SDHAF1 confers metabolic resilience to aging hematopoietic stem cells by promoting mitochondrial ATP production.

Aging generally predisposes stem cells to functional decline, impairing tissue homeostasis. Here, we report that hematopoietic stem cells (HSCs) acquire metabolic resilience that promotes cell survival. High-resolution real-time ATP analysis with glucose tracing and metabolic flux analysis revealed that old HSCs reprogram their metabolism to activate the pentose phosphate pathway (PPP), becoming more resistant to oxidative stress and less dependent on glycolytic ATP production at steady state. As a result, old HSCs can survive without glycolysis, adapting to the physiological cytokine environment in bone marrow. Mechanistically, old HSCs enhance mitochondrial complex II metabolism during stress to promote ATP production. Furthermore, increased succinate dehydrogenase assembly factor 1 (SDHAF1) in old HSCs, induced by physiological low-concentration thrombopoietin (TPO) exposure, enables rapid mitochondrial ATP production upon metabolic stress, thereby improving survival. This study provides insight into the acquisition of resilience through metabolic reprogramming in old HSCs and its molecular basis to ameliorate age-related hematopoietic abnormalities.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Oocyte aging in comparison to stem cells in mice.

To maintain homeostasis, many tissues contain stem cells that can self-renew and differentiate. Based on these functions, stem cells can reconstitute the tissue even after injury. In reproductive organs, testes have spermatogonial stem cells that generate sperm in men throughout their lifetime. However, in the ovary, oocytes enter meiosis at the embryonic stage and maintain sustainable oogenesis in the absence of stem cells. After birth, oocytes are maintained in a dormant state in the primordial follicle, which is the most premature follicle in the ovary, and some are activated to form mature oocytes. Thus, regulation of dormancy and activation of primordial follicles is critical for a sustainable ovulatory cycle and is directly related to the female reproductive cycle. However, oocyte storage is insufficient to maintain a lifelong ovulation cycle. Therefore, the ovary is one of the earliest organs to be involved in aging. Although stem cells are capable of proliferation, they typically exhibit slow cycling or dormancy. Therefore, there are some supposed similarities with oocytes in primordial follicles, not only in their steady state but also during aging. This review aims to summarise the sustainability of oogenesis and aging phenotypes compared to tissue stem cells. Finally, it focuses on the recent breakthroughs in vitro culture and discusses future prospects.

Generation of functional oocytes from male mice in vitro.

Sex chromosome disorders severely compromise gametogenesis in both males and females. In oogenesis, the presence of an additional Y chromosome or the loss of an X chromosome disturbs the robust production of oocytes1-5. Here we efficiently converted the XY chromosome set to XX without an additional Y chromosome in mouse pluripotent stem (PS) cells. In addition, this chromosomal alteration successfully eradicated trisomy 16, a model of Down's syndrome, in PS cells. Artificially produced euploid XX PS cells differentiated into mature oocytes in culture with similar efficiency to native XX PS cells. Using this method, we differentiated induced pluripotent stem cells from the tail of a sexually mature male mouse into fully potent oocytes, which gave rise to offspring after fertilization. This study provides insights that could ameliorate infertility caused by sex chromosome or autosomal disorders, and opens the possibility of bipaternal reproduction.

Dual role of Ovol2 on the germ cell lineage segregation during gastrulation in mouse embryogenesis.

In mammals, primordial germ cells (PGCs), the origin of the germ line, are specified from the epiblast at the posterior region where gastrulation simultaneously occurs, yet the functional relationship between PGC specification and gastrulation remains unclear. Here, we show that OVOL2, a transcription factor conserved across the animal kingdom, balances these major developmental processes by repressing the epithelial-to-mesenchymal transition (EMT) that drives gastrulation and the upregulation of genes associated with PGC specification. Ovol2a, a splice variant encoding a repressor domain, directly regulates EMT-related genes and, consequently, induces re-acquisition of potential pluripotency during PGC specification, whereas Ovol2b, another splice variant missing the repressor domain, directly upregulates genes associated with PGC specification. Taken together, these results elucidate the molecular mechanism underlying allocation of the germ line among epiblast cells differentiating into somatic cells through gastrulation. This article has an associated 'The people behind the papers' interview.

Generation of ovarian follicles from mouse pluripotent stem cells.

Oocytes mature in a specialized fluid-filled sac, the ovarian follicle, which provides signals needed for meiosis and germ cell growth. Methods have been developed to generate functional oocytes from pluripotent stem cell-derived primordial germ cell-like cells (PGCLCs) when placed in culture with embryonic ovarian somatic cells. In this study, we developed culture conditions to recreate the stepwise differentiation process from pluripotent cells to fetal ovarian somatic cell-like cells (FOSLCs). When FOSLCs were aggregated with PGCLCs derived from mouse embryonic stem cells, the PGCLCs entered meiosis to generate functional oocytes capable of fertilization and development to live offspring. Generating functional mouse oocytes in a reconstituted ovarian environment provides a method for in vitro oocyte production and follicle generation for a better understanding of mammalian reproduction.

Regulation of primordial follicle formation, dormancy, and activation in mice.

In female reproduction, the oocyte number is limited after birth. To achieve a continuous ovulatory cycle, oocytes are stored in primordial follicles. Therefore, the regulation of primordial follicle dormancy and activation is important for reproductive sustainability, and its collapse leads to premature ovarian insufficiency. In this review, we summarize primordial follicle development and the molecular mechanisms underlying primordial follicle maintenance and activation in mice. We also overview the mechanisms discovered through in vitro culture of functional oocytes, including the establishment of primordial follicle induction by environmental factors, which revealed the importance of hypoxia and compression by the extra cellular matrix (ECM) for primordial follicle maintenance in vivo.

D-Tryptophan enhances the reproductive organ-specific expression of the amino acid transporter homolog Dr-SLC38A9 involved in the sexual induction of planarian Dugesia ryukyuensis.

BACKGROUND: Many animals switch between asexual and sexual reproduction in nature. We previously established a system for the sexual induction of planarian Dugesia ryukyuensis by feeding asexual planarians with minced sexual planarians. We identified DL-tryptophan (Trp) as one of the sex-inducing substances. DL-Trp can induce ovarian development, the first and essential step of sexual induction. D-Trp must act as a principal bioactive compound in terms of ovarian development, because the ovary-inducing activity of D-Trp was 500 times more potent than that of L-Trp. However, how Trp controls sexual induction is still unknown. RESULTS: In this study, qRT-PCR analyses suggested that the putative amino acid transporter gene Dr-SLC38A9 is highly expressed in sexual worms, especially in the yolk glands. In situ hybridization analyses showed that Dr-SLC38A9 is expressed in the ovarian primordia of asexual worms and in the mature ovaries, testes, and yolk glands of sexual worms. In addition, Dr-SLC38A9 RNA interference during sexual induction resulted in the suppression of the development of reproductive organs. These results suggest that Dr-SLC38A9 is involved in the development of these organs. Moreover, we demonstrated that the reproductive organ-specific expression of Dr-SLC38A9 is enhanced by the addition of D-Trp. CONCLUSION: We propose that D-Trp activates the expression of Dr-SLC38A9 to promote sexual induction in the planarian D. ryukyuensis.

Germ cell-intrinsic effects of sex chromosomes on early oocyte differentiation in mice.

A set of sex chromosomes is required for gametogenesis in both males and females, as represented by sex chromosome disorders causing agametic phenotypes. Although studies using model animals have investigated the functional requirement of sex chromosomes, involvement of these chromosomes in gametogenesis remains elusive. Here, we elicit a germ cell-intrinsic effect of sex chromosomes on oogenesis, using a novel culture system in which oocytes were induced from embryonic stem cells (ESCs) harboring XX, XO or XY. In the culture system, oogenesis using XO and XY ESCs was severely disturbed, with XY ESCs being more strongly affected. The culture system revealed multiple defects in the oogenesis of XO and XY ESCs, such as delayed meiotic entry and progression, and mispairing of the homologous chromosomes. Interestingly, Eif2s3y, a Y-linked gene that promotes proliferation of spermatogonia, had an inhibitory effect on oogenesis. This led us to the concept that male and female gametogenesis appear to be in mutual conflict at an early stage. This study provides a deeper understanding of oogenesis under a sex-reversal condition.

Environmental factors for establishment of the dormant state in oocytes.

Guaranteeing the sustainability of gametogenesis is a fundamental issue for perpetuating the species. In the mammalian ovary, sustainability is accomplished by keeping a number of oocytes "stocked" in the dormant state. Despite the evident importance of this state, the mechanisms underlying the oocyte dormancy are not fully understood, although it is presumed that both intrinsic and extrinsic factors are involved. Here, we review environmental factors involved in the regulation of oocyte dormancy. Consideration of the environmental factors illustrates the nature of the ovarian compartment, in which primordial follicles reside. This should greatly improve our understanding of the mechanisms and also assist in reconstitution of the dormant state in culture. Accumulating knowledge on the dormant state of oocytes will contribute to a wide range of research in fields such as developmental biology, reproductive biology and regenerative medicine.

Lack of whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (WFDC2) causes neonatal death from respiratory failure in mice.

Respiratory failure is a life-threatening problem for pre-term and term infants, yet many causes remain unknown. Here, we present evidence that whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (Wfdc2), a protease inhibitor previously unrecognized in respiratory disease, may be a causal factor in infant respiratory failure. Wfdc2 transcripts are detected in the embryonic lung and analysis of a Wfdc2-GFP knock-in mouse line shows that both basal and club cells, and type II alveolar epithelial cells (AECIIs), express Wfdc2 neonatally. Wfdc2-null-mutant mice display progressive atelectasis after birth with a lethal phenotype. Mutant lungs have multiple defects, including impaired cilia and the absence of mature club cells from the tracheo-bronchial airways, and malformed lamellar bodies in AECIIs. RNA sequencing shows significant activation of a pro-inflammatory pathway, but with low-quantity infiltration of mononuclear cells in the lung. These data demonstrate that Wfdc2 function is vitally important for lung aeration at birth and that gene deficiency likely causes failure of the lung mucosal barrier.

Generation of Nanog reporter mice that distinguish pluripotent stem cells from unipotent primordial germ cells.

Nanog is a core transcription factor specifically expressed not only in the pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced PSCs (iPSCs), but also in the unipotent primordial germ cells (PGCs). Although Nanog promoter/enhancer regions are well characterized by in vitro analyses, direct correlations between the regulatory elements for Nanog expression and in vivo expression patterns of Nanog have not been fully clarified. In this study, we generated Nanog-RFP transgenic (Tg) mice in which expression of red fluorescent protein (RFP) is driven by a 5.2 kb Nanog promoter/enhancer region. As expected, RFP was expressed in the inner cell mass of blastocysts, ESCs, and iPSCs. However, RFP fluorescence was not observed in PGCs, although Nanog was expressed in PGCs. Because RFP fluorescence was visible in the PGC-derived pluripotent EGCs in culture, it was suggested that the reporter gene expression was specifically activated in PSCs. In conclusion, we have generated a novel Nanog-RFP Tg mouse line that can selectively tag PSCs over unipotent PGCs.

Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes.

The most immature oocytes remain dormant in primordial follicles in the ovary, ensuring the longevity of female reproductive life. Despite its biological and clinical importance, knowledge of mechanisms regulating the dormant state remains limited. Here, we show that mechanical stress plays a key role in maintaining the dormant state of the oocytes in primordial follicles in mice. Transcriptional and histological analyses revealed that oocytes were compressed by surrounding granulosa cells with extracellular matrix. This environmental state is functionally crucial, as oocytes became activated upon loosening the structure and the dormancy was restored by additional compression with exogenous pressure. The nuclei of oocytes in primordial follicles rotated in response to the mechanical stress. Pausing the rotation triggered activation of oocytes through nuclear export of forkhead box O3 (FOXO3). These results provide insights into the mechanisms by which oocytes are kept dormant to sustain female reproductive life.

Hypoxia induces the dormant state in oocytes through expression of Foxo3.

In mammals, most immature oocytes remain dormant in the primordial follicles to ensure the longevity of female reproductive life. A precise understanding of mechanisms underlying the dormancy is important for reproductive biology and medicine. In this study, by comparing mouse oogenesis in vivo and in vitro, the latter of which bypasses the primordial follicle stage, we defined the gene-expression profile representing the dormant state of oocytes. Overexpression of constitutively active FOXO3 partially reproduced the dormant state in vitro. Based on further gene-expression analysis, we found that a hypoxic condition efficiently induced the dormant state in vitro. The effect of hypoxia was severely diminished by disruption of the Foxo3 gene and inhibition of hypoxia-inducible factors. Our findings provide insights into the importance of environmental conditions and their effectors for establishing the dormant state.

Stem cells, in vitro gametogenesis and male fertility.

Reconstitution in culture of biological processes, such as differentiation and organization, is a key challenge in regenerative medicine, and one in which stem cell technology plays a central role. Pluripotent stem cells and spermatogonial stem cells are useful materials for reconstitution of germ cell development in vitro, as they are capable of differentiating into gametes. Reconstitution of germ cell development, termed in vitro gametogenesis, will provide an experimental platform for a better understanding of germ cell development, as well as an alternative source of gametes for reproduction, with the potential to cure infertility. Since germ cells are the cells for 'the next generation', both the culture system and its products must be carefully evaluated. In this issue, we summarize the progress in in vitro gametogenesis, most of which has been made using mouse models, as well as the future challenges in this field.

PRDM14 Drives OCT3/4 Recruitment via Active Demethylation in the Transition from Primed to Naive Pluripotency.

Primordial germ cells (PGCs) are specified from epiblast cells in mice. Genes associated with naive pluripotency are repressed in the transition from inner cell mass to epiblast cells, followed by upregulation after PGC specification. However, the molecular mechanisms underlying the reactivation of pluripotency genes are poorly characterized. Here, we exploited the in vitro differentiation of epiblast-like cells (EpiLCs) from embryonic stem cells (ESCs) to elucidate the molecular and epigenetic functions of PR domain-containing 14 (PRDM14). We found that Prdm14 overexpression in EpiLCs induced their conversion to ESC-like cells even in the absence of leukemia inhibitory factor in adherent culture. This was impaired by the loss of Kruppel-like factor 2 and ten-eleven translocation (TET) proteins. Furthermore, PRDM14 recruited OCT3/4 to the enhancer regions of naive pluripotency genes via TET-base excision repair-mediated demethylation. Our results provide evidence that PRDM14 establishes a transcriptional network for naive pluripotency via active DNA demethylation.

Reconstitution in vitro of the entire cycle of the mouse female germ line.

The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.

p38α Activates Purine Metabolism to Initiate Hematopoietic Stem/Progenitor Cell Cycling in Response to Stress.

Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. We do not yet have a clear understanding of how this metabolic activity changes during stress hematopoiesis, such as bone marrow transplantation. Here, we report a critical role for the p38MAPK family isoform p38α in initiating hematopoietic stem and progenitor cell (HSPC) proliferation during stress hematopoiesis in mice. We found that p38MAPK is immediately phosphorylated in HSPCs after a hematological stress, preceding increased HSPC cycling. Conditional deletion of p38α led to defective recovery from hematological stress and a delay in initiation of HSPC proliferation. Mechanistically, p38α signaling increases expression of inosine-5'-monophosphate dehydrogenase 2 in HSPCs, leading to altered levels of amino acids and purine-related metabolites and changes in cell-cycle progression in vitro and in vivo. Our studies have therefore uncovered a p38α-mediated pathway that alters HSPC metabolism to respond to stress and promote recovery.

Integrative Analysis of the Acquisition of Pluripotency in PGCs Reveals the Mutually Exclusive Roles of Blimp-1 and AKT Signaling.

Primordial germ cells (PGCs) are lineage-restricted unipotent cells that can dedifferentiate into pluripotent embryonic germ cells (EGCs). Here we performed whole-transcriptome analysis during the conversion of PGCs into EGCs, a process by which cells acquire pluripotency. To examine the molecular mechanism underlying this conversion, we focused on Blimp-1 and Akt, which are involved in PGC specification and dedifferentiation, respectively. Blimp-1 overexpression in embryonic stem cells suppressed the expression of downstream targets of the pluripotency network. Conversely, Blimp-1 deletion in PGCs accelerated their dedifferentiation into pluripotent EGCs, illustrating that Blimp-1 is a pluripotency gatekeeper protein in PGCs. AKT signaling showed a synergistic effect with basic fibroblast growth factor plus 2i+A83 treatment on EGC formation. AKT played a major role in suppressing genes regulated by MBD3. From these results, we defined the distinct functions of Blimp-1 and Akt and provided mechanistic insights into the acquisition of pluripotency in PGCs.

Telomerase reverse transcriptase has an extratelomeric function in somatic cell reprogramming.

Reactivation of the endogenous telomerase reverse transcriptase (TERT) catalytic subunit and telomere elongation occur during the reprogramming of somatic cells to induced pluripotent stem (iPS) cells. However, the role of TERT in the reprogramming process is unclear. To clarify its function, the reprogramming process was examined in TERT-KO somatic cells. To exclude the effect of telomere elongation, tail-tip fibroblasts (TTFs) from first generation TERT-KO mice were used. Although iPS cells were successfully generated from TERT-KO TTFs, the efficiency of reprogramming these cells was markedly lower than that of WT TTFs. The gene expression profiles of iPS cells induced from TERT-KO TTFs were similar to those of WT iPS cells and ES cells, and TERT-KO iPS cells formed teratomas that differentiated into all three germ layers. These data indicate that TERT plays an extratelomeric role in the reprogramming process, but its function is dispensable. However, TERT-KO iPS cells showed transient defects in growth and teratoma formation during continuous growth. In addition, TERT-KO iPS cells developed chromosome fusions that accumulated with increasing passage numbers, consistent with the fact that TERT is essential for the maintenance of genome structure and stability in iPS cells. In a rescue experiment, an enzymatically inactive mutant of TERT (D702A) had a positive effect on somatic cell reprogramming of TERT-KO TTFs, which confirmed the extratelomeric role of TERT in this process.