Gene expression profiling of human fresh and cryopreserved sperm
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PROVIDER: PRJNA934956 | ENA |
REPOSITORIES: ENA
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Project description:Cryopreservation process deregulates the expression of pathways important for fertility in human spermatozoa Study design, size, duration (max. 75 words): Semen samples were obtained from 20 normospermic men from April to May 2022. Each sample was divided in two aliquots. From one aliquot total RNA was immediately extracted. The second aliquot was slowly cryopreserved and after a week of storage in liquid nitrogen total RNA was extracted. A total of 13 paired RNA samples passed quality control and were analyzed after randomization into 4 pools, each of 6 patients. Participants/materials, setting, methods (max. 75 words): The men who participated in this study had a median age of 35.0 years (range: 29.0-46.0). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The paired Significance Analysis of Microarray (SAM) was performed using the limma, hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance (max. 200 words): The expression profiles of cryopreserved sperm significantly differed from those of fresh sperm in 219 down-regulated and 28 up-regulated unique transcripts. The gene ontology analysis disclosed that cryopreservation downregulates genes involved in sperm motility by the mitochondria function (CABS1, SPATA19) and the correct organization of the sperm midpiece and flagellum cytoskeleton (ACTL7A, AKAP4, C9ORF24, CAPZB, CCIN, IQCG, ODF2, SPATA6, SPATA6L, TBC1D21), in fertilization (ACTL7A, AKAP4, PLCZ1, PRSS37, TUBGCP3), early embryo development (AGT, CLMN, DCC, DHRS3, EGFLAM, FAM20A, FREM1, GPI, HEMGN, KRTDAP, IFT172, MICAL2, MLF1, NF2, PGRMC2, PHACTR1, POPDC3, RO60, ROBO1, RORA, SGCA, SPRR2D, TCF4, TNC, TNNI3), oxidant detoxification (TXNDC29) and DNA repair (BRIP1, ERCC6, TRIP12), calcium ion binding and homeostasis (AMY1C, ANO1, ANO2, ASGR1, CABS1, CPNE9, FREM1, KCNIP2, ITPR3, PKD2L1, PLCZ1, SELENOK, SYN3, TNNI3). Upregulated genes were enriched in pathways related to the negative regulation of DNA damage response (ALOX15B, CD74, RPL10, SNAI1, THBS1), cellular response to calcium ion (ALOX15B, FOSB, HLA-DQB1, S100A4, THBS1), regulation of transcription (CD74, FOSB, HLA-DRB1, MAMLD1, RASD1, RPL10, SNAI1, TCF15, TLE1), protein stabilization (CD74), protein ubiquitination (BAG1, DCAF6, ERCC6, HERPUD2, KLHL7, MARCHF8, NF2, PSMA6, RNF133, SERGEF, TRIP12, UBE2DNL, UBL3, UBQLN3, ZNRF3) and ubiquitin protein ligase binding (GPI, HSPA1L, PRKAR2A, STX8). The storage time of cryopreserved human spermatozoa does not affect pathways involved in fertility. Study design, size, duration This study included 24 normospermic men after their written informed consent, of whom 13 had cryostored semen for a short-time (1 week) and 11 had cryostored semen for a long-time (median 9 years, range 7-10). RNA was extracted from each frozen-thawed sperm sample and randomized into pools (4 pools for short-time samples and 3 pools for long-time samples), each of 5-6 patients. Wide-transcriptome comparison of short- and long-time pools were analyzed. Participants/materials, setting, methods The study population had a median age of 34.5 years (range: 22-46), without significant difference between the two groups (p-value = 0.147). RNA was extracted by miRNeasy Micro Kit (Qiagen), analyzed by High Sensitivity RNA on 2200 Tape Station system (Agilent), amplified by Ovation Pico WTA SystemV2 (Nugen), labeled and hybridized on GE 4x44K v2 microarrays (Agilent). The unpaired Significance Analysis of Microarray (SAM) was performed using the limma, hgug4112a.db and samr packages in R/BioConductor. Main results and the role of chance Comparison of gene expression profiles between thawed sperm after a short or long storage period in liquid nitrogen identified 5 unique transcripts significantly upregulated in the latter group (DCP1A, FAM217A, HUWE1, MSI2, PCSKIN). Functional annotation enrichment has shown that the dysregulated transcripts are involved in methylation and protein binding. In particular, HUWE1 encodes an E3 ubiquitin protein ligase required for protein ubiquitination. The protein encoded by DCP1A is a decapping enzyme, necessary for the degradation of mRNAs. The RNA binding protein MSI2 regulates the expression of target mRNAs at the translation level. Interestingly, HUWE1 serves as a central node in cellular stress responses, cell growth and death, signal transduction, etc. During early embryonic development, siRNA-mediated knockdown of HUWE1 induced apoptotic cell death and inhibited normal embryonic development. Consistent with this finding, decreased HUWE1 staining in human embryos was found to be associated with poor embryo quality, suggesting that HUWE1 plays important roles in promoting embryonic development. DCP1A is also essential for embryonic development. Overall, no critical pathways involved in fertility are dysregulated during storage of sperm in liquid nitrogen and upregulation of genes important for early embryonic development may compensate for sperm cryoinjury.
2024-04-09 | GSE225320 | GEO
Project description:Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been investigated previously. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit spermatozoa. Comparative analysis and identification of proteins were performed using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization mass (MALDI TOF/TOF) spectrometry. We found that 108 proteins differed in abundance in spermatozoa between fresh and frozen semen. Most of these proteins was involved in pathways related to energy metabolism and protein quality control under stress conditions, reproductive processes and mechanisms of cell death/survival regulation, resulting in a significant decrease of motility and viability of post-thawing rabbit sperm and its potential fertilizing ability.
2022-10-14 | PXD034832 | Pride
Project description:Cryoinjury and protein changes are a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, viability and fertilizing ability. However, potential proteomic changes in rabbit semen throughout the cryopreservation process have never been investigated previously. The aim of the present study was to compare the whole proteome of fresh and cryopreserved rabbit extracellular fluid of semen. Comparative analysis and identification of proteins were performed using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization mass (MALDI TOF/TOF) spectrometry. We found that 28 proteins differed in abundance between fresh and frozen extracellular fluid.
2022-10-13 | PXD034853 | Pride
Project description:Cryopreservation of blue catfish sperm can cause variable embryo hatch rates, which is the limiting factor for hybrid catfish breeding. In this study, we investigated gene expression changes caused by cryopreservation using transcriptome profiles of fresh sperm samples and cryopreserved sperm from the same set of blue catfish.
2022-06-26 | GSE204687 | GEO
Project description:Non-hematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils, lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable non-hematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LNSC cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and lymph nodes. The presence and spatial distribution of transcriptionally defined cell types was confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSC in human disease.
2023-05-16 | GSE224661 | GEO
Project description:Background: The cryopreservation of semen is crucial for conserving genetic diversity and reproductive success in endangered species like the Siberian sturgeon. However, this process can cause cryodamage, affecting quality and protein profile of spermatozoa. While cryoprotectants like dimethyl sulfoxide (DMSO) and methanol (MeOH) facilitate post-thaw motility recovery, DMSO-preserved spermatozoa exhibit reduced fertilizing ability. This study investigates how DMSO and MeOH impact the proteome of Siberian sturgeon spermatozoa and examines semen quality parameters. Two complementary approaches of quantitative proteomics, liquid chromatography-mass spectrometry (LC-MS) and two-dimensional difference in gel electrophoresis (2D-DIGE), were used to analyze the proteomic profiles of fresh and cryopreserved spermatozoa, as well as the extracellular medium (EM; n=7 for each group). Results: Cryopreservation led to a decline in motility parameters (MOT, VCL, PROG) and viability, along with an increase in ROS levels, membrane fluidity, and acrosome damage. Despite similar quality parameters between DMSO and MeOH-preserved sperm, DMSO-preserved sperm showed dramatically lower fertilization success (6.2% vs 51.2%). A total of 224 and 118 differentially abundant proteins in spermatozoa cryopreserved with MeOH and DMSO, respectively, were identified compared to fresh samples, with 342 and 363 proteins released into the EM. The most affected proteins by cryopreservation included H2A, CABYR, PYGM, ENO3, DBI and LTA4H. Additionally, 36 and 39 uniquely altered sperm-leakage proteins were identified for MeOH and DMSO cryopreserved samples, respectively. Bioinformatic analysis showed that MeOH-specific proteins were related to chromosomal structure and mitochondrial functionality, while DMSO-specific proteins were mainly involved in acrosome reaction, zona pellucida binding, flagella structure, and nuclear pore organization. These proteins are potentially involved in sturgeon sperm fertilizing ability. The expression of six proteins was verified by western blot analysis. Conclusions: This study provides the first comprehensive proteomic characterization of Siberian sturgeon spermatozoa after cryopreservation with DMSO and MeOH, revealing insights into proteomic changes that affect fertilizing ability and aiding conservation efforts for this endangered species.
2024-08-10 | PXD052170 | Pride
Project description:Antifreeze protein III (AFP III) has been used in the cryopreservation of germ cells in several kinds of animals, but it is not yet known what the specific cryoprotective mechanism of AFP III is, except for reducing the formation of ice crystals during cryopreservation. In order to study the cryoprotective mechanism of AFP III on sperm cryopreservation, the proteomic profile of Cynomolgus macaques sperm were identified after cryopreservation. Compared to fresh sperm, 141 and 32 proteins were differentially identified in Cynomolgus macaques sperm cryopreserved without and with 0.1µg/ml AFP III, respectively. These proteins were mainly involved in mitochondrial production of reactive oxygen species (ROS), glutathione (GSH) synthesis and cell apoptosis. According to the differential proteins, we supposed that AFP Ⅲ mainly reduce sperm lipid, protein and DNA damage, as well as the release of cytochrome c, and thereby reduce sperm apoptosis by modulating the production of ROS in mitochondria. The molecular mechanism that how AFP III acts with sperm proteins and protects cell from cryoinjuries needs further study.
2022-02-17 | PXD024836 | Pride
Project description:Umbilical cord blood banking is critical for the success of umbilical cord blood transplants. Here we analyzed transcriptomic differences between 27-year cryopreserved umbilical cord blood hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs) and those derived from fresh cord blood. We also leveraged differences in engraftment capacity to examine the transcriptomes of HSCs/HPCs defined by engraftment capacity, demonstrating the feasibility of this approach for identifying potency markers to aid in the selection of cord blood units for transplantation and revealing novel potential regulators of cord blood HSC/HPC engraftment.
2023-10-04 | GSE225699 | GEO
Project description:Gene expression profile at single cell level of stromal cells from fresh and cryopreserved tonsils and lymph node
| PRJNA932017 | ENA
Project description:We conducted a comparison of scRNA-seq on four paired fresh and cryopreserved atheroma samples, two from coronary arteries and two from carotid arteries, to determine whether it is possible to obtain comparable data from frozen samples. After enzymatic digestion into single cell suspensions, we sorted CD45+ cells from half of each sample and immediately processed these using the 10X Genomics scRNA-seq workflow after collection, while the other half was frozen in liquid nitrogen. After cryopreservation for an average of 7 days, the other half of each sample was thawed and sorted identically to the fresh samples.
2022-10-31 | GSE179159 | GEO