Throughout in vitro first spermatogenic wave: next-generation sequencing gene expression patterns of fresh and cryopreserved prepubertal mice testicular tissue explants
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ABSTRACT:
PROVIDER: PRJNA913915 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22812763.fastq.gz | Fastqsanger.gz | |||
| SRR22812764.fastq.gz | Fastqsanger.gz | |||
| SRR22812765.fastq.gz | Fastqsanger.gz | |||
| SRR22812766.fastq.gz | Fastqsanger.gz | |||
| SRR22812767.fastq.gz | Fastqsanger.gz |
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Project description:The in vitro spermatogenesis procedure, once optimized, should be proposed to survival prepubertal patients at high risk for malignant contamination in their testis, for example in case of acute leukaemia. Differentially expressed genes between the in vitro cultured testes and the aged-matched in vivo controls will be explored by a transcriptomic analysis using the RNA-Seq technology (single-read 50 bp, PolyA-enriched) at D0, D4, D16 and D30 of culture using our reference culture medium (one-step culture), and at the same time points after an initial culture step with growth factors (two-step culture). This approach will allow us to identify the signalling pathways or molecular mechanisms that are deregulated in vitro. Moreover, a comparison of transcriptomic analyses to cryopreserved testicular fragments will allow us to identify the potential impact of cryopreservation procedures for fragments after thawing (D0) and explants at the end of culture with our reference culture medium (D30).
2023-01-31 | GSE221333 | GEO
Project description:The present study explores the advantages of enriching the freezing medium with membrane lipids and antioxidants in improving the outcome of prepubertal testicular tissue cryopreservation. For the study, testicular tissue from Swiss albino mice of prepubertal age group (2 weeks) was cryopreserved by slow freezing method either in control freezing medium (CFM; containing DMSO and FBS in DMEM/F12) or test freezing medium (TFM; containing soy lecithin, phosphatidylserine, phosphatidylethanolamine, cholesterol, vitamin C, sodium selenite, DMSO and FBS in DMEM/F12 medium) and stored in liquid nitrogen (LN 2 ) for at least one week. The tissues were thawed and enzymatically digested to assess viability, DNA damage, and oxidative stress in the testicular cells. The results indicate that TFM significantly mitigated freeze-thaw-induced cell death, DNA damage, and lipid peroxidation compared to tissue cryopreserved in CFM. Further, a decrease in Cyt C, Caspase-3, and an increase in Gpx4 mRNA transcripts were observed in tissues frozen with TFM. Spermatogonial germ cells (SGCs) collected from tissues frozen with TFM exhibited higher cell survival and superior DNA integrity compared to those frozen in CFM. Proteomic analysis revealed that SGCs experienced a lower degree of freeze-thaw- induced damage when cryopreserved in TFM, as evident from an increase in the level of proteins involved in mitigating the heat stress response, and transcriptional and translational machinery. These results emphasize the beneficial role of membrane lipids and antioxidants in enhancing the cryosurvival of prepubertal testicular tissue offering a significant stride towards improving the clinical outcome of prepubertal testicular tissue cryopreservation.
2025-05-06 | PXD050080 | Pride
Project description:IntroductionSuitable cryopreservation procedures of pre-pubertal testicular tissue associated with efficient culture conditions are crucial in the fields of fertility preservation and restoration. In vitro spermatogenesis remains a challenging technical procedure to undergo a complete spermatogenesis.The number of haploid cells and more specifically the spermatic yield produced in vitro in mice is still extremely low compared to age-matched in vivo controls and this procedure has never yet been successfully transferred to humans.MethodsTo evaluate the impact of in vitro culture and freezing procedure, pre-pubertal testicular mice testes were directly cultured until day 4 (D4), D16 and D30 or cryopreserved by controlled slow freezing then cultured until D30. Testes composed of a panel of 6.5 dpp (days postpartum), 10.5 dpp, 22.5 dpp, and 36.5 dpp mice were used as in vivo controls. Testicular tissues were assessed by histological (HES) and immunofluorescence (stimulated by retinoic acid gene 8, STRA8) analyses. Moreover, a detailed transcriptome evaluation study has been carried out to study the gene expression patterns throughout the first in vitro spermatogenic wave.ResultsTranscriptomic analyses reveal that cultured tissues expression profiles are almost comparable between D16 and D30; highlighting an abnormal kinetic throughout the second half of the first spermatogenesis during in vitro cultures. In addition, testicular explants have shown dysregulation of their transcriptomic profile compared to controls with genes related to inflammation response, insulin-like growth factor and genes involved in steroidogenesis.DiscussionThe present work first shows that cryopreservation had very little impact on gene expression in testicular tissue, either directly after thawing or after 30 days in culture. Transcriptomic analysis of testis tissue samples is highly informative due to the large number of expressed genes and identified isoforms. This study provides a very valuable basis for future studies concerning in vitro spermatogenesis in mice.
| S-EPMC10063980 | biostudies-literature
Project description:WIN 18,446/RA treatment of neonatal mice was used to synchronize the initial wave of spermatogenesis and identify novel messages expressed within either germ or Sertoli cells as spermatogonia enter meiosis.
2014-05-01 | GSE54408 | GEO
Project description:WIN 18,446/RA treatment of neonatal mice was used to synchronize the initial wave of spermatogenesis and identify novel messages expressed within either germ or Sertoli cells as spermatogonia enter meiosis. germ cell-specific (Stra8-cre: RiboTag; or Ngn3-cre:RiboTag) and Sertoli cell-specific (Amh-Cre: RiboTag)
2014-05-01 | E-GEOD-54408 | biostudies-arrayexpress
Project description:The present study was undertaken to discover molecular markers in bovine cumulus cells predictive of oocyte competence and elucidate their functional significance. Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis. Four genes of interest encoding for the lysosomal cysteine proteinases cathepsin B, S, K and Z and displaying greater transcript abundance in cumulus cells surrounding oocytes harvested from prepubertal animals were chosen for further investigation. Greater mRNA abundance for such genes in cumulus cells of prepubertal oocytes was confirmed by real time RT-PCR. Elevated transcript abundance for cathepsins B, S and Z was also observed in cumulus cells surrounding adult metaphase II oocytes that developed to the blastocyst stage at a low percentage following parthenogenetic activation, versus those that developed at a high percentage. Functional significance of cumulus cell cathepsin expression to oocyte competence was confirmed by treatment of cumulus oocyte complexes during in vitro oocyte maturation with a cell permeable cysteine proteinase (cathepsin) inhibitor. Inhibitor treatment decreased apoptotic nuclei in the cumulus layer and enhanced development of parthenogenetically activated and in vitro fertilized adult oocytes to the blastocyst stage. Stimulatory effects of inhibitor treatment during meiotic maturation on subsequent embryonic development were not observed when oocytes were matured in the absence of cumulus cells. Results support a functional role for cumulus cell cathepsins in compromised oocyte competence and suggest that cumulus cell cathepsin mRNA abundance may be predictive of oocyte quality. Keywords: Bovine, microarray, cDNA, cumulus cells Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis. Total RNA from pools of cumulus cells (n = 4) collected from adult and prepubertal animals for microarray experiments was amplified. Two color microarray experiments were conducted using a bovine cDNA array containing expressed sequence tags (ESTs) representing approximately 15200 unique genes. Hybridizations were performed on duplicate slides (prepubertal versus adult) and incorporated a dye swap. The total number of slides used is eight.
2010-05-25 | E-GEOD-10819 | biostudies-arrayexpress
Project description:Purpose: Many young adults are in a state of stress due to social and psychological pressures, which may result in male reproductive dysfunction. To provide new insight into this phenomenon, we investigated the relationship between pathological changes in rat spermatogenic cells and the expression of genes specific to spermatogenic cell types under different stress conditions. Methods: After establishing rat stress models of different time durations, we observed pathological changes in testicular tissues through haematoxylin and eosin staining, and analysed gene expression in spermatogenic cells by RNA-seq, bioinformatic analysis, and reverse transcription qPCR (RT-qPCR).Three testicular samples were taken from each group to construct 12 cDNA libraries. For each sample, 3 μg RNA was used as the starting material. Ribosomal RNA was removed using the Epicentre Ribo-Zero™ Gold kit (Rat) (Epicentre, an Illumina company, Madison, WI, USA). Results:Compared with the control group, there were 1,194 DEGs in the 3-day RS+IS group , including 455 upregulated genes and 739 downregulated genes , 1,774 DEGs in the 14-day RS+IS group including 1,124 upregulated genes and 650 downregulated genes, and 2,267 DEGs in the 21-day RS+IS group including 1,366 upregulated genes and 901 downregulated genes. Mean-while, some same expression patterns were observed in three phenotypes.After comparison with single-cell sequencing data, 349 DEGs of spermatogenic cells at different developmental stages were found. Conclusions: Our study suggest that chronic psychosomatic stress can affect the spermatogenic transcriptome of the testes, leading to a significant change in the expression of the spermatogenic genes. At the same time, histopathological and immunohistochemical results showed that chronic stress may lead to pathological changes in spermatogenic cells and to a significant decrease in key regulatory proteins
2021-05-01 | GSE144485 | GEO
Project description:Defining prepubertal primate spermatogonial stem cells to address fertility preservation in prepubertal boys
| PRJNA917768 | ENA
Project description:chick intermediate neural explants
| PRJEB5921 | ENA
Project description:Developmental competences of oocytes derived from prepubertal heifers are lower than those derived from adult counterparts. The objective of this study was to identify a range of genes associated with reduced oocyte competence that are differentially expressed between adult versus prepubertal donors. Microarray experiments were conducted using total RNA isolated from GV and MII stages oocytes collected from adult and prepubertal animals using Affymetrix GeneChip Bovine Genome Array containing 24,072 probe sets representing over 23,000 transcripts. A total of 549 and 333 genes were differentially expressed between prepubertal versus adult bovine MII and GV stages oocytes respectively. Out of these, 312 and 176 genes were up-regulated, while 237 and 157 were down-regulated in prepubertal when compared with adult MII and GV oocytes respectively. Ontological classification of the differentially expressed genes revealed that up-regulated genes in adult oocytes were involved in signal transduction, regulation of transcription DNA-dependent, and transport. Results from the present study indicated that significant number of genes were differentially expressed (>2-fold, p<0.01) between the two groups. Thus the decreased developmental competence of oocytes from prepubertal heifers may be induced due to difference in gene expression abundance as observed in our study. In conclusion, transcript abundance analyses of oocytes using microarray approach have been carried out in bovine and several other species. However, to our knowledge, this is the first study carried out to examine genes expression differential abundance in oocytes derived from perpubertal versus adult Japanese Black Cattle. Bovine 4b PP biological rep1, Bovine 78b PP biological rep2, Bovine 79 PP biological rep3 represents GV stage oocytes derived from Prepubertal (PP) heifer group, while Bovine 74b A biological rep1, Bovine 80b A biological rep2, Bovine 81 A biological rep3 represents GV stage oocytes derived from Adult (A) cow group. Bovine 7 PP biological rep1, Bovine 53 PP biological rep2, Bovine 57 PP biological rep3 represents MII stage oocytes derived from Prepubertal heifer group, while Bovine 59 A biological rep1, Bovine 70 A biological rep2, Bovine 71 A biological rep3 represents MII stage oocytes from Adult cow group.
2012-06-01 | GSE24477 | GEO