The generation of properly functioning gametes in vitro requires reconstitution of the multistepped pathway of germ cell development. We demonstrate here the generation of primordial germ cell-like cells (PGCLCs) in mice with robust capacity for spermatogenesis. PGCLCs were generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pregastrulating epiblasts but distinct from epiblast stem cells (EpiSCs). Reflecting epiblast development, EpiLC induction from ESCs/iPSCs is a progressive process, and EpiLCs highly competent for the PGC fate are a transient entity. The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs meticulously capture those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-β3 and SSEA1 as markers that allow the isolation of PGCLCs with spermatogenic capacity from tumorigenic undifferentiated cells. Our findings provide a paradigm for the first step of in vitro gametogenesis.
Embryonic stem cells (ESCs) are apparently homogeneous self-renewing cells, but we observed heterogeneous expression of Stella in ESCs, which is a marker of pluripotency and germ cells. Here we show that, whereas Stella-positive ESCs were like the inner cell mass (ICM), Stella-negative cells were like the epiblast cells. These states were interchangeable, which reflects the metastability and plasticity of ESCs. The established equilibrium was skewed reversibly in the absence of signals from feeder cells, which caused a marked shift toward an epiblast-like state, while trichostatin A, an inhibitor of histone deactelylase, restored Stella-positive population. The two populations also showed different histone modifications and striking functional differences, as judged by their potential for differentiation. The Stella-negative ESCs were more like the postimplantation epiblast-derived stem cells (EpiSCs), albeit the stella locus was repressed by DNA methylation in the latter, which signifies a robust epigenetic boundary between ESCs and EpiSCs.
Epigenetic modifications of histones regulate gene expression and chromatin structure. Here we show that Meisetz (meiosis-induced factor containing a PR/SET domain and zinc-finger motif) is a histone methyltransferase that is important for the progression of early meiotic prophase. Meisetz transcripts are detected only in germ cells entering meiotic prophase in female fetal gonads and in postnatal testis. Notably, Meisetz has catalytic activity for trimethylation, but not mono- or dimethylation, of lysine 4 of histone H3, and a transactivation activity that depends on its methylation activity. Mice in which the Meisetz gene is disrupted show sterility in both sexes due to severe impairment of the double-stranded break repair pathway, deficient pairing of homologous chromosomes and impaired sex body formation. In Meisetz-deficient testis, trimethylation of lysine 4 of histone H3 is attenuated and meiotic gene transcription is altered. These findings indicate that meiosis-specific epigenetic events in mammals are crucial for proper meiotic progression.
Reconstitution of female germ cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells and induced pluripotent stem cells in mice are induced into primordial germ cell-like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, undergo X-reactivation, imprint erasure, and cyst formation, and exhibit meiotic potential. Upon transplantation under mouse ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which then contribute to fertile offspring after in vitro maturation and fertilization. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ cell development in vitro.
Induction of mouse germ cells occurs from the proximal epiblast at around embryonic day (E) 7.0. These germ cells then migrate to, and enter the gonads at about E10.5 after which they undergo epigenetic reprogramming including erasure of parental imprints. However, the epigenetic properties acquired by nascent germ cells and the potential remodeling of these epigenetic marks in the subsequent migratory period have been largely unexplored. Here we have used immunohistochemistry to examine several genome-wide epigenetic modifications occurring in germ cells from their specification to their colonization of the genital ridges. We show that at around E8.0, germ cells concomitantly and significantly reduce H3-K9 dimethylation and DNA methylation, two major repressive modifications for gene expression. These events are preceded by the transient loss of all the DNA methyltransferases from their nuclei. By contrast, germ cells substantially increase the levels of H3-K27 trimethylation, another repressive modification with more plasticity, at E8.5-9.0 and maintain this state until at least E12.5. H3-K4 methylation and H3-K9 acetylation, modifications associated with transcriptionally permissive/active chromatin, are similar in germ and surrounding somatic cells but germ cells transiently increase these marks sharply upon their entry into the genital ridge. H3-K9 trimethylation, a hallmark of centromeric heterochromatin, is kept relatively constant during the periods examined. We suggest that this orderly and extensive epigenetic reprogramming in premigratory and migratory germ cells might be necessary for their reacquisition of underlying totipotency, for subsequent specific epigenetic remodeling, including the resetting of parental imprints, and for the production of gametes with an appropriate epigenotype for supporting normal development.
Genetic and epigenetic mechanisms regulate the transition from the totipotent zygote to pluripotent primitive ectoderm cells in the inner cell mass of mouse blastocysts. These pluripotent cells can be propagated indefinitely in vitro, underpinned by a unique epigenetic state. Following implantation of the blastocyst, diverse epigenetic modifiers control differentiation of pluripotent epiblast cells into somatic cells, while specification of germ cells requires repression of the somatic program. Regenerating totipotency during development of germ cells entails re-expression of pluripotency-specific genes and extensive erasure of epigenetic modifications. Increasing knowledge of key underlying mechanisms heightens prospects for creating pluripotent cells directly from adult somatic cells.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the gyrB gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63°C. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63°C. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of M. tuberculosis complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 l of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 l of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.Because of their slow growth rate, identification of mycobacteria is a notorious problem for public health and clinical laboratories. To address the need for rapid and sensitive identification of Mycobacterium tuberculosis and other mycobacteria, various genotyping methods for routine diagnosis have been introduced during the past decade (1,2,4,9,10,(21)(22)(23)25). The recent trend in genetic testing is to make systems fully automatic with high-throughput analysis. Although this may be an ideal approach, it requires expensive equipment as well as a huge amount of space in routine diagnostic laboratories. As a result, it centralizes genetic testing in highly sophisticated facilities. If this trend continues, escalated pressure for transportation of clinical specimens will pose another problem. On the other hand, there are pressing needs for point-of-care testing at hospitals and primary care facilities.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method which relies on autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment (13,(15)(16)(17)...
BackgroundMicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear.Methodology/Principal FindingsIn this study, we first revealed the expression pattern of miRNAs by using a real-time PCR-based 220-plex miRNA expression profiling method. During development of germ cells, miR-17-92 cluster, which is thought to promote cell cycling, and the ES cell-specific cluster encoding miR-290 to -295 (miR-290-295 cluster) were highly expressed in primordial germ cells (PGCs) and spermatogonia. A set of miRNAs was developmentally regulated. We next analysed function of miRNA biogenesis in germ cell development by using conditional Dicer-knockout mice in which Dicer gene was deleted specifically in the germ cells. Dicer-deleted PGCs and spermatogonia exhibited poor proliferation. Retrotransposon activity was unexpectedly suppressed in Dicer-deleted PGCs, but not affected in the spermatogonia. In Dicer-deleted testis, spermatogenesis was retarded at an early stage when proliferation and/or early differentiation. Additionally, we analysed spermatogenesis in conditional Argonaute2-deficient mice. In contrast to Dicer-deficient testis, spermatogenesis in Argonaute2-deficient testis was indistinguishable from that in wild type.Conclusion/SignificanceThese results illustrate that miRNAs are important for the proliferation of PGCs and spermatogonia, but dispensable for the repression of retrotransposons in developing germ cells. Consistently, miRNAs promoting cell cycling are highly expressed in PGCs and spermatogonia. Furthermore, based on normal spermatogenesis in Argonaute2-deficient testis, the critical function of Dicer in spermatogenesis is independent of Argonaute2.
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