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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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Fractionation of sperm proteins confirms presence of the majority of basonuclin in the acrosome and sperm tail. Sperm were  isolated from the epididymis of adult mice. They were first stained with αBSN UC56 antibodies to confirm the presence of acrosomal  (ac) and middle piece (mp) staining in mature sperm (two sperm shown in A; bottom middle piece has a 90° kink). Sperm were then  fractionated by sonication and sucrose gradient centrifugation as described by Walensky and Snyder (1995). Proteins were solubilized in  10 mM DTT, 2% SDS, and samples were resolved by electrophoresis through 8.5% SDS–polyacrylamide gels. Gels were analyzed by either staining with Coomassie blue (B) to visualize total proteins or immunoblot analysis (C) with antibodies against basonuclin or  PLCβ1. E, total protein extract from epidermal keratinocytes; S, total sperm proteins; A, acrosomal fraction; T, tail fraction; N, nuclear  fraction. Note: Sperm centrioles were lost in the fractionation procedure, as judged by immunofluorescent staining of each fraction.
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Figure 9: Fractionation of sperm proteins confirms presence of the majority of basonuclin in the acrosome and sperm tail. Sperm were isolated from the epididymis of adult mice. They were first stained with αBSN UC56 antibodies to confirm the presence of acrosomal (ac) and middle piece (mp) staining in mature sperm (two sperm shown in A; bottom middle piece has a 90° kink). Sperm were then fractionated by sonication and sucrose gradient centrifugation as described by Walensky and Snyder (1995). Proteins were solubilized in 10 mM DTT, 2% SDS, and samples were resolved by electrophoresis through 8.5% SDS–polyacrylamide gels. Gels were analyzed by either staining with Coomassie blue (B) to visualize total proteins or immunoblot analysis (C) with antibodies against basonuclin or PLCβ1. E, total protein extract from epidermal keratinocytes; S, total sperm proteins; A, acrosomal fraction; T, tail fraction; N, nuclear fraction. Note: Sperm centrioles were lost in the fractionation procedure, as judged by immunofluorescent staining of each fraction.

Mentions: The pattern of BSN antibody staining was unexpected and diverse. To verify that the staining patterns reflected multiple locations for basonuclin protein, we conducted cell fractionation studies. Although procedures for isolation of centrosomes from testis tissue have not yet been developed, it is possible to dissociate isolated sperm into tail, acrosome, and headpiece by sonication and to subsequently resolve these fractions by sucrose gradient ultracentrifugation (Walensky and Snyder, 1995). We applied this procedure to mature sperm that we removed from the epididymis of adult mice. First, we verified that mature sperm, similar to spermatids, display αBSN UC56 immunofluorescence staining in the acrosome, middle piece of the tail, and centrosome. (Fig. 9 A; sperm centrosomal staining was more readily visible with the 372 antibody, which did not stain acrosomes so brightly.)


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)

Fractionation of sperm proteins confirms presence of the majority of basonuclin in the acrosome and sperm tail. Sperm were  isolated from the epididymis of adult mice. They were first stained with αBSN UC56 antibodies to confirm the presence of acrosomal  (ac) and middle piece (mp) staining in mature sperm (two sperm shown in A; bottom middle piece has a 90° kink). Sperm were then  fractionated by sonication and sucrose gradient centrifugation as described by Walensky and Snyder (1995). Proteins were solubilized in  10 mM DTT, 2% SDS, and samples were resolved by electrophoresis through 8.5% SDS–polyacrylamide gels. Gels were analyzed by either staining with Coomassie blue (B) to visualize total proteins or immunoblot analysis (C) with antibodies against basonuclin or  PLCβ1. E, total protein extract from epidermal keratinocytes; S, total sperm proteins; A, acrosomal fraction; T, tail fraction; N, nuclear  fraction. Note: Sperm centrioles were lost in the fractionation procedure, as judged by immunofluorescent staining of each fraction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139879&req=5

Figure 9: Fractionation of sperm proteins confirms presence of the majority of basonuclin in the acrosome and sperm tail. Sperm were isolated from the epididymis of adult mice. They were first stained with αBSN UC56 antibodies to confirm the presence of acrosomal (ac) and middle piece (mp) staining in mature sperm (two sperm shown in A; bottom middle piece has a 90° kink). Sperm were then fractionated by sonication and sucrose gradient centrifugation as described by Walensky and Snyder (1995). Proteins were solubilized in 10 mM DTT, 2% SDS, and samples were resolved by electrophoresis through 8.5% SDS–polyacrylamide gels. Gels were analyzed by either staining with Coomassie blue (B) to visualize total proteins or immunoblot analysis (C) with antibodies against basonuclin or PLCβ1. E, total protein extract from epidermal keratinocytes; S, total sperm proteins; A, acrosomal fraction; T, tail fraction; N, nuclear fraction. Note: Sperm centrioles were lost in the fractionation procedure, as judged by immunofluorescent staining of each fraction.
Mentions: The pattern of BSN antibody staining was unexpected and diverse. To verify that the staining patterns reflected multiple locations for basonuclin protein, we conducted cell fractionation studies. Although procedures for isolation of centrosomes from testis tissue have not yet been developed, it is possible to dissociate isolated sperm into tail, acrosome, and headpiece by sonication and to subsequently resolve these fractions by sucrose gradient ultracentrifugation (Walensky and Snyder, 1995). We applied this procedure to mature sperm that we removed from the epididymis of adult mice. First, we verified that mature sperm, similar to spermatids, display αBSN UC56 immunofluorescence staining in the acrosome, middle piece of the tail, and centrosome. (Fig. 9 A; sperm centrosomal staining was more readily visible with the 372 antibody, which did not stain acrosomes so brightly.)

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