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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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Detection of antibasonuclin immunofluorescence in the centrosomes of meiotic spermatocytes. Frozen sections (∼10 μm) of  mouse testis (2, 4, or 8 wk old, as indicated in the panels) were subjected to double or triple immunofluorescence, and sections were  viewed with a confocal microscope (Carl Zeiss, Inc.). Sections stained with anti-BSN antibodies (shown here are UC56 αBSN profiles)  were visualized by costaining with an FITC-conjugated anti–rabbit IgG secondary antibody (green). Sections stained with human H1 autoantisera (αH1) were visualized by costaining with a Texas red–conjugated anti–human IgG secondary antibody (red). Nuclei were labeled with DAPI (blue). (A) Seminiferous tubule at 2 wk, containing a mixture of mitotically active spermatogonia at the periphery and  primary spermatocytes in the midsection (Rugh, 1990); αBSN stained only midsection cells; (B and C) Seminiferous tubule at 4 wk, containing spermatogonia, primary spermatocytes, and secondary spermatocytes, located from the periphery to the centers of the tubules,  respectively; view is of midsection, double stained with: αBSN (B and C) and DAPI (C). Arrows denote typical single dot pattern. Inset  in B shows what is probably a secondary spermatocyte in late G2 or prophase, after duplication of the centrosomes; double labeling of  dots is with αBSN and αH1 antiserum. (D–F) Region of spermatocytes, triple labeled with αBSN (D and F), αH1 (E and F), and DAPI  (F). Yellow reveals costaining with αBSN and αH1. Portion of F to the left of the double arrows is the region shown in D and E. (G) 8-wk  sexually mature seminiferous tubule costained with αBSN, αH1, and DAPI. View is of spermatogonia, primary spermatocytes, and secondary spermatocytes. Note that spermatogonial centrosomes at the periphery only labeled with αH1 (arrowheads), while spermatocytes colabeled with αBSN and αH1 (arrows). Additional note: αBSN antibodies 372 and 176 gave similar immunofluorescence staining  patterns to those shown here. Dotted lines denote nuclear borders. Bars, 10 μm.
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Figure 7: Detection of antibasonuclin immunofluorescence in the centrosomes of meiotic spermatocytes. Frozen sections (∼10 μm) of mouse testis (2, 4, or 8 wk old, as indicated in the panels) were subjected to double or triple immunofluorescence, and sections were viewed with a confocal microscope (Carl Zeiss, Inc.). Sections stained with anti-BSN antibodies (shown here are UC56 αBSN profiles) were visualized by costaining with an FITC-conjugated anti–rabbit IgG secondary antibody (green). Sections stained with human H1 autoantisera (αH1) were visualized by costaining with a Texas red–conjugated anti–human IgG secondary antibody (red). Nuclei were labeled with DAPI (blue). (A) Seminiferous tubule at 2 wk, containing a mixture of mitotically active spermatogonia at the periphery and primary spermatocytes in the midsection (Rugh, 1990); αBSN stained only midsection cells; (B and C) Seminiferous tubule at 4 wk, containing spermatogonia, primary spermatocytes, and secondary spermatocytes, located from the periphery to the centers of the tubules, respectively; view is of midsection, double stained with: αBSN (B and C) and DAPI (C). Arrows denote typical single dot pattern. Inset in B shows what is probably a secondary spermatocyte in late G2 or prophase, after duplication of the centrosomes; double labeling of dots is with αBSN and αH1 antiserum. (D–F) Region of spermatocytes, triple labeled with αBSN (D and F), αH1 (E and F), and DAPI (F). Yellow reveals costaining with αBSN and αH1. Portion of F to the left of the double arrows is the region shown in D and E. (G) 8-wk sexually mature seminiferous tubule costained with αBSN, αH1, and DAPI. View is of spermatogonia, primary spermatocytes, and secondary spermatocytes. Note that spermatogonial centrosomes at the periphery only labeled with αH1 (arrowheads), while spermatocytes colabeled with αBSN and αH1 (arrows). Additional note: αBSN antibodies 372 and 176 gave similar immunofluorescence staining patterns to those shown here. Dotted lines denote nuclear borders. Bars, 10 μm.

Mentions: To assess the location of basonuclin within differentiating male germ cells, we conducted indirect immunofluorescence on frozen sections of developing mouse testes. Basonuclin protein was first detected in testis at 2 wk postnatally (Fig. 7 A). In contrast to BSN RNAs, which are expressed in mitotic spermatogonia, protein was not detected until the cells had differentiated into primary spermatocytes located at the midregion of the 2-wk seminiferous tubules. These cells, in the first meiotic phase of differentiating male germ cells, displayed a dotlike pattern of staining with the UC56 anti-BSN (αBSN) antibody. Double immunofluorescence with DAPI to stain chromatin indicated that the labeling was located near the nucleus (Fig. 7 A). Staining was more prevalent by 4 wk (Fig. 7, B and C), when primary and secondary spermatocytes exist (Rugh, 1990). While the majority of these spermatocytes contained single dots, a few seemingly contained double dots positioned at opposing sides of the nucleus (Fig. 7 B, inset). Similar staining patterns were observed with all three affinitypurified BSN antibodies, although antibodies UC56 (shown) and 372 gave the strongest staining. The pattern was not seen with secondary antibody alone.


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)

Detection of antibasonuclin immunofluorescence in the centrosomes of meiotic spermatocytes. Frozen sections (∼10 μm) of  mouse testis (2, 4, or 8 wk old, as indicated in the panels) were subjected to double or triple immunofluorescence, and sections were  viewed with a confocal microscope (Carl Zeiss, Inc.). Sections stained with anti-BSN antibodies (shown here are UC56 αBSN profiles)  were visualized by costaining with an FITC-conjugated anti–rabbit IgG secondary antibody (green). Sections stained with human H1 autoantisera (αH1) were visualized by costaining with a Texas red–conjugated anti–human IgG secondary antibody (red). Nuclei were labeled with DAPI (blue). (A) Seminiferous tubule at 2 wk, containing a mixture of mitotically active spermatogonia at the periphery and  primary spermatocytes in the midsection (Rugh, 1990); αBSN stained only midsection cells; (B and C) Seminiferous tubule at 4 wk, containing spermatogonia, primary spermatocytes, and secondary spermatocytes, located from the periphery to the centers of the tubules,  respectively; view is of midsection, double stained with: αBSN (B and C) and DAPI (C). Arrows denote typical single dot pattern. Inset  in B shows what is probably a secondary spermatocyte in late G2 or prophase, after duplication of the centrosomes; double labeling of  dots is with αBSN and αH1 antiserum. (D–F) Region of spermatocytes, triple labeled with αBSN (D and F), αH1 (E and F), and DAPI  (F). Yellow reveals costaining with αBSN and αH1. Portion of F to the left of the double arrows is the region shown in D and E. (G) 8-wk  sexually mature seminiferous tubule costained with αBSN, αH1, and DAPI. View is of spermatogonia, primary spermatocytes, and secondary spermatocytes. Note that spermatogonial centrosomes at the periphery only labeled with αH1 (arrowheads), while spermatocytes colabeled with αBSN and αH1 (arrows). Additional note: αBSN antibodies 372 and 176 gave similar immunofluorescence staining  patterns to those shown here. Dotted lines denote nuclear borders. Bars, 10 μm.
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Figure 7: Detection of antibasonuclin immunofluorescence in the centrosomes of meiotic spermatocytes. Frozen sections (∼10 μm) of mouse testis (2, 4, or 8 wk old, as indicated in the panels) were subjected to double or triple immunofluorescence, and sections were viewed with a confocal microscope (Carl Zeiss, Inc.). Sections stained with anti-BSN antibodies (shown here are UC56 αBSN profiles) were visualized by costaining with an FITC-conjugated anti–rabbit IgG secondary antibody (green). Sections stained with human H1 autoantisera (αH1) were visualized by costaining with a Texas red–conjugated anti–human IgG secondary antibody (red). Nuclei were labeled with DAPI (blue). (A) Seminiferous tubule at 2 wk, containing a mixture of mitotically active spermatogonia at the periphery and primary spermatocytes in the midsection (Rugh, 1990); αBSN stained only midsection cells; (B and C) Seminiferous tubule at 4 wk, containing spermatogonia, primary spermatocytes, and secondary spermatocytes, located from the periphery to the centers of the tubules, respectively; view is of midsection, double stained with: αBSN (B and C) and DAPI (C). Arrows denote typical single dot pattern. Inset in B shows what is probably a secondary spermatocyte in late G2 or prophase, after duplication of the centrosomes; double labeling of dots is with αBSN and αH1 antiserum. (D–F) Region of spermatocytes, triple labeled with αBSN (D and F), αH1 (E and F), and DAPI (F). Yellow reveals costaining with αBSN and αH1. Portion of F to the left of the double arrows is the region shown in D and E. (G) 8-wk sexually mature seminiferous tubule costained with αBSN, αH1, and DAPI. View is of spermatogonia, primary spermatocytes, and secondary spermatocytes. Note that spermatogonial centrosomes at the periphery only labeled with αH1 (arrowheads), while spermatocytes colabeled with αBSN and αH1 (arrows). Additional note: αBSN antibodies 372 and 176 gave similar immunofluorescence staining patterns to those shown here. Dotted lines denote nuclear borders. Bars, 10 μm.
Mentions: To assess the location of basonuclin within differentiating male germ cells, we conducted indirect immunofluorescence on frozen sections of developing mouse testes. Basonuclin protein was first detected in testis at 2 wk postnatally (Fig. 7 A). In contrast to BSN RNAs, which are expressed in mitotic spermatogonia, protein was not detected until the cells had differentiated into primary spermatocytes located at the midregion of the 2-wk seminiferous tubules. These cells, in the first meiotic phase of differentiating male germ cells, displayed a dotlike pattern of staining with the UC56 anti-BSN (αBSN) antibody. Double immunofluorescence with DAPI to stain chromatin indicated that the labeling was located near the nucleus (Fig. 7 A). Staining was more prevalent by 4 wk (Fig. 7, B and C), when primary and secondary spermatocytes exist (Rugh, 1990). While the majority of these spermatocytes contained single dots, a few seemingly contained double dots positioned at opposing sides of the nucleus (Fig. 7 B, inset). Similar staining patterns were observed with all three affinitypurified BSN antibodies, although antibodies UC56 (shown) and 372 gave the strongest staining. The pattern was not seen with secondary antibody alone.

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