Characterization of a testis specific protein localized to endoplasmic reticulum of spermatogenic cells

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.