Stallion spermatozoa express LDH isoforms A, B, and C, with LDHC playing a crucial role in sustaining sperm viability

Abstract

In brief: Three isoforms of lactate dehydrogenase (LDH) - LDHA (cytoplasmic), LDHB (mitochondrial), and LDHC (flagellar) - have been identified and localized in stallion spermatozoa. Functional inhibition assays indicate that these three isoforms constitute a lactate shuttle of crucial importance for sperm function.

Abstract: Stallion spermatozoa use different energy sources; while oxidative phosphorylation predominates, glycolysis and beta-oxidation of fatty acids are also present. Glycolysis depends on the availability of NAD+ as an electron acceptor. During glycolysis, NAD+ is reduced to NADH. To ensure glycolysis can continue, NAD+ must be regenerated. This regeneration typically occurs when NADH donates its electrons to the electron transport chain (specifically at Complex I), where it is oxidized back to NAD+. If mitochondria are damaged, the regeneration of NAD+ may be compromised, leading to reduced glycolysis and altering sperm metabolism. However, alternative ways to regenerate NAD+ may be present. We hypothesized that aerobic glycolysis is present in the stallion spermatozoa as a backup mechanism to regenerate NAD+. We incubated spermatozoa in two Tyrode's modified media with either 67 mM glucose and 1 mM pyruvate or 67 mM glucose and 10 mM pyruvate. The addition of 10 mM pyruvate improved sperm motility (P < 0.001). Spermatozoa incubated in 67 mM glucose and 1 mM pyruvate for 3 h at 37°C showed a significant decrease in motility (58.1 ± 1.8% vs 81.2 ± 1.8%, P < 0.0001). In contrast, spermatozoa incubated in 67 mM glucose and 10 mM pyruvate retained motility (77.1 ± 1.4%), viability, and mitochondrial membrane potential. We studied the metabolic proteome and metabolome and identified three different isoforms of the enzyme lactate dehydrogenase (LDH), LDHA (cytosolic), LDHB (mitochondrial, with higher affinity for pyruvate), and LDHC (cytosol, motile cilium). Functional experiments using a specific inhibitor of LDHC demonstrated that this isoform may be essential for sperm function. We concluded that activation of aerobic glycolysis in a high-glucose medium improves sperm survival through the regeneration of NAD+.

Keywords: LDH; NAD+; lactate; pyruvate; spermatozoa; stallion.

Figure 1

Effect of media composition on sperm motility and kinematics. Individual ejaculates were processed as described in material and methods and split samples were incubated for up to 3 h in different media. (A) Percentage of total motile spermatozoa, (B) percentage of linear motile spermatozoa, (C) circular velocity in μm/s, (D) straight line velocity (μm/s), (E) average velocity (μm/s), (G) BCF (Hz), TYR T0 = Tyrode’s beginning of the incubation period, TYR T3 = Tyrode’s after 3 h of incubation, 67 mM glucose 1 mM pyruvate after 3 h of incubation = 67 mM glucose + 1 mM pyruvate, 67 mM glucose + 10 mM pyruvate, 67 mM glucose and 10 mM pyruvate after 3 h of incubation, a-b-c- columns with different superscripts differ significantly P < 0.01.

Figure 2

Effect of media composition on sperm viability, membrane permeability, and apoptotic changes. Individual ejaculates were processed as described in material and methods, split samples were incubated for up to 3 h in different media, and flow cytometry analysis was conducted. (A) Percentage of live spermatozoa, (B) percentage of spermatozoa with increased membrane permeability, (C) percentage of necrotic spermatozoa, (D) expression of phosphatidylserine (PS) in the outer leaflet of the sperm membranes (relative fluorescence units), TYR T0 = Tyrode’s beginning of the incubation period, TYR T3 = Tyrode’s after 3 h of incubation, 67 mM glucose 1 mM pyruvate after 3 h of incubation = 67 mM glucose + 1 mM pyruvate, 67 mM glucose + 10 mM pyruvate, 67 mM glucose and 10 mM pyruvate after 3 h of incubation, a-b-c- columns with different superscripts differ significantly P < 0.01.

Figure 3

Effect of media composition on the percentage of spermatozoa with high mitochondrial membrane potential and mitochondrial mass. Individual ejaculates were processed as described in material and methods. Split samples were incubated for up to 3 h in different media, and flow cytometry analysis was conducted. (A) Percentage of spermatozoa showing high mitochondrial membrane potential, (B) changes in mitochondrial mass along the incubation period in different media, (C) flow cytometry dot plots showing the five-color panel in concatenated replicates. Density plots are presented. In column a, population 1 is the percentage of live spermatozoa, note that the density plot indicates an increase in the population of dead spermatozoa in spermatozoa incubated in the 67 mM glucose 1 mM pyruvate media (*). Column B dot plots showing changes in the mitochondrial membrane potential; gate 2 represents the population of dead spermatozoa with low mitochondrial membrane potential. Column C dot plots showing changes in the mitochondrial mass; population 3 shows live spermatozoa maintaining mitochondrial mass. Column C dot plots showing the transposition of phosphatidylserine (PS) to the outer membrane, a population of live spermatozoa with PS translocation to the outer membrane is depicted in 4. TYR T0 = Tyrode’s beginning of the incubation period, TYR T3 = Tyrode’s after 3 h of incubation, 67 mM glucose 1 mM pyruvate after 3 h of incubation = 67 mM glucose + 1 mM pyruvate, 67 mM glucose + 10 mM pyruvate, 67 mM glucose and 10 mM pyruvate after 3 h of incubation, a-b-c- columns with different superscripts differ significantly P < 0.01.

Figure 4

Enrichment analysis of the metabolic proteins detected in stallion spermatozoa. Equine orthologs were transformed into human orthologs and analyzed in the g profiler (https://biit.cs.ut.ee/gprofiler/gost) using default settings and in metascape (https://metascape.org/gp/index.html#/main/step1) using custom analysis interrogating the list of proteins against the terms ‘pyruvate’ and ‘lactate’. (A) Manhattan plot showing enriched terms, (B and C) enrichment of proteins matching membership terms: LACTATE and PYRUVATE. The outer pie shows the number and percentage of genes in the background associated with the membership (in black); the inner pie shows the number and percentage of genes in the individual input gene list associated with the membership. The P-value indicates whether the membership is statistically significantly enriched in the list.

Figure 5

Changes in the relative amounts of metabolic proteins in spermatozoa incubated in different media. Individual ejaculates were processed as described in material and methods. Split samples were incubated for up to 3 h in media containing different amounts of glucose and pyruvate, and proteomic analysis was performed as described in material and methods. TYR T0 = Tyrode’s at the beginning of the incubation period (T0), TYR T3 = Tyrode’s after 3 h of incubation, 67 mM glucose 1 mM pyruvate after 3 h of incubation = 67 mM glucose + 1 mM pyruvate, 67 mM glucose and 10 mM pyruvate after 3 h of incubation = 67 mM glucose + 10 mM pyruvate. Values with different superscripts differ statistically a-c P < 0.001.

Figure 6

Changes in the relative amounts of ATP synthase subunits alpha (A) and beta (B) along the incubation period in stallion spermatozoa. Individual ejaculates were processed as described in material and methods. Split samples were incubated for up to 3 h in different media, and proteomic analysis was performed as described in material and methods. TYR T0 = Tyrode’s at the beginning of the incubation period (T0), TYR T3 = Tyrode’s after 3 h of incubation, 67 mM glucose 1 mM pyruvate after 3 h of incubation = 67 mM glucose + 1 mM pyruvate, 67 mM glucose and 10 mM pyruvate after 3 h of incubation = 67 mM glucose + 10 mM pyruvate. Values with different superscripts differ statistically a-c P < 0.001.

Figure 7

Changes in the relative amounts of the three isoforms of LDH detected in stallion spermatozoa incubated up to 3 h in media with different concentrations of glucose and pyruvate. TYR T0 = Tyrode’s at the beginning of the incubation period (T0), TYR T3 = Tyrode’s after 3 h of incubation, 67 mM glucose 1 mM pyruvate after 3 h of incubation = 67 mM glucose + 1 mM pyruvate, 67 mM glucose and 10 mM pyruvate after 3 h of incubation = 67 mM glucose + 10 mM pyruvate. The structure of the proteins was downloaded from AlphaFold https://www.alphafold.ebi.ac.uk. (A) LDHA, (B) LDHB, (C) LDHC *P < 0.05, **P < 0.01, (D) Identification of the three isoforms of LDH A, B, and C in stallion sperm lysates.

Figure 8

Immunolocalization of the three isoforms of LDH in stallion spermatozoa. Stallion spermatozoa were processed as indicated in material and methods and evaluated using image flow cytometry (A) LDHA, this protein is expressed in the acrosomal region (B) LDH B is expressed in the midpiece, over the mitochondrial sheath (C) LDHC is expressed in the tail, especially in the principal piece.

Figure 9

Inhibition assay using oxamate (LDHA inhibitor) and EAA, a specific inhibitor of the C isoform of LDHC. Three independent ejaculates from three different stallions were used in the experiment (n = 9). Stallion spermatozoa were incubated for up to 3 h in the presence of 0, 20, and 40 mM oxamate and 0, 20, and 40 mM EAA. After 3 h of incubation, motility and circular velocity (VCL μm/s) were measured using CASA. The percentages of viable, apoptotic, and high mitochondrial potential spermatozoa were investigated using flow cytometry, b-d P < 0.01; b-c-d-e P < 0.00001, a-b P < 0.01.

Figure 10

UHPLC/MS/MS analysis of changes in the relative amounts of lactate, NAD+, ATP, and phosphoenolpyruvate in stallion spermatozoa incubated in two media differing in the amount of pyruvate, 67 mM glucose and 1 mM pyruvate, and 67 mM glucose and 10 mM pyruvate.