Adult Germ Cells Research Articles

Abstract LB-67: Global DNA methylation in fetal human germ cells and germ cell tumors: correlation with differentiation and cisplatin resistance

Abstract Differences in the global methylation pattern, i.e. hyper- as well as hypomethylation, are observed in cancer, including germ cell tumors (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n=103) and GCTs (n=251) by immunohistochemical staining for 5-mCytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, while female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, carcinoma in situ and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumors, choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam-2 before and after demethylation using 5-azacytidine. Exposure to 5-azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell specific marker VASA showed increased expression. Upon treatment with 5-azacytidine, TCam-2 cells were analyzed by high throughput methylation screen for changes in methylation sites of 14,000 genes. Among the genes revealing changes, interesting targets were identified, i.e. demethylation of KLF11, a putative tumor suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-67.

Abstract 4240: Transcription factor networks that regulate pluripotency and lineage differentiation in adult human male germ cell tumors

Abstract Recent interest and effort have been focused on identification of transcription factors (TFs) and TF networks that regulate pluripotency and lineage differentiation in human and murine cells. Yet, a complete repertoire of regulatory interactions supporting these processes remains elusive. Embryonic stem (ES) cells, induced pluripotent (iPS) cells, and embryonal carcinoma (EC) cells derived from germ cell tumors (GCTs) comprise the three classic pluripotent cell types. EC cells exhibit, in vivo and in vitro, many properties in common with ES cells, making them an excellent system to study pluripotency and lineage differentiation. We previously characterized the gene expression profile (GEP) of a panel of 135 GCT biopsies comprising all described in vivo differentiation lineages and 6 normal testes as controls, using the Affymetrix U133A+B arrays (Korkola et al. Cancer Res., 66: 820-7, 2006 and J. Clin. Oncol., 27: 5240-7, 2009). These GEP data were analyzed using the ARACNe reverse engineering algorithm (Basso et al., Nat. Genet., 37: 382-90, 2005) to reconstruct a global transcriptional interaction network regulating pluripotency and lineage differentiation in these tumors. The ensuing interactome, comprising 1312 TFs, displayed three strongly intra-connected modules: Module A, including 187 genes associated with regulation of development and differentiation, Module B, including 89 genes associated with regulation of immune response and B-cell development, and Module C, including 80 genes associated with regulation of transcription. Furthermore, the interactome recapitulated 830 regulatory targets of the core pluripotency TFs, POU5F1, NANOG, and SOX2 (369 of POU5F1, 375 of NANOG, and 306 of SOX2). Validation of these targets by ChIP-PCR and ChIP-seq in NT2/D1 (EC) and H9 (ES) cells revealed that all of the top 10 target genes of POU5F1 (NANOG, FAM46B, L1TD1, DND1, ZYG11, FRAT2, DPPA4, ARGEF1, KLRG2 and CBR3) and SOX2 (SEPHS1, PLCB2, ZNF518, ECE2, SLC26A3, GPM6B, PHLDA2, PATZ1, ADRB3 and MAP1A) could specifically bind the corresponding TF in their promoter regions, within both cell types. For NANOG, 9 of the top 10 targets (POU5F1, FAM46B, DPPA4, L1TD1, UTF1, FRAT2, RAB15, TEAD4 and ZYG11A) showed specific promoter binding of the TF in both cell types, while one target (DND1) showed specific binding only in H9 cells. ChIP-seq analysis was consistent with the results of ChIP-PCR. We sought functional validation of the targets by performing lentivirus-mediated shRNA silencing of POU5F1 in NT2/D1 cells. Results showed that the ARACNe-inferred targets were both positively and negatively regulated, consistently with regulation of pluripotency and/or differentiation. Taken together, these results define a novel and highly accurate regulatory model for the systems biology study of TFs involved in regulation of pluripotency and lineage differentiation in mammalian cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4240.

Partial Rescue of the KIT-Deficient Testicular Phenotype in KitW‑v/KitW‑v Tg(TSPY) Mice1

TSPY encodes the testis-specific protein Y-linked. In man, expression of TSPY is restricted to the testis, where TSPY is expressed in spermatogonia, primary spermatocytes, and round spermatids, and to the prostate gland. There is circumstantial evidence that TSPY is involved in spermatogonial proliferation and gonadal tumorigenesis. Because the laboratory mouse carries the Tspy gene in a naturally silenced state (Tspy-ps), we previously restored TSPY activity in mice and generated a TSPY transgenic mouse line in which the organization and expression of the human TSPY transgene follow the human pattern. In the present study, we generated TSPY transgenic KIT-deficient Kit(W-v)/Kit(W-v) mice and analyzed the histology of the testes and epididymides in order to contribute to understanding TSPY function in early germ cell development and spermatogenesis. The KIT receptor and its ligand KITL, previously called stem cell factor, have an indispensable role in hematopoiesis, melanogenesis, and gametogenesis. Homozygous Kit(W-v) mutant male mice on a C57BL/6J background with a mutation in the Kit gene are infertile due to an almost total loss of germ cells in the testes. In this study, histological analyses of testes and epididymides showed an increased number of meiotic and postmeiotic germ cells in Kit(W-v)/Kit(W-v) Tg(TSPY) mice compared with age-matched Kit(W-v)/Kit(W-v) controls. TSPY was able to restore fertility of some but not all TSPY transgenic Kit(W-v)/Kit(W-v) males. Our findings show that TSPY is able to partially rescue spermatogenesis and fertility of Kit(W-v)/Kit(W-v) mutants and thereby point to a putative role of TSPY in fetal and adult germ cell proliferation.

Differential localization and independent acquisition of the H3K9me2 and H3K9me3 chromatin modifications in the Caenorhabditis elegans adult germ line.

Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3) in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2) and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase) family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z) homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in this context. Further, these and other data support a model in which these two modifications function independently in adult C. elegans germ cells.

Testicular germline stem cells

Stem cells have the ability to both differentiate into other mature cell types and maintain an undifferentiated state by self-renewal. These unique properties form the basis for stem cell use in organ replacement and tissue regeneration in clinical medicine. Currently, embryonic stem cells are the best-studied stem cell type. However alternative stem cells such as induced pluripotent stem cells and other adult stem cells are also being actively investigated for their potential for cell-based therapy. Among adult stem cells, emerging research has focused on evaluating the pluripotency potential of testis stem cells. To date, stem cells with embryonic-like potential have been created from adult testis germ cells. These cells could provide patient-specific, non-embryo-derived stem cells for men in the future.

Current protocols in the generation of pluripotent stem cells: theoretical, methodological and clinical considerations

Pluripotent stem cells have been derived from various embryonic, fetal and adult sources. Embryonic stem cells (ESCs) and parthenogenic ESCs (pESCs) are derived from the embryo proper while embryonic germ cells (EGCs), embryonal carcinoma cells (ECCs), and germ-line stem cells (GSC) are produced from germ cells. ECCs were the first pluripotent stem cell lines established from adult testicular tumors while EGCs are generated in vitro from primordial germ cells (PGCs) isolated in late embryonic development. More recently, studies have also demonstrated the ability to produce GSCs from adult germ cells, known as spermatogonial stem cells. Unlike ECCs, the source of GSCs are normal, non-cancerous adult tissue. The study of these unique cell lines has provided information that has led to the ability to reprogram somatic cells into an ESC-like state. These cells, called induced pluripotent stem cells (iPSCs), have been derived from a number of human fetal and adult origins. With the promises pluripotent stem cells bring to cell-based therapies there remain several considerations that need to be carefully studied prior to their clinical use. Many of these issues involve understanding key factors regulating their generation, including those which define pluripotency. In this regard, the following article discusses critical aspects of pluripotent stem cell derivation and current issues about their therapeutic potential.

Family history of cancer and malignant germ cell tumors in children: A report from the Children’s Oncology Group

Family history of testicular cancer is an established risk factor for adult testicular germ cell tumors (GCT). We evaluated the association between family history of cancer and pediatric GCT in a Children's Oncology Group case-control study that included 274 GCT cases (195 female and 79 male) diagnosed < age 15 years and 418 controls frequency matched to cases on sex and age. Family history data were collected through telephone interviews with biological mothers and fathers and unconditional logistic regression was used to evaluate associations with GCT adjusting for potential confounders. A family history of cancer with onset < age 40 years was associated with a reduced risk of GCT among female cases (Odds Ratio (OR) = 0.50, 95% Confidence Interval (CI) 0.28-0.89) and an increased risk among male cases (OR = 2.56, 95% CI 1.02-6.44). Male cases were more likely to report family history of melanoma compared with male controls (OR = 4.65, 95% CI 1.40-15.4). There was an inverse association between family history of ovarian or uterine cancers and GCT in girls (OR = 0.46, 95% CI 0.22-0.96). These sex and cancer site specific associations should be confirmed in additional studies as they may provide clues to the etiology of pediatric GCT.

Cancer/testis (CT) antigens: Potential targets for immunotherapy

Cancer/testis (CT) antigens are protein antigens with normal expression restricted to adult testicular germ cells, and yet are aberrantly activated and expressed in a proportion of various types of human cancer. At least a subset of this group of antigens has been found to elicit spontaneous humoral and cell-mediated immune responses in cancer patients, raising the possibility that these antigens could be cancer vaccine targets. More than 100 CT antigen genes have been reported in the literature, with approximately 30 being members of multigene families on the X chromosome, so-called CT-X genes. Most CT-X genes are expressed at the spermatogonia stage of spermatogenesis, and their functions are mostly unknown. In cancer, the frequency of CT antigen expression is highly variable among different tumor types, but is more often expressed in high-grade late-stage cases in general. Cancer vaccine trials based on CT antigens MAGE-A3 and NY-ESO-1 are currently ongoing, and these antigens may also play a role in antigen-specific adoptive T-cell transfer and in the immunomodulation approach of cancer therapy.

Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells

dead end ( dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish ( Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.

Boule Is Present in Fish and Bisexually Expressed in Adult and Embryonic Germ Cells of Medaka

BackgroundThe DAZ family genes boule, daz and dazl encode RNA binding proteins essential for fertility of diverse animals including human. dazl has bisexual expression in both mitotic and meiotic germ cells, whereas daz has male premeiotic expression, and boule is largely a unisexual meiotic regulator. Although boule has been proposed as the ancestor for dazl/daz by gene duplication, it has been identified only in invertebrates and mammals. It has, however, remained unclear when and how the DAZ family has evolved in vertebrates.Methodology and Principal FindingsThis study was aimed at identifying and characterizing the DAZ family genes in fish as the basal vertebrate. We show that boule and dazl coexist in medaka and stickleback. Similar to the medaka dazl (Odazl), the medaka boule (Obol) is maternally supplied and segregates with primordial germ cells. Surprisingly, Obol is expressed in adult germ cells at pre-meiotic and meiotic stages of spermatogenesis and oogenesis. However, the maximal meiotic Obol expression in spermatocytes contrasts with the predominant pre-meiotic Odazl expression in spermatogonia, and the diffuse cytoplasmic Obol distribution in early oocytes contrasts with the Odazl concentration in the Balbinani's body.ConclusionsThe identification of fish boule and dazl genes provides direct evidence for the early gene duplication during vertebrate evolution. Our finding that Obol exhibits bisexual expression in both embryonic and adult germ cells considerably extends the diversity of boule expression patterns and offers a new insight into the evolutions of DAZ family members, expression patterns and functions in animal fertility.

A novel RRM domain protein is involved in the assembly of the tubulin cytoskeleton in Marsilea sperm cells

P granules, ribonucleoprotein complexes specific C. elegans germ cells, are implicated in post-transcriptional control of maternallytranscribed mRNAs. The Bennett laboratory studies the P granule component GLH-1 (Germline RNA Helicase-1). Through genetics we have shown that GLH-1 is essential for fertility. With immunoprecipitations and GST-pull-downs we report that GLH-1 and the riboendonuclease Dicer bind one another and their interaction is not RNAdependent. GLH-1 and DCR-1 also interact genetically. In the dcr-1 (ok247) null mutant, GLH-1 levels are dramatically reduced, both by western blot analysis and immunocytochemistry. Conversely, DCR-1 protein levels are reduced in the glh-1(gk100) loss-of-function mutant. Thus, GLH-1 and DCR-1 levels are mutually dependent. To determine if DCR-1 and GLH-1 co-localize, DCR-1-specific antibodieswere generated inmice and rabbits; these antibodiesdetectDicer inworms.We find that DCR-1 and GLH-1 co-localize in P granules in adult germ cells at the pachytene stage of oogenesis when most maternal RNAs are produced. Based on the GLH-1/Dicer association, we predict that these two proteins may work together in the miRNA pathway. How might GLH-1 levels depend upon Dicer? Microarray analysis of the dcr-1(ok247) mutant by the Bass lab reported glh-1mRNA significantly reducedwhen DCR-1 is missing, which our laboratory confirmed by qRT-PCR. Two miRNAs,miR-83 andmiR-67 are predicted to bind to the 3′UTR of glh-1 mRNA. Western blot analyses of protein from miR-83 and miR-67 mutants show that GLH-1 levels are also reduced. Therefore, these miRNAs may normally up-regulate translation of glh-1; thus, Dicer may actively promote its GLH-1 partner.

γH2AX marks the main events of the spermatogenic process

Recent findings reveal that chromatin reorganization requires formation and repair of DNA double strand breaks (DSBs). Therefore, dynamic changes in chromatin structure taking place along the mitotic and the meiotic processes should be accompanied by histone H2AX phosphorylation and formation of gamma H2AX, a prominent event occurring at DSB sites. Accordingly, gamma H2AX has been detected at multiple developmental steps in adult germ cells. Nevertheless, no comprehensive study focusing on the main biological events occurring in the seminiferous epithelium has been carried out in mammals. Here I analyze H2AX phosphorylation in immunostained testis sections comparing PAS/cresyl violet counterstained, noncounterstained, and immuno-fluorescence preparations and show several waves of H2AX phosphorylation/dephosphorylation coupled to various developmental phases of spermatogonia and spermatocytes as well as to spermatid differentiation. The combined detection of DNA replication or free 3'-OH DNA ends highlight the possible significance of gamma H2AX at these locations.

Differential Expression of Urocortin in Human Testicular Germ Cells in Course of Spermatogenesis: Role for Urocortin in Male Fertility?

To provide the first insights into the potential role of urocortin in mammalian spermatogenesis, we studied the expression of urocortin and corticotropin-releasing factor receptors 1 and 2 in the human testis. Urocortin is a bioactive peptide with antiapoptotic and antiproliferative properties. The proper regulation of apoptosis and proliferation is of high physiologic relevance in the control of spermatogenesis in adulthood and of the noncycling stage of gonocytes in fetal life. Using polymerase chain reaction analysis and immunohistochemistry, the expression and tissue localization of urocortin on mRNA and at the peptide level was studied in 9 normal adult, 5 fetal, and 5 Sertoli cell-only testicular specimens. Tissue localization of corticotropin-releasing factor receptors in normal adults was considered using immunohistochemistry. We found that urocortin mRNA was present in all adult specimens tested and that the urocortin peptide was expressed in elongating spermatids, mature spermatozoa, and peritubular myoid cells, and to a lesser extent in Leydig cells. Corticotropin-releasing factor receptor 1 was mainly expressed in spermatogonia, spermatocytes, and Leydig cells, and corticotropin-releasing factor receptor 2 was mostly expressed in spermatogonia. The antibodies against urocortin produced a nuclear staining in fetal gonocytes. No significant immunopositivity for urocortin could be observed in the Sertoli cell-only specimens. The expression of urocortin in normal adult and fetal testicular germ cells in the cell division arrest phases suggests a probable role for this peptide in the pathophysiology of germ cell differentiation and division. In human germ cells, the separate location of urocortin and its receptors indicates a receptor-independent action of urocortin in the course of spermatogenesis.

Comparisons for detecting NY-ESO-1 mRNA expression levels in hepatocellular carcinoma tissues

NY-ESO-1 is a cancer/testis (CT) antigen expressed in normal adult tissues solely in the testicular germ cells of normal adults and in various cancers. It induces specific humoral and cellular immunity in patients with NY-ESO-1-expressing cancer. We compared the expression of NY-ESO-1 mRNA in hepatocellular carcinoma (HCC) patients by using various primers and DNA polymerases to optimize RT-PCR conditions and to evaluate the correlations among the expression levels of NY-ESO-1, LAGE-1 and SSX-1 and clinical parameters. We determined differences in the abilities of the various primers and DNA polymerases to amplify the NY-ESO-1 gene at different exons. Primers designated as P3 detected targeted sequences better than primers P1 and P2; AmpliTaq Gold DNA polymerase was more effective than Platinum pfx DNA polymerase and Taq DNA polymerase. NY-ESO-1, LAGE-1 and SSX-1 mRNAs were detected in 29.7, 45.3 and 37.5%, respectively, of the 64 HCC specimens. No CT antigen mRNAs were detected in the 64-paired adjacent non-cancerous tissues. The frequency for the co-expression of one, two or three antigens of NY-ESO-1, LAGE-1 and SSX-1 was 57.8, 35.9 and 18.8%, respectively. We also analyzed the relationships among the CT antigen expression levels and several clinical parameters. There were no significant differences between CT antigen expression levels and clinical parameters, except the correlations between the expression of SSX-1 and the age of the patients.

170. TGFβ SIGNALING IN AN IN VITRO SEMINOMA MODEL

Testicular cancer, the second most common malignancy in young men, has a 95% cure rate but can result in infertility or subfertility. Its incidence has increased significantly in recent decades (1). This cancer is thought to arise during embryogenesis, based on the persistence of embryonic germ cell markers such as Blimp1 (2), Oct3/4 (3) and Nanog (3) in adult seminoma cells. TCam2 cells are a recently characterised in vitro seminoma model (4). We show by Q-PCR and immunofluorescence that they also express these early germ cell markers. TGFβ signaling plays a key role during germ cell development, and is implicated in the development of testicular cancers (5, 6). To investigate this further, we first determined whether the pathway is active in TCam2 cells. By Q-PCR we demonstrate expression of the TGFβ downstream transcription factors Smad 2, 3 and 4, and Activin type I and II receptors. Importantly, ActRIIA, which is undetectable in adult testicular germ cells, but readily detected in human foetal germ cells (7) and clinical seminoma samples (6), is readily detectable at both the mRNA and protein level in TCam2 cells. Furthermore, 24 hour treatment with Activin (5 and 50ng/ml) or BMP4 (5 and 50ng/ml) induces a 3-4 fold increase in ActRIIA mRNA levels, but not ActRIA, ActRIB or ActRIIB. Strikingly, in TCam2 cells BMP4 and to a lesser extent retinoic acid, but not activin, support survival and proliferation of TCam2 cells in the absence of serum. This is consistent with known roles of BMP4 and retinoic acid in enhancing murine foetal germ cell proliferation/self-renewal and survival (8, 9), and activin inhibition of foetal murine germ cell proliferation (10). This study is the first to demonstrate a functional response in seminoma cells consistent with their foetal germ cell-like identity and forms the basis for future mechanistic analyses of the role of TGFβ signaling in human testicular cancer.

Human Adult Testis-Derived Pluripotent Stem Cells: Revealing Plasticity from the Germline

A recent publication in Nature by Conrad et al. (2008) reports the generation of pluripotent stem cells from isolated human adult testicular germ cells. This work identifies a new source of stem cells for potential autologous therapeutic applications in men.

Clinical and biological significance of CXCL12 and CXCR4 expression in adult testes and germ cell tumours of adults and adolescents

Interaction between the chemokine CXCL12 (SDF1) and the G-protein coupled receptor CXCR4 is responsible for the maintenance of adult stem cell niches and is known to play an important role in utero in the migration of primordial germ cells. We demonstrate expression of CXCL12 by Sertoli cells and confirm CXCR4 expression by the germ cell population of the adult human testes. CXCR4 is also known to mediate organ-specific patterns of metastases in a range of common cancers. We identify consistent expression of CXCR4 mRNA and protein in testicular germ cell tumours (TGCT) that accounts for their patterns of relapse in sites of known CXCL12 expression. Extragonadal primary germ cell tumours express CXCR4 and their sites of occurrence are coincident with areas of known CXCL12 expression in utero. We show that CXCL12 stimulates the invasive migration of a TGCT cell line in vitro in a CXCR4-dependent fashion and activates ERK. Furthermore, we demonstrate that expression of CXCL12 in stage I non-seminomas is significantly associated with organ-confined disease post-orchidectomy and reduced risk of relapse (p = 0.003). This may be through the loss of CXCL12 gradients that might otherwise attract cells away from the primary tumour. We propose CXCL12 expression as a potential predictor of subsequent relapse that could lead to avoiding unnecessary treatment and associated late toxicities. Our observations support a role for CXCL12/CXCR4 in the adult germ cell population and demonstrate pathological function in germ cell tumour development and metastasis that may have clinical utility.

Regulation of adult human spermatogonial stem cells proliferation by estradiol in vitro

OBJECTIVE: Spermatogonial stem cells (SSCs), which are the only germline stem cells in adults, self-renew and produce a large number of differentiating germ cells. In the niche on the basement membrane of seminiferous tubules, SSCs are secluded from the outside world and affected by all kinds of nutrients, growth factors and hormones. Estrogens are now known to have profound effects on male reproductive systems and estrogen receptors (ERs) have been detected in adult human germ cells, thus the aim of the present study was to evaluate the role of 17β-estradiol, the main physiological estrogen, in SSCs proliferation in vitro.

Expression of the diaphanous‐related formin proteins mDia1 and mDia2 in the rat testis

Cytoskeletal alterations in both Sertoli cells and germ cells are important during many facets of mammalian spermatogenesis. Diaphanous-related formin proteins are known to control many aspects of actin-based cytoskeletal rearrangements, yet nothing is known regarding the expression of formins in the testis. Accordingly, here we present the first data describing mDia1 and mDia2 mRNA and protein expression in primary Sertoli cell isolates, established tissue culture cell lines often used as models for Sertoli cell analysis, and mixed populations of adult rat male germ cells. Furthermore, we have examined intact sections of rat testis. The results suggest strongly that mDia1 and mDia2 are indeed involved in the regulation of Sertoli cell and germ structure during mammalian spermatogenesis, and provide strong indications of the future directions for mechanistic studies.

Recovery of a low mutant frequency after ionizing radiation-induced mutagenesis during spermatogenesis

Humans are exposed to ionizing radiation (IR) under various circumstances, e.g. cosmic radiation, diagnostic X-rays and radiotherapy for cancer. It has been shown that IR can impair spermatogenesis and can cause mutations in germ cells. However, the mutagenic responses of germ cells exposed to IR at different stages of testicular maturation have not been examined by directly assessing the mutant frequency in defined spermatogenic cell types. This study was performed to address whether preadult exposure to IR can increase mutations in adult germ cells that could in turn have a major impact on adult reproductive function and the health of ensuing offspring. Male Lac I transgenic mice were irradiated with a single dose of 2.5 Gy of γ-ray at different ages before adulthood, reflecting different stages of testicular maturation, and then mutant frequency (MF) was determined directly in spermatogenic cell types emanating from the irradiated precursor cells. The results showed that (1) preadult exposure to IR did not significantly increase MF in adult epididymal spermatozoa; (2) spermatogenic stages immediately following the irradiated stage(s) displayed an elevated mutant frequency; but (3) the mutant frequency was restored to unirradiated levels in later stages of spermatogenesis. These findings provide evidence that there is a mechanism(s) to prevent spermatogenic cells with elevated mutant frequencies from progressing through spermatogenesis.