van Pelt A M, Morena A R, van Dissel-Emiliani F M, Boitani C, Gaemers I C, de Rooij D G, Stefanini M
Department of Cell Biology, Medical School, Utrecht University, The Netherlands.
Biol Reprod. 1996 Aug;55(2):439-44. 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.
本文描述了一种从成年维生素A缺乏（VAD）大鼠睾丸中分离A型精原细胞的方法。取出睾丸后，去除被膜并解剖曲细精管。首先用胶原酶、透明质酸酶和胰蛋白酶进行酶消化以去除大部分间质细胞。然后用胶原酶和透明质酸酶进行第二次消化以获得含有大量A型精原细胞的细胞悬液。通过在花生凝集素上预铺板并在不连续的Percoll梯度上分离，进一步富集细胞悬液中的A型精原细胞。通过该方法，可将悬液纯化至70 - 90%的A型精原细胞。在VAD大鼠的曲细精管中，仅存在支持细胞、A型精原细胞和一些前细线期精母细胞。在我们的大鼠中，A型精原细胞几乎都停滞在细胞周期的G1期，先于A1型精原细胞的S期，大概也先于它们分化为A1型精原细胞之前。给予维生素A后，精子发生从这些A型精原细胞开始同步进行。这些同步化的A型精原细胞的分离为研究A型精原细胞的分化和增殖调控以及后续类型A型精原细胞的生化特性开辟了道路。
