Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes

• Published in: Biology of reproduction 1996-08, Vol.55 (2), p.439-444
• Main Authors: Pelt, A.M.M. van (Utrecht University, Utrecht, The Netherlands.), Morena, A.R, Dissel-Emiliani, F.M.F. van, Boitani, C, Gaemers, I.C, Rooij, D.G. de, Stefanini, M
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; CARENCE EN VITAMINE; Cell Separation - methods; Cells, Cultured; Centrifugation, Density Gradient; Collagenases - metabolism; DEFICIENCIA DE VITAMINAS; DIFERENCIACION CELULAR; DIFFERENCIATION CELLULAIRE; ESPERMATOGENESIS; Female; Fundamental and applied biological sciences. Psychology; GAMETE; GAMETOS; Hyaluronoglucosaminidase - metabolism; Lectins; Male; Mammalian male genital system; MODELE ANIMAL; MODELOS ANIMALES; Morphology. Physiology; Peanut Agglutinin; Pregnancy; RAT; RATA; Rats; Rats, Wistar; RETINOL; SINCRONIZACION; SPERMATOGENESE; Spermatogonia - pathology; SYNCHRONISATION; TECHNIQUE DE L'ISOLEMENT; TECNICAS DE AISLAMIENTO; TESTICULE; TESTICULOS; Testis - pathology; Trypsin - metabolism; Vertebrates: reproduction; Vitamin A Deficiency - pathology
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod55.2.439

A method for isolating A spermatogonia from the adult vitamin A-deficient (VAD) rat testis is described. After removal, the testes were decapsulated and tubules were dissected. An enzymatic digestion with collagenase, hyaluronidase, and trypsin was performed first to eliminate most of the interstitial cells. A second digestion with collagenase and hyaluronidase was performed to obtain a cell suspension with a high number of A spermatogonia. The cell suspension was further enriched with A spermatogonia by preplating on peanut agglutinin and separating on a discontinuous Percoll gradient. By this procedure, purification of the suspension to 70-90% A spermatogonia was obtained. In the seminiferous tubules of the VAD rats, only Sertoli cells, A spermatogonia, and some preleptotene spermatocytes are present. In our rats, the A spermatogonia are almost all arrested in the G1 phase of the cell cycle before the S phase of A1 spermatogonia, and presumably before their differentiation into A1 spermatogonia. After administration of vitamin A, spermatogenesis starts synchronously from these A spermatogonia. The isolation of these synchronized A spermatogonia opens ways to investigate the regulation of differentiation and proliferation of A spermatogonia and the biochemical characteristics of the subsequent types of A spermatogonia