Dataset Information

Identification and validation of yak (Bos grunniens) frozen-thawed sperm proteins associated with the capacitation and acrosome reaction

ABSTRACT: We used a comprehensive quantitative proteomic profiling based on LC-MS/MS combined with TMT labeling strategy to analyze frozen-thawed Ashidan yak spermatozoa proteomic dynamic changes under three sequential conditions: density gradient centrifugation-based purification（FTH sperm）, incubation in capacitation medium(CAP sperm), and treatment with the calcium ionophore A23187 to induce the acrosome reaction process(AR sperm). FTH sperm Briefly, sperm-containing straws were thawed for 30 s at 37ºC prior to addition to a prepared 45-90% Percoll solution (Percoll P1644; 3.1mM KCl, 2.9mM NaH2PO4,80mM NaCl, 10mM HEPES, 2mM CaCl2·H2O, 0.4mM MgCl2·6H2O, 26mM Sodium DL-Lactate Solution, 25mM NaHCO3; pH 7.3-7.4) and diluted using modified Tyrode's Hepes buffered medium (Sp-TALPH, 114mM NaCl, 3.2mM KCl, 2mM NaHCO3, 0.4mM NaH2PO4·H2O, 10mM Na Lactate, 2mM CaCl2·H2O, 0.5mM MgCl2·6H2O, 10mM HEPES, 3mg/ml BSA, 0.2mM Na pyruvate, 7.5x10-3mg/ml Gentamicin; pH 7.3-7.4) followed by centrifugation for 10 min at 700 xg to enable the isolation of highly motile sperm. To ensure that the gradient material was completely eliminated from these samples, the sperm cells collected from the bottom layer were washed two times using supplemented Sp-TALPH medium and centrifuged for 5 min at 300 xg. Sperm were then isolated and adjusted to a concentration of 100x106/mL using modified Tyrode's bicarbonate buffered medium (Sp-TALP; 114mM NaCl, 3.2mM KCl, 25mM NaHCO3, 0.4mM NaH2PO4·H2O, 10mM Na Lactate, 2mM CaCl2·H2O, 0.5mM MgCl2·6H2O, 6mg/ml BSA, 0.2mM Na pyruvate, 5x10-3mg/ml Gentamicin; pH 7.3-7.4). A CASA approach was then used to assess sperm motility, with only those samples exhibiting >70% total motility being retained for further analyses, as per previously published protocols. An aliquot containing 100 million FTH sperm was taken from each sample for subsequent protein isolation, with the remaining sperm being incubated in capacitation medium. CAP sperm Capacitation medium was composed of Sp-TALP containing heparin (50 ug/ml) and caffeine (5 mM), and sperm were incubated in this medium for 30 min at 38.5ºC in a 5%CO2 incubator, as these conditions have previously been shown to be optimal for the induction of capacitation in frozen-thawed yak sperm. An aliquot containing 100 million CAP sperm was taken from each sample for subsequent protein isolation. AR sperm AR induction was achieved by treating 990 uL capacitated sperm samples with 10 µL of A23187 (1.0mM in DMSO, CSNpharm, IL, USA) to a final concentration of 10 µM, followed by a 15 min incubation at 38.5ºC, For individual replicates, aliquots of 100 million AR sperm were collected for subsequent protein analyses.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Bos Mutus Grunniens (wild Yak) (bos Grunniens)

TISSUE(S): Spermatozoon

SUBMITTER: zhang renzheng

LAB HEAD: Zhang Renzheng

PROVIDER: PXD035038 | Pride | 2023-03-11

REPOSITORIES: Pride

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Identification and Validation of Yak (<i>Bos grunniens</i>) Frozen-Thawed Sperm Proteins Associated with Capacitation and the Acrosome Reaction.

Zhang Renzheng R Guo Xian X Liang Chunnian C Pei Jie J Bao Pengjia P Yin Mancai M Wu Fude F Chu Min M Yan Ping P

Journal of proteome research 20221017 11

To achieve fertilization, mammalian spermatozoa must undergo capacitation and the acrosome reaction (AR) within the female reproductive tract. However, the effects of cryopreservation on sperm maturation and fertilizing potential have yet to be established. To gain insight into changes in protein levels within sperm cells prepared for use in the context of fertilization, a comprehensive quantitative proteomic profiling approach was used to analyze frozen-thawed Ashidan yak spermatozoa under thre ...[more]

PMID: 36251486

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Project description:U2OS osteosarcoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) and Raji B-cells in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS, Bio&Sell), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco) at 37°C at 8% or 5% CO2, respectively. Chromatin immunoprecipitation for ChIP-seq: Cells were crosslinked using a formaldehyde containing solution (10mM NaCl, 0,1mM EDTA pH 8, 0,05mM EGTA pH 8, 5mM HEPES pH 7.8 and 1% formaldehyde) for 10 minutes at 20°C, the reaction was quenched by the addition of glycin to a final concentration of 250uM for 5 minutes. Crosslinked cells were collected and washed twice with PBS before snap freezing in liquid nitrogen and storage at -80°C until subsequent use. Prior to sonication, the crosslinked cells were resuspended in lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100, 1X protease inhibitor cocktail) at 4°C for 20 minutes. Nuclei were collected by centrifugation and washed in a second buffer (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 1X protease inhibitor cocktail) for 10 minutes at 4°C then collected by centrifugation and resuspended in the shearing buffer (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1X protease inhibitor cocktail). Sonication was carried out in a Bioruptor Pico ultrasounds water bath (Diagenode B01060001) for 30 cycles of 30 sec ON and 30 sec OFF pulses in 4°C water. Sonicated extracts were centrifuged at high speed in the presence of 0,1% of Triton X-100 and snap frozen in liquid nitrogen then stored at -80°C until subsequent use. Prior to ChIP, the ZNF768 mAb was coupled to protein-G coated magnetic beads (Dynabeads, life technologies) by incubation in 0,5% BSA PBS overnight at 4°C. Pre-coated beads were then washed and incubated with the sonicated chromatin extracts, the ChIP was carried out overnight at 4°C on a rotating wheel. The equivalent of 10 × 107 cells sonicated extract was used for each ZNF ChIP experiment for both cell lines. After incubation, the beads were washed 7 times with Wash buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate, 1X protease inhibitor cocktail) followed by one wash with TE-NaCl buffer (10mM Tris pH 8 and 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatine was eluted by two sequential incubations with 100µl Elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) at 65°C for 15 minutes. The two eluates were pooled and incubated at 65°C for 12 hours to reverse-crosslink the chromatin followed by treatment with RNase A (0.2µg/mL) at 37°C for two hours and Proteinase K (0.2µg/mL) at 55°C for two hours. The DNA was isolated by phenol:chloroform: isoamylalcohol (25:24:1 pH 8) extranction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). At least 1ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol. The ChIP DNA was size selected using Ampure beads (Life technologies) to enrich for fragments < 400Bp prior to end-repair, 3’end adenylation and adapter ligation. Library fragments were then directly amplified by 10 cycles of PCR.

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