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An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Yang Z, Gallicano GI, Yu QC, Fuchs E - J. Cell Biol. (1997)

Bottom Line: In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes.Cell.Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The Howard Hughes Medical Institute, The University of Chicago, Illinois 60637, USA.

ABSTRACT
Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

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Related in: MedlinePlus

Electron and immunoelectron microscopy of mouse spermatids. Mouse testes from adult animals were fixed and processed  for either regular or immunoelectron microscopy as described in the Materials and Methods. For immunoelectron microscopy, we used  either αBSN (UC56) or anti–γ-tubulin (Shu and Joshi, 1995) antibodies. (A and B) Negatively stained sections of spermatid centrioles.  The tip of the proximal centriole (px) is always associated with the nuclear envelope through implantation foci (if). Its free tip is always  encased by a cloud of pericentriolar material; the distal centriole (dis) is always the site of 9 + 2 axoneme assembly and most sections revealed an attached appendage(s) or satellite structure(s) (arrowhead) located near the junction of the two centrioles and often in close  proximity to the nuclear envelope. The manchette (ma) of microtubules that surrounds the lower hemisphere of the nucleus is formed at  the acrosomal cap phase and disappears shortly after nuclear elongation and flagellar assembly (B). (C and D) Immunogold labeling of  centrioles with αBSN. Note that the appendages of the distal centrioles (arrowheads) labeled specifically with the antibody. Inset shows  a cross section of an appendage, revealing a hollow center to the structure. (E) Immunogold labeling of centrioles with anti–γ-tubulin.  Note that this antibody heavily labeled the pericentriolar cloud at the tip of the proximal centriole and, to a lesser extent, showed some  labeling near the distal appendages. (F) αBSN immunogold labeling of the acrosomal cap (ac) of a late-stage spermatid. Note that most  of the labeling is concentrated near the outer membrane (om) of the cap rather than the inner membrane (im) or acrosomal space. Note  the elongated nucleus, characteristic of nearly mature sperm. (G) Immunogold labeling of the sheath of mitochondria (mi) in the middle  piece surrounding the axoneme (Ax). Additional abbreviations: Nu, nucleus; A, annulus. Bar: (A–D) 0.2 μm; (E and F) 0.3 μm; (G) 0.4 μm.
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Figure 10: Electron and immunoelectron microscopy of mouse spermatids. Mouse testes from adult animals were fixed and processed for either regular or immunoelectron microscopy as described in the Materials and Methods. For immunoelectron microscopy, we used either αBSN (UC56) or anti–γ-tubulin (Shu and Joshi, 1995) antibodies. (A and B) Negatively stained sections of spermatid centrioles. The tip of the proximal centriole (px) is always associated with the nuclear envelope through implantation foci (if). Its free tip is always encased by a cloud of pericentriolar material; the distal centriole (dis) is always the site of 9 + 2 axoneme assembly and most sections revealed an attached appendage(s) or satellite structure(s) (arrowhead) located near the junction of the two centrioles and often in close proximity to the nuclear envelope. The manchette (ma) of microtubules that surrounds the lower hemisphere of the nucleus is formed at the acrosomal cap phase and disappears shortly after nuclear elongation and flagellar assembly (B). (C and D) Immunogold labeling of centrioles with αBSN. Note that the appendages of the distal centrioles (arrowheads) labeled specifically with the antibody. Inset shows a cross section of an appendage, revealing a hollow center to the structure. (E) Immunogold labeling of centrioles with anti–γ-tubulin. Note that this antibody heavily labeled the pericentriolar cloud at the tip of the proximal centriole and, to a lesser extent, showed some labeling near the distal appendages. (F) αBSN immunogold labeling of the acrosomal cap (ac) of a late-stage spermatid. Note that most of the labeling is concentrated near the outer membrane (om) of the cap rather than the inner membrane (im) or acrosomal space. Note the elongated nucleus, characteristic of nearly mature sperm. (G) Immunogold labeling of the sheath of mitochondria (mi) in the middle piece surrounding the axoneme (Ax). Additional abbreviations: Nu, nucleus; A, annulus. Bar: (A–D) 0.2 μm; (E and F) 0.3 μm; (G) 0.4 μm.

Mentions: To further examine basonuclin expression during spermiogenesis, we conducted electron and immunoelectron microscopy (Fig. 10). Fig. 10 A provides an example of the typical pair of centrioles that associates with the nuclear envelope during the acrosomal cap phase of spermiogenesis (also see diagram in Fig. 1 B). At this stage of differentiation, centrioles migrate to the nuclear pole opposing the acrosomal cap. The 9 + 2 axoneme assembly of the flagellum always initiates from the end of the distal centriole (Fig. 10, A and C, dis), which contains a prominent electron-dense satellite appendage often in close proximity to the nuclear envelope (Fig. 10 A and B, arrowheads). The proximal centriole (Fig. 10, A and C, px), laterally aligned with the nuclear envelope, is not directly involved in flagellar assembly, and its fate is unknown. Concomitant with the attachment of centrioles to the nuclear envelope, the cylinder of manchette microtubules forms around the lower half of the nucleus as it elongates (Fig. 10 B, ma).


An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Yang Z, Gallicano GI, Yu QC, Fuchs E - J. Cell Biol. (1997)

Electron and immunoelectron microscopy of mouse spermatids. Mouse testes from adult animals were fixed and processed  for either regular or immunoelectron microscopy as described in the Materials and Methods. For immunoelectron microscopy, we used  either αBSN (UC56) or anti–γ-tubulin (Shu and Joshi, 1995) antibodies. (A and B) Negatively stained sections of spermatid centrioles.  The tip of the proximal centriole (px) is always associated with the nuclear envelope through implantation foci (if). Its free tip is always  encased by a cloud of pericentriolar material; the distal centriole (dis) is always the site of 9 + 2 axoneme assembly and most sections revealed an attached appendage(s) or satellite structure(s) (arrowhead) located near the junction of the two centrioles and often in close  proximity to the nuclear envelope. The manchette (ma) of microtubules that surrounds the lower hemisphere of the nucleus is formed at  the acrosomal cap phase and disappears shortly after nuclear elongation and flagellar assembly (B). (C and D) Immunogold labeling of  centrioles with αBSN. Note that the appendages of the distal centrioles (arrowheads) labeled specifically with the antibody. Inset shows  a cross section of an appendage, revealing a hollow center to the structure. (E) Immunogold labeling of centrioles with anti–γ-tubulin.  Note that this antibody heavily labeled the pericentriolar cloud at the tip of the proximal centriole and, to a lesser extent, showed some  labeling near the distal appendages. (F) αBSN immunogold labeling of the acrosomal cap (ac) of a late-stage spermatid. Note that most  of the labeling is concentrated near the outer membrane (om) of the cap rather than the inner membrane (im) or acrosomal space. Note  the elongated nucleus, characteristic of nearly mature sperm. (G) Immunogold labeling of the sheath of mitochondria (mi) in the middle  piece surrounding the axoneme (Ax). Additional abbreviations: Nu, nucleus; A, annulus. Bar: (A–D) 0.2 μm; (E and F) 0.3 μm; (G) 0.4 μm.
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Related In: Results  -  Collection

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Figure 10: Electron and immunoelectron microscopy of mouse spermatids. Mouse testes from adult animals were fixed and processed for either regular or immunoelectron microscopy as described in the Materials and Methods. For immunoelectron microscopy, we used either αBSN (UC56) or anti–γ-tubulin (Shu and Joshi, 1995) antibodies. (A and B) Negatively stained sections of spermatid centrioles. The tip of the proximal centriole (px) is always associated with the nuclear envelope through implantation foci (if). Its free tip is always encased by a cloud of pericentriolar material; the distal centriole (dis) is always the site of 9 + 2 axoneme assembly and most sections revealed an attached appendage(s) or satellite structure(s) (arrowhead) located near the junction of the two centrioles and often in close proximity to the nuclear envelope. The manchette (ma) of microtubules that surrounds the lower hemisphere of the nucleus is formed at the acrosomal cap phase and disappears shortly after nuclear elongation and flagellar assembly (B). (C and D) Immunogold labeling of centrioles with αBSN. Note that the appendages of the distal centrioles (arrowheads) labeled specifically with the antibody. Inset shows a cross section of an appendage, revealing a hollow center to the structure. (E) Immunogold labeling of centrioles with anti–γ-tubulin. Note that this antibody heavily labeled the pericentriolar cloud at the tip of the proximal centriole and, to a lesser extent, showed some labeling near the distal appendages. (F) αBSN immunogold labeling of the acrosomal cap (ac) of a late-stage spermatid. Note that most of the labeling is concentrated near the outer membrane (om) of the cap rather than the inner membrane (im) or acrosomal space. Note the elongated nucleus, characteristic of nearly mature sperm. (G) Immunogold labeling of the sheath of mitochondria (mi) in the middle piece surrounding the axoneme (Ax). Additional abbreviations: Nu, nucleus; A, annulus. Bar: (A–D) 0.2 μm; (E and F) 0.3 μm; (G) 0.4 μm.
Mentions: To further examine basonuclin expression during spermiogenesis, we conducted electron and immunoelectron microscopy (Fig. 10). Fig. 10 A provides an example of the typical pair of centrioles that associates with the nuclear envelope during the acrosomal cap phase of spermiogenesis (also see diagram in Fig. 1 B). At this stage of differentiation, centrioles migrate to the nuclear pole opposing the acrosomal cap. The 9 + 2 axoneme assembly of the flagellum always initiates from the end of the distal centriole (Fig. 10, A and C, dis), which contains a prominent electron-dense satellite appendage often in close proximity to the nuclear envelope (Fig. 10 A and B, arrowheads). The proximal centriole (Fig. 10, A and C, px), laterally aligned with the nuclear envelope, is not directly involved in flagellar assembly, and its fate is unknown. Concomitant with the attachment of centrioles to the nuclear envelope, the cylinder of manchette microtubules forms around the lower half of the nucleus as it elongates (Fig. 10 B, ma).

Bottom Line: In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes.Cell.Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The Howard Hughes Medical Institute, The University of Chicago, Illinois 60637, USA.

ABSTRACT
Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

Show MeSH
Related in: MedlinePlus