The Epigenetic Mechanism Of Nuclear Uncondensation In Eriocheir Sinensis Spermatozoa

On the bases of the state of genetic material in spermatozoa nuclear, animal spermatozoa nuclei are classified into three types:condensed-, intermediate-condensed and uncondensed nuclear. The genetic material in mammal, bird and other animals'spermatozoa nuclei is compacted into the dense structure due to the replacement of histones by protamines and the tight coalescence of protamines with DNA, which involves the acetylation of histone replaced. The chromatin in the intermediate-condensed nuclei of Cerebratulus californeinsis, C Lacteus, Carassius auratus, and so on, are similar to the somatic type histone because they contain the histones in the nuclear. the nuclei of spermatozoa in Chinese crab Eriocheir sinensis (Decapoda, Crustacea) are uncondensed and present loose fibrous. However, the nucleogenesic mechanism of uncondensed nuclei is unclear.The process from the initiation of spermatogenesis to completion of acrosome reaction (AR) is a whole lifetime of the male reproductive cells. The exploration to the definition, distributions, changes and modifications in male germs of spermatozoa basic nuclear proteins (SBNP) in E. sinensis can illuminate the mechanisms of uncondensed nucleogenesis and protecting DNA in spermatozoa nuclei and analyze the evolutional history of Decapoda. In addition, the results have important economic value to the breeding of the crab because E. sinensis is an economically important crustacean.This study focused on the distribution, replacement and acetylation of SBNP during spermatogenesis and AR, and analyzed the varieties of histones in E. sinensis spermatozoa nuclei, and epigenetically explained the nucleogenesic mechanism of the uncondensed spermatozoa. The results show as below:1) Observating the microstructure and ultrastructure of various male germs during spermatogenesis and AR in E. sinensis, we found that there were a great deal of 30 nm granules and other size ones in the nuclear-round spermatids, which demonstrated that the uncondenstation of nuclei was initiated from the nuclear-round spermatids.2) With western blot, Tricine-SDS-PAGE, MS/MS (tandem mass spectrometry) analysis and immunohistochemistry (IHC), immunofluorescence (IF), immunoelectron microscopy (IEM) on the level of microstructure and ultrastructure, we identified that there were 4 core histones in the nuclei of mature spermatozoa and other germ cells (including acrosome-reacted spermatozoa) during spermatozoa and AR. Moreover, those histones also appeared out of the spermatozoa nuclei. In addition, they were transferred to the cytoplasm and then out of the cells from nuclei during their lifetime. Therefore, the histones in the mature spermatozoa were less than that of other germ cell and somatic cells.3) The results from westerrblot, Tricine-SDS-PAGE, MS/MS analysis also indicated that there were two variants or modifications of histone H4 in E. sinensis spermatozoa. Their amino acid sequences and composition were highly homology with various animal histone H4.4) The results from western blot, immunofluorescence and electron microscopy immunocytochemistry also showed that the acetyl-histone H3 was widely distributed in the various male germ cells during the spermatogenesis and AR in E. sinensis. The histone H3 was acetylated three times during the spermatogenesis, implemented in the nuclei of the interphase spermatogonia, the interphase spermatocytes and the new spermatids, respectively, and once in the nuclei during the AR. Especially the histone H3 in the new spermatids was dramatically acetylated, initiated at the new spermatid stage and terminated at the commencement of the early spermatid stage. The majority of acetyl-histone H3 was quickly removed out of the nuclei of early spermatids and a portion of rest was then gradually removed out of the nuclei during the stages from the mid spermatids to the mature spermatozoa. During and after the AR, the acetyl-histone H3 was less or not transferred out of the nuclei.5) The result of lysine and arginine specific dyeing showed that arginine only occurred in the spermatids and spermatozoa in the spermary but its content was less than in spermatozoa nuclei in mouse. The content of lysine in those stages was more than that in other stages, as well. They demonstrated that a new sperm-specific histone was produced during the spermatid stage and dramatically decreased at the resting stage; its basicity was slightly higher than somatic histones and significantly weaker the protamines in mouse spermatozoa. It might be a new sperm-specific histone H1 because the core histones, including the acetyl-histone H3, did not emerge so much in E. sinensis spermatozoa like it in the spermary.In conclusion, the uncondenstation of nuclei in E. sinensis spermatozoa was initiated from the nuclear-round spermatids. The replacement of the somatic histones H1 by the sperm-specific histone H1 and no replacement of the core histones, the acetylation and partial retainment of histones would contribute to the uncondensation of the nuclei in E. sinensis spermatozoa and the epigenetic factors might play irreplaceable roles in the regulating uncondensation of the nuclei in the crustacean spermatozoa.In addition, we also established a temperature difference method inducing AR of E. sinensis spermatozoa and an extraction method for uncondensed cup-shaped nuclear of spermatozoa during the process of experiments. We also developed the classification for spermary developmental cycles and classified the spermary into 7 stages.