Heat shock protein 70 kDa (HSP70) is involved in the maintenance of pig sperm function throughout liquid storage at 17 °C.

At present, liquid storage is the most efficient method for pig semen preservation. This approach relies upon reducing sperm metabolism, allowing for the maintenance of cell lifespan. In this context, the study of proteins that could protect sperm during liquid storage is of high relevance. The 70 kDa Heat Shock Protein (HSP70) is an anti-apoptotic protein that has been reported to be relevant to sperm survival. Thus, we explored the role of HSP70 during prolonged storage of pig semen at 17 °C. Six semen pools were incubated with YM-1 (0, 0.05, 0.1 and 0.2 µM), an HSP70 inhibitor, and stored at 17 °C for 21 days. On days 0, 4, 10, 14 and 21, sperm quality and function were evaluated through flow cytometry and Computer-Assisted Sperm Analysis (CASA), and HSP70 activity and chromatin condensation were also determined. While inhibition of HSP70 increased progressive motility, Ca2+ and Reactive Oxygen Species (ROS) levels, and mitochondrial activity during the first 10 days of storage, it had a detrimental effect on sperm motility after 14 and 21 days. In spite of this, sperm viability was not altered. We can conclude that HSP70 contributes to the liquid storage of pig semen because it keeps mitochondrial activity low, which is needed for the maintenance of sperm function.

Voltage-dependent anion channels are involved in the maintenance of pig sperm quality during liquid preservation.

Pigs are usually bred through artificial insemination with liquid semen preserved at 15-20 °C. While this method of preservation brings many benefits, including a greater reproductive performance compared to frozen-thawed sperm, the period of storage is a limiting factor. As the mitochondrion regulates many facets of sperm physiology, modulating its activity could have an impact on their lifespan. Aligned with this hypothesis, the present study sought to investigate whether inhibition of voltage-dependent anion channels (VDACs), which reside in the outer mitochondrial membrane and regulate the flux of ions between mitochondria and the cytosol in somatic cells, influences the resilience of pig sperm to liquid preservation at 17 °C. For this purpose, semen samples (N = 7) were treated with two different concentrations of TRO19622 (5 µM and 50 µM), an inhibitor of VDACs, and stored at 17 °C for 10 days. At days 0, 4 and 10, sperm quality and functionality parameters were evaluated by flow cytometry and computer-assisted sperm analysis (CASA). The effects of inhibiting VDACs depended on the concentration of the inhibitor. On the one hand, the greatest concentration of TRO19622 (50 µM) led to a decrease in sperm motility, viability and mitochondrial membrane potential, which could be related to the observed intracellular Ca2+ increase. In contrast, total sperm motility was higher in samples treated with 5 µM TRO19622 than in the control, suggesting that when VDACs channels are inhibited by the lowest concentration of the blocking agent the resilience of pig sperm to liquid storage increases. In conclusion, the current research indicates that mitochondrial function, as regulated by ion channels in the outer mitochondrial membrane like VDACs, is related to the sperm resilience to liquid preservation and may influence cell lifespan.

An intracellular, non-oxidative factor activates in vitro chromatin fragmentation in pig sperm.

BACKGROUND: In vitro incubation of epididymal and vas deferens sperm with Mn2+ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn2+ and Mg2+) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry. RESULTS: Incubation with Mn2+/Ca2+ activated SCF in a dose-dependent (P < 0.05) albeit not time-dependent manner (P > 0.05); in contrast, Mg2+/Ca2+ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn2+/Ca2+ or Mg2+/Ca2+, SCF generated DNA fragments of 33-194 Kb, compatible with the size of one or multiple toroids. Besides, Mn2+/Ca2+ affected sperm motility in a dose-dependent manner (P < 0.05), whereas Mg2+/Ca2+ only impaired this variable at high concentrations (P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn2+/Ca2+ or Mg2+/Ca2+ treatments. CONCLUSION: Mn2+/Ca2+ and Mn2+/Ca2+ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death.

Glutathione S-transferase Mu 3 is associated to in vivo fertility, but not sperm quality, in bovine.

In the dairy breeding industry, pregnancy of dairy cows is essential to initiate milk production, so that high fertility rates are required to increase their productivity. In this regard, sperm proteins that are indicative of sperm quality and/or fertility have become an important target of study. Glutathione S-transferase Mu 3 (GSTM3) has been established as a fertility and sperm quality parameter in humans and pigs and, consequently, it might be a potential biomarker in cattle. For this reason, the present work aimed to determine if GSTM3 could predict sperm quality and in vivo fertility in this species. Sperm quality was assessed with flow cytometry and computer-assisted sperm analysis. Immunoblotting and immunofluorescence analysis were performed to determine the presence and localisation pattern of sperm GSTM3. This enzyme was found to be present in bovine sperm and to be localised along the sperm tail and the equatorial segment of the head. No significant associations between sperm GSTM3 and sperm quality parameters were observed, except a negative association with morphologically abnormal sperm having a coiled tail. In addition, and more relevant, higher levels of GSTM3 in sperm were seen in bulls showing lower in vivo fertility rates. In conclusion, our data evidenced the presence of GSTM3 in bovine sperm. Moreover, we suggest that, despite not being associated with sperm quality, GSTM3 might be an in vivo subfertility biomarker in cattle sperm, and that high levels of this protein could be an indicative of defective spermatogenesis and/or epididymal maturation.