Regular Abstract Submission
Chloe Gardner, PhD
Graduate Student
University of Missouri
Cincinnati, Ohio, United States
Chloe Gardner1; Natalie Zelenkova1,2; Alexis Jones1; Erica Mantle1; Barbora Klusackova1,2; Peter Sutovsky1,3
1. Division of Animal Sciences, University of Missouri, Columbia, United States
2. Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Prague, Czech Republic
3. Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia, Missouri, United States
Abstract Text:
Post-fertilization sperm mitophagy is an essential process that prevents the inheritance of potentially harmful paternal mitochondrial DNA (mtDNA), ensuring proper embryonic development. We hypothesize that this targeted degradation of paternal mtDNA is mediated by the ubiquitin-proteasome system (UPS) and autophagy-related proteins such as Sequestosome 1 (SQSTM1) and valosin-containing protein (VCP). However, additional regulatory factors may also play key roles. In order to identify these key regulators, porcine in-vitro fertilization (IVF) and a novel mammalian cell-free system (CFS) composed of demembranated boar spermatozoa coincubated with porcine oocyte extracts were utilized to model early fertilization events. The CFS enables large-scale exposure of spermatozoa to oocyte cytosolic extracts, maximizing the number of observations of sperm structural changes induced by ooplasmic mitophagy factors. Together, these models provide a comprehensive approach to identifying candidate proteins involved in sperm mitophagy. Our previously published mass spectrometry study using the CFS identified 185 candidate proteins with significant changes in protein quantity across control and CFS-treated sperm samples. From this list, four were selected for further investigation in the present study: i) translocase of outer mitochondrial membrane 34 (TOMM34), a chaperone-like protein that imports preproteins into mitochondria; ii) FUN14 domain containing 1 (FUNDC1), a mitophagy receptor contributing to quality control following hypoxic stress; iii) dynamin 2 (DNM2), a protein implicated in the final steps of mitochondrial fission, a process that controls mitochondrial biogenesis but also the removal of damaged mitochondria; and iv) mitochondrial fission regulator 1 (MTFR1), a factor promoting mitochondrial fission and protection against oxidative stress. Using commercially available antibodies, these proteins were identified in both IVF zygotes and CFS-treated sperm samples. TOMM34 showed reduced signals in CFS-treated spermatozoa as well as an absence from paternal mitochondria in IVF zygotes, contributing to its categorization as a sperm-borne proteolytic substrate of oocyte autophagy factors. FUNDC1 was detected in both control CFS sperm samples and CFS-treated sperm samples, with a more prominent signal on the mitochondrial sheath (MS) of CFS-treated sperm. This may indicate its role as an ooplasm-modulated cofactor of mitophagy during the early events of fertilization. DNM2 was initially present throughout the sperm tail in CFS-treated samples but localized to the MS after 24 hours of CFS exposure. Similar results were found in the IVF zygotes, where DNM2 was detectable around the MS and sperm tail connecting piece. Such a pattern supports its suspected role as a facilitator in the process of mitochondrial fission. MTFR1 was also detected on the MS during IVF trials and localized to the sperm head post-acrosomal sheath and sperm tail at 4 hours in CFS samples. However, unlike DNM2, MTFR1 was absent from CFS-treated spermatozoa after 24 hours, suggesting a distinct role in early mitochondrial fission. The current investigation of these proteins with the addition of specific small molecule inhibitors of mitophagy and mitochondrial fission will help clarify their roles in post-fertilization sperm mitophagy. A deeper understanding of these mechanisms will contribute to advancements and improvements in assisted reproductive therapies for humans and agricultural species alike. Study funded by USDA-NIFA Animal Reproduction Program grant number 2021-67015-33404 (PS), seed funding from University of Missouri College of Agriculture, Food and Natural Resources (CAFNR; PS) and CAFNR Matching Assistantship Program (CMAP) graduate scholarship (CG).