Chromatin remodelling in vertebrate spermatozoa

Date

2009-12-24T16:45:16Z

Authors

Frehlick, Lindsay Jennifer

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Abstract

During spermatogenesis, one of the most drastic examples of chromatin remodelling takes place. In many organisms this coincides with drastic changes in chromatin composition, as histones are replaced by sperm nuclear basic proteins (SNBPs) of the protamine type (P-type). Due to their smaller size and higher charge, protamines compact sperm chromatin more efficiently. However, many organisms do not undergo this composition change and instead either retain histones similar to those in somatic cells in their sperm (H-type) or gain protamine-like proteins (PL-type), often in addition to histone. Fish and amphibian models are used in this thesis because they include genera with SNBPs representative of each of the three main types and provide a unique opportunity to study chromatin compaction. I focused on species that contain a partial or complete complement of histones in the sperm. Chapter 1 of this thesis is a review of the SNBP evolution, distribution and roles in chromatin compaction. In Chapter 2, the complete cDNA sequence of Xenopus laevis sperm specific proteins SP1 and SP2 is determined. Structural and functional analyses show that SP1/SP2 proteins are related to proteins of the histone H1 family, particularly to vertebrate histone H1x and are members of the protamine-like- I (PL-I) group of SNBPs. In H-type organisms that retain histones in their sperm, a remodelling of chromatin and a reduction in nuclear volume still occur during spermiogenesis. However, the factors that lead to the condensation of chromatin in these organisms are unknown and are addressed in Chapter 3. Ictalurus punctatus is determined to have sperm chromatin of the H-type, which is maximally compacted and organized into a highly repetitive structure indicative of uniformly condensed chromatin. Several histone variants and post-translational modifications (PTMs) are examined as a preliminary survey of factors potentially responsible for this compaction. Of the PTMs present in catfish testes, the most significant were histone H3 trimethylated at lysine 27, which is a well known marker of facultative heterochromatin, and histone H4 phosphorylated at serine 1, which has been documented to affect nuclear size and may help stabilize chromatin compaction in mice and yeast. A second extreme remodelling of the paternal pronucleus occurs following fertilization in order to convert the highly compacted, transcriptionally inert chromatin of the sperm into a substrate that is recognizable by the transcription and replication machinery of the zygote. Nucleoplasmin, a nuclear chaperone, participates in this remodelling in amphibians by displacing the specialized P-type and PL-type proteins from the sperm chromatin and by the transfer of H2A/H2B dimers. Nucleoplasmin was originally isolated from Xenopus (PL-type) and belongs to the nucleophosmin/nucleoplasmin (NPM) family of proteins, which have diverse functions in the cell (Reviewed in Chapter 4). The existence of H-type sperm raises uncertainty about the need for a nucleoplasmin-mediated removal process in these organisms. In Chapter 5, the presence of nucleoplasmin in Rana catesbeiana (H-type) and Bufo marinus (P-type) is assessed. The amphibian nucleoplasmins are shown to phylogenetically group with mammalian NPM2 proteins and the implications suggested by the presence of nucleoplasmin in organisms of all three SNBP-types are discussed.

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Keywords

Chromatin, Spermatozoa

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