The Autophagy Marker LC3 Is Processed during the Sperm Capacitation and the Acrosome Reaction and Translocates to the Acrosome Where It Colocalizes with the Acrosomal Membranes in Horse Spermatozoa

Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In somatic cells, this ratio indicates that autophagy has been activated and similar LC3 processing has been described in mammalian spermatozoa. The subcellular localization of autophagy-related proteins was assessed by immunofluorescence with specific antibodies that recognized Atg16, Beclin-1, and LC3. The colocalization of acrosomal membranes (PNA) and LC3 was studied by confocal microcopy, and the acrosome reacted cells were quantified by flow cytometry. The incubation of stallion sperm in capacitating conditions (BWW; 3 h) significantly increased LC3 processing. This increment was three to four times higher after the induction of the acrosome reaction in these cells. LC3 was mainly expressed in the head in mature ejaculated sperm showing a clear redistribution from the post-acrosomal region to the acrosome upon the incubation of sperm in capacitating conditions (BWW, 3 h). After the induction of the acrosome reaction, LC3 colocalized with the acrosome or the apical plasmalemma membranes in the head of the stallion spermatozoa. The inhibition or activation of autophagy-related pathways in the presence of autophagy activators (STF-62247) or inhibitors (E-64d, chloroquine) significantly increased LC3 processing and increased the percent of acrosome reacted cells, whereas 3-methyladenine almost completely inhibited LC3 processing and the acrosome reaction. In conclusion, we found that sperm capacitation and acrosome reaction could be regulated by autophagy components in sperm cells ex vivo by processes that might be independent of the intraluminal pH of the acrosome and dependent of LC3 lipidation. It can be speculated that, in stallion sperm, a form of noncanonical autophagy utilizes some components of autophagy machinery to facilitate the acrosome reaction.