Expression profiling of purified murine epididymal spermatozoa and murine leukocytes.
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE290696 | GEO | 2025/04/02
REPOSITORIES: GEO
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Project description:Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate with the loss of sperm function during cryopreservation remains unsolved. The present study attempted to clarify this issue evaluating differential changes in the proteome of pig spermatozoa retrieved from the cauda epididymis and the ejaculate, with clear differences in cryotolerance, comparing fresh and frozen-thawed cells. Sperm samples were collected from 10 healthy, sexually mature and fertile boars, and cryopreserved using a standard 0.5 mL straw protocol. Total and progressive motility, viability and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) cauda epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT sperm samples were analyzed using a LC-ESI-MS/MS-based SWATH approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein-protein networks highlighted a set of proteins directly involved in mitochondrial functionality among those quantitatively altered in ejaculated spermatozoa, which would explain the worse post-thaw quality of ejaculated pig spermatozoa.
2019-11-11 | PXD012984 | Pride
Project description:Proteins are essential for sperm function, including fertilizing capacity. Pig spermatozoa, emitted in well-defined ejaculate fractions, clearly vary in their functionality, which would be related to differences in protein composition. This study aimed firstly to update the porcine sperm proteome and secondly to identify proteins differentially expressed among mature spermatozoa from cauda epididymis and those fortuitously delivered in different ejaculate portions. Nine ejaculates from nine mature and fertile boars were manually collected in three separate ejaculate portions: the first 10 mL of sperm-rich ejaculate fraction (SRF), the rest of SRF and the post-SRF. The contents of cauda epididymides of the same boars was collected post-mortem by perfusion. All samples were centrifuged, pooling the resulting sperm pellets within the respective source-sample, which were later split to generate two technical replicates per source. The final eight sperm samples were subjected to iTRAQ-based 2D-LC-MS/MS for protein identification and quantification. A total of 1,723 proteins were identified (974 of Sus Scrofa taxonomy) and 1,602 of them were also quantified (960 of Sus Scrofa taxonomy). After an ANOVA test, 32 Sus Scrofa proteins showed quantitative differences (P < 0.01) among the sperm samples, which were particularly relevant for the functionality of spermatozoa fortuitously ejaculated in the post-SRF. The present study is the first showing quantitative differences in the protein profile of mature spermatozoa, involving proteins clearly implicated in sperm function, that prove the protein profile of boar spermatozoa is remodelled during ejaculation . These findings provide a valuable groundwork for further studies focused on identifying protein biomarkers of sperm fertility.
2018-10-02 | PXD010062 | Pride
Project description:Previously we have reported that cysteine rich secretory protein 2 (CRISP2) is involved in high molecular weight complexes in boar spermatozoa under a native state. In the current study, CRISP2 interacting partners were identified using proteomic technology and their interaction was investigated under non-capacitating conditions, after the induction of in vitro sperm capacitation and then subsequently during an ionophore A23187 induced acrosome reaction. Blue native gel electrophoresis and native immunoblots revealed that CRISP2 was present within a ~150 kDa protein complex under all three conditions. Interrogation of CRISP2-immunoreactive bands from blue native gels as well as CRISP2 immunoprecipitated products using mass spectrometry consistently revealed that acrosin and acrosin binding protein (ACRBP) were among the most abundant proteins and present under the three conditions. Co-immunoprecipitation and immunoblotting indicated that CRISP2 interacted with pro-acrosin (~53 kDa) and ACRBP under all three conditions and additionally to acrosin (~35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with CRISP2 was assessed via in situ proximity ligation assays. The colocalization signal of CRISP2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of CRISP2 and ACRBP colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post acrosomal sheath region upon capacitation. These results suggest that CRISP2 may act as a scaffold for protein complexes formation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.
2023-03-09 | PXD037181 | Pride
Project description:We reported the cell composition and segmental expression differences in three epididymis segments using single-cell RNA sequencing. A total of 13,949 cells were detected, which were further clustered into eight cell populations identified with all known constituent cells of mouse epididymis including principle cell, basal cell, clear/narrow cell and immune cells. Through the study of 35 known epididymal proteins linking to sperm function phenotypes (EPSFPs), we found they were mainly expressed in principle cells and also revealed their segmental specific expression patterns. Notably, a part of EPSFPs was associated with sperm protection and predominantly localized in caput principal cells, whereas some EPSFPs, highly expressed in corpus and cauda, were served as the regulating molecular for the sperm functions such as motility, capacitation or acrosome reactions.In addition, focused analyses uncovered 5 subtypes of the principal cell. Intriguingly, Prc5, the subpopulation of principle cells, was almost only existed in the caput epididymis.
2021-06-02 | GSE151998 | GEO
Project description:Expression profiling of purified murine epididymal spermatozoa and murine leukocytes.
| PRJNA1229584 | ENA
Project description:Mammalian spermatozoa are not utterly mature after ejaculation and must undergo additional functional and structural changes within female reproductive tracts to achieve subsequent fertilization, including both capacitation and acrosome reaction (AR), which are dominated by post-translational modifications (PTMs), especially phosphorylation. However, the mechanisms of protein phosphorylation during frozen-thawed sperm capacitation and AR has not yet been well studied. We employed phosphoproteomic approach based on TMT labeling combined with LC-MS/MS strategy to analyze frozen-thawed sperm in Ashidan yak under three sequential conditions (density gradient centrifugation-based purification, incubation in the capacitation medium and induction of AR processes by the calcium ionophore A23187 treatment).
2022-09-26 | PXD035751 | Pride
Project description:The male gamete is not completely mature after ejaculation and requires further events in the female genital tract to acquire fertilizing ability, including the processes of capacitation and acrosome reaction. In order to shed light on dynamic protein changes experienced by the sperm cell in preparation for fertilization, a comprehensive quantitative proteomic profiling based on isotopic peptide labeling and liquid chromatography followed by tandem mass spectrometry was performed on spermatozoa from three donors of proven fertility under three sequential conditions: purification with density gradient centrifugation, incubation with capacitation medium, and induction of acrosome reaction by the exposure to the calcium ionophore A23187. After applying strict selection criteria for peptide quantification and for statistical analyses, 36 proteins with significant changes in their relative abundance within sperm protein extracts were detected. Moreover, the presence of peptide residues potentially harboring sites for post-translational modification was revealed, suggesting that protein modification may be an important mechanism in sperm maturation. In this regard, increased levels of proteins mainly involved in motility and signaling, both regulated by protein modifiers, were detected in sperm lysates following incubation with capacitation medium. In contrast, less abundant proteins in acrosome-reacted cell lysates did not contain potentially modifiable residues, suggesting the possibility that all those proteins might be relocated or released during the process. Protein-protein interaction analysis revealed a subset of proteins potentially involved in sperm maturation, including ERLIN2, GGH, TMED 10, and ATP synthases. These results contribute to the current knowledge of the molecular basis of human fertilization and propose new candidates to be validated as modulators of male fertility.
2019-12-17 | PXD014871 | Pride
Project description:Following their production in the testis, spermatozoa enter the epididymis to gain their motility and fertilizing abilities. This post-testicular maturation coincides with sperm epigenetic profile changes that influence the progeny outcome. While recent studies underscored the dynamics of small non-coding RNAs in the maturing spermatozoa, little is known regarding sperm methylation changes and their impact at the post-fertilization level. To map out the sperm methylome dynamics, we purified spermatozoa by FACS from the testis and the different epididymal segments (i.e. caput, corpus and cauda) of CAG/su9-DsRed2; Acr3-EGFP transgenic mice. Reduced-Representation Bisulfite Sequencing (RRBS-Seq) performed on DNA from these respective sperm populations indicated that high methylation changes were observed between spermatozoa from the caput vs. testis with 5546 entries meeting our threshold values (q value < 0.01, methylation difference above 25 %). Most of these changes were transitory during epididymal sperm maturation according to the low number of entries identified between spermatozoa from cauda vs. testis.
2022-03-10 | GSE191051 | GEO
Project description:In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development leading to an increased appreciation of the importance of RNA interference pathways, and in particular microRNAs (miRNAs), as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon we employed next generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the dynamic post-testicular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Since sperm are not capable of de novo transcription these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to condition the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring. Examination of the microRNA expression profile in sperm thoughout the mouse epididymis and mouse using next generation sequencing in duplicate.
2015-08-05 | E-GEOD-70198 | biostudies-arrayexpress
Project description:In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development leading to an increased appreciation of the importance of RNA interference pathways, and in particular microRNAs (miRNAs), as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon we employed next generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the dynamic post-testicular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Since sperm are not capable of de novo transcription these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to condition the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring.
2015-08-05 | GSE70198 | GEO