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Production of fertile sperm from in vitro propagating enriched spermatogonial stem cells of farmed catfish, Clarias batrachus

Published online by Cambridge University Press:  15 July 2016

Swapnarani Nayak ,
Shajahan Ferosekhan ,
Sangram Ketan Sahoo ,
Jitendra Kumar Sundaray ,
Pallipuram Jayasankar  and
Hirak Kumar Barman
Swapnarani Nayak
Affiliation:
Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India.
Shajahan Ferosekhan
Affiliation:
Aquaculture Production and Environment Division, ICAR – Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India.
Sangram Ketan Sahoo
Affiliation:
Aquaculture Production and Environment Division, ICAR – Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India.
Jitendra Kumar Sundaray
Affiliation:
Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India.
Pallipuram Jayasankar
Affiliation:
Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India.
Hirak Kumar Barman*
Affiliation:
Fish Genetics and Biotechnology Division, ICAR – Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India. Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India.
*
All correspondence to: H. K. Barman. Fish Genetics and Biotechnology Division, ICAR – Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India. Tel: +91 6742465407; +91 6742465414. Fax: +91 6742465407. E-mail: Hirak.Barman@icar.gov.in; hkbarman68@hotmail.com
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Summary

Spermatogenesis is a highly co-ordinated and complex process. In vitro propagation of spermatogonial stem cells (SSCs) could provide an avenue in which to undertake in vivo studies of spermatogenesis. Very little information is known about the SSC biology of teleosts. In this study, collagenase-treated testicular cells of farmed catfish (Clarias batrachus, popularly known as magur) were purified by Ficoll gradient centrifugation followed by magnetic activated cell sorting using Thy1.2 (CD90.2) antibody to enrich for the spermatogonial cell population. The sorted spermatogonial cells were counted and gave ~3 × 106 cells from 6 × 106 pre-sorted cells. The purified cells were cultured in vitro for >2 months in L-15 medium containing fetal bovine serum (10%), carp serum (1%) and other supplements. Microscopic observations depicted typical morphological SSC features, bearing a larger nuclear compartment (with visible perinuclear bodies) within a thin rim of cytoplasm. Cells proliferated in vitro forming clumps/colonies. mRNA expression profiling by qPCR documented that proliferating cells were Plzf + and Pou2+, indicative of stem cells. From 60 days onwards of cultivation, the self-renewing population differentiated to produce spermatids (~6 × 107 on day 75). In vitro-produced sperm (2260 sperm/SSC) were free swimming in medium and hence motile (non-progressive) in nature. Of those, 2% were capable of fertilizing and generated healthy diploid fingerlings. Our documented evidence provides the basis for producing fertile magur sperm in vitro from cultured magur SSCs. Our established techniques of SSC propagation and in vitro sperm production together should trigger future in vivo experiments towards basic and applied biology research.

Keywords

Clarias batrachusFertile spermIn vitroMagnetic activated cell sortingSSC
Type
Research Article
Information
Zygote , Volume 24 , Issue 6 , December 2016 , pp. 814 - 824
Copyright
Copyright © Cambridge University Press 2016 

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References

Baazm, M., Abolhassani, F., Abbasi, M., Habibi Roudkenar, M., Amidi, F. & Beyer, C. (2013). An improved protocol for isolation and culturing of mouse spermatogonial stem cells. Cell Reprogram. 15, 329–36.CrossRefGoogle ScholarPubMed
Barman, H.K., Mohanta, R., Patra, S.K., Chakrapani, V., Panda, R.P., Nayak, S., Jena, S., Jayasankar, P. & Nandanpawar, P. (2015). The beta-actin gene promoter of rohu carp (Labeo rohita) drives reporter gene expressions in transgenic rohu and various cell lines, including spermatogonial stem cells. Cell. Mol. Biol. Lett. 20, 237–47.CrossRefGoogle ScholarPubMed
Buageaw, A., Sukhwani, M., Ben-Yehudah, A., Ehmcke, J., Rawe, V.Y., Pholpramool, C., Orwig, K.E. & Schlatt, S. (2005). GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes. Biol. Reprod. 73, 1011–6.CrossRefGoogle ScholarPubMed
Buom-yong, R., Orwig, K.E., Hiroshi, K., Avarbock, M.R. & Brinster, R.L. (2004). Phenotypic and functional characteristics of spermatogonial stem cells in rats. Dev. Biol. 274, 158–70.Google Scholar
Dym, M., Kokkinaki, M. & He, Z. (2009). Spermatogonial stem cells: mouse and human comparisons. Birth Defects Res. C Embryo Today 87, 27–34.CrossRefGoogle ScholarPubMed
Ebata, K.T., Zhang, X. & Nagano, M.C. (2005). Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development. Mol. Reprod. Dev. 72, 171–81.CrossRefGoogle ScholarPubMed
Feng, L.X., Chen, Y., Dettin, L., Reijo-Pera, R.A., Herr, J.C., Goldberg, E. & Dym, M. (2002). Generation and in vitro differentiation of a spermatogonial cell line. Science 297, 392–5.CrossRefGoogle ScholarPubMed
Gassei, K., Ehmcke, J. & Schlatt, S. (2009). Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting. Cell Tissue Res. 337, 177–83.CrossRefGoogle ScholarPubMed
He, Z., Kokkinaki, M., Jiang, J., Dobrinski, I. & Dym, M. (2010). Isolation, characterization, and culture of human spermatogonia. Biol. Reprod. 82, 363–72.CrossRefGoogle ScholarPubMed
Hermann, B.P., Sukhwani, M., Simorangkir, D.R., Chu, T., Plant, T.M. & Orwig, K.E. (2009). Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques. Hum. Reprod. 24, 1704–16.CrossRefGoogle ScholarPubMed
Hofmann, M.C., Hess, R.A., Goldberg, E. & Millan, J.L. (1994). Immortalized germ cells undergo meiosis in vitro . Proc. Natl. Acad. Sci. USA 91, 5533–7.CrossRefGoogle ScholarPubMed
Hofmann, M.C., Braydich-Stolle, L. & Dym, M. (2005). Isolation of male germline stem cells; influence of GDNF. Dev. Biol. 279, 114–24.CrossRefGoogle ScholarPubMed
Hong, Y., Liu, T., Zhao, H., Xu, H., Wang, W., Liu, R., Chen, T., Deng, J. & Gui, J. (2004). Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro . Proc. Natl. Acad. Sci. USA 101, 8011–6.CrossRefGoogle ScholarPubMed
Hua, J., Zhu, H., Pan, S., Liu, C., Sun, J., Ma, X., Dong, W., Liu, W. & Li, W. (2011). Pluripotent male germline stem cells from goat fetal testis and their survival in mouse testis. Cell. Reprogramming 13, 133–44.CrossRefGoogle ScholarPubMed
Ida, H., Murofushi, M., Fujiwara, S. & Fujino, K. (1978). Preparation of fish chromosomes by in vitro colchicine treatment. Jpn J. Ichthyol. 24, 281–4.Google Scholar
Izadyar, F., Ouden, K., Creemers, L.B., Posthuma, G., Parvinen, M. & Rooij, D.G. (2003). Proliferation and differentiation of bovine type a spermatogonia during long-term culture. Biol. Reprod. 68, 272–81.CrossRefGoogle ScholarPubMed
Kala, S., Kaushik, R., Singh, K.P., Kadam, P.H., Singh, M.K., Manik, R.S., Singla, S.K., Palta, P. & Chauhan, M.S. (2012). In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell. J. Assist. Reprod. Genet. 29, 1335–42.CrossRefGoogle ScholarPubMed
Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., Miki, H., Ogura, A., Toyokuni, S. & Shinohara, T. (2003). Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69, 612–6.CrossRefGoogle ScholarPubMed
Kanatsu-Shinohara, M., Toyokuni, S. & Shinohara, T. (2004). CD9 is a surface marker on mouse and rat male germline stem cells. Biol. Reprod. 70, 70–5.CrossRefGoogle ScholarPubMed
Kaucher, A.V., Oatley, M.J. & Oatley, J.M. (2012). NEUROG3 is a critical downstream effector for STAT3-regulated differentiation of mammalian stem and progenitor spermatogonia 1. Biol. Reprod. 86, 1–11.CrossRefGoogle Scholar
Kawasaki, T., Siegfried, K.R. & Sakai, N. (2016). Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture. Development 143, 566–74.Google ScholarPubMed
Kossack, N., Meneses, J., Shefi, S., Nguyen, H.N., Chavez, S., Nicholas, C., Gromoll, J., Turek, P.J. & Reijo-Pera, R.A. (2009). Isolation and characterization of pluripotent human spermatogonial stem cell-derived cells. Stem Cells 27, 138–49.CrossRefGoogle ScholarPubMed
Kubota, H., Avarbock, M.R. & Brinster, R.L. (2003). Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc. Natl. Acad. Sci. USA 100, 6487–6492.CrossRefGoogle Scholar
Liu, S., Tang, Z., Xiong, T. & Tang, W. (2011). Isolation and characterization of human spermatogonial stem cells. Reprod. Biol. Endocrinol. 9, 141.CrossRefGoogle ScholarPubMed
Loppion, G., Crespel, A., Martinez, A.S., Auvray, P. & Sourdaine, P. (2008). Study of the potential spermatogonial stem cell compartment in dogfish testis, Scyliorhinus canicula L. Cell Tissue Res. 332, 533–42.CrossRefGoogle ScholarPubMed
Mardanpour, P., Guan, K., Nolte, J., Lee, J.H., Hasenfuss, G., Engel, W. & Nayernia, K. (2008). Potency of germ cells and its relevance for regenerative medicine. J. Anat. 213, 26–9.CrossRefGoogle ScholarPubMed
Marh, J., Tres, L.L., Yamazaki, Y., Yanagimachi, R. & Kierszenbaum, A.L. (2003). Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes. Biol. Reprod. 69, 169–76.CrossRefGoogle ScholarPubMed
Meng, X., Lindahl, M., Hyvonen, M.E., Parvinen, M., de Rooij, D.G., Hess, M. W., Raatikainen-Ahokas, A., Sainio, K., Rauvala, H., Lakso, M., Pichel, J.G., Westphal, H., Saarma, M. & Sariola, H. (2000). Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 287, 1489–93.CrossRefGoogle ScholarPubMed
Miura, T., Yamauchi, K., Takahashi, H. & Nagahama, Y. (1991). Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica). Proc. Natl. Acad. Sci. USA 88, 5774–8.CrossRefGoogle ScholarPubMed
Mohanta, R., Jayasankar, P., Mahapatra, K.D., Saha, J.N. & Barman, H.K. (2014). Molecular cloning, characterization and functional assessment of the myosin light polypeptide chain 2 (mylz2) promoter of farmed carp, Labeo rohita . Transgenic Res. 23, 601–7.CrossRefGoogle ScholarPubMed
Mohapatra, C. & Barman, H.K. (2014). Identification of promoter within the first intron of Plzf gene expressed in carp spermatogonial stem cells. Mol. Biol. Rep. 41, 6433–40.CrossRefGoogle ScholarPubMed
Mohapatra, C., Barman, H.K., Panda, R.P., Kumar, S., Das, V., Mohanta, R., Mohapatra, S.D. & Jayasankar, P. (2010). Cloning of cDNA and prediction of peptide structure of Plzf expressed in the spermatogonial cells of Labeo rohita . Mar Genomics 3, 157–63.Google ScholarPubMed
Mohapatra, S.D., Kumar, K., Jayasankar, P. & Barman, H.K. (2013). Establishment of dry-down hypoxic stress treatment protocol for snakehead freshwater fish, Channa striatus . Int. J. Fish. Aquac. 1, 36–9.Google Scholar
Mohapatra, C., Patra, S.K., Panda, R.P., Mohanta, R., Saha, A., Das Mahapatra, K., Jayasankar, P. & Barman, H.K. (2014). Gene structure and identification of minimal promoter of Pou2 expressed in spermatogonial cells of rohu carp, Labeo rohita . Mol. Biol. Rep. 41, 4123–32.CrossRefGoogle ScholarPubMed
Momeni-Moghaddam, M., Matin, M.M., Boozarpour, S., Sisakhtnezhad, S., Mehrjerdi, H.K., Farshchian, M., Dastpak, M. & Bahrami, A.R. (2014). A simple method for isolation, culture, and in vitro maintenance of chicken spermatogonial stem cells. In Vitro Cell. Dev. Biol. Anim. 50, 155–61.CrossRefGoogle ScholarPubMed
Niu, Z., Goodyear, S.M., Rao, S., Wu, X., Tobias, J.W., Avarbock, M.R. & Brinster, R.L. (2011). MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 108, 12740–5.CrossRefGoogle ScholarPubMed
Panda, R.P., Barman, H.K. & Mohapatra, C. (2011). Isolation of enriched carp spermatogonial stem cells from Labeo rohita testis for in vitro propagation. Theriogenology 76, 241–51.CrossRefGoogle ScholarPubMed
Panda, R.P., Chakrapani, V., Patra, S.K., Saha, J.N., Jayasankar, P., Kar, B., Sahoo, P.K. & Barman, H.K. (2014). First evidence of comparative responses of Toll-like receptor 22 (TLR22) to relatively resistant and susceptible Indian farmed carps to Argulus siamensis infection. Dev. Comp. Immunol. 47, 25–35.CrossRefGoogle ScholarPubMed
Patra, S. K., Chakrapani, V., Panda, R.P., Mohapatra, C., Jayasankar, P. & Barman, H.K. (2015). First evidence of molecular characterization of rohu carp Sox2 gene being expressed in proliferating spermatogonial cells. Theriogenology 84, 268–76.CrossRefGoogle ScholarPubMed
Pradeep, P.J., Srijaya, T.C., Zain, R.B.M., Papini, A. & Chatterji, A.K. (2011). A Simple technique for chromosome preparation from embryonic tissues of teleosts for ploidy verification. Caryologia 64, 235–41.CrossRefGoogle Scholar
Pšenička, M., Saito, T., Linhartová, Z. & Gazo, L. (2015). Isolation and transplantation of sturgeon early-stage germ cells. Theriogenology 83, 1085–92.CrossRefGoogle ScholarPubMed
Rahman, M.A., Hwang, G.L., Razak, S.A., Sohm, F. & Maclean, N. (2000). Copy number related transgene expression and mosaic somatic expression in hemizygous and homozygous transgenic tilapia (Oreochromis niloticus). Transgenic Res. 9, 417–27.CrossRefGoogle ScholarPubMed
Reding, S.C., Stepnoski, A.L., Cloninger, E.W. & Oatley, J.M. (2010). THY1 is a conserved marker of undifferentiated spermatogonia in the prepubertal bull testis. Reproduction 139, 893–903.CrossRefGoogle ScholarPubMed
Rodriguez-Sosa, J.R., Dobson, H. & Hahnel, A. (2006). Isolation and transplantation of spermatogonia in sheep. Theriogenology 66, 2091–103.CrossRefGoogle ScholarPubMed
Ryu, B.Y., Orwig, K.E., Kubota, H., Avarbock, M.R. & Brinster, R.L. (2004). Phenotypic and functional characteristics of spermatogonial stem cells in rats. Dev. Biol. 274, 158–70.Google ScholarPubMed
Sanchez-Sanchez, A.V., Camp, E., Garcia-Espana, A., Leal-Tassias, A. & Mullor, J.L. (2010). Medaka Oct4 is expressed during early embryo development, and in primordial germ cells and adult gonads. Dev. Dyn. 239, 672–9.CrossRefGoogle ScholarPubMed
Schulz, R.W., Menting, S., Bogerd, J., Franca, L.R., Vilela, D.A. & Godinho, H.P. (2005). Sertoli cell proliferation in the adult testis—evidence from two fish species belonging to different orders. Biol. Reprod. 73, 891–8.CrossRefGoogle ScholarPubMed
Shang, M., Su, B., Lipke, E.A., Perera, D.A., Li, C., Qin, Z., Li, Y., Dunn, D.A., Cek, S., Peatman, E. & Dunham, R.A. (2015). Spermatogonial stem cells specific marker identification in channel catfish, Ictalurus punctatus and blue catfish, I. furcatus . Fish Physiol. Biochem. 41, 1545–56.CrossRefGoogle ScholarPubMed
Shinohara, T., Avarbock, M.R. & Brinster, R.L. (1999). b1- and a6-intergrin are surface markers and mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 96, 5504–9.CrossRefGoogle Scholar
Siraj, S.S., Sukardi, R., Imm, C.C.P., Mei, K.H., Vellasamy, S., Panandam, J.M. & Daud, S.K. (2009). Chromosome analysis of walking catfish, Clarias spp . Malays. Appl. Biol. 38, 61–4.Google Scholar
Stuart, G.W., Vielkind, J.R., McMurray, J.V. & Westerfield, M. (1990). Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression. Development 109, 577–84.CrossRefGoogle ScholarPubMed
Vilela, D.A.R., Silva, S.G.B., Peixoto, M.T.D., Godinho, H.P. & Franca, L.R. (2003). Spermatogenesis in teleost: insights from the Nile tilapia (Oreochromis niloticus) model. Fish Physiol. Biochem. 28, 187–90.CrossRefGoogle Scholar
Vlajkovi'c, S., Cukuranovi'c, R., Bjelakovi'c, M.D. & Stefanovi'c, V. (2012). Possible therapeutic use of spermatogonial stem cells in the treatment of male infertility: a brief overview. Scientific World J.CrossRefGoogle Scholar
WHO (2010). WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edition. Geneva, Switzerland: World Health Organization Press.Google Scholar
Wu, J., Song, W., Zhu, H., Niu, Z., Mua, H., Lei, A., Yang, C., Peng, S., Li, X., Li, G. & Hua, J. (2013). Enrichment and characterization of Thy1-positive male germline stem cells (mGSCs) from dairy goat (Capra hircus) testis using magnetic microbeads. Theriogenology 80, 1052–60.CrossRefGoogle ScholarPubMed
Yuan, Z., Hou, R. & Wu, J. (2009). Generation of mice by transplantation of an adult spermatogonial cell line after cryopreservation. Cell Proliferat. 42, 123–31.CrossRefGoogle ScholarPubMed
Zhao, H.B., Zhang, X.Y., Feng, G.Q., Guo, M.M., Chang, P., Qi, C., Zhong, X.P., Zhou, Q.C. & Wang, J.L. (2015). Expression of plzfa in embryo and adult of medaka Oryzias latipes . J. Fish Biol. 87, 231–40.CrossRefGoogle ScholarPubMed
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