Transition Of The Nuclear Basic Proteins And Acetylated H4 During Spermatogenesis And Acrosome Reaction Of Decapoda Crustacea

During spermatogenesis, primordial germ cells develop into spermatogonia, giving rise to spermatocytes that undergo two meiotic divisions to become spherical spermatids. These cells differentiate into spermatozoa during spermiogenesis. Spermatogenesis is a complex process for cell differentiation, which is characterized by several dramatically transitional stages in the nuclear basic proteins during premeiotic and meiotic stages and during spermiogenesis. Especially during spermiogenesis, the switch from a nucleosome-to a protamine-based chromatin structure is a characteristic feature of sperm maturation in many vertebrates including humans. First, testis-specific linker histones appear, and then histones are replaced by transition proteins (major types:TP1 and TP2), which in turn are replaced by protamines, leading to condensed chromatin with a doughnut structure.During spermatogenesis, the N-terminal domains of all four core histones are subject to reversible acetylation at certain lysine residues. This modification has been functionally linked to transcription, histone deposition at replication and to histone removal during spermatogenesis. H4 is the most conservative core histone in eucaryotic cells, ranging from fungi to man, and its acetylation is restricted only to the four lysines (residues 5,8,12, and 16). Therefore, the study of the presence of acetylated histone H4 during spermatogenesis can be used to evaluate its correlation with the events of gene transcription, histone deposition, and histone displacement.To date, a systematic study to the changes of basic proteins and the histone acetylation in crustacean spermatogenesis are not available. In this thesis, we have investigated the exchanges of basic proteins and the immunolocalization of acetylated H4 during crustacean spermatogenesis and have attempted to correlate these findings with the non-condensed sperm nucleus.Spermatogenesis of Fenneropenaeus chinensis was investigated using TEM, and the changes of histones during spermatogenesis of F. chinensis, Eriocheir sinensis and Macrobrachium nipponense were studied using microscopy after specific stain for lysine compared with histological of testis. The F. chinensis was further examined using TEM with ammoniacal silver reaction (ASR). The immunolocalization of acetylated H4 during spermatogenesis of all the three crustareans was appraised using TEM. Further more, the changes of basic proteins and the immunolocalization of acetylated H4 during acrosomal reaction of E. sinensis was assessed by specific stain for amino acid and using TEM with ASR, respectively. The results are summarized as follows: 1 There are two types of membrane complex during spermatogenesis of F. chinensis, one originating from nucleus exists inside the spermatocy, another from cytoplasm is formed during the anaphase of spermiogenesis and also exists in mature sperm.2 Analysis of the results between the commonly processed TEM samples and that only fixed with glutaraldehyde and dyed by uranyl acetate has showed that there are a lot of lipids in the spermmatozoa acrosome of F. chinensis.3 With specific stain of lysine, it is found that the blue reactions declined gradually from spermatocytes to spherical spermatids and to mature sperms in the three types of crustaceans, whereas there is still a blue reaction in mature sperm nucleuses, indicating the existence of some histones preserved in the nucleus.4 Using combined TEM observation via ammoniacal silver reaction (ASR), we have found evidence of some basal protein in the mature sperm nucleus of F. chinensis.5 Using the technique of post-embedding immunoelectron microscopy, we have assessed the changes of acetylated histone H4 during spermatogenesis for the three kinds of crustaceans. The results showed that a lot of acetylated histones H4 existed in the spermatogonium, and numerous acetylated histones H4 were transferred from nucleus into the acrosomal vesicle during spermiogenesis. However, some acetylated histones H4 still remained in mature sperm nucleus.6 During the acrosomal reaction of E. sinensis sperm, the acetylated histone H4 was observed using TEM with post-embedding immunolocalization, and the acetylated histone H4 was found in the nucleus.To summarize, during spermatogenesis of the crustaceans, a lot of histones were transferred from nucleus into the acrosome, and there is still some histones and acetylated histone H4 in the mature sperm nucleus by reservation and/or substitution. The existence of acetylated histone H4 in mature sperm nucleus suggests that DNA with a loose structure still has a transcription, which makes the mature sperm nucleus existing in a non-condensed state. The non-condensed sperm nucleus may facilitate the fertilization in crustacean.