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The dosage of X-linked genes is accurately regulated with the development of fetal germ cells (FGCs) 1,2 . How aberrant dosage of X-linked genes impairs FGC development in humans remains poorly understood. FGCs of patients with Klinefelter syndrome (KS), who have an extra X chromosome, provide natural models for addressing this issue 3 . Here we demonstrate that most human FGCs in KS are arrested at an early stage, characterized by the upregulation of genes related to pluripotency, the WNT pathway and the TGF-β pathway, along with the downregulation of genes involved in FGC differentiation. The limited KS FGCs that are capable of reaching the late stage remain relatively naive. X chromosomes are not inactivated and the dosage of X-linked genes is excessive in KS FGCs. X-linked genes dominate the differentially expressed genes and are enriched in critical biological processes associated with the developmental delay of KS FGCs. Moreover, aberrant interactions between Sertoli cells and FGCs disrupt the migration of late FGCs to the basement membrane in KS. Notably, inhibition of the TGF-β pathway improves the differentiation of KS FGCs. Our findings elucidate how the extra X chromosome impairs the development of male FGCs and reveal the initial molecular events preceding germ cell loss in KS., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Primordial germ cells (PGCs) are stem cells, from which only gametes develop. In birds, the female sex is heterogametic, thus female gene conservation necessitates preservation of PGCs. PGC transplantation can generate germline chimeras in a host organism and develop into gametes. However, competition between host and transplanted PGCs hinder efficiency of germline chimera generation. We hypothezised that in sterile hybrid recipients with no germ cells of its own, transplanted donor PGCs may exclusively form gametes. Advantages of sterile hybrids as host for PGCs is compliant with many national regulations on genetically modified organisms and technically simpler procedure than the use of busulphan. Therefore, we investigated whether sterile interspecific hybrids may be suitable as recipients for supporting donor PGCs by injecting green fluorescent protein-labelled chicken PGCs into 3-day-old Guinea fowl and domestic fowl hybrid embryos and monitoring PGC development. The injected PGCs colonized almost 100% of the recipient gonads and produced mature spermatozoa after 44 weeks. However, gamete production in these hybrids was initiated much slower than in domestic fowls. This delay may be caused by suboptimal hormonal regulation of gametogenesis in the hybrids. Our results suggest that sterile interspecific hybrids may be suitable hosts for PGCs for efficient gamete production., (© 2024. The Author(s).)

Germline segregation, essential for protecting germ cells against mutations, occurs during early embryogenesis in vertebrates, insects and nematodes. Highly regenerative animals (e.g., cnidarians), however, retain stem cells with both germinal and somatic potentials throughout adulthood, but their biology and evolution remain poorly understood. Among cnidarians (e.g., sea anemones, jellyfish), stem cells are only known in few hydrozoans (e.g., Hydra). Here, we identify and characterize a rare, multipotent population of stem and/or progenitor cells expressing the conserved germline and multipotency proteins Vasa2 and Piwi1 in the sea anemone Nematostella vectensis. Using piwi1 and vasa2 transgenic reporter lines, we reveal that the Vasa2+/Piwi1+ cell population generates not only gametes, but also a diversity of proliferative somatic cells, including neural progenitors, in juveniles and adults. Our work has uncovered a multipotent population of Vasa2+/Piwi1+ stem/progenitor cells that forms the cellular basis to understand body plasticity and regenerative capacities in sea anemones and corals., (© 2024. The Author(s).)

Avian primordial germ cells (PGCs) are important culture cells for the production of transgenic chickens and preservation of the genetic resources of endangered species; however, culturing these cells in vitro proves challenging. Although the proliferation of chicken PGCs is dependent on insulin, the underlying molecular mechanisms remain unclear. In the present study, we explored the expression of the PI3K/AKT signaling pathway in PGCs, investigated its effects on PGC self-renewal and biological properties, and identified the underlying mechanisms. Our findings indicated that although supplementation with the PI3K/AKT activator IGF-1 failed to promote proliferation under the assessed culture conditions, the PI3K/AKT inhibitor LY294002 resulted in retarded cell proliferation and reduced expression of germ cell-related markers. We further demonstrated that inhibition of PI3K/AKT regulates the cell cycle and promotes apoptosis in PGCs by activating the expression of BAX and inhibiting that of Bcl-2. These findings indicated that the PI3K/AKT pathway is required for cell renewal, apoptosis, and maintenance of the reproductive potential in chicken PGCs. This study aimed to provide a theoretical basis for the optimization and improvement of a culture system for chicken PGCs and provide insights into the self-renewal of vertebrate PGCs as well as potential evolutionary changes in this unique cell population., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Expression of the double homeobox 4 ( DUX4 ) transcription factor is highly regulated in early embryogenesis and is subsequently epigenetically silenced. Ectopic expression of DUX4 due to hypomethylation of the D4Z4 repeat array on permissive chromosome 4q35 alleles is associated with facioscapulohumeral muscular dystrophy (FSHD). In peripheral blood samples from 188 healthy individuals, D4Z4 methylation was highly variable, ranging from 19% to 76%, and was not affected by age. In 48 FSHD2 patients, D4Z4 methylation varied from 3% to 30%. Given that DUX4 is one of the earliest transcribed genes after fertilization, the D4Z4 array is expected to be unmethylated in mature germ cells. Deep bisulfite sequencing of 188 mainly normozoospermic sperm samples revealed an average methylation of 2.5% (range 0.3-22%). Overall, the vast majority (78%) of individual sperm cells displayed no methylation at all. In contrast, only 19 (17.5%) of 109 individual germinal vesicle (GV) oocytes displayed D4Z4 methylation <2.5%. However, it is not unexpected that immature GV oocytes which are not usable for assisted reproduction are endowed with D4Z4 (up to 74%) hypermethylation and/or abnormal ( PEG3 and GTL2 ) imprints. Although not significant, it is interesting to note that the pregnancy rate after assisted reproduction was higher for donors of sperm samples and oocytes with <2.5% methylation.

Chicken embryos serve as an important model for investigating germ cells due to their ease of accessibility and manipulation within the egg. Understanding the development of germ cells is particularly crucial, as they are the only cell types capable of transmitting genetic information to the next generation. Therefore, gene expression regulation in germ cells is important for genomic function. Epigenetic programming is a crucial biological process for the regulation of gene expression without altering the genome sequence. Although epigenetic programming is evolutionarily conserved, several differences between chickens and mammals have been revealed. In this review, we compared the epigenetic regulation of germ cells in chickens and mammals (mainly mice as a representative species). In mammals, migrating primordial germ cells (precursors for germ cells [PGCs]) undergo global DNA demethylation and persist until sexual differentiation, while in chickens, DNA is demethylated until reaching the gonad but remethylated when sexually differentiated. Prospermatogonia is methylated at the onset of mitotic arrest in mammals, while DNA is demethylated at mitotic arrest in chickens. Furthermore, genomic imprinting and inactivation of sex chromosomes are differentially regulated through DNA methylation in chickens and mammals. Chickens and mammals exhibit different patterns of histone modifications during germ cell development, and non-coding RNA, which is not involved in PGC differentiation in mice, plays an important role in chicken PGC development. Additionally, several chicken-specific non-coding RNAs have been identified. In conclusion, we summarized current knowledge of epigenetic gene regulation of chicken germ cells, comparing that of mammals, and highlighted notable differences between them., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The optimal cellular reservoir for the conservation and propagation of endangered poultry is acknowledged to be primordial germ cells (PGCs). This study aimed to evaluate and compare the efficacy of cryopreserving PGCs derived from purified sources and blood samples. The experimental design encompassed two treatment groups, each iterated six replication. Blood samples that had been filled with 500 µL of phosphate-buffered saline without Ca2+ and Mg2+ (PBS) underwent centrifugation at 1200 rpm for six minutes. The supernatant was discarded and the pellet was mixed with 500 µL 10% FBS-PBS and was then subdivided for cryopreservation and PGCs purification. Cryopreservation included the controlled freezing of blood and purified PGCs samples in Nalgene cryogenic vials with the incorporation of banker cells. Following thawing, statistical analyses revealed no significant variance in the mean PGCs count between purified PGCs and blood samples (p > 0.05). The percentages of PGCs mortality and the recovery rate demonstrated statistical significance (p < 0.05). Further investigations indicated that cryopreserving blood samples significantly increased the recovery rate of PGCs while decreasing post-thaw mortality. Morphology of PGCs from blood cryopreservation showed similar morphology to PGCs in general. The potential application of cell-based cryo-banking will contribute to the preservation of poultry genetic diversity for sustainability and adaptation to future poultry demands.

Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14-16 Hamburger-Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers ( SSEA1 , CVH , and DAZL ), as well as pluripotency markers ( OCT4 and NANOG ). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.

The function of germ cells in somatic growth and aging has been demonstrated in invertebrate models but remains unclear in vertebrates. We demonstrated sex-dependent somatic regulation by germ cells in the short-lived vertebrate model Nothobranchius furzeri . In females, germ cell removal shortened life span, decreased estrogen, and increased insulin-like growth factor 1 (IGF-1) signaling. In contrast, germ cell removal in males improved their health with increased vitamin D signaling. Body size increased in both sexes but was caused by different signaling pathways, i.e., IGF-1 and vitamin D in females and males, respectively. Thus, vertebrate germ cells regulate somatic growth and aging through different pathways of the endocrine system, depending on the sex, which may underlie the sexual difference in reproductive strategies.

Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Cytosine base editing achieves C•G-to-T•A substitutions and can convert four codons (CAA/CAG/CGA/TGG) into STOP-codons (induction of STOP-codons, iSTOP) to knock out genes with reduced mosaicism. iSTOP enables direct phenotyping in founders' somatic cells, but it remains unknown whether this works in founders' germ cells so as to rapidly reveal novel genes for fertility. Here, we initially establish that iSTOP in mouse zygotes enables functional characterization of known genes in founders' germ cells: Cfap43-iSTOP male founders manifest expected sperm features resembling human "multiple morphological abnormalities of the flagella" syndrome (i.e., MMAF-like features), while oocytes of Zp3-iSTOP female founders have no zona pellucida. We further illustrate iSTOP's utility for dissecting the functions of unknown genes with Ccdc183, observing MMAF-like features and male infertility in Ccdc183-iSTOP founders, phenotypes concordant with those of Ccdc183-KO offspring. We ultimately establish that CCDC183 is essential for sperm morphogenesis through regulating the assembly of outer dynein arms and participating in the intra-flagellar transport. Our study demonstrates iSTOP as an efficient tool for direct reproductive disease modeling and phenotyping in germ cells of the founder generation, and rapidly reveals the essentiality of Ccdc183 in fertility, thus providing a time-saving approach for validating genetic defects (like nonsense mutations) for human infertility., (© 2024. Science China Press.)

Male germ cells share a common origin across animal species, therefore they likely retain a conserved genetic program that defines their cellular identity. However, the unique evolutionary dynamics of male germ cells coupled with their widespread leaky transcription pose significant obstacles to the identification of the core spermatogenic program. Through network analysis of the spermatocyte transcriptome of vertebrate and invertebrate species, we describe the conserved evolutionary origin of metazoan male germ cells at the molecular level. We estimate the average functional requirement of a metazoan male germ cell to correspond to the expression of approximately 10,000 protein- coding genes, a third of which defines a genetic scaffold of deeply conserved genes that has been retained throughout evolution. Such scaffold contains a set of 79 functional associations between 104 gene expression regulators that represent a core component of the conserved genetic program of metazoan spermatogenesis. By genetically interfering with the acquisition and maintenance of male germ cell identity, we uncover 161 previously unknown spermatogenesis genes and three new potential genetic causes of human infertility. These findings emphasize the importance of evolutionary history on human reproductive disease and establish a cross- species analytical pipeline that can be repurposed to other cell types and pathologies. [ABSTRACT FROM AUTHOR]

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

Direct introduction of cryopreserved embryonic gonadal germ cells (GGC) into a sterile chicken surrogate host to reconstitute a chicken breed has been demonstrated as a feasible approach for preserving and utilizing chicken genetic resources. This method is highly efficient using male gonads; however, a large number of frozen female embryonic gonads is needed to provide sufficient purified GGC for the generation of fertile surrogate female hosts. Applying this method to indigenous chicken breeds and other bird species is difficult due to small flock numbers and poor egg production in each egg laying cycle. Propagating germ cells from the frozen gonadal tissues may be a solution for the biobanking of these birds. Here, we describe a simplified method for culture of GGC from frozen embryonic 9.5 d gonads. At this developmental stage, the germ cells are autonomously shed into medium, yielding hundreds to thousands of mitosis-competent germ cells. The resulting cultures of GGC have over 90% purity, uniformly express SSEA-1 and DAZL antigens and can re-colonize recipient's gonads. The GGC recovery rate from frozen gonads are 42% to 100%, depending on length of cryopreservation and the breed or line of chickens. Entire chicken embryos can also be directly cryopreserved for later gonadal isolation and culture. This storage method is a supplementary approach to safeguard local indigenous chicken breeds bearing valuable genetic traits and should be applicable to the biobanking of many bird species., Competing Interests: DISCLOSURES All authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Male germ cells share a common origin across animal species, therefore they likely retain a conserved genetic program that defines their cellular identity. However, the unique evolutionary dynamics of male germ cells coupled with their widespread leaky transcription pose significant obstacles to the identification of the core spermatogenic program. Through network analysis of the spermatocyte transcriptome of vertebrate and invertebrate species, we describe the conserved evolutionary origin of metazoan male germ cells at the molecular level. We estimate the average functional requirement of a metazoan male germ cell to correspond to the expression of approximately 10,000 protein-coding genes, a third of which defines a genetic scaffold of deeply conserved genes that has been retained throughout evolution. Such scaffold contains a set of 79 functional associations between 104 gene expression regulators that represent a core component of the conserved genetic program of metazoan spermatogenesis. By genetically interfering with the acquisition and maintenance of male germ cell identity, we uncover 161 previously unknown spermatogenesis genes and three new potential genetic causes of human infertility. These findings emphasize the importance of evolutionary history on human reproductive disease and establish a cross-species analytical pipeline that can be repurposed to other cell types and pathologies.

Prenatal somatic cell gene therapy (PSCGT) could potentially treat severe, early-onset genetic disorders such as spinal muscular atrophy (SMA) or muscular dystrophy. Given the approval of adeno-associated virus serotype 9 (AAV9) vectors in infants with SMA by the U.S. Food and Drug Administration, we tested the safety and biodistribution of AAV9-GFP (clinical-grade and dose) in fetal lambs to understand safety and efficacy after umbilical vein or intracranial injection on embryonic day 75 (E75) . Umbilical vein injection led to widespread biodistribution of vector genomes in all examined lamb tissues and in maternal uteruses at harvest (E96 or E140; term = E150). There was robust GFP expression in brain, spinal cord, dorsal root ganglia (DRGs), without DRG toxicity and excellent transduction of diaphragm and quadriceps muscles. However, we found evidence of systemic toxicity (fetal growth restriction) and maternal exposure to the viral vector (transient elevation of total bilirubin and a trend toward elevation in anti-AAV9 antibodies). There were no antibodies against GFP in ewes or lambs. Analysis of fetal gonads demonstrated GFP expression in female (but not male) germ cells, with low levels of integration-specific reads, without integration in select proto-oncogenes. These results suggest potential therapeutic benefit of AAV9 PSCGT for neuromuscular disorders, but warrant caution for exposure of female germ cells.

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., Competing Interests: Declaration of interests The authors declare no competing or financial interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Cryptorchidism in males constitutes a notable risk factor for both infertility and testicular cancer. Infertility in adulthood is closely linked to the germ cell status in childhood. Furthermore, the significance of germ cell status is important as more than 95% of all reported testicular malignancies are germ cell tumors. The review aims to elucidate the pathogenesis of germ cells in cryptorchid testes concerning their association with infertility and testicular malignancies. Impaired germ cell numbers are evident in cryptorchid testes even during antenatal and neonatal stages. In cryptorchidism there is a rapid decline in germ cell number within the first year of life, partially attributed to physiologic gonocyte apoptosis. Additionally, germ cells fail to differentiate normally during mini-puberty leading to reduced germ cell proliferation and delayed clearance of gonocytes from the seminiferous epithelium. Absence of germ cells in testicular biopsies occurs already 10 months of age and germ cell deterioration progressively worsens with approximately 50% of persisting cryptorchid testes lacking germ cells during puberty. The deficient germ cell maturation and proliferation leads to later infertility. Elevated temperature in the cryptorchid testes and also hormonal deficiency contribute to this phenomenon. Germ cell neoplasia in situ (GCNIS) originating during fetal development may manifest in rare cases associated with disorders of sexual development, chromosomal abnormalities in boys, specific syndromes, and teratomas that include cryptorchidism. In adults, the presence of GCNIS predominantly represents a new histology pattern before invasive germ cell cancer is demonstrated and is neither congenital nor related to abnormal gonocyte transformation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Thorup, Hildorf, Hildorf, Baastrup, Mamsen, Andersen, Olsen and Cortes.)

The transition from fetal primordial germ cells (PGCs) to spermatogonia (SPG) is critical for male germ cell development; however, the detailed transcriptomic dynamics and regulation underlying this transition remain poorly understood. Here by interrogating the comprehensive transcriptome atlas dataset of mouse male germ cells and gonadal cells development, we elucidated the regulatory networks underlying this transition. Our single‐cell transcriptome analysis revealed that the transition from PGCs to SPG was characterized by global hypertranscription. A total of 315 highly active regulators were identified to be potentially involved in this transition, among which a non‐transcription factor (TF) regulator TAGLN2 was validated to be essential for spermatogonial stem cells (SSCs) maintenance and differentiation. Metabolism profiling analysis also revealed dynamic changes in metabolism‐related gene expression during PGC to SPG transition. Furthermore, we uncovered that intricate cell–cell communication exerted potential functions in the regulation of hypertranscription in germ cells by collaborating with stage‐specific active regulators. Collectively, our work extends the understanding of molecular mechanisms underlying male germ cell development, offering insights into the recapitulation of germ cell generation in vitro. [ABSTRACT FROM AUTHOR]

Cannabis, the most consumed illicit psychoactive drug in the world, is increasingly used by pregnant women. However, while cannabinoid receptors are expressed in the early embryo, the impact of phytocannabinoids exposure on early embryonic processes is lacking. Here, we leverage a stepwise in vitro differentiation system that captures the early embryonic developmental cascade to investigate the impact of exposure to the most abundant phytocannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC). We demonstrate that Δ9-THC increases the proliferation of naive mouse embryonic stem cells (ESCs) but not of their primed counterpart. Surprisingly, this increased proliferation, dependent on the CB1 receptor binding, is only associated with moderate transcriptomic changes. Instead, Δ9-THC capitalizes on ESCs' metabolic bivalence by increasing their glycolytic rates and anabolic capabilities. A memory of this metabolic rewiring is retained throughout differentiation to Primordial Germ Cell-Like Cells in the absence of direct exposure and is associated with an alteration of their transcriptional profile. These results represent the first in-depth molecular characterization of the impact of Δ9-THC exposure on early stages of germline development.

Atrazine is currently one of the most commonly used agrochemicals in the United States and elsewhere. Here, we studied the immunoexpression of molecular markers of mammalian testicular functions: androgen receptor (AR), promyelocytic leukemia zinc finger (PLZF), GDNF family receptor alpha-1 (GFRA1), VASA/DDX4 (DEAD-Box Helicase 4) as well as the levels of intratesticular and intra-epididymal estradiol (E2) and dihydrotestosterone (DHT), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukins (IL-1β and IL-6, IL-10) and testicular chemokines (CXCL-1, CCL-2 and CCL3) in BalB/c mice after a sub-acute gavage treatment with a gonado-toxin, atrazine (50 mg/kg body wt.) for three days. We found high numbers of AR immunopositive Sertoli cells and low numbers of GFRA1, PLZF and VASA/DDX4-positive germ cells in the seminiferous tubule regions of the testes. While TNF-α level in the testes fell and remained unchanged in the epididymides, IFN-γ levels in the testes remained constant but increased in the epididymides. E2 and DHT concentrations remained unaltered in the testes but were changed in the epididymides. There were no significant changes in the levels of interleukins in the testis and epididymis. Intratesticular chemokines were also not significantly altered, except for CCL-4, which was increased in the testis. Light microscopy of the epididymis showed detached epithelium and some detached cells in the lumen. It is concluded that atrazine changed the inflammatory status of the gonads and highlighted Sertoli and undifferentiated spermatogonia as important targets for atrazine's toxic effects in the testis of mice. Concerning the epididymis, atrazine altered the epididymal hormonal concentrations and promoted histopathological modifications in its parenchyma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Abstract Primordial germ cells (PGCs) are stem cells, from which only gametes develop. In birds, the female sex is heterogametic, thus female gene conservation necessitates preservation of PGCs. PGC transplantation can generate germline chimeras in a host organism and develop into gametes. However, competition between host and transplanted PGCs hinder efficiency of germline chimera generation. We hypothezised that in sterile hybrid recipients with no germ cells of its own, transplanted donor PGCs may exclusively form gametes. Advantages of sterile hybrids as host for PGCs is compliant with many national regulations on genetically modified organisms and technically simpler procedure than the use of busulphan. Therefore, we investigated whether sterile interspecific hybrids may be suitable as recipients for supporting donor PGCs by injecting green fluorescent protein-labelled chicken PGCs into 3-day-old Guinea fowl and domestic fowl hybrid embryos and monitoring PGC development. The injected PGCs colonized almost 100% of the recipient gonads and produced mature spermatozoa after 44 weeks. However, gamete production in these hybrids was initiated much slower than in domestic fowls. This delay may be caused by suboptimal hormonal regulation of gametogenesis in the hybrids. Our results suggest that sterile interspecific hybrids may be suitable hosts for PGCs for efficient gamete production.

Abstract Germline segregation, essential for protecting germ cells against mutations, occurs during early embryogenesis in vertebrates, insects and nematodes. Highly regenerative animals (e.g., cnidarians), however, retain stem cells with both germinal and somatic potentials throughout adulthood, but their biology and evolution remain poorly understood. Among cnidarians (e.g., sea anemones, jellyfish), stem cells are only known in few hydrozoans (e.g., Hydra). Here, we identify and characterize a rare, multipotent population of stem and/or progenitor cells expressing the conserved germline and multipotency proteins Vasa2 and Piwi1 in the sea anemone Nematostella vectensis. Using piwi1 and vasa2 transgenic reporter lines, we reveal that the Vasa2+/Piwi1+ cell population generates not only gametes, but also a diversity of proliferative somatic cells, including neural progenitors, in juveniles and adults. Our work has uncovered a multipotent population of Vasa2+/Piwi1+ stem/progenitor cells that forms the cellular basis to understand body plasticity and regenerative capacities in sea anemones and corals.

Rationale: Spermatogenesis is a highly organized cell differentiation process in mammals, involving mitosis, meiosis, and spermiogenesis. DIS3L2, which is primarily expressed in the cytoplasm, is an RNA exosome-independent ribonuclease. In female mice, Dis3l2 -deficient oocytes fail to resume meiosis, resulting in arrest at the germinal vesicle stage and complete infertility. However, the role of DIS3L2 in germ cell development in males has remained largely unexplored. Methods: We established a pre-meiotic germ cell conditional knockout mouse model and investigated the biological function of DIS3L2 in spermatogenesis and male fertility through bulk RNA-seq and scRNA-seq analyses. Results: This study unveils that conditional ablation of Dis3l2 in pre-meiotic germ cells with Stra8-Cre mice impairs spermatogonial differentiation and hinders spermatocyte meiotic progression coupled with cell apoptosis. Such conditional ablation leads to defective spermatogenesis and sterility in adults. Bulk RNA-seq analysis revealed that Dis3l2 deficiency significantly disrupted the transcriptional expression pattern of genes related to the cell cycle, spermatogonial differentiation, and meiosis in Dis3l2 conditional knockout testes. Additionally, scRNA-seq analysis indicated that absence of DIS3L2 in pre-meiotic germ cells causes disrupted RNA metabolism, downregulated expression of cell cycle genes, and aberrant expression of spermatogonial differentiation genes, impeding spermatogonial differentiation. In meiotic spermatocytes, loss of DIS3L2 results in disturbed RNA metabolism, abnormal translation, and disrupted meiotic genes that perturb meiotic progression and induce cell apoptosis, leading to subsequent failure of spermatogenesis and male infertility. Conclusions: Collectively, these findings highlight the critical role of DIS3L2 ribonuclease-mediated RNA degradation in safeguarding the correct transcriptome during spermatogonial differentiation and spermatocyte meiotic progression, thus ensuring normal spermatogenesis and male fertility., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: The maintenance of genome stability in primordial germ cells (PGCs) is crucial for the faithful transmission of genetic information and the establishment of reproductive reserve. Numerous studies in recent decades have linked the Fanconi anemia (FA) pathway with fertility, particularly PGC development. However, the role of FAAP100, an essential component of the FA core complex, in germ cell development is unexplored., Results: We find that FAAP100 plays an essential role in R-loop resolution and replication fork protection to counteract transcription-replication conflicts (TRCs) during mouse PGC proliferation. FAAP100 deletion leads to FA pathway inactivation, increases TRCs as well as cotranscriptional R-loops, and contributes to the collapse of replication forks and the generation of DNA damage. Then, the activated p53 signaling pathway triggers PGC proliferation defects, ultimately resulting in insufficient establishment of reproductive reserve in both sexes of mice., Conclusions: Our findings suggest that FAAP100 is required for the resolution of TRCs in PGCs to safeguard their genome stability., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Objective The objective of this study was to investigate the regulation relationship of Ten-eleven translocation 1 (Tet1) in DNA demethylation and the proliferation of primordial germ cells (PGCs) in chickens. Methods siRNA targeting Tet1 was used to transiently knockdown the expression of Tet1 in chicken PGCs, and the genomic DNA methylation status was measured. The proliferation of chicken PGCs was detected by flow cytometry analysis and cell counting kit-8 assay when activation or inhibition of Wnt4/β-catenin signaling pathway. And the level of DNA methylation and hisotne methylation was also tested. Results Results revealed that knockdown of Tet1 inhibited the proliferation of chicken PGCs and downregulated the mRNA expression of Cyclin D1 and cyclin-dependent kinase 6 (CDK6), as well as pluripotency-associated genes (Nanog, PouV, and Sox2). Flow cytometry analysis confirmed that the population of PGCs in Tet1 knockdown group displayed a significant decrease in the proportion of S and G2 phase cells, which meant that there were less PGCs entered the mitosis process than that of control. Furthermore, Tet1 knockdown delayed the entrance to G1/S phase and this inhibition was rescued by treated with BIO. Consistent with these findings, Wnt/β-catenin signaling was inactivated in Tet1 knockdown PGCs, leading to aberrant proliferation. Further analysis showed that the methylation of the whole genome increased significantly after Tet1 downregulation, while hydroxymethylation obviously declined. Meanwhile, the level of H3K27me3 was upregulated and H3K9me2 was downregulated in Tet1 knockdown PGCs, which was achieved by regulating Wnt/β-catenin signaling pathway. Conclusion These results suggested that the self-renewal of chicken PGCs and the maintenance of their characteristics were regulated by Tet1 mediating DNA demethylation through the activation of Wnt4/β-catenin signaling pathway.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level. [ABSTRACT FROM AUTHOR]

Germ cells (GCs) serve as indispensable carriers in both animals and plants, ensuring genetic continuity across generations. While it is generally acknowledged that the timing of germline segregation differs significantly between animals and plants, ongoing debates persist as new evidence continues to emerge. In this review, we delve into studies focusing on male germ cell specifications in plants, and we summarize the core gene regulatory circuits in germ cell specification, which show remarkable parallels to those governing meristem homeostasis. The similarity in germline establishment between animals and plants is also discussed.

Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells, migratory PGCs exhibit a latent pluripotency characterized by the ability to derive embryonic germ cells (EGCs) and form teratoma. Here, we show that inhibition of p38 mitogen-activated protein kinase (MAPK) by chemical compounds in mouse migratory PGCs enables derivation of chemically induced Embryonic Germ-like Cells (cEGLCs) that do not require conventional growth factors like LIF and FGF2/Activin-A, and possess unique naïve pluripotent-like characteristics with epiblast features and chimera formation potential. Furthermore, cEGLCs are regulated by a unique PI3K-Akt signaling pathway, distinct from conventional naïve pluripotent stem cells described previously. Consistent with this notion, we show by performing ex vivo analysis that inhibition of p38 MAPK in organ culture supports the survival and proliferation of PGCs and also potentially reprograms PGCs to acquire indefinite proliferative capabilities, marking these cells as putative teratoma-producing cells. These findings highlight the utility of our ex vivo model in mimicking in vivo teratoma formation, thereby providing valuable insights into the cellular mechanisms underlying tumorigenesis. Taken together, our research underscores a key role of p38 MAPK in germ cell development, maintaining proper cell fate by preventing unscheduled pluripotency and teratoma formation with a balance between proliferation and differentiation.

Background: SOX17 has been identified as a critical factor in specification of human primordial germ cells, but whether SOX17 regulates development of germ cells after sex differentiation is poorly understood., Methods: We collected specimens of gonadal ridge from an embryo (n=1), and ovaries of foetuses (n=23) and adults (n=3). Germ cells were labelled with SOX17, VASA (classic germ cells marker), phosphohistone H3 (PHH3, mitosis marker) and synaptonemal complex protein 3 (SCP3, meiosis marker)., Results: SOX17 was detected in both cytoplasm and nucleus of oogonia and oocytes of primordial and primary follicles from 15 to 28 gestational weeks (GW). However, it was exclusively expressed in cytoplasm of oogonia at 7 GW, and in nucleus of oocytes in secondary follicles. Co-expression rates of SOX17 in VASA + germ cells ranged from 81.29% to 97.81% in foetuses. Co-staining rates of SOX17 and PHH3 or SCP3 were 0%-34% and 0%-57%, respectively. Interestingly, we distinguished a subpopulation of SOX17 + VASA - germ cells in fetal ovaries. These cells clustered in the cortex and could be co-stained with the mitosis marker PHH3 but not the meiosis marker SCP3., Conclusions: The dynamic expression of SOX17 was detected in human female germ cells. We discovered a population of SOX17 + VASA - germ cells clustering at the cortex of ovaries. We could not find a relationship between mitosis or meiosis and SOX17 or VASA staining in germ cells. Our findings provide insight into the potential role of SOX17 involving germ cells maturation after specification, although the mechanism is unclear and needs further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Luo, Jie, Huang, Cai, Zhou, Liang, Zhou and Mai.)

Human germ cell development is a highly regulated process beginning soon after embryo implantation with the specification of primordial germ cells (PGCs) and ending in adulthood with the differentiation of gametes. Here, we show that fibroblast growth factor receptor 3 (FGFR3) is expressed by human PGCs during the first and second trimester, becoming repressed as PGCs differentiate into primordial oocytes. Using fluorescence-activated cell sorting (FACS) with antibodies that recognize FGFR3 followed by single-cell RNA sequencing, we show that isolating FGFR3-positive cells enriches for human PGCs. Taken together, FGFR3 could be used in future studies as a strategy to identify maturing PGCs in vitro.

The function of germ cells in somatic growth and aging has been demonstrated in invertebrate models but remains unclear in vertebrates. We demonstrated sex-dependent somatic regulation by germ cells in the short-lived vertebrate model Nothobranchius furzeri. In females, germ cell removal shortened life span, decreased estrogen, and increased insulin-like growth factor 1 (IGF-1) signaling. In contrast, germ cell removal in males improved their health with increased vitamin D signaling. Body size increased in both sexes but was caused by different signaling pathways, i.e., IGF-1 and vitamin D in females and males, respectively. Thus, vertebrate germ cells regulate somatic growth and aging through different pathways of the endocrine system, depending on the sex, which may underlie the sexual difference in reproductive strategies. [ABSTRACT FROM AUTHOR]

Abstract YAP plays a vital role in controlling growth and differentiation in various cell lineages. Although the expression of YAP in mice testicular and spermatogenic cells suggests its role in mammalian spermatogenesis, the role of YAP in the development of human male germ cells has not yet been determined. Using an in vitro model and a gene editing approach, we generated human spermatogonia stem cell-like cells (hSSLCs) from human embryonic stem cells (hESCs) and investigated the role of YAP in human spermatogenesis. The results showed that reducing YAP expression during the early stage of spermatogenic differentiation increased the number of PLZF+ hSSLCs and haploid spermatid-like cells. We also demonstrated that the up-regulation of YAP is essential for maintaining spermatogenic cell survival during the later stages of spermatogenic differentiation. The expression of YAP that deviates from this pattern results in a lower number of hSSLCs and an increased level of spermatogenic cell death. Taken together, our result demonstrates that the dynamic expression pattern of YAP is essential for human spermatogenesis. Modulating the level of YAP during human spermatogenesis could improve the production yield of male germ cells derived from hESCs, which could provide the optimization method for in vitro gametogenesis and gain insight into the application in the treatment of male infertility.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The development of oocytes occurs over a broad time frame, starting at the earliest stages of embryogenesis and continuing into adulthood. Conditional knockout technologies such as the Cre/loxP recombination system are useful for analyzing oocyte development at specific stages, but not every time frame has appropriate Cre drivers, for instance, during oocyte meiotic initiation through early prophase I in the embryo. Here, we generated a novel knockin mouse line that produces a bicistronic transcript from the endogenous Stra8 locus that includes a "self-cleaving" 2A peptide upstream of cre. This allows for high efficiency cleavage and production of both proteins individually and results in expression of cre in both male and female gonads at the biologically relevant stage. Fluorescent reporter analysis confirms that this line recapitulates endogenous Stra8 expression in both sexes and does not affect fertility of heterozygous nor homozygous mice. This line, named Stra8P2Acre, adds to the repertoire of germ-cell specific cre driver lines and, importantly, allows for deletion of target genes during key embryonic oocyte developmental stages, including early events in meiosis. Summary Sentence Generation of a novel cre recombinase knockin to the Stra8 locus allows production of Stra8 and cre without affecting fertility., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The cultivation and expansion of chicken primordial germ cells (cPGCs) are of critical importance for both biotechnological applications and the management of poultry genetic biodiversity. The feeder-free culture system has become the most popular approach for the cultivation and expansion of cPGCs. However, despite some success in the cultivation of cPGCs, the reproducibility of culture conditions across different laboratories remains a challenge. This study aimed to compare two defined and enriched media for the growth of cPGCs originating from the Hubbard JA57 broiler. To this end, cPGCs were isolated from the embryonic blood of Hamburger-Hamilton (HH) stages 14-16 and cultured at various time points. The Growth properties and characteristics of these cells were evaluated in two different culture conditions (the defined or enriched medium) and their migratory properties were assessed after genetic engineering and injection into the vasculature of 2.5-day-old chicken embryos. The main finding of this study was that the use of an enriched medium (the defined medium with Knock-Out Serum Replacement; KOSR) resulted in improved growth properties of cPGCs originating from the Hubbard JA57 broiler compared to a defined medium. The ability to cultivate and expand cPGCs is crucial for the generation of both genetically engineered birds and breeds of interest from local or commercial origins. Therefore, these results highlight the importance of choosing an appropriate culture medium for cPGCs growth and expansion., (© 2023. The Author(s).)

In mammals, a near complete resetting of DNA methylation (DNAme) is observed during germline establishment. This wave of epigenetic reprogramming is sensitive to the environment, which could impair the establishment of an optimal state of the gamete epigenome, hence proper embryo development. Yet, we lack a comprehensive understanding of DNAme dynamics during spermatogenesis, especially in rats, the model of choice for toxicological studies. Using a combination of cell sorting and DNA methyl-seq capture, we generated a stage-specific mapping of DNAme in nine populations of differentiating germ cells from perinatal life to spermiogenesis. DNAme was found to reach its lowest level at gestational day 18, the last demethylated coding regions being associated with negative regulation of cell movement. The following de novo DNAme displayed three different kinetics with common and distinct genomic enrichments, suggesting a non-random process. DNAme variations were also detected at key steps of chromatin remodeling during spermiogenesis, revealing potential sensitivity. These methylome datasets for coding sequences during normal spermatogenesis in rat provide an essential reference for studying epigenetic-related effects of disease or environmental factors on the male germline., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

In recent years, the Chinese hook snout carp (Opsariichthys bidens) is becoming increasingly popular due to its high nutritional value and delicious taste. However, anthropogenic influences have caused a rapid reduction in its population. Considering long-term development sustainability, it is essential to conduct research on its reproductive development. In this study, the germ-cell related genes dazl (Obdazl) and dnd (Obdnd) were cloned, and their expression patterns were revealed. RT-PCR analysis indicated that Obdazl and Obdnd were specifically expressed in gonads. In the testis, Obdazl and Obdnd RNAs have a similar expression pattern: their transcripts were abundant in spermatogonia, gradually decreased, and eventually disappeared during spermatogenesis. Early oocytes in the ovary exhibited strong expression of Obdazl and Obdnd RNAs and reduced remarkably after peak expression at III oocytes. Surprisingly, Obdazl RNA was dynamically found in perinuclear spots that finally aggregated into the Balbiani body (Bb) in the early oocytes. Moreover, Obdazl 3′ UTR enabled the reporter gene to be expressed stably in medaka PGCs, suggesting that dazl was identified as the germ cell marker in O. bidens, which may provide perspectives for further studies on the development of PGCs in the future. [ABSTRACT FROM AUTHOR]

The high plasticity, or the ability to differentiate into various cell types, and capacity to become both gametes of early-stage germ cells (eGCs) allows them to be used for the long-term storage and recovery of genetic information. In this study, species-specific protocols (isolation period, enzymatic separation solution, incubation period, appropriate cryoprotectant, and cryopreservation protocol) were established for the isolation and long-term preservation of Black Sea trout eGCs. In addition, the difference between the application of cryopreservation to the whole gonad tissue and to the enzymatically separated cells has been shown in terms of viability and the number of cells obtained. According to the cell count made from the histological sections of the testicular tissue throughout the year, the period with the highest concentration of eGCs in the tissue is between May and June. To identify the optimal enzymatic dissociation solution, we subjected testicular tissues to digestion using various combinations of trypsin (T) and collagenase (C) (0.3-0.5% T and 0.1-0.3% C). Our findings revealed that the 0.3% C provided the highest yield of viable cells (90.9%). We cryopreserved the enzymatically dissociated cells (EDCs) using six different cryoprotectants (Dimethyl sulfoxide, Glycerol, and CryoSOfree™, in both L-15 and phosphate-buffered saline - PBS mediums). Additionally, whole tissue exclusively with CryoSOfree™. After storing the samples at -152 °C for two years, the group of EDCs cryopreserved in L15+Glycerol exhibited the highest viability at 93.3%. Meanwhile, the whole tissue group yielded the greatest number of cells per ml after thawing, with a count of 33 210 000 cells. When considering both the viability (91.11%) and the number of cells obtained (33 210 000 cells/ml), the whole tissue cryopreservation group outperformed all the other methods.