The Role Of E3 Ubiquitin Ligase Cullin During Spermatogenesis In Chinese Mitten Crab Eriocheir Sinensis

The Chinese mitten crab (Eriocheir sinensis), also known as the fresh water crab, which is a seasonally breeding species and one of the important aquaculture species in China. The special male reproductive development and sperm protection system of E. sinensis provides us with a good research model. To study the mechanism of its molecular biology can directly interpret the mechanism of sperm quality control during spermatogenesis, clarify the reproductive mechanism of crustaceans and provide theoretical basis and guiding significance for the development of artificial insemination technology. This paper focuses on and studies the molecular regulation of E3 ubiquitin ligase CRLs on spermatogenesis of E. sinensis. The ubiquitin-proteasome system plays a fundamental role in the control of numerous cellular processes, including cell cycle progression, gene transcription, signal transduction, proliferation, and differentiation. However, their contribution to male gonad development, especially how the Cullin gene is involved in the regulation of spermatogenesis, is not well understood.Combined with genomic and transcriptome data analysis, molecular cloning, testis cell culture, histological analysis, quantitative real time PCR, western blot, immunofluorescence and other molecular technology have been used for the E. sinensis-in the following six studies:(1) Microscopic assessments and hematoxylin and eosin" (H&E) staining were used to describe all spermatogenic stages for differences in structure, testicular development and staging are generally based on the relative proportions of germ cells. Spermatogenesis in the seminiferous tubules can be divided into five stages:?, spermatogonium stage (May-June); ?, spermatocyte stage (July-August); ?, spermatid stage (August-October); ?, sperm stage (October to April); and ?, rest stage (April-May). (2) To observe the morphological changes during spermatogenesis, we successfully initiated primary cell culture using testis tissue of E. sinensis, and spermiogenesis in Chinese mitten crab E. sinensis is marked by three stages (early, middle and late) of spermatid differentiation and maturation of sperm. (3) We identified five evolutionarily conserved Cullins (Cull, Cul2, Cul3, Cul4 and Cul5) from the transcriptome (-2.31 Gb) and genome (?439.3 Gb) of E. sinensis, and obtained the full-length cDNAs. (4) a phylogenetic tree was constructed using MrBayes with Bayesian inference (BI) methods, and the Chinese mitten crab Cullins are evolutionally conserved. (5) To investigate the level of Cullin mRNA expression, qRT-PCR was performed to characterize the expression profiles of Cullins, which with a similar tissue expression pattern. (6) The aim of the current study was to determine the mechanisms of Cullin4's effects on spermatogenesis, the main regulatory mechanisms are as follows:We used the proliferation marker (PCNA) to investigate the role of Cullins in regulating spermatogenesis. PCNA is a well-known, highly conserved 36-kDa nuclear protein that is associated with DNA replication and damage repair and is expressed variably at different stages of the cell cycle, with a maximum in the S phase. Cul4 and the cell cycle proteins p21, p27, p53, and PCNA show very similar expression pattern and intensities in the different development stages of the testis in E. sinensis. They are all expressed intensely in the primary spermatocyte stage (August), whereas these proteins were severely reduced during the development and maturation of testis. In order to clarify the reasons for this phenomenon, we also investigated the accumulation of Cullin substrates by treatment with inhibitor of NEDD8-activating enzyme MLN4924 in primary cell culture using testis tissue. Recent studies showed that MLN4924 was a potent inhibitor of NAE, blocks Cullin neddylation and causes the subsequent accumulation of several key regulatory substrates of CRLs, and consequently induces cell apoptosis, senescence, and autophagy. MLN4924 treatment (1 ?M) caused the levels of the cell cycle regulator p21 to increase substantially after 36 and 48 h compared with the dimethyl sulfoxide (DMSO) vehicle control, and p27 accumulation was observed prominently after 24 and 36 h. The CDK inhibitor p21 has a central role as an effector of cell cycle arrest and is transactivated by the p53 tumor suppressor protein in response to DNA damage. Previously, p21 was shown to bind PCNA, and this interaction was suggested as a mechanism to inactivate PCNA after DNA damage. 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 of E. sinensis. Given its widespread evolutionary conservation, Cullin4 may regulate germ line development similarly in other organisms.