Male Germline Cells Research Articles

Cancer–testis antigen HCA587/MAGE-C2 interacts with BS69 and promotes its degradation in the ubiquitin–proteasome pathway

HCA587, also known as MAGE-C2, belonging to the MAGE gene family which is characterized by a conserved MAGE Homology Domain, is active in various types of tumors and silent in normal tissues except in male germ-line cells. The biological function of HCA587 is largely unknown. To analyze it, we attempted to identify protein partners of HCA587. We immunopurified HCA587-containing complex from HEK293 cells and identified BS69, a potential tumor suppressor, as an associated protein by mass spectrometry, and the following Immunoprecipitation and GST pull-down assays confirmed HCA587 interaction with BS69. Interestingly, overexpression of HCA587 promoted ubiquitination and the proteasomal degradation of BS69 whereas knockdown of endogenous HCA587 increased the protein level of BS69. Consistent with a functional role for BS69 in negatively regulating LMP1-induced NF-κB activation, overexpression of HCA587 resulted in a significant enhancement of LMP1-induced IL-6 production. These data indicate that HCA587 is a new negative regulator of BS69.

Regulatory non‐coding transcripts in spermatogenesis: shedding light on ‘dark matter’

Global rise in male infertility over the past decades as a result of falling sperm count and quality has been pointed out by different investigations. Therefore, it is important to understand the molecular mechanism of spermatogenesis and its regulation. Mammalian spermatogenesis, a streamlined process through which male germline cells divide and differentiate into mature spermatozoa, is strictly regulated by phase-specific gene expression which, in turn, is controlled by myriads of regulatory non-coding RNAs (ncRNAs). Rapid advancement in genome mining technologies has identified role of ncRNAs including microRNAs, PIWI-interacting RNAs, endogenous small-interfering RNAs and long non-coding RNAs as controller of gene expression at transcriptional as well as post-transcriptional level in different biological context and disease processes. Here, we discuss the recent progress in our understanding about the involvement of these molecules in spermatogenesis. In addition, we describe here the possible roles of long non-coding RNAs in controlling this process which is not delved so far.

Triploid planarian reproduces truly bisexually with euploid gametes produced through a different meiotic system between sex

Although polyploids are common among plants and some animals, polyploidization often causes reproductive failure. Triploids, in particular, are characterized by the problems of chromosomal pairing and segregation during meiosis, which may cause aneuploid gametes and results in sterility. Thus, they are generally considered to reproduce only asexually. In the case of the Platyhelminthes Dugesia ryukyuensis, populations with triploid karyotypes are normally found in nature as both fissiparous and oviparous triploids. Fissiparous triploids can also be experimentally sexualized if they are fed sexual planarians, developing both gonads and other reproductive organs. Fully sexualized worms begin reproducing by copulation rather than fission. In this study, we examined the genotypes of the offspring obtained by breeding sexualized triploids and found that the offspring inherited genes from both parents, i.e., they reproduced truly bisexually. Furthermore, meiotic chromosome behavior in triploid sexualized planarians differed significantly between male and female germ lines, in that female germ line cells remained triploid until prophase I, whereas male germ line cells appeared to become diploid before entry into meiosis. Oocytes at the late diplotene stage contained not only paired bivalents but also unpaired univalents that were suggested to produce diploid eggs if they remained in subsequent processes. Triploid planarians may therefore form euploid gametes by different meiotic systems in female and male germ lines and thus are be able to reproduce sexually in contrast to many other triploid organisms.

Toward a more precise and informative nomenclature describing fetal and neonatal male germ cells in rodents.

The germ cell lineages are among the best characterized of all cell lineages in mammals. This characterization includes precise nomenclature that distinguishes among numerous, often subtle, changes in function or morphology as development and differentiation of germ cells proceed to form the gametes. In male rodents, there are at least 41 distinct cell types that occur during progression through the male germ cell lineage that gives rise to spermatozoa. However, there is one period during male germ cell development-that which occurs immediately following the primordial germ cell stage and prior to the spermatogonial stage-for which the system of precise and informative cell type terminology is not adequate. Often, male germ cells during this period are referred to simply as "gonocytes." However, this term is inadequate for multiple reasons, and it is suggested here that nomenclature originally proposed in the 1970s by Hilscher et al., which employs the terms M-, T1-, and T2-prospermatogonia, is preferable. In this Minireview, the history, proper utilization, and advantages of this terminology relative to that of the term gonocytes are described.

Bisphenol A: An endocrine and metabolic disruptor

Bisphenol A (BPA), initially designed, like diethylstilbestrol, as a synthetic estrogen, has been rapidly and widely used for its cross-linking properties in the manufacture of polycarbonate plastics and epoxy resins. Because of incomplete polymerization and degradation of the polymers by exposure to higher than usual temperatures, BPA leaches out from food and beverage containers, as well as from dental sealants. In humans, free active unconjugated BPA is metabolized by rapid glucurono- or sulfo-conjugation and eliminated via renal clearance. However, exposure to environmental nanomolar concentrations of BPA is ubiquitous and continuous via different routes: oral, air, skin. In rodents, fetal and perinatal exposure to such environmentally relevant doses of BPA has been shown to affect the brain, liver, gut, adipose tissue, endocrine pancreas, mammary gland and reproductive tract and function. Similar concentrations are also able in vitro to impact human malignant breast, prostate, male germ or adipocyte cell lines (with a promoting effect and by interfering with chemotherapy drugs), or to stimulate pancreatic β cell insulin secretion. High levels of BPA have recently been correlated with obesity, diabetes, cardiovascular diseases, polycystic ovarian disease or low sperm count. However, before the real impact of BPA on human health can be clearly assessed, prospective longitudinal epidemiological studies are needed as well as characterization of selective biomarkers to verify long-term exposure and selective imprinting.

The nuclear import factor importin α4 can protect against oxidative stress

The importin (IMP) superfamily of nuclear transport proteins is essential to key developmental pathways, including in the murine testis where expression of the 6 distinct IMPα proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMPα4 is unique in showing a striking nuclear localization, a property we previously found to be linked to maintenance of pluripotency in embryonic stem cells and to the cellular stress response in cultured cells. Here we examine the role of IMPα4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMPα4–EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMPα4 expression in spermatids. IMPα4 overexpression did not affect overall fertility, testis morphology/weight or spermatogenic progression under normal conditions, but conferred significantly (>30%) increased resistance to oxidative stress specifically in the spermatid subpopulation expressing the transgene. Consistent with a cell-specific role for IMPα4 in protecting against oxidative stress, haploid germ cells from IMPα4 null mice were significantly (c. 30%) less resistant to oxidative stress than wild type controls. These results from two unique and complementary mouse models demonstrate a novel protective role for IMPα4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity.

Females and males contribute in opposite ways to the evolution of gene order in Drosophila.

An intriguing association between the spatial layout of chromosomes within nuclei and the evolution of chromosome gene order was recently uncovered. Chromosome regions with conserved gene order in the Drosophila genus are larger if they interact with the inner side of the nuclear envelope in D. melanogaster somatic cells. This observation opens a new door to understand the evolution of chromosomes in the light of the dynamics of the spatial layout of chromosomes and the way double-strand breaks are repaired in D. melanogaster germ lines. Chromosome regions at the nuclear periphery in somatic cell nuclei relocate to more internal locations of male germ line cell nuclei, which might prefer a gene order-preserving mechanism to repair double-strand breaks. Conversely, chromosome regions at the nuclear periphery in somatic cells keep their location in female germ line cell nuclei, which might be inaccessible for cellular machinery that causes gene order-disrupting chromosome rearrangements. Thus, the gene order stability for genome regions at the periphery of somatic cell nuclei might result from the active repair of double-strand breaks using conservative mechanisms in male germ line cells, and the passive inaccessibility for gene order-disrupting factors at the periphery of nuclei of female germ line cells. In the present article, I find evidence consistent with a DNA break repair-based differential contribution of both D. melanogaster germ lines to the stability/disruption of gene order. The importance of germ line differences for the layout of chromosomes and DNA break repair strategies with regard to other genomic patterns is briefly discussed.

Muscle glycogen synthase isoform is responsible for testicular glycogen synthesis: Glycogen overproduction induces apoptosis in male germ cells

Glycogen is the main source of glucose for many biological events. However, this molecule may have other functions, including those that have deleterious effects on cells. The rate-limiting enzyme in glycogen synthesis is glycogen synthase (GS). It is encoded by two genes, GYS1, expressed in muscle (muscle glycogen synthase, MGS) and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase, LGS). Expression of GS and its activity have been widely studied in many tissues. To date, it is not clear which GS isoform is responsible for glycogen synthesis and the role of glycogen in testis. Using RT-PCR, Western blot and immunofluorescence, we have detected expression of MGS but not LGS in mice testis during development. We have also evaluated GS activity and glycogen storage at different days after birth and we show that both GS activity and levels of glycogen are higher during the first days of development. Using RT-PCR, we have also shown that malin and laforin are expressed in testis, key enzymes for regulation of GS activity. These proteins form an active complex that regulates MGS by poly-ubiquitination in both Sertoli cell and male germ cell lines. In addition, PTG overexpression in male germ cell line triggered apoptosis by caspase3 activation, proposing a proapoptotic role of glycogen in testis. These findings suggest that GS activity and glycogen synthesis in testis could be regulated and a disruption of this process may be responsible for the apoptosis and degeneration of seminiferous tubules and possible cause of infertility.

Generation of a novel germline stem cell line expressing a germline‐specific reporter in the mouse

Germline stem (GS) cells are stem cell lines derived from postnatal male germline cells. Remarkably, GS cells can form functional spermatozoa when transplanted into infertile host mouse testes, indicating that GS cells have spermatogonial stem cell (SSC) activity. As GS cells are the only type with SSC activity, they are most suitable for in vitro studies on male germ cell differentiation. However, GS cells can deviate from the germ cell state to become other types of cells, depending on culture conditions. Therefore, it is desirable to have a monitor system to ensure that GS cells are kept at the germ cell state in culture. Here, we established GS cell lines from neonatal testes of transgenic mice that express the fluorescent protein, Venus, whose gene expression is driven by the promoter of Mvh (mouse Vasa homolog), a gene highly specific to mammalian germ cells. This novel cell line has genuine GS cell properties equivalent to existing GS lines, including the ability to generate viable offspring. This Mvh-Venus GS cell line, to our knowledge, is the first one expressing a germ cell-specific reporter. This valuable resource should provide new opportunities for studies on male germ cell differentiation.

BMP4 can generate primordial germ cells from bone‐marrow‐derived pluripotent stem cells

Evidence of germ cell derivation from embryonic and somatic stem cells provides an in vitro model for the study of germ cell development, associated epigenetic modification and mammalian gametogenesis. More importantly, in vitro derived gametes also represent a potential strategy for treating infertility. In mammals, male and female gametes, oocyte and sperm, are derived from a specific cell population, PGCs (primordial germ cells) that segregate early in embryogenesis. We have isolated pluripotent SSEA-1+ (stage-specific embryonic antigen-1+) cells from mice bone marrow using a MACS (magnetic-activated cell sorting) system. SSEA-1+ cells were directly separated from the suspension of MMCs (murine mononuclear cells) harvested from bone marrow of 2-4-week-old mice. Flow-cytometry assay immediately after sorting and culturing under undifferentiated condition showed 55±7% and 87±4% purity respectively. RT-PCR (reverse transcription-PCR) analysis after differentiation of SSEA-1+ cells into derivations of three germ layers showed the pluripotency properties of isolated cells. SSEA-1+ cells were induced to differentiate along germ cell lineage by adding BMP4 (bone morphogenic factor-4) to the medium. Regarding the expression of germ cell markers (PGCs, male and female germ cell lineage), it was found that adding exogenous BMP4 to culture medium could differentiate pluripotent SSEA-1+ cells isolated from an adult tissue into gamete precursors, PGCs. Differentiated cells expressed specific molecular markers of PGCs, including Oct4, fragilis, Stella and Mvh (mouse vasa homologue). Therefore BMP4 is insufficient to induce SSEA-1+ cells derived from PGCs to develop further into late germ cells in vitro.

MASTR: A Technique for Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination Using Conditional Floxed Alleles in Mice

Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination), which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26(MASTR)) was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26(MASTR) mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial) and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.

Chromatin associated Sin3A is essential for male germ cell lineage in the mouse

Spermatogenesis is a complex process that requires coordinated proliferation and differentiation of male germ cells. The molecular events that dictate this process are largely unknown, but are likely to involve highly regulated transcriptional control. In this study, we investigate the contribution of chromatin associated Sin3A in mouse germ cell lineage development. Genetic inactivation of Sin3A in the male germline leads to sterility that results from the early and penetrant apoptotic death observed in Sin3A-deleted germ cells, coincident with the reentry in mitosis. Sin3A-deleted testes exhibit a Sertoli-cell only phenotype, consistent with the absolute requirement for Sin3A in germ cells' development and/or viability. Interestingly, transcripts analysis revealed that the expression program of Sertoli cells is altered upon inactivation of Sin3A in germ cells. These studies identified a central role for the mammalian Sin3-HDAC complex in the germ cell lineage, and point to an exquisite transcriptional crosstalk between germ cells and their niche to support fertility in mammals.

Spatio‐temporal patterns of apoptotsis and mitosis during organismal regeneration in a basal chordate

Ascidians are marine invertebrate chordates that are closely related to vertebrates. The colonial ascidian Botryllus schlosseri represents a model system for the study of homeostasis and regeneration. During its weekly budding cycle known as blastogenesis, new buds are generated by asexual reproduction, and as they reach functional maturity, replace the old asexual generation of adult zooids. During this phase of blastogenesis (called takeover), the entire generation of adult zooids undergoes apoptotic programmed cell death (PCD) followed by its resorption and clearance by circulating phagocytes within 24–36 hours. The goal of the present study was to investigate the spatio‐temporal relationship between PCD and mitosis using polyester wax‐embedded serial sections from colonies at various phases of the blastogenetic cycle. We have used an immunofluorescence microscopy approach in conjunction with the use of an antibody against phospho histone H3 (pHH3) to detect mitotic cells. Here, we demonstrate that mitosis is largely restricted to tissues and organs of developing buds as well as male germline cells of both buds and adult zooids. We are currently using pHH3 immunoreactivity simultaneously with terminal deoxynucleotidyl transferase dUTP end‐labeling (TUNEL assay) to respectively visualize mitotic and apoptotic cells respectively. Experiments are currently underway to determine how these processes quantitatively differ during growth and death phases of a B. schlosseri colony.

Drosophila Carrying Pex3 or Pex16 Mutations Are Models of Zellweger Syndrome That Reflect Its Symptoms Associated with the Absence of Peroxisomes

The peroxisome biogenesis disorders (PBDs) are currently difficult-to-treat multiple-organ dysfunction disorders that result from the defective biogenesis of peroxisomes. Genes encoding Peroxins, which are required for peroxisome biogenesis or functions, are known causative genes of PBDs. The human peroxin genes PEX3 or PEX16 are required for peroxisomal membrane protein targeting, and their mutations cause Zellweger syndrome, a class of PBDs. Lack of understanding about the pathogenesis of Zellweger syndrome has hindered the development of effective treatments. Here, we developed potential Drosophila models for Zellweger syndrome, in which the Drosophila pex3 or pex16 gene was disrupted. As found in Zellweger syndrome patients, peroxisomes were not observed in the homozygous Drosophila pex3 mutant, which was larval lethal. However, the pex16 homozygote lacking its maternal contribution was viable and still maintained a small number of peroxisome-like granules, even though PEX16 is essential for the biosynthesis of peroxisomes in humans. These results suggest that the requirements for pex3 and pex16 in peroxisome biosynthesis in Drosophila are different, and the role of PEX16 orthologs may have diverged between mammals and Drosophila. The phenotypes of our Zellweger syndrome model flies, such as larval lethality in pex3, and reduced size, shortened longevity, locomotion defects, and abnormal lipid metabolisms in pex16, were reminiscent of symptoms of this disorder, although the Drosophila pex16 mutant does not recapitulate the infant death of Zellweger syndrome. Furthermore, pex16 mutants showed male-specific sterility that resulted from the arrest of spermatocyte maturation. pex16 expressed in somatic cyst cells but not germline cells had an essential role in the maturation of male germline cells, suggesting that peroxisome-dependent signals in somatic cyst cells could contribute to the progression of male germ-cell maturation. These potential Drosophila models for Zellweger syndrome should contribute to our understanding of its pathology.

Insights on PRAME and osteosarcoma by means of gene expression profiling

BackgroundOsteosarcoma (OS) is the most frequent bone tumor in children and adolescents. Tumor antigens are encoded by genes that are expressed in many types of solid tumors but are silent in normal tissues, with the exception of placenta and male germ-line cells. It has been proposed that antigen tumors are potential tumor markers. ObjectivesThe premise of this study is that the identification of novel OS-associated transcripts will lead to a better understanding of the events involved in OS pathogenesis and biology. MethodsWe analyzed the expression of a panel of seven tumor antigens in OS samples to identify possible tumor markers. After selecting the tumor antigen expressed in most samples of the panel, gene expression profiling was used to identify osteosarcoma-associated molecular alterations. A microarray was employed because of its ability to accurately produce comprehensive expression profiles. ResultsPRAME was identified as the tumor antigen expressed in most OS samples; it was detected in 68% of the cases. Microarray results showed differences in expression for genes functioning in cell signaling and adhesion as well as extracellular matrix-related genes, implying that such tumors could indeed differ in regard to distinct patterns of tumorigenesis. ConclusionsThe hypothesis inferred in this study was gathered mostly from available data concerning other kinds of tumors. There is circumstantial evidence that PRAME expression might be related to distinct patterns of tumorigenesis. Further investigation is needed to validate the differential expression of genes belonging to tumorigenesis-related pathways in PRAME-positive and PRAME-negative tumors.

Small RNA molecules and regulation of spermatogenesis

Small RNA molecules (small RNAs) have recently emerged as important regulators of gene expression at the post-transcriptional or translation level. Significant progress has recently been made in utilizing small RNAs to elucidate the molecular mechanisms regulating spermatogenesis. There are three major small RNAs: small interfering RNAs (siRNAs), microRNAs (miRNAs), and piwi-interacting RNAs (piRNAs). Small-RNAs have diverse biological functions in meiosis and spermatogenesis, In vitro or in vivo, use of siRNA to knockdown genes is a way to study the function of genes of interest in spermatogenesis. miRNA can be involved in the regulation of mitosis, meiosis, and postmeiosis in spermatogenesis. piRNAs are mainly involved in regulating the process of meiosis and postmeiosis, and repressing retrotransposon transposition in male germline cells. In this paper, we reviewed recent works on the synthesis, mechanism, function, and outlook of small RNAs.

Vinblastine-Induced Cytogenotoxicity in Spermatogonia and Its Transmission in the Germline Cells of Swiss Mice

Vinblastine, a cytotoxic anti-neoplastic drug and a known mitotic spindle inhibitor, has reportedly induced numerical and structural alterations in chromosome complements of treated animals. In the present study, cytogenotoxic effects of three different doses of vinblastine (0.5, 1.0, and 1.5 mg/kg body weight) were assessed from mouse spermatogonia at 24 hours after treatment after a single intraperitoneal exposure. The transmission potential of such effects in the male germline of mice was also assessed from primary spermatocytic chromosome analysis and sperm morphology assay at weeks 4 and 8 after treatments, respectively. Induction of statistically significant percentages of aberrant spermatogonial metaphases (P ≤ 0.01) and chromosomal aberrations (excluding gaps) (P ≤ 0.05) in vinblastine-treated mice indicated its clastogenicity. Induction of significant percentages of aberrant primary spermatocytes with atypical bivalents (P ≤ 0.01) and different categories of abnormal sperm, although not with significant variation in frequency, indicated the transmission of vinblastine-induced cytogenotoxic effects from spermatogonia to spermatocyte to sperm. We conclude that vinblastine is cytogenotoxic to mouse spermatogonia and that such induced effects are transmissible in the male germline cells of Swiss mice. Potential transmission of such cytogenotoxic effects, from cancer survivors of reproductive age with vinblastine pretreatment through gametes, is a serious concern.

What a difference a day makes! The contribution of intrinsic FGF9 signalling to germline masculinisation

Although the key discovery of sex-determining region Y (SRY) 20 years ago as the essential sex-determining gene emerged from studies of sex-reversed humans,1, 2 most of our knowledge of how male and female germ cells diverge after sex determination comes from studies of mice. This is demonstrated in a recent publication from Bowles and colleagues in Developmental Cell, which documents the functional importance of fibroblast growth factor 9 (FGF9) signalling in murine fetal germ cells.3 The study reveals events immediately following sex determination which influence how germline cells in the testis respond to FGF9 in a manner that reinforces the process of masculinisation. The picture that is coming into focus is of a signalling network which mediates the correct timing of progressive changes, thereby ensuring that germ cells and the somatic cells which create their niche develop together. Retinoic acid (RA) produced by the mesonephros adjacent to the fetal gonad has recently been identified as the key stimulant for fetal germ cell entry into meiosis: its degradation by the Cyp26b1 enzyme within the testis ensures that most male germ cells continue mitosis and do not enter cell cycle arrest until postnatal processes trigger the first wave of spermatogenesis.4, 5 The additional and antagonistic involvement of FGF9 in driving male germline development was discerned by Bowles and colleagues from several experimental observations of germ cells in Cyp26b1-null testes. These XY germ cells are exposed to high levels of RA but do not fully adopt the phenotype of their female counterparts. Transcripts encoding meiotic markers that are usually upregulated in XX germ cells are only partially increased in XY Cyp26b1-null germ cells, and quite remarkably, the levels of the pluripotency markers Oct4 and Sox2 persist as usual for male germline cells. This provided a clue to the existence of another factor which sustained male pathway development, and FGF9 was an obvious candidate considering that previous studies had shown how its genetic deletion from mice caused male to female sex reversal.6 A remarkable observation reported in this manuscript was the identification in isolated embryonic day (E) 11.5 germ cells of a transcript encoding a fibroblast growth factor receptor isoform that binds FGF9 with high affinity. This FGFR2-IIIc transcript was characterized through reverse transcription-PCR. The authors have deduced, by extrapolation from previous publications, that this highlights a switch in receptor isoform synthesis between E10.5 germ cells7 and E11.5, the time frame identified for germline gender commitment in the testis.8 This isoform switch, which is expected to render the cells highly responsive to FGF9, is mediated by alternative mRNA splicing which alters the structure of the receptors' extracellular domain. Isoform switching in FGF receptors is a well-documented phenomenon that is known to trigger changes over a similar, short time span in other developmental processes, is linked with oncogenic changes and can be induced by exposure to other FGF ligands (e.g., Ref. 9). Identification of the local factor(s) which mediate this change in transcript processing within the developing testis will enhance our understanding of the processes of germline masculinisation. The results of this study have reinforced the knowledge that male and female germline developments are mutually exclusive. The authors have shown that FGF9 actions are antagonistic to, and independent of, RA signalling. FGF9 is initially present in both male and female gonads but it is greatly elevated in the testis by E11.5. The addition of FGF9 to urogenital ridge cultures did not alter Cyp26b1 transcript levels, nor did the addition of RA or absence of Cyp26b1 diminish FGF9 mRNA levels. However, the exposure of E11.5 XX urogenital ridges to FGF9 does diminish synthesis of the direct RA target, Stra8, and this appears to be by diminishing the responsiveness of XX germline cells to RA. As the field of in vitro germline genesis progresses in parallel, information about the pathway by which FGF9 drives male fate may offer the potential for efficiencies and gender selectivity in gamete derivation from embryonic and induced pluripotent stem cells. Similarly, the importance of learning how pluripotency is sustained is of clear importance to testicular cancer and may even be applicable to harnessing spermatogonial stem cells as a potential source of therapeutic materials. The study by Bowles, Koopman and colleagues8 represents another piece in the puzzle of sex determination which has recently progressed from being focussed on somatic cell events to one in which germline events are now being delineated. The use of mice to understand the fundamental events which shape our genetic material is essential, due to the ethical concerns and practical restrictions that limit work on relevant human samples. What do we know about FGF9 in human gonad biology? The study presented here suggests clear starting points for learning how diseases arising from intrinsic germline defects may emerge due to disregulation of FGF signalling.

Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl

Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient's testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa. Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens.

Epigenetic Mechanisms Regulate Stem Cell Expressed Genes Pou5f1 and Gfra1 in a Male Germ Cell Line

Male fertility is declining and an underlying cause may be due to environment-epigenetic interactions in developing sperm, yet nothing is known of how the epigenome controls gene expression in sperm development. Histone methylation and acetylation are dynamically regulated in spermatogenesis and are sensitive to the environment. Our objectives were to determine how histone H3 methylation and acetylation contribute to the regulation of key genes in spermatogenesis. A germ cell line, GC-1, was exposed to either the control, or the chromatin modifying drugs tranylcypromine (T), an inhibitor of the histone H3 demethylase KDM1 (lysine specific demethylase 1), or trichostatin (TSA), an inhibitor of histone deacetylases, (HDAC). Quantitative PCR (qPCR) was used to identify genes that were sensitive to treatment. As a control for specificity the Myod1 (myogenic differentiation 1) gene was analyzed. Chromatin immunoprecipitation (ChIP) followed by qPCR was used to measure histone H3 methylation and acetylation at the promoters of target genes and the control, Myod1. Remarkably, the chromatin modifying treatment specifically induced the expression of spermatogonia expressed genes Pou5f1 and Gfra1. ChIP-qPCR revealed that induction of gene expression was associated with a gain in gene activating histone H3 methylation and acetylation in Pou5f1 and Gfra1 promoters, whereas CpG DNA methylation was not affected. Our data implicate a critical role for histone H3 methylation and acetylation in the regulation of genes expressed by spermatogonia – here, predominantly mediated by HDAC-containing protein complexes.