GSTM3, but not IZUMO1, is a cryotolerance marker of boar sperm

Abstract

Background Cryopreservation is currently the most efficient method for long-term preservation of mammalian gametes and is extensively used in swine artificial insemination (AI) centres. However, it is well-known that cryopreservation procedures induce changes in the water phase in both intra and extracellular compartments, which alter the content and localisation of several proteins and ends up curtailing the structural integrity of functional sperm (i.e., cryoinjuries). Alterations and deficiencies of sperm-oocyte binding proteins during gamete recognition are one of the causes of reproductive failure both in vitro and in vivo . In this sense, characterisation of cryopreservation effects upon oocyte-binding proteins of sperm, such as IZUMO1 and GSTM3, is essential when assessing the impact of this technique in swine reproduction. Results Cryopreservation was found to induce changes in the localisation of IZUMO1 and GSTM3 in boar sperm. However, the relative content of both proteins was not altered after thawing. Furthermore, whereas IZUMO1 content was found not to be related to the cryotolerance of boar sperm, GSTM3 content was observed to be higher in poor (PFE) than in good (GFE) freezability ejaculates in both pre-frozen (1.00 INT·mm 2 ± 0.14 INT·mm 2 vs. 0.72 INT·mm 2 ± 0.15 INT·mm 2 ; P < 0.05) and post-thawed (0.96 INT·mm 2 ± 0.20 INT·mm 2 vs. 70 INT·mm 2 ± 0.19 INT·mm 2 ; P < 0.05) samples. Moreover, GSTM3 levels were found to be higher in those spermatozoa that exhibited low mitochondrial activity, high reactive oxygen species (ROS) production, and high membrane lipid disorder post-thaw ( P < 0.05). Conclusions The difference in GSTM3 content between GFE and PFE, together with this protein having been found to be related to poor sperm quality post-thaw, suggests that it could be used as a cryotolerance marker of boar spermatozoa. Furthermore, both IZUMO1 and GSTM3 relocate during cryopreservation, which could contribute to the reduced fertilising capacity of frozen-thawed boar sperm. Electronic supplementary material The online version of this article (10.1186/s40104-019-0370-5) contains supplementary material, which is available to authorized users.

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

“…Since immunoblotting analysis found GSTM3 content to be reduced during semen storage, it becomes apparent that such enzyme is lost rather than relocalised from the mid-piece during liquid-storage. Contrary to the results of the present study, GSTM3 was reported to relocalise to the mid-piece following boar sperm cryopreservation [17].…”

“…Sperm GSTM3 was localised in the mid, principal, and end pieces of the tail and the equatorial subdomain of the head of samples at 0 h of liquid-storage. This localisation pattern is similar to that found in the boar [17] and other species, such as the buffalo [41]. Moreover, the localisation of GSTM3 in the sperm tail would contribute to explaining the dramatic effect of GSTs-inhibition upon sperm motility and mitochondrial function.…”

“…Mounting evidence in the literature supports the relationship between GSTM3 and mitochondrial function, since GSTM3 content is known to be higher in mitochondrial-altered sperm of men [42]. Moreover, recent studies demonstrated that GSTM3 content in fresh sperm is highly correlated to the mitochondrial activity of frozen-thawed sperm, and relocalisation of this enzyme from the entire tail to the mid-piece occurs during cryopreservation of boar [17] and buffalo [41] sperm. Therefore, the relationship between GSTM3 content and mitochondrial activity found in the present study strengthens the hypothesis of a tight molecular relationship between sperm GSTs and mitochondrial function.…”

“…However, sperm liquid-preservation may result in impaired motility, viability, membrane stability, OS, and apoptotic-like changes [3,4]. GSTs in sperm are membrane-attached, detoxifying enzymes [14], which have been considered to be fertility [16], and cryotolerance [17] biomarkers in boar sperm. Furthermore, previous studies have shown that extender supplementation with glutathione decreases OS and improves the quality of boar semen during liquid storage at 17 • C [33].…”

“…Therefore, the relationship between GSTM3 content and mitochondrial activity found in the present study strengthens the hypothesis of a tight molecular relationship between sperm GSTs and mitochondrial function. Moreover, GSTM3 is clearly related to sperm quality, as it has been established as a quality [42][43][44], fertility [16], and cryotolerance [17] biomarker in both boar and human sperm. Hence, one could suggest that the GSTM3 content in fresh boar semen may be used as a biomarker of sperm quality during liquid preservation.…”