Lack of Acrosome Formation in Hrb-Deficient Mice Ningling Kang-Decker, George T. Mantchev, Subhash C. Juneja, Mark A. McNiven, and Jan M. A. van Deursen

Generation of knockout mice. The Hrb targeting vector contained a 12.0 kb Hrb 129Sv/J genomic DNA fragment in which a unique ~500 bp Spe I/Stu I fragment had been replaced by a promoterless IRES-lacZ-neo selection cassette (1), interrupting the Hrb gene at amino acid 108. We electroporated this targeting vector into E14 ES cells and performed drug selections as previously described (1). We identified correctly targeted ES cell clones by Southern blot analysis using a 3' external probe on Kpn I-cut genomic DNA. Mutant mice were derived from these targeted ES cell clones as previously described in detail (2).

Hrb antibody production. To generate Hrb-specific antibodies, human Hrb sequences encoding amino acids 386-562 were cloned into pQE31 (Qiagen). HIS-tagged recombinant protein was expressed in E. coli DH12S cells, purified with Ni-NTA agarose (Qiagen) and injected into rabbits. Hrb antiserum was affinity purified using HIS-Hrb (386-562) recombinant protein immobilized on ProBlot (Perkin Elmer, Life Sciences) as previously described (1). Western blotting was performed as described previously (3).

Electron microscopy. Histological analyses of testis were according to standard procedures (4). For electron microscopic examinations, testis biopsies were fixed for 4 hours with 2.5% gluteraldehyde/0.6% formaldehyde (buffered with sodium cacodylate, pH 7.2), and then post-fixed with 2% buffered OsO₄. Samples were dehydrated in graded ethanol and then embedded in Spurr. Thin sections were examined by transmission electron microscopy using a JEOL 1200 EXII. For IEM, we isolated testis from Hrb^+/+ mice, removed the testis capsules and minced the seminiferous tubules in PBS. Pre-embedding immunogold labelings with affinity-purified Hrb (386-562) antibody on the resulting cell suspensions were essentially as described in detail by Cordes and coworkers (5). For SEM, cauda epididymal spermatozoa from Hrb^+/+ and Hrb^-/- males were isolated, processed and examined as previously described (6).

Indirect immunofluorescence and confocal microscopy. For immunostainings, we collected testis of Hrb^+/+ and Hrb^-/- mice, removed the capsule tissue and minced the seminiferous tubules in PBS. Suspended cells were pelleted by centrifugation at 1000 rpm for 5 min, fixed in 3% paraformaldehyde, 0.1 M PIPES, pH 6.95, 1 mM EGTA, 3 mM MgSO₄ for 20 min at 37°C. Cells were washed 3 times with PBS and attached to polyethylene amine coated slides. Immunostainings were as previously described (3). The following primary antibodies were used: purified anti-Hrb (386-562) (1:800); rabbit antibody to Eps15 (C-20; SC-534, Santa Cruz, 1:25); mouse monoclonal antibody to Ap1 (anti--adaptin, Transduction laboratories, 1:800); mouse monoclonal antibodies to Ap2 [anti--adaptin (clone AC1-M11), Affinity Bioreagents Inc., 1:100; anti--adaptin, Transduction laboratories, 1:100]; mouse monoclonal antibody to Golgi protein 58K (Sigma, 1:50). Appropriate species-specific secondary antibodies (Molecular Probes) were applied at 1:250 dilutions. For triple labeling, rabbit Eps15 antibody was covalently linked to Alexa-680 according to the manufacturer (Molecular Probes) and used at 1:25 dilution. Fluorescence was visualized with a Zeiss confocal microscope. Mitochondrial sheaths were visualized using the mitochondrion-specific vital dye MitoTracker green FM (Molucular Probes) as previously described (7).

In vitro fertilization. Egg collection, spermatozoa retrieval and capacitation, and in vitro fertilization were as previously described in detail (8). Eggs were incubated with a drop of 5 x 10⁵ spermatozoa per ml. Sperm-egg interaction and fertilization were determined by microcopy at respectively 2 and 24 hours post-insemination.

Co-immunoprecipitations. We collected testis of Hrb^+/+ mice, removed the capsules and minced the seminiferous tubules in PBS. We prepared lysates from 5 x 10⁶ testis cells and performed immunoprecipitations with affinity-purified Hrb (386-562) antibody as described previously (3). We utilized purified Rae1 (188-347) antibody as a control (9). Anti-Eps15 (C-20) rabbit IgG (SC-534, Santa Cruz) was used for detection of coimmunoprecipitated Eps15 by Western blotting.

References

1. J. van Deursen, J. Boer, L. Kasper, G. Grosveld, EMBO J. 15, 5574 (1996).
2. J. van Deursen et al., Cell 74, 621 (1993).
3. L. H. Kasper et al., Mol. Cell. Biol. 19, 764 (1999).
4. B. Hogan, R. Beddington, F. Constantini, E. Lacy, Manipulating the Mouse Embryo (Cold Spring Harbor Press, Cold Spring Harbor, NY, ed. 2, 1994).
5. V. C. Cordes, A. Gajewski, S. Stumpp, G. Krohne, Differentiation 58, 307 (1995).
6. B. Bartoov et al., Ultramorphological Characteristics of Human Sperm Cells (Plenum, New York, 1990), pp. 493-513.
7. V. Y. Rawe, G. D. Galaverna, A. A. Acosta, S. B. Olmedo, H. E. Chemes, Hum. Reprod. 16, 879 (2001).
8. S. C. Juneja, T. L. Pfeifer, X.-M. Tang, R. S. Williams, N. Chegini, Endocrine 3, 69 (1995).
9. C. E. Pritchard, M. Fornerod, L. H. Kasper, J. M. van Deursen, J. Cell. Biol. 145, 237 (1999).

Supplemental Figure 1. Generation and characterization of Hrb-deficient mice. (A) Targeted disruption of the Hrb gene. Indicated is part of the murine Hrb locus (top), the targeting vector (middle), and the disrupted Hrb allele (bottom). Kpnl (K) restriction sites and a 3' DNA probe (open bar) used for Southern blot identification of wild-type (Wt) and mutant (Mt) Hrb alleles are shown. (B) Southern blot containing DNAs extracted from a tail biopsy of a Hrb^+/+, and Hrb^+/+ and a Hrb^-/- mouse. (C) Hrb protein detection in a variety of tissues from wild-type and knockout mice by Western blot analysis. (D) Analysis of Hrb gene activity in testicular from 12-week-old males by histochemical staining for -galactosidase. Because in heterozygous mutants the -galactosidase gene is under control of the endogenous Hrb promoter, -galactosidase activity indirectly reveals Hrb gene activity. Wild-type mice lack the -galactosidase gene cassette and yield only background staining. Sections were counterstained with nuclear fast red. (E) Testis sections from adult Hrb^+/+ and Hrb^-/- males stained with hematoxylin and eosin. The seminiferous tubule diameter in Hrb^+/+ and Hrb^-/- mice is comparable. The normal tubule has many spermatids with elongated nuclei, whereas the mutant tubule only contains spermatids with round nuclei.

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