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We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 μM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.

Spermatozoa undergo energy- and metabolism-dependent processes to successfully fertilize the oocyte. AMP-activated protein kinase, AMPK, is a sensor of cell energy. We recently showed that AMPK controls spermatozoa motility. Our aims are i) to investigate the intracellular localization of AMPK in boar spermatozoa by immunofluorescence, ii) to study whether AMPK plays a role in other relevant processes of spermatozoa: mitochondrial membrane potential (∆Ψm), plasma membrane lipid disorganization, outward phosphatidylserine (PS) exposure, acrosome integrity and induced-acrosome reaction by flow cytometry and iii) to investigate intracellular AMPK pathways by western blot. Spermatozoa were incubated under different conditions in the presence or absence of compound C (CC, 30μM), an AMPK inhibitor and/or cAMP analog 8Br-cAMP. AMPKα protein is expressed at the entire acrosome and at the midpiece of spermatozoa flagellum, whereas phospho-Thr172-AMPK is specifically localized at the apical part of acrosome and at flagellum midpiece. CC treatment rapidly confers head-to-head aggregation-promoting property to spermatozoa. Long term AMPK inhibition in spermatozoa incubated in TCM significantly reduces high ∆Ψm. Moreover, AMPK inhibition significantly induces plasma membrane lipid disorganization and simultaneously reduces outward PS translocation at plasma membrane in a time-dependent manner. Acrosomal integrity in TCM is significantly enhanced when AMPK is inhibited. However, neither acrosome reaction nor membrane lipid disorganization induced by ionophore A23187 are affected by CC. AMPK phosphorylation is potently stimulated upon PKA activation in spermatozoa. This work suggests that AMPK, lying downstream of PKA, regulates at different levels mammalian spermatozoa membrane function.

Boar semen preservation for later use in artificial insemination is performed by diluting semen in an appropriate medium and then lowering the temperature to decrease spermatozoa metabolism. The adenosine monophosphate-activated kinase, AMPK, is a key cell energy sensor that controls cell metabolism and recently has been identified in boar spermatozoa. Our aim was to investigate the role of AMPK in spermatozoa functional parameters including motility, mitochondrial membrane potential, plasma membrane integrity, acrosome integrity, and cell viability during long-term boar semen storage at 17 � C in Beltsville thawing solution. Boar seminal doses were diluted in Beltsville thawing solution in the presence or absence of different concentrations of AMPK inhibitor, compound C (1, 10, and 30 mM) and evaluations were performed at 1, 2, 4, 7, or 10 days. Data demonstrate that AMPK becomes phosphorylated at threonine 172 (active) during storage of boar semen reaching maximum levels at Day 7. Moreover, AMPK inhibition during boar semen storage causes: (1) a potent inhibition of spermatozoa motility; (2) a reduction in the percentage of spermatozoa showing high mitochondria membrane potential; (3) a rise in the percentage of spermatozoa displaying high plasma membrane scrambling; and (4) a loss of acrosomal membrane integrity. Our study suggests that AMPK activity plays an important role in the maintenance of the spermatozoa quality during long-term storage of boar semen.