Abstract
The degradation of sperm-borne mitochondria after fertilization is a conserved event. This process known as post-fertilization sperm mitophagy, ensures exclusively maternal inheritance of the mitochondria-harbored mitochondrial DNA genome. This mitochondrial degradation is in part carried out by the ubiquitin-proteasome system. In mammals, ubiquitin-binding pro-autophagic receptors such as SQSTM1 and GABARAP have also been shown to contribute to sperm mitophagy. These systems work in concert to ensure the timely degradation of the sperm-borne mitochondria after fertilization. We hypothesize that other receptors, cofactors, and substrates are involved in post-fertilization mitophagy. Mass spectrometry was used in conjunction with a porcine cell-free system to identify other autophagic cofactors involved in post-fertilization sperm mitophagy. This porcine cell-free system is able to recapitulate early fertilization proteomic interactions. Altogether, 185 proteins were identified as statistically different between control and cell-free-treated spermatozoa. Six of these proteins were further investigated, including MVP, PSMG2, PSMA3, FUNDC2, SAMM50, and BAG5. These proteins were phenotyped using porcine in vitro fertilization, cell imaging, proteomics, and the porcine cell-free system. The present data confirms the involvement of known mitophagy determinants in the regulation of mitochondrial inheritance and provides a master list of candidate mitophagy co-factors to validate in the future hypothesis-driven studies.