Abstract
During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.
Highlights
Histones comprise a group of basic proteins that are responsible for DNA packing and chromatin condensation in the nuclei of almost all eukaryotic cells
The basic proteins associated with DNA undergo continuous change throughout spermatogenesis of most animal species: somatic-typed histones are partly replaced by spermspecific histones, which are successively replaced by transition proteins and protamines
A band of about 408 bp was obtained from E. sinensis testes DNA by Polymerase chain reaction (PCR) with primers (H3F1 and H3R1) that were designed based on the high conservation of histone H3 sequence and codon preference of E. sinensis through 1% agarose gel electrophoresis (Fig 1A)
Summary
Histones comprise a group of basic proteins that are responsible for DNA packing and chromatin condensation in the nuclei of almost all eukaryotic cells In one group of decapod crustaceans the mature sperm nuclei differ markedly from those in other taxa in which the chromatin has a decondensed, loosely arranged organization [5,6,7,8]. Earlier researchers reported that these decapod crustacean sperm nuclei did not contain basic proteins, and that their chromatin was uncondensed [17]. Recent studies on Cancer pagurus and Cancer magister [18], Maja brachydactyla [19] and Portunus pelagicus [20] provided new evidence regarding the longstanding question of histones in mature crustacean spermatozoa These sperm nuclei contain relatively low contents of whole or parts of histones. The results showed that not all histones are lost during spermatogenesis This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4
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