The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions

Abstract

Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of autophagy activation. The main objective of this study was to investigate whether LC3 is processed during cooling, freezing and the stressful conditions associated with these technologies. A secondary objective was to determine if LC3 processing can be modulated and if that may improve the quality of cryopreserved semen. LC3 processing was studied by Western blot with a specific antibody that recognized both LC3-I and LC3-II. Viability was assessed by flow cytometry. Modulation of LC3-I to LC3-II was studied with known autophagy activators (STF-62247 and rapamycin) or inhibitors (chloroquine and 3-MA) used in somatic cells. The results showed that conversion of LC3-I to LC3-II increased significantly during cooling at 4°C, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress and changes in temperature). STF-62247 and rapamycin increased the LC3-II/LC3-I ratio and decreased the viability of equine sperm, whereas chloroquine and 3-MA inhibited LC3 processing and maintained the percentage of viable cells after 2 h of incubation at 37°C. Finally, refrigeration at 4°C for 96 h and freezing at −196°C in the presence of chloroquine and 3-MA resulted in higher percentages of viable cells. In conclusion, results showed that an 'autophagy-like' mechanism may be involved in the regulation of sperm viability during equine semen cryopreservation. Modulation of autophagy during these reproductive technologies may result in an improvement of semen quality and therefore in higher fertility rates.Keywords: cryopreservation, refrigeration, viability, autophagy, sperm ImplicationsReproductive technologies with cooled and frozen semen have unquestionable advantages in animal breeding; however, semen preservation results in important injuries in the sperm cells, compromising their viability and hence the pregnancy rates. In the last decade, the scientific community has increased its efforts to improve current cryopreservation protocols. The results of the current study demonstrate that LC3, a protein involved in autophagy, is activated under stressful conditions during cryopreservation, and plays a role in regulating viability. Therefore, this study is of interest not only from a scientific point of view, but also for the improvement of semen quality, which could result in high fertility and finally increased economic gain.

Diet-induced obesity impairs spermatogenesis: a potential role for autophagy

“…Autophagy was also found to be associated with sperm survival545556, and a recent study also showed that autophagy and apoptosis work synergistically to induce germ cell death during mouse spermatogenesis30. However, there are no related reports about the role of autophagy in HFD-induced male spermatogenesis impairment.…”

Elevation of autophagy rescues spermatogenesis by inhibiting apoptosis of mouse spermatocytes

“…Conversion of LC3-I to LC3-II increases significantly during cooling at 4°C, freezing/thawing and several other stressful conditions (UV irradiation, oxidative stress, osmotic stress and changes in temperature). Moreover, exposure to STF-62247, an autophagic agonist, and rapamycin (an autophagic agonist and an antagonist of mammalian target of rapamycin (mTOR)), increases the LC3-II/LC3-I ratio, thereby maintaining the percentage of viable cells, whereas exposure to chloroquine and 3-MA inhibits LC3 processing, thereby decreasing the viability (Aparicio et al 2016). In addition, xeno-estrogen 4-nonylphenol (NP) induces reproductive dysfunction in male rats by inducing caspase-dependent apoptosis and necrosis.…”

Regulation of Autophagy-Related Protein and Cell Differentiation by High Mobility Group Box 1 Protein in Adipocytes

“…A cytosolic form of LC3 (LC3I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3II), which is recruited to autophagosomal membranes [34, 35]. Thus, the formation of LC3II appears to be correlated with the induction of autophagy [36]. P62 serves as a link between LC3 and ubiquitinated substrates, and the inhibition of autophagy correlates with the increased levels of p62.…”