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Fu J.,Chinese Institute of Basic Medical Sciences | Zong S.-D.,National Research Institute for Family Planning Beijing | Miao S.-Y.,Chinese Institute of Basic Medical Sciences | Song W.,Chinese Institute of Basic Medical Sciences
Acta Academiae Medicinae Sinicae | Year: 2013

Objective: To obtain recombinant sperm-protein actin-like protein 7a (ACTL7a) and detect the damage seminiferous tubules in mouse testis caused by anti-sperm antibodies generated by purified ACTL7a active immunization. Methods: The recombinant expression plasmid pET30a-ACTL7a was constructed and then transformed into E. coli Rosseta (DE3). The protein expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the protein was purified by nickel ions chelating resin. Finally, the protein was separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) and harvested by excising the gel containing target. ICR (Institute of Cancer Research) mice were then immunized using purified ACTL7a protein. The antibody titers were determined by ELISA and the development of seminiferous tubules after active immunization was stained by PAS staining. Results: Induced by IPTG, the target protein ACTL7a was expressed in E. coli. After purification, it was used to immunize the ICR mice. As shown by PAS staining, spermatid expulsion, pyknotic cells, absence of germ cells, and germ cells degenerated were seen in the seminiferous tubules in the immunized testes. Conclusions: The ACTL7a prokaryotic expression vector was successfully constructed. High-purity target protein was obtained after induction and purification. After the active immunization with the target protein, the seminiferous tubules in the mouse testes will be severely damaged. Source

Wang H.,Tsinghua University | Wang H.,Shanxi Medical University | Song W.,Tsinghua University | Hu T.,Tsinghua University | And 4 more authors.
Cellular and Molecular Life Sciences | Year: 2011

Regulation of apoptosis at various stages of differentiation plays an important role in spermatogenesis. Therefore, the identification and characterisation of highly expressed genes in the testis that are involved in apoptosis is of great value to delineate the mechanism of spermatogenesis. Here, we reported that Fank1, a novel gene highly expressed in testis, functioned as an anti-apoptotic protein that activated the activator protein 1 (AP-1) pathway. We found that Jab1 (Jun activation domain-binding protein 1), a co-activator of AP-1, specifically interacted with Fank1. Reporter analyses showed that Fank1 activated AP-1 pathway in a Jab1-dependent manner. Fank1 overexpression also increased the expression and activation of endogenous c-Jun. Further study showed that Fank1 inhibited cell apoptosis by upregulating and activating endogenous c-Jun and its downstream target, Bcl-3. This process was shown to be Jab1 dependent. Taken together, our results indicated that by interacting with Jab1, Fank1 could suppress cell apoptosis by activating the AP-1-induced anti-apoptotic pathway. © 2010 Springer Basel AG. Source

Cheng X.,Peking Union Medical College | Liang J.,Peking Union Medical College | Teng Y.,Capital Medical University | Fu J.,Peking Union Medical College | And 3 more authors.
FEBS Letters | Year: 2012

Spermatogenesis is an extremely intricate process that is tightly regulated and orchestrated by a series of well-coordinated gene expression programmes. Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine kinase that functions in a wide variety of developmental events. Nevertheless, the function of NLK in spermatogenesis has not been investigated. In this study, we found that the distribution of NLK in mice exhibited a dynamic change during testicular development and gradually became concentrated in the acrosomes of elongated spermatids. NLK overexpression promoted etoposide-induced apoptosis of male germ cell-derived GC-1 cells, while knockdown of NLK by RNA interference (RNAi) attenuated etoposide-induced apoptosis. Our findings suggest that NLK plays an important role in etoposide-induced germ cell apoptosis and may be associated with spermatogenesis. © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Source

Wu H.,Peking Union Medical College | Chen Y.,Peking Union Medical College | Miao S.,Peking Union Medical College | Zhang C.,National Research Institute for Family Planning Beijing | And 3 more authors.
FEBS Letters | Year: 2010

cAMP response element modulator (CREM)-mediated gene expression is an essential regulatory mechanism for germ cell differentiation. CREM and its coactivator in testis, ACT, activate the transcription of many essential genes for spermatogenesis. Sperm associated antigen 8 (SPAG8) is a testis-specific component that is expressed during germ cell differentiation. In this study, we found the pattern of SPAG8 expression largely overlapped with that of ACT during spermatogenesis and verified the association of SPAG8 with ACT. Furthermore, we showed that SPAG8 enhanced the transcriptional activation of ACT-mediated CREMτ by strengthening the binding of ACT to CREMτ. These results indicate that SPAG8 acts as a regulator of ACT and plays an important role in CREM-ACT-mediated gene transcription during spermatogenesis. Structured summary: MINT-7892874: CREM tau (uniprotkb:. P27699-1) physically interacts (MI:. 0915) with ACT (uniprotkb:. Q9WTX7) by anti tag coimmunoprecipitation (MI:. 0007). MINT-7892809: ACT (uniprotkb:. Q9WTX7) physically interacts (MI:. 0915) with SPAG8 (uniprotkb:. B9EKF1) by pull down (MI:. 0096). MINT-7892820, MINT-7892840, MINT-7892987, MINT-7892854: ACT (uniprotkb:. Q9WTX7) physically interacts (MI:. 0915) with SPAG8 (uniprotkb:. B9EKF1) by anti tag coimmunoprecipitation (MI:. 0007). © 2010 Federation of European Biochemical Societies. Source

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