Project description:Self-renewal and differentiation of spermatogonial stem cells (SSCs) provides the foundation for testis homeostasis, yet mechanisms that control their functions in mammals are poorly defined. We used microarray transcript profiling to identify specific genes whose expression are augmented in the SSC-enriched Thy1+ germ cell fraction of mouse pup testes. Comparisons of gene expression in the Thy1+ germ cell fraction to the Thy1-depeleted testis cell population identified 202 genes that are expressed 10-fold or higher in Thy1+ cells. This database provided a mining tool to investigate specific characteristics of SSCs and identify novel mechanisms that potentially influence their functions.
Project description:MicroRNAs (miRs) play a key role in the control of gene expression in a wide array of tissue systems where their functions include the regulation of self-renewal, cellular differentiation, proliferation, and apoptosis. However, the functional importance of individual miRs in controlling spermatogonial stem cell (SSC) homeostasis has not been investigated. Using high-throughout sequencing, we profiled the expression of miRs in the Thy1+ testis cell population, which is highly enriched for SSCs, and the Thy1- cell population, composed primarily of testis somatic cells. In addition, we profiled the global expression of miRs in cultured germ cells, also enriched for SSCs. Our results demonstrate that miR-21, along with miR-34c, -182, -183, -146a, -465a-3p, -465b-3p, -465c-3p, and -465c-5p are preferentially expressed in the Thy1+ SSC-enriched population, as compared to Thy1- somatic cells, and we further observed that Thy1+ SSC-enriched testis cells and SSC-enriched cultured germ cells share remarkably similar miR expression profiles. Spermatogonial Stem Cell enriched cell populations (freshly isolated and short-term cultured) and somatic cell populations were isolated from C57B/L6 mouse donors and subjected to small RNA isolation and sequencing.
Project description:MicroRNAs (miRs) play a key role in the control of gene expression in a wide array of tissue systems where their functions include the regulation of self-renewal, cellular differentiation, proliferation, and apoptosis. However, the functional importance of individual miRs in controlling spermatogonial stem cell (SSC) homeostasis has not been investigated. Using high-throughout sequencing, we profiled the expression of miRs in the Thy1+ testis cell population, which is highly enriched for SSCs, and the Thy1- cell population, composed primarily of testis somatic cells. In addition, we profiled the global expression of miRs in cultured germ cells, also enriched for SSCs. Our results demonstrate that miR-21, along with miR-34c, -182, -183, -146a, -465a-3p, -465b-3p, -465c-3p, and -465c-5p are preferentially expressed in the Thy1+ SSC-enriched population, as compared to Thy1- somatic cells, and we further observed that Thy1+ SSC-enriched testis cells and SSC-enriched cultured germ cells share remarkably similar miR expression profiles.
Project description:Mouse spermatogonial stem cells (SSCs) continuously self-renew on the feeder layers in serum-free culture medium supplemented with glial cell line-derived neurotrophic factor and fibroblast growth factor 2. To identify novel nuclear proteins involved in SSC maintenance, comparative proteomic profiling of nuclear proteins was performed between self-renewing and differentiation-initiated SSCs in culture. The self-renewing SSC cultures were established from C57BL/6 mouse testes. Nuclear fractions from self-renewing SSC cultures treated with ethanol as a vehicle control (spermatogonial stem cells) and differentiation-initiated SSC cultures treated with 0.3 μM retinoic acid for 24 h (spermatogonial progenitor cells) were isolated for proteomic analysis.
Project description:To investigate the role of DDX20 in spermatogenesis, we generated Ddx20 flox/flox Ddx4-Cre mice to make a germ cell-specific Ddx20 knockout, and isolated spermatogonia from four-day-old mouse testes by THY1 magnetic beads.We then performed gene expression profiling analysis using data obtained from RNA-seq of THY1 + spermatogonia.
Project description:Male FVB strain mice aged 12-days-old through 26-days-old were administered daily intraperitoneal injections of rapamycin (4mg/kg body weight) or control vehicle (5% Tween-80, 5% PEG-400), beginning at postnatal day (P)12. Mice were euthanized at P26 and their testes were isolated for germ cell enrichment. Single cell suspensions of germ cells were prepared from isolated testes and subjected to magnetic-activated cell sorting (MACS). This procedure enriches the undifferentiated spermatogonia fraction, which represents the spermatogonial stem cell (SSC) population. Total RNA from cells double-positive for the SSC surface markers thymus cell antigen 1, theta (THY1) and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRA1) was isolated for gene expression microarray analysis. Magnetic-activated cell sorting (MACS) was used to enrich undifferentiated spermatogonia from a pool of primary testicular cells isolated from 5 littermate rapamycin (RM)-treated male mice and from 5 littermate control vehicle (VEH)-treated male mice. With MACS-enriched cells from pool generating RNA, no technical replicates performed.
Project description:Spermatogonial stem cells are the foundation of spermatogenesis and as such can serve as a tool for the treatment of infertility in prepubertal cancer survivors. Spermatogonial stem cells are unique as they develop from primordial germ cells (PGCs), which colonize the developing tubules as immature SSC precursors. It has been controversial, when SSCs are maturing to an adult-like stem cell and recent research has found that prepubertal SSCs are actually metabolically distinct from adult SSCs until puberty. Sertoli cells picture a major part of the SSC niche and undergo drastic changes with puberty and polarize to compartmentalize the seminiferous epithelium with formation of tight junctions to a tight basal part where SSCs reside and an apical part with more differentiated stages of spermatogenesis. In the study were mapping the progression of Sertoli cells maturation events to the metabolic changes SSCs undergo during prepubertal development.
Project description:Male FVB strain mice aged 12-days-old through 26-days-old were administered daily intraperitoneal injections of rapamycin (4mg/kg body weight) or control vehicle (5% Tween-80, 5% PEG-400), beginning at postnatal day (P)12. Mice were euthanized at P26 and their testes were isolated for germ cell enrichment. Single cell suspensions of germ cells were prepared from isolated testes and subjected to magnetic-activated cell sorting (MACS). This procedure enriches the undifferentiated spermatogonia fraction, which represents the spermatogonial stem cell (SSC) population. Total RNA from cells double-positive for the SSC surface markers thymus cell antigen 1, theta (THY1) and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRA1) was isolated for gene expression microarray analysis.
Project description:We have used SmartSeq2 to sequence single phenotypic skeletal stem cell (SSCs) populations purified via FACS from adult mouse long bones. Two putative SSC populations were isolated based on their previously reported surface marker profiles. An osteochondrogenic SSC (ocSSC, CD45-CD31-Tie2-Ter119-CD51+6C3-Thy1-CD105-) and a perivascular SSC (pvSSC, CD45-CD31-Pdgfra+Sca1+CD24+) were investigated.
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.