Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

doi:10.1083/jcb.74.1.68

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Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

AR Bellve, JC Cavicchia, CF Millette, DA O'Brien, YM Bhatnagar, and M Dym

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).

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Habasque, C., Satie, A.-P., Aubry, F., Jegou, B., Samson, M. (2003). Expression of fractalkine in the rat testis: molecular cloning of a novel alternative transcript of its gene that is differentially regulated by pro-inflammatory cytokines. Mol Hum Reprod 9: 449-455 [Abstract] [Full Text]
Brinster, C. J., Ryu, B.-Y., Avarbock, M. R., Karagenc, L., Brinster, R. L., Orwig, K. E. (2003). Restoration of Fertility by Germ Cell Transplantation Requires Effective Recipient Preparation. Biol. Reprod. 69: 412-420 [Abstract] [Full Text]
Bourguiba, S., Chater, S., Delalande, C., Benahmed, M., Carreau, S. (2003). Regulation of Aromatase Gene Expression in Purified Germ Cells of Adult Male Rats: Effects of Transforming Growth Factor {beta}, Tumor Necrosis Factor {alpha}, and Cyclic Adenosine 3',5'-Monosphosphate. Biol. Reprod. 69: 592-601 [Abstract] [Full Text]
Saito, H., Takeda, K., Yasumoto, K.-i., Ohtani, H., Watanabe, K.-i., Takahashi, K., Fukuzaki, A., Arai, Y., Yamamoto, H., Shibahara, S. (2003). Germ Cell-Specific Expression of Microphthalmia-Associated Transcription Factor mRNA in Mouse Testis. J Biochem 134: 143-150 [Abstract] [Full Text]
Yu, Z., Guo, R., Ge, Y., Ma, J., Guan, J., Li, S., Sun, X., Xue, S., Han, D. (2003). Gene Expression Profiles in Different Stages of Mouse Spermatogenic Cells During Spermatogenesis. Biol. Reprod. 69: 37-47 [Abstract] [Full Text]
Brown, P. R., Miki, K., Harper, D. B., Eddy, E. M. (2003). A-Kinase Anchoring Protein 4 Binding Proteins in the Fibrous Sheath of the Sperm Flagellum. Biol. Reprod. 68: 2241-2248 [Abstract] [Full Text]
Wakayama, T., Koami, H., Ariga, H., Kobayashi, D., Sai, Y., Tsuji, A., Yamamoto, M., Iseki, S. (2003). Expression and Functional Characterization of the Adhesion Molecule Spermatogenic Immunoglobulin Superfamily in the Mouse Testis. Biol. Reprod. 68: 1755-1763 [Abstract] [Full Text]
Com, E., Evrard, B., Roepstorff, P., Aubry, F., Pineau, C. (2003). New Insights into the Rat Spermatogonial Proteome: Identification of 156 Additional Proteins. Mol. Cell. Proteomics 2: 248-261 [Abstract] [Full Text]
Hsia, K.-T., Millar, M. R., King, S., Selfridge, J., Redhead, N. J., Melton, D. W., Saunders, P. T. K. (2003). DNA repair gene Ercc1 is essential for normal spermatogenesis and oogenesis and for functional integrity of germ cell DNA in the mouse. Development 130: 369-378 [Abstract] [Full Text]
Anway, M. D., Li, Y., Ravindranath, N., Dym, M., Griswold, M. D. (2003). Expression of Testicular Germ Cell Genes Identified by Differential Display Analysis. J Androl 24: 173-184 [Abstract] [Full Text]