Expression and Localization of NANOS1 in Spermatogenic Cells during Spermatogenesis in Rat


Expression and localization of NANOS1 in the spermatogenic cells of rat testis were studied by immunofluorescence and immunoelectron microscopy. Using immunofluorescence techniques, NANOS1 was localized in the cytoplasm and discrete granules of spermatocytes and spermatids. The staining intensity of NANOS1 signal varied depending upon the stage of the cycle of seminiferous epithelium. Double immunofluorescence staining with antibodies against NANOS1 and DDX4 showed that several DDX4-positive compartments of nuage were also stained for NONOS1. Immunoelectron microscopy revealed that the major subcellular localization sites for NANOS1 were the chromatoid body (CB) and satellite body (SB), and the minor sites were the rest of the nuage compartments, including the irregularly-shaped perinuclear granules (ISPG), and intermitochondrial cement (IMC). Non-nuage structures such as mitochondria-associated granules (MAG), granulated body (GB), and reticulated body (RB) were also labeled by theNANOS1 antibody. In addition, NANOS1 was found in the outer dense fibers of flagella of spermatids at steps 12-19, and in the head cap of late spermatids after step 15. These results suggest that NANOS1 is one of the nuage proteins and functions mainly in the CBs as well as in the cytoplasm. NANOS1 may also have additional functions in non-nuage structures such as MAGs, GBs and RBs.

Share and Cite:

S. Yokota and Y. Onohara, "Expression and Localization of NANOS1 in Spermatogenic Cells during Spermatogenesis in Rat," CellBio, Vol. 2 No. 1, 2013, pp. 1-10. doi: 10.4236/cellbio.2013.21001.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] S. Kobayashi, M. Yamada, M. Asaoka and T. Kitamura, “Essential Role of the Posterior Morphogen Nanos for Germline Development in Drosophila,” Nature, Vol. 380, No. 6576, 1996, pp. 708-711. doi:10.1038/380708a0
[2] K. Subramaniam and G. Seydoux, “Nos-1 and Nos-2, Two Genes Related to Drosophila Nanos, Regulate Primordial Germ Cell Development and Survival in Caenorhabditis elegans,” Development, Vol. 126, No. 21, 1999, pp. 4861-4871.
[3] Z. Wang and H. Lin, “Nanos Maintains Germline Cell Self-Renewal by Preventing Differentiation,” Science, Vol. 303, No. 5666, 2004, pp. 2016-2019. doi:10.1126/science.1093983
[4] D. Curtis, J. Apfeld and L. Lehmann, “Nanos Is an Evolutionarily Conserved Organizer of Anterior-Posterior Polarity,” Development, Vol. 121, No. 6, 1995, pp. 1899-1910.
[5] R. Lehmann and C. Nüsslein-Volhard, “The Maternal Gene Nanos Has a Central Role in Posterior Pattern Formation of the Drosophila embryo,” Development, Vol. 112, No. 3, 1991, pp. 679-691.
[6] C. Wang, K. K. Dickinson and R. Lehman, “Genetics of nanos Localization in Drosophila,” Developmental Dynamics, Vol. 199, No. 2, 1999, pp. 103-115. doi:10.1002/aja.1001990204
[7] Y. Wang, R. M. Zayas T. Guo and P. A. Newmark, “Nanos Function Is Essential for Development and Regeneration of Planarian Germ Cells,” Proceedings of National Academy of Sciences of the United States of America, Vol. 104, No. 14, 2007, pp. 5901-5906. doi:10.1073/pnas.0609708104
[8] G. Deshpande, G. Calhoun, J. L. Yanowitzand and P. D. Schedl, “Novel Functions of nanos in Down Regulating Mitosis and Transcription during the Development of the Drosophila Germline,” Cell, Vol. 99, No. 3, 1999, pp. 271-181. doi:10.1016/S0092-8674(00)81658-X
[9] Y. Hayashi, M. Hayashi and S. Kobayashi, “Nanos Suppresses Somatic Cell Fate in Drosophila Germ Line,” Proceedings of National Academy of Sciences of the United States of America, Vol. 101, No. 28, 2004, pp. 10338-10342. doi:10.1073/pnas.0401647101
[10] M.Tsuda, Y. Sasaoka, M. Kiso, K. Abe, S. Haraguchi, S. Kobayashi and Y. Saga, “Conserved Role of nanos Proteins in Germ Cell Development,” Science, Vol. 301, No. 5637, 2003, pp. 1239-1241. doi:10.1126/science.1085222
[11] K. Sato, Y. Hayashi, Y. Ninomiya, S. Shigenobu, K. Arita, M. Mukai and S. Kobayashi, “Maternal Nanos Represses hid/skl-Dependent Apoptosis to Maintain the Germ Line in Drosophila Embryos,” Proceedings of National Academy of Sciences of the United States of America, Vol. 104, No. 18, 2007, pp. 7455-7460. doi:10.1073/pnas.0610052104
[12] S. Haraguchi, M. Tsuda, S. Kitajima, Y. Sasaoka, A. Nomura-Kitabayashi, K. Kurokawa and Y. Saga, “nanos1: A Mouse nanos Gene Expressed in the Central Nervous System Is Dispensable for Normal Development,” Mechanism of Development, Vol. 120, No. 6, 2003, pp. 721-731. doi:10.1016/S0925-4773(03)00043-1
[13] A. Suzuki, M. Tsuda and Y. Saga, “Functional Redundancy among Nanos Proteins and a Distinct Role of Nanos2 during Male Germ Cell Development,” Development, Vol. 134, No. 1, 2007, pp. 77-83. doi:10.1242/dev.02697
[14] A. Suzuki and Y. Saga, “Nanos2 Suppresses Meiosis and Promotes Male Germ Cell Differentiation,” Genes & Development, Vol. 22, No. 4, 2008, pp. 430-435. doi:10.1101/gad.1612708
[15] V. T. A. Julaton and R. A. R. Pera, “NANOS3 Function in Human Germ Cell Development,” Human Molecular Genetics, Vol. 20, No. 11, 2011, pp. 2238-2250. doi:10.1093/hmg/ddr114
[16] A. Forbes and R. Lehmann, “Nanos and Pumilio Have critical Roles in the Development and Function of Drosophila Germline Stem Cells,” Development, Vol. 125, 1998, pp. 679-690.
[17] F. Lolicato, R. Marino, M. P. Paronetto, M. Pellegrini, S. Dolci, R. Geremia and P. Grimaldi, “Potential Role Nanos3 in Maintaining the Undifferentiated Spermatogonia Population,” Developmental Biology, Vol. 313, No. 2, 2008, pp. 725-738. doi:10.1016/j.ydbio.2007.11.011
[18] J. Jaruzelska, M. Kotecki, K. Kusz, A. Spik, M. Firpo and R. A. R. Pera, “Conservation of a Pumilio-Nanos Complex from Drosophila Germ Plasm to Human Germ Cells,” Development Genes and Evolution, Vol. 213, No. 3, 2003, pp. 120-126.
[19] B. Ginter-Matuszewska, K. Kusz, A. Spik, D. Grzeszkowiak, A. Rembiszewska, J. Kupryjanczyk and J. Jaruzelska, “NANOS1 and PUMILIO2 Bind MicroRNA Biogenesis Factor GEMIN3, within Chromatoid Body in Human Germ Cells,” Histochemistry and Cell Biology, Vol. 136, No. 3, 2011, pp. 279-287. doi:10.1007/s00418-011-0842-y
[20] B. Ginter-Matuszewska, A. Spik, A. Rembiszewska, C. Koyias, J. Kupryjańczyk and J. Jaruzelska, “The SNARE-Associated Component SNAPIN Binds PUMILIO2 and NANOS1 Proteins in Human Male Germ cells,” Molecular Human Reproduction, Vol. 15, No. 3, 2009, pp. 173-179. doi:10.1093/ molehr/gap004
[21] Y. Onohara and S. Yokota, “Expression of DDX25 in Nuage Components of Mammalian Spermatogenic Cells: Immunofluorescence and Immunoelectron Microscopic Study,” Histochemistry and Cell Biology, Vol. 137, No. 1, 2012, pp. 37-51. doi:10.1007/s00418-011-0875-2
[22] H. Yonetamari, Y. Onohara and S. Yokota, “Localization of BRUNOL2 in Rat Spermatogenic Cells Revealed by Immunofluorescence and Immunoelectron Microscopic Techniques,” Open Journal of Cell Biology, Vol. 2, No. 2, 2012, pp. 11-20. doi:10.4236/ojcb.2012.22002
[23] Y. Onohara, T. Fujiwara, T. Yasukochi, M. Himeno and S. Yokota, “Localization of Mouse Vasa Homolog Protein in Chromatoid Body and Related Nuage Structures of Mammalian Spermatogenic Cells during Spermatogenesis,” Histochemistry and Cell Biology, Vol. 133, No. 6, 2010, pp. 627-639. doi:10.1007/s00418-010-0699-5
[24] C. M. Haraguchi, T. Mabuchi and S. Yokota, “Localization of a Mitochondrial Type of NADP-Dependent Isocitrate Dehydrogenase in Kidney and Heart of Rat: An Immunocytochemical and Biochemical Study,” Journal of Histochemistry and Cytochemistry, Vol. 51, No. 2, 2003, pp. 215-226. doi:10.1177/002215540305100210
[25] C. de Roe, P. J. Courtoy and P. Baudhuin, “A Model of Protein-Colloidal Gold Interactions,” Journal of Histochemistry and Cytochemistry, Vol. 35, No. 11, 1987, pp. 1191-1198. doi:10.1177/ 35.11.3655323
[26] D. Rickwood and T. C. Ford, “Preparation and Fractionation of Nuclei, Nucleoli and Deoxyribonucleoproteins,” In: D. Rickwood Ed., Iodinated Density Gradient Media, Practical Approach, IRL Press, Oxford, Washington DC, 1983, pp. 69-89.
[27] L. D. Russell, R. A. Ettlin, A. S. P. Hikimand and E. D. Clegg, “Histological and Histopathological Evaluation of Testis,” Cache River Press, Clearwater, 1990.
[28] L. Russell and B. Frank, “Ultrastructural Characterization of Nuage in Spermatocytes of the Rat Testis,” Anatomical Record, Vol. 190, No. 1, 1978, pp. 79-97. doi:10.1002/ar.1091900108
[29] Y. Clermont, R. Oko and L. Hermo, “Immunocyto-chemical Localization of Proteins Utilized in the Formation of Outer Dense Fibers and Fibrous Sheath in Rat Spermatids: An Electron Microscope Study,” Anatomical Record, Vol. 227, No. 4, 1990, pp. 447-457. doi:10.1002/ar.1092270408
[30] K. M. Kusz, L. Tomczyk, M. Sajek, A. Spik, A. Latos-Bielenska, P. Jedrzejczak, L. Pawelczyk and J. Jaruzelska, “The Highly Conserved NANOS2 Protein: Testis-Specific Expression and Significance for the Human Male Reproductionm,” Molecular Human Reproduction, Vol. 15, No. 3, 2009, pp. 165-171. doi:10.1093/molehr/gap003
[31] C. M. Haraguchi, T. Mabuchi, S. Hirata, T. Shoda, K. Hoshi, K. Akasaki and S. Yokota, “Chromatoid Bodies: Aggresome-Like Characteristics and Degradation Sites for Organelles of Spermiogeniccells,” Journal of Histochemistry and Cytochemistry, Vol. 53, No. 4, 2000, pp. 455-465. doi:10.1369/ jhc.4A6520.2005
[32] C. Barreau, L. Paillard, A. Méreau and H. B. Osborne, “Mammalian CELF/Bruno-Like RNA-Binding Proteins: Molecular Characteristics and Biological Functions,” Biochemie, Vol. 88, No. 5, 2006, pp. 515-525. doi:10.1016/j.biochi.2005.10.011
[33] Y. Sheng, C.-H. Tsai-Morris, R. Gutti, Y. Maeda and M. L. Dufau, “Gonadotropin-Regulated Testicular RNA Helicase (GRTH/Ddx25) Is a Transport Protein Involved in Gene-Specific mRNA Export and Protein Translation during Spermatogenesis,” Journal of Biological Chemistry, Vol. 281, No. 46, 2006, pp. 35048-35056. doi:10.1074/jbc.M605086200

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.