PURIFICATION OF RAT SPERMATOGENIC CELLS AND PRELIMINARY BIOCHEMICAL-ANALYSIS OF THESE CELLS
PURIFICATION OF RAT SPERMATOGENIC CELLS AND PRELIMINARY BIOCHEMICAL-ANALYSIS OF THESE CELLS
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DOI：
10.1095/biolreprod25.5.1065
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发表时间：
1981-01-01
影响因子：
3.6
通讯作者：
MACE, ML
中科院分区：
文献类型：
--
作者：
MEISTRICH, ML;LONGTIN, J;MACE, ML
本发明描述了一种用于获得高度纯化的大鼠睾丸细胞级分的方法。采用离心淘洗法分离成年大鼠睾丸单细胞悬液。粗线期初级精母细胞，早期精子细胞和从晚期精子细胞分离的胞质中富集的馏分。通过在Percoll梯度上的平衡密度离心进一步分离这些级分。以这种方式，3 × 3的分数。107个粗线期精母细胞（纯度98%），1.1 × 10 - 6个。108个早期精子细胞（93%纯度）和1.1X.处死大鼠后6 h内获得108个细胞质（纯度98%）。细胞形态完整，保持其生化完整性。聚丙烯酰胺凝胶电泳分析的酸溶性核蛋白表明，组蛋白4在粗线期阶段合成，并证实，睾丸特异性组蛋白在此阶段合成。从纯化粗线期精母细胞和纯化的早期精子细胞的微粒体RNA制剂的分析表明，完整的核糖体RNA（rRNA）可以从纯化的细胞。这两种细胞类型都在合成具有广泛沉降值的假定信使RNA（mRNA）方面具有活性，但未检测到明显的rRNA合成。
A method for obtaining highly purified fractions of rat testicular cells is described. Single cell suspensions from adult rat testes were separated by centrifugal elutriation. Fractions enriched in pachytene primary spermatocytes, early spermatids and cytoplasts detached from late spermatids were obtained. These fractions were further separated by equilibrium density centrifugation on gradients of Percoll. In this manner fractions of 3 .times. 107 pachytene spermatocytes (98% purity), 1.1 .times. 108 early spermatids (93% purity) and 1.1 .times. 108 cytoplasts (98% purity) were obtained within 6 h after sacrificing the rats. The cells were morphologically intact and retained their biochemical integrity. Analysis of acid-soluble nuclear proteins by polyacrylamide gel electrophoresis showed that histone 4 is synthesized during the pachytene stage and confirmed that testis-specific histones are synthesized during this stage. Analysis of a microsomal RNA preparation from purified pachytene spermatocytes and purified early spermatids by sucrose gradients indicated that intact ribosomal RNA (rRNA) can be obtained from purified cells. Both cell types are active in synthesizing persumptive messenger RNA (mRNA) with a wide range of sedimentation values, but no appreciable rRNA synthesis was detected.