Comparative Proteomics Elucidates the Potential Mechanism of Sperm Capacitation of Chinese Mitten Crabs (Eriocheir sinensis).

Sperm capacitation is broadly defined as a suite of biochemical and biophysical changes resulting from the acquisition of fertilization ability. To gain insights into the regulation mechanism of crustacean sperm capacitation, 4D label-free quantitative proteomics was first applied to analyze the changes of sperm in Eriocheir sinensis under three sequential physiological conditions: seminal vesicles (X2), hatched with the seminal receptacle content (X3), and incubated with egg water (X5). In total, 1536 proteins were identified, among which 880 proteins were quantified, with 82 and 224 proteins significantly altered after incubation with the seminal receptacle contents and egg water. Most differentially expressed proteins were attributed to biological processes by Gene Ontology annotation analysis. As the fundamental bioenergetic metabolism of sperm, the oxidative phosphorylation, glycolysis, and the pentose phosphate pathway presented significant changes under the treatment of seminal receptacle contents, indicating intensive regulation for sperm in the seminal receptacle. Additionally, the seminal receptacle contents also significantly increased the oxidation level of sperm, whereas the enhancement of abundance in superoxide dismutase, peroxiredoxin 1, and glutathione S-transferase after incubation with egg water significantly improved the resistance against oxidation. These results provided a new perspective for reproduction studies in crustaceans.

A pattern recognition receptor interleukin-1 receptor is involved in reproductive immunity in Macrobrachium nipponense ovary.

The family of TIR domain-containing receptors includes numerous proteins involved in innate immunity. In this study, a member of this family was characterized from the ovary of the oriental river prawn Macrobrachium nipponense and identified as interleukin-1 receptor (MnIL-1R). Meanwhile, to elucidate the conservation of IL-1R, its orthologous were identified in several crustacean species as well. In addition, the expression pattern of MnIL-1R in various adult tissues and post different pathogen-associated molecular patterns (PAMPs) challenge in ovary was analyzed with qRT-PCR technology. Finally, the roles of MnIL-1R in the ovary were analyzed by RNAi technology. The main results are as follows: (1) MnIL-1R comprises a 1785 bp ORF encoding 594 amino acids and is structurally composed of five domains: a signal peptide, two immunoglobulin (IG) domains, a transmembrane region, and a TIR-2 domain; (2) the TIR domain showed a high conservation among analyzed crustacean species; (3) MnIL-1R is widely detected in all tested tissues including ovary; (4) MnIL-1R showed a positive response to challenges with LPS, PGN, and polyI:C in the ovary; (5) its IG domain showed strong binding ability to LPS and PGN, confirming its role as a pattern recognition receptor; (6) the expression patterns of several members of the Toll signaling pathway (Myd88, TRAF-6, Dorsal, and Relish) was similar to that of MnIL-1R after challenges with LPS, PGN, and polyI:C in the ovary; (7) the silencing of MnIL-1R resulted in down-regulation of theses gene' (Myd88, TRAF-6, Dorsal, and Relish) expression level in the ovary. These results suggest that MnIL-1R can activate the Toll signaling pathway in the ovary by directly recognizing LPS and PGN through its IG domain, thereby contributing to the immune response in the ovary of M. nipponense.

Sperm acrosomal released proteome reveals MDH and VDAC3 from mitochondria are involved in acrosome formation during spermatogenesis in Eriocheir sinensis.

Acrosome is inextricably related to membranous organelles. The origin of acrosome is still controversial, one reason is that limited articles were reported about the proteomic analysis of the acrosome. Mitochondrial proteins were found exist in the acrosome, nevertheless, only limited attention has been paid to the function of mitochondrial proteins in the acrosome formation. Eriocheir sinensis sperm has a large acrosome, which makes it an ideal model to study acrosome formation. Here, we firstly compared the rate of acrosome reaction induced by the calcium ionophore A23187 and ionomycin. The rate of acrosome reaction induced by ionomycin is higher (95.8%) than A23187 (58.7%). Morphological changes were observed using light, confocal and transmission electron microscopy. Further more, proteins released during the acrosome reaction as induced by ionomycin were collected for LC-MS/MS analysis. A total of 945 proteins, including malate dehydrogenase (MDH) and voltage-dependent anion channel 3 (VDAC3), were identified in the acrosomal released proteome. The number of proteins from mitochondria (17.57%) was higher compared with endoplasmic reituculum (1.59%) and lysosomes (1.8%). To investigate the functions of target mitochondrial proteins during spermatogenesis, poly-antibodies of MDH in E. sinensis were prepared. The characteristics, further analyzed using immunofluorescence, of two mitochondrial proteins during acrosome formation showed that MDH and VDAC3 were independently involved in the formation of acrosomal membrane. These findings illustrate the acrosomal released proteome and provide important data resource for understanding the relationship between mitochondria and the acrosome in Decapoda crustacean.

Rab2 and Rab6 are Implicated in Acrosome Formation during Spermatogenesis in Eriocheir sinensis: Based on Sperm Proteome.

BACKGROUND: Rab proteins are GTP-dependent small proteins that function as regulators of intracellular vesicle transport, fusion, and localization. However, few studies have investigated their function in Decapoda reproduction. The Eriocheir sinensis sperm has no tail and the nuclei are uncondensed. With the acrosome forming the majority of the sperm mass, it provides an ideal model for studying acrosome formation. METHODS: We firstly analyzed the sperm proteome using LC-MS/MS. To study the functions of Rab2 and Rab6, related to the Golgi apparatus, in the acrosome formation during spermatogenesis, the genes of Rab2 and Rab6 were cloned based on the testis transcriptome of E.sinensis and poly-clonal antibodies were prepared. The presence of 2 Rab proteins was confirmed in the testis and sperm by western blot. We further observed the characteristics of target 2 Rab proteins using immunofluorescence (IF). RESULTS: A total of 1247 proteins including 7 Rab proteins, Rab1, Rab2, Rab5, Rab6, Rab11, Rab14, and Rab18 were identified in the sperm proteome. The IF results showed that Rab2 co-localizes with GM130, a cis-Golgi matrix protein, in the spermatagonia and spermatocytes. In the early spermatids, Rab2 and Rab6 participate in the formation of pre-acrosomal vesicles. In maturing spermatids, both Rab2 and Rab6 settle on the acrosomal membrane but present different characteristics wrapping the pre-acrosome. In the mature sperm, Rab2 localizes in the perinuclear theca surrounding the nuclei cup, while Rab6 remains on the acrosomal membrane. CONCLUSIONS: Our research found 7 Rab proteins based on the analysis of the sperm proteome in E.sinensis, and confirmed the involvement of Rab2 and Rab6 in acrosome formation. These findings provide a foundation for studying the functions of Rab proteins during spermatogenesis in Decapoda animals.

Assessment of genetic diversity and population structure of Neocaridina denticulata sinensis in the Baiyangdian drainage area, China, using microsatellite markers and mitochondrial cox1 gene sequences.

Neocaridina denticulata sinensis is a crustacean of major economic significance in the Baiyangdian drainage area. In this study, the first assessment of N. denticulata sinensis genetic diversity and population structure was performed based on sequence analysis of nine polymorphic microsatellite loci and the mitochondrial cytochrome oxidase subunit I (cox1) gene. Samples (n = 192) were collected from four different regions in the Baiyangdian drainage area i.e., Baiyangdian Lake, Jumahe River, Xidayang Reservoir, and Fuhe River. Microsatellite loci analysis identified high levels of genetic diversity represented by observed heterozygosity (Ho) of 0.6865 âˆ¼ 0.9583, expected heterozygosity (He) of 0.7151 âˆ¼ 0.8723, and polymorphism information content (PIC) of 0.6676 âˆ¼ 0.8585. Based on the analysis of cox1 sequences, haplotype diversity (Hd) ranged from 0.568 to 0.853 while nucleotide diversity (π) ranged from 0.0029 to 0.2236. Furthermore, there was no evidence of expansion events in the N. denticulata sinensis populations. Pairwise FST revealed pronounced genetic differentiation, and clustering analyses showed defined genetic structures within the N. denticulata sinensis population. Three groups were identified from four sampled stocks, with Xidayang Reservoir, and Fuhe River populations clustered in the same group. This work identified novel molecular markers and provided an important reference to guide management strategies to assist conservation of N. denticulata sinensis resources.

Microsatellite markers reveal genetic diversity and population structure of Portunus trituberculatus in the Bohai Sea, China.

The swimming crab, Portunus trituberculatus, is one of the main aquaculture species in Chinese coastal regions due to its palatability and high economic value. To obtain a better understanding of the genetic diversity of P. trituberculatus in the Bohai Sea, the present study used 40 SSR loci to investigate the genetic diversity and population structure of 420 P. trituberculatus individuals collected from seven populations in the Bohai Sea. Genetic parameters revealed a low level of genetic diversity in the cultured population (SI = 1.374, He = 0.687, and PIC = 0.643) in comparison with wild populations (SI ≥ 1.399, He ≥ 0.692, and PIC ≥ 0.651). The genetic differentiation index (Fst) and gene flow (Nm) ranged from 0.001 to 0.060 (mean: 0.022) and 3.917 to 249.750 (mean: 31.289) respectively, showing a low differentiation among the seven populations of P. trituberculatus. Population structure analysis, phylogenetic tree, and principal component analysis (PCA) demonstrated that the seven groups of P. trituberculatus were divided into four subpopulations (K = 4), but the correlation between genetic structure and geographical distribution was not obvious. These results are expected to provide useful information for the fishery management of wild swimming crabs.

Genome-wide investigation of toll-like receptor genes (TLRs) in Procambarus clarkia and their expression pattern in response to black may disease.

As a crucial component of pattern-recognition receptors (PRRs) that recognizing pathogen-associated molecular patterns (PAMPs) and defending against invading pathogens, the Toll-like receptors (TLRs) have been paid extensive attention. While the identification and functional roles of TLRs in innate immunity have been reported in a plenty of organisms, the systematic knowledge of TLRs is still lacking in the red swamp crayfish (Procambarus clarkia). In current study, a total of 7 tlr genes were identified in P. clarkia based on the published transcriptome and genome data. The PcTLRs length varied from 939 to 1517aa and contain typical domains of TLR protein, including transmembrane region, varied LRR and TIR domains. 7 Pctlr genes were distributed in 5 chromosomes and 2 scaffolds. The expression pattern of different Pctlr genes in different tissues (hepatopancreas, gill and muscle) and in response to black may disease (BMD) showed significant difference. In addition, 5 proteins that might interact with PcTLR-2 were predicted, among them the expression pattern of dorsal and relish was consistent with Pctlr-2 in three tissues, while the other genes were not. The PcTLR-2-Dorsal/Relish pathway might play crucial roles in response to BMD infection. The results provided a theoretical foundation for further studies on the molecular mechanisms of TLRs in BMD infection in the red swamp crayfish and provided reference for the research of other crustacean species.

Development of Microsatellite Markers Based on Transcriptome Sequencing and Evaluation of Genetic Diversity in Swimming Crab (Portunus trituberculatus).

P. trituberculatus is an economically important mariculture species in China. Evaluating its genetic diversity and population structure can contribute to the exploration of germplasm resources and promote sustainable aquaculture production. In this study, a total of 246,243 SSRs were generated by transcriptome sequencing of P. trituberculatus. Among the examined 254,746 unigenes, 66,331 had more than one SSR. Among the different SSR motif types, dinucleotide repeats (110,758, 44.98%) were the most abundant. In 173 different base repeats, A/T (96.86%), AC/GT (51.46%), and ACC/GGT (26.20%) were dominant in mono-, di-, and trinucleotide, respectively. GO annotations showed 87,079 unigenes in 57 GO terms. Cellular process, cell, and binding were the most abundant terms in biological process, cellular component, and molecular function categories separately. A total of 34,406 annotated unigenes were classified into 26 functional categories according to the functional annotation analysis of KOG, of which "general function prediction only" was the biggest category (6,028 unigenes, 17.52%). KEGG pathway annotations revealed the clustering of 34,715 unigenes into 32 different pathways. Nineteen SSRs were identified as polymorphic and, thus, used to assess the genetic diversity and structure of 240 P. trituberculatus individuals from four populations in the Bohai Sea. Genetic parameter analysis showed a similar level of genetic diversity within wild populations, and the cultured population indicated a reduction in genetic diversity compared with wild populations. The pairwise FST values were between 0.001 and 0.04 with an average of 0.0205 (p < 0.05), suggesting a low but significant level of genetic differentiation among the four populations. Structure analysis demonstrated that the four populations were classified into two groups including the cultured group and other populations. The phylogenetic tree and PCA revealed that a vast number of samples were clustered together and that cultivated individuals were distributed more centrally than wild individuals. The findings contribute to the further assessment of germplasm resources and assist to provide valuable SSRs for marker-assisted breeding of P. trituberculatus in the future.

Microsatellite analysis of genetic diversity in wild and cultivated Portunus trituberculatus in Bohai Bay.

BACKGROUND: Previous studies have revealed a reduction in the genetic diversity of P. trituberculatus in Bohai Sea. However, because swimming crabs have been released into this area for some time, it is unclear whether the release of cultured populations from the national breeding farms of swimming crabs in Bohai Bay have affected the population genetics of wild populations of P. trituberculatus. METHODS AND RESULTS: In this study, the genetic diversity and population structure of 120 P. trituberculatus specimens in Bohai Bay were investigated using six microsatellite loci, including one wild population and one cultivated population. A total of 132 alleles were identified for all loci. The mean expected (He) and observed heterozygosity (Ho) were 0.8185 and 0.7759, respectively, thus indicating high levels of genetic diversity for these two populations. Molecular variance analysis (AMOVA) (FST = 0.0180), genetic distance (D = 0.1168) and similarity (S = 0.8898) indicated that these two populations could not be distinguished genetically. Structural analysis, phylogenetic tree construction, and principal component analysis, showed no significant distinction between the wild and cultivated populations. Finally, we investigated genetic exchange between the two populations by analyzing migration rate (M) and gene flow (Nm), thus demonstrating significant flow of genetic information. CONCLUSIONS: These findings contribute information for further breeding schemes for P. trituberculatus, evaluation of its genetic potential and programs for the protection of wild resources.

Dynamics of hyperacetylated histone H4 (H4Kac) during spermatogenesis in four decapod crustaceans.

During spermatogenesis, the transition from histone to protamine is highly conserved in most invertebrates and vertebrates. Thus far, a large and growing body of literature has demonstrated that histones and histone modifications still exist in the sperm nucleus of decapod crustaceans. H4Kac is believed to play an important role in the process of sperm chromatin condensation. However, the dynamics of hyperacetylated histone H4 (H4Kac) during spermatogenesis in decapoda are still unknown. In this paper, the distribution of H4Kac in four decapod crustaceans (Eriocheir sinensis, Charybdis japonica, Procambarus clarkii, and Macrobrachium nipponense) were investigated via immunofluorescence. Our results indicated that H4Kac was visible in the mature sperm nucleus of E. sinensis, C. japonica, and M. nipponense. Unlike the other three species, H4Kac was translocated from the nuclei to cytoplasm in mid-spermatids of P. clarkii. Eventually, H4Kac were not present in mature spermatozoa of P. clarkii. Importantly, we observed for the first time that H4Kac was distributed outside the nucleus, which reminds us that H4Kac may participate in the formation of acrosome structure in decapod crustaceans and may be a prerequisite for proper chromatin decondensation.

Spermiogenic histone transitions and chromatin decondensation in Decapoda.

Decapoda are among of the most diverse groups of Crustacea with an important economic value, and have thus been the focus of various reproductive biology studies. Although spermatozoa are morphologically diverse, decapod spermatozoa possess common features, such as being non-motile and having uncondensed nuclear chromatin. Many scholars have studied uncondensed chromatin in decapod spermatozoa; however, the role of biologically regulated decondensation in spermatozoa remains unclear. In this study, histone changes in the spermatozoa of five commercially relevant aquatic crustacean species (Eriocheir sinensis, Scylla paramamosain, Procambarus clarkii, Fenneropenaeus chinensis, and Macrobrachium nipponense) were studied via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunofluorescence. The LC-MS/MS results confirmed that all four core histones were present in the sperm nuclei of the five Decapoda species. Positive fluorescent signals from histones H2A, H2B, H3, and H4 were detected in the spermatozoa nuclei of E. sinensis, S. paramamosain and M. nipponense via immunofluorescence. Histone H2A was first identified in the membrane sheets or cytoplasm of mature sperm in P. clarkii and F. chinensis, whereas H3 and H4 were generally distributed in the nucleus of the spermatozoa. Histone H2B gradually disappeared during spermiogenesis and was not found in the sperm of P. clarkii and F. chinensis eventually. Our data suggest that core histones are instructive and necessary for chromatin decondensation in decapods spermatozoa. Thus, our results may help resolve the complex sperm histone code and provide a reference for the study of spermatozoa evolution in Decapoda.

H4S1ph, an alternative epigenetic marker for sperm maturity.

Histone phosphorylation, an epigenetic post-translational modification, plays essential roles in male gamete chromatin compaction during spermatogenesis and sperm maturity. Previously, we studied the epigenetic marker of phosphorylated serine 1 of histone H2A and H4 (HS1ph) during spermatogenesis in mice and crabs, which was shown to be closely related to the sperm maturity. To further investigate the correlation between phosphorylated serine 1 of histone H4 (H4S1ph) and sperm maturation, a comparison study was conducted in this work between the healthy and the precocious crabs. It was discovered that the distribution of H4S1ph was similar for the two groups of crabs during spermatogenesis before maturity, but totally different in the sperm nuclei. H4S1ph vanished in the nuclei of healthy crab spermatozoa mostly, while retained in the precocious crabs just like what it was in elongated spermatid of both kinds of crabs. The results showed that a high level of H4S1ph conservation was closely associated with immaturity and might indicate inefficient fertility of male precocious crabs. Thus, H4S1ph was suggested to be an epigenetic marker of sperm maturity.

Ultrastructure of spermiogenesis and the distribution of spermatozoal nuclear histones in the Japanese mantis shrimp, Oratosquilla oratoria (Crustacea: Stomatopoda).

The Japanese mantis shrimp Oratosquilla oratoria (Stomatopoda; Crustacea) is one of the most economically important aquatic species of Pacific shrimp and it is distributed from Japan to the coast of China, the Philippines, the Malay Peninsula, and the Hawaiian Islands. Early studies described certain characteristics of spermatogenesis and the sperm ultrastructure in Stomatopoda, but the composition of sperm basic nuclear proteins (SBNPs) remains completely unknown. We studied the sperm ultrastructure of O. oratoria using transmission electron microscopy and the histone composition using immunofluorescence and immunoelectron microscopy. We found that the spherical nucleus is adjacent to the electron translucent external coat, which occurs in early spermatids. The acrosomal structure begins to form at the junction of the nucleus and the external coat. At the mid-spermatid stage, part of the chromatin appears to be more electron-dense than the external coat side. The aflagellate sperm of O. oratoria, are rounded or slightly ovoid in shape and have a consistent granular nucleus, an acrosome structure of pushpin shape and a spherical vesicular body in which faintly granular material is scattered. The acrosome consists of an acrosomal vesicle, perforatorium, and subacrosomal material. The sperm contains histones H2A, H2B, H3, H4, H3.3, H2AX, and H2AZ as well as some histone modifications, that is, H3K9me3, H3K4me2, H3S10ph, H4Kac, and H2A + H4S1ph. Histones are localized not only in the nucleus of the sperm but also in other structures outside the nucleus. The results may provide new perspectives for systematic studies of crustaceans and their sperm chromatin components. These findings extend the study of the sperm structure of Stomatopoda and provide basic data to elucidate the epigenetic mechanism of fertilization.

H3K9ac involved in the decondensation of spermatozoal nuclei during spermatogenesis in Chinese mitten crab Eriocheir sinensis.

As a well-known crustacean model species, the Chinese mitten crab Eriocheir sinensis presents spermatozoa with decondensed DNA. Our aim was to analyze structural distribution of the histone H3 and its acetylated lysine 9 (H3K9ac) during spermatogenesis for the mechanistic understanding of the nuclear decondensation of the spermatozoa in E. sinensis. Using specific antibodies, we followed the structural distribution and acetylated lysine 9 of the histone H3 during spermatogenesis, especially spermiogenesis, of E. sinensis. Various spermary samples at different developmental stages were used for histological immunofluorescence and ultrastructural immunocytochemistry. Our results demonstrate a wide distribution of the histone H3 and H3K9ac during spermatogenesis, including spermatogonia, spermatocytes, spermatids, and immature and mature spermatozoa except for absence of H3K9ac in the secondary spermatocytes. Especially during the initial stage of nuclear decondensation, histone H3 lysine 9 was acetylated and then an amount of H3K9ac was removed from within to outside of the nuclei of late spermatids. The portion of remaining H3K9ac was gradually transferred from the nuclei during the stages of spermatozoa maturation. Our findings suggest both the acetylation of histone H3 lysine 9 and the remain of H3K9ac to contribute to the nuclear decondensation in spermatozoa of E. sinensis.

Dynamics of histone H2A, H4 and HS1ph during spermatogenesis with a focus on chromatin condensation and maturity of spermatozoa.

Histones and histone phosphorylation play vital roles during animal spermatogenesis and spermatozoa maturation. The dynamic distribution of histones H2A and H4 and phosphorylated H2A and H4 at serine 1 (HS1ph) was explored in mammalian and Decapoda germ cells, with a special focus on the distribution of H2A, H4 and HS1ph between mouse condensed spermatozoa chromatin and crab non-condensed spermatozoa chromatin. The distribution of histone marks was also analysed in mature spermatozoa with different chromatin structures. Histone H2A and H4 marks were closely associated with the relatively loose chromatin structure in crab spermatozoa. The significant decrease in the HS1ph signal during spermatogenesis suggests that eliminating most of these epigenetic marks in the nucleusis closely associated with spermatozoa maturity.

Histone H2B gene cloning, with implication for its function during nuclear shaping in the Chinese mitten crab, Eriocheir sinensis.

Spermatogenesis in animals is the process by which male spermatogonia develop into mature spermatozoa. In most taxa, the process involves changes in the basic proteins associated with DNA. Somatic-type histones are partially or totally replaced by transition proteins, which in turn are replaced by protamines producing compact packaging of the genome. Sperm chromatin in the Chinese mitten crab (Eriocheir sinensis) has a noncompacted loosely arranged organization. However, its formation during spermatogenesis is not clear. In this study, a cDNA sequence encoding histone H2B was cloned by polymerase chain reaction amplification, and its recombinant protein was expressed and purified. Protein alignment studies demonstrated that this histone H2B had 80.80%, 95.12%, 80.16%, 91.87%, 81.75%, 77.78% and 99.19% identity with its counterparts in zebrafish, fruit fly, human, prawn, mouse, African clawed frog, and crayfish, respectively. Western blotting indicated that the recombinant protein could be recognized by an anti-H2B antibody and confirmed that histone H2B exists in sperm nuclei. Immunofluorescence demonstrated that histone H2B was present in the nuclei of spermatogonia, spermatocytes, spermatids, and mature spermatozoa. This is the first report that the mature sperm nucleus of E. sinensis contains histone H2B. This work complements a previous study of sperm histones of this species and provides a basis for further study of the noncondensed sperm nuclei of decapod crustaceans.

Effects of Atrazine on the Development of Neural System of Zebrafish, Danio rerio.

By comparative analysis of histomorphology and AChE activity, the changes of physiological and biochemical parameters were determined in zebrafish embryos and larvae dealt with atrazine (ATR) at different concentrations (0.0001, 0.001, 0.01, 0.1, and 1 mg/L). This study showed that the development of the sarcomere and the arrangement of white muscle myofibers were affected by ATR significantly and the length of sarcomere shortened. Further analysis of the results showed that the AChE activity in juvenile fish which was treated with ATR was downregulated, which can indicate that the innervation efficiency to the muscle was impaired. Conversely, the AChE activity in zebrafish embryos which was treated with ATR was upregulated. A parallel phenomenon showed that embryonic primary sensory neurons (Rohon-Beard cells), principally expressing AChE in embryos, survived the physiological apoptosis. These phenomena demonstrated that the motor integration ability of the zebrafish was damaged by ATR which can disturb the development of sensory neurons and sarcomere and the innervations of muscle.

Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping 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 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.

[Histone phosphorylation and spermatogenesis].

Histone phosphorylation is one of the most important post-translational modifications (PTMs) which play a key role in the chromatin remodeling process during spermatogenesis. Histone phosphorylation is related to distinct important biological processes, such as mitotic/meiotic chromosome condensation, chromatin function regulation, transcriptional activation or inactivation, double-strand DNA break repair, and other metabolic reactions. This review discusses the correlation between histone phosphorylation and spermatogenesis in different organisms from literatures published in the last decade. The critical functions of histone phosphorylation during spermatogenesis (sporogenesis) are summarized, such as binding sites involved in protein interaction, regulation of meiotic replication and/or recombination, correct chromatin remodeling, and chromatin compaction in the sperm nucleus (spore) after post-meiotic process. These findings will help build a solid foundation for further study, and also deepen our understanding of the roles for histones and their modifications in male germ cell development and differentiation.

Transition of basic protein during spermatogenesis of Fenneropenaeus chinensis (Osbeck, 1765).

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed.