STAGE-SPECIFIC PROTEIN-SYNTHESIS BY ISOLATED SPERMATOGENIC CELLS THROUGHOUT MEIOSIS AND EARLY SPERMIOGENESIS IN THE MOUSE
STAGE-SPECIFIC PROTEIN-SYNTHESIS BY ISOLATED SPERMATOGENIC CELLS THROUGHOUT MEIOSIS AND EARLY SPERMIOGENESIS IN THE MOUSE
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DOI：
10.1095/biolreprod37.1.147
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发表时间：
1987-08-01
影响因子：
3.6
通讯作者：
OBRIEN, DA
中科院分区：
文献类型：
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作者：
OBRIEN, DA
通过单位重力沉降从青春期前和成年小鼠中分离的生精细胞已被用于检测在减数分裂和早期精子发生过程中以特定阶段的方式合成的蛋白质。从17日龄小鼠中分离prelepptotene、lepptotene /zygotene和pachytene精母细胞。从成熟动物中分离出成年粗线精细胞和圆形精细胞。然后将这些生殖细胞在含有[35S]蛋氨酸（[35S]met）的培养基中培养4-5小时。对于每种细胞类型，测定[35S]met的相对掺入量，并通过二维（2D）聚丙烯酰胺凝胶电泳和放射自显影法比较标记蛋白。分离的生殖细胞中[35S]met的结合水平与先前精子发生过程中蛋白质合成的放射自显影估计密切相关（Monesi, 1967）。当以cpm/-106细胞或cpm/mg蛋白表达时，来自青春期前小鼠的粗筋素精母细胞含有最高水平的[35S]met。二维自x线图的比较表明，许多蛋白质，包括肌动蛋白和微管蛋白，在所有检查阶段的合成水平大致相同。其他蛋白质，包括热休克蛋白和多种质膜成分，在细粒/zygotene精母细胞、粗粒精母细胞和圆形精母细胞中以特定阶段的方式合成。这些研究确定了在减数分裂粗线期之前监测分离的生精细胞中蛋白质合成的条件，提供了在这些减数分裂早期阶段合成的蛋白质的二维图，并用生化和免疫学方法检查了先前在二维凝胶上鉴定的几种蛋白质的合成。
Spermatogenic cells isolated from prepubertal and adult mice by unit gravity sedimentation have been used to examine proteins synthesized in a stage-specific manner throughout meiosis and early spermiogenesis. Preleptotene, leptotene/zygotene, and pachytene spermatocytes were isolated from 17-day-old mice. Adult pachytene spermatocytes and round spermatids were isolated from mature animals. These germ cells were then cultured in defined medium with [35S]methionine ([35S]met) for 4-5 h. For each cell type, relative [35S]met incorporation was determined and labeled proteins were compared by two-dimensional (2D) polyacrylamide gel electrophoresis and autoradiography. Levels of [35S]met incorporation by isolated germ cells correlate closely with previous autoradiographic estimates of protein synthesis during spermatogenesis (Monesi, 1967). Pachytene spermatocytes from prepubertal mice incorporate the highest levels of [35S]met, when expressed either as cpm/-106 cells or cpm/mg protein. Comparisons of 2D autoradiograms indicated that many proteins, including actin and tubulins, are synthesized at approximately equal levels in all stages examined. Other proteins, including heat-shock proteins and multiple plasma membrane constitutents, are synthesized in a stage-specific manner in leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. These studies define conditions for monitoring protein synthesis in isolated spermatogenic cells prior to the pachytene stage of meiosis, provide a 2D map of proteins synthesized at these earlier meiotic stages, and examine the synthesis of several proteins previously identified on 2D gels with biochemical and immunological methods.