{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/checksum.txt"],"Mzml":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep2.mzML","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep2.mzML","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep3.mzML","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep1.mzML","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep1.mzML","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep3.mzML"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep1.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep3.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep1.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep3.raw"],"Mzid":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep2.mzid","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep2.mzid","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep3.mzid","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep1.mzid","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep1.mzid","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep3.mzid"],"Fasta":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SwissProt_Mouse_03232022.fasta"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep3.pdResult","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep2.pdResult","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep3.pdResult","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermT_Rep1.pdResult","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD079474/SpermC_Rep1.pdResult"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["svijayar@kent.edu"],"submitter":["Belinda Willard"],"technology_type":["Mass Spectrometry","Bottom-up proteomics"],"disease":["Male Infertility"],"software":[""],"submitter_keywords":["Male contraceptive","Glycogen synthase kinase 3 alpha","Sperm maturation","Phosphoproteomics","Spermatogenesis","Brd0705"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD079474"],"tissue":["Testis","Sperm"],"sample_protocol":["Lyophilized mouse sperm samples were reconstituted in urea lysis buffer (20 mM HEPES, 9 M urea, 1× HALT protease/phosphatase inhibitor, pH 8.0). Protein concentration was determined using the Pierce BCA assay, and proteins from the same experimental group were pooled to obtain sufficient material for phosphoproteomic analysis. Two pooled samples were subjected to in-solution digestion. Proteins were reduced with dithiothreitol (DTT), alkylated with iodoacetamide (IAA), and digested overnight with trypsin (Promega #V5113). Digestion was quenched with trifluoroacetic acid (final concentration 0.5–1%), and peptides were desalted using Sep-Pak® C18 cartridges (Waters #WAT023590). A total of 400 µg of each digest was dried and enriched for phosphopeptides using the High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific #A32992). Enriched peptides were dried, reconstituted in 25 µL of 0.1% formic acid, and filtered using a 0.22 µm Ultrafree-MC centrifugal filter (MilliporeSigma #UFC30GVNB) prior to LC-MS/MS analysis. The LC-MS system was a ThermoScientific Exploris 480 mass spectrometer equipped with a Vanquish Neo uHPLC system. The HPLC column was a 50 cm x 75 µm id Easy Spray Pepmap NEO C18, 2μm, 100 Å reversed- phase capillary chromatography column. Five μL volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 0.3 μL/min were introduced into the source of the mass spectrometer on-line. The digest was analyzed using the data dependent multitask capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans.    LC-MS Method Instrument Exploris 480 Vanquish NEO UHPLC  Method DDA_120min_noFAIMS_low-load  Trapping Column ThermoScientific PepMap NEO C18 30µm x 5mm, 5µm  Analytical Column ThermoScientific Easy Spray PepMap Neo: 75µm x 500mm, C18, 2µm, 100Å  Solvent A 0.1 Formic Acid in H¬2O  Solvent B 0.1 Formic Acid in Acetonitrile  Gradient Time (%B) 0 (3%) 2 (3%) 107 (48%) 117 (99%) 132 (99%)  MS1 Resolution 120000 (profile)  MS1 Range 350-1400 Da  RF Lens 50%  AGC Target 300%  Max Inj Time 25 msec  Microscans 1  MS2 Method HCD, Orbitrap  Minimum MS1 Intensity 8.0e3  Charge for MS2 2 to 6  MS2 Isolation Window 2.0 Da  MS2 Collision Energy 30% nCE  MS2 resolution 15000 (centroid)  MS2 AGC Custum, 50%  First Mass 120 Da  Max Inj. Time 40 ms  Microscans 1  DDA settings Top 15   Dynamic Exclusion 1 repeat 10 ppm window 20 s exclusion"],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["The data were analyzed by using all CID spectra collected in the experiment to search the mouse SwissProtKB database using the program Sequest bundled into PD2.4.    Search Parameters Program: Protein ID Proteome Discoverer v2.4  Database Mouse SwissProtKB Downloaded on 3-23-2022 Entries 17552  Protease Trypsin, full  Missed Cleavages 3  Mass Accuracy 10 ppm MS1 0.06 Da MS2  Variable Modifications Oxidation of Met N-terminal Acetyl: Protein Phosphorylation S,T,Y  PTM site localization ptmRS node  Static Modifications Carbamidomethylation of Cis  Program: Protein/Peptide validation Percolator  Protein ID Requirements 1 Peptides  FDR Rate: Peptide Peptide Prophet 1%  FDR Rate: Protein Peptide Prophet 1%   LFQ Quantitation   Alignment Minora Feature Detector node  Normalization Total peptides  Peptides All  Imputation Impute using a normal distribution (Perseus)  Matrix Reduction Remove proteins with less than 2 valid values in at least one group (Perseus)"],"omics_type":["Proteomics"],"labhead":["Srinivasan Vijayaraghavan, PhD"],"instrument_platform":[""],"submission_type":["COMPLETE"],"labhead_affiliation":["Department of Biological Sciences, Kent State University, Kent, OH, United States."],"species":["Mus Musculus (mouse)"],"submitter_mail":["willarb@ccf.org"],"publication":["Not available"],"submitter_affiliation":["Cleveland Clinic"],"submitter_country":["United States"],"doi":["10.6019/PXD079474"],"additional_accession":[]},"is_claimable":false,"name":"Physiological basis of contraceptive function of a GSK3α-selective inhibitor, manifested during epididymal sperm maturation","description":"Glycogen synthase kinase 3 alpha (GSK3α) has emerged as a prospective target to develop male contraceptives. The GSK3α paralog is highly conserved and present in the sperm of placental mammals.  Global or testis-specific loss of GSK3α, but not GSK3β, results in male infertility. A preliminary study showed that in vivo administration of the GSK3α-specific inhibitor, BRD0705, to heterozygous (Gsk3a +/-) male mice results in reversible male infertility. Previous studies demonstrated that the GSK3α-selective inhibitor BRD0705 induces reversible infertility in heterozygous mice. Here, we show that the treatment of BRD0705 in wild-type male mice also results in equally effective male sterility. Here, we show that BRD0705 induces similar infertility in wild-type male mice. Superovulation in female mice does not alter fertility outcomes when mated with BRD0705-treated males. Further, no evidence of fetal resorption was observed in these females. Sperm numbers in treated mice were not altered; however, morphological defects appeared during their passage through the epididymis. The reversibility of BRD0705 treatment was also established by performing a 3-month in vivo fertilisation study in male mice after cessation of BRD0705 treatment. The GSK3α inhibitor affected pathways related to the enzyme's catalytic activity in epididymal sperm. Phospho-proteome analysis of sperm from inhibitor-treated mice showed that phosphorylation of over 50 proteins involved in motility, metabolism and fertility, among others, was Phosphoproteome analysis of sperm from inhibitor-treated mice showed that phosphorylation of over 50 proteins involved in motility, metabolism, and fertility, among others, was significantly altered. Among the highly hypophosphorylated proteins, four are likely substrates of GSK3 (viz., AKAP4, NT5C1B, SPATA18, and TRA2B), as they show reduced phosphorylation within the consensus GSK3 phosphorylation sequence. Analysis of the seminiferous tubules of BRD0705-treated mice showed normal spermatogenesis, suggesting that the drug did not affect developing spermatogenic cells.  However, in vitro exposure of post-meiotic germ cells demonstrated their susceptibility to this inhibitor. These findings support GSK3α inhibition via BRD0705 as a non-hormonal, on-demand, and reversible male contraceptive strategy.","dates":{"publication":"2026-07-29","submission":"2026-06-09"},"accession":"PXD079474","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:111205","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:1584","NEWT:36630","NEWT:383379","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:111225","NEWT:7719","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:2065263","NEWT:177437","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:237610","NEWT:95486","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:906914","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:550","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:595495","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:1613","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:298176","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:40674","NEWT:1194669","NEWT:51329","NEWT:443906","NEWT:13037","NEWT:519","NEWT:2510939","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:105487","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:300641","NEWT:39655","NEWT:9554","NEWT:38323","NEWT:475932","NEWT:256318","NEWT:206411","NCBITaxon:6191","NEWT:229533","NEWT: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