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J. Biol. Chem., Vol. 278, Issue 34, 32431-32438, August 22, 2003

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Somatic Histones Are Components of the Perinuclear Theca in Bovine Spermatozoa^*

P. Ronald Tovich and Richard J. Oko 

From the Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada

The perinuclear theca is a non-ionic detergent-resistant, electron-dense layer surrounding the condensed nucleus of mammalian sperm. The known proteins originating from the perinuclear theca have implicated the structure in a variety of important cellular processes during spermiogenesis and fertilization. Nonetheless, the composition of the perinuclear theca remains largely unexplored. We have isolated a group of low molecular mass (14–19 kDa) perinuclear theca-derived proteins from acrosome-depleted bovine sperm heads by salt (1 M KCl) extraction and have identified them as core somatic histones. N-terminal sequencing and immunoblotting with anti-histone antibodies confirmed the presence of both intact and proteolytically cleaved somatic histones H3, H2B, H2A, and H4. Identical proteins were isolated using 2% SDS or 1 N HCl extractions. Subsequent acid and SDS extractions of intact bovine sperm revealed the presence of all four intact histone subtypes, with minimal proteolysis. Two-dimensional acid/urea/Triton-SDS-PAGE, coupled with immunoblotting analysis, confirmed the somatic nature of these perinuclear theca-derived histones. Estimates of the abundance of perinuclear theca-derived histones showed that up to 0.2 pg per sperm of each histone subtype was present. Immunogold labeling at the ultrastructural level localized all four core somatic histones to the post-acrosomal sheath region of bovine epididymal sperm, when probed with affinity-purified anti-histone antibodies. Little immunoreactivity was detected in residual perinuclear theca structures following the extractions. Taken together, these findings indicate the unprecedented and stable localization of non-nuclear somatic histones in bovine sperm perinuclear theca.

Received for publication, April 11, 2003 , and in revised form, May 28, 2003.

^* This work was supported by a grant and a studentship from the Canadian Institute of Health Research (to R. J. O. and P. R. T., respectively). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, Queen's University, Botterell Hall, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2858; Fax: 613-533-2566; E-mail: ro3{at}post.queensu.ca.

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