Research Article: Roles of three Es-Caspases during spermatogenesis and Cadmium-induced apoptosis in Eriocheir sinensis

Date Published: May 24, 2018

Publisher: Impact Journals

Author(s): Ya-Ru Xu, Wan-Xi Yang.


Functions of Caspases remain obscure in Crustacea. We studied the existence and participations of apoptosis-related factors in Eriocheir sinensis testis. Three Es-Caspases (Es-Caspase 3/ 7/ 8) in E. sinensis were cloned and characterized. We observed that three es-caspases mRNA had specific expression patterns during spermiogenesis, with weak signal around the nucleus and invaginated acrosomal vesicle in early-stage spermatids, became stronger in middle-stage, finally focused on the acrosomal tube and nucleus in mature sperm. We then investigated the immunostaining intensity and positional alterations of Es-Caspase 3, Es-Caspase 8 and p53 during spermatogenesis, which were correlated with the differential tendencies of cells to undergo apoptosis and specific organelles shaping processes. After apoptotic induction by Cadmium, Es-Caspase 8 increased gradually, while Es-Caspase 3 increased firstly and then decreased, Es-p53 initially decreased and then increased. These results implies that Es-Caspase 3/ Es-Caspase 8/ p53 may play roles in Cadmium-induced apoptosis during spermatogenesis, and Caspase 8-Caspase 3-p53 pathway may interact with extrinsic or intrinsic pathways to regulate the destiny of sperm cells. Our study revealed the indispensable roles of Caspases during spermatogenesis and the possible molecular interactions in response to the Cadmium-induced apoptosis in E. sinensis, which filled the gap of apoptotic mechanisms of crustacean.

Partial Text

Apoptotic programmed cell death (PCD) is a primary type of cell death, initiated and executed jointly by a series of apoptosis-related factors, like a class of cysteine-dependent aspartate-directed proteases (Caspases) [1,2]. Lots of biochemical and morphological characteristics typically occur during apoptosis like chromatin and nuclear condensation, DNA cleavage, organelles destruction, membrane blebbing, apoptotic bodies formation and so on [3–5]. The above apoptosis-related events are caused by extrinsic apoptotic pathway and intrinsic apoptotic pathway. Specific stimuli induce the binding of death receptors (like tumor necrosis factor receptor-TNFR) on the cell membrane with their corresponding ligands (like tumor necrosis factor-TNF) outside the cell, which will trigger the extrinsic Caspase-cascade [6]. Bcl-2 family members or Caspase 2 may facilitate the releasing of Cytochrome c from mitochondrial intermembrane space, and result in cell death via mitochondrial pathway [7,8]. Many factors are responsible for apoptosis, including domestic and external factors, inherited and genetic factors, etc. For example, drugs or metal elements treatment, growth factors shortage, radiation induction, oncogene overexpression or mutation convergence, can all cause cell death [9–11]. The apoptotic cysteine proteases had been found to play indispensable roles in the process of apoptosis since the first discovery in 1985 [12]. They are divided into initiator Caspases and effector Caspases according to the specific protein domains and their functions. Caspase 8 and/or Caspase 2 are classified into the initiator Caspase family as the death effector domains (DED) or caspase-recruitment domains (CARD) in the N-teminus [13]. DED or CARD could mediate signals dimerization and/or upstream adapter molecules recognition. Caspase 3 is the canonical effector Caspase, which could react to both death-inducing signaling complex (DISC)-mediated extrinsic pathway and Bcl-2 family-Caspase 9-mediated intrinsic pathway. Substrates of Caspases affect all aspects of cell life, such as transcriptional factors, cytoskeletal proteins, kinases, and so on [13,14].

Testicular apoptosis function to sweep away the damaged, defective, superfluous or inadequately supported germ cells [43,44]. Such process could help maintain the structural integrity of the spermatogenic epithelium and keep a healthy environment for testis development. In the present study, three caspase family members (es-caspase 3/ es-caspase 7/ es-caspase 8) were identified and characterized in the testis of the Chinese mitten crab E. sinensis. In this study, on the base of former research of morphological and biochemical performance in Cd2+-induced germ cell apoptosis, we attempt to analyze the functions and potential action mechanisms of Caspases and p53 in testis, particularly during meiotic germ cell division.

In conclusion, three crucial apoptosis-related proteases in E. sinensis (Es-Caspase 3/ Es-Caspase 7/ Es-Caspase 8) were isolated and annotated. Three Caspases had a wide distribution and were evolutionarily conserved. In this study, we noted that the variations in Es-Caspase 3/ Es-Caspase 8/ p53 immunostaining patterns were associated with distinct nuclear alterations and acrosome formation. The potential molecular mechanisms of Cd2+-induced apoptosis were explored for the first time. We found that the extrinsic pathway involved Es-Caspase 8-Es-Caspase 3 and the intrinsic pathway involved Es-p53 jointly to take effect during spermatogenesis after Cd2+-handling. The possible apoptotic signal network triggered by Cd2+ is summarized in Figure 7. Figuring out such network in more detail will provide insights into the apoptosis occurrence in crustaceans, and will identify strategies to improve the sperm quality and output quantity in the future. In addition, this work did provide an important foundation for the research of apoptosis in crustaceans, although the molecular mechanisms of action need more detailed investigations.




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