Stable isotope labelling mass spectrometry analysis of isolated mouse sperm

Metabolism provides sperm with the energy needed to swim to and fertilize the oocyte. While mammalian sperm become motile during ejaculation and undergo maturation in the female genital tract, their energy demand increases. Investigations into the metabolism of sperm and the capacitation-induced increase in energy demand have been stymied by a lack of appropriate methodologies. Here, we present a detailed methodology to perform stable isotope labeling mass spectrometry in isolated mouse sperm, allowing to follow the fate of exogenous energy substrates through their metabolic pathways. As an example, mouse sperm are exposed to ubiquitously and positionally labelled 13C-glucose and the rate of accumulation of 13C in different metabolites is detected and analyzed. Using this assay in the presence of different exogenous energy substrates, with sperm from different species, genetically modified mouse lines, and/or pharmacological activators and/or inhibitors can provide important insight into the contribution of different metabolic enzymes and pathways to sperm energy homeostasis.