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There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2−/− sperm, the short one (Mw 36 kDa) is mis-localized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in Prm2−/− sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/2/3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 proteins or SUN4 expression in Prm2−/− testes. Furthermore, the Prm2−/− sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone–protamine 1/2 ratio, modified levels of cytoskeletal proteins and we detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions. In conclusion, our findings present potential interaction between Septin 12 and Protamine 2 or Lamin B2/3 and describe a new connection between their expression and localization, contributing likely to low sperm motility and morphological abnormalities.

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.

Preeclampsia (PE) is characterized by maternal hypertension and placental dysfunction, often leading to fetal growth restriction (FGR). It is associated with an overexpression of the anti-angiogenic sFLT1 protein, which originates from the placenta and serves as a clinical biomarker to predict PE. To analyze the impact of sFLT1 on placental function and fetal growth, we generated transgenic mice with placenta-specific human sFLT1 (hsFLT1) overexpression. Immunohistochemical, morphometrical, and molecular analyses of the placentas on 14.5 dpc and 18.5 dpc were performed with a focus on angiogenesis, nutrient transport, and inflammation. Additionally, fetal development upon placental hsFLT1 overexpression was investigated. Dams exhibited a mild increase in serum hsFLT1 levels upon placental hsFLT1 expression and revealed growth restriction of the fetuses in a sex-specific manner. Male FGR fetuses expressed higher amounts of placental hsFLT1 mRNA compared to females. FGR placentas displayed an altered morphology, hallmarked by an increase in the spongiotrophoblast layer and changes in labyrinthine vascularization. Further, FGR placentas showed a significant reduction in placental glycogen storage and nutrient transporter expression. Moreover, signs of hypoxia and inflammation were observed in FGR placentas. The transgenic spongiotrophoblast-specific hsFLT1 mouse line demonstrates that low hsFLT1 serum levels are sufficient to induce significant alterations in fetal and placental development in a sex-specific manner.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population—embryonal carcinoma (EC)—is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra‐embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three‐dimensional (3D) co‐cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho‐ and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin‐resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

Synthetic embryo models have arisen as an approach to probe early development in vitro, facilitating the study of difficult to access stages. Here the authors present a simple system for generating embryo-like structures that resemble peri-implantation mouse embryos.

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O2-dependent shuttle of oxidizable fatty acid substrates. MB’s impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively.

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis, and metastasis of tumor cells. The testes-specific PFN3 is localized in the acroplaxome–manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3–/– males are subfertile, displaying a type II globozoospermia. We revealed that Pfn3–/– sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3–/– sperm showed reduced sperm motility resulting from flagellum deformities. We show that acrosome biogenesis is impaired starting from the Golgi phase, and mature sperm seems to suffer from a cytoplasm removal defect. An RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed, resulting in the inhibition of autophagy. This dysregulation of AMPK/mTOR affected the autophagic flux, which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3–/– testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3–ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3–/– testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicate this protein as a candidate for male factor infertility in humans.

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis.

In December 2019, the first case of COVID-19 was reported and since then several groups have already published that the virus can be present in the testis. To study the influence of SARS-CoV-2 which cause a dysregulation of the androgen receptor (AR) level, thereby leading to fertility problems and inducing germ cell testicular changes in patients after the infection. Formalin-Fixed-Paraffin-Embedded (FFPE) testicular samples from patients who died with or as a result of COVID-19 (n = 32) with controls (n = 6), inflammatory changes (n = 9), seminoma with/without metastasis (n = 11) compared with healthy biopsy samples (n = 3) were analyzed and compared via qRT-PCR for the expression of miR-371a-3p. An immunohistochemical analysis (IHC) and ELISA were performed in order to highlight the miR-371a-3p targeting the AR. Serum samples of patients with mild or severe COVID-19 symptoms (n = 34) were analyzed for miR-371a-3p expression. In 70% of the analyzed postmortem testicular tissue samples, a significant upregulation of the miR-371a-3p was detected, and 75% of the samples showed a reduced spermatogenesis. In serum samples, the upregulation of the miR-371a-3p was also detectable. The upregulation of the miR-371a-3p is responsible for the downregulation of the AR in SARS-CoV-2-positive patients, resulting in decreased spermatogenesis. Since the dysregulation of the AR is associated with infertility, further studies have to confirm if the identified dysregulation is regressive after a declining infection.

Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer accounting for 80% of all renal cancers as well as the majority of renal cancer-associated deaths. During the last decade, the treatment paradigm for ccRCC has radically changed. In particular, the recent development of immune checkpoint inhibitors (ICI) has led to an increased overall survival in the metastatic setting. Moreover, novel immune therapies targeting the tumor microenvironment have been developed. In this rapidly evolving treatment landscape, precise tools for personalized cancer therapy are needed. Here, we collected fresh tissue from 42 patients who underwent surgical resection for renal cell carcinoma. Part of the tissue was used to obtain formalin-fixed, paraffin-embedded samples or RNA. The remaining tissue was minced and cultured in a collagen-based three-dimensional, air-liquid interface (ALI) culture system. The generated patient-derived tumor organoids (ALI PDOs) were characterized by immunohistochemistry staining and RNA sequencing to validate their close similarity to the matched tumor. Immune cells and stromal cells within the microenvironment could be identified. Finally, we treated 10 ALI PDOs with the commonly used targeted cancer drug cabozantinib or the ICI nivolumab. Interestingly, we observed varying responses of ALI PDOs to these treatments and future studies are needed to investigate whether the ALI PDO approach could inform about treatment responses in patients. In conclusion, this three-dimensional ccRCC culture model represents a promising, facile tool for monitoring tumor responses to different types of therapies in a controlled manner, yet, still preserves the key features of the tumor of origin.

The anti-angiogenic soluble fms-like tyrosine kinase 1 (sFLT1) is one of the candidates in the progression of preeclampsia, often associated with fetal growth restriction (FGR). Therapeutic agents against preeclampsia with/without FGR, as well as adequate transgenic sFLT1 mouse models for testing such agents, are still missing. Much is known about sFLT1–mediated endothelial dysfunction in several tissues; however, the influence of sFLT1 on placental and fetal development is currently unknown. We hypothesize that sFLT1 is involved in the progression of FGR by influencing placental differentiation and vascularization and is a prime candidate for interventional strategies. Therefore, we generated transgenic inducible human sFLT1/reverse tetracycline-controlled transactivator (hsFLT1/rtTA) mice, in which hsFLT1 is ubiquitously overexpressed during pregnancy in dams and according to the genetics in hsFLT1/rtTA homozygous and heterozygous fetuses. Induction of hsFLT1 led to elevated hsFLT1 levels in the serum of dams and on mRNA level in all placentas and hetero-/homozygous fetuses, resulting in FGR in all fetuses at term. The strongest effects in respect to FGR were observed in the hsFLT1/rtTA homozygous fetuses, which exhibited the highest hsFLT1 levels. Only fetal hsFLT1 expression led to impaired placental morphology characterized by reduced placental efficiency, enlarged maternal sinusoids, reduced fetal capillaries, and impaired labyrinthine differentiation, associated with increased apoptosis. Besides impaired placental vascularization, the expression of several transporter systems, such as glucose transporter 1 and 3 (Glut-1; Glut-3); amino acid transporters, solute carrier family 38, member one and two (Slc38a1; Slc38a2); and most severely the fatty acid translocase Cd36 and fatty acid binding protein 3 (Fabp3) was reduced upon hsFLT1 expression, associated with an accumulation of phospholipids in the maternal serum. Moreover, the Vegf pathway showed alterations, resulting in reduced Vegf, Vegfb, and Plgf protein levels and increased Bad and Caspase 9 mRNA levels. We suggest that hsFLT1 exerts an inhibitory influence on placental vascularization by reducing Vegf signaling, which leads to apoptosis in fetal vessels, impairing placental differentiation, and the nutrient exchange function of the labyrinth. These effects were more pronounced when both the dam and the fetus expressed hsFLT1 and ultimately result in FGR and resemble the preeclamptic phenotype in humans.

Filamins are large dimeric F-actin cross-linking proteins, crucial for the mechanosensitive properties of a number of cell types. Due to their interaction with a variety of different proteins, they exert important regulatory functions. However, in the human testis the role of filamins has been insufficiently explored. Immunohistochemical staining of human testis samples identified filamin A (FLNA) in spermatogonia and peritubular myoid cells. Investigation of different testicular tumor samples indicated that seminoma also express FLNA. Moreover, mass spectrometric analyses identified FLNA as one of the most abundant proteins in human seminoma TCam-2 cells. We therefore focused on FLNA in TCam-2 cells, and identified by co-immunoprecipitation LAD1, RUVBL1 and DAZAP1, in addition to several cytoskeletal proteins, as interactors of FLNA. To study the role of FLNA in TCam-2 cells, we generated FLNA-deficient cells using the CRISPR/Cas9 system. Loss of FLNA causes an irregular arrangement of the actin cytoskeleton and mechanical instability, impaired adhesive properties and disturbed migratory behavior. Furthermore, transcriptional activity of typical stem cell factors is increased in the absence of FLNA. In summary, our data suggest that FLNA is crucially involved in balancing stem cell characteristics and invasive properties in human seminoma cells and possibly human testicular germ cells.

Protamines are the safeguards of the paternal sperm genome. They replace most of the histones during spermiogenesis, resulting in DNA hypercondensation, thereby protecting its genome from environmental noxa. Impaired protamination has been linked to male infertility in mice and humans in many studies. Apart from impaired DNA integrity, protamine-deficient human and murine sperm show multiple secondary effects, including decreased motility and aberrant head morphology. In this study, we use a Protamine-2 (Prm2)-deficient mouse model in combination with label-free quantitative proteomics to decipher the underlying molecular processes of these effects. We show that loss of the sperm’s antioxidant capacity, indicated by downregulation of key proteins like Superoxide dismutase type 1 (SOD1) and Peroxiredoxin 5 (PRDX5), ultimately initiates an oxidative stress-mediated destruction cascade during epididymal sperm maturation. This is confirmed by an increased level of 8-OHdG in epididymal sperm, a biomarker for oxidative stress-mediated DNA damage. Prm2-deficient testicular sperm are not affected and initiate the proper development of blastocyst stage preimplantation embryos in vitro upon intracytoplasmic sperm injection (ICSI) into oocytes. Our results provide new insight into the role of Prm2 and its downstream molecular effects on sperm function and present an important contribution to the investigation of new treatment regimens for infertile men with impaired protamination.

Activating mutations in the human KIT receptor is known to drive severe hematopoietic disorders and tumor formation spanning various entities. The most common mutation is the substitution of aspartic acid at position 816 to valine (D816V), rendering the receptor constitutively active independent of ligand binding. As the role of the KIT receptor in placental signaling cascades is poorly understood, we analyzed the impact of KITD816V expression on placental development using a humanized mouse model. Placentas from KITD816V animals present with a grossly changed morphology, displaying a reduction in labyrinth and spongiotrophoblast layer and an increase in the Parietal Trophoblast Giant Cell (P-TGC) layer. Elevated differentiation to P-TGCs was accompanied with reduced differentiation to other Trophoblast Giant Cell (TGC) subtypes and by severe decrease in proliferation. The embryos display growth retardation and die in utero. KITD816V-trophoblast stem cells (TSC) differentiate much faster compared to wild type (WT) controls. In undifferentiated KITD816V-TSCs, levels of Phosphorylated Extracellular-signal Regulated Kinase (P-ERK) and Phosphorylated Protein Kinase B (P-AKT) are comparable to wildtype cultures differentiating for 3–6 days. Accordingly, P-TGC markers Placental Lactogen 1 (PL1) and Proliferin (PLF) are upregulated as well. The results reveal that KIT signaling orchestrates the fine-tuned differentiation of the placenta, with special emphasis on P-TGC differentiation. Appropriate control of KIT receptor action is therefore essential for placental development and nourishment of the embryo.

Trophoblast stem cells (TSCs) are in vitro equivalents to the precursor cells of the placenta. TSCs are cultured in serum-rich medium with fibroblast growth factor 4, heparin, and embryonic-fibroblast-conditioned medium. Here, we developed a simple medium consisting of ten chemically defined ingredients for culture of TSCs on Matrigel or synthetic substrates, named TX medium. Gene expression and DNA methylation profiling demonstrated the faithful propagation of expression profiles and epigenomic characteristics of TSCs cultured in TX. Further, TX medium supported the de novo derivation of TSC lines. Finally, TSCs cultured in TX differentiate into all derivatives of the trophectodermal lineage in vitro, give rise to hemorrhagic lesions in nude mice, and chimerize the placenta, indicating that they retained all hallmarks of TSCs. TX media formulation no longer requires fetal bovine serum and conditioned medium, which facilitates and standardizes the culture of this extraembryonic lineage.

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.

An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms.14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells (eBM-MSC). Expression stabilities of the reference genes were evaluated using geNorm, NormFinder, and BestKeeper. Similar results were obtained by the three approaches for the most and least stably expressed genes. The R-based packages NormqPCR, SLqPCR and the NormFinder for R script gave identical gene rankings. Interestingly, different outputs were obtained between the original software packages and the RefFinder tool, which is based on raw Cq values for input. When the raw data were reanalysed assuming 100% efficiency for all genes, then the outputs of the original software packages were similar to the RefFinder software, indicating that RefFinder outputs may be biased because PCR efficiencies are not taken into account.This report shows that assay efficiency is an important parameter for reference gene validation. New software tools that incorporate these algorithms should be carefully validated prior to use.

Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tfap2c in embryonic stem cells and primordial germ cell-like cells. We show that loss of Tfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation of maturation markers and induction of markers indicative for somatic differentiation, cell cycle, epigenetic remodeling and pluripotency. Chromatin-immunoprecipitation analyses demonstrated binding of TFAP2C to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l) suggesting that these genes are direct transcriptional targets of TFAP2C in primordial germ cells. Since Tfap2c deficient primordial germ cell-like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tfap2c develop with high incidence germ cell cancer resembling human pediatric germ cell tumors. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate that mice with a heterozygous deletion of the TFAP2C target gene Nanos3 are also prone to develop teratomas. These data highlight TFAP2C as a critical and dose-sensitive regulator of germ cell fate.

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 5 mC 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 (IDH1 (R132)) and IDH2 (IDH2 (R172)) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected.

BackgroundThe transcription factor Tcfap2c has been demonstrated to be essential for various processes during mammalian development. It has been found to be upregulated in various undifferentiated tumors and is implicated with poor prognosis. Tcfap2c is reported to impinge on cellular proliferation, differentiation and apoptosis. However, the physiological consequences of Tcfap2c-expression remain largely unknown.Methodology/principal findingsTherefore we established a gain of function model to analyze the role of Tcfap2c in development and disease. Induction of the transgene led to robust expression in all tissues (except brain and testis) and lead to rapid mortality within 3-7 days. In the liver cellular proliferation and apoptosis was detected. Accumulation of microvesicular lipid droplets and breakdown of major hepatic metabolism pathways resulted in steatosis. Serum analysis showed a dramatic increase of enzymes indicative for hepatic failure. After induction of Tcfap2c we identified a set of 447 common genes, which are deregulated in both liver and primary hepatocyte culture. Further analysis showed a prominent repression of the cytochrome p450 system, PPARA, Lipin1 and Lipin2. These data indicate that in the liver Tcfap2c represses pathways, which are responsible for fatty acid metabolism. In the intestine, Tcfap2c expression resulted in expansion of Sox9 positive and proliferative active epithelial progenitor cells resulting in dysplastic growth of mucosal crypt cells and loss of differentiated mucosa.ConclusionsThe transgenic mice show that ectopic expression of Tcfap2c is not tolerated. Due to the phenotype observed, iTcfap2c-mice represent a model system to study liver failure. In intestine, Tcfap2c induced cellular hyperplasia and suppressed terminal differentiation indicating that Tcfap2c serves as a repressor of differentiation and inducer of proliferation. This might be achieved by the Tcfap2c mediated activation of Sox9 known to be expressed in intestinal and hepatic stem/progenitor cell populations.