Metabolic Shift in Porcine Spermatozoa during Sperm Capacitation-Induced Zinc Flux

Abstract

Mammalian spermatozoa rely on glycolysis and mitochondrial oxidative phosphorylation for energy leading up to fertilization. Sperm capacitation involves a series of well-regulated biochemical steps that are necessary to give spermatozoa the ability to fertilize the oocyte. Additionally, zinc ion (Zn²⁺) fluxes have recently been shown to occur during mammalian sperm capacitation. Semen from seven commercial boars was collected and analyzed using image-based flow cytometry before, after, and with the inclusion of 2 mM Zn²⁺ containing in vitro capacitation (IVC) media. Metabolites were extracted and analyzed via Gas Chromatography-Mass Spectrometry (GC-MS), identifying 175 metabolites, with 79 differentially abundant across treatments (p < 0.05). Non-capacitated samples showed high levels of respiration-associated metabolites including glucose, fructose, citric acid, and pyruvic acid. After 4 h IVC, these metabolites significantly decreased, while phosphate, lactic acid, and glucitol increased (p < 0.05). With zinc inclusion, we observed an increase in metabolites such as lactic acid, glucitol, glucose, fructose, myo-inositol, citric acid, and succinic acid, while saturated fatty acids including palmitic, dodecanoic, and myristic acid decreased compared to 4 h IVC, indicating regulatory shifts in metabolic pathways and fatty acid composition during capacitation. These findings underscore the importance of metabolic changes in improving artificial insemination and fertility treatments in livestock and humans.

Keywords: capacitation; glycolysis; image-based flow cytometry; metabolites; metabolomics; spermatozoa.

Figure 1

Image-based flow cytometry validation of in vitro capacitation. (a–c) Histogram overlays between 0 h (shown in red, filled in solid) and 4 h IVC (shown in green, not filled in), showing changes in the fluorescence of biomarkers between the two treatments. (a) FluoZin 3 AM intensity reporting zinc ion localization, (b) PI intensity reflecting plasma membrane integrity/remodeling, and (c) lectin PNA conjugated to AlexaFluor 647 intensity reflecting acrosomal integrity/remodeling. Boar #2 is displayed here for illustrative purposes. (d–g) Images taken from the Cytek^® Amnis^® ImageStream^®X MkII image-based flow cytometer (Fremont, CA, USA) validating capacitation status. Columns are denoted by channel number and fluorescent probe/target, and rows are single-cell images of different spermatozoa. Channels used: (1) Brightfield (BF), (2) FluoZin 3 AM reflecting zinc (Zn) ion localization, (4) propidium iodide (PI), (7) Hoescht 33342 nuclear stain, and (11) lectin peanut agglutin (PNA)-Alexa Fluor 647™. Examples of (d) zinc signature 1 spermatozoa, (e) zinc signature 2, (f) zinc signature 3, and (g) zinc signature 4. Total percentages of sperm per biomarker classification are in Table S1.

Figure 2

Changes in abundance in key metabolites associated with cellular respiration after in vitro capacitation. A total of 79 metabolites involved in cellular respiration pathways were identified as differentially abundant following capacitation (p < 0.05; complete heatmap of those identified displayed in Figure S2). Eight of the well-known metabolites that are represented here are integral to well-known cellular respiration processes. Non-capacitated (0 h) is shown on the left in red, capacitated (4 h) is shown in the middle in green, and capacitated +2 mM zinc (4 h + Zn) is shown on the right in blue, for each respective metabolite.

Figure 3

Heat map representing top 25 differentially abundant metabolites in sperm metabolome of all sires individually across treatments. Fold change is illustrated by a gradient from dark blue (negative) to dark red (positive). 0 h (non-capacitated) are in red, 4 h IVC are in green, and 4 h IVC + 2 mM zinc are in blue, by class. Metabolites starting with the name “RI=” are unidentified features that have been given a retention-index-based name for tracking purposes. Total 79 statistically significant metabolites are reported in Figure S2.

Figure 4

Principal component analysis. PCA analysis was employed to visualize the metabolic differentiation between non-capacitated spermatozoa (red triangles), capacitated spermatozoa (green squares), and capacitated spermatozoa supplemented with 2 mM zinc (blue circles). Each symbol represents an individual sample plotted against the first two principal components. Colored ellipses represent confidence regions, indicating the clustering of samples within each group, with each color corresponding to a different treatment.

Figure 5

Volcano plots representing top 25 differentially abundant metabolites in sperm metabolome across treatments. Fold change (log2(FC)) is represented on the X-axis, and the statistical significance (−log10(p-value)) is represented on the Y-axis. Metabolites with labels are the top 25 differentially abundant within treatment groups. Varying size in circles in (a,b) represents the relative abundance of each metabolite (a) Comparison before and after 4 h of in vitro capacitation: 23 metabolites were less abundant and 19 metabolites were more abundant after 4 h of IVC. (b) Changes in sperm metabolome abundances between 4 h IVC and 4 h IVC + 2 mM Zinc: 6 metabolites were less abundant and 13 metabolites were more abundant after 4 h of IVC + 2 mM zinc inclusion. Metabolites starting with the name “RI=” are unidentified features that have been given a retention-index-based name for tracking purposes.