Histones Gene Cloning And Dynamic Distribution During Spermatogenesis Of The Chinese Mitten Crab, Eriocheir Sinensis

During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by spermatoza-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of spermatozoa basic nuclei proteins(SBNPs), sperm 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.To study histones dynamic distribution during spermatogenesis of E. sinensis, first, we cloned the sequences encoding H2 A, H2 B, H3 and H4 through PCR. The encoding sequence of H2 B consists of 369 bp, encoding 123 amino acids with a predicted molecular weight of 13.6 Ku, H2 A encoding sequence is 369 bases pairs long, encoding 123 amino acids with an approximate molecular weight of 13.1 Ku, H3 encoding area contains 408 bp and encodes 136 amino acids with a predicted molecular weight of 15.3 Ku, and H4 encoding area contains 309 bp, encoding 103 amino acids with a predicted molecular weight of 11.3 Ku. Then we constructed the corresponding prokaryotic expression vector p ET-30a-H2 B, p ET-30a-H2 A, p ET-30a-H3 and p ET-30a-H4, which were then transferred into Escherichia coli Rosetta(DE3) and induced to express the recombinant proteins by isopropyl β-D-thiogalactoside(IPTG). Meanwhile, the recombinant proteins were purified through Ni-NTA Agarose affinity chromatography and purified protein were used as antigen to inoculate Oryctolagus cuniculus to produce antiserum. And we successfully produced H2 B and H2 A polyclonal antibodies.Second, the dynamic distribution of H2 A, H2 B, H3 and H4 during spermatogenesis of E. sinensis were studied by immunofluorescence and immune electron microscopy technology. The results were shown as follows:1. Histone H2 B existed in the nuclei of spermatogonia, spermatocytes, early and middle spermatids, during late stage of spermiogenesis, parts of histone H2 B migrated from nucleus into acrosomal vesicle. At last, histone H2 B was mainly in acrosomal vesicle in mature sperm.2. Histone H3 was present in the nuclei of various male germ cells during the spermatogenesis of E. sinensis even the mature sperm, which didn’t happen discard and transference.3. Histone H4 existed in the nuclei of spermatogonia, spermatocytes, spermatids and mature sperm during spermatogenesis. And there was no transfer into or out of the nuclei.4. Histone H2 A existed in the nuclei and cytoplasms of spermatogonia, spermatocytes and spermatids. In mature sperm, histone H2 A was mainly in acrosomal vesicle and inner side of cup-shaped nucleus.Summarizing the above results, we speculated that the conservation of histones H3 and H4 in mature nuclei and the migration into acrosomal vesicle of histones H2 A and H2 B may have some relationship to the uncondensed sperm chromatin of E. sinensis.