A single-cell view of spermatogonial stem cells

Spermatogonial stem cells (SSCs) are essential for long-term spermatogenesis and are the subject of considerable clinical interest, as ‘SSC therapy’ has the potential to cure some forms of male infertility. Recently, we have learned more about SSCs and spermatogenesis in general from a plethora of studies that performed single-cell RNA sequencing (scRNAseq) analysis on dissociated cells from human, macaque, and/or mice testes. Here, we discuss what scRNAseq analysis has revealed about SSC precursor cells, the initial generation of SSCs during perinatal development, and their heterogeneity once established. scRNAseq studies have also uncovered unexpected heterogeneity of the larger class of cells that includes SSCs — undifferentiated spermatogonia. This raises the controversial possibility that multiple SSC subsets exist, which has implications for mechanisms underlying spermatogenesis and future SSC therapeutic approaches.     

Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells

Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation.     

Deciphering the heterogeneity in DNA methylation patterns during stem cell differentiation and reprogramming

Background

Human induced pluripotent stem cells (iPSCs) have a wide range of applications throughout the fields of basic research, disease modeling and drug screening. Epigenetic instable iPSCs with aberrant DNA methylation may divide and differentiate into cancer cells. Unfortunately, little effort has been taken to compare the epigenetic variation in iPSCs with that in differentiated cells. Here, we developed an analytical procedure to decipher the DNA methylation heterogeneity of mixed cells and further exploited it to quantitatively assess the DNA methylation variation in the methylomes of adipose-derived stem cells (ADS), mature adipocytes differentiated from ADS cells (ADS-adipose) and iPSCs reprogrammed from ADS cells (ADS-iPSCs).

Results

We observed that the degree of DNA methylation variation varies across distinct genomic regions with promoter and 5’UTR regions exhibiting low methylation variation and Satellite showing high methylation variation. Compared with differentiated cells, ADS-iPSCs possess globally decreased methylation variation, in particular in repetitive elements. Interestingly, DNA methylation variation decreases in promoter regions during differentiation but increases during reprogramming. Methylation variation in promoter regions is negatively correlated with gene expression. In addition, genes showing a bipolar methylation pattern, with both completely methylated and completely unmethylated reads, are related to the carbohydrate metabolic process, cellular development, cellular growth, proliferation, etc.

Conclusions

This study delivers a way to detect cell-subset specific methylation genes in a mixed cell population and provides a better understanding of methylation dynamics during stem cell differentiation and reprogramming.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-978) contains supplementary material, which is available to authorized users.     

Stem cell and niche development in the postnatal rat testis

Adult tissue stem cells self-renew and differentiate in a way that exactly meets the biological demand of the dependent tissue. We evaluated spermatogonial stem cell (SSC) activity in the developing rat testis and the quality and accessibility of the stem cell niche in wild type, and two busulfan-treated models of rat pup recipient testes using an SSC transplantation technique as a functional assay. While our results revealed a 69-fold increase in stem cell activity during rat testis development from neonate to adult, only moderate changes in SSC concentration were observed, and stem cells from neonate, pup, and adult donor testes produce spermatogenic colonies of similar size. Analysis of the stem cell niche in recipient rat testes demonstrated that pup testes support high levels of donor stem cell engraftment when endogenous germ cells are removed or compromised by busulfan treatment. Fertility was established when rat pup donor testis cells were transplanted into fetal- or pup-busulfan-treated recipient rat pup testes, and the donor genotype was transmitted to subsequent generations. These results provide insight into stem cell/niche interactions in the rat testis and demonstrate that techniques originally developed in mice can be extended to other species for regenerative medicine and germline modification.     

Rising from the crypt: decreasing DNA methylation during differentiation of the small intestine

The differentiation of intestinal stem cells involves few DNA methylation changes, assayed by bisulfite sequencing, in contrast to other adult somatic stem cell hierarchies.Please see related Research article: http://genomebiology.com/2013/14/5/R50     

探索Sertoli细胞对去除精原细胞的动态反应

目的探索Sertoli细胞对去除小鼠精原细胞后睾丸的动态反应。方法采用15、30和44 mg/kg的白消安腹腔注射法建立不同程度去除精原细胞的动物模型,处理后5 d和28 d时对睾丸进行组织学检测,评价精子发生状态,并运用实时定量荧光PCR技术检测这两个时期睾丸GDNF、PLZF、Nanog和GFRα1基因mRNA的表达量。结果在白消安处理后第5天,GDNF出现显著升高,且呈剂量依赖趋势,而PLZF与GFRɑ1并无显著变化,睾丸组织学观察亦无明显变化。在白消安处理后28 d时,GDNF、PLZF、Nanog、GFRɑ1基因mRNA相对表达量均出现大幅度的升高,睾丸组织学切片观察显示随着给药剂量的增加,精子发生受到的损伤愈加严重。结论 Sertoli细胞早在白消安处理后第5天就对精原细胞的变化发生了反应,Sertoli细胞分泌GDNF的能力发生代偿性增加,进而刺激精原干细胞自我更新速度加快,体现在Nanog和PLZF水平提高,从而实现精子发生的重建。     

DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells

Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.     

Isolation of enriched carp spermatogonial stem cells from Labeo rohita testis for in vitro propagation

The in vitro culture system for spermatogonial stem cells (SSCs) is a powerful tool for exploring molecular mechanisms of male gametogenesis and gene manipulation. Very little information is available for fish SSC biology. Our aim was to isolate highly pure SSCs from the testis of commercially important farmed carp, Labeo rohita. The minced testis of L. rohita was dissociated with collagenase. Dissociated cells purified by two-step Ficoll gradient centrifugation followed by magnetic activated cell sorting (MACS) using Thy1.2 (CD90.2) antibody dramatically heightened recovery rate for spermatogonial cells. The purified cells were cultured in vitro conditions for more than two months in L-15 media containing 10% fetal bovine serum (FBS), 1% carp serum, and other nutrients. The proliferative cells were dividing as validated by 5-bromo-2′-deoxyuridine (BrdU) incorporation assay and formed colonies/clumps with the typical characteristics of SSCs A majority of enriched cell population represented a Vasa⁺, Pou5f1/pou5f1⁺, Ssea-1⁺, Tra-1-81⁺, plzf⁺, Gfrα1/gfrα1⁻, and c-Kit/c-kit⁻ as detected by immunocytochemical and/or quantitative real-time polymerase chain reaction (RT-PCR) analyses. Thus, Thy1⁺ SSCs were enriched with greater efficiency from the mixed population of testicular cells of L. rohita. A population of enriched spermatogonial cells could be cultured in an undifferentiated state. The isolated SSCs could provide avenue for undertaking research on basic and applied reproductive biology.     

DNA methylation in mouse embryonic stem cells and development

Mammalian development is associated with considerable changes in global DNA methylation levels at times of genomic reprogramming. Normal DNA methylation is essential for development but, despite considerable advances in our understanding of the DNA methyltransferases, the reason that development fails when DNA methylation is deficient remains unclear. Furthermore, although much is known about the enzymes that cause DNA methylation, comparatively little is known about the mechanisms or significance of active demethylation in early development. In this review, we discuss the roles of the various DNA methyltransferases and their likely functions in development.     

Spermatogonial stem cell transplantation and testicular function

Spermatogonial stem cells (SSCs) are responsible for the continual production of spermatozoa throughout adult life. Interactions between SSCs and the surrounding cells in the seminiferous tubules regulate the biological activity of these cells. Factors involved in the regulation of SSCs are beginning to be defined by animal models and the culture of SSCs in defined media. A critical development in the characterization of SSCs has been the development of the germ cell transplantation technique, which provides the only assay for the presence of SSCs in a population of cells, and which allows the determination of whether SSCs are proliferating or differentiating in culture. This approach has accelerated SSC-focused research and promises to provide a better understanding of the factors and mechanisms that regulate these cells. The knowledge provided by this work is also critical to an appraisal of the components of the SSC niche in the seminiferous epithelium. Thus, many aspects of testicular function can be defined by the investigation of SSCs and the factors, cells, and environment that regulate SSCs, thereby leading to a more comprehensive understanding of spermatogenesis.     

Establishment of a proteomic profile associated with gonocyte and spermatogonial stem cell maturation and differentiation in neonatal mice

Initiation of the first wave of spermatogenesis in the neonatal mouse testis is characterized by differentiation of a transient population of germ cells called gonocytes in the center of the seminiferous tubules. After resuming mitotic activity, gonocytes relocate on the basement membrane, giving rise to spermatogonial stem cells (SSCs). These processes begin from birth in mice, and differentiated type A spermatogonia first appear by day 6 postpartum. During these processes, Sertoli cells within the seminiferous tubules and Leydig cells in the interstitial tissue form the stem cell “niche,” and influence SSC fate decisions. Thus, we collected whole mouse testis tissues during the first wave of spermatogenesis at specific time points (days 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 postpartum) and constructed a comparative proteomic profile. We identified 252 differentially expressed proteins classified into three clusters based on expression, and bioinformatics analysis correlated each protein pattern to specific cell processes. Expression patterns of nine selected proteins were verified via Western blot, and cellular localizations of three proteins with little known information in testes were further investigated during spermatogenesis. Taken together, the results provide an important reference profile of a functional proteome during neonatal mouse gonocyte and SSC maturation and differentiation.     

Phenotypic and functional characteristics of spermatogonial stem cells in rats

Spermatogonial stem cells (SSCs) are at the foundation of the highly productive spermatogenic process that continuously produces male gametes throughout postnatal life. However, experimental evaluation of SSCs in postnatal testes is complicated because these cells are extremely rare and few defining morphology or biochemical characteristics are known. In this study, we used the spermatogonial transplantation functional assay, combined with fluorescence-activated cell sorting (FACS) analysis to identify cellular, biochemical and surface antigenic characteristics of SSCs in rat testes during development. Our results demonstrated that forward scatter (FSc)(hi), side scatter (SSc)(hi), mitochondria membrane potential (DeltaPsim)(lo), Ep-CAM(+), Thy-1(+), beta3-integrin(+) stem cells in neonate rat testes become SSc(lo), DeltaPsim(hi), Ep-CAM(+), Thy-1(lo), beta3-integrin(-) stem cells in pup rat testes. Furthermore, prospective identification of rat testis cell populations (Ep-CAM(+)), highly enriched for SSCs (1 in 13 for neonate; 1 in 8.5 for pup) enabled us to predict the Thy-1 and beta3-integrin status of stem cells in neonate and pup testes, which was subsequently confirmed by transplantation analyses. Systematic characterization of SSCs enabled the production of testis cell populations highly enriched (up to 120-fold) for SSCs and will facilitate future investigations of functional and genomic characteristics.     

A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse

In whole mounts of seminiferous tubules of C3H/101 F₁ hybrid mice, spermatogonia were counted in various stages of the epithelial cycle. Furthermore, the total number of Sertoli cells per testis was estimated using the disector method. Subsequently, estimates were made of the total numbers of the different spermatogonial cell populations per testis.

The results of the cell counts indicate that the undifferentiated spermatogonia are actively proliferating from stage XI until stage IV. Three divisions of the undifferentiated spermatogonia are needed to obtain the number of A₁ plus undifferentiated spermatogonia produced each epithelial cycle. Around stage VIII almost two-thirds of the A_pr and all of the A_al spermatogonia differentiate into A₁ spermatogonia. It was estimated that there are 2.5 × 10⁶ differentiating spermatogonia and 3.3 × 10⁵ undifferentiated spermatogonia per testis. There are about 35,000 stem cells per testis, constituting about 0.03% of all germ cells in the testis. It is concluded that the undifferentiated spermatogonia, including the stem cells, actively proliferate during about 50% of the epithelial cycle.     

Alteration in genes expression patterns during in vitro differentiation of mouse spermatogonial cells into neuroepithelial-like cells

Pluripotent stem cells derived from testis is a new, natural, and unlimited source for cell therapy in regenerative medicine and represent a possible alternative to replacing of all cells in the body. Here, we designed a simple co-culture system of spermatogonia cells with Sertoli cells for the generation of embryonic stem-like cells from mouse testis. The importance of our simple method will be clear when we compared it with other complex and time-consuming methods. Embryonic stem-like colonies with sharp border confirmed by real-time PCR, immunocytochemistry and flow cytometry assessments. Embryonic stem-like colonies were immunopositive for pluripotency markers. Transition of spermatogonia cells to embryonic stem-like cells was accompanied by extensive changes in gene expression. These changes included significant increase in pluripotency genes expression and significant decrease in germ cell-specific genes expression. Also, we proved the differentiation capacity of embryonic stem-like cells to neuroepithelial-like cells which were immunoreactive to Nestin and Neurofilament 68. Evaluation of genes expression during in vitro differentiation into neuroepithelial-like cells showed high-level expression of Nestin whether this gene approximately has no expression in undifferentiated embryonic stem-like cells. Also, expression of pluripotency genes has significantly decreased in neuroepithelial-like cells compared with embryonic stem-like cells. This study shows that embryonic stem-like cells derived from testis are capable to differentiate into neuroepithelial-like cells that may provide a cellular reservoir usable for neurodegenerative disorders.     

Common and distinct factors regulate expression of mRNA for ETV5 and GDNF, Sertoli cell proteins essential for spermatogonial stem cell maintenance

Ets variant gene 5 (ETV5) and glial cell-derived neurotrophic factor (GDNF) are produced in Sertoli cells and required for maintenance and self-renewal of spermatogonial stem cells (SSCs) in mice. Fibroblast growth factors (FGFs) have been reported to stimulate Etv5 mRNA expression, and FSH was shown to stimulate Gdnf mRNA in Sertoli cell cultures, but there is no other information on factors that regulate these key Sertoli cell proteins necessary for stem cell maintenance. In this study, we investigated regulation of ETV5 and GDNF using the TM4 murine Sertoli cell line. FGF2 stimulated a time- and dose-dependent increase in Etv5 mRNA expression, with a maximal 8.3-fold increase at 6 h following 25 ng/ml FGF2 treatment. This FGF2 dose also stimulated Gdnf mRNA at 48 h. FGF2 effects on Etv5 and Gdnf mRNA were partially mediated through mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-signaling cascades. Specific inhibitors of MAPK (PD98059) and PI3K (wortmannin) pathways reduced Etv5 and Gdnf mRNA expression in FGF2-treated cells. Epidermal growth factor (EGF) stimulated Etv5 mRNA but not Gdnf mRNA. TNFalpha and IL-1beta stimulated Gdnf mRNA, but had no effect on Etv5 mRNA. Other hormonal regulators of Sertoli cells such as testosterone, triiodothyronine and activin A did not affect Etv5 or Gdnf mRNA expression. Results with primary Sertoli cell cultures confirmed findings obtained with the TM4 cell line, validating the use of the TM4 model to examine regulation of Etv5 and Gdnf mRNA expression. In conclusion, we have identified common and unique pathways that regulate Etv5 and Gdnf mRNA in Sertoli cells, and FGFs are emerging as key regulators of the Sertoli cell proteins that control SSCs.     

5-aza deoxycytidine-induced inhibition of differentiation of spermatogonia into spermatocytes in the mouse

In order to explore the significance of DNA methylation in proliferation and differentiation of germ cells in testis, 5-aza,2′-deoxycytidine (5-azaCdR), a hypomethylating agent, was administered in vivo to neonatal mice having only spermatogonial (premeiotic) cells. End-labeling of the Mspl, Hpall, and Hhal digested DNA revealed considerable loss of methylation following the treatment. Cellular and histological preparations of the testis showed complete inhibition of differentiation into spermatocytic stage. Analysis of protein synthesis in the treated and control testis by growing the cells in ³⁵S-Methionine medium and resolving the lysate by SDS-PAGE revealed that the programme of expression of at least 5 polypeptides (35.0, 31.5, 27.0, 22.5, and 18.0 KD) was altered as a result of 5-azaCdR incorporation. It appears that DNA methylation plays a critical role in the differentiation of gonia into primary spermatocytes. © 1995 wiley-Liss, Inc.     

Identification and IVC of spermatogonial stem cells in prepubertal buffaloes

Development of suitable selective marker for buffalo spermatogonial stem cells (SSCs), optimization of long-term IVC conditions, and their pluripotent retention capacity in buffaloes can be of prime importance in selective genetic modifications of this species. In the present study, we identified CDH1 as a specific marker for buffalo SSCs and revealed that it existed in two protein isoforms (large [135 kDa] and small [90 kDa] subunits) in the buffalo testis; furthermore, immunohistochemical analysis revealed that CDH1 expression was present in spermatogonia but absent in the somatic cells of 4-month-old buffalo testis. After 7 days of enrichment, expression of CDH1 was also detectable in IVC colonies (∼53% enrichment efficiency by Fluorescence-activated cell sorting (FACS)). For long-term culture of SSCs, proliferation studies with different factors showed that combination of 20 ng/mL GDNF, 10 ng/mL FGF2, and 1000 U/mL LIF could significantly promote number of colonies (∼two folds) and proliferation of buffalo SSCs (∼three folds) compared with those of control or single-treatment groups; furthermore, addition of these combination growth factors significantly upregulated the messenger RNA level of spermatogonial-specific and pluripotency-related markers (BCL6B, GFRA1, and POU5F1), whereas downregulated receptor tyrosine kinase (KIT). For confirmation of their stem cell potential, Dolichos biflorus agglutinin–stained cells were identified in the basal membrane of seminiferous tubules of xenotransplanted mice testis. These findings indicate the identification of a new buffalo SSCs marker; furthermore, it may help in establishing long-term culture that would assist in genetic modification of these buffaloes.