Sur, V. P. | bioRxiv Science

bioRxiv2024

Bioenergetics of human spermatozoa in patients with testicular germ cell tumour

In testicular germ cell tumour (TGCT) patients, sperm cryopreservation prior to anti-cancer treatment represents the main fertility preservation approach. However, it is associated with low sperm recovery rate after thawing. Since sperm is a high-energy demanding cell, which is supplied by glycolysis and oxidative phosphorylation (OXPHOS), mitochondrial dysfunctionality can directly result in sperm anomalies. In this study, we investigated the bioenergetic pattern of cryopreserved sperm of TGCT patients in comparison with normozoospermic samples using two state-of-the-art methods; the Extracellular Flux Analyzer (XF Analyzer) and Two-Photon Fluorescence Lifetime imaging (2P-FLIM), in order to assess the contributions of OXPHOS and glycolysis to energy provision. A novel protocol for combined measurement of OXPHOS (Oxygen Consumption Rate - OCR) and glycolysis (Extracellular Acidification Rate - ECAR) using the XF Analyzer was developed together with a unique customized AI-based approach for semiautomated processing of 2P-FLIM images. Our study delivers optimized Low-HEPES modified Human Tubal Fluid media (mHTF) for sperm handling during pre-analytical and analytical phases to maintain sperm physiological parameters and optimal OCR, equivalent of OXPHOS. The negative effect of cryopreservation was signified by deterioration of both bioenergetic pathways represented by modified OCR and ECAR curves and the derived parameters. This was true for normozoospermic as well as TGCT samples, which showed even a stronger damage within the respiratory chain compared to the level of glycolytic activity impairment. The impact of cryopreservation and pathology are supported by 2P-FLIM analysis showing a significant decrease in bound NADH in contrast to unbound NAD(P)H which reflects decreased metabolic activity in samples from TGCT patients. Our study provides novel insight into the impact of TGCT on sperm bioenergetics and delivers a verified protocol to be used for assessment of human sperm metabolic activity, which can be a valuable tool for further research and clinical andrology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/595824v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@130b9eeorg.highwire.dtl.DTLVardef@1fec841org.highwire.dtl.DTLVardef@d401forg.highwire.dtl.DTLVardef@1eccff8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Transcriptomic response in pyroxsulam-resistant and susceptible Bromus sterilis identified three distinct mechanisms of resistance

Bromus sterilis has evolved into a more predominant weed in the Czech Republics winter wheat fields, owing largely to the widespread application of pyroxsulam for its management. In this study, we report a biotype that has developed resistance to pyroxsulam and has also shown cross- resistance to other herbicides. Although no differences in ploidy levels or no mutations of acetolactate synthase (ALS) were detected, a significant elevation of ALS enzyme activity was observed in the R biotype. Through combined analysis of enzyme inhibition and total transcript expression (RNA-Seq), we have identified differentially expressed transcripts that potentially contribute to pyroxsulam metabolism. Furthermore, we observed a significant increase in the expression of genes involved in redox mechanisms and transporters that could contribute to enhanced resistance to pyroxulam in the R biotype. Our results present a novel understanding of herbicide resistance in B. sterilis through three distinct resistance mechanisms (ALS gene overexpression, enhanced metabolism and reduced translocation) without mutation in the herbicide target protein. This understanding is the foundation for improving management strategies for herbicide resistant B. sterilis.

plant biology↗