogenesis and sex reversal in protogynous hermaphroditic fish. J Exp Zool Part A Ecol Genet Physiol 307:207–219

84 Ye J, Fang L, Zheng HK, Zhang Y, Chen J, Zhang ZJ, Wang J, Li ST, Li RQ, Bolund L (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:W293–W297

85 Yin YK, Tang HP, Liu Y, Chen Y, Li GF, Liu XC, Lin HR (2017) Targeted disruption of aromatase reveals dual functions of cyp19a1a during sex differentiation in zebrafish. Endocrinology 158:3030–3041

86 Yuan J, Zhang Y, Sheng Y, Fu XZ, Cheng HH, Zhou RJ (2015) MYBL2 guides autophagy suppressor VDAC2 in the developing ovary to inhibit autophagy through a complex of VDAC2-BECN1-BCL2L1 in mammals. Autophagy 11:1081–1098