Ohsako S, Bunick D, Hess R A, Nishida T, Kurohmaru M, Hayashi Y
Department of Veterinary Anatomy, University of Tokyo, Japan.
Anat Rec. 1994 Mar;238(3):335-48. doi: 10.1002/ar.1092380308.
In order to understand the mechanism of spermiogenesis, it is important to characterize germ cell specific genes and proteins expressed during spermatogenesis. We previously reported that a mouse monoclonal antibody, 1C9, raised against golden hamster testis homogenate, recognized a 103 kDa protein in hamster spermatogenic cells (Ohsako et al.; J. Vet. Med. Sci., 53:969-974, 1991). In the present study, we have determined the precise stage and intracellular localization of this protein.
Hamster, mouse, and rat tissues were used for immunocytochemistry, SDS-PAGE, and immunoblotting. Immunoelectron microscopy was performed using Lowicryl K4M embedded hamster testis and colloidal gold conjugated second antibody. Furthermore, immuno-affinity purification was carried out using a 1C9-Sepharose column.
In immunoblot analysis, 1C9 also recognized a 103 kDa protein and a 101 kDa protein in the rat and the mouse testes, respectively. Ten different hamster tissues other than testis did not show reactivity against 1C9. In immunostained paraffin sections of hamster testis, the initial staining appeared in middle pachytene spermatocytes and persisted until maturation phase spermatids (step 15). However, it was no longer detectable in the subsequent steps of spermatids. In addition, strong staining was observed in the post-nuclear region of elongated spermatids. Immunoelectron microscopic analysis showed that the protein was localized to the endoplasmic reticulum (ER) and nuclear envelope of spermatogenic cells, but not in the other organelles, such as Golgi apparatus and acrosome of the spermatids. This protein appears to be associated with ER membrane. Furthermore, this protein is found exclusively in the testicular microsomal fraction, not in the cytosol. By affinity purification, approximately 320 micrograms of the 103 kDa protein was obtained from 10 hamster testes. The purified 103 kDa protein was unaffected by N-glycanase, indicating it does not have asparagine-linked glycoconjugates.
These results indicate that the protein recognized by 1C9 appears to be a unique protein that is localized in the ER and nuclear envelope of spermatogenic cells.
为了解精子发生的机制，鉴定精子发生过程中表达的生殖细胞特异性基因和蛋白质非常重要。我们之前报道过，一种针对金黄仓鼠睾丸匀浆产生的小鼠单克隆抗体1C9，可识别仓鼠生精细胞中的一种103 kDa蛋白质（大迫等；《兽医医学杂志》，53:969 - 974，1991）。在本研究中，我们确定了这种蛋白质的确切阶段和细胞内定位。
仓鼠、小鼠和大鼠组织用于免疫细胞化学、SDS - PAGE和免疫印迹分析。使用用Lowicryl K4M包埋的仓鼠睾丸和胶体金偶联二抗进行免疫电子显微镜分析。此外，使用1C9 - 琼脂糖柱进行免疫亲和纯化。
在免疫印迹分析中，1C9还分别在大鼠和小鼠睾丸中识别出一种103 kDa蛋白质和一种101 kDa蛋白质。除睾丸外的十种不同仓鼠组织对1C9无反应性。在仓鼠睾丸的免疫染色石蜡切片中，最初的染色出现在粗线期中期精母细胞，并持续到成熟阶段的精子细胞（第15步）。然而，在精子细胞的后续阶段中不再能检测到。此外，在延长型精子细胞的核后区域观察到强染色。免疫电子显微镜分析表明，该蛋白质定位于生精细胞的内质网（ER）和核膜，但不存在于其他细胞器，如精子细胞的高尔基体和顶体中。这种蛋白质似乎与ER膜相关。此外，这种蛋白质仅存在于睾丸微粒体部分，而不存在于胞质溶胶中。通过亲和纯化，从10个仓鼠睾丸中获得了约320微克的103 kDa蛋白质。纯化的103 kDa蛋白质不受N - 聚糖酶的影响，表明它没有天冬酰胺连接的糖缀合物。
这些结果表明，1C9识别的蛋白质似乎是一种独特的蛋白质，定位于生精细胞的内质网和核膜。
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