DOI: 10.5061/dryad.t4b8gtj5v
title: Porcine cell-free system mass spectrometry compiled data sets

description: 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.
description: <strong><em>Sperm Priming for Cell-Free System</em></strong> Boar spermatozoa were washed with phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na<sub>2</sub>HPO<sub>4</sub>, 1.8 mM KH<sub>2</sub>HPO<sub>4</sub>, pH = 7.2) containing 0.1% (w/v) PVA (PBS-PVA) two times by centrifugation at 800 × g for 5 min. The sperm mitochondria were labeled with MitoTracker<sup>®</sup> Red CMXRos for 10 min at 37°C. At the previously tested concentration of 400 nM, the probe specifically stains boar sperm mitochondria but is also taken up by the sperm head structures (W. H. Song et al., 2016). To prime sperm mitochondrial sheaths for cell-free studies, spermatozoa pre-labeled with MitoTracker were demembranated/permeabilized with 0.05% (w/v) L-α-lysophosphatidylcholine in KMT (20 mM KCl, 5 mM MgCl<sub>2</sub>, 50 mM TRIS∙HCl, pH = 7.0) for 10 min at 37°C and washed twice with the KMT for 5 min by centrifugation, to terminate the reaction. The spermatozoa were subsequently incubated with 1.0 mM dithiothreitol (DTT) diluted in KMT, pH = 8.2 for 20 min at 37°C and washed twice with KMT for 5 min by centrifugation, to terminate the reaction. <strong><em>Preparation of Porcine Oocyte Extracts</em></strong> Cumulus cells of matured COCs were removed with 0.1% (w/v) hyaluronidase in TL-HEPES-PVA medium. The oocytes were then searched for mature MII oocytes as designated by the presence of a polar body. Mature oocytes were then washed three times with TL-HEPES-PVA medium. Zonae pellucidae (ZP) were removed by 0.1% (w/v) pronase (Sigma) in TL-HEPES-PVA. The ZP-free, mature MII oocytes were transferred into an extraction buffer (50 mM KCl, 5 mM MgCl<sub>2</sub>, 5 mM ethylene glycol-bis[β-aminoethyl ether]-N,N,N’,N’-tetraacetic acid [EGTA], 2 mM β-mercaptoethanol, 0.1 mM PMSF, protease inhibitor cocktail [78410, ThermoFischer Scientific], 50 mM HEPES, pH = 7.6) containing an energy-regenerating system (2 mM ATP, 20 mM phosphocreatine, 20 U/mL creatine kinase, and 2 mM GTP), and submerged three times into liquid nitrogen for 5 min each. Next, the frozen-thawed oocytes were crushed by high-speed centrifugation at 16,650 × g for 20 min at 4°C in a Sorvall Biofuge Fresco (Kendro Laboratory Products). Batches of oocyte extract were made from 1,000 oocytes in 100 µL of extract. The supernatants were harvested, transferred into a 1.5 mL tube, and stored in a deep freezer (–80°C). <strong><em>Co-Incubation of Permeabilized Mammalian Spermatozoa with Porcine Oocyte Extracts</em></strong> The permeabilized boar spermatozoa were added to porcine oocyte extracts at a concentration of 1x10<sup>4 </sup>spermatozoa/10 μL of an extract and co-incubated for 4–24 h in an incubator at 38.5°C, with 5% CO<sub>2 </sub>in the air. After co-incubation, spermatozoa were washed 3x with KMT. At which point the spermatozoa were prepared for mass spectrometry analysis. <strong><em>Mass Spectrometry Sample Preparation</em></strong> Cell-free system exposed spermatozoa, spermatozoa controls, and oocyte extract underwent protein precipitation using a TCA protein precipitation protocol from Dr. Luis Sanchez. These samples were then resuspended in acetone and submitted to the University of Missouri Gehrke Proteomics Center for MALDI-TOF Mass Spectrometry analysis. At the Proteomics Center, these samples were washed by 80% cold acetone twice. Then 10 µl 6M urea 2M thiourea and 100mM ammonium bicarbonate was added to the protein pellet. Solubilized protein was reduced by DTT and alkylated by iodoacetamide. Then trypsin was added for disgestion overnight. The digested peptides were C18 ziptip deslated, lyophilized and resuspended in 10 µL 5/0.1% acetonitrile/formic acid. A volume of 1 µL of suspended peptides was loaded onto a C18 column with a step gradient of acetonitrile at 300 nL/min. A Bruker nanoElute system was connected to a timsTOF pro mass spectrometer. The loaded peptide was eluted at a flow rate of 300 nl/min with the initial gradient of 3% B (A: 0.1% formic acid in water, B: 99.9% acetonitrile, 0.1% formic acid), followed by 11 min ramp to 17%B, 17-25% B over 21 min, 25-37% B over 10 min, 37-80% B over 4 min, holding at 80% B for 9 min, 80-3% B in 1 min, and holding at 3% B for 3 min. Total running time was 60 min. Raw data was searched using PEAKs (version X+) with UniProt <em>Sus scrofa </em>protein<em> </em>database downloaded March 01, 2019 with 88374 items. Samples were searched with trypsin as enzyme, 4 missed cleavages allowed; carbamidomethyl cysteine as a fixed modification; oxidized methionine and acetylation on protein N terminus as variable modification. 50 ppm mass tolerance on precursor ions, 0.1Da on fragment ions. For the protein identification, the following criteria were used: peptide FDR and protein FDR &lt; 1%, and &gt;=4 spectrum per protein in each sample. Samples were submitted in triplicate for both the 4- and 24-hours cell-free system trials. <strong><em>Mass Spectrometry Data Statistical Analysis</em></strong> Prior to statistical analysis, the primed and cell-free treated sperm samples were normalized based on the content of outer dense fiber proteins (ODF) 1, 2, and 3. To further reduce batch variance, the protein spectrum counts were also subject to normalization by means. After these normalization steps, the primed and cell-free extract treated sperm samples were statistically compared using a paired T-test. This T-test was comparing the relative normalized protein abundance between our primed control and cell-free treated samples. <em>P</em>&lt;0.1 and <em>P</em>&lt;0.2 was considered to indicate statistical  significance. <strong><em>Protein Classification</em></strong> Both the 4-hour and 24-hour protein inventories were divided into three different classes. Class 1 proteins were detected only in the oocyte extract (not in the vehicle control or primed control spermatozoa) and found on the spermatozoa only after extract co-incubation. These proteins are interpreted as ooplasmic mitophagy receptors/determinants and nuclear/centrosomal remodeling factors (p&lt;0.2). Class 2 proteins were detected in the primed spermatozoa but increased in the spermatozoa exposed to cell-free system co-incubation (p&lt;0.1). Class 3 proteins were present in both the gametes or only the spermatozoa, but are decreased in the spermatozoa after co-incubation, interpreted as sperm-borne mitophagy determinants and/or sperm-borne proteolytic substrates of the oocyte autophagic system (p&lt;0.1). 
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