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BOR - Papers in Press, published online ahead of print June 9, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030445
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BIOLOGY OF REPRODUCTION 71, 1182–1194 (2004)
DOI: 10.1095/biolreprod.104.030445
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Novel Aspect of Perinuclear Theca Assembly Revealed by Immunolocalization of Non-Nuclear Somatic Histones During Bovine Spermiogenesis1

P. Ronald Tovich3, Peter Sutovsky4, and Richard J. Oko2,3

Department of Anatomy and Cell Biology,3 Queen's University, Kingston, Ontario, Canada K7L3N6 Departments of Animal Science and Obstetrics & Gynecology,4 University of Missouri-Columbia, Missouri 65211-5300

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-ß-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.

1 Supported by a studentship and grant from the Canadian Institute of Health Research (P.R.T. and R.J.O., respectively), NRI research grant 2002-02069 from USDA/NRI (P.S.) and from the Food for the 21st Century Program of the University of Missouri-Columbia (P.S.).

2 Correspondence. FAX: 613 533 2566; ro3{at}post.queensu.ca






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Copyright © 2004 by the Society for the Study of Reproduction.