EXPRESSION OF C-KIT RECEPTOR AND ITS AUTOPHOSPHORYLATION IN IMMATURE RAT TYPE-A SPERMATOGONIA
EXPRESSION OF C-KIT RECEPTOR AND ITS AUTOPHOSPHORYLATION IN IMMATURE RAT TYPE-A SPERMATOGONIA
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DOI：
10.1095/biolreprod52.1.8
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发表时间：
1995-01-01
影响因子：
3.6
通讯作者：
RAVINDRANATH, N
中科院分区：
文献类型：
--
作者：
DYM, M;JIA, MC;RAVINDRANATH, N
本研究的目的是检测大鼠a型精原细胞中kit配体（干细胞因子，钢铁因子）的特异性受体c-kit受体的表达和激活。从9日龄大鼠中获得睾丸，去囊，然后进行顺序酶消化。然后用单位重力下的沉降速度分离睾丸细胞类型的混合物。对分离的A型精原细胞进行了光镜和电镜观察。它们表现出含有几个核仁的球形细胞核和相关的染色质团块和细胞器，通常位于核周位置，与在体内9天大的睾丸中发现的相似。通过将总RNA与小鼠c-kit受体特异性cDNA杂交，建立了精原细胞对c-kit受体的合成。观察到两个mRNA转录本在4.8 kb和12 kb处迁移。c-Lit受体在分离细胞中的定位通过免疫细胞化学使用c-kit蛋白抗体来确定。在分离的A型精原细胞细胞质中观察到c-kit受体特异性染色。此外，使用相同的抗体，Western blot分析证实了c-kit受体蛋白在精原细胞中的存在。该抗体识别的c-kit受体与160 kDa相似。为了确定该受体是否具有功能意义，我们研究了kit配体对c-kit受体磷酸化的影响。c-rt受体似乎在低基础水平上对酪氨酸进行了组成性自磷酸化，并且在kit配体的刺激下，磷酸化蛋白的数量显着增加。这些观察结果表明，kit配体诱导c-kit受体的自磷酸化，这可能导致负责精原细胞增殖和/或分化的其他细胞靶蛋白的激活。
The objective of this study was to examine the expression and activation of the c-kit receptor, a specific receptor for kit ligand (stem cell factor, steel factor), in rat type A spermatogonia. Testes were obtained from 9-day-old rats, decapsulated, and then subjected to sequential enzymatic digestion. The mixture of testicular cell types was then separated by sedimentation velocity at unit gravity. The isolated type A spermatogonia were characterized by light and electron microscopy. They exhibited spherical nuclei containing several nucleoli and associated chromatin clumps and organelles generally in a perinuclear location similar to that found in the in vivo 9-day-old testis. The synthesis of the c-kit receptor by the spermatogonia was established by hybridization of total RNA with a specific cDNA for mouse c-kit receptor. Two mRNA transcripts migrating at 4.8 kb and 12 kb were observed. Localization of the c-Lit receptor in the isolated cells was determined by immunocytochemistry using an antibody to c-kit protein. Specific staining for c-kit receptor was observed in the cytoplasm of the isolated type A spermatogonia. Furthermore, the presence of the c-kit receptor protein in the spermatogonia was confirmed by Western blot analysis using the same antibody. The antibody recognized the c-kit receptor at similar to 160 kDa. In an attempt to determine whether this receptor has a functional significance, we examined the effect of kit ligand on the phosphorylation of the c-kit receptor. The c-rt ir receptor appeared to be constitutively autophosphorylated on tyrosine at low basal levels, and upon stimulation with kit ligand, the amount of phosphorylated protein increased significantly. These observations indicate that kit ligand induces autophosphorylation of the c-kit receptor, which may lead to the activation of other cellular target proteins responsible for spermatogonial proliferation and/or differentiation.