The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination.
References
[1]
Wibbels, T. Critical approaches to sex determination in Turtles. In The Biology of Sea Turtles; Lutz, P.L., Musik, J.A., Wineken, J., Eds.; CRC Press Inc: Boca Ratón, FL, USA, 2003; Volume 2, pp. 103–134.
[2]
Pieau, C.; Dorizzi, M. Oestrogens and temperatura-dependent sex determination in reptiles: All is in the gonads. J. Endocrinol. 2004, 181, 367–377, doi:10.1677/joe.0.1810367.
[3]
Koopman, P.; Gubbay, J.; Vivian, N.; Goodfellow, P.; Lovell-Badge, R. Male development of chromosomally female mice transgenic for Sry. Nature 1991, 351, 117–121, doi:10.1038/351117a0.
[4]
Shoemaker, C.; Crews, D. Analyzing the coordinated gene network underlying temperature-dependent sex determination in Reptiles. Semin. Cell. Dev. Biol. 2009, 20, 293–303, doi:10.1016/j.semcdb.2008.10.010.
[5]
Rhen, T.; Schroeder, A. Molecular mechanisms of sex determination in reptiles. Sex. Dev. 2010, 4, 16–28, doi:10.1159/000282495.
[6]
Chue, J.; Smith, C.A. Sex determination and sexual differentiation in the avian model. FEBS J. 2011, 278, 1027–1034, doi:10.1111/j.1742-4658.2011.08032.x.
[7]
Kent, J.; Wheatley, S.C.; Andrews, J.E.; Sinclair, A.H.; Koopman, P. A male-specific role for SOX9 in vertebrate sex determination. Development 1996, 122, 2813–2822.
[8]
Morais da Silva, S.; Hacker, A.; Harley, V.; Goodfellow, P.; Swain, A.; Lovell-Badge, R. Sox9 expression during gonadal development implies a conserved role for gene in testis differentiation in mammals and birds. Nat. Genet. 1996, 14, 62–68.
[9]
Western, P.S.; Harry, J.L.; Graves, J.A.M.; Sinclair, A.H. Temperature-dependent sex determination: Upregulation of SOX9 expression after commitment to male development. Dev. Dyn. 1999, 214, 171–177, doi:10.1002/(SICI)1097-0177(199903)214:3<171::AID-AJA1>3.0.CO;2-S.
[10]
Moreno-Mendoza, N.; Harley, V.R.; Merchant-Larios, H. Differential expression of SOX9 in gonads of the sea turtle Lepidochelys olivacea at male- or female- promoting temperatures. J. Exp. Zool. 1999, 284, 705–710, doi:10.1002/(SICI)1097-010X(19991101)284:6<705::AID-JEZ12>3.0.CO;2-1.
[11]
Shoemaker, C.M.; Queen, J.; Crews, D. Response of candidate sex-determinig genes to changes in temperature reveals their involvement in the molecular network undelying temperature-dependent sex determination. Mol. Endocrinol. 2007, 21, 2750–2763, doi:10.1210/me.2007-0263.
[12]
Shoemaker, C.; Ramsey, M.; Queen, J.; Crews, D. Expression of Sox9, Mis, and Dmrt1, in the gonad of a species with temperature-dependent sex determination. Dev. Dyn. 2007, 236, 1055–1063, doi:10.1002/dvdy.21096.
[13]
Barske, L.A.; Capel, B. Estrogen represses SOX9 during sex determination in the red-eared slider turtle Trachemys scripta. Dev. Biol. 2010, 341, 305–314, doi:10.1016/j.ydbio.2010.02.010.
[14]
Harley, V.R.; Clarkson, M.J.; Argentaro, A. The molecular action and regulation of the testis-determining factors, SRY (sex determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9]. Endocrinol. Rev. 2003, 24, 466–487, doi:10.1210/er.2002-0025.
[15]
Sim, H.; Argentaro, A.; Harley, V.R. Boys, girls and shuttling of SRY and SOX9. Trends Endocrinol. Metab. 2008, 19, 213–222, doi:10.1016/j.tem.2008.04.002.
[16]
Sekido, R.; Lovell-Badge, R. Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer. Nature 2008, 453, 930–934, doi:10.1038/nature06944.
[17]
Vidal, V.P.; Chaboissier, M.C.; de Rooji, D.G.; Schedl, A. Sox9 induces testis development in XX transgenic mice. Nat. Genet. 2001, 28, 216–217.
[18]
Torres-Maldonado, L.C.; Moreno-Mendoza, N.; Landa, A.; Merchant-Larios, H. Timing of SOX9 downregulation and female sex determination in gonads of the sea turtle Lepidochelys olivacea. J. Exp. Zool. 2001, 290, 498–503, doi:10.1002/jez.1093.
[19]
Torres-Maldonado, L.C.; Landa-Piedra, A.; Moreno-Mendoza, N.; Marmolejo-Valencia, A.; Meza-Martínez, A.; Merchant-Larios, H. Expression profiles of Dax1, Dmrt1, and Sox9 during temperature sex determination in gonads of the sea turtle Lepidochelys olivacea. Gen. Comp. Endocrinol. 2002, 129, 20–26, doi:10.1016/S0016-6480(02)00511-7.
[20]
Moreno-Mendoza, N.; Harley, V.R.; Merchant-Larios, H. Temperature regulates SOX9 expression in cultured gonads of Lepidochelys olivacea, a species with temperature sex determination. Dev. Biol. 2001, 229, 319–326, doi:10.1006/dbio.2000.9952.
[21]
Shoemaker-Daly, C.M.; Jackson, K.; Yatsu, R.; Matsumoto, Y.; Crews, D. Genetic network underlying temperature-dependent sex determination is endogenously regulated by temperature in isolated cultured Trachemys scripta gonads. Dev. Dyn. 2010, 239, 1061–1075, doi:10.1002/dvdy.22266.
[22]
De Santa Barbara, P.; Bonneaud, N.; Boizet, B. Direct interaction of SRY-related protein SOX9 and Steroidogenic factor 1 regulates transcription of the human anti-Mullerian hormone gene. Mol. Cell Biol. 1998, 18, 6653–6718.
[23]
Behringer, R.R.; Finegold, M.J.; Cate, R.L. Müllerian-inhibiting substance, function during mammalian sexual development. Cell 1994, 79, 415–425, doi:10.1016/0092-8674(94)90251-8.
[24]
Merchant-Larios, H.; Díaz-Hernández, V.; Marmolejo-Valencia, A. Gonadal morphogenesis and gene expression in reptiles with temperature-dependent sex determination. Sex. Dev. 2010, 4, 50–61, doi:10.1159/000276768.
[25]
Oreal, E.; Pieau, C.; Mattei, M.G.; Josso, N.; Picard, J.Y.; Eusébe, D.C.; Magre, S. Early expression of Amh in chicken embryonic gonads precedes testicular Sox9 expression. Dev. Dyn. 1998, 212, 522–532, doi:10.1002/(SICI)1097-0177(199808)212:4<522::AID-AJA5>3.0.CO;2-J.
[26]
Western, P.S.; Sinclair, A.H. Sex, genes and heat: Triggers of diversity. J. Exp. Zool. 2001, 290, 624–631, doi:10.1002/jez.1113.
[27]
Sifuentes-Romero, I.; Milton, L.S.; García-Gasca, A. Post-trasncriptional gene silencing by RNA interferance in non-mammalian vertebrate systems: Where do we stand? Mut. Res. 2011, 728, 158–171, doi:10.1016/j.mrrev.2011.09.001.
[28]
Davies, J.A.; Ladomery, M.; Hohenstein, P.; Michael, L.; Shafe, A.; Spraggon, L.; Hastie, N. Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation. Hum. Mol. Genet. 2004, 13, 235–246, doi:10.1093/hmg/ddh251.
[29]
Wang, N.; Zhang, P.; Guo, X.; Zhou, Z.; Sha, J. Hnrnpk, a protein differentially expressed in immature rat ovarian development, is required for normal primordial follicle assembly and development. Endocrinology 2011, 152, 1024–1035, doi:10.1210/en.2010-0797.
[30]
Nayak, G.; Prentice, H.M.; Milton, S.L. Role of neuroglobin in regulating reactive oxygen species in the brain of the anoxic-tolerant turtle Trachemys scripta. J. Neurochem. 2009, 110, 603–612, doi:10.1111/j.1471-4159.2009.06157.x.
[31]
Miller, D. Embryology of marine Turtles. In Biology of the Reptilia; Gans, C., Billet, F., Maderson, P.F.A., Eds.; Wiley: New York, NY, USA, 1985; pp. 270–328.
[32]
Díaz-Hernández, V.; Marmolejo-Valencia, A.; Harfush, M.; Merchant-Larios, H. Formation of the genital ridges is preceded by a domain of ectopic Sox9-expressing cells in Lepidochelys olivacea. Dev. Biol. 2012, 361, 156–166, doi:10.1016/j.ydbio.2011.10.001.
[33]
Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2？2ΔΔCT method. Methods 2001, 25, 402–408, doi:10.1006/meth.2001.1262.
[34]
Lance, V.A. Is regulation of Aromatase expression in reptiles the key to understanding temperature-dependent sex determination? J. Exp. Zool. 2009, 311, 314–322, doi:10.1002/jez.465.
[35]
Josso, N.; Belville, C.; di Clemente, N.; Picard, J.Y. AMH and AMH receptor defects in persistent Müllerian duct syndrome. Hum. Reprod. Update 2005, 11, 351–356, doi:10.1093/humupd/dmi014.
[36]
Desvages, G.; Pieau, C. Aromatase activity in gonads of turtle embryos as a function of the incubation temperature of eggs. J. Steroid Biochem. Mol. Biol. 1992, 41, 851–853, doi:10.1016/0960-0760(92)90437-N.
[37]
Desvages, G.; Girondot, M.; Pieau, C. Sensitive stages for the effects of temperature on gonadal aromatase activity in embryos of marine turtle Dermochelys coriacea. Gen. Comp. Endocrinol. 1993, 92, 54–61, doi:10.1006/gcen.1993.1142.
[38]
Jeyasuria, P.; Roosenburg, W.M.; Place, A.R. Role of P-450 Aromatase in sex determination of the diamondback terrapin, Malaclemys terrapin. J. Exp. Zool. 1994, 270, 95–111, doi:10.1002/jez.1402700111.
[39]
Rhen, T.; Metzger, K.; Schroeder, A.; Woodward, R. Expression of putative sex-determining genes during the thermosensitive period of gonad development in the snapping turtle, Chelydra serpentina. Sex Dev. 2007, 1, 255–270, doi:10.1159/000104775.
[40]
Ramsey, M.; Crews, D. Adrenal-kidney-gonad complex measurements may not predict gonad-specific changes in gene expression patterns during temperature-dependent sex determination in the red-eared slider turtle (Trachemys scripta elegans). J. Exp. Zool. A Ecol. Gen. Phys. 2007, 307, 463–470, doi:10.1002/jez.399.
[41]
Valenzuela, N.; Neuwald, J.L.; Literman, R. Transcriptional evolution underlying vertebrate sexual development. Dev. Dyn. 2012, 242, 307–326, doi:10.1002/dvdy.23897.
[42]
Ryan, J.; Ludbrook, L.; Wilhelm, D.; Sinclair, A.; Koopman, P.; Bernard, P.; Harley, V.R. Analysis of gene function in cultured embryonic mouse gonads using nucleofection. Sex Dev. 2011, 5, 7–15, doi:10.1159/000322162.
[43]
Veitia, R.A. FoxL2 versus Sox9: A lifelong “battle of the sexes”. BioEssays 2010, 32, 375–380, doi:10.1002/bies.200900193.
