Abstract
Activin receptor type IIB (ActRIIB) is a transmembrane serine/threonine kinase receptor which plays a pivotal role in regulating the reproduction in vertebrates including teleost. Earlier studies have documented its importance in governing gonadal maturation in higher vertebrates. However, reports on the regulation of fish reproductive system by ActRIIB gene are still limited. Here, we report the identification and characterization of ActRIIB cDNA of Labeo rohita, a commercially important fish species of the Indian subcontinent. The full-length gene encoding rohu ActRIIB was cloned and found to be of 1674 bp in length. Functional similarities were evident from evolutionary analysis across vertebrates. Real-time PCR to measure the expression of ActRIIB transcript in rohu revealed significant mRNA levels in gonads followed by non-reproductive tissues, including the brain, pituitary and muscle. With respect to different gonadal maturation stages, predominant expression of ActRIIB mRNA was observed during the pre-spawning phase of both sexes. To further delineate its role in rohu reproduction, a recombinant protein of the extracellular domain of ActRIIB (rECD-ActRIIB) was produced, and polyclonal antibody is raised against the protein for its immuno-localization studies during different gonadal maturation stages. Strong immunoreactivity was noticed in the pre-vitellogenic oocytes which decreased dramatically in the fully mature oocytes. Similarly, the strong and intense immunoreactivity was found in the spermatids and spermatocytes of the immature testis, and eventually the intensity reduced with the progression of the maturation stage. These results provide the first evidence of the presence of ActRIIB in rohu gonadal tissues. Taken together, our observations lay the groundwork for further understanding and investigating on the potential role of ActRIIB in fish reproduction system in the event of gonadal maturation.
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Data availability
The ActRIIB cDNA sequence was submitted in the GenBank with accession number KX710215.
References
Anderson RA, Cambray N, Hartley PS, McNeilly AS (2002) Expression and localization of inhibin alpha, inhibin/activin betaA and betaB and the activin type II and inhibin beta-glycan receptors in the developing human testis. Reprod 123(6):779–788. https://doi.org/10.1530/rep.0.1230779
Attisano L, Wrana JL, Cheifetz S, Massague J (1992) Novel activin receptors: distinct genes and alternative mRNA splicing generate a repertoire of serine/threonine kinase receptors. Cell 68:97–108. https://doi.org/10.1016/0092-8674(92)90209-u
Cameron VA, Nishimura ER, Mathews LS, Lewis KA, Sawchenko PE, Vale WW (1994) Hybridization histochemical localization of activin receptor subtypes in rat brain, pituitary, ovary, and testis. Endocrinology 134(2):799–808. https://doi.org/10.1210/endo.134.2.8299574
Campanella JJ, Bitincka L, Smalley J (2003) MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinform 4:29. https://doi.org/10.1186/1471-2105-4-29
Carpio Y, Acosta J, Morales R, Santisteban Y, Sanchéz A, Estrada MP (2009) Regulation of body mass growth through activin type IIB receptor in teleost fish. Gen Comp Endocrinol 160:158–167. https://doi.org/10.1016/j.ygcen.2008.11.009
Cheng FYG, Yuen CW, Ge W (2007) Evidence for the existence of a local activin–follistatin negative feedback loop in the goldfish pituitary and its regulation by activin and gonadal steroids. J Endocrinol 195:373–384. https://doi.org/10.1677/JOE-07-0265
Dijke PT, Miyazono K, Heldin CH (1996) Signaling via hetero-oligomeric complexes of type I and type II serine/threonine kinase receptors. Curr Opin Cell Biol 8:139-l45. https://doi.org/10.1016/s0955-0674(96)80058-5
DiMuccio T, Mukai ST, Clelland E, Kohli G, Cuartero M, Wu T, Peng C (2005) Cloning of a second form of activin-betaA cDNA and regulation of activin-betaA subunits and activin type II receptor mRNA expression by gonadotropin in the zebrafish ovary. Gen Comp Endocrinol 143:287–299. https://doi.org/10.1016/j.ygcen.2005.04.003
Drummond AE, Le MT, Ethier JF, Dyson M, Findlay JK (2002) Expression and localization of activin receptors, Smads, and βglycan to the postnatal rat ovary. Endocrinology 143(4):1423–1433. https://doi.org/10.1210/endo.143.4.8728
Ethier JF, Houde A, Lussier JG, Silversides DW (1994) Bovine activin receptor type II cDNA: cloning and tissue expression. Mol Cell Endocrinol 106:1–8. https://doi.org/10.1016/0303-7207(94)90179-1
Funkenstein B, Krol E, Esterin E, Kim YS (2012) Structural and functional characterizations of activin type 2B receptor (acvr2b) ortholog from the marine fish, gilthead sea bream, Sparus aurata: evidence for gene duplication of acvr2b in fish. J Mol Endocrinol 49(3):175. https://doi.org/10.1530/JME-12-0075
Garg RR, Bally-Cuif L, Lee SE, Gong Z, Ni X, Hew CL, Peng C (1999) Cloning of zebrafish activin type IIB receptor (ActRIIB) cDNA and mRNA expression of ActRIIB in embryos and adult tissues. Mol Cell Endocrinol 153(1–2):169–181. https://doi.org/10.1016/s0303-7207(99)00044-1
Goebel EJ, Corpina RA, Hinck CS, Czepnik M, Castonguay R, Grenha R, Boisvert A, Milklossy G, Fullerton PT, Matzuk MM, Idone VJ, Economides AN, Kumar R, Hink AP, Thompson TB (2019) Structural characterization of an activin class ternary receptor complex reveals a third paradigm for receptor specificity. PNAS 116(31):15505–15513. https://doi.org/10.1073/pnas.1906253116
Greenwald J, Fischer WH, Vale WW, Choe S (1999) Three-finger toxin folds for the extracellular domain of the type II activin receptor serine kinase. Nat Struct Biol 6:18–22. https://doi.org/10.1038/4887
Hall TA (1999) BioEdit: a user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–8. https://doi.org/10.14601/Phytopathol_Mediterr-14998u1.29
Huang F, Chen YG (2012) Regulation of TGF-β receptor activity. Cell Bio Sci 2:9. https://doi.org/10.1155/2014/874065
Josso N, di Clemente N (1997) Serine/threonine kinase receptors and ligands. Curr Opin Genetics Dev 7(3):371–377. https://doi.org/10.1016/s0959-437x(97)80151-7
Kingsley DM (1994) The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8(2):133–146. https://doi.org/10.1101/gad.8.2.133
Kubiczkova L, Sedlarikova L, Hajek R, Sevcikova S (2012) TGF-β–an excellent servant but a bad master. J Transl Med 10(1):1–24. https://doi.org/10.1186/1479-5876-10-183
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. https://doi.org/10.1038/227680a0
Lin SJ, Lerch TF, Cook RW, Jardetzky TS, Woodruff TK (2006) The structural basis of TGF-β, bone morphogenetic protein and activin ligand binding. Reproduction 132(2):179–190. https://doi.org/10.1530/rep.1.01072
Mathews LS (1994) Activin receptors and cellular signaling by the receptor serine kinase family. Endocr Rev 15:310–325. https://doi.org/10.1210/edrv-15-3-310
Mathews LS, Vale WW (1991) Expression cloning of an activin receptor, a predicted transmembrane serine kinase. Cell 65:973–982. https://doi.org/10.1016/0092-8674(91)90549-e
Matsuzaki T, Hanai S, Kishi H, Liu Z, Bao Y, Kikuchi A, Tsuchida K, Sugino H (2002) Regulation of endocytosis of activin type II receptors by a novel PDZ protein through Ral/Ral-binding protein 1-dependent pathway. J Biol Chem 277(21):19008–19018. https://doi.org/10.1074/jbc.M112472200
Miles DC, Wakeling SI, Stringer JM, Van den Bergen JA, Wilhelm D, Sinclair AH, Western PS (2013) Signaling through the TGF beta-activin receptors ALK4/5/7 regulates testis formation and male germ cell development. PLoS One 8(1):e54606. https://doi.org/10.1371/journal.pone.0054606
Nagaso H, Suzuki A, Tada M, Ueno N (1999) Dual specificity of activin type II receptor ActRIIb in dorso ventral patterning during zebrafish embryogenesis. Dev Growth Differ 4:119–133. https://doi.org/10.1046/j.1440-169x.1999.00418.x
Østbye TKK, Bardal T, Vegusdal A, Frang OT, Kjørsvik E, Andersen Ø (2007) Molecular cloning of the Atlantic salmon activin receptor IIB cDNA–localization of the receptor and myostatin in vivo and in vitro in muscle cells. Comp Biochem Physiol Part D Genomics Proteomics 2(2):101–111. https://doi.org/10.1016/j.cbd.2006.12.003
Otto A, Patel K (2010) Signaling and the control of skeletal muscle size. Exp Cell Res 316(18):3059–3066. https://doi.org/10.1016/j.yexcr.2010.04.009
Pangas SA, Rademaker AW, Fishman DA, Woodruff TK (2002) Localization of the activin signal transduction components in normal human ovarian follicles: implications for autocrine and paracrine signaling in the ovary. J Clin Endocrinol Metab 87(6):2644–2657. https://doi.org/10.1210/jcem.87.6.8519
Patnaik S, Mohanty M, Bit A, Sahoo L, Das S, Jayasankar P, Das P (2017) Molecular characterization of activin receptor type IIA and its expression during gonadal maturation and growth stages in rohu carp. Comp Biochem Physiol B 203:1–10. https://doi.org/10.1016/j.cbpb.2016.08.005
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45. https://doi.org/10.1093/nar/29.9.e45
Shav-Tal Y, Lapter S, Parameswaran R, Zipori D (2001) Activin receptors. Cytokine Reference. https://doi.org/10.1006/rwcy.1906
Shoji H, Tsuchida K, Kishi H, Yamakawa N, Matsuzaki T, Liu Z, Nakamura T, Sugino H (2000) Identification and characterization of a PDZ protein that interacts with activin type II receptors. J Biol Chem 275(8):5485–5492. https://doi.org/10.1074/jbc.275.8.5485
Silva JR, Van den Hurk R, Van Tol HT, Roelen BA, Figueiredo JR (2004) Gene expression and protein localization for activin-A, follistatin and activin receptors in goat ovaries. J Endocrinol 183(2):405–415. https://doi.org/10.1677/joe.1.05756
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Bio Evol 30(12):2725–2729. https://doi.org/10.1093/molbev/mst197
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76(9):4350–4354. https://doi.org/10.1073/pnas.76.9.4350
Tsuchida K, Lewis KA, Mathews LS, Vale WW (1993) Molecular characterization of rat transforming growth factor- β typeII receptor. Biochem Biophys Res Commun 191(3):790–795. https://doi.org/10.1006/bbrc.1993.1286
Tsuchida K, Nakatani M, Matsuzaki T, Yamakawa N, Liu Z, Bao Y, Arai KY, Murakami T, Takehara Y, Kurisaki A, Sugino H (2004) Novel factors in regulation of activin signaling. Mol Cell Endocrinol 225(1):1–8. https://doi.org/10.1016/j.mce.2004.02.006
Van den Hurk R, Van de Pavert SA (2001) Localization of an activin/activin receptor system in the porcine ovary. Mol Reprod Dev 60(4):463–471. https://doi.org/10.1002/mrd.1111
Walton KL, Makanji Y, Harrison CA (2012) New insights into the mechanisms of activin action and inhibition. Mol Cell Endocrinol 359(1):2–12. https://doi.org/10.1016/j.mce.2011.06.030
Wu T, Patel H, Mukai S, Melino C, Garg R, Ni X, Chang J, Peng C (2000) Activin, inhibin, and follistatin in zebrafish ovary: expression and role in oocyte maturation. Biol Reprod 62(6):1585–1592. https://doi.org/10.1095/biolreprod62.6.1585
Wu TC, Jih MH, Wang L, Wan YJ (1994) Expression of activin receptor II and IIB mRNA isoforms in mouse reproductive organs and oocytes. Mol Reprod Dev 38:9–15. https://doi.org/10.1002/mrd.1080380103
Yamashita H, Dijke PT, Franzen P, Miyazono K, Heldin CH (1994) Formation of hetero-oligomeric complexes of type I and type II receptors for transforming growth factor-beta. J Biol Chem 269(31):20172–20178
Yuen CW, Ge W (2004) Follistatin suppresses FSH-beta but increases LH-beta expression in the goldfish – evidence for an activin-mediated autocrine/paracrine system in fish pituitary. Gen Comp Endocrinol 135:108–115. https://doi.org/10.1016/j.ygcen.2003.08.012
Acknowledgements
The authors are grateful to the Department of Biotechnology, Government of India, New Delhi, and ICAR-Central Institute of Freshwater Aquaculture for financial support. The authors are thankful to Dr. K. D. Mahapatra and Dr. P. Routray, Principal Scientists, ICAR-CIFA, for generously providing a part of the experimental samples.
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PD, SD and PJ conceived the project; JNS performed animal rearing and tissue sampling; SP, MM and LS carried out the laboratory experiments; AB and PKM carried out the bioinformatics analysis; SP, LS, PD, SD and PJ wrote and reviewed the MS. All authors read and approved the manuscript.
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All handling of fish was carried out following the guidelines for control and supervision of experiments on animals by the Government of India and approved by Institutional Animal Ethics Committee (AEC) of ICAR-CIFA.
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Patnaik, S., Sahoo, L., Mohanty, M. et al. Activin receptor type IIB in rohu (Labeo rohita): molecular characterization, tissue distribution and immunohistochemical localization during different stages of gonadal maturation. Fish Physiol Biochem 47, 1353–1367 (2021). https://doi.org/10.1007/s10695-021-00973-2
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DOI: https://doi.org/10.1007/s10695-021-00973-2