Human Germ Cell Research Articles

The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells

Germ cells are vital for transmitting genetic information from one generation to the next and for maintaining the continuation of species. Here, we analyze the transcriptome of human primordial germ cells (PGCs) from the migrating stage to the gonadal stage at single-cell and single-base resolutions. Human PGCs show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes, with a subset of them displaying developmental-stage-specific features. Furthermore, we analyze the DNA methylome of human PGCs and find global demethylation of their genomes. Approximately 10 to 11 weeks after gestation, the PGCs are nearly devoid of any DNA methylation, with only 7.8% and 6.0% of the median methylation levels in male and female PGCs, respectively. Our work paves the way toward deciphering the complex epigenetic reprogramming of the germline with the aim of restoring totipotency in fertilized oocytes.

A Unique Gene Regulatory Network Resets the Human Germline Epigenome for Development

SummaryResetting of the epigenome in human primordial germ cells (hPGCs) is critical for development. We show that the transcriptional program of hPGCs is distinct from that in mice, with co-expression of somatic specifiers and naive pluripotency genes TFCP2L1 and KLF4. This unique gene regulatory network, established by SOX17 and BLIMP1, drives comprehensive germline DNA demethylation by repressing DNA methylation pathways and activating TET-mediated hydroxymethylation. Base-resolution methylome analysis reveals progressive DNA demethylation to basal levels in week 5–7 in vivo hPGCs. Concurrently, hPGCs undergo chromatin reorganization, X reactivation, and imprint erasure. Despite global hypomethylation, evolutionarily young and potentially hazardous retroelements, like SVA, remain methylated. Remarkably, some loci associated with metabolic and neurological disorders are also resistant to DNA demethylation, revealing potential for transgenerational epigenetic inheritance that may have phenotypic consequences. We provide comprehensive insight on early human germline transcriptional network and epigenetic reprogramming that subsequently impacts human development and disease.

US science academies take on human-genome editing

National Academy of Sciences and National Academy of Medicine to develop guidelines for rapidly advancing technology to modify human embryos and germ cells.

This Month in Investigative Urology

No AccessJournal of UrologyThis Month in Investigative Urology1 Aug 2015This Month in Investigative Urology Karl-Erik Andersson Karl-Erik AnderssonKarl-Erik Andersson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.05.031AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "This Month in Investigative Urology." The Journal of Urology, 194(2), pp. 276–277 References 1 : Bone metastasis in renal cell carcinoma is preprogrammed in the primary tumor and caused by AKT and integrin α5 signaling. J Urol2015; 194: 539. Link, Google Scholar 2 : Synergy of histone-deacetylase inhibitor AR-42 with cisplatin in bladder cancer. J Urol2015; 194: 547. Link, Google Scholar 3 : Human fetal testicular tissue xenotransplantation: a platform to study the effect of gonadotropins on human germ cell development in utero. J Urol2015; 194: 585. Link, Google Scholar 4 : Abnormalities in expression of structural, barrier and differentiation related proteins, and chondroitin sulfate in feline and human interstitial cystitis. J Urol2015; 194: 571. Link, Google Scholar © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 194Issue 2August 2015Page: 276-277 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karl-Erik Andersson More articles by this author Expand All Advertisement PDF downloadLoading ...

Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation.

Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility.

Human Primordial Germ Cells in a Dish

Researchers have cultured cells that look and act like human primordial germ cells (PGCs)—and they have done it reproducibly, in abundance. Analysis of these cells, derived from both human embryonic stem cells and induced pluripotent cells, reveals some of the factors that drive PGC specification and development [1]. Researchers have previously produced mouse PGCs in a dish, but this transformation has proven more recalcitrant from human stem cell lines, which give rise to PGCs only at low frequency. Jacob Hanna and M. Azim Surani overcame this barrier, starting with a protocol they had previously developed to generate embryonic stem cells or induced pluripotent stem cells. Their protocol yields ‘‘naive’’ pluripotent cells that resemble the blastocyst inner cell mass, in contrast with previous methods, which yielded cells with a slightly more differentiated state, akin to cells of the epithelial postimplantation epiblast. The researchers could handily generate PGC-like cells by applying a molecular cocktail similar to that used to derive PGC-like cells from mouse stem cells. The resulting cells had similar RNA profiles as human germline tumor cells and primordial germ cells derived from aborted human fetuses. The cultured cells also showed signs of epigenetic changes—DNA demethylation—known to occur in PGCs. Further analysis, including gene knockout studies, identified some differences and similarities with mouse PGCs. For instance, the researchers found that the transcriptional regulator SOX17 is a key regulator of PGC-like fate in human cells—a role that it does not seem to serve in mice, where it is better known for specifying endoderm. The researchers identified some of the factors downstream of SOX17, including BLIMP1, which seems to repress somatic genes during specification of PGCs. BLIMP1 is also involved in mouse PGC fate. The findings open the door to the possibility of generating sperm and egg cells in cell culture. But for the more immediate future, this experimental system should be invaluable for answering basic questions about human development at its earliest stages.

MiR-888: A Novel Cancer-Testis Antigen that Targets the Progesterone Receptor in Endometrial Cancer

Cancer-testis (CT) antigens are a large family of genes that are selectively expressed in human testis germ cells, overexpressed in a variety of tumors and predominantly located on the X chromosome. To date, all known CT antigens are protein-coding genes. Here, we identify miR-888 as the first miRNA with features characteristic of a CT antigen. In a panel of 21 normal human tissues, miR-888 expression was high in testes and minimal or absent in all other examined tissues. In situ hybridization localized miR-888 expression specifically to the early stages of sperm development within the testes. Using The Cancer Genome Atlas database, we discovered that miR-888 was predominately expressed in endometrial tumors, with a significant association to high-grade tumors and increased percent invasion. In a separate panel of endometrial tumor specimens, we validated overexpression of miR-888 by real-time polymerase chain reaction. In addition, miR-888 expression was highest in endometrial carcinosarcoma, a rare and aggressive type of endometrial tumor. Moreover, we identified the progesterone receptor (PR), a potent endometrial tumor suppressor, as a direct target of miR-888. These data define miR-888 as the first miRNA CT antigen and a potential mediator of an aggressive endometrial tumor phenotype through down-regulation of PR.

Male germ-like cell differentiation potential of human umbilical cord Wharton's jelly-derived mesenchymal stem cells in co-culture with human placenta cells in presence of BMP4 and retinoic acid.

Mesenchymal stem cells (MSCs) derived from Wharton's jelly (WJ-MSCs) are now much more appealing for cell-based infertility therapy. Hence, WJ-MSCs differentiation toward germ layer cells for cell therapy purposes is currently under intensive study. MSCs were isolated from human Wharton's jelly and treated with BMP4, retinoic acid (RA) or co-cultured on human amniotic epithelial (HAE) and chorionic plate (HCP) placenta feeder cells. profile of POU5F1, Fragilis, Plzf, DDX4, Piwil2, Stra8, Dazl, β1- and α6-integrins (ITΒ1, ITA6) genes expression as germ cell markers were analyzed using RT-PCR and real-time PCR. Immunocytochemistry of surface markers were conducted. After 3 weeks treatment with different reagents and co-culture system, morphology of WJ-MSCs changed to shiny clusters and germ cell specific markers in mRNA were up-regulated in both placental feeder + RA and BMP4 + RA. Induction of hWJ-MSCs with BMP4 in presence of RA resulted in significant up-regulation (P≤0.05) of all germ cell specific genes (c-Kit; 2.84±0.59, DDX4; 1.69±0.39, Piwil2; 1.14±0.21, Dazl; 0.65±0.25, α6 integrin; 1.26±0.53, β1 integrins; 1.18±0.65) compared to control and placental feeder cells + RA. Our results indicated that HAE and HCP followed by RA treatment were involved in human germ cell development. We demonstrated that under the right conditions, hWJ-MSCs have the ability to differentiate to germ cells and this provides an excellent pattern to study infertility cause and treatment.

Assessing reproductive toxicity of two environmental toxicants with a novel in vitro human spermatogenic model

Environmental influences and insults by reproductive toxicant exposure can lead to impaired spermatogenesis or infertility. Understanding how toxicants disrupt spermatogenesis is critical for determining how environmental factors contribute to impaired fertility. While current animal models are available, understanding of the reproductive toxic effects on human fertility requires a more robust model system. We recently demonstrated that human pluripotent stem cells can differentiate into spermatogonial stem cells/spermatogonia, primary and secondary spermatocytes, and haploid spermatids; a model that mimics many aspects of human spermatogenesis. Here, using this model system, we examine the effects of 2-bromopropane (2-BP) and 1,2,dibromo-3-chloropropane (DBCP) on in vitro human spermatogenesis. 2-BP and DBCP are non-endocrine disrupting toxicants that are known to impact male fertility. We show that acute treatment with either 2-BP or DBCP induces a reduction in germ cell viability through apoptosis. 2-BP and DBCP affect viability of different cell populations as 2-BP primarily reduces spermatocyte viability, whereas DBCP exerts a much greater effect on spermatogonia. Acute treatment with 2-BP or DBCP also reduces the percentage of haploid spermatids. Both 2-BP and DBCP induce reactive oxygen species (ROS) formation leading to an oxidized cellular environment. Taken together, these results suggest that acute exposure with 2-BP or DBCP causes human germ cell death in vitro by inducing ROS formation. This system represents a unique platform for assessing human reproductive toxicity potential of various environmental toxicants in a rapid, efficient, and unbiased format.

The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1.

The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis.

Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile.

Primordial germ cells (PGCs) develop only into sperm and oocytes in vivo. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of in vitro models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of de novo DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3–6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine.

Current proceedings of cerebral palsy.

Cerebral palsy (CP) is a complicated disease with varying causes and outcomes. It has created significant burden to both affected families and societies, not to mention the quality of life of the patients themselves. There is no cure for the disease; therefore, development of effective therapeutic strategies is in great demand. Recent advances in regenerative medicine suggest that the transplantation of stem cells, including embryonic stem cells, neural stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells, umbilical cord blood cells, and human embryonic germ cells, focusing on the root of the problem, may provide the possibility of developing a complete cure in treating CP. However, safety is the first factor to be considered because some stem cells may cause tumorigenesis. Additionally, more preclinical and clinical studies are needed to determine the type of cells, route of delivery, cell dose, timing of transplantation, and combinatorial strategies to achieve an optimal outcome.

Human Fetal Testicular Tissue Xenotransplantation: A Platform to Study the Effect of Gonadotropins on Human Germ Cell Development In Utero

We examined the effects of long-term hCG stimulation on germ cell maturation, and Sertoli and Leydig cell function in a xenotransplantation model of the human fetal testis. A total of 20 human fetal testes were ectopically xenografted on 20 castrated NCr male nude mice. Grafts were collected for analysis 24 weeks later. Mice were treated with saline as the control or with hCG beginning 4 weeks after the grafts were transplanted. Of the grafts 65% survived at 24 weeks. In contrast to untreated pregrafted samples, hCG stimulated xenografts showed significantly increased density of seminiferous tubule formation with Sertoli cell migration to the basement membrane. Germ cell proliferation and differentiation from gonocytes (M2A(+)) to prespermatogonia (MAGE-4A(+)) were observed in graft samples recovered from the hCG and nonhCG treated groups at 24 weeks of treatment. Leydig cells in hCG treated grafts produced significantly more testosterone than nonhCG treated grafts. Although further studies are required to investigate the potential for further differentiation and maturation of xenografted human fetal testes, normal in utero testicular development was reproduced under long-term hCG stimulation. This model represents a means to study long-term effects of gonadotoxins or hormonal stimulation on the maturation of human fetal testes.

Interrogation of a context-specific transcription factor network identifies novel regulators of pluripotency.

The predominant view of pluripotency regulation proposes a stable ground state with coordinated expression of key transcription factors (TFs) that prohibit differentiation. Another perspective suggests a more complexly regulated state involving competition between multiple lineage-specifying TFs that define pluripotency. These contrasting views were developed from extensive analyses of TFs in pluripotent cells in vitro. An experimentally validated, genome-wide repertoire of the regulatory interactions that control pluripotency within the in vivo cellular contexts is yet to be developed. To address this limitation, we assembled a TF interactome of adult human male germ cell tumors (GCTs) using the Algorithm for the Accurate Reconstruction of Cellular Pathways (ARACNe) to analyze gene expression profiles of 141 tumors comprising pluripotent and differentiated subsets. The network (GCT(Net)) comprised 1,305 TFs, and its ingenuity pathway analysis identified pluripotency and embryonal development as the top functional pathways. We experimentally validated GCT(Net) by functional (silencing) and biochemical (ChIP-seq) analysis of the core pluripotency regulatory TFs POU5F1, NANOG, and SOX2 in relation to their targets predicted by ARACNe. To define the extent of the in vivo pluripotency network in this system, we ranked all TFs in the GCT(Net) according to sharing of ARACNe-predicted targets with those of POU5F1 and NANOG using an odds-ratio analysis method. To validate this network, we silenced the top 10 TFs in the network in H9 embryonic stem cells. Silencing of each led to downregulation of pluripotency and induction of lineage; 7 of the 10 TFs were identified as pluripotency regulators for the first time.

Stem cells. Human primordial germ cells in a dish.

A robust method to induce primordial germ cells from human pluripotent stem cells has been developed, highlighting the importance of SOX17 in the specification of the human germline.

Nonylphenol affects activity of human reproductive cells

Nonylphenol (NP) is an industrial compound belonging to Endocrine Disrupting Chemicals (EDCs) with xenoestrogenic activity abundantly present in the environment. Its xenoestrogenic activity was demonstrated both in vitro and in vivo. Estrogens play an important role in development and growth of human endometrium and prostate. Since NP mimics endogenous estrogens, it could have a negative influence on normal physiology of both organs. In this study we examined the effects of NP and 17-β-estradiol (E2) on human prostate adenocarcinoma epithelial cell line (LNCaP) and endometrial adenocarcinoma cell line Ishikawa. Firstly, we performed a MTT assay in order to see cell proliferation. We found that both NP and E2 stimulated prostate and endometrial cell proliferation but in different manner. In fact, on human prostate cells NP induced a non-monotonic increase of cell proliferation whereas on endometrial cells its effect was dose-dependent. Secondly, we studied expression and localization of estrogen receptor alpha (ERα) in both LNCaP and Ishikawa cells. Immunofluorescence and western blot analyses revealed that both NP and E2 induced cytoplasm-nucleus translocation of ERα even if E2 was able to induce an early nuclear translocation respect to NP. Finally through qPCR analysis we studied changing in gene expression of genes involved in proliferation pathways. We observed that in prostate cells NP and E2 induced upregulation of cyclin D1, ki-67, cytochine IL-8 and IL-1β together with a downregulation of cyclin E. On the contrary, in human endometrial cells gene upregulation was present only after E2 treatment whereas NP seem have no effects on these specific gene targets. These results showed that NP has estrogenic activity on human reproductive cells both in male and female system, but probably it may also interfere with other molecular pathways to induce a deregulation of normal cell physiology as seen in human endometrial cells.

NODAL secreted by male germ cells regulates the proliferation and function of human Sertoli cells from obstructive azoospermia and nonobstructive azoospermia patients.

This study was designed to explore the regulatory effects of male germ cell secreting factor NODAL on Sertoli cell fate decisions from obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) patients. Human Sertoli cells and male germ cells were isolated using two-step enzymatic digestion and SATPUT from testes of azoospermia patients. Expression of NODAL and its multiple receptors in human Sertoli cells and male germ cells were characterized by reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry. Human recombinant NODAL and its receptor inhibitor SB431542 were employed to probe their effect on the proliferation of Sertoli cells using the CCK-8 assay. Quantitative PCR and Western blots were utilized to assess the expression of Sertoli cell functional genes and proteins. NODAL was found to be expressed in male germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ACTR-IIB were detected in Sertoli cells and germ cells, suggesting that NODAL plays a regulatory role in Sertoli cells and germ cells via a paracrine and autocrine pathway, respectively. Human recombinant NODAL could promote the proliferation of human Sertoli cells. The expression of cell cycle regulators, including CYCLIN A, CYCLIN D1 and CYCLIN E, was not remarkably affected by NODAL signaling. NODAL enhanced the expression of essential growth factors, including GDNF, SCF, and BMP4, whereas SB431542 decreased their levels. There was not homogeneity of genes changes by NODAL treatment in Sertoli cells from OA and Sertoli cell-only syndrome (SCO) patients. Collectively, this study demonstrates that NODAL produced by human male germ cells regulates proliferation and numerous gene expression of Sertoli cells.

Insights into female germ cell biology: from in vivo development to in vitro derivations.

Understanding the mechanisms of human germ cell biology is important for developing infertility treatments. However, little is known about the mechanisms that regulate human gametogenesis due to the difficulties in collecting samples, especially germ cells during fetal development. In contrast to the mitotic arrest of spermatogonia stem cells in the fetal testis, female germ cells proceed into meiosis and began folliculogenesis in fetal ovaries. Regulations of these developmental events, including the initiation of meiosis and the endowment of primordial follicles, remain an enigma. Studying the molecular mechanisms of female germ cell biology in the human ovary has been mostly limited to spatiotemporal characterizations of genes or proteins. Recent efforts in utilizing in vitro differentiation system of stem cells to derive germ cells have allowed researchers to begin studying molecular mechanisms during human germ cell development. Meanwhile, the possibility of isolating female germline stem cells in adult ovaries also excites researchers and generates many debates. This review will mainly focus on presenting and discussing recent in vivo and in vitro studies on female germ cell biology in human. The topics will highlight the progress made in understanding the three main stages of germ cell developments: namely, primordial germ cell formation, meiotic initiation, and folliculogenesis.

Genes at stake.

We are living in the era of genetic engineering and looking forward to its eventual clinical application to life-threatening human diseases. In this context, we appreciate the UK government's decision in February to allow the clinical application of mitochondrial DNA transfer techniques to women who carry defective mitochondrial genes. Such genes can produce unhealthy offspring. Now we face another report from daring researchers in China (doi: 10.1007/s13238-015-0153-5). This month, Dr. Junjiu Huang and colleagues at Sun Yat-Sen University in Guangzhou performed human embryo genome editing to correct β-thalassemia by adopting the clustered regularly interspaced short palindromic repeat-associated system (CRISPR/Cas9). Although they found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB), the efficiency of homologous recombination-directed repair of HBB was low and the edited embryos were mosaic. They concluded that gene editing by this method is not practical for clinics, highlighting the pressing need to improve the accuracy of gene editing technology. Previous human genetic modification efforts have been focused on non-reproductive somatic cells using zinc-finger nucleases, DNA-binding proteins that can be engineered to induce a double-strand break in a section of DNA incorporating a new stretch of DNA into a selected location, to fix a mutation that could cause disease. CRISPR/Cas9 is the newest and simplest method to use RNA molecules that recognize specific human DNA sequences. Human embryonic research using CRISPR/Cas9 raises anxiety because it shows that we are still not ready to precisely control our reproductive cells. In light of the outcomes reported by Dr. Huang and colleagues, we should recognize the urgent need for guidelines on genomic editing studies on human germ cells. Our knowledge about genes remains limited, and the outcome of embryonic gene editing can be confirmed after birth only. We must first and foremost clarify whether and when genomic editing research in reproductive cells should take place. As responsible scientists, we can expect vigorous debate over the benefit to patients.

Synthesis, characterization and biological evaluation of N-(2,3-dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H-pyrazol-4-yl)benzamides

Comparison of active site residues of modelled h-GCAP with template h-PLAP.