• spermatogenesis
• pluripotency
• meiosis

• Animals
• Male
• Spermatogenesis/genetics
• Mice
• Wnt Signaling Pathway/genetics
• Mice, Knockout
• Cell Differentiation/genetics
• Germ Cells/metabolism
• Germ Cells/cytology
• Embryonic Stem Cells/metabolism
• Embryonic Stem Cells/cytology
• Testis/metabolism
• Testis/cytology
• Gene Expression Regulation, Developmental
• Spermatocytes/metabolism
• Spermatocytes/cytology
• Histones/metabolism
• Promoter Regions, Genetic

• C32583GG New York State Stem Cell Science (NYSTEM)
• R21 HD106263 NICHD NIH HHS
• R01 HD095938 NICHD NIH HHS
• R35 GM154906 NIGMS NIH HHS
• S10 OD020056 NIH HHS
• R01 GM129157 NIGMS NIH HHS
• UL1 TR001873 NCATS NIH HHS
• HD095938 U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)
• GM129157 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)
• S10 RR027050 NCRR NIH HHS
• R01 HD097268 NICHD NIH HHS
• HD097268 U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)
• C32569GG New York State Stem Cell Science (NYSTEM)
• R01 HD114122 NICHD NIH HHS
• P30 CA013696 NCI NIH HHS
• U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)
• U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)

• Decellularization
• Extracellular matrix
• Ovarian organoid
• Pluripotent stem cells
• Synthetic matrices

• Organoids/cytology
• Organoids/metabolism
• Humans
• Female
• Ovary/cytology
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/metabolism
• Cell Differentiation
• Regenerative Medicine/methods
• Extracellular Matrix/metabolism
• Tissue Engineering/methods
• Animals
• Tissue Scaffolds/chemistry

• BJ-2021-236 National High Level Hospital Clinical Research Funding
• BJ-2023-182 National High Level Hospital Clinical Research Funding

• Humans
• Single-Cell Analysis/methods
• Transcriptome
• Cell Line, Tumor
• Male
• Drug Resistance, Neoplasm/genetics
• Neoplasms/genetics
• Neoplasms/drug therapy
• Neoplasms/pathology
• Gene Expression Profiling/methods
• Prostatic Neoplasms, Castration-Resistant/genetics
• Prostatic Neoplasms, Castration-Resistant/drug therapy
• Prostatic Neoplasms, Castration-Resistant/pathology
• Tumor Microenvironment/genetics
• Antineoplastic Agents/pharmacology
• Rhabdomyosarcoma/genetics
• Rhabdomyosarcoma/drug therapy
• Rhabdomyosarcoma/pathology
• Carcinoma, Pancreatic Ductal/genetics
• Carcinoma, Pancreatic Ductal/drug therapy
• Carcinoma, Pancreatic Ductal/pathology
• Pancreatic Neoplasms/genetics
• Pancreatic Neoplasms/drug therapy
• Pancreatic Neoplasms/pathology
• Sequence Analysis, RNA/methods
• RNA-Seq

• R01 CA229618 NCI NIH HHS
• R01 CA204856 NCI NIH HHS
• 75N91019D00024 NCI NIH HHS
• A-PReP Clinical and Translational Science Institute, University of Minnesota (CTSI)
• OACA grants University of Minnesota (UMN)
• R01CA229618 National Cancer Institute (NCI)
• R01CA204856 National Cancer Institute (NCI)
• BICB Fellowship University of Minnesota (UMN)
• IDF Graduate School, University of Minnesota (The Graduate School, University of Minnesota)
• DDF Graduate School, University of Minnesota (The Graduate School, University of Minnesota)

• autophagy
• male germ cells
• reactive oxygen species
• titanium dioxide nanoparticles

• Titanium/toxicity
• Male
• Autophagy/drug effects
• Animals
• Acetylcysteine/pharmacology
• Mice
• Reactive Oxygen Species/metabolism
• Cell Line
• Cell Proliferation/drug effects
• Metal Nanoparticles/toxicity
• Spermatogonia/drug effects
• Nanoparticles/toxicity

• chicken
• embryonic stem cell
• primordial germ cell
• ubiquitome

• Animals
• Ubiquitination
• Chickens
• Germ Cells/metabolism
• Proteome
• Cell Differentiation
• Tandem Mass Spectrometry
• Embryonic Stem Cells
• Chromatography, Liquid/veterinary

• R01 CA204856 NCI NIH HHS
• R01 CA229618 NCI NIH HHS

• Cell interactions
• Embryo
• Gastruloid
• Primordial germ cell
• Stem cell

• Male
• Mice
• Animals
• Semen
• Germ Cells
• Endoderm
• Cells, Cultured
• Signal Transduction
• Cell Differentiation/genetics

• CC2186 Wellcome Trust
• CC2186 Medical Research Council
• CC2186 Cancer Research UK
• 105031/D/14/Z Wellcome Trust
• Wellcome Trust
• CC2186 Arthritis Research UK
• Francis Crick Institute

• Blimp1
• Nanog
• Otx2
• PGC
• PGCLC
• Prdm1
• Psmd3
• Psmd7
• Tex10
• Wnt signaling
• infertility
• spermatogenesis

• S10 RR027050 NCRR NIH HHS
• R01 HD097268 NICHD NIH HHS
• R01 HD095938 NICHD NIH HHS
• S10 OD020056 NIH HHS
• P30 CA013696 NCI NIH HHS
• R01 GM129157 NIGMS NIH HHS
• UL1 TR001873 NCATS NIH HHS

• MAPK
• TGFβ
• chromatin accessibility
• primordial germ cell
• telomere

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X‐chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X‐inactivation and reactivation dynamics using a tailor‐made in vitro system of primordial germ cell‐like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X‐inactivation in PGCLCs in vitro and in germ cell‐competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X‐inactivation is followed by step‐wise X‐reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X‐inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine‐tuned X‐chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.

• X-chromosome inactivation
• epigenetic reprogramming
• in vitro model
• meiosis
• primordial germ cells

Cryopreservation and in vitro culture of germ cells are key techniques for the genetic resource preservation of the declining population of American shad. Two types of cryopreserved samples, namely testis pieces and testicular cells of American shad, were comparatively analyzed for cell viability. The results showed that the cell viability of the cryopreserved testis pieces was much higher than that of the cryopreserved testicular cells. The viability of the cells from the cryopreserved testis pieces ranged from 65.2 ± 2.2 (%) to 93.8 ± 0.6 (%), whereas the viability of the dissociated cells after cryopreservation was 38.5 ± 0.8 (%) to 87.1 ± 2.6 (%). Moreover, the testicular cells isolated from the post-thaw testicular tissue could be cultured and propagated in vitro. Our findings would benefit further investigations on genetic resource preservation and other manipulations of germ cells in a commercially and ecologically important fish species.

Germ cells, as opposed to somatic cells, can transmit heredity information between generations. Cryopreservation and in vitro culture of germ cells are key techniques for genetic resource preservation and cellular engineering breeding. In this study, two types of cryopreserved samples, namely testis pieces and testicular cells of American shad, were comparatively analyzed for cell viability. The results showed that the cell viability of the cryopreserved testis pieces was much higher than that of the cryopreserved testicular cells. The viability of cells from the cryopreserved testis pieces ranged from 65.2 ± 2.2 (%) to 93.8 ± 0.6 (%), whereas the viability of the dissociated cells after cryopreservation was 38.5 ± 0.8 (%) to 87.1 ± 2.6 (%). Intriguingly, the testicular cells from the post-thaw testicular tissue could be cultured in vitro. Likewise, most of the cultured cells exhibited germ cell properties and highly expressed Vasa and PCNA protein. This study is the first attempt to effectively preserve and culture the male germ cells through freezing tissues in the American shad. The findings of this study would benefit further investigations on genetic resource preservation and other manipulations of germ cells in a commercially and ecologically important fish species.

• American shad
• cell culture
• cryopreservation of genetic resource
• male germ cells

Reproduction is a key event in life guaranteeing the propagation and evolution of a species. Infertility caused by abnormal germ cell development is a topic of extensive concern. Herein, in vitro germline specification studies provide a modeling platform to investigate gametogenesis. The differentiation of pluripotent stem cells (PSCs) into germ cells has been studied for more than 30 years, and there have been many astonishing breakthroughs in the last decade. Fertile sperm and oocytes can be obtained from mouse embryonic stem cells (ESCs) through a primordial germ cell (PGC)-based method. Moreover, human PGC-like cells (PGCLCs) can be derived with a similar strategy as that used for mouse PGCLC derivation. In this review, we describe the reconstitution of PGCs and the subsequent meiosis, as well as the signaling pathways and factors involved in these processes.

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.

Proper specification of primordial germ cells (PGCs) is crucial as PGCs serve as the precursors of germline stem cells. To specify PGC fate, invertebrates rely upon cell autonomous preformation involving maternally deposited germ plasm. In Drosophila melanogaster, to insulate newly formed PGCs from the adverse effects of the cell-cell signaling pathways, germ plasm determinants silence transcription and attenuate the cell cycle. However, our data on the BMP signaling pathway challenge this long-held view of PGC specification and suggest that appropriate specification of embryonic PGCs is sensitive to the BMP ligand, decapentaplegic (dpp), and its cognate receptor, thickveins. We find that PGCs are not only capable of responding to BMP signals from the soma, but also that these signals impact the proper determination of the germ cells. Based on these unanticipated similarities between mammals and flies, we propose a model integrating contribution of both the cell-autonomous (preformation) and non-autonomous (epigenesis) pathways during PGC determination. Consistent with the model, we have observed dominant genetic interactions between, oskar, the maternal determinant of PGC fate, and the BMP pathway ligand dpp.

• Animals
• Blastoderm
• Body Patterning
• Bone Morphogenetic Proteins/metabolism
• Cell Differentiation
• Drosophila Proteins/metabolism
• Drosophila melanogaster/embryology
• Drosophila melanogaster/genetics
• Drosophila melanogaster/metabolism
• Epigenesis, Genetic
• Female
• Germ Cells/metabolism
• Germ Cells/physiology
• Male
• Signal Transduction

• R35 GM126975 NIGMS NIH HHS
• national institute of child health and human development
• national institute of general medical sciences
• national science foundation
• burroughs wellcome fund

Primordial germ cells (PGCs) are common ancestors of all germline cells. However, mechanistic understanding of how PGC specification occurs is limited. Here, we identified transcription factor CP2-like 1 (Tfcp2l1), an important pluripotency factor, as a pivotal factor for PGC-like cell (PGCLC) specification. High-throughput sequencing and quantitative real-time PCR analysis showed that Tfcp2l1 expression is gradually increased during mouse and human epiblast differentiation into PGCLCs in vivo and in vitro. Consequently, overexpression of Tfcp2l1 can enhance the specification efficiency even without inductive cytokines in mouse epiblast-like cells derived from embryonic stem cells, while knockdown of Tfcp2l1 significantly inhibits PGCLC generation. Mechanistic studies revealed that Tfcp2l1 exerts its function partially through the direct induction of PR domain zinc finger protein 14, a key PGC marker, as downregulation of the PR domain zinc finger protein 14 transcript can impair the ability of Tfcp2l1 to direct PGCLC commitment. Importantly, we finally demonstrated that the crucial role of the human homolog Tfcp2l1 in promoting PGCLC specification is conserved in human pluripotent stem cells. Together, our data uncover a novel function of Tfcp2l1 in PGCLC fate determination and facilitate a better understanding of germ cell development.

• Prdm14
• TFCP2l1
• embryonic stem cells
• primordial germ cell–like cells

• Animals
• Cell Line
• Germ Cells
• Humans
• Mice
• Pluripotent Stem Cells/metabolism
• Protein Domains
• Repressor Proteins/genetics
• Repressor Proteins/metabolism
• Transcription Factors/biosynthesis
• Transcription Factors/genetics

During early mammalian development, the pluripotent cells of the embryo are exposed to a combination of signals that drive exit from pluripotency and germ layer differentiation. At the same time, a small population of pluripotent cells give rise to the primordial germ cells (PGCs), the precursors of the sperm and egg, which pass on heritable genetic information to the next generation. Despite the importance of PGCs, it remains unclear how they are first segregated from the soma, and if this involves distinct responses to their signaling environment. To investigate this question, we mapped BMP, MAPK and WNT signaling responses over time in PGCs and their surrounding niche in vitro and in vivo at single-cell resolution. We showed that, in the mouse embryo, early PGCs exhibit lower BMP and MAPK responses compared to neighboring extraembryonic mesoderm cells, suggesting the emergence of distinct signaling regulatory mechanisms in the germline versus soma. In contrast, PGCs and somatic cells responded comparably to WNT, indicating that this signal alone is not sufficient to promote somatic differentiation. Finally, we investigated the requirement of a BMP response for these cell fate decisions. We found that cell lines with a mutation in the BMP receptor (Bmpr1a^−/−), which exhibit an impaired BMP signaling response, can efficiently generate PGC-like cells revealing that canonical BMP signaling is not cell autonomously required to direct PGC-like differentiation.

Summary: A subpopulation of pluripotent cells of the embryo give rise to the primordial germ cells (PGCs), the precursors of the sperm and egg, which pass on heritable genetic information to the next generation. To determine how PGCs are first segregated from the soma, we investigated BMP, MAPK and WNT signaling over time in PGCs and their surrounding niche in vitro and in vivo at single-cell resolution.

• BMP
• MAPK
• Mouse embryo
• Primordial germ cell
• WNT

• Cell adhesion
• Embryonic stem cells
• Mesh substrates
• Pluripotency
• Self-organization
• Tissue topography

Understanding the mechanisms of human pluripotent stem cells (hPSCs) specification, development and differentiation to gametes are useful for elucidating the causes of infertility and potential treatment. This study aims to examine whether hPSCs can be induced to DDX4 extracellularly expressing primordial germ cell-like cells (DDX4^ec PGCLCs) and further into ovarian follicle stage in a combined in vitro and in vivo model. The transcriptional signatures show that these DDX4^ec PGCLCs are characteristic of PGCs and express ovarian folliculogenesis markers. We also verify that keratin (KRT)-8 is highly expressed in the DDX4^ec PGCLCs and plays a crucial role in germ cell migration. By co-culturing DDX4^ec PGCLCs with human granulosa cells (GCs), these cells are further induced into ovarian follicle-like structures in a xenograft mice model. This approach can in the future design practical strategies for treating germ cell-associated issues of infertility.

•

hPSC-derived DDX4 PGCLCs participate ovarian follicle-like structure formation

• Cell Biology
• Developmental Biology
• Molecular Biology
• Stem Cell Research

In the present work, we described the expression and activity of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cells (PGCs) from 8.5–14.5 days post coitum (dpc) and investigated whether these kinases play a role in regulating the various processes of PGC development. Using immunofluorescence and immunoblotting to detect the active phosphorylated form of ERK1-2 (p-ERK1-2), we found that the kinases were present in most proliferating 8.5–10.5 dpc PGCs, low in 11.5 dpc PGCs, and progressively increasing between 12.5–14.5 dpc both in female and male PGCs. In vitro culture experiments showed that inhibiting activation of ERK1-2 with the MEK-specific inhibitor U0126 significantly reduced the growth of 8.5 dpc PGCs in culture but had little effect on 11.5–12.5 dpc PGCs. Moreover, we found that the inhibitor did not affect the adhesion of 11.5 dpc PGCs, but it significantly reduced their motility features onto a cell monolayer. Further, while the ability of female PGCs to begin meiosis was not significantly affected by U0126, their progression through meiotic prophase I was slowed down. Notably, the activity of ERK1-2 was necessary for maintaining the correct expression of oocyte-specific genes crucial for germ cells survival and the formation of primordial follicles.

• Extracellular signal-regulated kinases (ERKs)
• Gene expression
• Meiosis
• Oogenesis
• Primordial germ cells

• differentiation
• folliculogenesis
• mesenchymal stem cells
• oocyte-like cells
• oogenesis
• primordial germ cells
• transfection

• Cell Differentiation
• Female
• Humans
• Infertility, Female/therapy
• Oocytes/cytology
• Ovary/cytology
• Stem Cell Transplantation/trends
• Stem Cells/cytology

• ChIP-seq
• Embryonic stem cells
• Oct4
• Prdm14
• Primordial germ cells
• Targeted DamID

• ESCs
• FGF8
• MAPK/ERK signaling pathway
• PGCs
• SSCs
• chicken

Mouse embryonic stem cells (ESCs) are maintained in serum with leukemia inhibitory factor (LIF) to maintain self-renewal and pluripotency. Recently, a 2i culture method was reported using a combination of MEK inhibition (MEKi) and GSK3 inhibition (GSK3i) with LIF to maintain ESCs in a naive ground state. How 2i maintains a ground state of ESCs remains elusive. Here we show that MEKi and GSK3i maintain the ESC ground state by downregulating global DNA methylation through two distinct mechanisms. MEK1 phosphorylates JMJD2C for ubiquitin-mediated protein degradation. Therefore, MEKi increased JMJD2C protein levels but decreased DNMT3 expression. JMJD2C promotes TET1 activity to increase 5-hydroxymethylcytosine (5hmC) levels. GSK3i suppressed DNMT3 expression, thereby decreasing DNA methylation without affecting 5hmC levels. Furthermore, 2i increased PRDM14 expression to inhibit DNMT3A/B protein expression by promoting G9a-mediated DNMT3A/B protein degradation. Collectively, 2i allows ESCs to maintain a naive ground state through JMJD2C-dependent TET1 activation and PRDM14/G9a-mediated DNMT3A/B protein degradation.

•

MEKi increases JMJD2C protein levels and decreases DNMT3 expression in ESCs

• 2i
• G9a
• GSK3i
• JMJD2C
• MEKi
• PRDM14
• TET1
• methylation
• phosphorylation

• Basic fibroblast growth factor
• Embryonic germ stem cell
• Primordial germ cell
• Reprogramming

• epigenetic
• gametes
• infertility
• primordial germ cells
• reprogramming
• transdifferentiation

Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors.

• Primordial germ cell
• Embryonic germ cell
• Germ cell tumor
• Reprogramming
• Induced pluripotent stem cell
• Small molecule compound
• Gene
• Signal
• Transcription factor