The E3 Ubiquitin Ligase CRL4 Regulates Proliferation and Progression Through Meiosis in Chinese Mitten Crab Eriocheir sinensis.

The Cullin-RING E3s are multisubunit ubiquitin ligases composed of a scaffold protein known as Cullin, a RING finger protein that regulates diverse cellular pathways; however, their contribution to male gonad development, especially the spermatogenesis of the Chinese mitten crab (Eriocheir sinensis), is not well understood. We identified five evolutionarily conserved Cullins from the transcriptome and genome ofE. sinensis that are potentially involved in regulating male gonad development. The aim of the current study was to determine the mechanisms of Cullin4's effects on spermatogenesis. We observed that Cullin4, p53, and proliferating cell nuclear antigen had a specific expression and localization in primary spermatocytes. We also investigated the accumulation of Cullin substrates by treatment with inhibitor of NEDD8-activating enzyme MLN4924 in vitro. Cell cycle inhibitors p27 and p21 accumulated significantly after 24 and 36 h, respectively. We speculated that p53-mediated spontaneous germ cell apoptosis acts as a quality control mechanism to eliminate defective germ cells and that the Cullin4 complex maintains p53, p21, and p27 homeostasis in primary spermatocytes to regulate spermatogenesis ofE. sinensis Given its widespread evolutionary conservation, Cullin4 may regulate germ line development similarly in other organisms.

Profiling microRNAs in the testis during sexual maturation stages in Eriocheir sinensis.

MicroRNAs (miRNAs) have been implicated in several cellular processes of reproductive tissues through post-transcriptional regulation of protein coding genes. The presence of miRNAs, their expression patterns and processing machinery genes in different stages of testicular and ovarian cellular development have demonstrated the potential role of miRNAs in testicular and ovarian physiology. The Chinese mitten crab (Eriocheir sinensis) is one of the most important aquaculture species in China and has high commercial value as a food source. The molecular mechanisms underlying testis development in these and other crustaceans, which migrate to a new habitat to breed, remain poorly understood. We focus on the gonad of E. sinensis and systematically examined the expression profile of miRNAs in testes during sexual maturation stages using the Illumina Solexa sequencing technology. We found that the microRNA transcriptome exhibited dynamic expression during crab testis development. Subsequent bioinformatic analysis on both conserved and 15 novel miRNAs illustrated that some miRNAs demonstrated a tissue-specific expression pattern and were associated with target genes involved in reproductive function. Our study illustrates how detailed profiling of miRNA expression during stages of sexual maturation and in different tissues can lead to elucidate the role of miRNAs in regulating the development and differentiation of reproductive organs.

ERK is involved in the process of acrosome reaction in vitro of the Chinese mitten crab, Eriocheir sinensis.

Mitogen-activated protein kinases (MAPKs), also termed extracellular signal-regulated kinases (ERKs), are cytoplasmic and nuclear serine/threonine kinases involved in signal transduction of several extracellular effectors. In mammals, ERKs participate in the regulation of spermatogenesis, mature spermatozoa motility, hyperactivation, and the acrosome reaction. To investigate ERK functions in Eriocheir sinensis reproduction, we successfully cloned the full-length ERK from the testis of E. sinensis (ES-ERK). The 1098-nucleotide open reading frame encodes a 365-amino-acid protein with a predicted molecular weight of 42 kDa. Expressions of ES-ERK in different tissues and testis development stages were detected by the quantitative RT-PCR and Western blotting. ES-ERK is expressed relatively highly in the testis. The expression of ES-ERK protein gradually increased in the spermatid stage, reaching a peak in sperm stage. Western blotting showed a similar expression pattern for the total ES-ERK protein, but phospho-ERK (p-ERK) showed the higher expression in spermatid than sperm stage. We also used trypan blue and hematoxylin and eosin staining to identify structural changes in E. sinensis spermatozoa during the process of acrosome reaction (AR). After stimulating the process of AR, the ES-ERK has translocated from the nucleus to the acrosomal tubule. This result suggested that the ERK MAPK might be involved in the regulation of the E. sinensis acrosome reaction.

P38 participates in spermatogenesis and acrosome reaction prior to fertilization in Chinese mitten crab Eriocheir sinensis.

P38 mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine protein kinases that play important roles in cellular responses to inflammatory cytokines and environmental stresses. These kinases are involved in controlling cell division, differentiation and death in mammalian testes and therefore are critical to spermatogenesis. To explore their functions in male reproduction of Chinese mitten crabs, Eriocheir sinensis p38 (Es-p38) protein expression was determined in different tissues including testes at different developmental stages by Western blot. Es-p38 was expressed in various tissues, with higher levels in the heart, stomach, gills and testes. Total Es-p38 protein levels increased gradually during spermatogenesis, but phosphorylated Es-p38 was much higher in the spermatid (August-October) than the spermatocyte (July-August) and sperm (October-January) stages. Trypan blue staining and hematoxylin/eosin staining were both used to detect sperm motility and changes in sperm morphology during the acrosome reaction (AR) induced by pre-incubation with A23187 in vitro, activated Es-p38 proteins detected by fluorescent microscopy were translocated gradually to nuclear and apical cap regions, accumulating at the anterior of the acrosomal tubule. The results suggest the involvement of p38 MAPK in spermatogenesis and the AR in E. sinensis.