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SQSTM1-dependent autophagy pathway and VCP-mediated proteasomal proteolysis contribute to sperm mitophagy after mammalian fertilization
Mitochondrial and mitochondrial DNA (mtDNA) is maternally inherited in humans and most animals. The degradation of sperm-borne mitochondria after fertilization assures normal preimplantation embryo development and may prevent mitochondrial diseases derived from heteroplasmy. Although it has been known that ubiquitin-proteasome system (UPS) is the major degradation pathway of post-fertilization sperm mitochondria in mammals, it is unclear how the UPS, which is able to get rid of single protein molecule at a time, can eliminate whole sperm mitochondrial organelle. We considered that the autophagy receptors [sequestosome 1(SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), and gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the non-traditional mitophagy pathways involving UPS and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP) may act independently or in concert during post-fertilization sperm mitophagy. We found that the association of SQSTM1 with sperm mitochondria was displayed in both pig and rhesus monkey zygotes after fertilization. Sperm mitochondrial proteins [mitochondrial trifunctional enzyme subunit alpha (HADHA), mitochondrial aconitase 2 (ACO2), and mitochondrial ATP synthase H+ transporting F1 complex β-subunit (ATP5B)] co-purified with the synthetic, SQSTM1-derived, ubiquitin-binding UBA domain were identified. Also, the accumulation of GABARAP-positive protein aggregates was observed around sperm mitochondrial sheaths in fertilized oocytes, which reflects autophagosome formation. Furthermore, the inhibition of VCP delayed the process of sperm mitophagy and completely blocked it when embryos were co-injected with autophagy-targeting antibodies, such as anti-SQSTM1 and/or anti-GABARAP. Thus, both SQSTM1-dependent autophagy pathway and VCP-mediated proteasomal proteolysis facilitate post-fertilization sperm mitophagy in mammals. This explains how the proteolytic pathway can coordinate autophagy pathway to degrade the sperm mitochondrial sheath inside the fertilized oocyte.
