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Background: The majority of testicular germ cell cancers develop through a pre-invasive carcinoma in situ (CIS) stage. The CIS cell is a neoplastic counterpart of foetal germ cells. During their development, foetal germ cells undergo extensive and essential epigenetic modifications, but little is known about epigenetic patterns in CIS cells., Methods: Immunohistochemistry was used to investigate epigenetic patterns in CIS, germ cell tumours, normal adult and foetal testicular tissue., Results: CIS cells show low levels of DNA methylation and repressive histone modifications H3K9me2 and H3K27me3, but high levels of H3K9 acetylation, H3K4 methylation and H2A.Z, which all are associated with an activated and accessible chromatin structure. Collectively this renders a permissive chromatin structure and in accordance high levels of RNA polymerase II activity and proliferation (Ki-67 and mitotic index) is observed in CIS cells. Epigenetic patterns similar to that of CIS cells were observed in human gonocytes present within sex cords in foetal testes but correspond to migrating primordial germ cell in mice. Development of overt tumours involves epigenetic repression of the chromatin., Conclusion: CIS cells have a permissive and foetal-like chromatin structure, which is associated with a high transcriptional and proliferative activity, likely empowering neoplastic transformation. Developmental epigenetic cues in foetal germ cells are substantially different between humans and mice.

The germline cells are essential for the propagation of human beings, thus essential for the survival of mankind. The germline stem cells, as a unique cell type, generate various states of germ stem cells and then differentiate into specialized cells, spermatozoa and ova, for producing offspring, while self-renew to generate more stem cells. Abnormal development of germline stem cells often causes severe diseases in humans, including infertility and cancer. Primordial germ cells (PGCs) first emerge during early embryonic development, migrate into the gentile ridge, and then join in the formation of gonads. In males, they differentiate into spermatogonial stem cells, which give rise to spermatozoa via meiosis from the onset of puberty, while in females, the female germline stem cells (FGSCs) retain stemness in the ovary and initiate meiosis to generate oocytes. Primordial germ cell-like cells (PGCLCs) can be induced in vitro from embryonic stem cells or induced pluripotent stem cells. In this review, we focus on current advances in these embryonic and adult germline stem cells, and the induced PGCLCs in humans, provide an overview of molecular mechanisms underlying the development and differentiation of the germline stem cells and outline their physiological functions, pathological implications, and clinical applications., (© 2022. The Author(s).)

Background and Objectives: The presence and contribution of senescent cells in premalignant lesions is well documented, but not in germ cell neoplasia in situ. The purpose of this study is to identify the presence of senescent cells in pre-malignant testicular conditions and in different histological types of testicular cancer. Materials and Methods: Thirty patients who underwent orchiectomy due to testicular tumors were included. Formalin-fixed paraffin-embedded (FFPE) testicular tissue for each patient was available. Sections from these specimens were examined by immunohistochemical analysis with the following markers: GL13 for cellular senescence, p21WAF1/Cip1 for cell cycle arrest, and Ki67 for cell proliferation. Results: Thirteen (43.3%) suffered from seminoma with a mean total proportion of GCNIS senescence of 20.81 ± 6.81%. In the group of embryonal testicular tumors, nine (30%) patients were included, with an average rate of 6.64 ± 5.42% of senescent cells in GCNIS. One (3.3%) patient suffered from chondrosarcoma in which 7.9% of GL13+ cells were detected in GCNIS. Four (13.4%) patients suffered from teratoma and three (10%) from yolk sac tumors, while GCNIS senescence was detected in a range of 4.43 ± 1.78% and 3.76 ± 1.37%, respectively. Conclusions: Cellular senescence was detected in both germ cell neoplasia in situ and testicular cancer, but was more prevalent within the premalignant lesions. [ABSTRACT FROM AUTHOR]

Background: Testicular germ cell tumours (TGCTs) represent a clinical challenge; they are most prevalent in young individuals and are triggered by molecular mechanisms that are not fully understood. The origin of TGCTs can be traced back to primordial germ cells that fail to mature during embryonic development. These cells express high levels of pluripotency factors, including the transcription factor NANOG which is highly expressed in TGCTs. Gain or amplification of the NANOG locus is common in advanced tumours, suggesting a key role for this master regulator of pluripotency in TGCT stemness and malignancy. Methods: In this study, we analysed the expression of microRNAs (miRNAs) that are regulated by NANOG in TGCTs via integrated bioinformatic analyses of data from The Cancer Genome Atlas and NANOG chromatin immunoprecipitation in human embryonic stem cells. Through gain-of-function experiments, MIR9-2 was further investigated as a novel tumour suppressor regulated by NANOG. After transfection with MIR9-2 mimics, TGCT cells were analysed for cell proliferation, invasion, sensitivity to cisplatin, and gene expression signatures by RNA sequencing. Results: For the first time, we identified 86 miRNAs regulated by NANOG in TGCTs. Among these, 37 miRNAs were differentially expressed in NANOG-high tumours, and they clustered TGCTs according to their subtypes. Binding of NANOG within 2 kb upstream of the MIR9-2 locus was associated with a negative regulation. Low expression of MIR9-2 was associated with tumour progression and MIR9-2-5p was found to play a role in the control of tumour stemness. A gain of function of MIR9-2-5p was associated with reduced proliferation, invasion, and sensitivity to cisplatin in both embryonal carcinoma and seminoma tumours. MIR9-2-5p expression in TGCT cells significantly reduced the expression of genes regulating pluripotency and cell division, consistent with its functional effect on reducing cancer stemness. Conclusions: This study provides new molecular insights into the role of NANOG as a key determinant of pluripotency in TGCTs through the regulation of MIR9-2-5p, a novel epigenetic modulator of cancer stemness. Our data also highlight the potential negative feedback mediated by MIR9-2-5p on NANOG expression, which could be exploited as a therapeutic strategy for the treatment of TGCTs. [ABSTRACT FROM AUTHOR]

Redox balance plays an important role in testicular homeostasis. While lots of antioxidant molecules have been identified as widely expressed, the understanding of the critical mechanisms for redox management in male germ cells is inadequate. This study identified LanCL2 as a major male germ cell-specific antioxidant gene that is important for testicular homeostasis. Highly expressed in the brain and testis, LanCL2 expression correlates with testicular maturation and brain development. LanCL2 is enriched in spermatocytes and round spermatids of the testis. By examining LanCL2 knockout mice, we found that LanCL2 deletion did not affect postnatal brain development but injured the sperm parameters of adult mice. With histopathological analysis, we noticed that LanCL2 KO caused a pre-maturation and accelerated the self-renewal of spermatogonial stem cells in the early stage of spermatogenesis. In contrast, at the adult stage, LanCL2 KO damaged the acrosomal maturation in spermiogenesis, resulting in spermatogenic defects with a reduced number and motility of spermatozoa. Furthermore, we show that this disruption of testicular homeostasis in the LanCL2 KO testis was due to dysbalanced testicular redox homeostasis. This study demonstrates the critical role of LanCL2 in testicular homeostasis and redox balance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Testicular germ cell tumors (TGCTs) represent the most frequent malignancy in young adult men and have one the highest heritability rates among all cancers. A recent multicenter case–control study identified CHEK2 as the first moderate-penetrance TGCT predisposition gene. Here, we analyzed CHEK2 in 129 TGCT cases unselected for age of onset, histology, clinical outcome, and family history of any cancer, and the frequency of identified variants was compared to findings in 27,173 ancestry-matched cancer-free men. We identified four TGCT cases harboring a P/LP variant in CHEK2 (4/129, 3.10%), which reached statistical significance (p = 0.0191; odds ratio (OR), 4.06; 95% CI, 1.59–10.54) as compared to the control group. Cases with P/LP variants in CHEK2 developed TGCT almost 6 years earlier than individuals with CHEK2 wild-type alleles (5.67 years; 29.5 vs. 35.17). No association was found between CHEK2 status and further clinical and histopathological characteristics, including histological subtypes, the occurrence of aggressive TGCT, family history of TGCT, and family history of any cancer. In addition, we found significant enrichment for the low-penetrance CHEK2 variant p.Ile157Thr (p = 0.0259; odds ratio (OR), 3.69; 95% CI, 1.45–9.55). Thus, we provide further independent evidence of CHEK2 being a moderate-penetrance TGCT predisposition gene. [ABSTRACT FROM AUTHOR]

Minipuberty is a transient activity period of the hypothalamic-pituitary-gonadal (HPG) axis in the postnatal and infant period in humans and non-human primates. Hallmarks of this period are surging serum concentrations of reproductive hormones. While in females, the role of minipuberty seems to be dispensable for future fertility, in males, it is significantly associated with reproductive function in later life. In males, this activity period promotes further masculinization, including testicular and penile growth, as well as completion of testicular descent if not already achieved at birth. At the testicular level, both, somatic and germ cells undergo proliferation and partial maturation during this period. Minipuberty is thought to prime male gonadal tissue for subsequent growth and maturation. Notably, perturbed or absent minipuberty is associated with reduced male reproductive function in adulthood. While the sustained HPG axis activity during adulthood is known to control reproductive function, minipuberty appears to be a prerequisite for obtaining full male reproductive function in later life, thereby determining future fertility potential, i.e. the ability to father a child. This review maps the role of male minipuberty for reproductive function and presents suitable animal models to study minipuberty. Also, it describes the development and maturation of testicular cell types, discusses short- and long-term effects of minipuberty and highlights future research perspectives. [ABSTRACT FROM AUTHOR]

Male germ cells undergo a complex sequence of developmental events throughout fetal and postnatal life that culminate in the formation of haploid gametes: the spermatozoa. Errors in these processes result in infertility and congenital abnormalities in offspring. Male germ cell development starts when pluripotent cells undergo specification to sexually uncommitted primordial germ cells, which act as precursors of both oocytes and spermatozoa. Male-specific development subsequently occurs in the fetal testes, resulting in the formation of spermatogonial stem cells: the foundational stem cells responsible for lifelong generation of spermatozoa. Although deciphering such developmental processes is challenging in humans, recent studies using various models and single-cell sequencing approaches have shed new insight into human male germ cell development. Here, we provide an overview of cellular, signaling and epigenetic cascades of events accompanying male gametogenesis, highlighting conserved features and the differences between humans and other model organisms. [ABSTRACT FROM AUTHOR]

Approximately 8% of the human genome, over four times more than its protein-coding regions, comprises sequences of viral origin that are known as human endogenous retroviral elements (HERVs). Present in the genome of all human cells, HERVs resulted from the integration of now-extinct exogenous retroviruses into mammalian ancestor germ cells or their precursors on several occasions, sometimes as long as tens of millions of years ago. Most HERVs have become silenced because of mutations such as substitutions, insertions, or deletions, and as a result of epigenetic changes, and are vertically transmitted in the population. Considered for a long time to be part of the "junk DNA," HERVs were shown, in more recent years, to perform critical functions in the host. Two of the very few HERVs known to encode functional proteins, syncytin-1 and syncytin-2, are critical during embryogenesis, when they contribute to the formation of the placenta and facilitate tolerance of the maternal immune system toward the developing fetus. Homologs of syncytin-encoding genes were described in several other species, and it appears that during evolution they were stably endogenized into the respective genomes on multiple occasions and became co-opted for critical physiological functions. The aberrant expression of HERVs has been linked to conditions that include infectious, autoimmune, malignant, and neurological diseases. HERVs, our genomic fossils and storytellers, provide a fascinating and somewhat mysterious insight into our co-evolution with viruses, and will undoubtedly offer many teachings, surprises, and paradigm changes for years to come. [ABSTRACT FROM AUTHOR]

Human prepubertal testicular tissues are rare, but organ culture conditions to develop a system for human in vitro-spermatogenesis are an essential option for fertility preservation in prepubertal boys subjected to gonadotoxic therapy. To avoid animal testing in line with the 3Rs principle, organ culture conditions initially tested on human adult testis tissue were applied to prepubertal samples (n = 3; patient ages 7, 9, and 12 years). Tissues were investigated by immunostaining and transmission electron microscopy (TEM), and the collected culture medium was profiled for steroid hormones by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Culture conditions proved suitable for prepubertal organ culture since SSCs and germ cell proliferation could be maintained until the end of the 3-week-culture. Leydig cells (LCs) were shown to be competent for steroid hormone production. Three additional testis tissues from boys of the same age were examined for the number of germ cells and undifferentiated spermatogonia (SPG). Using TEM micrographs, eight tissues from patients aged 1.5 to 13 years were examined, with respect to the sizes of mitochondria (MT) in undifferentiated SPG and compared with those from two adult testicular tissues. Mitochondrial sizes were shown to be comparable between adults and prepubertal boys from approximately 7 years of age, which suggests the transition of SSCs from normoxic to hypoxic metabolism at about or before this time period. [ABSTRACT FROM AUTHOR]

This concise review summarises tissue and serum markers useful for differential diagnosis of germ cell tumours (GCT), with focus on the most common testicular GCT (TGCT). GCT are characterised by phenotypic heterogeneity due to largely retained embryonic pluripotency and aberrant somatic differentiation. TGCT that occur in young men are divided into two main types, seminoma and nonseminoma, both derived from a pre-invasive germ cell neoplasia in situ (GCNIS), which originates from transformed foetal gonocytes. In severely dysgenetic gonads, a GCNIS-resembling lesion is called gonadoblastoma. GCT occur rarely in young children (infantile GCT) in whom the pathogenesis is different (no GCNIS/gonadoblastoma stage) but the histopathological features are similar to the adult GCT. The rare spermatocytic tumour of older men is derived from post-pubertal spermatogonia that clonally expand due to gain-of function mutations in survival-promoting genes (e.g. FGFR3, HRAS), thus this tumour has a different expression profile than GCNIS-derived TGCT. Clinically most informative immunohistochemical markers for GCT, except teratoma, are genes expressed in primordial germ cells/gonocytes and embryonic pluripotency-related factors, such as placental-like alkaline phosphatase (PLAP), OCT4 (POU5F1), NANOG, AP-2¿ (TFAP2C) and LIN28, which are not expressed in normal adult germ cells. Some of these markers can also be used for immunocytochemistry to detect GCNIS or incipient tumours in semen samples. Gene expression in GCT is regulated in part by DNA and histone modifications, and the epigenetic profile of these tumours is characterised by genome-wide demethylation, except nonseminomas. In addition, a recently discovered mechanism of post-genomic gene expression regulation involves small non-coding RNAs, predominantly micro-RNA (miR). Testicular GCT display micro-RNA profiles similar to embryonic stem cells. Targeted miRNA-based blood tests for miR-371-3 and miR-367 clusters are currently under development and hold a great promise for the future. In some patients miR-based tests may be even more sensitive than the classical serum tumour markers, ß -chorio-gonadotrophin (ß-hCG), ¿-fetoprotein (AFP) and lactate dehydrogenase (LDH), which are currently used in the clinic. In summary, research advances have provided clinicians with a panel of molecular markers, which allow specific diagnosis of various subtypes of GCT and are very useful for early detection at the precursor stage and for monitoring of patients during the follow-up. [ABSTRACT FROM AUTHOR]

Background: Screening programmes for contralateral carcinoma in situ (CIS) testis in patients with unilateral germ-cell cancer (GCC) have never been evaluated. We investigated the effect of screening for contralateral CIS in a large nationwide, population-based study. Patients and methods: A contralateral single-site biopsy was offered to 4130 patients in whom GCC had been diagnosed in 1984-2007 (screened cohort); 462 patients in whom GCC was diagnosed in 1984-1988 comprised the unscreened cohort. Cases with CIS were offered radiotherapy. Initially CIS-negative biopsies in patients with metachronous GCC were revised according to today's standards. Risk for metachronous GCC was estimated using cumulative incidence and the Cox proportional hazards model. Results: In the screened cohort, contralateral CIS was found in 181 (4.4%) patients. The cumulative incidence of metachronous GCC after 20 years was 1.9% in the screened cohort and 3.1% in the unscreened cohort (P = 0.097), hazard ratio (HR) for the unscreened cohort: 1.59 (P = 0.144). Expert revision with contemporary methodology of CIS-negative biopsy samples from patients with metachronous cancer revealed CIS in 17 out of 45 (38%) cases. Decreased risks for metachronous GCC were related to older age at diagnosis (HR 0.52 per 10 years, P < 0.001) and chemotherapy (HR 0.35, P = 0.002). Limitations include the small number of patients in the unscreened cohort and the retrospective study design. Conclusions: Our evaluation of a national population-based screening programme for contralateral CIS in patients with testicular cancer showed no significant difference in the risk for metachronous GCC between a screened and an unscreened cohort. Single-site biopsy including modern immunohistochemistry does not identify all cases of CIS. [ABSTRACT FROM AUTHOR]

The pathways of gametogenesis encompass elaborate cellular specialization accompanied by precise partitioning of the genome content in order to produce fully matured spermatozoa and oocytes. Transcription factors are an important class of molecules that function in gametogenesis to regulate intrinsic gene expression programs, play essential roles in specifying (or determining) germ cell fate and assist in guiding full maturation of germ cells and maintenance of their populations. Moreover, in order to reinforce or redirect cell fate in vitro, it is transcription factors that are most frequently induced, over-expressed or activated. Many reviews have focused on the molecular development and genetics of gametogenesis, in vivo and in vitro, in model organisms and in humans, including several recent comprehensive reviews: here, we focus specifically on the role of transcription factors. Recent advances in stem cell biology and multi-omic studies have enabled deeper investigation into the unique transcriptional mechanisms of human reproductive development. Moreover, as methods continually improve, in vitro differentiation of germ cells can provide the platform for robust gain- and loss-of-function genetic analyses. These analyses are delineating unique and shared human germ cell transcriptional network components that, together with somatic lineage specifiers and pluripotency transcription factors, function in transitions from pluripotent stem cells to gametes. This grand theme review offers additional insight into human infertility and reproductive disorders that are linked predominantly to defects in the transcription factor networks and thus may potentially contribute to the development of novel treatments for infertility. [ABSTRACT FROM AUTHOR]

Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior. [ABSTRACT FROM AUTHOR]

Study Question: Does pituitary response to a GnRH stimulation test differ according to the different FSHB-211 G/T genotypes?Summary Answer: The promoter polymorphism FSHB-211 G > T affects the pituitary response to exogenous GnRH stimulation by reducing FSH and increasing LH outputs.What Is Known Already: The FSHB-211 G > T single nucleotide polymorphism (SNP) is known to affect pituitary FSH output by impairing the transcriptional activity of FSHB.Study Design, Size, Duration: This was a cross-sectional, retrospective study on 67 male subjects (mean age: 24.6 ± 10.3 years) undergoing a GnRH stimulation test for diagnostic purposes in cases of secondary hypogonadism.Participants/materials, Setting, Methods: A GnRH stimulation test was performed by administering an i.v. bolus of 100 µg of the GnRH-analogue gonadorelin acetate to all patients, with blood samples drawn from the cubital vein immediately prior to injection (T0) and 30 (T1) and 45 minutes (T2) after. Clinical and genetic data were retrieved from a computerized database. Linear longitudinal mixed-effect models were used to assess the effects of SNP genotype on FSH and LH levels over time via additive and recessive models.Main Results and the Role Of Chance: An overall marked increase in serum FSH and LH following administration i.v. of 100 µg of an LHRH-analogue was found (P < 0.0001 for linear trend, both models). Peak levels of LH were significantly higher in TT carriers than in GT and GG carriers (P = 0.012); no significant between-groups difference was found concerning stimulated FSH levels. In both the additive and recessive model, the main effect of T allele(s) did not reach statistical significance concerning FSH levels (P = 0.9502 and P = 0.8576, respectively), yet interaction effects over time demonstrated an attenuated response in T-allele carriers compared to the GG-allele carriers (P = 0.0219 and P = 0.0276). Main and interaction effects for LH were significant in both the additive (P = 0.0022 and P = 0.0013, respectively) and recessive model (P = 0.0025 and P = 0.0016, respectively).Limitations, Reasons For Caution: Given the retrospective nature of the study and the small number of TT carriers, results should be interpreted with caution.Wider Implications Of the Findings: The FSHB c.-211G>T polymorphism might result in an impaired response to endogenous, as well as exogenous, GnRH stimulation. This finding might contribute to the clinical phenotype of reduced testicular volume and sperm count for patients carrying one or two T alleles.Study Funding/competing Interest(s): Parts of the study were supported by the German Research Foundation (CRU326 Male Germ Cells). On behalf of all authors, the corresponding author states that there is no conflict of interest.Trial Registration Number: NA. [ABSTRACT FROM AUTHOR]

In each generation, the germline is tasked with producing somatic lineages that form the body, and segregating a population of cells for gametogenesis. During animal development, when do cells of the germline irreversibly commit to producing gametes? Integrating findings from diverse species, we conclude that the final commitment of the germline to gametogenesis -- the process of germ cell determination -- occurs after primordial germ cells (PGCs) colonize the gonads. Combining this understanding with medical findings, we present amodel whereby germcell tumors arise fromcells that failed to undertake germcell determination, regardless of their having colonized the gonads.We propose that the diversity of cell types present in these tumors reflects the broad developmental potential of migratory PGCs. [ABSTRACT FROM AUTHOR]

Spermatogenesis is characterized by unique epigenetic programs that enable chromatin remodeling and transcriptional regulation for proper meiotic divisions and germ cells maturation. Paternal lifestyle stressors such as diet, drug abuse, or psychological trauma can directly impact the germ cell epigenome and transmit phenotypes to the next generation, pointing to the importance of epigenetic regulation during spermatogenesis. It is established that environmental perturbations can affect the development and behavior of the offspring through epigenetic inheritance, including changes in small non-coding RNAs, DNA methylation, and histones post-translational modifications. But how male germ cells react to lifestyle stressors and encode them in the paternal epigenome is still a research gap. Most lifestyle stressors activate catecholamine circuits leading to both acute and long-term changes in neural functions, and epigenetic mechanisms show strong links to both long-term and rapid, dynamic gene expression regulation during stress. Importantly, the testis shares a molecular and transcriptional signature with the brain tissue, including a rich expression of catecholaminergic elements in germ cells that seem to respond to stressors with similar epigenetic and transcriptional profiles. In this minireview, we put on stage the action of catecholamines as possible mediators between paternal stress responses and epigenetic marks alterations during spermatogenesis. Understanding the epigenetic regulation in spermatogenesis will contribute to unravel the coding mechanisms in the transmission of the biological impacts of stress between generations. [ABSTRACT FROM AUTHOR]

Background: Fatty acid oxidation plays an important role in a variety of developing and mature organ systems. However, the role of this metabolic pathway in different stages of testis development remains unknown. Here, we elucidate the mechanisms by which fatty acid oxidation regulates the maintenance and differentiation of gonocytes and spermatogonial stem cells. Results: During E13.5-E15.5, male germ cells gradually enter the mitotic arrest phase, while the expression of CPT1A, a rate-limiting enzyme for fatty acid oxidation, gradually increases. Therefore, we treated pregnant mice (E13.5 to E15.5) with etomoxir, which is an inhibitor of CPT1A. Etomoxir-treated mice showed no difference in embryonic morphology; however, etomoxir-treated male gonocytes exited mitotic arrest, and cells of the gonad underwent apoptosis. In addition, etomoxir-treated mice at P7 displayed impaired homing of spermatogonia and increased cell apoptosis. We further demonstrated that inhibition of fatty acid oxidation in gonads was associated with gonocyte differentiation events and the histone modification H3K27ac. Conclusions: Inhibiting fatty acid oxidation can specifically reduce the level of H3K27ac in the reproductive crest, which may be the cause of the down-regulation of male differentiation-specific gene expression, which ultimately leads to the male primordial germ cells exited from mitotic arrest. Our work uncovers metabolic reprogramming during male gonadal development, revealing that it plays an important role in the maintenance of gonocytes in a differentiated and quiescent state during foetal testis development. [ABSTRACT FROM AUTHOR]

The G protein-coupled estrogen receptor (GPER), also known as GPR30, is a widely conserved 7-transmembrane-domain protein which has been identified as a novel 17β-estradiol-binding protein that is structurally distinct from the classic oestrogen receptors (ERα and ERβ). There are still conflicting data regarding the exact role and the natural ligand of GPER/GPR30 in reproductive tracts as both male and female knock-out mice are fertile and have no abnormalities of reproductive organs. Testicular germ cell cancers (TGCCs) are the most common malignancy in young males and the most frequent cause of death from solid tumors in this age group. Clinical and experimental studies suggested that estrogens participate in the physiological and pathological control of male germ cell proliferation. In human seminoma cell line, while 17β-estradiol (E2) inhibits in vitro cell proliferation through an ERβ-dependent mechanism, an impermeable E2 conjugate (E2 coupled to BSA), in vitro cell proliferation is stimulated by activating ERK1/2 and protein kinase A through a membrane GPCR that we further identified as GPER/GPR30. The same effect was observed with low but environmentally relevant doses of BPA, an estrogenic endocrine disrupting compound. Furthermore, GPER/GPR30 is specifically overexpressed in seminomas but not in non-seminomas and this overexpression is correlated with an ERβ-downregulation. This GPER/GPR30 overexpression could be linked to some genetic variations, as single nucleotide polymorphisms, which was also reported in other hormone-dependent cancers. We will review here the implication of GPER/GPR30 in TGCCs pathophysiology and the arguments to consider GPER/GPR30 as a potential therapeutic target in humans. [ABSTRACT FROM AUTHOR]

Azoospermia is a condition defined as the absence of spermatozoa in the ejaculate, but the testicular phenotype of men with azoospermia may be very variable, ranging from full spermatogenesis, through arrested maturation of germ cells at different stages, to completely degenerated tissue with ghost tubules. Hence, information regarding the cell-type-specific expression patterns is needed to prioritise potential pathogenic variants that contribute to the pathogenesis of azoospermia. Thanks to technological advances within next-generation sequencing, it is now possible to obtain detailed cell-type-specific expression patterns in the testis by single-cell RNA sequencing. However, to interpret single-cell RNA sequencing data properly, substantial knowledge of the highly sophisticated data processing and visualisation methods is needed. Here we review the complex cellular structure of the human testis in different types of azoospermia and outline how known genetic alterations affect the pathology of the testis. We combined the currently available single-cell RNA sequencing datasets originating from the human testis into one dataset covering 62,751 testicular cells, each with a median of 2637 transcripts quantified. We show what effects the most common data-processing steps have, and how different visualisation methods can be used. Furthermore, we calculated expression patterns in pseudotime, and show how splicing rates can be used to determine the velocity of differentiation during spermatogenesis. With the combined dataset we show expression patterns and network analysis of genes known to be involved in the pathogenesis of azoospermia. Finally, we provide the combined dataset as an interactive online resource where expression of genes and different visualisation methods can be explored (https://testis.cells.ucsc.edu/). [ABSTRACT FROM AUTHOR]

Study Question: Is WNT signalling functional in normal and/or neoplastic human male germ cells?Summary Answer: 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.What Is Known Already: 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.Study Design, Size, Duration: 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.Participants/materials, Setting, Methods: 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.Main Results and the Role Of Chance: 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.Large Scale Data: No large-scale datasets were generated in this study.Limitations, Reasons For Caution: 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.Wider Implications Of the Findings: 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.Study Funding and Competing Interest(s): 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. [ABSTRACT FROM AUTHOR]

Klinefelter syndrome (KS) is the most common sex chromosome aneuploidy. A distinctive characteristic of KS is oligozoospermia. Despite multiple studies that have described the natural history of the degenerative process of germ cells in patients with KS, the molecular mechanisms that initiate this process are not well characterized. MicroRNA (miRNA)-mediated post-transcriptional control mechanisms have been increasingly recognized as important regulators of spermatogenesis; however, only a few studies have evaluated the role of miRNAs in the gonadal failure of these patients. Here, we describe a differential expression profile for the miRNAs in testicular tissue samples taken from KS patients. We analysed testicular tissue samples from 4 KS patients and 5 control patients (obstructive azoospermia) through next-generation sequencing, which can provide information about the mechanisms involved in the degeneration of germ cells. A distinctive differential expression profile was identified for 166 miRNAs in the KS patients: 66 were upregulated, and 100 were downregulated. An interactome analysis was performed for 7 of the upregulated and the 20 downregulated miRNAs. The results showed that the target genes are involved in the development, proliferation, and differentiation processes of spermatogenesis, which may explain their role in the development of infertility. This is the first report of a miRNA expression profile generated from testicular tissue samples of KS patients. [ABSTRACT FROM AUTHOR]

Background: Testicular germ cell tumours (TGCTs) are characterised by an overall high cisplatin-sensitivity which has been linked to their continued expression of pluripotency factors. Recently, the Nodal signalling pathway has been implicated in the regulation of pluripotency factor expression in fetal germ cells, and the pathway could therefore also be involved in regulating expression of pluripotency factors in malignant germ cells, and hence cisplatin-sensitivity in TGCTs.Methods: We used in vitro culture of the TGCT-derived cell line NTera2, ex vivo tissue culture of primary TGCT specimens and xenografting of NTera2 cells into nude mice in order to investigate the consequences of manipulating Nodal and Activin signalling on pluripotency factor expression, apoptosis, proliferation and cisplatin-sensitivity.Results: The Nodal signalling factors were markedly expressed concomitantly with the pluripotency factor OCT4 in GCNIS cells, seminomas and embryonal carcinomas. Despite this, inhibition of Nodal and Activin signalling either alone or simultaneously did not affect proliferation or apoptosis in malignant germ cells in vitro or ex vivo. Interestingly, inhibition of Nodal signalling in vitro reduced the expression of pluripotency factors and Nodal pathway genes, while stimulation of the pathway increased their expression. However, cisplatin-sensitivity was not affected following pharmacological inhibition of Nodal/Activin signalling or siRNA-mediated knockdown of the obligate co-receptor CRIPTO in NTera2 cells in vitro or in a xenograft model.Conclusion: Our findings suggest that the Nodal signalling pathway may be involved in regulating pluripotency factor expression in malignant germ cells, but manipulation of the pathway does not appear to affect cisplatin-sensitivity or tumour cell proliferation. [ABSTRACT FROM AUTHOR]

In chickens, the embryonic ovary differentiates into two distinct domains before meiosis: a steroidogenic core (the female medulla), overlain by the germ cell niche (the cortex). The differentiation of the medulla is a cell-autonomous process based on chromosomal sex identity (CASI). In order to address the extent to which cortex differentiation depends on intrinsic or extrinsic factors, we generated models of gonadal intersex by mixing ZW (female) and ZZ (male) cells in gonadal chimeras, or by altering oestrogen levels of ZW and ZZ embryos. We found that CASI does not apply to the embryonic cortex. Both ZWand ZZ cells can form the cortex and this can happen independently of the phenotypic sex of the medulla as long as oestrogen is provided. We also show that the cortex-promoting activity of oestrogen signalling is mediated via estrogen receptor alpha within the left gonad epithelium. However, the presence of a medulla with an 'intersex' or male phenotype may compromise germ cell progression into meiosis, causing cortical germ cells to remain in an immature state in the embryo. [ABSTRACT FROM AUTHOR]

Background: Testicular germ cell cancer (TGCC) develops from pre-malignant germ neoplasia in situ (GCNIS) cells. GCNIS originates from fetal gonocytes (POU5F1+/MAGE-A4-), which fail to differentiate to pre-spermatogonia (POU5F1-/MAGE-A4+) and undergo malignant transformation. Gankyrin is an oncogene which has been shown to prevent POU5F1 degradation and specifically interact with MAGE-A4 in hepatocellular carcinoma (HCC) cells. We aimed to investigate the role of Gankyrin in progression from gonocyte to pre-invasive GCNIS and subsequent invasive TGCC.Methods: We determined Gankyrin expression in human fetal testicular tissue (gestational weeks 9-20; n = 38), human adult testicular tissue with active spermatogenesis (n = 9), human testicular tissue with germ cell maturation delay (n = 4), testicular tissue from patients with pre-invasive GCNIS (n = 6), and invasive TGCC including seminoma (n = 6) and teratoma (n = 7). Functional analysis was performed in-vitro by siRNA knock-down of Gankyrin in the NTera2 cells (derived from embryonal carcinoma).Results: Germ cell expression of Gankyrin was restricted to a sub-population of prespermatogonia in human fetal testes. Nuclear Gankyrin was also expressed in GCNIS cells of childhood and adult pre-invasive TGCC patients, and in GCNIS from seminoma and non-seminoma patients. Cytoplasmic expression was observed in seminoma tumour cells and NTera2 cells. Gankyrin knock-down in NTera2 cells resulted in an increase in apoptosis mediated via the TP53 pathway, whilst POU5F1 expression was unaffected. Furthermore, Gankyrin knock-down in NTera2 cells increased cisplatin sensitivity with an increase in cell death (13%, p < 0.05) following Gankyrin knock-down, when compared to cisplatin treatment alone, likely via BAX and FAS. Our results demonstrate that Gankyrin expression changes in germ cells during normal transition from gonocyte to prespermatogonia. In addition, changes in Gankyrin localisation are associated with progression of pre-invasive GCNIS to invasive TGCC. Furthermore, we found that Gankyrin is involved in the regulation of NTera2 cell survival and that a reduction in Gankyrin expression can modulate cisplatin sensitivity.Conclusions: These results suggest that manipulation of Gankyrin expression may reduce the cisplatin dose required for the treatment of TGCC, with benefits in reducing dose-dependent side effects of chemotherapy. Further studies are required in order to assess the effects of modulating Gankyrin on GCNIS/TGCC using in vivo models. [ABSTRACT FROM AUTHOR]

Testicular development from the initially bipotential gonad is a tightly regulated process involving a complex signalling cascade to ensure proper sequential expression of signalling factors and secretion of steroid hormones. Initially, Sertoli cell specification facilitates differentiation of the steroidogenic fetal Leydig cells and establishment of the somatic niche, which is critical in supporting the germ cell population. Impairment of the somatic niche during fetal life may lead to development of male reproductive disorders, including arrest of gonocyte differentiation, which is considered the first step in the testicular cancer pathogenesis. In this review, we will outline the signalling pathways involved in fetal testis development focusing on the Nodal pathway, which has recently been implicated in several aspects of testicular differentiation in both mouse and human studies. Nodal signalling plays important roles in germ cell development, including regulation of pluripotency factor expression, proliferation and survival. Moreover, the Nodal pathway is involved in establishment of the somatic niche, including formation of seminiferous cords, steroidogenesis and Sertoli cell function. In our outline of fetal testis development, important differences between human and mouse models will be highlighted to emphasise that information obtained from mouse studies cannot always be directly translated to humans. Finally, the implications of dysregulated Nodal signalling in development of the testicular cancer precursor, germ cell neoplasia in situ, and testicular dysgenesis will be discussed - none of which arise in rodents, emphasising the importance of human models in the effort to increase our understanding of origin and early development of these disorders. [ABSTRACT FROM AUTHOR]

Human primordial germ cells (PGCs) have been described in the yolk sac wall around the beginning of the third week. From week 4 to 5, they migrate under control of SCF/c-KIT signaling pathway to the genital ridge, where they become gonocytes. PGCs and gonocytes express classic pluripotency markers, such as KIT, NANOG, and OCT3/4 that, during spermatogonia differentiation, are gradually suppressed, and substituted by the expression of some germ cell specific genes, such as VASA, SOX17, and TSPY. These genes, during normal development of germ cells, are tightly regulated by epigenetic modification, in terms of microRNA expression and DNA methylation. In adolescents and young adults, testicular germ cell tumors (TGCT) have a common precursor, the germ cell neoplasia in situ (GCNIS); the hypothesis of their origin from PGCs or gonocytes, whose maturation is altered, is widely accepted. The origin of TGCT, probably starting at early stages of embryogenesis, seems to be a part of the Testicular Dysgenesis Syndrome (TDS) where some early PGC/gonocytes, for still unclear reasons, are blocked in their differentiation, retaining their early marker profile. In this paper, current knowledge on the combination of epidemiological and genomic factors, involved in the development of testicular germ cell tumors, is reviewed. [ABSTRACT FROM AUTHOR]

Study Question: Does loss of DMRT1 in human fetal testis alter testicular development and result in testicular dysgenesis?Summary Answer: DMRT1 repression in human fetal testis alters the expression of key testicular and ovarian determining genes, and leads to focal testicular dysgenesis.What Is Known Already: Testicular dysgenesis syndrome (TDS) is associated with common testicular disorders in young men, but its etiology is unknown. DMRT1 has been shown to play a role in the regulation of sex differentiation in the vertebrate gonad. Downregulation of DMRT1 in male mice results in trans-differentiation of Sertoli cells into granulosa (FOXL2+) cells resulting in an ovarian gonadal phenotype.Study Design, Size, Duration: To determine the effect of DMRT1 repression on human fetal testes, we developed a novel system for genetic manipulation, which utilizes a Lentivral delivered miRNA during short-term in vitro culture (2 weeks). A long-term (4-6 weeks) ex vivo xenograft model was used to determine the subsequent effects of DMRT1 repression on testicular development and maintenance. We included first and second-trimester testis tissue (8-20 weeks gestation; n = 12) in the study.Participants/materials, Setting, Methods: Human fetal testes were cultured in vitro and exposed to either of two DMRT1 miRNAs (miR536, miR641), or to scrambled control miRNA, for 24 h. This was followed by a further 14 days of culture (n = 3-4), or xenografting (n = 5) into immunocompromised mice for 4-6 weeks. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence and quantitative RT-PCR. Endpoints included histological evaluation of seminiferous cord integrity, mRNA expression of testicular, ovarian and germ cell genes, and assessment of cell number and protein expression for proliferation, apoptosis and pluripotency factors. Statistical analysis was performed using a linear mixed effect model.Main Results and the Role Of Chance: DMRT1 repression (miR536/miR641) resulted in a loss of DMRT1 protein expression in a sub-population of Sertoli cells of first trimester (8-11 weeks gestation) human fetal testis; however, this did not affect the completion of seminiferous cord formation or morphological appearance. In second-trimester testis (12-20 weeks gestation), DMRT1 repression (miR536/miR641) resulted in disruption of seminiferous cords with absence of DMRT1 protein expression in Sertoli (SOX9+) cells. No differences in proliferation (Ki67+) were observed and apoptotic cells (CC3+) were rare. Expression of the Sertoli cell associated gene, SOX8, was significantly reduced (miR536, 34% reduction, P = 0.031; miR641 36% reduction, P = 0.026), whilst SOX9 expression was unaffected. Changes in expression of AMH (miR536, 100% increase, P = 0.033), CYP26B1 (miR641, 38% reduction, P = 0.05) and PTGDS (miR642, 30% reduction, P = 0.0076) were also observed. Amongst granulosa cell associated genes, there was a significant downregulation in R-spondin 1 expression (miR536, 76% reduction, P < 0.0001; miR641, 49% reduction, P = 0.046); however, there were no changes in expression of the granulosa cell marker, FOXL2. Analysis of germ cell associated genes demonstrated a significant increase in the expression of the pluripotency gene OCT4 (miR536, 233%, P < 0.001). We used the xenograft system to investigate the longer-term effects of seminiferous cord disruption via DMRT1 repression. As was evident in vitro for second-trimester samples, DMRT1 repression resulted in focal testicular dysgenesis similar to that described in adults with TDS. These dysgenetic areas were devoid of germ cells, whilst expression of FOXL2 within the dysgenetic areas, indicated trans-differentiation from a male (Sertoli cell) to female (granulosa cell) phenotype.Limitations, Reasons For Caution: Human fetal testis tissue is a limited resource; however, we were able to demonstrate significant effects of DMRT1 repression on the expression of germ and somatic cell genes, in addition to the induction of focal testicular dysgenesis, using these limited samples. In vitro culture may not reflect all aspects of human fetal testis development and function; however, the concurrent use of the xenograft model which represents a more physiological system supports the validity of the in vitro findings.Wider Implications Of the Findings: Our findings have important implications for understanding the role of DMRT1 in human testis development and in the origin of testicular dysgenesis. In addition, we provide validation of a novel system that can be used to determine the effects of repression of genes that have been implicated in gonadal development and associated human reproductive disorders.Study Funding/competing Interest(s): This project was funded by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) awarded to RTM. LBS was supported by MRC Programme Grant MR/N002970/1. RAA was supported by MRC Programme Grant G1100357/1. RMS was supported by MRC Programme Grant G33253. This work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. The funding bodies had no input into the conduct of the research or the production of this manuscript. The authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Study Question: When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome (KS)?Summary Answer: In KS, germ cell loss is not observed in testicular tissue from fetuses in the second semester of pregnancy but present at a prepubertal age when the testicular architecture is still normal, while fibrosis is highly present at an adolescent age.What Is Known Already: Most KS patients are azoospermic at adult age because of a massive germ cell loss. However, the timing when this germ cell loss starts is not known. It is assumed that germ cell loss increases at puberty. Therefore, testicular sperm extraction (TESE) at an adolescent age has been suggested to increase the chances of sperm retrieval at onset of spermatogenesis. However, recent data indicate that testicular biopsies from peripubertal KS patients contain only a few germ cells.Study Design, Size, Duration: In this study, we give an update on fertility preservation in adolescent KS patients and evaluate whether fertility preservation would be beneficial at prepubertal age. The possibility of retrieving testicular spermatozoa by TESE was evaluated in adolescent and adult KS men. The presence of spermatogonia and the degree of fibrosis were also analysed in testicular biopsies from KS patients at different ages. The patients were divided into four age groups: foetal (n = 5), prepubertal (aged 4-7 years; n = 4), peripubertal (aged 12-16 years; n = 20) and adult (aged 18-41 years; n = 27) KS patients.Participants/materials, Setting, Methods: In peripubertal and adult KS patients, retrieval of spermatozoa was attempted by semen analysis after masturbation, vibrostimulation, electroejaculation or by TESE. MAGE-A4 immunohistochemistry was performed to evaluate the presence of germ cells in testicular biopsies from foetal, prepubertal, peripubertal and adult KS patients. Tissue morphology was evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining.Main Results and the Role Of Chance: Testicular spermatozoa were collected by TESE in 48.1% of the adult KS patients, while spermatozoa were recovered after TESE in only one peripubertal patient (5.0%). Germ cells were detectable in testicular biopsies from 21% of adult men for whom no spermatozoa could be retrieved by TESE and in 31.5% of peripubertal KS boys. Very small numbers of spermatogonia (0.03-0.06 spermatogonia/tubule) were detected in three out of four (75%) prepubertal patients. At a foetal age, the number of germ cells was similar for KS and control samples. Increased signs of fibrosis were not present at foetal and prepubertal ages, but peripubertal and adult KS patients showed high levels of fibrosis.Large Scale Data: N/A.Limitations, Reasons For Caution: Only four prepubertal biopsies were included in this study, but they all showed a very low germ cell number. A high variability in the number of spermatogonia per mm2 was observed in the limited (n = 5) number of foetal biopsies. However, testicular biopsies from prepubertal and foetal Klinefelter patients are difficult to obtain.Wider Implications Of the Findings: Testicular tissue banking at a prepubertal age has been suggested as a potential method for fertility preservation in early diagnosed KS boys. However, our results show that a reduction in germ cell number has already taken place in childhood. Therefore, offering testicular tissue banking in young KS boys to prevent subsequent sterility might be a questionable strategy. However, this should be confirmed in a larger study population.Study Funding/competing Interest(s): This project was funded by the scientific Fund Willy Gepts from the UZ Brussel (D.V.S., J.D.S.), grants from the Vrije Universiteit Brussel (E.G.) and a Methusalem grant (K.S.). D.V.S is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared. [ABSTRACT FROM AUTHOR]

BACKGROUND: Analgesic exposure during pregnancy may affect aspects of fetal gonadal development that are targeted by endocrine disruptors. OBJECTIVES: We investigated whether therapeutically relevant doses of acetaminophen and ibuprofen affect germ cell (GC) development in human fetal testes/ovaries using in vitro and xenograft approaches. METHODS: First-trimester human fetal testes/ovaries were cultured and exposed to acetaminophen or ibuprofen (7 d). Second-trimester human fetal testes were xenografted into mice and exposed to acetaminophen (1 or 7 d), or ibuprofen (7 d). To determine mechanism of action, a human GC tumor-derived cell line (NTera2) exhibiting fetal GC characteristics was used in addition to in vitro and in vivo rat models. RESULTS AND DISCUSSION: Gonocyte (TFAP2C+ ) number was reduced relative to controls in first-trimester human fetal testes exposed in vitro to acetaminophen (-28%) or ibuprofen (-22%) and also in ovaries exposed to acetaminophen (-43%) or ibuprofen (-49%). Acetaminophen exposure reduced gonocyte number by 17% and 30% in xenografted second-trimester human fetal testes after treatment of host mice for 1 or 7 d, respectively. NTera2 cell number was reduced following exposure to either analgesic or prostaglandin E2 (PGE2) receptor antagonists, whereas PGE2 agonists prevented acetaminophen-induced reduction in NTera2 cell number. Expression of GC pluripotency genes, and genes that regulate DNA/histone methylation, also differed from controls following analgesic and PGE2 receptor antagonist exposures. Gene expression changes were observed in rat fetal testis/ovary cultures and after in vivo acetaminophen exposure of pregnant rats. For example, expression of the epigenetic regulator TET1, was increased following exposure to acetaminophen in human NTera2 cells, rat fetal testis/ovary cultures, and in fetal testes and ovaries after in vivo exposure of pregnant rats, indicating translatability across experimental models and species. CONCLUSIONS: Our results demonstrate evidence of PGE2-mediated effects of acetaminophen and ibuprofen on GC/NTera2 cells, which raises concerns about analgesic use during human pregnancy that warrant further investigation. [ABSTRACT FROM AUTHOR]

Testicular teratomas result from anomalies in embryonic germ cell development. In 129 inbred mice, teratoma initiation coincides with germ cell sex-specific differentiation and the mitotic-meiotic switch: XX and XY germ cells repress pluripotency, XX germ cells initiate meiosis, and XY germ cells activate male-specific differentiation and mitotic arrest. Here, we report that expression of Nanos2, a gene that is crucial to male sex specification, is delayed in teratomasusceptible germ cells. Decreased expression of Nanos2 was found to be due, in part, to the Nanos2 allele present in 129 mice. In teratoma-susceptible germ cells, diminished expression of genes downstream of Nanos2 disrupted processes that were crucial to male germ cell differentiation. Deficiency for Nanos2 increased teratoma incidence in 129 mice and induced developmental abnormalities associated with tumor initiation in teratoma-resistant germ cells. Finally, in the absence of commitment to the male germ cell fate, we discovered that a subpopulation of teratoma-susceptible germ cells transition into embryonal carcinoma (EC) cells with primed pluripotent features. We conclude that delayed male germ cell sex-specification facilitates the transformation of germ cells with naïve pluripotent features into primed pluripotent EC cells. [ABSTRACT FROM AUTHOR]

Background: Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models. Methods: Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks. Results: With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice. Conclusions: Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures. [ABSTRACT FROM AUTHOR]

Background: Aberrant overexpression of PIWI/piRNA pathway proteins is shown for many types of tumors. Interestingly, these proteins are downregulated in testicular germ cell tumors (TGCTs) compared to normal testis tissues. Here, we used germline and TGCT markers to assess the piRNA biogenesis and function in TGCTs and their precursor germ cell neoplasia in situ (GCNIS).Methods: We used small RNA deep sequencing, qRT-PCR, and mining public RNAseq/small RNA-seq datasets to examine PIWI/piRNA gene expression and piRNA biogenesis at four stages of TGCT development: (i) germ cells in healthy testis tissues, (ii) germ cells in testis tissues adjacent to TGCTs, (iii) GCNIS cells and (iv) TGCT cells. To this end, we studied three types of samples: (a) healthy testis, (b) testis tissues adjacent to two types of TGCTs (seminomas and nonseminomas) and containing both germ cells and GCNIS cells, as well as (c) matching TGCT samples.Results: Based on our analyses of small RNA-seq data as well as the presence/absence of expression correlation between PIWI/piRNA pathway genes and germline or TGCT markers, we can suggest that piRNA biogenesis is intact in germ cells present in healthy adult testes, and adjacent to TGCTs. Conversely, GCNIS and TGCT cells were found to lack PIWI/piRNA pathway gene expression and germline-like piRNA biogenesis. However, using an in vitro cell line model, we revealed a possible role for a short PIWIL2/HILI isoform expressed in TGCTs in posttranscriptional regulation of the youngest members of LINE and SINE classes of transposable elements. Importantly, this regulation is also implemented without involvement of germline-like biogenesis of piRNAs.Conclusions: Though further studies are warranted, these findings suggest that the conventional germline-like PIWI/piRNA pathway is lost in transition from germ cells to GCNIS cells. [ABSTRACT FROM AUTHOR]

So far, the understanding of germ cell cancer (GCC) pathogenesis is based on a model, where seminomas and non-seminomas represent distinct entities although originating from a common precursor termed germ cell neoplasia in situ (GCNIS). Embryonal carcinomas (ECs), the stem cell population of the non-seminomas, is pluri- to totipotent and able to differentiate into cells of all three germ layers, giving rise to teratomas or tumours mimicking extraembryonic tissues (yolk sac tumours, choriocarcinomas). With regard to gene expression, (epi)genetics and histology, seminomas are highly similar to GCNIS and primordial germ cells, but limited in development. It remains elusive, whether this block in differentiation is controlled by cell intrinsic mechanisms or by signals from the surrounding microenvironment. Here, we reviewed the recent literature emphasizing the plasticity of GCCs, especially of seminomas. We propose that this plasticity is controlled by the microenvironment, allowing seminomas to transit into an EC or mixed non-seminoma and vice versa. We discuss several mechanisms and routes of reprogramming that might be responsible for this change in the cell fate. We finally integrate this plasticity into a new model of GCC pathogenesis, allowing for an alternative view on the dynamics of GCC development and progression. [ABSTRACT FROM AUTHOR]

The hypothesis that developmental estrogenic exposure induces a constellation of male reproductive tract abnormalities is supported by experimental and human evidence. Experimental data also suggest that some induced effects persist in descendants of exposed males. These multi- and transgenerational effects are assumed to result from epigenetic changes to the germline, but few studies have directly analyzed germ cells. Typically, studies of transgenerational effects have involved exposing one generation and monitoring effects in subsequent unexposed generations. This approach, however, has limited human relevance, since both the number and volume of estrogenic contaminants has increased steadily over time, intensifying rather than reducing or eliminating exposure. Using an outbred CD-1 mouse model, and a sensitive and quantitative marker of germline development, meiotic recombination, we tested the effect of successive generations of exposure on the testis. We targeted the germline during a narrow, perinatal window using oral exposure to the synthetic estrogen, ethinyl estradiol. A complex three generation exposure protocol allowed us to compare the effects of individual, paternal, and grandpaternal (ancestral) exposure. Our data indicate that multiple generations of exposure not only exacerbate germ cell exposure effects, but also increase the incidence and severity of reproductive tract abnormalities. Taken together, our data suggest that male sensitivity to environmental estrogens is increased by successive generations of exposure. [ABSTRACT FROM AUTHOR]

Over the past few decades, several studies have attempted to decipher the biology of mammalian germline stem cells (GSCs). These studies provide evidence that regulatory mechanisms for germ cell specification and migration are evolutionarily conserved across species. The characteristics and functions of primate GSCs are highly distinct from rodent species; therefore the findings from rodent models cannot be extrapolated to primates. Due to limited availability of human embryonic and testicular samples for research purposes, two non-human primate models (marmoset and macaque monkeys) are extensively employed to understand human germline development and differentiation. This review provides a broader introduction to the in vivo and in vitro germline stem cell terminology from primordial to differentiating germ cells. Primordial germ cells (PGCs) are the most immature germ cells colonizing the gonad prior to sex differentiation into testes or ovaries. PGC specification and migratory patterns among different primate species are compared in the review. It also reports the distinctions and similarities in expression patterns of pluripotency markers (OCT4A, NANOG, SALL4 and LIN28) during embryonic developmental stages, among marmosets, macaques and humans. This review presents a comparative summary with immunohistochemical and molecular evidence of germ cell marker expression patterns during postnatal developmental stages, among humans and non-human primates. Furthermore, it reports findings from the recent literature investigating the plasticity behavior of germ cells and stem cells in other organs of humans and monkeys. The use of non-human primate models would enable bridging the knowledge gap in primate GSC research and understanding the mechanisms involved in germline development. Reported similarities in regulatory mechanisms and germ cell expression profile in primates demonstrate the preclinical significance of monkey models for development of human fertility preservation strategies. [ABSTRACT FROM AUTHOR]

Adult male germline stem cells (spermatogonia) proliferate by mitosis and, after puberty, generate spermatocytes that undertake meiosis to produce haploid spermatozoa. Germ cells are under evolutionary constraint to curtail mutations and maintain genome integrity. Despite constant turnover, spermatogonia very rarely form tumors, so-called spermatocytic tumors (SpT). In line with the previous identification of FGFR3 and HRAS selfish mutations in a subset of cases, candidate gene screening of 29 SpTs identified an oncogenic NRAS mutation in two cases. To gain insights in the etiology of SpT and into properties of the male germline, we performed whole-genome sequencing of five tumors (4/5 with matched normal tissue). The acquired single nucleotide variant load was extremely low (~0.2 per Mb), with an average of 6 (2–9) non-synonymous variants per tumor, none of which is likely to be oncogenic. The observed mutational signature of SpTs is strikingly similar to that of germline de novo mutations, mostly involving C>T transitions with a significant enrichment in the ACG trinucleotide context. The tumors exhibited extensive aneuploidy (50–99 autosomes/tumor) involving whole-chromosomes, with recurrent gains of chr9 and chr20 and loss of chr7, suggesting that aneuploidy itself represents the initiating oncogenic event. We propose that SpT etiology recapitulates the unique properties of male germ cells; because of evolutionary constraints to maintain low point mutation rate, rare tumorigenic driver events are caused by a combination of gene imbalance mediated via whole-chromosome aneuploidy. Finally, we propose a general framework of male germ cell tumor pathology that accounts for their mutational landscape, timing and cellular origin. [ABSTRACT FROM AUTHOR]

Fetal germ cell development is tightly regulated by the somatic cell environment, and is characterised by cell cycle states that differ between XY and XX gonads. In the testis, gonocytes enter G1/G0 arrest from 12.5 days post coitum (dpc) in mice and maintain cell cycle arrest until after birth. Failure to correctly maintain G1/G0 arrest can result in loss of germ cells or, conversely, germ cell tumours. High mobility group box containing transcription factor 1 (HBP1) is a transcription factor that was previously identified in fetal male germ cells at the time of embryonic cell cycle arrest. In somatic cells, HBP1 is classified as a tumour suppressor protein, known to regulate proliferation and senescence. We therefore investigated the possible role of HBP1 in the initiation and maintenance of fetal germ cell G1/G0 arrest using the mouse model. We identified two splice variants of Hbp1, both of which are expressed in XY and XX fetal gonads, but only one of which is localised to the nucleus in in vitro assays. To investigate Hbp1 loss of function, we used embryonic stem (ES) cells carrying a Genetrap mutation for Hbp1 to generate mice lacking Hbp1 function. We found that Hbp1-genetrap mouse mutant germ cells proliferated correctly throughout development, and adult males were viable and fertile. Multiple Hbp1-LacZ reporter mouse lines were generated, unexpectedly revealing Hbp1 embryonic expression in hair follicles, eye and limbs. Lastly, in a model of defective germ cell G1/G0 arrest, the Rb1-knockout model, we found no evidence for Hbp1 mis-regulation, suggesting that the reported RB1-HBP1 interaction is not critical in the germline, despite co-expression. [ABSTRACT FROM AUTHOR]

Study Question: Should fertility preservation be offered to children with Klinefelter syndrome (KS)?Summary Answer: Current evidence shows that fertility preservation should not be offered to adolescents with KS younger than 16 years because of lower retrieval rates for germ cells by testicular sperm extraction (TESE) compared with retrieval rates for adolescents and adults between 16 and 30 years.What Is Known Already: KS, the most common chromosomal disorder in men leading to non-obstructive azoospermia, is caused by the presence of at least one additional X chromosome. The onset of puberty in adolescents with KS leads to progressive degeneration of the testicular environment. The impact of the subsequent tissue degeneration on fertility potential of patients with KS is unknown, but in previous literature it has been suggested that fertility preservation should be started in adolescents as early as possible. However spermatozoa can be found by TESE in about 50% of adults with KS despite severe testicular degeneration. This review discusses the current evidence for fertility preservation in children and adolescents and possible prognostic markers for fertility treatment in KS.Study Design, Size, Duration: An extensive literature search was conducted, searching Pubmed, Embase, Cinahl and Web of Science from origin until April 2016 for 'Klinefelter syndrome' and 'fertility' and various synonyms. Titles and abstracts have been scanned manually by the authors for eligibility.Participants/materials, Setting, Methods: In total 76 studies were found to be eligible for inclusion in this review. Information from the papers was extracted separately by two authors.Main Results and the Role Of Chance: Various studies have shown that pre-pubertal children with KS already have a reduced number of germ cells despite a normal hormonal profile during childhood. The presence of spermatozoa in the ejaculate of adolescents with KS is extremely rare. Using TESE, the retrieval rates of spermatozoa for adolescents younger than 16 years old are much lower (0-20%) compared with those for adolescents and young adults between 16 and 30 years old (40-70%). Although spermatogonia can be found by TESE in about half of the peri-pubertal adolescents, there are currently no clinically functional techniques for their future use. Children and adolescents need to be informed that early fertility preservation before the age of 16 cannot guarantee fertility later in life and may even reduce the chances for offspring by removing functional immature germ cells which may possibly develop into spermatozoa after puberty. Furthermore, except for the age of patients with KS, there are no identified factors that can reliably be used as a predictive marker for fertility preservation.Limitations, Reasons For Caution: Most of the evidence presented in this review is based on studies including a small number of adolescents with KS. Therefore, the studies may have been underpowered to detect clinically significant differences for their various outcomes, especially for potential predictive factors for fertility preservation, such as hormone levels. Furthermore, the population of patients with KS diagnosed during childhood might be different from the adult population with KS where the diagnosis is based on infertility. Results based on comparisons between the two groups must be interpreted with caution.Wider Implications Of the Findings: Despite the limitations, this review summarizes the current evidence for managing fertility preservation in patients with KS to provide optimal health care.Study Funding/competing Interests: There was no funding for this study. S.F., Y.H., K.D., W.L.M.N., D.S., H.L.C.-v.d.G. and L.R. declare to have no conflicts of interests. D.D.M.B. reports grants from Merck Serono, grants from Ferring and grants from MSD, outside the submitted work. K.F. reports personal fees from MSD (commercial sponsor), personal fees from Ferring (commercial sponsor), grants from Merck-Serono (commercial sponsor), grants from Ferring (commercial sponsor) and grants from MSD (commercial sponsor), outside the submitted work. [ABSTRACT FROM AUTHOR]

Solid tumors are characterized by a plethora of epigenetic changes. In particular, patterns methylation of cytosines at the 5-position (5mC) in the context of CpGs are frequently altered in tumors. Recent evidence suggests that 5mC can get converted to 5-hydroxylmethylcytosine (5hmC) in an enzymatic process involving ten eleven translocation (TET) protein family members, and this process appears to be important in facilitating plasticity of cytosine methylation. Here we evaluated the global levels of 5hmC using a validated immunohistochemical staining method in a large series of clear cell renal cell carcinoma (n = 111), urothelial cell carcinoma (n = 55) and testicular germ cell tumors (n = 84) and matched adjacent benign tissues. Whereas tumor-adjacent benign tissues were mostly characterized by high levels of 5hmC, renal cell carcinoma and urothelial cell carcinoma showed dramatically reduced staining for 5hmC. 5hmC levels were low in both primary tumors and metastases of clear cell renal cell carcinoma and showed no association with disease outcomes. In normal testis, robust 5hmC staining was only observed in stroma and Sertoli cells. Seminoma showed greatly reduced 5hmC immunolabeling, whereas differentiated teratoma, embryonal and yolk sack tumors exhibited high 5hmC levels. The substantial tumor specific loss of 5hmC, particularly in clear cell renal cell carcinoma and urothelial cell carcinoma, suggests that alterations in pathways involved in establishing and maintaining 5hmC levels might be very common in cancer and could potentially be exploited for diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Study Question: What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads?Summary Answer: RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures.What Is Known Already: One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo.Study Design, Size, Duration: This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation.Participants/materials, Setting, Methods: Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay.Main Results and the Role Of Chance: A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P < 0.05) and DMRT1-positive oogonia initiating meiosis (P < 0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P < 0.05), a reduced percentage of proliferating gonocytes (P < 0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology.Limitations, Reasons For Caution: The number of samples included in this study was relatively small due to the limited availability of human fetal tissue.Wider Implications Of the Findings: The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects.Study Funding/competing Interests: This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and Aase & Ejnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest. [ABSTRACT FROM AUTHOR]

Type II germ cell cancers (GCC) can be subdivided into seminomas and non-seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as seminomas with a non-seminoma component, raising the question if a CIS gives rise to seminomas and ECs at the same time or whether seminomas can be reprogrammed to ECs. In this study, we utilized the seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (GDF3, NODAL, DNMT3B, DPPA3, GAL, AK3L1) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state. [ABSTRACT FROM AUTHOR]

Background: Malignant germ cell tumours are the most common malignant tumours in young men. They are histologically divided into seminomas and non-seminomas. Non-seminomas are further subdivided into embryonic carcinomas, yolk sac tumours, chorionic carcinomas, and teratomas. For the therapeutic management it is essential to differentiate between these histological subtypes. Methods: Investigated cases included normal testis (n = 50), intratubular germ cell neoplasia (n = 25), seminomas (n = 67), embryonic carcinomas (n = 56), yolk sac tumours (n = 29), chorionic carcinomas (n = 2), teratomas (n = 7) and four metastases of YST's for their CK19 expression. In addition Leydig cell- (n = 10) and Sertoli cell- tumours (n = 4) were included in this study. Results: All investigated seminomas, embryonic carcinomas as well as normal testis and intratubular germ cell neoplasias did not express CK19. In contrast, all investigated yolk sac tumours strongly expressed CK19 protein. These findings became also evident in mixed germ cell tumours consisting of embryonic carcinomas and yolk sac tumours, although CK19-expression could also be observed in analysed chorionic carcinomas and epithelial components of teratomas. Conclusion: CK19 proved to be a sensitive marker to identify yolk sac tumours of the testis and to distinguish them from other germ cell tumours, especially seminomas and embryonic carcinomas. [ABSTRACT FROM AUTHOR]

Background: Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma In Situ (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments. Results: This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape. Conclusion: The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to exhibit a more germ cell-like profile, whereas NCCIT shows a more pluripotent phenotype. The results provide insight into the functional genome in GCC cell lines. [ABSTRACT FROM AUTHOR]

During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of IDH1 (IDH1R132) and IDH2 (IDH2R172) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected. [ABSTRACT FROM AUTHOR]

Testicular germ cell cancer (TGCC) is the most common type of cancer in young men. Seminomas account for around half of them and are characterized by a pronounced infiltration of immune cells. So far, the impact of the tumor microenvironment (TME) on disease progression, especially the interaction of individual immune cell subtypes with the tumor cells, remains unclear. To address this question, we used an in vitro TME model involving the seminoma-derived cell line Tcam-2 and immune cell subsets purified from human peripheral blood. T cells and monocytes were strongly activated when individually cocultured with Tcam-2 cells as revealed by increased expression of activation markers and pro-inflammatory cytokines both on the mRNA and protein level. Importantly, the interaction between tumor and immune cells was mutual. Gene expression of pluripotency markers as well as markers of proliferation and cell cycle activity were upregulated in Tcam-2 cells in cocultures with T cells, whereas gene expression of SOX17, a marker for seminomas, was unaltered. Interestingly, the impact of monocytes on gene expression of Tcam-2 cells was less pronounced, indicating that the effects of individual immune cell subsets on tumor cells in the TME are highly specific. Collectively, our data indicate that seminoma cells induce immune cell activation and thereby generate a strong pro-inflammatory milieu, whereas T cells conversely increase the proliferation, metastatic potential, and stemness of tumor cells. Although the employed model does not fully mimic the physiological situation found in TGCC in vivo, it provides new insights potentially explaining the connection between inflammatory infiltrates in seminomas and their tendency to burn out and metastasize. [ABSTRACT FROM AUTHOR]