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Characterization of eight novel proteins with male germ cell-specific expression in mouse.

Baek N, Woo JM, Han C, Choi E, Park I, Kim do H, Eddy EM, Cho C - Reprod. Biol. Endocrinol. (2008)

Bottom Line: Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes.The authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed.We analyzed eight novel germ cell-specific proteins, providing new and inclusive information about their developmental and cellular characteristics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science and Research Center for Biomolecular Nanotechnology, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea. NamHo.Baek@celltrion.com

ABSTRACT

Background: Spermatogenesis and fertilization are highly unique processes. Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes. We investigated eight proteins encoded by novel spermatogenic cell-specific genes previously identified from the mouse round spermatid UniGene library.

Methods: Polyclonal antibodies were generated against the novel proteins and western blot analysis was performed with various protein samples. Germ cell specificity was investigated using testes from germ cell-less mutant mice. Developmental expression pattern was examined in testicular germ cells, testicular sperm and mature sperm. Subcellular localization was assessed by cell surface biotin labeling and trypsinization. Protein localization and properties in sperm were investigated by separation of head and tail fractions, and extractabilities by a non-ionic detergent and urea.

Results: The authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed. In examining the developmental expression patterns, we found the presence of four proteins only in testicular germ cells, a single protein in testicular germ cells and testicular sperm, and three proteins in the testicular stages and mature sperm from the epididymis. Further analysis of the three proteins present in sperm disclosed that one is located at the surface of the acrosomal region and the other two are associated with cytoskeletal structures in the sperm flagellum. We name the genes for these sperm proteins Shsp1 (Sperm head surface protein 1), Sfap1 (Sperm flagellum associated protein 1) and Sfap2 (Sperm flagellum associated protein 2).

Conclusion: We analyzed eight novel germ cell-specific proteins, providing new and inclusive information about their developmental and cellular characteristics. Our findings will facilitate future investigation into the biological roles of these novel proteins in spermatogenesis and sperm functions.

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Characterization of Shsp1/Mm.87328, Sfap1/Mm.386907 and Sfap2/Mm.157049. (A) Acrosome reaction of sperm from the epididymis and vas deferens was induced by calcium ionophore A23187. Acrosome-intact and -reacted sperm were subjected to western blot anaylsis with the anti-Shsp1/Mm.87328 antibody. AI, acrosome-intact sperm; AR, acrosome-reacted sperm. (B) Sperm from the epididymis and vas deferens were treated with 1% Triton X-100 and urea with different concentrations (2, 3, 4 and 6 M). Soluble and insoluble fractions after centrifugation of the treated sperm were subjected to western blot anaylsis with the anti-Sfap1/Mm.386907 and anti-Sfap2/Mm.157049 antibodies. Sup, supernatant after centrifugation; Pt, pellet after centrifugation.
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Figure 6: Characterization of Shsp1/Mm.87328, Sfap1/Mm.386907 and Sfap2/Mm.157049. (A) Acrosome reaction of sperm from the epididymis and vas deferens was induced by calcium ionophore A23187. Acrosome-intact and -reacted sperm were subjected to western blot anaylsis with the anti-Shsp1/Mm.87328 antibody. AI, acrosome-intact sperm; AR, acrosome-reacted sperm. (B) Sperm from the epididymis and vas deferens were treated with 1% Triton X-100 and urea with different concentrations (2, 3, 4 and 6 M). Soluble and insoluble fractions after centrifugation of the treated sperm were subjected to western blot anaylsis with the anti-Sfap1/Mm.386907 and anti-Sfap2/Mm.157049 antibodies. Sup, supernatant after centrifugation; Pt, pellet after centrifugation.

Mentions: To further examine the characteristics and localization of Shsp1/Mm.87328, we compared the protein between acrosome-intact and -reacted sperm. During acrosome reaction, plasma membrane overlying the acrosome fuses with outer acrosomal membrane and consequently the two membranes are lost from the sperm head. We found that Shsp1/Mm.87328, present in acrosome-intact sperm, is absent from acrosome-reacted sperm (Fig. 6A). Thus, together with the finding on subcellular localization (Fig. 5A), this suggests that Shsp1/Mm.87328 is localized on the plasma membrane of the acrosomal region.


Characterization of eight novel proteins with male germ cell-specific expression in mouse.

Baek N, Woo JM, Han C, Choi E, Park I, Kim do H, Eddy EM, Cho C - Reprod. Biol. Endocrinol. (2008)

Characterization of Shsp1/Mm.87328, Sfap1/Mm.386907 and Sfap2/Mm.157049. (A) Acrosome reaction of sperm from the epididymis and vas deferens was induced by calcium ionophore A23187. Acrosome-intact and -reacted sperm were subjected to western blot anaylsis with the anti-Shsp1/Mm.87328 antibody. AI, acrosome-intact sperm; AR, acrosome-reacted sperm. (B) Sperm from the epididymis and vas deferens were treated with 1% Triton X-100 and urea with different concentrations (2, 3, 4 and 6 M). Soluble and insoluble fractions after centrifugation of the treated sperm were subjected to western blot anaylsis with the anti-Sfap1/Mm.386907 and anti-Sfap2/Mm.157049 antibodies. Sup, supernatant after centrifugation; Pt, pellet after centrifugation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2500023&req=5

Figure 6: Characterization of Shsp1/Mm.87328, Sfap1/Mm.386907 and Sfap2/Mm.157049. (A) Acrosome reaction of sperm from the epididymis and vas deferens was induced by calcium ionophore A23187. Acrosome-intact and -reacted sperm were subjected to western blot anaylsis with the anti-Shsp1/Mm.87328 antibody. AI, acrosome-intact sperm; AR, acrosome-reacted sperm. (B) Sperm from the epididymis and vas deferens were treated with 1% Triton X-100 and urea with different concentrations (2, 3, 4 and 6 M). Soluble and insoluble fractions after centrifugation of the treated sperm were subjected to western blot anaylsis with the anti-Sfap1/Mm.386907 and anti-Sfap2/Mm.157049 antibodies. Sup, supernatant after centrifugation; Pt, pellet after centrifugation.
Mentions: To further examine the characteristics and localization of Shsp1/Mm.87328, we compared the protein between acrosome-intact and -reacted sperm. During acrosome reaction, plasma membrane overlying the acrosome fuses with outer acrosomal membrane and consequently the two membranes are lost from the sperm head. We found that Shsp1/Mm.87328, present in acrosome-intact sperm, is absent from acrosome-reacted sperm (Fig. 6A). Thus, together with the finding on subcellular localization (Fig. 5A), this suggests that Shsp1/Mm.87328 is localized on the plasma membrane of the acrosomal region.

Bottom Line: Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes.The authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed.We analyzed eight novel germ cell-specific proteins, providing new and inclusive information about their developmental and cellular characteristics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science and Research Center for Biomolecular Nanotechnology, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea. NamHo.Baek@celltrion.com

ABSTRACT

Background: Spermatogenesis and fertilization are highly unique processes. Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes. We investigated eight proteins encoded by novel spermatogenic cell-specific genes previously identified from the mouse round spermatid UniGene library.

Methods: Polyclonal antibodies were generated against the novel proteins and western blot analysis was performed with various protein samples. Germ cell specificity was investigated using testes from germ cell-less mutant mice. Developmental expression pattern was examined in testicular germ cells, testicular sperm and mature sperm. Subcellular localization was assessed by cell surface biotin labeling and trypsinization. Protein localization and properties in sperm were investigated by separation of head and tail fractions, and extractabilities by a non-ionic detergent and urea.

Results: The authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed. In examining the developmental expression patterns, we found the presence of four proteins only in testicular germ cells, a single protein in testicular germ cells and testicular sperm, and three proteins in the testicular stages and mature sperm from the epididymis. Further analysis of the three proteins present in sperm disclosed that one is located at the surface of the acrosomal region and the other two are associated with cytoskeletal structures in the sperm flagellum. We name the genes for these sperm proteins Shsp1 (Sperm head surface protein 1), Sfap1 (Sperm flagellum associated protein 1) and Sfap2 (Sperm flagellum associated protein 2).

Conclusion: We analyzed eight novel germ cell-specific proteins, providing new and inclusive information about their developmental and cellular characteristics. Our findings will facilitate future investigation into the biological roles of these novel proteins in spermatogenesis and sperm functions.

Show MeSH
Related in: MedlinePlus