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Preferential localization of rat GAPDS on the ribs of fibrous sheath of sperm flagellum and its expression during flagellar formation.

Tanii I, Yagura T, Inagaki N, Nakayama T, Imaizumi K, Yoshinaga K - Acta Histochem Cytochem (2007)

Bottom Line: To obtain information on the assembly of the glycolytic enzyme into the flagellum, the precise localization of rat GAPDS in the flagellum and the stage of incorporation into the flagellum were examined using a monoclonal antibody.Immunoreactivity was first detected in the cytoplasm and flagella of the step-16 spermatids during the final step of FS formation.Together with the expression of other FS proteins, the present results indicate the sequential assembly of FS components, suggesting that the expression and transport of GAPDS is regulated in a coordinated manner during sperm flagellar formation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan.

ABSTRACT
The proper assembly of sperm flagellar proteins is fundamental for sperm motility. The sperm- and spermatid-specific isoform of glyceraldehyde 3-phosphate dehydrogenase, GAPDS, is a flagellar protein indispensable for sperm flagellar movement. To obtain information on the assembly of the glycolytic enzyme into the flagellum, the precise localization of rat GAPDS in the flagellum and the stage of incorporation into the flagellum were examined using a monoclonal antibody. Immunolocalization of rat GAPDS was restricted to the fibrous sheath (FS) in the sperm flagellum, and was predominant in the circumferential ribs rather than the longitudinal columns. Immunoreactivity was first detected in the cytoplasm and flagella of the step-16 spermatids during the final step of FS formation. Together with the expression of other FS proteins, the present results indicate the sequential assembly of FS components, suggesting that the expression and transport of GAPDS is regulated in a coordinated manner during sperm flagellar formation.

No MeSH data available.


Related in: MedlinePlus

Partial purification and identification of the antigenic 64-kDa protein. A: Partial purification by MC321-immunoaffinity chromatography. Total proteins (a) or proteins isolated by MC321-immunoaffinity chromatography (b, c) were resolved in reducing SDS gels, and visualized with Coomassie brilliant blue (a, b), or examined by Western blotting with MC321 (c). Numbers on left indicate molecular weight markers (×10−3). B: MALDI-TOF mass spectrum of the 64-kDa protein after in-gel trypsin digestion. Asterisk labeled peptides matched the rat GAPDS sequence. A tryptic peptide is indicated by T. C: Rat GAPDS sequence. Peptides in bold have been matched to the peptides in MALDI MS.
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Figure 3: Partial purification and identification of the antigenic 64-kDa protein. A: Partial purification by MC321-immunoaffinity chromatography. Total proteins (a) or proteins isolated by MC321-immunoaffinity chromatography (b, c) were resolved in reducing SDS gels, and visualized with Coomassie brilliant blue (a, b), or examined by Western blotting with MC321 (c). Numbers on left indicate molecular weight markers (×10−3). B: MALDI-TOF mass spectrum of the 64-kDa protein after in-gel trypsin digestion. Asterisk labeled peptides matched the rat GAPDS sequence. A tryptic peptide is indicated by T. C: Rat GAPDS sequence. Peptides in bold have been matched to the peptides in MALDI MS.

Mentions: To isolate the antigens, testicular cytosolic proteins from adult rats were loaded onto an MC321-immunoaffinity column, and the proteins specifically bound to the column were then eluted and analyzed by SDS-PAGE and Western blotting. In the gel stained by Coomassie brilliant blue (CBB), the 64-kDa protein was predominant, while several minor proteins with relative molecular masses of 67 kDa and 39 kDa were detected in the eluate (Fig. 3A). Among them, the 64-kDa protein was immunoreactive while the other proteins showed no immunoreactivity. An immunoreactive band of 59 kDa was detected in the Western blots, but no corresponding band was found in the CBB stained gel. Neither the CBB stained band nor immunoreactive band for the 77-kDa protein was detectable in the eluate. To identify the 64-kDa protein, a portion of the 64-kDa gel band was excised and submitted for proteolytic digestion and peptide identification by MALDI-TOF MS. As a result, the 64-kDa protein was unambiguously identified as GAPDS (GenBank accession no. AJ297631) (Fig. 3B, C).


Preferential localization of rat GAPDS on the ribs of fibrous sheath of sperm flagellum and its expression during flagellar formation.

Tanii I, Yagura T, Inagaki N, Nakayama T, Imaizumi K, Yoshinaga K - Acta Histochem Cytochem (2007)

Partial purification and identification of the antigenic 64-kDa protein. A: Partial purification by MC321-immunoaffinity chromatography. Total proteins (a) or proteins isolated by MC321-immunoaffinity chromatography (b, c) were resolved in reducing SDS gels, and visualized with Coomassie brilliant blue (a, b), or examined by Western blotting with MC321 (c). Numbers on left indicate molecular weight markers (×10−3). B: MALDI-TOF mass spectrum of the 64-kDa protein after in-gel trypsin digestion. Asterisk labeled peptides matched the rat GAPDS sequence. A tryptic peptide is indicated by T. C: Rat GAPDS sequence. Peptides in bold have been matched to the peptides in MALDI MS.
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Related In: Results  -  Collection

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Figure 3: Partial purification and identification of the antigenic 64-kDa protein. A: Partial purification by MC321-immunoaffinity chromatography. Total proteins (a) or proteins isolated by MC321-immunoaffinity chromatography (b, c) were resolved in reducing SDS gels, and visualized with Coomassie brilliant blue (a, b), or examined by Western blotting with MC321 (c). Numbers on left indicate molecular weight markers (×10−3). B: MALDI-TOF mass spectrum of the 64-kDa protein after in-gel trypsin digestion. Asterisk labeled peptides matched the rat GAPDS sequence. A tryptic peptide is indicated by T. C: Rat GAPDS sequence. Peptides in bold have been matched to the peptides in MALDI MS.
Mentions: To isolate the antigens, testicular cytosolic proteins from adult rats were loaded onto an MC321-immunoaffinity column, and the proteins specifically bound to the column were then eluted and analyzed by SDS-PAGE and Western blotting. In the gel stained by Coomassie brilliant blue (CBB), the 64-kDa protein was predominant, while several minor proteins with relative molecular masses of 67 kDa and 39 kDa were detected in the eluate (Fig. 3A). Among them, the 64-kDa protein was immunoreactive while the other proteins showed no immunoreactivity. An immunoreactive band of 59 kDa was detected in the Western blots, but no corresponding band was found in the CBB stained gel. Neither the CBB stained band nor immunoreactive band for the 77-kDa protein was detectable in the eluate. To identify the 64-kDa protein, a portion of the 64-kDa gel band was excised and submitted for proteolytic digestion and peptide identification by MALDI-TOF MS. As a result, the 64-kDa protein was unambiguously identified as GAPDS (GenBank accession no. AJ297631) (Fig. 3B, C).

Bottom Line: To obtain information on the assembly of the glycolytic enzyme into the flagellum, the precise localization of rat GAPDS in the flagellum and the stage of incorporation into the flagellum were examined using a monoclonal antibody.Immunoreactivity was first detected in the cytoplasm and flagella of the step-16 spermatids during the final step of FS formation.Together with the expression of other FS proteins, the present results indicate the sequential assembly of FS components, suggesting that the expression and transport of GAPDS is regulated in a coordinated manner during sperm flagellar formation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan.

ABSTRACT
The proper assembly of sperm flagellar proteins is fundamental for sperm motility. The sperm- and spermatid-specific isoform of glyceraldehyde 3-phosphate dehydrogenase, GAPDS, is a flagellar protein indispensable for sperm flagellar movement. To obtain information on the assembly of the glycolytic enzyme into the flagellum, the precise localization of rat GAPDS in the flagellum and the stage of incorporation into the flagellum were examined using a monoclonal antibody. Immunolocalization of rat GAPDS was restricted to the fibrous sheath (FS) in the sperm flagellum, and was predominant in the circumferential ribs rather than the longitudinal columns. Immunoreactivity was first detected in the cytoplasm and flagella of the step-16 spermatids during the final step of FS formation. Together with the expression of other FS proteins, the present results indicate the sequential assembly of FS components, suggesting that the expression and transport of GAPDS is regulated in a coordinated manner during sperm flagellar formation.

No MeSH data available.


Related in: MedlinePlus