Localization of BRUNOL2 in Rat Spermatogenic Cells as Revealed by Immunofluorescence and Immunoelectron Microscopic Techniques


Distribution and localization of a RNA-binding protein, BRUNOL2 in rat spermatogenic cells were studied by dot blotting of cell fractions, immunofluorescence (IF), and immunoelectron microscopy (IEM). BRUNOL2 distributed in nuclear (23%), mitochondrial (19%), microsomal (15%), and cytosol fractions (43%). BRUNOL2 was detected in all spermatogenic cells. In the cytoplasm and nucleoplasm of the spermatogonia, spermatocytes and spermatids, both diffuse and granular staining patterns were observed. Many cytoplasmic granules were stained also for DDX4 and DDX25. Large granules in the cytoplasm of elongated spermatids were stained for BRUNOL2 but not for the nuage proteins. IEM showed that gold signals for BRUNOL2 were concentrated in nuage components including loose aggregates of small particles, chromatoid body (CB), intermitochondrial cement (IMC), and satellite body (SB). In addition, many non-nuage structures such as ER-attached small granules, less dense material surrounding connecting piece of flagellum, reticulated body, mitochondria-associated granules (MAG), granulated body, ribosome aggregate, and manchette, were stained for BRUNOL2 with different staining intensities. In the nucleus, gold signals were concentrated in heterochromatin area and nucleolus. The results suggest that BRUNOL2 is one of the nuage proteins and also associated with the other non-nuage structures, suggesting multiple functions of this protein.

Share and Cite:

H. Yonetamari, Y. Onohara and S. Yokota, "Localization of BRUNOL2 in Rat Spermatogenic Cells as 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.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] A. N. Ladd, N. Charlet-B. and T. A. Cooper, “The CELF Family of RNA Binding Proteins Is Implicated in Cell-Specific and Developmentally Regulated Alternative Splicing,” Molecular and Cellular Biology, Vol. 21, No. 4, 2001, pp. 1285-1296. doi:10.1128/MCB.21.4.1285-1296.2001
[2] H. Suzuki, Y. Jin, H. Otani, K. Yasuda and K. Inoue, “Regulation of Alternative Splicing of α-Actinin Transcript by Bruno-Like Proteins,” Genes to Cells, Vol. 7, No. 2, 2002, pp. 133-141. doi:10.1046/j.1356-9597.2001.00506.x
[3] C. Barreau, L. Paillard, A. Méreau and H. B. Osborne, “Mammalian CELF/Bruno-Like RNA-Binding Proteins: Molecular Characteristics and Biological Functions,” Biochimie, Vol. 88, No. 5, 2006, pp. 515-525.
[4] L. T. Timchenko, J. W. Miller, N. A. Timchenko, D. R. DeVore, K. V. Datar, L. Lin, R. Roberts, C. T. Caskey and M. S. Swanson, “Identification of a (CUG)n Triplet Repeat RNA-Binding Protein and Its Expression in Myotonic Dystrophy,” Nucleic Acids Research, Vol. 24, No. 22, 1996, pp. 4407-4414. doi:10.1093/nar/24.22.4407
[5] X. H. Lu, N. A. Timchenko and L. T. Timchenko, “Cardiac Elav-Type RNA-Binding Protein (ETR-3) Binds to RNA CUG Repeats Expanded in Myotonic Dystrophy,” Human Molecular Genetics, Vol. 8, No. 1, 1999, pp. 53-60. doi:10.1093/hmg/8.1.53
[6] R. Roberts, N. A. Timchenko, J. W. Miller, S. Reddy, C. T. Caskey, M. S. Swanson and L. T. Timchenko, “Altered Phosphorylation and Intracellular Distribution of a (CUG)n Triplet Repeat RNA-Binding Protein in Patients with Myotonic Dystrophy and in Myotonin Kinase Knockout Mice,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 24, 1997, pp. 13221-13226. doi:10.1073/pnas.94.24.13221
[7] A. N. Ladd and T. A. Cooper, “Multiple Domains Control the Subcellular Localization and Activity of ETR-3, a Regulator of Nuclear and Cytoplasmic RNA Processing Events,” Journal of Cell Science, Vol. 117, No. 16, 2004, pp. 3519-3529. doi:10.1242/jcs.01194
[8] J. Wu, C. Li, S. Zhao and B. Mao, “Differential Expression of the Brunol/CELF Family Genes during Xenopus Laevis Early Development,” The International Journal of Experimental Biology, Vol. 54, 2010, pp. 209-214. doi:10.1387/ijdb.082685jw
[9] L. T. Timchenko, E. Sailsbury, G.-L. Wang, H. Nguyen, J. H. Albrecht, J. W. B. Hershey and N. A. Timchencko, “Age-Specific CUGBP1-eIF2 Complex Increases Translation of CCAAT/Enhancer-Binding Protein β in Old Liver,” The Journal of Biological Chemistry, Vol. 281, No. 43, 2006, pp. 32806-32819. doi:10.1074/jbc.M605701200
[10] C. Kress, C. Gautier-Courteille, H. B. Osborne, C. Babinet and L. Pillard, “Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice,” Molecular and Cellular Biology, Vol. 27, No. 3, 2007, pp. 1146-1157. doi:10.1128/MCB.01009-06
[11] 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
[12] C. de Roe, P. J. Courtoy and P. Baudhuin, “A Model of Protein Colloidal Gold Interactions,” Journal of Histochemistry & Cytochemistry, Vol. 35, No. 11, 1987, pp. 1191-1198. doi:10.1177/35.11.3655323
[13] L. D. Russell, R. A. Ettlin, A. S. P. Hikim and E. D. Clegg, “Histological and Histopathological Evaluation of Testis,” Cache River Press, Clearwater, 1990.
[14] Y. Toyooka, N. Tsunekawa, Y. Matsui, M. Satoh and T. Noce, “Expression and Intracellular Localization of Mouse Vasa-Homologue during Germ Cell Development,” Mechanisms of Development, Vol. 93, No. 1-2, 2000, pp. 139-149. doi:10.1016/S0925-4773(00)00283-5
[15] T. Noce, S. Okamoto-Ito and N. Tsunekawa, “Vasa Homolog Genes in Mammalian Germ Cell Development,” Cell Structure and Function, Vol. 26, No. 3, 2001, pp. 131-136. doi:10.1247/csf.26.131
[16] C.-H. Tsai-Morris, Y. Sheng, E. Lee, K.-J. Lei and M. L. Dufau, “Gonadotropin-Regulated Testicular RNA Helicase (GRTH/Ddx25) is Essential for Spermatid Development and Completion of Spermatogenesis,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, No. 17, 2004, pp. 6373-6378. doi:10.1073/pnas.0401855101
[17] S. Chuma, M. Hiyoshi, A. Yamamoto, M. Hosokawa, K. Takamune and N. Nakatsuji, “Mouse Tudor Repeat1(MTR-1) is a Novel Component of Chromatoid Bodies/Nuages in Male Germ Cells and Forms a Complex with snRNPs,” Mechanisms of Development, Vol. 120, No. 9, 2003, pp. 970-990.
[18] M. Hosokawa, M. Shoji, K. Kitamura, T. Tanaka, T. Noce, S. Chuma and N. Nakatsuji, “Tudor-Related Protein TDRD 1/MTR-1, TDRD6 and TDR7/TRAP: Domain Composition, Intracellular Localization, and Function in Male Germ Cells in Mice,” Developmental Biology, Vol. 301, No. 1, 2007, pp. 38-52. doi:10.1016/j.ydbio.2006.10.046
[19] J. Pan, M. Goodheart, S. Chuma, N. Nakatsuji, D. C. Page and P. J. Wang, “RNF17, a Component of the Mammalian Germ Cell Nuage, is Essential for Spermiogenesis,” Development, Vol. 132, 2005, pp. 4029-4039. doi:10.1242/dev.02003
[20] N. Kotaja, H. Lin, M. Parvienen and P. Sassone-Corsi, “Interplay of PIWI/Argonaute Protein MIWI and Kinesin KIF17b in Chromatoid Bodies of Male Germ Cells,” Journal of Cell Science, Vol. 119, No. 119, 2006, pp. 2819-2825. doi:10.1242/jcs.03022
[21] S. F. C. Soper, G. W. van der Heijden, T. C. Hardiman, M. Goodheart, S. L. Marten, P. de Boer and A. Bortvin, “Mouse Maelstrom, a Component of Nuage, is Essential for Spermatogenesis and Transposon Repression in Meiosis,” Developmental Cell, Vol. 15, No. 2, 2008, pp. 285-297. doi:10.1016/j.devcel.2008.05.015
[22] 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, 2011, pp. 37-51.
[23] Y. Clermont, R. Oko and L. Hermo, “Cell Biology of Mammalian Spermatogenesis,” In: C. Desjardins and L. L. Ewing, Eds., Cell and Molecular Biology of the Testis, Oxford University Press, New York, 1993, pp. 332-376.
[24] Y. Clermont, R. Oko and L. Hermo, “Immunocytoche mical Localization of Proteins Utilized in the Formation of Outer Dense Fibers and Fibrous Sheath in Rat Spermatids: An Electron Microscopic Study,” The Anatomical Record, Vol. 227, No. 4, 1990, pp. 447-457. doi:10.1002/ar.1092270408
[25] A. L. Kierszenbaum, “Intramanchette Transport (IMT): Managing the Making of the Spermatid Head, Centrosome, and Tail,” Molecular Reproduction and Development, Vol. 63, No. 1, 2002, pp. 1-4. doi:10.1002/mrd.10179
[26] L. L. Tres and A. L. Kierszenbaum, “Sak57, an Acidic Keratin Initially Present in the Spermatid Manchette before Becoming a Compound of Paraaxonemal Structures of the Developing Tail,” Molecular Reproduction and Development, Vol. 44, No. 3, 1996, pp. 395-407. doi:10.1002/(SICI)1098-2795(199607)44:3<395::AID-MRD13>3.0.CO;2-#
[27] P. D. Taulman, C. J. Hycraft, D. F. Balkovetz and B. K. Yoder; “Polaris, a Protein Involved in Left-Right Axis Patterning, Localizes to Basal Bodies and Cilia,” Molecular Biology of the Cell, Vol. 12, No. 3, 2001, pp. 589-599.
[28] E. Rivkin, E. B. Cullinan, L. L. Tres and A. L. Kierszenbaum, “A Protein Associated with the Manchette during Rat Spermiogenesis is Encoded by a Gene of the TBP-1-Like Subfamily with Highly Conserved ATPase and Protease Domain,” Molecular Reproduction and Development, Vol. 48, No. 1, 1997, pp. 77-89. doi:10.1002/(SICI)1098-2795(199709)48:1<77::AID-MRD10>3.0.CO;2-T
[29] A. Junco, B. Bhullar, H. A. Tarnasky and F. A. van der Hoorn, “Kinesin Light Chain KLC3 Expression in Testis is Restricted to Spermatids,” Biology of Reproduction, Vol. 64, No. 5, 2001, pp. 1320-1330. doi:10.1095/biolreprod64.5.1320
[30] M. G. Miller, D. J. Mulholand and W. A. Vogt, “Rat Testis Motor Proteins Associated with Spermatid Translocation (Dynein) and Spermatid Flagella (Kinesin-II),” Biology of Reproduction, Vol. 60, No. 4, 1999, pp. 1047-1056. doi:10.1095/biolreprod60.4.1047
[31] D. L. Spector, “Nuclear Organization and Gene Expression,” Experimental Cell Research, Vol. 229, No. 2, 1996, pp. 189-197. doi:10.1006/excr.1996.0358
[32] M. Labrador and V. G. Corces, “Setting the Boundaries of Chromatin Domains and Nuclear Organization,” Cell, Vol. 111, No. 2, 2002, pp. 151-154. doi:10.1016/S0092-8674(02)01004-8

Copyright © 2024 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.