Early Germ Cell Development Research Articles

A translation regulator that drives spermatogonial stem cell formation.

Spermatogonial stem cells (SSCs) are essential for adult spermatogenesis. Themolecular mechanisms driving SSC generation are poorly understood. Zou et al. reported that the precursor cells that give rise to SSCs-prospermatogonia (ProSG) require the RNA-binding protein, DDX20, in orderto undergo the obligatory proliferative re-activation step that proceeds SSC formation. Literature search. We summarize the authors' discovery that the RNA-binding protein, DDX20, iscritical for driving the proliferative re-activation of ProSG, a key step that proceeds SSC generation in vivo. They provide evidence that DDX20 performs this role through its ability to promote the translation of mRNAs encoding proteins known to be essential for cell-cycle and spermatogonial homeostasis. It remains to be determined whether this role is conserved inhumans. It will also be interesting to elucidate whether other post-transcriptional regulators also have roles in early germ cell development. More broadly, it will be fascinating to determine whether post-transcriptional regulators workin concert with transcriptional regulators to drive germ-cell development.

Data Analysis Pipeline for scRNA-seq Experiments to Study Early Oogenesis.

Germ cells as the means for the transmission of genetic information between generations have been a hot topic of research for decades. The analysis of the transcriptomes, that is of the RNA transcripts produced by the genotype at a given time, of germ cells and the surrounding somatic cells, is essential to unravel the cellular and molecular processes regulating gametogenesis. However, the asynchronized differentiation of germ cells and high cellular heterogeneity in the developing ovary or testis represent two unsurmountable challenges for delineating the transcription regulation mechanism of germ cells using traditional bulk RNA sequencing. By performing single-cell RNA sequencing (scRNA-seq), it is now possible to dissect the transcriptome of germ cell development at single-cell resolution, and apply powerful bioinformatics methods to translate raw sequencing data into meaningful information. Here, using the 10× Genomic platform and the most widely cited bioinformatics tools, we describe how to analyze early female germ cell development using scRNA-seq data generated from mouse E11.5 to E14.5 ovaries. This pipeline will provide a guide for exploring the processes of early germ cell development at single-cell resolution.

Single-cell RNA sequencing identifies eggplant as a regulator of germ cell development in Drosophila.

Drosophila ovarian germline stem cells (GSCs) are a powerful model for stem cell research. In this study, we use single-cell RNA sequencing (scRNA-seq), an RNAi screen and bioinformatic analysis, to identify genes involved in germ cell differentiation, including 34 genes with upregulated expression during early germ cell development and 19 genes that may regulate germ cell differentiation. Among these, a gene we have named eggplant (eggpl) is highly expressed in GSCs and downregulated in early daughter cells. RNAi knockdown of eggpl causes germ cell proliferation and differentiation defects. In flies fed a rich yeast diet, the expression of eggpl is significantly lower and knockdown or knockout of eggpl phenocopies a rich diet. In addition, eggpl knockdown suppresses the reduction in germ cell proliferation caused by inhibition of the insulin effector PI3K. These findings suggest that downregulation of eggpl links nutritional status to germ cell proliferation and differentiation. Collectively, this study provides new insights into the signaling networks that regulate early germ cell development and identifies eggpl as a key player in this process.

The Synchronized Progression from Mitosis to Meiosis in Female Primordial Germ Cells between Layers and Broilers.

Layer and broiler hens show a dramatic difference in the volume and frequency of egg production. However, it is unclear whether the intrinsic competency of oocyte generation is also different between the two types of chicken. All oocytes were derived from the primordial germ cells (PGC) in the developing embryo, and female PGC proliferation (mitosis) and the subsequent differentiation (meiosis) determine the ultimate ovarian pool of germ cells available for future ovulation. In this study, we systematically compared the cellular phenotype and gene expression patterns during PGC mitosis (embryonic day 10, E10) and meiosis (E14) between female layers and broilers to determine whether the early germ cell development is also subjected to the selective breeding of egg production traits. We found that PGCs from E10 showed much higher activity in cell propagation and were enriched in cell proliferation signaling pathways than PGCs from E14 in both types of chicken. A common set of genes, namely insulin-like growth factor 2 (IGF2) and E2F transcription factor 4 (E2F4), were identified as the major regulators of cell proliferation in E10 PGCs of both strains. In addition, we found that E14 PGCs from both strains showed an equal ability to initiate meiosis, which was associated with the upregulation of key genes for meiotic initiation. The intrinsic cellular dynamics during the transition from proliferation to differentiation of female germ cells were conserved between layers and broilers. Hence, we surmise that other non-cell autonomous mechanisms involved in germ-somatic cell interactions would contribute to the divergence of egg production performance between layers and broilers.

SETDB1 Regulates Porcine Spermatogonial Adhesion and Proliferation through Modulating MMP3/10 Transcription.

The transition from gonocytes into spermatogonia takes place during the homing process. A subpopulation of undifferentiated spermatogonia in niche then shifts to spermatogonial stem cells (SSCs), accompanied by the self-renewal ability to maintain life-long fertility in males. Enormous changes in cell morphology, gene expression, and epigenetic features have been reported during spermatogenesis. However, little is known about the difference of these features in SSCs during aging. Here, we examined the dynamics of SET domain bifurcated 1 (SETDB1) expression in porcine testes. SETDB1 was expressed in postnatal undifferentiated spermatogonia, while gradually disappeared after being packed within the basal compartment of seminiferous tubules. In addition, the cell-adhesion ability, proliferative activity, and trimethylation of the histone H3 lysine 9 (H3K9me3) level were significantly altered in SETDB1-deficient porcine SSCs. Moreover, the matrix metalloproteinases 3/10 (MMP3/10) was upregulated at both mRNA and protein levels. These results illustrate the significance of SETDB1 in modulating early male germ cell development.

Differentiation of Human-Induced Pluripotent Stem Cells (hiPSCs) into Human Primordial Germ Cell-like Cells (hPGCLCs) In Vitro.

In humans, germ cells are specified in the extraembryonic yolk sac, at proximity of allantois, around the second week of gestation. Derivation of human germ cell-like cells (hPGCLCs) from human pluripotent cells in vitro is of a great importance for research purposes, such as disease modeling, or studying the early human germ cell development and the effect of environmental factors on this development. As it is not possible to access human embryos at early developmental stages, a two-step protocol has been proposed by Sasaki and colleagues to differentiate hPGCLCs in vitro from human pluripotent stem cells. Here, we report a detailed protocol for in vitro hPGCLCs differentiation from induced pluripotent stem cells (iPSCs).

Human embryonic development: from peri-implantation to gastrulation.

The basic body plan of the mammalian embryo is established through gastrulation, a pivotal early postimplantation event during which the three major germ layers (endoderm, ectoderm, and mesoderm) are specified with cellular and spatial diversity. Despite its basic and clinical importance, human embryo development from peri-implantation to gastrulation remains shrouded in mystery. Recent advances in the elongated in vitro culture of rodent and non-primate embryos and the construction of embryo-like structures have helped to improve understanding of the mechanisms of human early embryonic development. Here, we review the recent advances and possible future directions in the development of in vitro models to better understand human embryogenesis from peri-implantation to gastrulation.

The Majority of Boys Having Orchidopexy for Congenital Nonsyndromic Cryptorchidism during Minipuberty Exhibited Normal Reproductive Hormonal Profiles

The activation of the hypothalamic-pituitary-gonadal axis that occurs in male minipuberty during the first 5 months of life is important for early germ cell development. Orchidopexy during minipuberty may improve fertility potential as the germinative epithelium may benefit from the naturally occurring gonadotropin stimulation. We hypothesize that most boys with congenital nonsyndromic cryptorchidism display normal reproductive hormonal profiles and histological findings during minipuberty. We included boys with congenital nonsyndromic cryptorchidism who underwent orchidopexy at less than 160 days of age, having no potential for spontaneous resolution clinically. At surgery, testicular biopsies and reproductive hormones were collected and compared with normal reference values. We measured the germ cells (G/T) and type A dark spermatogonia number per tubule. Thirty-five boys aged 37 to 159 (median age: 124) days at orchidopexy were included, five were bilateral. G/T was below the normal lower range in 26% (9/35) of the cases. In six of these cases, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were above 97.5 percentile, whereas one case had FSH below 2.5 percentile. Totally, 97% (33/34, one FSH was missing) exhibited a normal LH/FSH ratio. LH was more often above 97.5 percentile than FSH: 34% (12/35) versus 3% (1/34, p < 0.001). Inhibin B was below 2.5 percentile in 17% (6/35) of cases who all proved FSH above normal mean and four had LH above normal mean. Generally, reproductive hormonal profiles of the cryptorchid boys exhibited normal minipubertal pattern. Thus, 26% of the boys had reduced germ cell number, and transient hypogonadotropic hypogonadism could be suspected in few cases.

NANOS3 downregulation in Down syndrome hiPSCs during primordial germ cell-like cell differentiation.

Human infertility is a complex disorder at the genetic, molecular, cellular, organ, and hormonal levels. New developing technology based on the generation of human primordial germ cell-like cells (hPGCLCs) from induced pluripotent stem cells (hiPSCs) might improve understanding of early germ cell development (specification, migration, gametogenesis, and epigenetic reconstitutions), as well as offering a solution for infertility and hereditary disorders. In this study, we differentiated hiPSCs with trisomy 21 into hPGCLCs. In vitro-derived germ cells from hiPSCs with Down syndrome (DS) express hPGCLC core circuitry, EOMES, SOX17, and PRDM14 at relatively low levels. TFAP2C and PRDM1 were expressed and remained elevated, whereas NANOS3 and NANOG were downregulated in BMP4-induced hiPSCs with DS. The low level of NANOG and NANOS3 expression might negatively influence hPGCLC generation in DS hiPSCs. We suggest that DS hPGCLCs could be a suitable model for studying human early germ cell development, the epigenetic and molecular mechanisms of PGC specification and formation, as well as related infertility disorders, such as azoospermia and teratozoospermia.

Examining the Developmental Trajectory of an in Vitro Model of Mouse Primordial Germ Cells following Exposure to Environmentally Relevant Bisphenol A Levels.

Background:Animal-based studies indicate that bisphenol A (BPA) exposure is detrimental to reproductive health, but its impact on the earliest stages of germ cell development remains poorly defined.Objectives:Using a murine in vitro model of early germ cell specification and differentiation, we sought to assess whether exposure to low levels of BPA prior to formation of primordial germ cells (PGCs) alters their differentiation trajectory and unique molecular program.Methods:We used an established method of in vitro differentiation of mouse embryonic stem cells (ESCs) into epiblast-like cells (EpiLCs) followed by PGC-like cells (PGCLCs), which together recapitulate defined stages of early germ cell development. Cellular consequences were determined using hemocytometer-based cell counting, fixation, and intracellular staining, followed by flow cytometry/fluorescence-activated cell sorting (FACS) of cells exposed to increasing concentrations (range: ) of BPA. To interrogate and characterize gene expression differences resulting from BPA exposure, we also generated RNA-seq libraries from RNA extracted from FACS-purified PGCLCs and performed transcriptome analysis using bioinformatics-based approaches.Results:Exposure of EpiLCs to BPA resulted in higher numbers of cells that were associated with a higher proportion of cells in S-phase as well as a lower proportion undergoing apoptosis; this difference occurred in a concentration-dependent manner. Exposure also resulted in a greater fraction of EpiLCs showing signs of DNA damage. Remarkably, EpiLC exposure did not negatively affect PGC specification and resulted in a concentration-dependent effect on PGCLC proliferation in XX but not XY cells. PGCLC transcriptome analysis revealed an aberrant program with significant deregulation of X-linked genes and retrotransposon expression. Differential gene expression analysis also revealed the deregulation of genes associated with lipid metabolism as well as deregulated expression of genes associated with later stages of gametogenesis.Conclusions:To the best of our knowledge our findings represent the first characterization of the consequences of early BPA exposure on a model of mammalian PGC development, highlighting altered cell behavior, altered underlying pathways, and altered molecular processes. https://doi.org/10.1289/EHP8196

Metabolo-epigenetics: the interplay of metabolism and epigenetics during early germ cells development

Metabolites control epigenetic mechanisms, and conversly, cell metabolism is regulated at the epigenetic level in response to changes in the cellular environment. In recent years, this metabolo-epigenetic control of gene expression has been implicated in the regulation of multiple stages of embryonic development. The developmental potency of stem cells and their embryonic counterparts is directly determined by metabolic rewiring. Here, we review the current knowledge on the interplay between epigenetics and metabolism in the specific context of early germ cell development. We explore the implications of metabolic rewiring in primordial germ cells in light of their epigenetic remodeling during cell fate determination. Finally, we discuss the relevance of concerted metabolic and epigenetic regulation of primordial germ cells in the context of mammalian transgenerational epigenetic inheritance.

Cloning, pattern of gonadal soma-derived factor mRNA in the orange-spotted grouper, Epinephelus coioides

The orange-spotted grouper (Epinephelus coioides), as a protogynous hermaphrodite, provides a novel model for understanding the mechanisms of sex determination and differentiation in teleosts. Gonadal soma-derived factor (gsdf), involved in early germ cell development, is a new member of the transforming growth factor-beta (TGF-β) superfamily. In this study, a 2364-bp long gsdf gene was cloned from E. coioides and there was further analysis of its tissue distribution and patterns of gene expression during the female phase, bisexual gonads stage and testis stage. The cellular localization of Gsdf during the early sexual differentiation stage was also analyzed. In addition, the expression levels of gsdf, dmrt1, foxl2 and cyp19a1a in gonads during the early sexual differentiation stage were analyzed by real-time PCR. Tissue distribution analysis showed that gsdf was only expressed in the testis and ovary. The gene expression showed that gsdf was expressed significantly higher in testes and bisexual gonads compared to ovaries. The early-stage ovary showed a significant fluorescence signal in the supporting cells surrounding gonium and oogonium, but not pre-vitellogenic oocytes, indicating that gsdf expression was restricted to morphologically undifferentiated supporting cells in the early stages of oogenesis. During the early sexual differentiation stage, Real-time PCR showed that both the expression levels of gsdf and dmrt1 were progressively increased and the expression levels of foxl2 and cyp19a1a were growing slowly. These results suggest that gsdf could have roles in the early sexual differentiation in hermaphroditic, E. coioides.

Oxygen tension modulates the mitochondrial genetic bottleneck and influences the segregation of a heteroplasmic mtDNA variant in vitro

Most humans carry a mixed population of mitochondrial DNA (mtDNA heteroplasmy) affecting ~1–2% of molecules, but rapid percentage shifts occur over one generation leading to severe mitochondrial diseases. A decrease in the amount of mtDNA within the developing female germ line appears to play a role, but other sub-cellular mechanisms have been implicated. Establishing an in vitro model of early mammalian germ cell development from embryonic stem cells, here we show that the reduction of mtDNA content is modulated by oxygen and reaches a nadir immediately before germ cell specification. The observed genetic bottleneck was accompanied by a decrease in mtDNA replicating foci and the segregation of heteroplasmy, which were both abolished at higher oxygen levels. Thus, differences in oxygen tension occurring during early development likely modulate the amount of mtDNA, facilitating mtDNA segregation and contributing to tissue-specific mutation loads.

Advances in Female Germ Cell Induction from Pluripotent Stem Cells.

Germ cells are capable of maintaining species continuity through passing genetic and epigenetic information across generations. Female germ cells mainly develop during the embryonic stage and pass through subsequent developmental stages including primordial germ cells, oogonia, and oocyte. However, due to the limitation of using early human embryos as in vivo research model, in vitro research models are needed to reveal the early developmental process and related mechanisms of female germ cells. After birth, the number of follicles gradually decreases with age. Various conditions which damage ovarian functions would cause premature ovarian failure. Alternative treatments to solve these problems need to be investigated. Germ cell differentiation from pluripotent stem cells in vitro can simulate early embryonic development of female germ cells and clarify unresolved issues during the development process. In addition, pluripotent stem cells could potentially provide promising applications for female fertility preservation after proper in vitro differentiation. Mouse female germ cells have been successfully reconstructed in vitro and delivered to live offspring. However, the derivation of functional human female germ cells has not been fully achieved due to technical limitations and ethical issues. To provide an updated and comprehensive information, this review centers on the major studies on the differentiation of mouse and human female germ cells from pluripotent stem cells and provides references to further studies of developmental mechanisms and potential therapeutic applications of female germ cells.

Perinatal exposure to Bisphenol A disturbs the early differentiation of male germ cells

Understanding the effects of Bisphenol A (BPA) on early germ cell differentiation and their consequences in adult life is an area of growing interest in the field of endocrine disruption. Herein, we investigate whether perinatal exposure to BPA affects the differentiation of male germ cells in early life using a transgenic mouse expressing the GFP reporter protein under the Oct4 promoter. In this model, the expression of GFP reflects the expression of the Oct4 gene. This pluripotency gene is required to maintain the spermatogonial stem cells in an undifferentiated stage. Thus, GFP expression was used as a parameter to evaluate the effect of BPA on early germ cell development. Female pregnant transgenic mice were exposed to BPA by oral gavage, from embryonic day 5.5 to postnatal day 7 (PND7). The effects of BPA on male germ cell differentiation were evaluated at PND7, while sperm quality, testicular morphology, and protein expression of androgen receptor and proliferating cell nuclear antigen were studied at PND130.We found that perinatal/lactational exposure to BPA up-regulates the expression of Oct4-driven GFP in testicular cells at PND7. This finding suggests a higher proportion of undifferentiated spermatogonia in BPA-treated animals compared with non-exposed mice. Moreover, in adulthood, the number of spermatozoa per epididymis was reduced in those animals perinatally exposed to BPA.This work shows that developmental exposure to BPA disturbed the normal differentiation of male germ cells early in life, mainly by altering the expression of Oct4 and exerted long-lasting sequelae at the adult stage, affecting sperm count and testis.

Single-cell RNA sequencing reveals the landscape of early female germ cell development.

Meiosis initiation is a crucial step for the production of haploid gametes, which occurs from anterior to posterior in fetal ovaries. The asynchrony of the transition from mitosis to meiosis results in heterogeneity in the female germ cell populations, which limits the studies of meiosis initiation and progression at a higher resolution level. To dissect the process of meiosis initiation, we investigated the transcriptional profiles of 19363 single germ cells collected from E12.5, E14.5, and E16.5 mouse fetal ovaries. Clustering analysis identified seven groups and defined dozens of corresponding transcription factors, providing a global view of cellular differentiation from primordial germ cells toward meiocytes. Furthermore, we explored the dynamics of gene expression within the developmental trajectory with special focus on the critical state of meiosis. We found that meiosis initiation occurs as early as E12.5 and the cluster of oogonia_4 is the critical state between mitosis and meiosis. Our data provide key insights into the transcriptome features of peri-meiotic female germ cells, which offers new information not only on meiosis initiation and progression but also on screening pathogenic mutations in meiosis-associated diseases.

WNT signalling in the normal human adult testis and in male germ cell neoplasms.

Is WNT signalling functional in normal and/or neoplastic human male germ cells? Regulated WNT signalling component synthesis in human testes indicates that WNT pathway function changes during normal spermatogenesis and is active in testicular germ cell tumours (TGCTs), and that WNT pathway blockade may restrict seminoma growth and migration. Regulated WNT signalling governs many developmental processes, including those affecting male fertility during early germ cell development at embryonic and adult (spermatogonial) ages in mice. In addition, although many cancers arise from WNT signalling alterations, the functional relevance and WNT pathway components in TGCT, including germ cell neoplasia in situ (GCNIS), are unknown. The cellular distribution of transcripts and proteins in WNT signalling pathways was assessed in fixed human testis sections with normal spermatogenesis, GCNIS and seminoma (2-16 individuals per condition). Short-term (1-7 h) ligand activation and long-term (1-5 days) functional outcomes were examined using the well-characterised seminoma cell line, TCam-2. Pathway inhibition used siRNA or chemical exposures over 5 days to assess survival and migration. The cellular localisation of WNT signalling components was determined using in situ hybridisation and immunohistochemistry on Bouin's- and formalin-fixed human testis sections with complete spermatogenesis or germ cell neoplasia, and was also assessed in TCam-2 cells. Pathway function tests included exposure of TCam-2 cells to ligands, small molecules and siRNAs. Outcomes were measured by monitoring beta-catenin (CTNNB1) intracellular localisation, cell counting and gap closure measurements. Detection of nuclear-localised beta-catenin (CTNNB1), and key WNT signalling components (including WNT3A, AXIN2, TCF7L1 and TCF7L2) indicate dynamic and cell-specific pathway activity in the adult human testis. Their presence in germ cell neoplasia and functional analyses in TCam-2 cells indicate roles for active canonical WNT signalling in TGCT relating to viability and migration. All data were analysed to determine statistical significance. No large-scale datasets were generated in this study. As TGCTs are rare and morphologically heterogeneous, functional studies in primary cancer cells were not performed. Functional analysis was performed with the only well-characterised, widely accepted seminoma-derived cell line. This study demonstrated the potential sites and involvement of the WNT pathway in human spermatogenesis, revealing similarities with murine testis that suggest the potential for functional conservation during normal spermatogenesis. Evidence that inhibition of canonical WNT signalling leads to loss of viability and migratory activity in seminoma cells suggests that potential treatments using small molecule or siRNA inhibitors may be suitable for patients with metastatic TGCTs. This study was funded by National Health and Medical Research Council of Australia (Project ID 1011340 to K.L.L. and H.E.A., and Fellowship ID 1079646 to K.L.L.) and supported by the Victorian Government's Operational Infrastructure Support Program. None of the authors have any competing interests.

Subfamily-specific quantification of endogenous mouse L1 retrotransposons by droplet digital PCR

Long interspersed element type 1 (LINE-1; L1) mobilizes during early embryogenesis, neurogenesis, and germ cell development, accounting for 25% of disease-causing heritable insertions and 98% of somatic insertions in cancer. To better understand the regulation and impact of L1 mobilization in the genome, reliable methods for measuring L1 copy number variation (CNV) are needed. Here we present a comprehensive analysis of a droplet digital PCR (ddPCR) based method for quantifying endogenous mouse L1. We provide experimental evidence that ddPCR assays can be designed to target specific L1 subfamilies using diagnostic single nucleotide polymorphisms (SNPs). The target and off-target L1 subfamilies form distinct droplet clusters, which were experimentally verified using both synthetic gene fragments and endogenous L1 derived plasmid clones. We further provide a roadmap for in silico assay design and evaluation of target specificity, ddPCR testing, and optimization for L1 CNV quantification. The assay can achieve a sensitivity of 5% CNV with 8 technical replicates. With 24 technical replicates, it can detect 2% CNV because of the increased precision. The same approach will serve as a guide for the development of ddPCR based assays for quantifying human L1 copy number and any other high copy genomic target sequences.

Positive Roles of Resveratrol in Early Development of Testicular Germ Cells against Maternal Restraint Stress in Mice.

Simple SummaryMaternal stress during pregnancy affected the early programming of the brain in the fetus with changes in neuroendocrine regulation, and offspring behavior was proven in the literature. In addition, resveratrol (RES) was reported to play positive roles against stress. However, how maternal stress affects testicular development and whether RES has potential protecting roles is unknown.Our present study was designed to evaluate the effects of resveratrol (RES) in Swiss mice by exposing them to prenatal stress. Twenty-four Swiss mice were divided into four groups: control (C), maternal restraint stress (MRS), maternal restraint stress + resveratrol (MRS + RES) 2 mg, and maternal restraint stress + resveratrol (MRS + RES) 20 mg. Dams were exposed to stress by restraint in plastic tubes for four hours a day from 12–18 days of gestation. The results showed that male pups of MRS were significantly decreased in the testis weight, anogenital distance, area of seminiferous tubules, diameter of seminiferous tubules, area of the lumen, diameter of the lumen, and epithelial height of seminiferous tubules. However, the anomalies of the reproductive tract produced under restraint stress were neutralized by the use of RES 2 mg/kg. A significant difference was observed between terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)- positive germ cells in MRS and MRS + RES 20 mg/kg groups, while it was non-significant between MRS + RES 2 mg/kg and C groups. Apart from these effects, blood glucose levels were increased in MRS and MRS + RES 20 mg/kg groups, while experimental animals of the MRS + RES 2 mg/kg group significantly recovered. These results suggested that a lower dose of RES could cure the adverse effects of prenatal stress in early age male progeny. Thus, our study suggests, for the first time, practical values for a lower dose of RES 2 mg/kg as a safe and effective agent in the first week age of prenatally stressed mice.

Cannabinoid Receptors Signaling in the Development, Epigenetics, and Tumours of Male Germ Cells.

Endocannabinoids are natural lipid molecules whose levels are regulated by specific biosynthetic and degradative enzymes. They bind to and activate two main cannabinoid receptors type 1 (CB1) and type 2 (CB2), and together with their metabolizing enzymes form the “endocannabinoid system” (ECS). In the last years, the relevance of endocannabinoids (eCBs) as critical modulators in various aspects of male reproduction has been pointed out. Mammalian male germ cells, from mitotic to haploid stage, have a complete ECS which is modulated during spermatogenesis. Compelling evidence indicate that in the testis an appropriate “eCBs tone”, associated to a balanced CB receptors signaling, is critical for spermatogenesis and for the formation of mature and fertilizing spermatozoa. Any alteration of this system negatively affects male reproduction, from germ cell differentiation to sperm functions, and might have also an impact on testicular tumours. Indeed, most of testicular tumours develop during early germ-cell development in which a maturation arrest is thought to be the first key event leading to malignant transformation. Considering the ever-growing number and complexity of the data on ECS, this review focuses on the role of cannabinoid receptors CB1 and CB2 signaling in male germ cells development from gonocyte up to mature spermatozoa and in the induction of epigenetic alterations in these cells which might be transmitted to the progeny. Furthermore, we present new evidence on their relevance in testicular cancer.