Glutathione S-Transferases Play a Crucial Role in Mitochondrial Function, Plasma Membrane Stability and Oxidative Regulation of Mammalian Sperm

Abstract

Glutathione S-transferases (GSTs) are essential sperm antioxidant enzymes involved in cell protection against oxidative stress and toxic chemicals, preserving sperm function and fertilising ability. Artificial insemination (AI) in pigs is commonly carried out through the use of liquid-stored semen at 17 °C, which not only reduces sperm metabolic activity but also sperm quality and AI-farrowing rates within the 72 h of storage. While one may reasonably suggest that such enzymes are implicated in the physiology and maintenance of mammalian sperm function during liquid-storage, no previous studies conducted on any species have addressed this hypothesis. Therefore, the objective of the present work was to characterise the presence and function of sperm GSTs in mammalian sperm, using the pig as a model. In this regard, inhibition of such enzymes by ethacrynic acid (EA) during semen storage at 17 °C was performed to evaluate the effects of GSTs in liquid-preserved boar sperm by flow cytometry, immunofluorescence, and immunoblotting analysis. The results of this study have shown, for the first time in mammalian species, that the inhibition of GSTs reduces sperm quality and functionality parameters during their storage at 17 °C. These findings highlight the key role of such enzymes, especially preserving mitochondrial function and maintaining plasma membrane stability. In addition, this study has identified and localised GSTM3 in the tail and equatorial subdomain of the head of boar sperm. Finally, this study has set grounds for future investigations testing supplementation of semen extenders with GSTs, as this may improve fertility outcomes of swine AIs.

Keywords: Boar semen; Ethacrynic acid; GSTM3; GSTs; Liquid-storage; Mammalian sperm.

Figure 1

(A) percentages of total motile sperm, (B) percentages of progressive motile sperm, (C) average pathway velocity (VAP; μm/s), and (D) percentages of high ΔΨm sperm (JC1_agg sperm) of semen samples treated with ethacrynic acid (EA), a glutathione S-transferases (GSTs) inhibitor, and the control group, assessed at different evaluation times during liquid storage at 17 °C (0, 24, 48, and 72 h). Different letters (a, b) indicate significant differences (p < 0.05) between treatments within storage time.

Figure 2

Percentages of (A) total viable sperm (SYBR14⁺/PI^-), (B) viable sperm with high membrane lipid disorder (M540⁺/YO-PRO-1^-), (C) viable apoptotic-like sperm (AnnexinV⁺/PI^-) and (D) viable acrosome membrane-intact sperm (PNA-FITC^-/PI^-) of semen samples treated with ethacrynic acid (EA), a glutathione S-transferases (GSTs) inhibitor, and the control group, assessed at different evaluation times during liquid storage at 17 °C (0, 24, 48, and 72 h). Different letters (a, b) indicate significant differences (p < 0.05) between treatments within storage time.

Figure 3

(A) Percentages of viable spermatozoa showing high intracellular calcium levels in the mid-piece and head (Fluo3⁺/PI⁻), (B) mean Fluo3⁺ fluorescence intensity of viable spermatozoa showing high intracellular calcium levels in the mid-piece and head, (C) percentages of viable spermatozoa showing high intracellular calcium levels in the head (Rhod5⁺/YO-PRO-1^–), and (D) mean Rhod5⁺ fluorescence intensity of viable spermatozoa showing high intracellular calcium levels in the sperm head of semen samples treated with ethacrynic acid (EA), a glutathione S-transferases (GSTs) inhibitor, and the control group, assessed at different evaluation times during liquid storage at 17 °C (0, 24, 48, and 72 h). Different letters (a, b) indicate significant differences (p < 0.05) between treatments within storage time.

Figure 4

(A) Percentages of viable spermatozoa showing high superoxide (O₂^-•) levels (E⁺/YO-PRO-1⁻), (B) mean E⁺ fluorescence intensity of viable spermatozoa showing high superoxide levels, (C) percentages of viable spermatozoa showing high peroxide (H₂O₂) levels (DCF⁺/PI⁻), and (D) mean DCF⁺ fluorescence intensity of viable spermatozoa showing high peroxide levels of semen samples treated with ethacrynic acid (EA), a glutathione S-transferases (GSTs) inhibitor, and the control group, assessed at different evaluation times during liquid storage at 17 °C (0, 24, 48, and 72 h). Different letters (a, b) indicate significant differences (p < 0.05) between treatments within storage time.

Figure 5

Immunolocalisation of sperm GSTM3. (A,B) control group at 0 h of storage at 17 °C, (C,D) control group at 72 h of liquid-storage, (E,F) Ethacrynic acid (EA)-treated spermatozoa at 72 h of liquid-storage, (G) negative control, and (H) peptide competition assay. White arrows indicate the sperm midpiece. The nucleus is shown in blue colour (DAPI), whereas GSTM3 is shown in green (fluorescein isothiocyanate, FITC). Scale bars: A, C, E, H: 30 μm; D, G: 15 μm; B, F: 10 μm.

Figure 6

(A) Representative Western blots resulting from the incubation with the GSTM3 antibody (Anti-GSTM3) and its loading control (α-tubulin). (B) Western blots resulting from incubation with the GSTM3 antibody with GSTM3-blocking peptide (Anti-GSTM3 + blocking peptide) and its loading control (α-tubulin). Lanes MW: molecular weight. Lanes C0: control at 0 h of sperm liquid storage. Lanes C72: control at 72 h of sperm liquid storage. Lanes E72: EA-treated samples at 72 h of liquid storage.

Figure 7

(A) Representative Western blot resulting from incubation with the GSTM3 antibody and (B) its loading control (α-tubulin). (C) Relative abundances of ~25 (GSTM3-A) and ~28 (GSTM3-B) kDa bands as mean ± standard error of the mean in all treatments. Values were normalised using the α-tubulin protein as an internal standard. Each sperm sample (n = 10) was evaluated two times. CNT 0 h: control at 0 h of sperm liquid storage; CNT 72 h: control at 72 h of sperm liquid storage; EA 72 h: Ethacrynic acid (EA)-treated samples at 72 h of liquid storage. Different letters (a, b) indicate significant differences (p < 0.05) between treatments.