O-134 Down regulation of Clock gene in testicle may affect energy metabolism of sertoli cell
Authors: Shan X , Cheng S , Zhou J 
Published: July 2024

Abstract

Study question
Whether decrease expression of Clock gene in the testis could affect the energy metabolism of Sertoli cells and what is the possible mechanism?

Summary answer
Down-regulating of Clock gene could regulate the expression of PIK3CB affect AMPK signaling pathways, significantly weakening the energy supply of sertoli cells in mouse testicular.

What is known already
The causes of Nonobstructive azoospermia (NOA) and other male infertility diseases are diverse and complex, including spermatogenesis and germ cell maturation at different stages of meiosis, the expression level of Clock gene maybe is one reason of which. In the testis of NOA patients, expression level of Clock gene was significantly decreased compared with Obstructive azoospermia (OA), meanwhile, the decrease of Clock gene expression can result in the decrease of male mouse fetal number. The decrease of Clock gene expression cause some kind of male infertility problem is confirmed, but the process and mechanism is not clear.

Study design, size, duration
Differential expression genes related to low expression of Clock and pathways related to spermatogenesis were screened out through transcriptome analysis on testicular tissue samples from patients with NOA(n = 24) and OA(n = 17). The model was established by transfecting mouse testicular Sertoli cell line (TM4 cells) with lentivirus containing Clock knock-down gene and energy metabolism effects were detected by Seahorse tests. Adifluorescein reporter gene was constructed to detect the binding of CLOCK and candidated differential expressed gene.

Participants/materials, setting, methods
Normal testis sequencing data were obtained from GTEX database as control. Combined with sequencing data, the differentially expressed genes were obtained as candidate gene, which expression was verified by RT-qPCR, Western-blotting. Energy metabolism were detected by Seahorse mitochondrial-stress test, glycolysis-rate test. Adifluorescein reporter gene for the promoter regions of candidate gene was constructed and transfected into TM4 cells to detect whether CLOCK could regulate candidate gene by binding to the E-box of the promoter region.

Main results and the role of chance
After GO(GeneOntology) and KEGG(Kyoto Encyclopedia of Genes and Genomes) enrichment analysis, PIK3CB which involved in AMPK signaling pathway was selected for further verification and molecular mechanism study. In Seahorse tests, the basal respiratory capacity, maximal respiratory capacity OCR, spare respiratory capacity and ATP-related respiratory capacity of TM4 cells were significantly decreased after Clock gene knockdown, but there were no significant differences in glycolytic rate, basal glycolysis, compensatory glycolysis level and glycolytic rate. RT-qPCR and Western-blotting confirmed that after knock down of Clock gene, the expression level of Pik3cb gene was down-regulated. That imply when Clock gene is decreased, it regulates the expression of PIK3CB affects AMPK signaling pathways, weakening the ability of mouse testicular sertoli cells to supply energy to various levels of spermatogenic cells and sperm cells, which may be one reason for the occurrence of nonobstructive azoospermia. During this process, there were significant changes in the mitochondrial energy supply of testicular supporting cells, but no significant changes in glycolysis. Finally, the dual luciferin report showed that the Clock gene could bind to the promoter regions of Pik3cb to regulate the expression of PIK3CB, and that the promoter of PIK3CB was activated by the transcription factor CLOCK.

Limitations, reasons for caution
Although we identified that Clock gene could bind to the promoter regions of Pik3cb to regulate the expression of PIK3CB and affect AMPK signaling pathways, weakening the ability of mouse testicular sertoli cells to supply energy, the available expression level and whether it will be work in vivo is unknown.

Wider implications of the findings
The results imply that the main energy changes affect the occurrence and development of spermatogonia and primary spermatocytes near the base of the seminiferous tubules are related to the influence of Clock on genes involved in regulating mitochondrial energy supply. It need more further studies.

Trial registration number
not applicable