However, the knockdown of ACLY significantly reduced the effects of the culture supernatant on sperm motility and LIN (Figure 7K–N). In particular, the enhancement of oleic acid secretion by testosterone was inhibited by ACLY knockdown (Figure 7H–J). According to the GC–MS quantification results, the amount of fatty acids secreted into the culture medium was also reduced by ACLY knockdown. Therefore, we performed shRNA knockdown experiments on ACLY to clarify the impact of ACLY on the cellular metabolic shift.
(A, B) The glucose uptake and lipid measurements were normalized based on cell count and viability. These data suggest that GLUT4 was partially localized at the plasma membrane and contributed to glucose uptake. As expected from the results of flux metabolic analyses, testosterone treatment significantly increased glucose uptake (Figure 6A). To determine whether these changes were accompanied by changes in gene expression of specific metabolic-related enzymes, we analyzed gene expression levels. Overall, these data indicate that testosterone promotes the redistribution of metabolic flux. We therefore examined cellular energy metabolism with a flux analyzer, anticipating that testosterone would elevate glycolytic flux, thereby producing more pyruvate from phosphoenolpyruvate.
The fluorescence intensity of sperm detected by flow cytometry significantly increased in a dose-dependent manner (Fig.7B, C). Quantitative analysis of GLUT4 relative to α-tubulin obtained from Western blot. Specifically, the expression of genes encoding a group of enzymes of the electron transport chain was significantly upregulated by continuous flutamide administration in vivo, whereas the gene expression of Acly and Acc was significantly decreased by flutamide administration. However, the expression of enzymes involved in the electron transport chain was significantly decreased.
ACLY was a critical factor in this metabolic change, which produces oleic acid and enhances their fertilization ability in vivo. This section collects any data citations, data availability statements, or supplementary materials included in this article. (3) This reviewer does not see the merit in including a lipid mixture motility experiment compared to using OA alone. Specifically, decapacitation factors temporarily stabilize the sperm membrane, preventing early capacitation.
Curvi-Linear Velocity; VCL, Straight-Line Velocity; VSL and Linearity; LIN is the ratio of VSL to VCL of sperm incubated in HTF medium one hour. (A) The baseline information for the epididymal sperm used in Fig 1 and Supplemental Fig 2A. Additional information needed to reanalyze the data reported in this paper is available from the principal contact upon request.