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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

Analysis of precise stage of protein expression during the seminiferous cycle in adult testis. Seminiferous tubules in different stages were immunostained with MC321. A: Stage II. B: Stage III. C: Stage V. D: Stage VII. E: Stage XIII. The sections were counterstained with hematoxylin. Note that the immunoreactivity in the cytoplasm and flagella of the spermatids varies with the spermatogenic cycle stage. Bars=50 µm (A–E). F: Control section with the preimmune serum without counterstaining. DIC images. Bar=200 µm.
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Figure 5: Analysis of precise stage of protein expression during the seminiferous cycle in adult testis. Seminiferous tubules in different stages were immunostained with MC321. A: Stage II. B: Stage III. C: Stage V. D: Stage VII. E: Stage XIII. The sections were counterstained with hematoxylin. Note that the immunoreactivity in the cytoplasm and flagella of the spermatids varies with the spermatogenic cycle stage. Bars=50 µm (A–E). F: Control section with the preimmune serum without counterstaining. DIC images. Bar=200 µm.

Mentions: We next determined the distribution and change in expression of GAPDS in adult testes by immunohistochemistry. Immunoreactivity was detected in the cytoplasm and flagella of the elongating spermatids, as well as at the sites near the basal membrane and around the head caps of the elongating spermatids (Fig. 5A–E). Although multiple sites were immunoreactive, judging from the results of the experiment with the busulfan-treated testes (Fig. 2), the immunoreactivity in the cytoplasm and flagella of the elongating spermatids corresponds to GAPDS. The immunoreactivity around the head cap is assumed to be localized either to spermatid and/or Sertoli cells. To determine the precise localization, post-embedding immunogold staining was done in the testis. Gold particles were restricted to the actin layer of ectoplasmic specialization in Sertoli cells (Fig. 6). No or scarcely any gold labeling was found on the nucleus and acrosome of the elongating spermatids. Control experiments showed no distinct gold labeling. The immunoreactive sites near the basal membrane correspond to the blood-testis barrier which is formed by the inter-Sertoli tight junctions. In this study, we focused on the temporal sequence of the GAPDS expression appearing in the cytoplasm and flagellum of the elongating spermatids. In the seminiferous tubules at stage II, a weak immunoreaction was first detectable in both the cytoplasm and the flagella of the early step-16 spermatids (Fig. 5A). In the earlier stages, no immunoreaction was detectable in either the flagella or cytoplasm of the spermatids (Fig. 5E). The intensity of the immunoreaction increased as spermiogenesis proceeded (Fig. 5B, C), reached a maximum in the late step-17 spermatids in the stage-V seminiferous tubules (Fig. 5C), and persisted until the terminal stage of spermiogenesis (Fig. 5D). No immunoreactivity was found in the nucleus of any cells in the testis, nor in the cytoplasm of spermatogonia, spermatocytes, peritubular myoid cells, Leydig cells, or blood vessels. The control sections with the preimmune serum showed no immunoreactivity (Fig. 5F).


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)

Analysis of precise stage of protein expression during the seminiferous cycle in adult testis. Seminiferous tubules in different stages were immunostained with MC321. A: Stage II. B: Stage III. C: Stage V. D: Stage VII. E: Stage XIII. The sections were counterstained with hematoxylin. Note that the immunoreactivity in the cytoplasm and flagella of the spermatids varies with the spermatogenic cycle stage. Bars=50 µm (A–E). F: Control section with the preimmune serum without counterstaining. DIC images. Bar=200 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1828079&req=5

Figure 5: Analysis of precise stage of protein expression during the seminiferous cycle in adult testis. Seminiferous tubules in different stages were immunostained with MC321. A: Stage II. B: Stage III. C: Stage V. D: Stage VII. E: Stage XIII. The sections were counterstained with hematoxylin. Note that the immunoreactivity in the cytoplasm and flagella of the spermatids varies with the spermatogenic cycle stage. Bars=50 µm (A–E). F: Control section with the preimmune serum without counterstaining. DIC images. Bar=200 µm.
Mentions: We next determined the distribution and change in expression of GAPDS in adult testes by immunohistochemistry. Immunoreactivity was detected in the cytoplasm and flagella of the elongating spermatids, as well as at the sites near the basal membrane and around the head caps of the elongating spermatids (Fig. 5A–E). Although multiple sites were immunoreactive, judging from the results of the experiment with the busulfan-treated testes (Fig. 2), the immunoreactivity in the cytoplasm and flagella of the elongating spermatids corresponds to GAPDS. The immunoreactivity around the head cap is assumed to be localized either to spermatid and/or Sertoli cells. To determine the precise localization, post-embedding immunogold staining was done in the testis. Gold particles were restricted to the actin layer of ectoplasmic specialization in Sertoli cells (Fig. 6). No or scarcely any gold labeling was found on the nucleus and acrosome of the elongating spermatids. Control experiments showed no distinct gold labeling. The immunoreactive sites near the basal membrane correspond to the blood-testis barrier which is formed by the inter-Sertoli tight junctions. In this study, we focused on the temporal sequence of the GAPDS expression appearing in the cytoplasm and flagellum of the elongating spermatids. In the seminiferous tubules at stage II, a weak immunoreaction was first detectable in both the cytoplasm and the flagella of the early step-16 spermatids (Fig. 5A). In the earlier stages, no immunoreaction was detectable in either the flagella or cytoplasm of the spermatids (Fig. 5E). The intensity of the immunoreaction increased as spermiogenesis proceeded (Fig. 5B, C), reached a maximum in the late step-17 spermatids in the stage-V seminiferous tubules (Fig. 5C), and persisted until the terminal stage of spermiogenesis (Fig. 5D). No immunoreactivity was found in the nucleus of any cells in the testis, nor in the cytoplasm of spermatogonia, spermatocytes, peritubular myoid cells, Leydig cells, or blood vessels. The control sections with the preimmune serum showed no immunoreactivity (Fig. 5F).

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