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The chromatin state affects the level of transcription at the initial stage of gene expression. Therefore, it is important to determine chromatin accessibility in regions of the genome that contain regulatory elements. To this end, several methods have been developed. The ATAC-seq method outperforms the other methods in profiling chromatin accessibility for every single cell, and standardized library preparation kits are now commercially available. In this chapter, we describe the ATAC-seq method that we have applied to mouse embryonic germ cells as well as to mouse neural stem cells, which are relatively rare. We also developed new method to examine the changes in global accessibility during their developmental process using spike-in normalization with fly cells.

Primordial germ cells (PGCs) must complete a complex and dynamic developmental program during embryogenesis to establish the germline. This process is highly conserved and involves a diverse array of tasks required of PGCs, including migration, survival, sex differentiation, and extensive epigenetic reprogramming. A common theme across many organisms is that PGC success is heterogeneous: only a portion of all PGCs complete all these steps while many other PGCs are eliminated from further germline contribution. The differences that distinguish successful PGCs as a population are not well understood. Here, we examine variation that exists in PGCs as they navigate the many stages of this developmental journey. We explore potential sources of PGC heterogeneity and their potential implications in affecting germ cell behaviors. Lastly, we discuss the potential for PGC development to function as a multistage selection process that assesses heterogeneity in PGCs to refine germline quality.

Mammalian germline cells, embryonic stem cells, and adult stem cells hold the potential for defining, constructing, and maintaining a unique individual through the respective processes of gametogenesis, embryogenesis, and tissue repair over the span of a lifetime. Autophagy carries out critical functions in these cells using a variety of mechanisms. Of particular importance to the integrity of germline and stem cells are (1) the removal of damaged and/or excess mitochondria, thereby reducing the potential for generating reactive oxygen species that can damage genomic content; (2) the removal and recycling of cellular structures to facilitate the extensive remodeling that occurs during the initial establishment and subsequent differentiation of these cell populations; and (3) the supply of substrates and energy sources when needed under stress and in the environment of the “hypoxic niche” that these cells often occupy.

The germ cells (sperm and ovum) have a distinct epigenetic signature varies according to personal, environment, psychological, behavioral, and nutrition criteria and experiences for parental. Epigenetic parental memory is transmitted to the offspring at the time of fertilization and is maintained throughout life through spermatozoa or ova. So, we postulate that sperm and the ovum have a memory and reminiscences. Trauma and negative memories can be passed on from parents to children through sperm and ovum during sexual intercourse. Contrariwise, if offspring from traumatized lineages are put into enriched and positive environments, the process can be reversed and the transfer process of trauma will go away. So, it needs to be reinforced the life parental so that their life is positive, because that reshape the epigenetic signature of spermatozoa and ovulation in human trauma to the best.

Drosophila melanogaster oogenesis is a versatile model system to address many fundamental questions of cell and developmental biology, such as stem cell biology, mitosis, meiosis or cell polarity. Many mutagenesis and powerful genetic tools have contributed massively to identify and dissect in vivo gene functions in a stage and tissue specific manner. However, the small number of germ cells during the early steps of oogenesis have hampered a systematic description of RNA and protein contents at each stage. We describe here a protocol for isolating and comparing two small subpopulations of cells in the ovary for the purpose of RNA sequence profiling. The method is based on fluorescence-activated cell sorting (FACS) of GFP- and RFP-labeled proteins that are expressed in distinct and mostly non-overlapping regions of the germline. We used a transgene expressing a GFP-tagged Bam protein driven by its own promoter, labeling specifically the mitotic region of the germarium. We also took advantage of the short-lived Wicked protein tagged with RFP and expressed under the nanos promoter to label the meiotic region. We generated flies expressing both markers and were able to sort enough cells from each region to extract total RNAs and small RNAs. Total RNA or small RNA extracted from sorted cells were then used to generate deep-sequencing libraries that show specificity toward each compartment. This method of isolating a very small number of cells and the data generated from comparing distinct cell populations within the germline should further our understanding of these conserved steps of oogenesis.

Primordial germ cells (PGCs) are embryonic precursors of sperm and egg that pass on genetic and epigenetic information from one generation to the next. In mammals, they are induced from a subset of cells in peri-implantation epiblast by BMP signaling from the surrounding tissues. PGCs then initiate a unique developmental program that involves comprehensive epigenetic resetting and repression of somatic genes. This is orchestrated by a set of signaling molecules and transcription factors that promote germ cell identity. Here we review significant findings on mammalian PGC biology, in particular, the genetic basis for PGC specification in mice and human, which has revealed an evolutionary divergence between the two species. We discuss the importance and potential basis for these differences and focus on several examples to illustrate the conserved and divergent roles of critical transcription factors in mouse and human germline.

The origin and development of the precursors of gametes termed primordial germ cells (PGCs) have always fascinated the scientists. Today is largely accepted that in Mammals the germ cell lineage is specified in extraembryonic tissues and that after determination PGCs move into the embryo proper to colonize the gonadal primordia. Although the mechanisms of such processes have been partly clarified in the mouse experimental model, in humans they remain still elusive. Likewise, the epigenetic modifications and sex differentiation events accompanying PGC migration and following gonad colonization by such cells are only partly known in the mouse and little characterized in other mammalian species including humans. Here is a brief account of the main progresses that in the last about thirty years have been done in studying the cellular and molecular events of the PGC origin and development in mouse and man.

Primordial germ cells (PGCs) are the embryonic precursors of the totipotent germline stem cells that form the eggs and sperm required for sexual reproduction. Proper PGC specification, development, and sexual identity are fundamental for fertility and the generational inheritance of traits. Drosophila melanogaster serves as a valuable model system to study conserved mechanisms of PGC development.

In mammals, germ cell development is a long process starting in the fetus and going on along the whole reproductive lifespan of the individual. There are major differences between male and female gametogenesis at the organ, tissue cell, and subcellular levels. Such differences have a large influence on the final impact that occupational, dietary, and lifestyle exposure may have on the quality and quantity of mature gametes. Adverse effects of environmental exposures on the germ cells may concern a reduction of their number or fertilizing capacity, which will have an impact on the individual fertility, as well as genetic and epigenetic alterations which will entail a risk of heritable diseases.

Publisher Summary Embryonic germ (EG) cells are pluripotent stem cells derived from primordial germ cells (PGCs) that arise in the late embryonic and early fetal period of development. Embryonic stem (ES) cells were first derived from the inner cell mass of mouse pre-implantation embryos, and EG cells were initially derived from mouse PGCs. Subsequently, EG cells have been derived from chicken, pig, and human PGCs. Pig, chicken, and mouse EG cells have been demonstrated to contribute to experimentally produced chimeric animals, including germline transmission in the latter two species. Human EG cells can be derived from PGCs by using methods similar to those used to derive mouse EG cultures. Like mouse embryonic stem and EG cells, human EG cells require leukemia inhibitory factor (LIF) for proliferation as undifferentiated stem cells. Unlike mouse EG cells, however, human EG cells do not readily lose their dependence on exogenous cytokines and factors supplied by the feeder layer, and they have a higher frequency of spontaneous differentiation into embryoid bodies (EBs). Although EBs are a loss to the pluripotent stem cell population, they are a source of cells expressing markers of mature cellular phenotypes, as well as their presumed progenitors and precursors. Cells that retain a high capacity for cell proliferation and express makers of multiple lineages can be isolated from EBs, and can be used in a variety of in vitro and in vivo differentiation paradigms. The current challenges are to match individual EB-derived (EBD) cultures to desired endpoints, and to enrich or purify populations of cells within EBD cultures to more specifically address biological requirements.

To date, stem cells have been derived from three sources of germ cells. These include embryonic germ cells (EGCs), embryonal carcinoma cells (ECCs), and multipotent germ line stem cells (GSCs). EGCs are derived from primordial germ cells that arise in the late embryonic and early fetal period of development. ECCs are derived from adult testicular tumors whereas GSCs have been derived by culturing spermatogonial stem cells from mouse neonates and adults. For each of these lines, their pluripotency has been demonstrated by their ability to differentiate into cell types derived from the three germ layers in vitro and in vivo and in chimeric animals, including germ line transmission. These germ line-derived stem cells have been generated from many species including human, mice, porcine, and chicken albeit with only slight modifications. This chapter describes general considerations regarding critical aspects of their derivation compared with their counterpart, embryonic stem cells (ESCs). Detailed protocols for EGC derivation and maintenance from human and mouse primordial germ cells (PGCs) will be presented.

Publisher Summary In mammals, three types of pluripotent stem cells have been identified and isolated into culture; these are embryonic stem (ES) cells, embryonic germ (EG) cells, and embryonal carcinoma (EC) cells. These pluripotent stem cells share two important properties. First, they can be maintained indefinitely in culture as an essentially immortal cell line. Second, they are capable of giving rise to every cell type in the body. These features make such cells potentially important tools for the treatment of human disease because the differentiated derivatives of pluripotent stem cells could be used to replace damaged or diseased cells via transplantation. Pluripotent stem cells and their derivatives will also likely generate important information about embryonic development. Many of the standard techniques used for the culture of ES cells—such as culture medium, feeding regimen, and subculture technique—can be used for the culture of EG cells. The major difference lies in the initial isolation of the cells. EG cell lines have been derived from both mouse and human embryos using the same culture conditions. These observations suggest that some of the factors regulating primordial germ cell (PGC) growth and differentiation have been conserved during evolution and, therefore, that the techniques for isolating EG cells described here may be applicable to other mammalian and nonmammalian species. This chapter outlines the protocols developed for the culture of mouse PGCs and their conversion into EG cells, but the protocols may be applicable to the culture of the same cells from a variety of different species, including birds and humans.

The foundation for spermatogenesis is provided by the actions of an undifferentiated spermatogonial population that consists of stem cell and progenitor pools. The spermatogonial stem cell (SSC) pool is a reservoir from which cohorts of progenitor spermatogonia arise that transiently amplify in number prior to committing to a pathway of terminal differentiation. A primary undifferentiated spermatogonial population is established from quiescent gonocyte precursors during early postnatal development, and interaction with Sertoli cells is crucial for this process. Emerging evidence suggests that during neonatal development, contributions from Sertoli cells influence the resumption of mitotic cell cycle progression in gonocytes and guide gonocyte migration from the center of seminiferous cords to the basement membrane. Continued Sertoli cell contributions provide the cues required for establishment of an SSC pool and formation of initial progenitor spermatogonia. In addition, an initial population of differentiating spermatogonia arises directly from a subset of gonocytes to constitute the first round of spermatogenesis. This process is known to occur in the testes of rodents, and an evolving model indicates that Sertoli cell secreted retinoic acid is the major inducer. Miscues in Sertoli cell interaction with gonocytes and newly formed subsets of spermatogonia can have dire consequences for male fertility. Impaired formation of the SSC pool leads to loss of the germ line, and infertility ensues.

Despite the global reprogramming of most epigenetic memories related to cell lineage and cell fate, in the course of the production of somatic clones by nuclear transfer (NT), genomic imprinting memories in donor somatic cells are not erased and faithfully maintained in each individual. This is a crucial factor for successful birth of somatic clones, because the correct parental imprints are essential for normal mammalian development. Thus, germ cell cloning, especially the production of cloned embryos from primordial germ cells (PGCs) and gonocytes, was used for the elucidation of the erasing and re-establishing processes as well as the imprinting-free or default states of the genomic imprinting memories which occurred in specific stages of the PGCs and gonocytes. An important finding was that the PGC clones with the default state of genomic imprinting could not develop to term, and exhibited early embryonic lethality. All these results have contributed to establish the current understanding of the genomic imprinting mechanism and also provided a strong evidence for the essential nature of genomic imprinting in mammals.

Current dogma is that mouse primordial germ cells (PGCs) segregate within the allantois, or source of the umbilical cord, and translocate to the gonads, differentiating there into sperm and eggs. In light of emerging data on the posterior embryonic-extraembryonic interface, and the poorly studied but vital fetal-umbilical connection, we have reviewed the past century of experiments on mammalian PGCs and their relation to the allantois. We demonstrate that, despite best efforts and valuable data on the pluripotent state, what is and is not a PGC in vivo is obscure. Furthermore, sufficient experimental evidence has yet to be provided either for an extragonadal origin of mammalian PGCs or for their segregation within the posterior region. Rather, most evidence points to an alternative hypothesis that PGCs in the mouse allantois are part of a stem/progenitor cell pool that exhibits all known PGC "markers" and that builds/reinforces the fetal-umbilical interface, common to amniotes. We conclude by suggesting experiments to distinguish the mammalian germ line from the soma.

Genomic imprinting is an epigenetic phenomenon in which either the paternal or the maternal allele of imprinted genes is expressed in somatic cells. It is unique to eutherian mammals, marsupials, and flowering plants. It is absolutely required for normal mammalian development. Dysregulation of genomic imprinting can cause a variety of human diseases. About 150 imprinted genes have been identified so far in mammals and many of them are clustered such that they are coregulated by a cis-acting imprinting control region, called the ICR. One hallmark of the ICR is that it contains a germ line-derived differentially methylated region that is methylated on the paternal chromosome or on the maternal chromosome. The DNA methylation imprint is reset in the germ line and differential methylation at an ICR is restored upon fertilization. The DNA methylation imprint is resistant to a genome-wide demethylation process in early embryos and is stably maintained in postimplantation embryos. Maintenance of the DNA methylation imprint is dependent on two distinct maternal effect genes (Zfp57 and PGC7/Stella). In germ cells, around midgestation, the DNA methylation imprint is erased and undergoes another round of the DNA methylation imprint cycle that includes erasure, resetting, restoration, and maintenance of differential DNA methylation.

A number of major adaptations in animals have been mediated by alteration of germ cells and their immediate derivatives, the gametes. Here, several such cases are discussed, including examples from echinoderms, vertebrates, insects, and nematodes. A feature of germ cells that make their development (and hence evolution) distinct from the soma is the prominent role played by posttranscriptional controls of mRNA translation in the regulation of proliferation and differentiation. This presents a number of special challenges for investigation of the evolution of germline development. Caenorhabditis nematodes represent a particularly favorable system for addressing these challenges, both because of technical advantages and (most importantly) because of natural variation in mating system that is rooted in alterations of germline sex determination. Recent studies that employ comparative genetic methods in this rapidly maturing system are discussed, and likely areas for future progress are identified.

Publisher Summary The germ cell lineage in the mouse is not predetermined, but is established during gastrulation, in response to signaling molecules acting on a subset of epiblast cells that move through the primitive streak together with extra-embryonic mesoderm precursors. The germ cell lineage terminates in the differentiation of the gametes (eggs and spermatozoa). In mammals the lineage arises in the extraembryonic mesoderm at the posterior end of the primitive streak. During this period, they proliferate at a steady rate and are known as primordial germ cells (PGCs). PGCs do not at any stage constitute a stem cell population: each of the cell divisions that they undergo (9 or 10 in the mouse, more in the human) moves them further along their developmental trajectory. After migration to the site of the future gonads, germ cell sex determination is achieved, with germ cell phenotype in male and female embryos diverging. Site-specific DNA methylation of imprinted genes is erased in germ cells at about the time of entry into the future gonads, and new imprints are established later. Germ cells respond to certain growth factors by proliferating indefinitely. These immortalized embryonic germ cell lines are chromosomally stable and pluripotent, closely resembling the embryonic stem cell lines derived from blastocyst-stage embryos.

In mammals, germ cells are induced from a population of cells at the base of the allantois. This regulative mechanism of germ line induction depends on Bmp signals and a combination of epigenetic changes that silence somatic differentiation genes and activate pluripotency genes. RNA binding proteins are a conserved feature of germ cell development in mammals, and play critical roles in the establishment and maintenance of pluripotency. After their specification, germ cells move through the gut to the gonads under the influence of migratory and attractive cues. In the gonad, germ cells initiate sex-specific differentiation. Germ cells that arrive in the ovary enter meiosis, whereas germ cells that arrive in the testis undergo mitotic arrest. Entry into meiosis is controlled by retinoic acid signals that are blocked in the testis. The signals regulating mitotic arrest in the testis are still not completely understood, but likely involve RNA-binding proteins. Epigenetic reprograming occurs during specification, migratory stages, and sex-specific stages, when maternal and paternal imprints are established. The facility of transitions between germ cells and stem cells suggests a close relationship among their genomic programs.

Primordial germ cells, which carry the responsibility for perpetuation of the species, are set aside from their somatic neighbors very early in mammalian embryonic development. The founder population of germ cells is rare and difficult to identify and isolate in quantities suitable for molecular and biochemical analysis, thereby highlighting the importance of an in vitro system for deriving germ cells from embryonic stem cells. This chapter details methods for in vitro derivation of germ lineage elements and discusses potential applications of these techniques in germ cell research.

Microinsemination has been widely used in basic reproductive research and in human-assisted reproductive technology for treating infertility. Historically, microinsemination in mammals started with research on the golden hamster; since then, it has provided invaluable information on the mechanisms of mammalian fertilization. Thanks to advances in animal genetic engineering and germ-cell technologies, microinsemination techniques are now used extensively to identify the biological significance of genes of interest or to confirm the genetic normality of gametes produced by experimental manipulations in vitro. Fortunately, in mice, high rates of embryo development to offspring can be obtained so long as postmeiotic spermatogenic cells are used as male gametes-that is, round spermatids, elongated spermatids, and spermatozoa. For some other mammalian species, using immature spermatogenic cells significantly decreases the efficiency of microinsemination. Physically unstable chromatin and low oocyte-activating capacity are the major causes of fertilization failure. The youngest male germ cells, including primordial germ cells and gonocytes, can be used in the construction of diploid embryos by nuclear-transfer cloning. The cloned embryos obtained in this way provide invaluable information on the erasure and reestablishment of genomic imprinting in germ cells.

Surface cells in adult ovaries represent germ line-competent embryonic stem cells. They are a novel type of totipotent progenitors for distinct cell types including female germ cells/oocytes, with the potential for use in the autologous treatment of ovarian infertility and stem cell therapy. Ovarian infertility and stem cell therapy are complex scientific, therapeutic, and socioeconomic issues, which are accompanied by legal restrictions in many developed countries. We have described the differentiation of distinct cell types and the production of new eggs in cultures derived from adult human ovaries. The possibility of producing new eggs from ovarian surface epithelium representing totipotent stem cells supports new opportunities for the treatment of premature ovarian failure, whether idiopathic or after cytostatic chemotherapy treatment, as well as infertility associated with aged primary follicles, and infertility after natural menopause. The stem cells derived from adult human ovaries can also be used for stem cell research and to direct autologous stem cell therapy. This chapter describes general considerations regarding the egg origin from somatic progenitor cells, oogenesis and follicle formation in fetal and adult human ovaries (follicular renewal), including the promotional role of the immune system-related cells in vivo, and possible causes of ovarian infertility. It then provides detailed protocols for the separation and cultivation of adult ovarian stem cells.

Germ cells are the embryonic precursors of the gametes. They are set aside from the somatic cell lineages early in the development of most species. In the mouse, the germ cells, once they have formed, migrate through the tissues of the embryo to the gonad primordia, where they coassemble with somatic gonadal cells to form the sex cords. The sex cords are the forerunners of the seminiferous tubules of the male gonad or the ovarian follicles of the female gonad. Germ cells that do not enter the gonad primordia can develop into germ line tumors later in life. Errors in germ cell differentiation can lead to infertility. Germ cells are the only cells in the body to undergo meiotic cell divisions during their differentiation. This leads to haploidy of the gametes and also generates genetic differences between individuals.

Major modifications in chromatin organization occur in spermatid nuclei, resulting in a high degree of DNA packaging within the spermatozoon head. However, before arrest of transcription during midspermiogenesis, high levels of mRNA are found in round spermatids. Some transcripts are the product of genes expressed ubiquitously, whereas some are generated from male germ cell-specific gene homologs of somatic cell genes. Others are transcript variants derived from genes with expression regulated in a testis-specific fashion. The haploid genome of spermatids also initiates the transcription of testis-specific genes. Various general transcription factors, distinct promoter elements, and specific transcription factors are involved in transcriptional regulation. After meiosis, spermatids are genetically but not phenotypically different, because of transcript and protein sharing through cytoplasmic bridges connecting spermatids of the same generation. Interestingly, different types of mRNAs accumulate in the sperm cell nucleus, raising the question of their origin and of a possible role after fertilization.

Publisher Summary This chapter discusses the expression and function of c-mos in mammalian germ cells. The c-mos proto-oncogene is unique in being specifically expressed in male and female germ cells where it appears to play a central role in regulating the meiotic cell cycle. In somatic cells, c-mos are either silent or possibly expressed at very low levels, indicating that expression of this proto-oncogene is subject to stringent tissue-specific regulation. The chapter identifies two distinct regulatory elements that activate c-mos transcription in oocytes and repress its transcription in somatic cells. C-mos appears to play a unique role in germ cells, so studies of its expression and function offer the promise of novel insights into the mechanisms that control germ cell development and the meiotic cell cycle. The proteins encoded by most proto-oncogenes are normally expressed in a variety of differentiated cell types, where they generally function as components of signaling pathways that regulate cell growth and differentiation. The c-mos gene also encodes a protein-serine/threonine kinase (Mos). Both the regulation and function of c-mos thus pose important issues with respect to understanding the molecular mechanisms that control mammalian development.

Structural and experimental studies on the germ plasms of Drosophila (Insecta) and Rana and Xenopus (Amphilia) are reviewed. Major studies implicating germ plasm in formation of primordial germ cell involve destruction or deletion of the plasm. UV-sensitive germ cell determinants are present in germ plasms of both these major groups. The polar granules (Drosophila) contain stainable RNA which is no longer detectable after the blastoderm stage. In amphibians, there is less evidence for the presence of RNA in germinal granules. In both groups, however, associated ribosomes are implicated in protein synthesis by the granules. One hypothesis is that such bodies are the sites where maternal messenger RNA which codes for proteins specific to primordial germ cells are localized. An alternative hypothesis involves ''protective'' action of germ plasm whereby there is differentiation of the mitotically inhibited, embryonic germ cells. Unequivocal evidence comes from studies of Drosophila pole plasm and pole cell transfers which show that material in the pole plasm induces formation of the distinct germ cells which become functional gametes. The germ plasm provides the clearest example of cytoplasmic localizations which play a determinative role in early organogenesis. The nature, origin, and mode of action of germ cell determinants remainmore » problematical. Progress has been made by identifying within the germinal plasm region discrete electron dense bodies which are correlated with germ cell determination. What remains to be done is direct demonstration of whether these structures alone direct germ cell formation. By isolating the dense bodies and directly testing their activity, it should be possible to identify both their biological function as well as their chemical composition.« less

PIWI-interacting RNAs (piRNAs) are small RNAs bound by PIWI-clade Argonaute proteins that function to silence transposable elements (TEs). Following mouse Primordial Germ Cell (mPGC) specification around E6.25, fetal piRNAs subsequently emerge in male gonocytes from E13.5 onwards. The in vitro differentiation of mPGC-Like Cells (mPGCLCs) from mESCs has raised the tantalizing prospect of studying the fetal piRNA pathway in greater depth. However, using single-cell RNA-seq and RT-qPCR along mPGCLC differentiation, we find that piRNA pathway factors are not yet expressed in D6 mPGCLCs. Moreover, we do not detect piRNAs across a panel of D6 mPGCLC lines using small RNA-seq. Our combined efforts from two laboratories highlight that in vitro differentiated D6 mPGCLCs do not yet resemble E13.5 or later mouse gonocytes where the piRNA pathway is active, in contrast to a prior report.

Publisher Summary This chapter discusses the effects of irradiation on germ cells and embryonic development in teleosts. Sterility and weight reduction of testes after irradiation are mainly because of actual cell loss, and the order of decreasing sensitivity of spermatogenic cells is from the most sensitive spermatogonia, through primary and secondary spermatocytes and spermatids, to sperm, which are quite radioresistant. Even in morphologically homogeneous populations of spermatogonia, heterogeneity of sensitivity exists. The results of local irradiation of the testes are different from those of irradiation of the ovaries. The radiosensitivity of gametogenesis in the ovary and testis cannot be directly compared, except probably from the viewpoint of genetic effects of radiation. Most studies concerning radiosensitivity of fish embryos have so far been performed for the end points of hatchability and lethality during development. This is mainly because of the simplicity of the technique. Many irradiated larvae that appear normal at hatching carry radiation damage that becomes evident at some future stage.

Maternal nutrition and physiology are intimately associated with reproductive success in diverse organisms. Despite decades of study, the molecular mechanisms linking maternal diet to the production and quality of oocytes remain poorly defined. Nuclear receptors (NRs) link nutritional signals to cellular responses and are essential for oocyte development. The fruit fly, Drosophila melanogaster, is an excellent genetically tractable model to study the relationship between NR signaling and oocyte production. In this review, we explore how NRs in Drosophila regulate the earliest stages of oocyte development. Long-recognized as an essential mediator of developmental transitions, we focus on the intrinsic roles of the Ecdysone Receptor and its ligand, ecdysone, in oogenesis. We also review recent studies suggesting broader roles for NRs as regulators of maternal physiology and their impact specifically on oocyte production. We propose that NRs form the molecular basis of a broad physiological surveillance network linking maternal diet with oocyte production. Given the functional conservation between Drosophila and humans, continued experimental investigation into the molecular mechanisms by which NRs promote oogenesis will likely aid our understanding of human fertility.

Sea urchin gametes have been historically used to demonstrate fertilization and early development in student laboratories. Large amounts of egg and sperm are easily acquired, and the conspicuous changes in egg surface morphology, indicative of sperm-egg fusion and egg activation, are readily observed in the classroom. However, less often incorporated into teaching labs are exercises that demonstrate the dramatic metabolic changes that accompany egg activation. One example is the massive up-regulation of various essential transport activities in the embryo’s plasma membrane, including xenobiotic transporter activity. Here we outline a laboratory that incorporates this concept into a teaching lab, capitalizing on the magnitude and uniformity of the xenobiotic transporter activation event in certain species of sea urchins. The introduction of this chapter provides background information for the instructor, and the remainder serves as a laboratory manual for students. The experiments detailed within the manual can be completed in a total of 4–8h spread over one or two lab periods. The lab manual guides students through a modified version of the United States Environmental Protection Agency (EPA) toxicity test, a novel undergraduate-level laboratory on xenobiotic transporters, and analysis of microscope data using ImageJ. We have found this lab to be of interest to a wide range of biology and environmental science undergraduates, and effective in teaching underlying concepts in developmental biology, physiology and toxicology.

The various symptoms associated with hereditary defects in the DNA damage response (DDR), which range from developmental and neurological abnormalities and immunodeficiency to tissue-specific cancers and accelerated aging, suggest that DNA damage affects tissues differently. Mechanistic DDR studies are, however, mostly performed in vitro, in unicellular model systems or cultured cells, precluding a clear and comprehensive view of the DNA damage response of multicellular organisms. Studies performed in intact, multicellular animals models suggest that DDR can vary according to the type, proliferation and differentiation status of a cell. The nematode Caenorhabditis elegans has become an important DDR model and appears to be especially well suited to understand in vivo tissue-specific responses to DNA damage as well as the impact of DNA damage on development, reproduction and health of an entire multicellular organism. C. elegans germ cells are highly sensitive to DNA damage induction and respond via classical, evolutionary conserved DDR pathways aimed at efficient and error-free maintenance of the entire genome. Somatic tissues, however, respond differently to DNA damage and prioritize DDR mechanisms that promote growth and function. In this mini-review, we describe tissue-specific differences in DDR mechanisms that have been uncovered utilizing C. elegans as role model. (C) 2015 Elsevier B.V. All rights reserved.

During mammalian embryonic development, the trophectoderm and primitive endoderm give rise to extraembryonic tissues, while the epiblast differentiates into all somatic lineages and the germline. Remarkably, only a few classes of signaling pathways induce the differentiation of these progenitor cells into diverse lineages. Accordingly, the functional outcome of a particular signal depends on the developmental competence of the target cells. Thus, developmental competence can be defined as the ability of a cell to integrate intrinsic and extrinsic cues to execute a specific developmental program toward a specific cell fate. Downstream of signaling, there is the combinatorial activity of transcription factors and their cofactors, which is modulated by the chromatin state of the target cells. Here, we discuss the concept of developmental competence, and the factors that regulate this state with reference to the specification of mammalian primordial germ cells.

In Xenopus, the germline is specified by the inheritance of germ-plasm components synthesized at the beginning of oogenesis. Only the cells in the early embryo that receive germ plasm, the primordial germ cells (PGCs), are competent to give rise to the gametes. Thus, germ-plasm components continue the totipotent potential exhibited by the oocyte into the developing embryo at a time when most cells are preprogrammed for somatic differentiation as dictated by localized maternal determinants. When zygotic transcription begins at the mid-blastula transition, the maternally set program for somatic differentiation is realized. At this time, genetic control is ceded to the zygotic genome, and developmental potential gradually becomes more restricted within the primary germ layers. PGCs are a notable exception to this paradigm and remain transcriptionally silent until the late gastrula. How the germ-cell lineage retains full potential while somatic cells become fate restricted is a tale of translational repression, selective degradation of somatic maternal determinants, and delayed activation of zygotic transcription.

Many have contributed to the various discoveries that brought recognition of the enormous potential of cells of early embryonic origin for genetic modification of organisms, regenerative medicine, and investigation of facets of development that are difficult to explore in vivo. Historically, the work of two researchers stands out as forming a foundation for our current understanding of embryonic stem cells and their potential. Leroy Stevens and Barry Pierce were pioneers in the study of tumors that arose from germ cells in mice: teratomas and teratocarcinomas, from which embryonic carcinomas cells (or EC cells) were eventually identified.

Primordial germ cells (PGCs) are the embryonic precursors of the gametes and represent the founder cells of the germline. Specification of PGCs is a critical divergent point during embryogenesis. Whereas the somatic lineages will ultimately perish, cells of the germline have the potential to form a new individual and hence progress to the next generation. It is therefore critical that the genome emerges intact and carrying the appropriate epigenetic information during its passage through the germline. To ensure this fidelity of transmission, PGC development encompasses extensive epigenetic reprogramming. The low cell numbers and relative inaccessibility of PGCs present a challenge to those seeking mechanistic understanding of the crucial developmental and epigenetic processes in this most fascinating of lineages. Here, we present an overview of PGC development in the mouse and compare this with the limited information available for other mammalian species. We believe that a comparative approach will be increasingly important to uncover the extent to which mechanisms are conserved and reveal the critical steps during PGC development in humans.

12 pages, 7 figures, Vasa is a protein expressed mainly in germ cells and conserved across taxa. However, sex-related differences and environmental influences on vasa expression have not been documented. This study characterized the cDNA of a vasa homolog in the European sea bass, Dicentrarchuslabrax (sb-vasa), a gonochoristic fish with temperature influences on gonadogenesis. The 1911 bp open reading frame predicted a 637-amino acid protein with the eight conserved domains typical of Vasa proteins. Comparisons of the deduced amino acid sequence with those of other vertebrates and invertebrates revealed the highest homology (68–85%) with those of other teleosts. An updated tree with the full-length sequences for Vasa proteins in 66 species belonging to six different phyla was constructed, establishing the evolutionary relationships of Vasa amino acid sequences. European sea bass vasa was highly expressed in gonads with little or no expression in other tissues. Real time RT-PCR quantification of the temporal expression of sb-vasa from early development throughout sex differentiation showed that mRNA levels were high in unfertilized eggs, decreased during larval development and increased again during the period of germ cell proliferation. Rearing of fish at high temperature resulted in further increased sb-vasa levels, most likely reflecting temperature effects on both somatic and gonadal growth. Differences in expression were also found well before sex differentiation and persisted until the end of the first year, with higher levels present in females. These differences in expression demonstrate the implication of vasa during the initial stages of fish sex differentiation and gametogenesis and suggest that, through its helicase activity, it might be implicated in the translational regulation of mRNAs involved in the specification and differentiation of gonadal-specific cell types, This work was supported by the EU grant PROBASS (Q5RS-2000-31365) to F.P and C.C.M. Research at the lab of F.P. is partially funded by project Consolider ‘‘Aquagenomics” CDS2007-0002. M. Blázquez was supported by a postdoctoral contract from the EU and a Ramón y Cajal contract from the Spanish Ministry of Science and Technology

Despite its significant role in oocyte generation and hormone production in adulthood, the ovary, with regard to its formation, has received little attention compared to its male counterpart, the testis. With the exception of germ cells, which undergo a female-specific pattern of meiosis, morphological changes in the fetal ovary are subtle. Over the past 40 years, a number of hypotheses have been proposed for the organogenesis of the mammalian ovary. It was not until the turn of the millennium, thanks to the advancement of genetic and genomic approaches, that pathways for ovary organogenesis that consist of positive and negative regulators have started to emerge. Through the action of secreted factors (R-spondin1, WNT4, and follistatin) and transcription regulators (beta-catenin and FOXL2), the developmental fate of the somatic cells is directed toward ovarian, while testicular components are suppressed. In this chapter, we review the history of studying ovary organogenesis in mammals and present the most recent discoveries using the mouse as the model organism.

All metazoan genomes encode multiple RAS GTPase activating proteins (RasGAPs) that negatively regulate the conserved RAS/MAPK signaling pathway. In mammals, several RasGAPs exhibit tumor suppressor activity by preventing excess RAS signal transduction. We have identified gap-3 as the to date missing Caenorhabditis elegans member of the p120 RasGAP family. By studying the genetic interaction of gap-3 with the two previously identified RasGAPs gap-1 and gap-2, we find that different combinations of RasGAPs are used to repress LET-60 RAS signaling depending on the cellular context. GAP-3 is the predominant negative regulator of RAS during meiotic progression of the germ cells, while GAP-1 is the key inhibitor of RAS during vulval induction. In other tissues such as the sex myoblasts or the chemosensory neurons, all three RasGAPs act in concert. The C. elegans RasGAPs have thus undergone partial specialization after gene duplication to allow the differential regulation of the RAS/MAPK signaling pathway in different cell types. A similar tissue specialization of the human tumor suppressor genes may explain the strong bias in the type of cancer they promote when mutated.