Expression data from aging study on mouse spermatogonial stem/progenitor cell population
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ABSTRACT: In mouse, spermatogonial stem/progenitor cells are the progenitor cell which develop to mature sperms through a series of mitotic and meiotic divisions and differentiation. Gfra1 is an established surface marker for mouse spermatogonial stem/progenitor cells. In this study, we used a transcriptomic approach to investigate the effect of aging on Gfra1-positive and -negative populations of mouse male germ cells. Spermatogonial stem/progenitor cells were isolated from testes of mice at ages 6-day, 21-day, 60-day, and 8-months by magnetic assisted cell sorting (MACS) technique using Gfra1 marker. Transcriptomes among different ages and Gfra1 status were compared.
Project description:In mouse, spermatogonial stem/progenitor cells are the progenitor cell which develop to mature sperms through a series of mitotic and meiotic divisions and differentiation. Gfra1 is an established surface marker for mouse spermatogonial stem/progenitor cells. In this study, we used a transcriptomic approach to investigate the effect of aging on Gfra1-positive and -negative populations of mouse male germ cells.
Project description:Spermatogenesis is a multi-step yet tightly regulated developmental process to produce male gemetes for reproduction. In mouse spermatogenesis, a sub-group of type A spermatogonia (Spga) known as spermatogonial stem cells (SSCs) maintain their population by self-renewal, and differentiate through mitosis and meiosis to form tetraploid (4n) pacchytene spermatocytes (Spcy) and haploid (n) round spermatids (Sptd), which further differentiate into functional sperms. In this study, we selected three representative stages of mouse spermatogenesis, namely Spga, Spcy and Sptd, to study their transcriptomic charteristics using whole genome tiling microarray. Three biological replicates of each selected stage. Spga were isolated from 6-day-old Balb/c mice, Spcy and Sptd from 60-day-old Balb/c mice. Transcriptomic landscapes of these three cell types were analysed with whole genome tiling array.
Project description:Maintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. In this manuscript we have identified a key role for the nucleosome remodelling protein Chromodomain Helicase DNA binding protein 4 (CHD4) in regulating SSC function. Gene expression analyses revealed that CHD4 expression is largely restricted to spermatogonia in the mouse testis, and is particularly enriched in SSCs. Using spermatogonial transplantation techniques and RNAi mediated knockdown it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, resulting in a ~50% reduction in colonisation of recipient testes. A single cell RNA-seq comparison depicted reduced expression of ‘self-renewal’ genes such as Gfra1 and Pten following Chd4 knockdown, along with increased expression of signature progenitor genes, Neurog3 and Dazl. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only though its traditional association with the remodelling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated fundamental role for CHD4 in controlling fate decisions in the spermatogonial pool.
Project description:Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides and have little or no potential for translation. More and more studies have domenstrated mammalian lncRNAs are intrinsically functional and growing data indicate lncRNAs are determinants of stem cell fate by regulating potency, self-renewal and differentiation. There is no report of lncRNAs in spermatogonial stem cells?SSCs?, and importance of lncRNAs in germline linage stem cell has not been investigated yet. here, we presents a large –scale profiling of all lncRNAs in SSCs as well as those lncRNAs regulated by SSC dependent growth factor GDNF through high throughput sequencing. Spermatogonial stem cells were first suffered from an 18 hr GDNF withdrawal followed by refreshment with GDNF for 8 hrs and RNA samples were collected from normal culture wells (N), GDNF 18hr withdrawal wells (0hr), and GDNF 8 hr refreshed cells (8hr). After all GDNF treatment, cultured germ cell clumps were gently blowed with a 200?l pipette, and followed by Total RNA isolation and sequencing by Illumina HiSeqTM 2000
Project description:Here, gene profiles in rat spermatogonial stem cell lines are compared to publicly available mouse, monkey and human spermatogonial gene profiles. Interestingly, rat spermatogonia expressed metabolic control factors Foxa1, Foxa2 and Foxa3. Germline Foxa2 was enriched in Gfra1Hi and Gfra1Low undifferentiated A-single spermatogonia. Foxa2-bound loci in spermatogonial chromatin were over-represented by conserved stemness genes (Dusp6, Gfra1, Etv5, Rest, Nanos2, Foxp1) that intersect bioinformatically with conserved glutathione/pentose phosphate metabolism genes (Tkt, Gss, Gclc, Gclm, Gpx1, Gpx4, Fth), marking elevated spermatogonial GSH:GSSG. Cystine-uptake and intracellular conversion to cysteine typically couple glutathione biosynthesis to pentose phosphate metabolism. Rat spermatogonia, curiously, displayed poor germline stem cell viability in cystine-containing media, and, like primate spermatogonia, exhibited reduced transsulfuration pathway markers. Exogenous cysteine, cysteine-like mercaptans, somatic testis cells and ferroptosis inhibitors counteracted the cysteine starvation-induced spermatogonial death and stimulated spermatogonial growth factor activity in vitro.
Project description:Defective germline reprogramming in Miwi2- and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises due to defective germline reprogramming rather than a function for the respective factors within spermatogonia. In both Miwi2-/- and Dnmt3l-/- spermatogonia the intracisternal-A particle (IAP) family of endogenous retroviruses is de-repressed, but in contrast to meiotic cells DNA damage is not observed. Instead we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring host genes. In summary, defective reprogramming deregulates the spermatogonial transcriptome that may underlie spermatogonial dysfunction.
Project description:GFRa1-GFP positive, Miwi2-tdTom negative and GFRa1-GFP negative, Miwi2-tdTom positive single cells from the testes of 2 adult, 2 aged and 3 busulfan-treated mice were sorted with FACS and cDNA libraries prepared with the SMARTseq2 protocol. Single-cell libraries were sequenced
Project description:Cortical interneurons originating from the medial ganglionic eminence (MGE) are among the most diverse cells within the CNS. Different pools of proliferating progenitor cells are thought to exist in the ventricular zone of the MGE, but whether the underlying subventricular and mantle regions of the MGE are spatially patterned has not yet been addressed. Here, we combined laser-capture microdissection and multiplex RNA-sequencing to map the transcriptome of MGE cells at a spatial resolution of 50 M-BM-5m. Distinct groups of progenitor cells showing different stages of interneuron maturation were identified and topographically mapped based on their genome-wide transcriptional pattern. One 50 M-BM-5m coronal section from the MGE was taken from each of two wildtype and one GFRa1 mutant E12.5 C57bl6/J mouse. Each section was laser microdissected into approximately 100 cubes, covering the whole MGE, and each cube was further processed for RNA-seq analysis.
Project description:Samples were prepared from 100 GFRa1-GFP positive, Miwi2-tdTom negative and 100 GFRa1-GFP negative, Miwi2-tdTom positive cells that were sorted with FACS. cDNA libraries were prepared with the SMARTseq2 protocol. Adult, control samples had four biological replicates while the aged and busulfan-treated samples were in triplicate. There were 2 technical replicates per biological sample.