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Differential motility parameters and identification of proteomic profiles of human sperm cryopreserved with cryostraw and cryovial.

ABSTRACT: Background:Although sperm cryopreservation has been widely used in human reproductive medicine as an integral infertility management in infertility clinics and for banking sperm in sperm banks, the freezing/thawing protocols are not optimal. The freezing and thawing processes result in changes at both structural and molecular levels, some even detrimental, in human sperm when compared with fresh sperm. The change of sperm proteins after cryopreservation may play negative roles for fertilization and early embryo development. Conventionally, cryostraws (CS) and cryovials (CV) are the most widely used cryopreservation carriers (CPCs) for human sperm cryopreservation accompanied with the use of egg yolk free commercial media. However, the influence of cryopreservation on the proteomic profile of human sperm preserved with the two CPCs is unknown. Therefore the purpose of the present study was to compare the frozen-thawed motility, investigate the proteomic profile of human sperm cryopreserved with the two types of CPCs, and identify the susceptible proteins that play key roles for sperm function and fertility. Methods:The present study compared the cryosurvival of human sperm frozen with the two different CPCs and identified the sperm proteomic changes by using the isobaric tags for relative and absolute quantification labeling technique coupled with 2D LC-MS/MS analysis after freezing and thawing. Results:Our results indicated that sperm cryopreserved with CV showed higher values for percentage of motile sperm and forward activity rate than those with CS. Compared to fresh sperm, 434 and 432 proteins were differentially identified in human sperm cryopreserved with CS and CV, respectively. Conclusion:The proteomic profiles of human sperm are greatly affected by cryopreservation with either type of CPC. GO analysis revealed that most of the differentially identified sperm proteins enriched in the extracellular membrane-bounded organelles, cytoplasm and cytosol. In addition, 106 susceptible proteins having known identities related to sperm functions were identified. In general, cryovial seems to be the preferred CPC for human sperm cryopreservation based on the post-thaw motility parameters and the effect on sperm proteomic profiles. These results are beneficial for the insight into the understanding of the cryoinjury mechanism of sperm and the development of human sperm cryopreservation strategies.

SUBMITTER: Li S

PROVIDER: S-EPMC6582484 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Differential motility parameters and identification of proteomic profiles of human sperm cryopreserved with cryostraw and cryovial.

Li Shanshan S Ao Lei L Yan Yaping Y Jiang Jiang J Chen Bingbing B Duan Yanchao Y Shen Fei F Chen Jinbao J Inglis Briauna B Ni Renmin R Ji Weizhi W Si Wei W

Clinical proteomics 20190619

<h4>Background</h4>Although sperm cryopreservation has been widely used in human reproductive medicine as an integral infertility management in infertility clinics and for banking sperm in sperm banks, the freezing/thawing protocols are not optimal. The freezing and thawing processes result in changes at both structural and molecular levels, some even detrimental, in human sperm when compared with fresh sperm. The change of sperm proteins after cryopreservation may play negative roles for fertil ...[more]

PMID: 31244561

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Project description:PurposeThe advent of proteomics provides new opportunities to investigate the molecular mechanisms underlying male infertility. The selection of relevant targets based on a single analysis is not always feasible, due to the growing number of proteomic studies with conflicting results. Thus, this study aimed to systematically review investigations comparing the sperm proteome of normozoospermic and infertile men to define a panel of proteins with the potential to be used to evaluate sperm quality.Materials and methodsA literature search was conducted on PubMed, Web of Science, and Scopus databases following the PRISMA guidelines. To identify proteins systematically reported, first the studies were divided by condition into four groups (asthenozoospermia, low motility, unexplained infertility, and infertility related to risk factors) and then, all studies were analysed simultaneously (poor sperm quality). To gain molecular insights regarding identified proteins, additional searches were performed within the Human Protein Atlas, Mouse Genome Informatics, UniProt, and PubMed databases.ResultsThirty-two studies were included and divided into 4 sub-analysis groups. A total of 2752 proteins were collected, of which 38, 1, 3 and 2 were indicated as potential markers for asthenozoospermia, low motility, unexplained infertility and infertility related to risk factors, respectively, and 58 for poor sperm quality. Among the identified proteins, ACR, ACRBP, ACRV1, ACTL9, AKAP4, ATG3, CCT2, CFAP276, CFAP52, FAM209A, GGH, HPRT1, LYZL4, PRDX6, PRSS37, REEP6, ROPN1B, SPACA3, SOD1, SPEM1, SPESP1, SPINK2, TEKT5, and ZPBP were highlighted due to their roles in male reproductive tissues, association with infertility phenotypes or participation in specific biological functions in spermatozoa.ConclusionsSperm proteomics allows the identification of protein markers with the potential to overcome limitations in male infertility diagnosis and to understand changes in sperm function at the molecular level. This study provides a reliable list of systematically reported proteins that could be potential targets for further basic and clinical studies.

Project description:At present, there are few reports about the proteomics changes provoked by butylated hydroxytoluene (BHT) supplementation on cryopreserved semen in mammals. Thus, we aimed to evaluate the effects of different concentrations of BHT on goat sperm and to investigate the proteomics changes of adding BHT to cryopreserved goat (Capra hircus) sperm. Firstly, semen samples were collected from four goats, and frozen in the basic extenders containing different concentrations of BHT (0.5 mM, 1.0 mM, 2.0 mM) and a control without BHT, respectively. After thawing, the protective effects of dose-dependent replenished BHT to the freezing medium on post-thaw sperm motility, integrities of plasma membrane and acrosome, reactive oxygen species levels were confirmed, with 0.5 mM BHT being the best (B group) as compared to the control (without BHT, C group). Afterwards, TMT-based quantitative proteomic technique was performed to profile proteome of the goat sperm between C group and B group. Parallel reaction monitoring was used to confirm reliability of the data. Overall, 2,476 proteins were identified and quantified via this approach. Comparing the C and B groups directly (C vs. B), there were 17 differentially abundant proteins (DAPs) po-tentially associated with sperm characteristics and functions were identified, wherein three were upregulated and 14 were downregulated, respectively. GO annotation analysis demonstrated the potential involvement of the identified DAPs in metabolic process, multi-organism process, reproduction, reproductive process, and cellular process. KEGG enrichment analysis further indicated their potential roles in renin-angiotensin system and glutathione metabolism pathways. Together, this novel study clearly shows that BHT can effectively improve quality parameters and fertility potential of post-thawed goat sperm at the optimal concentration, and its cryoprotection may be realized through regulation of sperm metabolism and antioxidative capability from the perspective of sperm proteomic modification.

Project description:Graphical abstractAbstractSperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw is vital to ensure recovery of the highest quality spermatozoa with minimal iatrogenic damage. This not only enhances the chances of successful conception but also reduces the risk of genetic damage in the embryo. To address this issue, human semen samples were cryopreserved using a slow freezing protocol and Quinn's Advantage™ Sperm Freeze medium. The samples were subsequently thawed and subjected to three types of sperm isolation procedures: direct swim-up, density gradient centrifugation, and electrophoretic separation using the Felix™ device. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with increases in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility which was particularly evident with the swim-up and Felix™ procedures. The latter also significantly improved sperm vitality. There were no differences between sperm separation techniques with respect to morphology, and mitochondrial reactive oxygen species generation remained essentially unchanged when cell vitality was taken into account. By contrast, major differences were observed in DNA integrity and lipid aldehyde formation, where Felix™ isolated cells exhibiting significantly less DNA damage than the other isolation procedures as well as lower levels of 4-hydroxynonenal formation. Electrophoretic sperm isolation, therefore, offers significant advantages over alternative separation strategies, in terms of the quality of the gametes isolated and the time taken to achieve the isolation.Lay summaryLong-term storage of sperm is vital to assisted reproductive technology because it permits the preservation of fertility that might be compromised as a result of factors such as chemotherapy or vasectomy. This goal can be achieved via cryopreservation - the freezing of cells to -196°C. When the sperm are subsequently required for conception, they must be carefully separated from the cryopreservation medium in a manner that maximizes the chances of successful conception and minimizes the risk of genetic defects in the offspring. In this paper, three isolation techniques were compared for their ability to separate ideal sperm from semen and media following cryopreservation. It was found that cryopreservation led to lower levels of motility and vitality and created higher levels of DNA and cell membrane damage. Of the three techniques compared, only cells separated on the basis of their size and electric charge (electrophoretic isolation) exhibited significantly lower levels of DNA fragmentation.

Project description:Pacific abalone, Haliotis discus hannai, is a highly commercial seafood in Southeast Asia. The present study aimed to determine the influence of saccharides and vitamins on post-thaw sperm quality, ATP content, fertilization capacity, hatching capacity, and mRNA content of motility and fertilization-associated genes of Pacific abalone. Sperm cryopreserved using saccharides improved the post-thaw sperm quality including motility, acrosome integrity (AI), plasma membrane integrity (PMI), and mitochondrial membrane potential (MMP). However, vitamins (l-ascorbic acid) did not result in any significant improvement in sperm quality. Sperm cryopreserved using saccharides also improved ATP content, DNA integrity, and mRNA content of motility and fertilization-associated genes of post-thaw sperm than sperm cryopreserved without saccharides. Among sperm cryopreserved using different saccharides, post-thaw sperm quality indicators (except PMI) and mRNA content of motility and fertilization-associated genes did not show significant differences between sperm cryopreserved using 3% sucrose (S) combined with 8% dimethyl sulfoxide (DMSO) and sperm cryopreserved using 1% glucose (G) combined with 8% ethylene glycol (EG). However, sperm cryopreserved using 3% S + 8% DMSO showed higher post-thaw sperm quality (motility: 58.4 ± 2.9%, AI: 57.1 ± 3.2%, PMI: 65.3 ± 3.3%, and MMP: 59.1 ± 3.2%), ATP content (48.4 ± 1.8 nmol/ml), and % DNA in tail (2.09 ± 0.20%) than sperm cryopreserved using other saccharides. When sperms were cryopreserved using 3% S + 8% DMSO, the mRNA content of motility (heat shock protein 70, HSP70; heat shock protein 90, HSP90; protein kinase A, PKA-C; axonemal protein 66.0, Axpp66.0; and tektin-4) and fertilization-associated (sperm protein 18 kDa, SP18 kDa) genes were higher than in sperm cryopreserved using other saccharides. However, changes in the mRNA contents of these genes were insignificant between sperm cryopreserved using 3% S + 8% DMSO and 1% G + 8% EG. Taken together, these results indicate that cryopreservation using 3% S + 8% DMSO can improve post-thaw sperm quality and mRNA contents better than other examined cryoprotectants. The present study suggests that 3% S + 8% DMSO is a suitable cryoprotectant for sperm cryopreservation and molecular conservation of this valuable species.

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.

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Differential motility parameters and identification of proteomic profiles of human sperm cryopreserved with cryostraw and cryovial.

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Differential motility parameters and identification of proteomic profiles of human sperm cryopreserved with cryostraw and cryovial.

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