Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2024
Publication Date: 7/19/2024
Citation: Weide, T., Mills, K.M., Breitzman, M., Kerns, K. 2024. Metabolic shift in porcine spermatozoa during sperm capacitation-induced zinc flux.. International Journal of Molecular Sciences. Vol. 25 Issue 14 ; 7919. https://doi.org/10.3390/ijms25147919.
DOI: https://doi.org/10.3390/ijms25147919

Interpretive Summary: In mammalian reproduction, there are a series of physiologic changes that sperm must undergo to successfully fertilize an egg. The phenomenon that begins this process is known as capacitation. Capacitation prepares the sperm cell for fertilization by allowing the plasma membrane to become more fluid so DNA can be passed from the sperm cell to the egg. Although this process has been well known for many years, the exact pathways that regulate capacitation remain poorly understood. Identifying and understanding how these pathways work together can help researchers improve artificial insemination techniques and fertility treatment modalities in humans and livestock. With the advancement of Mass Spectrometry, we can better understand the physiologic changes associated with capacitation by evaluating compounds that sperm cells use and produce for energy known as metabolites. Therefore, the objective of this study was to observe and characterize changes in metabolite abundance following capacitation. As expected, we observed shifts of metabolites used and produced after cell energy production and those that protect the sperm cell while undergoing these changes (antioxidants). These findings allow us to understand how specific metabolites work together within the sperm cell and will be integral to the advancement of assisted reproductive technologies, artificial insemination, as well as germplasm cryopreservation techniques for all mammalian species.

Technical Abstract: Mammalian spermatozoa bioenergetics utilize glycolysis and mitochondrial oxidative phosphorylation as the main energy producing pathways for sperm capacitation and fertilization. Sperm capacitation is one of the final maturation events that prepares mammalian spermatozoa for fertilization and is characterized by an influx of bicarbonate and calcium ions, removal of decapacitating factors, intracellular pH changes, and changes in various sperm protease activities. Our laboratory recently described the importance of zinc efflux and zinc regulation during capacitation, however, the regulatory mechanisms of other metabolic pathways involved with capacitation remain poorly understood. Therefore, the objective of this study was to observe changes within the sperm cell metabolome following capacitation to characterize and better understand the capacitation event. Fresh semen samples from seven commercial boars were capacitated and analyzed on the flow cytometer before capacitation, after in vitro capacitation (IVC), and IVC with 2 mM Zinc. Metabolites were extracted from semen samples and gas chromatography-mass spectrometry (GC-MS) was used for metabolome analysis. We identified a total of 175 metabolites, with 79 being identified as differentially abundant across all three treatments (P<0.05). In the non-capacitated samples, we observed metabolites associated with cellular respiration pathways with high abundance, metabolites such as Glucose, Fructose, Citric Acid, and Pyruvic Acid. After four hours of IVC, we observed a decrease in these metabolites coupled with an increase in substrates such as Phosphate, Lactic Acid, and Glucitol (P <0.05). In the 4-hour IVC with 2 mM Zinc, we observed an increase of Lactic Acid, Glucitol, Glucose, Fructose, Myo-Inositol, Citric Acid, and Succinic Acid compared to 4-hour IVC, and a decrease in saturated fatty acids such as Palmitic Acid, Dodecanioc Acid, and Myristic Acid, potentially hinting at the regulation of metabolic pathways and fatty acid composition during capacitation events. The data obtained in this study demonstrates the importance of evaluating the changes of these pathway intermediates to ultimately improve artificial insemination techniques and fertility treatment modalities in livestock and humans.