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Spermatogonial stem cells (SSCs) are the foundation of life-long spermatogenesis. While SSC research has advanced greatly over the past two decades, characterization of SSCs during postnatal development has not been well documented. Using the mouse as a model, in this study, we defined the immunophenotypic profiles of testis cells during the course of postnatal development using multi-parameter flow cytometry with up to five cell-surface antigens. We found that the profiles progress over time in a manner specific to developmental stages. We then isolated multiple cell fractions at different developmental stages using fluorescent-activated cell sorting (FACS) and identified specific cell populations with prominent capacities to regenerate spermatogenesis upon transplantation and to initiate long-term SSC culture. The data indicated that the cell fraction with the highest level of regeneration capacity exhibited the most prominent potential to initiate SSC culture, regardless of age. Interestingly, refinement of cell fractionation using GFRA1 and KIT did not lead to further enrichment of regenerative and culture-initiating stem cells, suggesting that when a high degree of SSC enrichment is achieved, standard markers of SSC self-renewal or commitment may lose their effectiveness to distinguish cells at the stem cell state from committed progenitors. This study provides a significant information resource for future studies and practical applications of mammalian SSCs.

Plasticizers give flexibility to a wide range of consumer and medical plastic products. Among them, phthalate esters are recognized as endocrine disruptors that target male reproductive functions. With this notion, past studies designed and produced alternative plasticizers that could replace phthalates with limited toxicity to the environment and to male reproductive functions. Here, we focused on one reproductive cell type that was not investigated in past studies—spermatogonial stem cells (SSCs)—and examined in vitro the effects on 22 compounds (seven plasticizers currently in use and 15 newly synthesized potential alternative plasticizers) for their effects on SSCs. Our in vitro compound screening analyses showed that a majority of the compounds examined had a limited level of toxicity to SSCs. Yet, some commercial plasticizers and their derivatives, such as DEHP (di-(2-ethylhexyl) phthalate) and MEHP (mono-(2-ethylhexyl) phthalate), were detrimental at 10−5 to 10−4 M. Among new compounds, some of maleate- and fumarate-derivatives showed toxic effects. In contrast, no detrimental effects were detected with two new compounds, BDDB (1,4 butanediol dibenzoate) and DOS (dioctyl succinate). Furthermore, SSCs that were exposed to BDDB and DOS in vitro successfully established spermatogenic colonies in testes of recipient mice after transplantation. These results demonstrate that SSC culture acts as an effective platform for toxicological tests on SSC function and provide novel information that two new compounds, BDDB and DOS, are alternative plasticizers that do not have significant negative impacts on SSC integrity.

Spermatogenesis requires that a careful balance be maintained between the self-renewal of spermatogonial stem cells (SSCs) and their commitment to the developmental pathway through which they will differentiate into spermatozoa. Recently, a series of studies employing various in vivo and in vitro models have suggested a role of the wingless-related MMTV integration site gene family/beta-catenin (WNT/CTNNB1) pathway in determining the fate of SSCs. However, conflicting data have suggested that CTNNB1 signaling may either promote SSC self-renewal or differentiation. Here, we studied the effects of sustained CTNNB1 signaling in SSCs using the Ctnnb1 tm1Mmt/+ ; Ddx4 -Cre Tr/+ (Δ Ctnnb1 ) mouse model, in which a stabilized form of CTNNB1 is expressed in all germ cells. Δ Ctnnb1 mice were found to have reduced testis weights and partial germ cell loss by 4 months of age. Germ cell transplantation assays showed a 49% reduction in total functional SSC numbers in 8 month-old transgenic mice. In vitro , Thy 1-positive undifferentiated spermatogonia from Δ Ctnnb1 mice formed 57% fewer clusters, which was associated with decreased cell proliferation. A reduction in mRNA levels of genes associated with SSC maintenance ( Bcl6b , Gfra1 , Plzf ) and increased levels for markers associated with progenitor and differentiating spermatogonia ( Kit , Rarg , Sohlh1 ) were detected in these cluster cells. Furthermore, RNAseq performed on these clusters revealed a network of more than 900 genes regulated by CTNNB1, indicating that CTNNB1 is an important regulator of spermatogonial fate. Together, our data support the notion that CTNNB1 signaling promotes the transition of SSCs to undifferentiated progenitor spermatogonia at the expense of their self-renewal.

Proper regulation of spermatogonial stem cells (SSCs) is crucial for sustaining steady-state spermatogenesis. Previous work has identified several paracrine factors involved in this regulation, in particular, glial cell line-derived neurotrophic factor and fibroblast growth factor 2, which promote long-term SSC self-renewal. Using a SSC culture system, we have recently reported that Wnt5a promotes SSC self-renewal through a β-catenin-independent Wnt mechanism whereas the β-catenin-dependent Wnt pathway is not active in SSCs. In contrast, another study has reported that Wnt3a promotes SSC self-renewal through the β-catenin-dependent pathway, as it can stimulate the proliferation of a spermatogonia cell line. To reconcile these two contradictory reports, we assessed Wnt3a effects on SSCs and progenitor cells, rather than a cell line, in vitro. We observed that Wnt3a induced β-catenin-dependent signalling in a large subset of germ cells and increased SSC numbers. However, further investigation revealed that cell populations with greater β-catenin-signalling activity contained fewer SSCs. The increased maintenance of SSCs by Wnt3a coincided with more active cell cycling and the formation of germ cell aggregates, or communities, under feeder-free conditions. Therefore, the results of this study suggest that Wnt3a selectively stimulates proliferation of progenitors that are committed to differentiation or are in the process of exiting the SSC state, leading to enhanced formation of germ cell communities, which indirectly support SSCs and act as an in vitro niche.

The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

Constitutive activation of the WNT signaling effector CTNNB1 (β-catenin) in the Sertoli cells of the Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) mouse model results in progressive germ cell loss and sterility. In this study, we sought to determine if this phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) and wild-type mice showed that SSC activity is lost in Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) testes over time, whereas the mutant testes could not support colonization by wild-type SSCs. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway, which was also found to occur in Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) testes. One CTNNB1 target gene encoded the secreted signaling molecule WNT4. We therefore tested the effects of WNT4 on SSC-enriched germ cell cultures, and found that WNT4 induced cell death and reduced SSC activity without affecting cell cycle. Conversely, conditional inactivation of Wnt4 in the Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Furthermore, WNT4 was found to signal via the CTNNB1 pathway in Sertoli cells, suggesting a self-reinforcing positive feedback loop. Collectively, these data indicate for the first time that ectopic activation of a signaling cascade in the stem cell niche depletes SSC activity through a paracrine factor. These findings may provide insight into the pathogenesis of male infertility, as well as embryonic gonadal development.

Proper regulation of spermatogonial stem cells (SSCs) is crucial for sustaining steady-state spermatogenesis. Previous work has identified several paracrine factors involved in this regulation, in particular, glial cell line-derived neurotrophic factor and fibroblast growth factor 2, which promote long-term SSC self-renewal. Using a SSC culture system, we have recently reported that Wnt5a promotes SSC self-renewal through a [beta]-catenin-independent Wnt mechanism whereas the [beta]-catenin-dependent Wnt pathway is not active in SSCs. In contrast, another study has reported that Wnt3a promotes SSC self-renewal through the [beta]-catenin-dependent pathway, as it can stimulate the proliferation of a spermatogonia cell line. To reconcile these two contradictory reports, we assessed Wnt3a effects on SSCs and progenitor cells, rather than a cell line, in vitro. We observed that Wnt3a induced [beta]-catenin-dependent signalling in a large subset of germ cells and increased SSC numbers. However, further investigation revealed that cell populations with greater [beta]-catenin-signalling activity contained fewer SSCs. The increased maintenance of SSCs by Wnt3a coincided with more active cell cycling and the formation of germ cell aggregates, or communities, under feeder-free conditions. Therefore, the results of this study suggest that Wnt3a selectively stimulates proliferation of progenitors that are committed to differentiation or are in the process of exiting the SSC state, leading to enhanced formation of germ cell communities, which indirectly support SSCs and act as an in vitro niche.

Black phosphorus (BP) is a promising two-dimensional layered semiconductor material for next-generation electronics and optoelectronics, with a thickness-dependent tunable direct bandgap and high carrier mobility. Though great research advantages have been achieved on BP, lateral synthesis of high quality BP films still remains a great challenge. Here, we report the direct growth of large-scale crystalline BP films on insulating silicon substrates by a gas-phase growth strategy with an epitaxial nucleation design and a further lateral growth control. The optimized lateral size of the achieved BP films can reach up to millimeters, with the ability to modulate thickness from a few to hundreds of nanometers. The as-grown BP films exhibit excellent electrical properties, with a field-effect and Hall mobility of over 1200 cm 2 V −1 s −1 and 1400 cm 2 V −1 s −1 at room temperature, respectively, comparable to those exfoliated from BP bulk crystals. Our work opens the door for broad applications with BP in scalable electronic and optoelectronic devices. Direct synthesis of large area crystalline black phosphorus films is still challenging. Here, the authors report growth of high-quality black phosphorus films on insulating silicon substrates through a gas-phase epitaxial growth strategy with field-effect and Hall mobilities of over 1200 and 1400 cm2 /Vs at room temperature, respectively and a current on/off ratio of up to 10 6 , comparable to the exfoliated flakes.

Relatively low mobility and thermal conductance create challenges for application of tungsten diselenide (WSe 2 ) in high performance devices. Dielectric interface is of extremely importance for improving carrier transport and heat spreading in a semiconductor device. Here, by near-equilibrium plasma-enhanced chemical vapour deposition, we realize catalyst-free growth of poly-crystalline two-dimensional hexagonal-boron nitride (2D-BN) with domains around 20~ 200 nm directly on SiO 2 /Si, quartz, sapphire, silicon or SiO 2 /Si with three-dimensional patterns at 300 °C. Owing to the atomically-clean van-der-Walls conformal interface and the fact that 2D-BN can better bridge the vibrational spectrum across the interface and protect interfacial heat conduction against substrate roughness, both improved performance and thermal dissipation of WSe 2 field-effect transistor are realized with mobility around 56~ 121 cm 2  V −1 s −1 and saturated power intensity up to 4.23 × 10 3  W cm −2 . Owing to its simplicity, conformal growth on three-dimensional surface, compatibility with microelectronic process, it has potential for application in future two-dimensional electronics. Plasma-enhanced chemical vapour deposition (PECVD) is an industrially compatible microelectronics technology. Here, the authors use PECVD to obtain low-temperature, catalyst-free growth of poly-crystalline two-dimensional hexagonal-boron nitride, thus enabling superior thermal dissipation in WSe 2 field-effect transistors with mobility up to 121 cm 2  V −1 s −1 .

O-GlcNAcylation has been implicated in the tumorigenesis of various tissue origins, but its function in liver tumorigenesis is not clear. Here, we demonstrate that O-GlcNAcylation can enhance the expression, stability and function of Yes-associated protein (YAP), the downstream transcriptional regulator of the Hippo pathway and a potent oncogenic factor in liver cancer. O-GlcNAcylation induces transformative phenotypes of liver cancer cells in a YAP-dependent manner. An O-GlcNAc site of YAP was identified at Thr241, and mutating this site decreased the O-GlcNAcylation, stability, and pro-tumorigenic capacities of YAP, while increasing YAP phosphorylation. Importantly, we found via in vitro cell-based and in vivo mouse model experiments that O-GlcNAcylation of YAP was required for high-glucose-induced liver tumorigenesis. Interestingly, a positive feedback between YAP and global cellular O-GlcNAcylation is also uncovered. We conclude that YAP O-GlcNAcylation is a potential therapeutic intervention point for treating liver cancer associated with high blood glucose levels and possibly diabetes. Yap is a transcriptional factor involved in tumorigenesis. Here the authors show that a previously unknown post-translational modification of Yap, O-GlcNAcylation, increases its transcriptional activity and is required for high glucose-induced liver cancer development.