Identification and validation of ram sperm proteins associated with cryoinjuries caused by the cryopreservation process

Jia, Baoyu; Allai, Larbi; Lv, Chunrong; Liang, Jiazhong; Xiang, Decai; Zhang, Bin; Fang, Yi; Shen, Wei; Wu, Guoquan; Quan, Guobo

doi:10.1016/j.theriogenology.2022.03.015

Cited by 13 publications

(13 citation statements)

References 65 publications

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“…We do not know whether such phenomena could be a possible explanation for the abundance of TXNRD1 and HSPA4L proteins, as well as the electrophoretic protein fractions in FT sperm. Previous studies have demonstrated that the expression of several proteins that are involved in sperm capacitation, signal transduction, response to stress, energy status and sperm-oocyte fusion was increased in FT semen of boars, humans and rams [ 38 , 45 , 46 ]. In our previous study, we demonstrated that increased oxidative stress was triggered by the up-regulation of inflammatory and apoptosis-related genes in sperm from boars with low cryotolerance [ 21 ].…”

Section: Discussionmentioning

confidence: 99%

Expression of TXNRD1, HSPA4L and ATP1B1 Genes Associated with the Freezability of Boar Sperm

Mańkowska

Gilun

Zasiadczyk

et al. 2022

IJMS

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Cryopreservation is associated with increased oxidative stress, which is responsible for sperm damage. We analyzed the effect of cryopreservation on mRNA and protein expression of thioredoxin reductase 1 (TXNRD1), heat shock protein family A (HSP 70) member 4 like (HSPA4L) and sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1) genes in boar sperm with different freezability. Boars were classified as having good and poor semen freezability (GSF and PSF, respectively), according to the assessment of post-thaw sperm motility. Total RNA was isolated from fresh pre-freeze (PF) and frozen-thawed (FT) sperm from five boars of the GSF and PSF groups, respectively. Quantification of TXNRD1, HSPA4L and ATP1B1 gene expression was performed by RT-qPCR analysis. Proteins extracted from sperm were subjected to Western blotting and SDS-PAGE analyses. Poor freezability ejaculates were characterized by significantly higher relative mRNA expression levels of TXNRD1 and HSPA4L in FT sperm compared with the fresh PF sperm. Furthermore, the relative mRNA expression level of ATP1B1 was significantly higher in the fresh PF sperm of the GSF group. Western blotting analysis revealed significantly higher relative expression of TXNRD1 protein in the fresh PF sperm of the GSF group, while HSPA4L protein expression was markedly increased in FT sperm of the PSF group. Electrophoretic and densitometric analyses revealed a higher number of proteins in the fresh PF and FT sperm of the PSF and GSF groups, respectively. The results of this study indicate that ATP1B1 mRNA expression in the fresh PF sperm is a promising cryotolerance marker, while the variations of TXNRD1 and HSPA4L protein expression in the fresh PF or FT sperm provide useful information that may help to elucidate their biological significance in cryo-damage.

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Section: Discussionmentioning

confidence: 99%

Expression of TXNRD1, HSPA4L and ATP1B1 Genes Associated with the Freezability of Boar Sperm

Mańkowska

Gilun

Zasiadczyk

et al. 2022

IJMS

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“…Spermatozoa were separated from the seminal plasma by centrifugation at 4°C for 20 min at 3000 g, then washed with phosphate buffered saline (PBS) three times and stored at -80°C until analysis. The protein extraction, trypsin digestion, iTRAQ labeling and mass spectrometry procedures were performed as previously described [15]. Tryptic peptides were analyzed by LC-MS/MS on the Q Exactive HF mass spectrometer coupled with a NanoLC-1000 HPLC system (Thermo, USA).…”

Section: Itraq-based Proteomics Analysismentioning

confidence: 99%

“…Compared with 2-dimensional SDS-PAGE and label free quanti cation, isobaric tags for relative and absolute quantitation (iTRAQ) has the advantages of robustness to variations in sensitivity and chromatographic reproducibility [9]. iTRAQ-coupled LC-MS/MS technology is currently being used to track various biomarkers in spermatogenesis [10,11], sperm maturation [12], concentration and motility changes of ejaculates [13], cryoinjury [14,15] and fertility [16,17]. The parallel reaction monitoring (PRM) technology is recently developed in the use of targeted mass spectrometry.…”

Section: Introductionmentioning

confidence: 99%

“…The parallel reaction monitoring (PRM) technology is recently developed in the use of targeted mass spectrometry. The precision, resolution and sensitivity of this method have signi cant improvement when compared to either selected reaction monitoring or WB [15]. Recently, PRM has been used for the identi cation and quanti cation of target proteins, which was also validated for proteomics data [15].…”

Section: Introductionmentioning

confidence: 99%

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iTRAQ-based comparative proteomics reveal an enhancing role of PRDX6 in the freezability of Mediterranean buffalo sperm

Luo

Liang

Huang

et al. 2023

Preprint

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Background Semen cryopreservation is a critical tool for breed improvement and preservation of biodiversity. However, instability of sperm freezability affects its application. The Mediterranean buffalo is one of the river-type buffaloes with the capacity for high milk production. Until now, there is no specific cryopreservation system for Mediterranean buffalo, which influences the promotion of excellent cultivars. To improve the semen freezing extender used in cryopreservation of Mediterranean buffalo, different protein datasets relating to freezability sperm were analyzed by iTRAQ-based proteomics. This study will be beneficial for further understanding the sperm freezability mechanism and developing new cryopreservation strategy for buffalo semen. Results 2652 quantified proteins were identified, including 248 significantly differentially expressed proteins (DEP). Gene Ontology (GO) analysis indicated that many these were mitochondrial proteins, enriched in the molecular function of phospholipase A2 activity and enzyme binding, and biological processes of regulation of protein kinase A signaling and motile cilium assembly. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 17 significant pathways, including oxidative phosphorylation (OXPHOS). Furthermore, 7 DEPs were verified using parallel reaction monitoring or western blot, which confirmed the accuracy of the iTRAQ data. Peroxiredoxin 6 (PRDX6), which was expressed 1.72-fold higher in GFE compared to PFE sperm, was selected to explore the function in sperm freezability by adding recombinant PRDX6 protein into the semen freezing extender. The results showed that the motility, mitochondrial function and in vitro fertilization capacity of frozen-thawed sperm were significantly increased, while the oxidation level was significantly decreased when 0.1mg/L PRDX6 was added compared with blank control. Conclusions Above results revealed the metabolic pattern of freezability of Mediterranean buffalo sperms was negatively associated with OXPHOS, and PRDX6 had protective effect on cryo-damage of frozen-thawed sperms.

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“…Compared with 2-dimensional SDS-PAGE and label free quantification, isobaric tags for relative and absolute quantitation (iTRAQ) have the advantages of robustness to variations in sensitivity and chromatographic reproducibility [ 9 ]. iTRAQ-coupled LC-MS/MS technology is currently being used to track various biomarkers in spermatogenesis [ 10 , 11 ], sperm maturation [ 12 ], concentration and motility changes of ejaculates [ 13 ], cryoinjury [ 14 , 15 ] and fertility [ 16 , 17 ]. The parallel reaction monitoring (PRM) technology is recently developed in the use of targeted mass spectrometry.…”

Section: Introductionmentioning

confidence: 99%

iTRAQ-based comparative proteomics reveal an enhancing role of PRDX6 in the freezability of Mediterranean buffalo sperm

et al. 2023

View full text Add to dashboard Cite

Background Semen cryopreservation is a critical tool for breed improvement and preservation of biodiversity. However, instability of sperm freezability affects its application. The Mediterranean buffalo is one of the river-type buffaloes with the capacity for high milk production. Until now, there is no specific cryopreservation system for Mediterranean buffalo, which influences the promotion of excellent cultivars. To improve the semen freezing extender used in cryopreservation of Mediterranean buffalo, different protein datasets relating to freezability sperm were analyzed by iTRAQ-based proteomics. This study will be beneficial for further understanding the sperm freezability mechanism and developing new cryopreservation strategy for buffalo semen. Results 2652 quantified proteins were identified, including 248 significantly differentially expressed proteins (DEP). Gene Ontology (GO) analysis indicated that many these were mitochondrial proteins, enriched in the molecular function of phospholipase A2 activity and enzyme binding, and biological processes of regulation of protein kinase A signaling and motile cilium assembly. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 17 significant pathways, including oxidative phosphorylation (OXPHOS). Furthermore, 7 DEPs were verified using parallel reaction monitoring or western blot, which confirmed the accuracy of the iTRAQ data. Peroxiredoxin 6 (PRDX6), which expressed 1.72-fold higher in good freezability ejaculate (GFE) compared to poor freezability ejaculate (PFE) sperms, was selected to explore the function in sperm freezability by adding recombinant PRDX6 protein into the semen freezing extender. The results showed that the motility, mitochondrial function and in vitro fertilization capacity of frozen-thawed sperm were significantly increased, while the oxidation level was significantly decreased when 0.1 mg/L PRDX6 was added compared with blank control. Conclusions Above results revealed the metabolic pattern of freezability of Mediterranean buffalo sperms was negatively associated with OXPHOS, and PRDX6 had protective effect on cryo-damage of frozen-thawed sperms.

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Identification and validation of ram sperm proteins associated with cryoinjuries caused by the cryopreservation process

Cited by 13 publications

References 65 publications

Expression of TXNRD1, HSPA4L and ATP1B1 Genes Associated with the Freezability of Boar Sperm

Expression of TXNRD1, HSPA4L and ATP1B1 Genes Associated with the Freezability of Boar Sperm

iTRAQ-based comparative proteomics reveal an enhancing role of PRDX6 in the freezability of Mediterranean buffalo sperm

iTRAQ-based comparative proteomics reveal an enhancing role of PRDX6 in the freezability of Mediterranean buffalo sperm

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