Ectopic expression of Cvh (Chicken Vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate

When they are derived from blastodermal cells of the pre-primitive streak in vitro, the pluripotency of Chicken Embryonic Stem Cells (cESC) can be controlled by the cPouV and Nanog genes. These cESC can differentiate into derivatives of the three germ layers both in vitro and in vivo, but they only weakly colonize the gonads of host embryos. By contrast, non-cultured blastodermal cells and long-term cultured chicken primordial germ cells maintain full germline competence. This restriction in the germline potential of the cESC may result from either early germline determination in the donor embryos or it may occur as a result of in vitro culture. We are interested in understanding the genetic determinants of germline programming. The RNA binding protein Cvh (Chicken Vasa Homologue) is considered as one such determinant, although its role in germ cell physiology is still unclear. Here we show that the exogenous expression of Cvh, combined with appropriate culture conditions, induces cESC reprogramming towards a germ cell fate. Indeed, these cells express the Dazl, Tudor and Sycp3 germline markers, and they display improved germline colonization and adopt a germ cell fate when injected into recipient embryos. Thus, our results demonstrate that Vasa can drive ES cell differentiation towards the germ cell lineage, both in vitro and in vivo.     

On the formation of germ cells: The good,the bad and the ugly

Mammalian germ cells are powerful cells, the only ones that transmit information to the next generation ensuring the continuation of the species. But “with great power, comes great responsibility”, meaning that germ cells are only a few steps away from turning carcinogenic. Despite recent advances little is known about germ cell formation in mammals, predominantly because of the inaccessibility of these cells. Moreover, it is difficult to pin down what in essence is characteristic of a germ cell, as germ cells keep changing place, morphology, expression markers and epigenetic identity. Formation of (primordial) germ cells in primate ES cell cultures would therefore be helpful to identify molecular signalling pathways associated with germ cell differentiation and to study epigenetic changes in germ cells. In addition, the in vitro derivation of functional germ cells from ES cells could be used in combination with therapeutic cloning to generate patient-specific ES cell lines, and can have applications in animal breeding. In this review we present the state-of-the-art on how mouse and human germ cells are formed in vivo (the good), we discuss the link between germ cells, pluripotency and germ cell tumours (the bad) and show that despite continuous progress in trying to differentiate germ cells in vitro (the ugly) the generation of functional germ cells is still a real challenge.     

Sxl in the germline of Drosophila: A target for somatic late induction

In Drosophila, the sex of germ cells is determined by autonomous and inductive signals. Somatic inductive signals can drive XX germ cells into oogenesis or into spermatogenesis. An autonomous signal makes XY germ cells male and unresponsive to sex determination by induction. The elements forming the X:A ratio in the soma and the genes tra, tra2, dsx, and ix that determine the sex of somatic cells have no similar role in the germline. The gene Sxl, however, is required for female differentiation of somatic and germ cells. Inductive signals that are dependent on somatic tra and dsx expression already affect the sex-specific development of germ cells of first instar larvae. At this early stage, however, germline expression of Sxl does not appear to affect the sexual characteristics of germ cells. Since inductive signals dependent on tra and dsx nevertheless influence the choice of sex-specific splicing of Sxl, it can be concluded that Sxl is a target of the inductive signal, but that its product is required late for oogenesis. Other genes must therefore control the early sexual dimorphism of larval germ cells. © 1994 Wiley-Liss, Inc.     

Blurring the germline: Genome editing and transgenerational epigenetic inheritance

Sperm, eggs and embryos are made up of more than genes, and there are indications that changes to non-genetic structures in these elements of the germline can also be inherited. It is, therefore, a mistake to treat phrases like ‘germline inheritance’ and ‘genetic inheritance’ as simple synonyms, and bioethical discussion should expand its focus beyond alterations to the genome when considering the ethics of germline modification. Moreover, additional research on non-genetic inheritance draws attention to a variety of means whereby differences can be inherited in offspring generations that do not rely on differences in germline structures. Research on these diverse forms of inheritance challenges the notion that there is some special form of ethical concern that falls on germline interventions in general, and on interventions to the nuclear genome within the germline in particular.     

The role of longitudinal cohort studies in epigenetic epidemiology: challenges and opportunities

ABSTRACT: Longitudinal cohort studies are ideal for investigating how epigenetic patterns change over time and relate to changing exposure patterns and the development of disease. We highlight the challenges and opportunities in this approach.     

The planarian <Emphasis Type="Italic">nanos</Emphasis>-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration

Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Germline-derived DNA methylation and early embryo epigenetic reprogramming: The selected survival of imprints

DNA methylation is an essential epigenetic mechanism involved in many essential cellular processes. During development epigenetic reprograming takes place during gametogenesis and then again in the pre-implantation embryo. These two reprograming windows ensure genome-wide removal of methylation in the primordial germ cells so that sex-specific signatures can be acquired in the sperm and oocyte. Following fertilization the majority of this epigenetic information is erased to give the developing embryo an epigenetic profile coherent with pluripotency. It is estimated that ∼65% of the genome is differentially methylated between the gametes, however following embryonic reprogramming only parent-of-origin methylation at known imprinted loci remains. This suggests that trans-acting factors such as Zfp57 can discriminate imprinted differentially methylated regions (DMRs) from the thousands of CpG rich regions that are differentially marked in the gametes. Recently transient imprinted DMRs have been identified suggesting that these loci are also protected from pre-implantation reprograming but succumb to de novo remethylation at the implantation stage. This highlights that “ubiquitous” imprinted loci are also resilient to gaining methylation by protecting their unmethylated alleles. In this review I examine the processes involved in epigenetic reprograming and the mechanisms that ensure allelic methylation at imprinted loci is retained throughout the life of the organism, discussing the critical differences between mouse and humans.This article is part of a Directed Issue entitled: Epigenetics Dynamics in development and disease.     

Expression of the tudor-related gene Tdrd5 during development of the male germline in mice

Homologues of Drosophila germ cell determinant genes such as vasa, nanos and tudor have recently been implicated in development of the male germline in mice. In the present study, the mouse gene encoding Tudor domain containing protein 5 (TDRD5) was isolated from a 12.5-13.5 days post coitum (dpc) male-enriched subtracted cDNA library. Whole-mount in situ hybridization analysis of Tdrd5 expression in the mouse embryonic gonad indicated that this gene is upregulated in the developing testis from 12.5 dpc, with expression levels remaining higher in testis than ovary throughout embryogenesis. Expression of Tdrd5 was absent in testes isolated from We/We embryos, which lack germ cells. In situ hybridization (ISH) on cryosectioned 13.5 dpc testes suggests that expression of Tdrd5, like that of Oct4, is restricted to germ cells. Northern hybridization analysis of expression in adult tissues indicated that Tdrd5 is expressed in the testis only, implying that expression of this gene is restricted to the male germline throughout development to adulthood.     

The multiple hats of Vasa: its functions in the germline and in cell cycle progression

Vasa, an ATP-dependent RNA helicase, is broadly conserved among various organisms from cnidarians to mammals. It has a rich history of utility as a germline marker, and is believed to function as a positive translational regulator in the determination and maintenance of germline cells. Studies in non-model organisms, however, revealed that Vasa is also present in somatic cells of many tissues. In many cases these cells are multipotent, are non-germline associated, and give rise to a variety of different tissue types. Recent work now also demonstrates that Vasa functions in the regulation of the cell cycle. Here, we discuss this newly described function of Vasa in mitotic and meiotic cell cycles, and we address the conundrum created within these observations, that is, that most cells are mitotically independent of Vasa, yet when Vasa is present in a cell, it appears to be essential for cell cycle progression.     

Morphological,histochemical and biochemical studies on germ cell mitochondria of normal rats

Summary Morphological changes in rat germ cell mitochondria are described. In diplotene and secondary spermatocytes and in the spermatids of the Golgi, cap and acrosomal phases, the mitochondria take on a rounded appearance with the inner space containing the matrix flattened against the outer membrane and the intracristal spaces considerably swollen (condensed mitochondria).Functional studies on condensed mitochondria isolated from the germ cells of normal rats have been performed. The following parameters have been evaluated: ADP/O ratio, respiratory control ratio (RCR) and ADP affinity. The ADP/O values found in the presence of various substrates are in agreement with the theoretical figures. The RCR is remarkably high. Moreover, the ADP affinity of these mitochondria is very high, as demonstrated by the low values of the apparent Km. These biochemical findings, which demonstrate a high oxidative capacity coupled with a marked phosphorylation, suggest that the condensed appearance of germ cell mitochondria is the expression of an active functional state.The work was partially supported by a grant from The Consiglio Nazionale delie Ricerche (C.N.R.), Rome, Italy     

The relationship between genotype, developmental stability and mating performance: disentangling the epigenetic causes

Developmental stability (DS) may confer an advantage in competition for mates. The present study tests this hypothesis using Drosophila immigrans, and proposes a novel approach to help broadly define the epigenetic factors causing such an effect. We first estimated the magnitude of isofemale heritability in sternopleural bristle fluctuating asymmetry (FA), using replicate genetic lines extracted from nature. Positional FA (PFA) exhibited significant among-line variation, and the heritability estimate of 0.10 (0.046 s.e.m.) was statistically significant. Among individual males, there was a significant positive relationship between PFA and copulation latency (time elapsed between introduction of females and copulation) and duration, but not copulation frequency. Moreover, high-DS lines exhibited significantly shorter copulation latency and duration compared with low-DS lines. When these components of sexual performance were again contrasted between lines with among-individual differences in bristle asymmetry controlled statistically, significant line effects on copulation latency and duration disappeared. The results suggest that deficits in the developmental apparatus underlying one particular trait can compromise individual sexual performance, and weaken the hypothesis that FA is a cue of overall 'genetic quality'.     

The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken

A novel system has been developed to determine the origin and development of primordial germ cells (PGCs) in avian embryos directly. Approximately 700 cells were removed from the center of the area pellucida, the outer of the area pellucida, and the area opaca of the stage X blastoderm (Eyal-Giladi and Kochav, 1976; Dev Biol 49:321–337). When the cells were removed from the center of the area pellucida, the mean number of circulating PGCs per 1 μl of blood was significantly decreased to 13 (P < 0.05) in the embryo at stage 15 (Hamburger and Hamilton, 1951: J Morphol 88:49–92) as compared to intact embryos of 51. When the removed recipient cells from the center of the area pellucida were replenished with 500 donor cells, no reduction in the PGC number was observed. The removal of cells from the outer of area pellucida or from the area opaca had no effect on the number of PGCs. When another set of the manipulated embryos were cultured ex vivo to hatching and reared to sexual maturity, the absence of germ cells and the degeneration of seminiferous tubules were observed in resulting chickens derived from the blastoderm from which the cells were removed from the center of the area pellucida. Chimeric embryos produced by the male donor cells and the female recipient contained the female-derived cells at 97.2% in the whole embryo and 94.3% in the erythrocytes at 5 days of incubation. At 5–7 days of incubation, masculinization was observed in about one half of the mixed-sex embryos. The proportions of the female-derived cells in the whole embryo and in the erythrocytes were 76.5% and 80.2% at 7 days to 55.7% and 62.5% at 10 days of incubation, respectively. When the chimeras reached their sexual maturity, they were test mated to assess donor contribution to their germline. Five of six male chimeras (83%) and three of five female chimeras (60%) from male donor cells and a female recipient embryo from which 700 cells at the center of area pellucida were removed were germline chimeras. Three of the five male germline chimeras (60%) and one of the three female germline chimeras (33%) transmitted exclusively (100%) donor-derived gametes into the offspring. When embryonic cells were removed from the outer of area pellucida or area opaca, regardless of the sex combination of the donor and the recipient, the transmission of the donor-derived gametes was essentially null. The findings in the present studies demonstrated, both in vivo and in vitro, that the PGCs originate in the central part of the area pellucida and that the developmental fate to germ cell (PGCs) had been destined at stage X blastoderm in chickens. Mol. Reprod. Dev. 48:501–510, 1997. © 1997 Wiley-Liss, Inc.     

Translational repression restricts expression of the C. elegans Nanos homolog NOS-2 to the embryonic germline

Members of the nanos gene family are evolutionarily conserved regulators of germ cell development. In several organisms, Nanos protein expression is restricted to the primordial germ cells (PGCs) during early embryogenesis. Here, we investigate the regulation of the Caenorhabditis elegans nanos homolog nos-2. We find that the nos-2 RNA is translationally repressed. In the adult germline, translation of the nos-2 RNA is inhibited in growing oocytes, and this inhibition depends on a short stem loop in the nos-2 3'UTR. In embryos, nos-2 translation is repressed in early blastomeres, and this inhibition depends on a second region in the nos-2 3'UTR. nos-2 RNA is also degraded in somatic blastomeres by a process that is independent of translational repression and requires the CCCH finger proteins MEX-5 and MEX-6. Finally, the germ plasm component POS-1 activates nos-2 translation in the PGCs. A combination of translational repression, RNA degradation, and activation by germ plasm has also been implicated in the regulation of nanos homologs in Drosophila and zebrafish, suggesting the existence of conserved mechanisms to restrict Nanos expression to the germline.     

Fate of the initial follicle pool: empirical and mathematical evidence supporting its sufficiency for adult fertility

The importance of the initial follicle pool in fertility in female adult mammals has recently been debated. Utilizing a mathematical model of the dynamics of follicle progression (primordial to primary to secondary), we examined whether the initial follicle pool is sufficient for adult fertility through reproductive senescence in CD1 mice. Follicles in each stage were counted from postnatal day 6 through 12 months and data were fit to a series of first-order differential equations representing two mechanisms: an initial pool of primordial follicles as the only follicle source (fixed pool model), or an initial primordial follicle pool supplemented by germline stem cells (stem cell model). The fixed pool model fit the experimental data, accurately representing the maximum observed primary follicle number reached by 4-6 months of age. Although no germline stem cells could be identified by SSEA-1 immunostaining, the stem cell model was tested using a range of de novo primordial follicle production rates. The stem cell model failed to describe the observed decreases in follicles over time and did not parallel the accumulation and subsequent reduction in primary follicles during the early fertile lifespan of the mouse. Our results agree with established dogma that the initial endowment of ovarian follicles is not supplemented by an appreciable number of stem cells; rather, it is sufficient to ensure the fertility needs of the adult mouse.