The E3 Ubiquitin Ligase CRL4 Regulates Proliferation and Progression Through Meiosis in Chinese Mitten Crab Eriocheir sinensis1

Abstract

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.

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

“…In mammals, the role of p53 in testis is to modulate DNA repair and cell apoptosis to control quality and quantity of mature spermatozoa during spermatogenesis [ 32 ]. Studies found that p53 homeostasis was maintained by the E3 ubiquitin ligase Cul4 complex in primary spermatocytes to maintain male fertility, as p53 could induce cell cycle arrest to allow DNA meiotic reshuffling and correction of DNA damage [ 33 ]. The cell cycle gene p53 also played a critical role in the maintenance of undifferentiated state by inhibiting a mammalian target of the rapamycin complex 1 (mTORC1) in spermatogonia [ 56 ].…”

“…In mammals, p53 is an active participant in DNA repair and DNA damage response pathways [ 32 ]. In E. sinensis testis, p53 was also found to mediate spontaneous germ cell apoptosis to eliminate defective germ cells [ 33 ]. What's more, the mitochondria-dependent apoptotic pathway involving caspase 3 and caspase 9 was initiated.…”

A core component of the CUL4 ubiquitin ligase complexes, DDB1, regulates spermatogenesis in the Chinese mitten crab, Eriocheir sinensis

“…To check the accuracy of the predicted gene model, we designed primers to re-clone the full-length cDNA. The gene organization (intron/exon boundaries) was predicted using Genscan software (http://genes.mit.edu/GENSCAN.html) (Burge and Karlin, 1997) and the schematic diagram of the Es-DBB1 gene organization was constructed according to the predicted results (Wang et al, 2016).…”