High glucose in the extender impacts the metabolic phosphoproteome and modifies the phosphorylation of AKAP4 in stallion spermatozoa.

Overview

• This study investigates how high glucose levels in semen extender solutions affect the phosphorylation of proteins in stallion spermatozoa, particularly focusing on AKAP4, a protein important for sperm function.
• The research found that excessive glucose alters protein phosphorylation negatively, but increasing pyruvate concentration in the extender can prevent these alterations and improve sperm motility.

Background and Rationale

• Commercial extenders used for preserving equine semen typically contain high glucose concentrations (67 mM), which are much higher than physiological levels.
• There is emerging evidence that these supraphysiological glucose levels can negatively impact spermatozoa, potentially compromising their function.
• Because spermatozoa lack the ability to make new proteins (translational inactivity), their regulation is largely controlled through post-translational protein modifications, such as phosphorylation.
• Supraphysiological glucose has been known to disrupt normal cellular regulatory mechanisms, likely through altering these post-translational modifications.
• The researchers hypothesized that the high glucose in the extender affects sperm protein phosphorylation and that increasing pyruvate, an energy substrate, might prevent this deregulation.

Experimental Design and Methods

• Stallion semen samples were divided and incubated in two different media formulations for three hours at 38ºC:
• One with 67 mM glucose and 1 mM pyruvate (standard high glucose, low pyruvate condition).
• The other with 67 mM glucose and an increased 10 mM pyruvate concentration.
• Post incubation, aliquots of spermatozoa were prepared for:
• Proteomic analysis to detect changes in protein phosphorylation patterns.
• Kinematic analysis to assess sperm motility and movement efficiency.

Main Findings

• Spermatozoa incubated in the high glucose, low pyruvate medium (67 mM glucose, 1 mM pyruvate) showed significant changes in the phosphorylation status of AKAP4, a key protein linked to sperm function (P < 0.01).
• These changes were associated with alterations in gene ontology terms related to sperm functionality, indicating broader effects on protein regulation pathways.
• However, incubation with the higher pyruvate concentration (67 mM glucose, 10 mM pyruvate) prevented these phosphorylation changes in AKAP4.
• Additional benefits observed with increased pyruvate included improved sperm kinematic parameters, suggesting better motility and functional competence.

Conclusions and Implications

• High glucose concentrations in semen extenders cause disruption of important post-translational modifications, specifically the phosphorylation of proteins vital for sperm performance such as AKAP4.
• This disruption may impair sperm function, potentially lowering fertility outcomes when using standard commercial extenders with high glucose.
• Increasing pyruvate levels in the extender can offset these negative effects, restoring phosphorylation balance and enhancing sperm motility.
• These findings suggest that adjusting extender composition—specifically increasing pyruvate concentration—may improve stallion semen preservation practices by protecting sperm biochemistry and functional quality.