Assuntos
Linfadenite Histiocítica Necrosante , Linfo-Histiocitose Hemofagocítica , Humanos , Linfadenite Histiocítica Necrosante/complicações , Linfadenite Histiocítica Necrosante/diagnóstico , Linfo-Histiocitose Hemofagocítica/complicações , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/tratamento farmacológicoRESUMO
Mammalian spermatogenesis is a well-organized process of cell development and differentiation. Meiosis expressed gene 1 (MEIG1) plays an essential role in the regulation of spermiogenesis. To explore potential mechanisms of MEIG1's action, a yeast two-hybrid screen was conducted, and several potential binding partners were identified; one of them was membrane occupation and recognition nexus repeat containing 3 (MORN3). MORN3 mRNA is only abundant in mouse testis. In the testis, Morn3 mRNA is highly expressed in the spermiogenesis stage. Specific anti-MORN3 polyclonal antibody was generated against N-terminus of the full-length MORN3 protein, and MORN3 expression and localization was examined in vitro and in vivo. In transfected Chinese hamster ovary cells, the antibody specifically crossed-reacted the full-length MORN3 protein, and immunofluorescence staining revealed that MORN3 was localized throughout the cytoplasm. Among multiple mouse tissues, about 25 kDa protein, was identified only in the testis. The protein was highly expressed after day 20 of birth. Immunofluorescence staining on mixed testicular cells isolated from adult wild-type mice demonstrated that MORN3 was expressed in the acrosome in germ cells throughout spermiogenesis. The protein was also present in the manchette of elongating spermatids. The total MORN3 expression and acrosome localization were not changed in the Meig 1-deficient mice. However, its expression in manchette was dramatically reduced in the mutant mice. Our studies suggest that MORN3 is another regulator for spermatogenesis, probably together with MEIG1.
Assuntos
Acrossomo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Infertilidade Masculina/metabolismo , Infertilidade Masculina/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , RNA Mensageiro/genética , Espermatogênese/genética , Testículo/metabolismoRESUMO
OBJECTIVE: To construct a mammalian expression plasmid of the BC022687 gene and investigate the expression and localization of the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The BC022687 coding sequence was amplified by polymerase chain reaction (PCR) and subcloned into the pEGFP-C1 vector carrying the gene of green fluorescence protein (GFP). After the target region was sequenced, the recombinant plasmid was transfected into CHO cells, and its expression in the CHO cells was determined by Western blot. The localization of GFP-tagged BC022687 in the CHO cells was observed by laser scanning confocal microscopy. RESULTS: BC022687 was successfully cloned into the mammalian expression vector pEGFP-C1, with the restriction fragment length of 950 bp. The expression of the fusion protein was confirmed, with the relative molecular weight of 64 000. The GFP-tagged BC022687 protein was mainly localized in the cytoplasm, and also presented in the centrioles in the transfected CHO cells. CONCLUSION: The successful construction of the plasmid expressing BC022687 in CHO cells has laid a foundation for further studies on the role of this protein in ciliogenesis.
