P-001 ClpX is required in maintaining mitochondrial functions during meiosis and spermatogenesis

Abstract

Abstract Study question What is the role of ClpX in spermatogonia differentiation and spermatogenesis? Summary answer ClpX is required to maintain normal mitochondrial functions in spermatocytes, deficiency of ClpX resulted in meiosisarrest and diruption of spermatogenesis in mice. What is known already ClpX is mitochondria-specific quality control protease, it maintains proteostasis via degrading misfolded or damagedproteins in mitochondrial matrix. ClpX is a AAA+ protease that uses the energy of ATP binding and hydrolysis to degradeunfolded or misfolded proteins. ClpX is reported working as a complex with ClpP, the complex consists of a hexamer ofClpX and a tetradecamer of ClpP. ClpX recognizes the protein substrates via binding with their unstructured peptidesequences, termed as degradation tags or recognition signals. The fragments of the cleaved polypeptides can then exitthe chamber and be further degraded. Study design, size, duration Study design: we generated a germ cell specific ClpX knockout mice line and evaluated size, weight, inner structure oftestis tissue. We also applied biochemical techniques and high-throughput sequencing techniques to investigate themechanism of ClpX in regulating spermatogenesis. Size: more than 50 mice were used in this study, including the control mice. Duration: 2 years Participants/materials, setting, methods Participants/materials: we generated a ClpX conditional knockout (cKO) mice line by CRISPR-gene editing and Cre-LoxP system, which specifically knockout ClpX in the germ cells of male mice. Setting: the siblings of ClpX WT mice and ClpX cKO mice were compared in various of experiments. Mehods: we performed morphology comparement to check the weight and size of tetes in the control and ClpX cKOmice, and applied immunofluorescence, western blot, histological study, high-throughput sequencing andpharmocological treatment. Main results and the role of chance We found ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply duringmeiosis and attenuated zygotene-pachytene transformation of the male germ cells. The affected spermatocytesexhibited disorder of chromosome synapsis and cross-over events. Their telomeres failed to attach to the nuclearenvelop, which was associated with failure of α-tubulin formation in the ClpX deficient spermatocytes. The dysregulatedspermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within theseminiferous tubules. Both transcriptome analysis and m6A-methylation sequencing analysis highlighted dysregulationof metabolic pathways including the mTOR signaling in the isolated spermatocytes. We confirmed over-activation of themTORC1 pathway with increased expression of phosphorylated S6 and 4EBP after deletion of ClpX in spermatocytes.Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis in theseminiferous tubules with much less apoptotic germ cells and formation of late stage of meiotic germ cells, such asround spermatids and elongated spermatozoon. The data reveal the novel roles of ClpX in regulating meiosis andspermatogenesis. Limitations, reasons for caution This study is based on animal experiments, thus, these results only indicated the important roles of ClpX in micespermatogenesis rather than human beings. Clinical data has not yet found a Clpx gene mutation in reproductivedisease. Wider implications of the findings Clinical report has already found the mutation in ClpP can induce autosomal recessive Perrault syndrome, while themutation of ClpX has not been reported yet from clinical data. Our resutls demonstrated severe effect onspermatogenesis after knockingout of ClpX, this might give some insights to the clinic. Trial registration number not applicable