Effect of exogenous hormones on R-spondin 1 (RSPO1) gene expression and embryo development in Pelodiscus sinensis
Hongwei Liang A B D , Yan Meng
B , Lihuan Cao B , Xiang Li C and Guiwei Zou B D
+ Author Affiliations
- Author Affiliations
A Key Laboratory of Aquatic Genomics, Ministry of Agriculture, Yangtze River Fisheries Research Institute, Chinese Academy of Fisheries Science, Wuhan, Hubei 430223, China.
B Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, China.
C Anhui Xijia Agricultural Development Co. Ltd, Bengbu, Anhui 233700, China.
D Corresponding authors. Emails: lianghw@yfi.ac.cn; zougw@yfi.ac.cn
Reproduction, Fertility and Development 31(9) 1425-1433 https://doi.org/10.1071/RD19045
Submitted: 9 May 2018 Accepted: 24 February 2019 Published: 1 May 2019
Abstract
Little is known about sex determination and differentiation in the Chinese soft-shelled turtle Pelodiscus sinensis. R-Spondin 1 (RSPO1), a candidate sex-determining gene, is an important regulator of ovarian differentiation in animals. Exogenous drugs can affect sex differentiation. In this study we cloned the RSPO1 gene from P. sinensis (psRSPO1) and analysed its expression profile. The psRSPO1 gene exhibited sequence identity with RSPO1 genes from other species. RSPO1 protein-based phylogenetic analysis showed that psRSPO1 in P. sinensis is closely related to RSPO1 proteins from other turtles. psRSPO1 showed abundant expression in adult brain and gonads, with higher levels in females than males. We also evaluated the effects of three finaconcentration of 2.5, 5.0 and 10 mg mL−1 exogenous oestradiol (E2) and aromatase inhibitor (letrozole) on the expression of psRSPO1, external embryo morphology, growth status of embryos and the sex ratio when the drugs were injected to eggs during incubation. The expression of psRSPO1 was upregulated and downregulated by exogenous oestradiol and letrozole respectively, despite inconsistent expression trends at different embryo development times. External embryo morphology, growth status and sex ratio were affected by both exogenous oestradiol and the aromatase inhibitor. Feminisation was induced by oestradiol, but inhibited by letrozole. These results will contribute to studies of the potential molecular mechanisms underlying sex differentiation and sex control in the Chinese soft-shelled turtle.
Additional keywords: aromatase inhibitor, Chinese soft-shelled turtle, exogenous oestradiol, sex reversal.
References
Barlian, A., Vanawati, N., and Ayuningtyas, F. D. (2015). The patterns of sex determination and differentiation genes in green sea turtle (Chelonia mydas). J. Biol. Res. (Thessalon.) 20, 15–20.| The patterns of sex determination and differentiation genes in green sea turtle (Chelonia mydas).Crossref | GoogleScholarGoogle Scholar |
Barske, L. A., and Capel, B. (2010). Estrogen represses SOX9 during sex determination in the red-eared slider turtle Trachemys scripta. Dev. Biol. 341, 305–314.
| Estrogen represses SOX9 during sex determination in the red-eared slider turtle Trachemys scripta.Crossref | GoogleScholarGoogle Scholar | 20153744PubMed |
Cai, C., Yu, Q. C., Jiang, W. M., Liu, W., Song, W., Yu, H., Zhang, L., Yang, Y., and Zeng, Y. A. (2014). R-spondin1 is a novel hormone mediator for mammary stem cell self-renewal. Genes Dev. 28, 2205–2218.
| R-spondin1 is a novel hormone mediator for mammary stem cell self-renewal.Crossref | GoogleScholarGoogle Scholar | 25260709PubMed |
Chassot, A. A., Ranc, F., Gregoire, E. P., Roepers-Gajadien, H. L., Taketo, M. M., Camerino, G., de Rooij, D. G., Schedl, A., and Chaboissier, M. C. (2008). Activation of beta-catenin signaling by rspo1 controls differentiation of the mammalian ovary. Hum. Mol. Genet. 17, 1264–1277.
| Activation of beta-catenin signaling by rspo1 controls differentiation of the mammalian ovary.Crossref | GoogleScholarGoogle Scholar | 18250098PubMed |
Cheek, A., Brouwer, T. H., Carroll, S., Manning, S., Brouwer, M., and McLachlan, J. A. (2001). Developmental exposure to anthracene and estradiol alters reproductive success in medaka (Oryzias latipes). Environ. Sci. 8, 31–45.
Chikae, M., Ikeda, R., Hasan, Q., Morita, Y., and Tamiya, E. (2004). Effects of tamoxifen, 17α-ethynylestradiol, flutamide, and methyltestosterone on plasma vitellogenin levels of male and female Japanese medaka (Oryzias latipes). Environ. Toxicol. Pharmacol. 17, 29–33.
| Effects of tamoxifen, 17α-ethynylestradiol, flutamide, and methyltestosterone on plasma vitellogenin levels of male and female Japanese medaka (Oryzias latipes).Crossref | GoogleScholarGoogle Scholar | 21782710PubMed |
Devlin, R. H., and Nagahama, Y. (2002). Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208, 191–364.
| Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences.Crossref | GoogleScholarGoogle Scholar |
Ezaz, T., Azad, B., O’Meally, D., Young, M. J., Matsubara, K., Edwards, M. J., Zhang, X. W., Holleley, C. E., Deakin, J. E., Graves, J. A. M., Georges, A., Edwards, S. V., and Sarre, S. D. (2013). Sequence and gene content of a large fragment of a lizard sex chromosome and evaluation of candidate sex differentiating gene R-spondin 1. BMC Genomics 14, 899.
| Sequence and gene content of a large fragment of a lizard sex chromosome and evaluation of candidate sex differentiating gene R-spondin 1.Crossref | GoogleScholarGoogle Scholar | 24344927PubMed |
Fang, M. D., Zhang, Y. M., and Wang, X. J. (2015). Rspo1 function on sex differentiation of female vertebrates. Chin. J. Biochem. Mol. Biol. 31, 801–805.
Flament, S., Chardard, D., Chesnel, A., and Dumond, H. (2011). Sex determination and sex differentiation in amphibians. In ‘Hormones and Reproduction of Vertebrates, Amphibians’. Vol. 2. (Eds D. O. Norris and K. H. Lopez.) pp. 1–19. (Academic Press: London.)
Fritz, U., Gong, S. P., Auer, M., Kuchling, G., Schneeweiß, N., and Hundsdörfer, A. K. (2010). The world’s economically most important chelonians represent a diverse species complex (Testudines: Trionychidae: Pelodiscus). Org. Divers. Evol. 10, 227–242.
| The world’s economically most important chelonians represent a diverse species complex (Testudines: Trionychidae: Pelodiscus).Crossref | GoogleScholarGoogle Scholar |
Gong, S., Vamberger, M., Auer, M., Praschag, P., and Fritz, U. (2018). Millennium-old farm breeding of chinese softshell turtles (pelodiscus spp.) results in massive erosion of biodiversity. Naturwissenschaften 105, 34.
| Millennium-old farm breeding of chinese softshell turtles (pelodiscus spp.) results in massive erosion of biodiversity.Crossref | GoogleScholarGoogle Scholar | 29728774PubMed |
Han, Y. Q., Geng, J., Shi, H. T., Zhang, X. M., Du, L. L., Liu, F. T., Li, M. M., Wang, X. T., Wang, Y. Y., and Yang, G. Y. (2015). Molecular cloning and tissue distribution profiles of the chicken R-spondin1 gene. Genet. Mol. Res. 14, 3090–3097.
| Molecular cloning and tissue distribution profiles of the chicken R-spondin1 gene.Crossref | GoogleScholarGoogle Scholar | 25966073PubMed |
Hu, Q., Meng, Y., Tian, H., Zhang, Y., and Xiao, H. (2016). Sexually dimorphic expression of Foxl2 and Ftz-F1 in Chinese giant salamander Andrias davidianus. J. Exp. Zool. B Mol. Dev. Evol. 326, 363–374.
| Sexually dimorphic expression of Foxl2 and Ftz-F1 in Chinese giant salamander Andrias davidianus.Crossref | GoogleScholarGoogle Scholar | 27527384PubMed |
Ji, X., Chen, F., Du, W. G., and Chen, H. L. (2003). Incubation temperature affects hatchling growth but not sexual phenotype in the Chinese soft-shelled turtle, Pelodiscus sinensis (Trionychidae). J. Zool., Lond. 261, 409–416.
| Incubation temperature affects hatchling growth but not sexual phenotype in the Chinese soft-shelled turtle, Pelodiscus sinensis (Trionychidae).Crossref | GoogleScholarGoogle Scholar |
Kazanskaya, O., Glinka, A., del Barco Barrantes, I., Stannek, P., Christof Niehrs, C., and Wu, W. (2004). R-Spondin2 is a secreted activator of Wnt/β-catenin signaling and is required for Xenopus myogenesis. Dev. Cell 7, 525–534.
| R-Spondin2 is a secreted activator of Wnt/β-catenin signaling and is required for Xenopus myogenesis.Crossref | GoogleScholarGoogle Scholar | 15469841PubMed |
Kim, K. A., Zhao, J., Andarmani, S., Kakitani, M., Oshima, T., Binnerts, M. E., Abo, A., Tomizuka, K., and Funk, W. (2006). R-spondin proteins: a novel link to β-catenin activation. Cell Cycle 5, 23–26.
| R-spondin proteins: a novel link to β-catenin activation.Crossref | GoogleScholarGoogle Scholar | 16357527PubMed |
Kim, K. A., Wagle, M., Tran, K., Zhan, X. M., Dixon, M., Liu, S. C., Gros, D., Korver, W., Yonkovich, S., Tomasevic, N., Binnerts, M. E., and Abo, A. (2008). R-Spondin family members regulate the Wnt pathway by a common mechanism. Mol. Biol. Cell 19, 2588–2596.
| R-Spondin family members regulate the Wnt pathway by a common mechanism.Crossref | GoogleScholarGoogle Scholar | 18400942PubMed |
Kocer, A., Pinheiro, I., Pannetier, M., Renault, L., Parma, P., Radi, O., Kim, K. A., Camerino, G., and Pailhoux, E. (2008). R-spondin1 and FOXL2 act into two distinct cellular types during goat ovarian differentiation. BMC Dev. Biol. 8, 36.
| R-spondin1 and FOXL2 act into two distinct cellular types during goat ovarian differentiation.Crossref | GoogleScholarGoogle Scholar | 18384673PubMed |
Koopman, P. (1995). The molecular biology of SRY and its role in sex determination in mammals. Reprod. Fertil. Dev. 7, 713–722.
| The molecular biology of SRY and its role in sex determination in mammals.Crossref | GoogleScholarGoogle Scholar | 8711208PubMed |
Lau, W. B. D., Berend, S., and Clevers, H. C. (2012). The R-spondin protein family. Genome Biol. 13, 242.
| The R-spondin protein family.Crossref | GoogleScholarGoogle Scholar |
Li, H. J. (2012). Study on the differentiation of gonad and the effects of temperatures on it in Trionyx sinensis. Master Dissertation (M. Sc), Hebei University, Baoding.
Li, G. L., Deng, S. P., Sun, J., Wang, W. D., Shi, S. L., and Zhu, C. H. (2013). Effects of aromatase inhibitor letrozole on sex differentiation and related gene expression in Clarias fuscus. J. Fish. Sci. Chin 20, 911–917.
Liu, J., Liu, T., Niu, J., Wu, X., Zhai, J., Zhang, Q., and Qi, J. (2018). Expression pattern and functional analysis of R-spondin1 in tongue sole Cynoglossus semilaevis. Gene 642, 453–460.
| Expression pattern and functional analysis of R-spondin1 in tongue sole Cynoglossus semilaevis.Crossref | GoogleScholarGoogle Scholar | 29155330PubMed |
Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25, 402–408.
| Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method.Crossref | GoogleScholarGoogle Scholar | 11846609PubMed |
Matsumoto, Y., and Crews, D. (2012). Molecular mechanisms of temperature-dependent sex determination in the context of ecological developmental biology. Mol. Cell. Endocrinol. 354, 103–110.
| Molecular mechanisms of temperature-dependent sex determination in the context of ecological developmental biology.Crossref | GoogleScholarGoogle Scholar | 22037450PubMed |
Matsumoto, Y., Yatsu, R., Taylor, C., and Crews, D. (2013). Changes in gonadal gene network by exogenous ligands in temperature-dependent sex determination. J. Mol. Endocrinol. 50, 389–400.
| Changes in gonadal gene network by exogenous ligands in temperature-dependent sex determination.Crossref | GoogleScholarGoogle Scholar | 23532621PubMed |
Nagahama, Y. (2005). Molecular mechanisms of sex determination and gonadal sex differentiation in fish. Fish Physiol. Biochem. 31, 105–109.
| Molecular mechanisms of sex determination and gonadal sex differentiation in fish.Crossref | GoogleScholarGoogle Scholar | 20035442PubMed |
Nam, J. S., Turcotte, T. J., Smith, P. F., Choi, S., and Yoon, J. K. (2006). Mouse Cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate-catenin-dependent gene expression. J. Biol. Chem. 281, 13247–13257.
| Mouse Cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate-catenin-dependent gene expression.Crossref | GoogleScholarGoogle Scholar | 16543246PubMed |
Ortega, I., Sokalska, A., Villanueva, J. A., Cress, A. B., Wong, D. H., Stener-Victorin, E., Stanley, S. D., and Duleba, A. J. (2013). Letrozole increases ovarian growth and Cyp17a1 gene expression in the rat ovary. Fertil. Steril. 99, 889–896.
| Letrozole increases ovarian growth and Cyp17a1 gene expression in the rat ovary.Crossref | GoogleScholarGoogle Scholar | 23200686PubMed |
Pannetier, M., Chassot, A. A., Chaboissier, M. C., and Pailhouxa, E. (2016). Involvement of FOXL2 and RSPO1 in ovarian determination, development, and maintenance in mammals. Sex Dev. 10, 167–184.
| Involvement of FOXL2 and RSPO1 in ovarian determination, development, and maintenance in mammals.Crossref | GoogleScholarGoogle Scholar | 27649556PubMed |
Parma, P., Radi, O., Vidal, V., Chaboissier, M. C., Dellambra, E., Valentini, S., and Camerino, G. (2006). R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat. Genet. 38, 1304–1309.
| R-spondin1 is essential in sex determination, skin differentiation and malignancy.Crossref | GoogleScholarGoogle Scholar | 17041600PubMed |
Phuge, S. K., and Gramapurohit, N. P. (2015). Sex hormones alter sex ratios in the Indian skipper frog, Euphlyctis cyanophlyctis: determining sensitive stages for gonadal sex reversal. Gen. Comp. Endocrinol. 220, 70–77.
| Sex hormones alter sex ratios in the Indian skipper frog, Euphlyctis cyanophlyctis: determining sensitive stages for gonadal sex reversal.Crossref | GoogleScholarGoogle Scholar | 24815042PubMed |
Pinto, P. I. S., Teodósio, H. R., Galay-Burgos, M., Power, D. M., Sweeney, G. E., and Canário, A. V. M. (2006). Identification of estrogen-responsive genes in the testis of sea bream (Sparus auratus) using suppression subtractive hybridization. Mol. Reprod. Dev. 73, 318–329.
| Identification of estrogen-responsive genes in the testis of sea bream (Sparus auratus) using suppression subtractive hybridization.Crossref | GoogleScholarGoogle Scholar |
Piprek, R. P., Pecio, A., Laskowska-Kaszub, K., Kubiak, J. Z., and Szymura, J. M. (2013). Sexual dimorphism of AMH, DMRT1 and RSPO1 localization in the developing gonads of six anuran species. Int. J. Dev. Biol. 57, 891–895.
| Sexual dimorphism of AMH, DMRT1 and RSPO1 localization in the developing gonads of six anuran species.Crossref | GoogleScholarGoogle Scholar | 24623081PubMed |
Shoemaker, C. M., and Crews, D. (2009). Analyzing the coordinated gene network underlying temperature-dependent sex determination in reptiles. Semin. Cell Dev. Biol. 20, 293–303.
| Analyzing the coordinated gene network underlying temperature-dependent sex determination in reptiles.Crossref | GoogleScholarGoogle Scholar | 19022389PubMed |
Smith, C. A., Shoemaker, C. M., Roeszler, K. N., Queen, J., Crews, D., and Sinclair, A. H. (2008). Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development. BMC Dev. Biol. 8, 72.
| Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development.Crossref | GoogleScholarGoogle Scholar | 18651984PubMed |
Sun, X. Z. (2009). Study on the relationship between sex differentiation and incubation temperature of Chinese softshell turtles. Master Dissertation (M. Sc), Shaanxi Normal University, Yangling.
Tang, Y. (2015). Study on the gender identity of Trionyx sinensis and the impact of exogenous hormones. Master Dissertation (M. Sc), Southwest University, Chongqing.
Tomaselli, S., Megiorni, F., Lin, L., Mazzilli, M. C., Gerrelli, D., Majore, S., Grammatico, P., and Achermann, J. C. (2011). Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling. PLoS One 6, e16366.
| Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling.Crossref | GoogleScholarGoogle Scholar | 21297984PubMed |
Tomizuka, K., Horikoshi, K., Kitada, R., Sugawara, Y., Iba, Y., Kojima, A., Yoshitome, A., Yamawaki, K., Amagai, M., Inoue, A., Oshima, T., and Kakitani, M. (2008). R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling. Hum. Mol. Genet. 17, 1278–1291.
| R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling.Crossref | GoogleScholarGoogle Scholar | 18250097PubMed |
Wang, Q., Guo, Z. L., Li, Y. S., Yun, D., Huang, B., and Zhou, Z. L. (2012). Effects of letrozole on gonadal differentiation and related gene expression in gonad of Takifugu obscurus. Dongwuxue Zazhi 5, 16–23.
Wu, L., Yang, P., Luo, F., Wang, D., and Zhou, L. (2016). R-spondin1 signaling pathway is required for both the ovarian and testicular development in a teleosts, Nile tilapia (Oreochromis niloticus). Gen. Comp. Endocrinol. 230–231, 177–185.
| R-spondin1 signaling pathway is required for both the ovarian and testicular development in a teleosts, Nile tilapia (Oreochromis niloticus).Crossref | GoogleScholarGoogle Scholar | 27044511PubMed |
Wu, Q. S., Wang, X. Q., Zeng, Y. Y., Ma, X., and He, H. Z. (2011). Influence on sexual differentiation in Trionyx sinesis by temperature. Feed Rev. 2, 42–44.
Ye, Y. Z., Huang, W. Y., and Wu, C. J. (1993). A study on induction effects of methyl testosterone on morphological traits of the Chinese softshell turtle (Trionyx sinensis). J. Huazhong Agric. Univ. 18, 72–74.
Zhang, Y., Li, F., Sun, D., Liu, J., Liu, N., and Yu, Q. (2011). Molecular analysis shows differential expression of R-spondin1 in zebrafish (Danio rerio) gonads. Mol. Biol. Rep. 38, 275–282.
| Molecular analysis shows differential expression of R-spondin1 in zebrafish (Danio rerio) gonads.Crossref | GoogleScholarGoogle Scholar | 20349143PubMed |
Zhou, L., Charkraborty, T., Yu, X., Wu, L., Liu, G., Mohapatra, S., Wang, D., and Nagahama, Y. (2012). R-spondins are involved in the ovarian differentiation in a teleost, medaka (Oryzias latipes). BMC Dev. Biol. 12, 36.
| R-spondins are involved in the ovarian differentiation in a teleost, medaka (Oryzias latipes).Crossref | GoogleScholarGoogle Scholar | 23217106PubMed |
