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Helicase POLQ‐like (HELQ) is a DNA helicase essential for the maintenance of genome stability. A recent study identified two HELQ missense mutations in some cases of infertile men. However, the functions of HELQ in the process of germline specification are not well known and whether its function is conserved between mouse and human remains unclear. Here, we revealed that Helq knockout (Helq−/−) could significantly reduce the efficiency of mouse primordial germ cell‐like cell (PGCLC) induction. In addition, Helq−/− embryonic bodies exhibited a severe apoptotic phenotype on day 6 of mouse PGCLC induction. p53 inhibitor treatment could partially rescue the generation of mouse PGCLCs from Helq mutant mouse embryonic stem cells. Finally, the genetic ablation of HELQ could also significantly impede the induction of human PGCLCs. Collectively, our study sheds light on the involvement of HELQ in the induction of both mouse and human PGCLCs, providing new insights into the mechanisms underlying germline differentiation and the genetic studies of human fertility.

Helicase POLQ‐like (HELQ) is a DNA helicase essential for the maintenance of genome stability. A recent study identified two HELQ missense mutations in some cases of infertile men. However, the functions of HELQ in the process of germline specification are not well known and whether its function is conserved between mouse and human remains unclear. Here, we revealed that Helq knockout (Helq−/−) could significantly reduce the efficiency of mouse primordial germ cell‐like cell (PGCLC) induction. In addition, Helq−/− embryonic bodies exhibited a severe apoptotic phenotype on day 6 of mouse PGCLC induction. p53 inhibitor treatment could partially rescue the generation of mouse PGCLCs from Helq mutant mouse embryonic stem cells. Finally, the genetic ablation of HELQ could also significantly impede the induction of human PGCLCs. Collectively, our study sheds light on the involvement of HELQ in the induction of both mouse and human PGCLCs, providing new insights into the mechanisms underlying germline differentiation and the genetic studies of human fertility. [ABSTRACT FROM AUTHOR]

Abstract In vitro gametogenesis (IVG) has been a topic of great interest in recent years not only because it allows for further exploration of mechanisms of germ cell development, but also because of its prospect for innovative medical applications especially for the treatment of infertility. Elucidation of the mechanisms underlying gamete development in vivo has inspired scientists to attempt to recapitulate the entire process of gametogenesis in vitro. While earlier studies have established IVG methods largely using pluripotent stem cells of embryonic origin, the scarcity of sources for these cells and the ethical issues involved in their use are serious limitations to the progress of IVG research especially in humans. However, with the emergence of induced pluripotent stem cells (iPSCs) due to the revolutionary discovery of dedifferentiation and reprogramming factors, IVG research has progressed remarkably in the last decade. This paper extensively reviews developments in IVG using iPSCs. First, the paper presents key concepts from groundwork studies on IVG including earlier researches demonstrating that IVG methods using embryonic stem cells (ESCs) also apply when using iPSCs. Techniques for the derivation of iPSCs are briefly discussed, highlighting the importance of generating transgene-free iPSCs with a high capacity for germline transmission to improve efficacy when used for IVG. The main part of the paper discusses recent advances in IVG research using iPSCs in various stages of gametogenesis. In addition, current clinical applications of IVG are presented, and potential future applications are discussed. Although IVG is still faced with many challenges in terms of technical issues, as well as efficacy and safety, novel IVG methodologies are emerging, and IVG using iPSCs may usher in the next era of reproductive medicine sooner than expected. This raises both ethical and social concerns and calls for the scientific community to cautiously develop IVG technology to ensure it is not only efficacious but also safe and adheres to social and ethical norms.

In vitro gametogenesis (IVG) has been a topic of great interest in recent years not only because it allows for further exploration of mechanisms of germ cell development, but also because of its prospect for innovative medical applications especially for the treatment of infertility. Elucidation of the mechanisms underlying gamete development in vivo has inspired scientists to attempt to recapitulate the entire process of gametogenesis in vitro. While earlier studies have established IVG methods largely using pluripotent stem cells of embryonic origin, the scarcity of sources for these cells and the ethical issues involved in their use are serious limitations to the progress of IVG research especially in humans. However, with the emergence of induced pluripotent stem cells (iPSCs) due to the revolutionary discovery of dedifferentiation and reprogramming factors, IVG research has progressed remarkably in the last decade. This paper extensively reviews developments in IVG using iPSCs. First, the paper presents key concepts from groundwork studies on IVG including earlier researches demonstrating that IVG methods using embryonic stem cells (ESCs) also apply when using iPSCs. Techniques for the derivation of iPSCs are briefly discussed, highlighting the importance of generating transgene-free iPSCs with a high capacity for germline transmission to improve efficacy when used for IVG. The main part of the paper discusses recent advances in IVG research using iPSCs in various stages of gametogenesis. In addition, current clinical applications of IVG are presented, and potential future applications are discussed. Although IVG is still faced with many challenges in terms of technical issues, as well as efficacy and safety, novel IVG methodologies are emerging, and IVG using iPSCs may usher in the next era of reproductive medicine sooner than expected. This raises both ethical and social concerns and calls for the scientific community to cautiously develop IVG technology to ensure it is not only efficacious but also safe and adheres to social and ethical norms. [ABSTRACT FROM AUTHOR]

According to estimations, approximately about 15% of couples worldwide suffer from infertility, in which individuals with azoospermia or oocyte abnormalities cannot be treated with assisted reproductive technology. The skin-derived stem cells (SDSCs) differentiation into primordial germ cell-like cells (PGCLCs) is one of the major breakthroughs in the field of stem cells intervention for infertility treatment in recent years. However, the cellular origin of SDSCs and their dynamic changes in transcription profile during differentiation into PGCLCs in vitro remain largely undissected. Here, the results of single-cell RNA sequencing indicated that porcine SDSCs are mainly derived from multipotent dermal fibroblast progenitors (MDFPs), which are regulated by growth factors (EGF/bFGF). Importantly, porcine SDSCs exhibit pluripotency for differentiating into three germ layers and can effectively differentiate into PGCLCs through complex transcriptional regulation involving histone modification. Moreover, this study also highlights that porcine SDSC-derived PGCLCs specification exhibit conservation with the human primordial germ cells lineage and that its proliferation is mediated by the MAPK signaling pathway. Our findings provide substantial novel insights into the field of regenerative medicine in which stem cells differentiate into germ cells in vitro, as well as potential therapeutic effects in individuals with azoospermia and/or defective oocytes. [ABSTRACT FROM AUTHOR]

In vitro differentiation of stem cells into functional gametes remains of great interest in the biomedical field. Skin‐derived stem cells (SDSCs) are an adult stem cells that provides a wide range of clinical applications without inherent ethical restrictions. In this paper, porcine SDSCs were successfully differentiated into primordial germ cell‐like cells (PGCLCs) in conditioned media. The PGCLCs were characterized in terms of cell morphology, marker gene expression, and epigenetic properties. Furthermore, we also found that 25 μM melatonin (MLT) significantly increased the proliferation of the SDSC‐derived PGCLCs while acting through the MLT receptor type 1 (MT1). RNA‐seq results found the mitogen‐activated protein kinase (MAPK) signaling pathway was more active when PGCLCs were cultured with MLT. Moreover, the effect of MLT was attenuated by the use of S26131 (MT1 antagonist), crenolanib (platelet‐derived growth factor receptor inhibitor), U0126 (mitogen‐activated protein kinase kinase inhibitor), or CCG‐1423 (serum response factor transcription inhibitor), suggesting that MLT promotes the proliferation processes through the MAPK pathway. Taken together, this study highlights the role of MLT in promoting PGCLCs proliferation. Importantly, this study provides a suitable in vitro model for use in translational studies and could help to answer numerous remaining questions related to germ cell physiology. [ABSTRACT FROM AUTHOR]

Parthenogenetic embryos, created by activation and diploidization of oocytes, arrest at mid-gestation for defective paternal imprints, which impair placental development. Also, viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells (pESCs) derived from parthenogenetic embryos, presumably attributable to their aberrant imprinting. We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring. Moreover, normal expression of imprinted genes is found in the germ cells and the mice. pESCs exhibited imprinting consistent with exclusively maternal lineage, and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background. pESCs differentiated into primordial germ cell-like cells (PGCLCs) and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function. The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs, consistent with efficient reprogramming of methylation and genomic imprinting. These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting, offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function. [ABSTRACT FROM AUTHOR]

Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency in vitro by forming embryonic germ (EG) cells and in vivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation in vivo establishes a latent form of pluripotency that restricts chimera contribution. [Display omitted] • Injected PGCs can survive, proliferate, and integrate within the host inner cell mass • ES cell lines can be derived from injected apoptosis-deficient PGCs • Apoptosis-deficient PGCs do not functionally contribute to later embryogenesis • In vitro -derived PGC-like cells can contribute to mid-gestation chimeras Sepulveda-Rincon et al. present a detailed investigation of the potency of primordial germ cells (PGCs). Following injection into early mouse embryos, PGCs can display properties akin to naive pluripotent cells, but they do not functionally contribute to later embryogenesis, supporting the notion that PGCs exhibit a latent form of pluripotency. [ABSTRACT FROM AUTHOR]

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells in vitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid ( RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein ( BMP) and RA synergistically induce primordial germ cells ( PGCs)/ PGC-like cells ( PGCLCs) derived from embryonic stem cells ( ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression in vivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/ PGCLCs, but not in PGCs/ PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans. [ABSTRACT FROM AUTHOR]

Different formative pluripotent stem cells harboring similar functional properties have been recently established to be lineage neutral and germline competent yet have distinct molecular identities. Here, we show that WNT/β-catenin signaling activation sustains transient mouse epiblast-like cells as epiblast-like stem cells (EpiLSCs). EpiLSCs display metastable formative pluripotency with bivalent cellular energy metabolism and unique transcriptomic features and chromatin accessibility. We develop single-cell stage label transfer (scSTALT) to study the formative pluripotency continuum and reveal that EpiLSCs recapitulate a unique developmental period in vivo , filling the gap of the formative pluripotency continuum between other published formative stem cells. WNT/β-catenin signaling activation counteracts differentiation effects of activin A and bFGF by preventing complete dissolution of naive pluripotency regulatory network. Moreover, EpiLSCs have direct competence toward germline specification, which is further matured by an FGF receptor inhibitor. Our EpiLSCs can serve as an in vitro model for mimicking and studying early post-implantation development and pluripotency transition. [Display omitted] • EpiLSCs are molecularly distinct from other formative pluripotency stem cells • Single-cell stage label transfer (scSTALT) enables integrated pseudotime analysis • WNT activation sustains metastable formative pluripotency of EpiLSCs • EpiLSCs have competence for germline induction with enhanced maturation by PD Luo et al. report mouse epiblast-like stem cells (EpiLSCs) displaying a metastable formative pluripotency and recapitulating a particular developmental period. EpiLSCs serve as an in vitro model for post-implantation development and pluripotency transition. [ABSTRACT FROM AUTHOR]

The generation of artificial gametes is a real challenge for the scientific community today. In vitro development of human eggs and sperm will pave the way for the understanding of the complex process of human gametogenesis and will provide with human gametes for the study of infertility and the onset of some inherited disorders. However, the great promise of artificial gametes resides in their future application on reproductive treatments for all these people wishing to have genetically related children and for which gamete donation is now their unique option of parenthood. This is the case of infertile patients devoid of suitable gametes, same sex couples, singles and those fertile couples in a high risk of transmitting serious diseases to their progeny. In the search of the best method to obtain artificial gametes, many researchers have successfully obtained human germ cell-like cells from stem cells at different stages of differentiation. In the near future, this field will evolve to new methods providing not only viable but also functional and safe artificial germ cells. These artificial sperm and eggs should be able to recapitulate all the genetic and epigenetic processes needed for the correct gametogenesis, fertilization and embryogenesis leading to the birth of a healthy and fertile newborn. [ABSTRACT FROM AUTHOR]