Cyp26b1 Expression in Murine Sertoli Cells Is Required to Maintain Male Germ Cells in an Undifferentiated State during Embryogenesis

In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.     

Epigenetic regulation of germ cell differentiation

Germ cells and somatic cells have the identical genome. However, unlike the mortal fate of somatic cells, germ cells have the unique ability to differentiate into gametes that retain totipotency and produce an entire organism upon fertilization. The processes by which germ cells differentiate into gametes, and those by which gametes become embryos, involve dramatic cellular differentiation accompanied by drastic changes in gene expression, which are tightly regulated by genetic circuitries as well as epigenetic mechanisms. Epigenetic regulation refers to heritable changes in gene expression that are not due to changes in primary DNA sequence. The past decade has witnessed an ever-increasing understanding of epigenetic regulation in many different cell types/tissues during embryonic development and adult homeostasis. In this review, we focus on recent discoveries of epigenetic regulation of germ cell differentiation in various metazoan model organisms, including worms, flies, and mammals.     

Epigenetic transitions in germ cell development and meiosis

Germ cell development is controlled by unique gene expression programs and involves epigenetic reprogramming of histone modifications and DNA methylation. The central event is meiosis, during which homologous chromosomes pair and recombine, processes that involve histone alterations. At unpaired regions, chromatin is repressed by meiotic silencing. After meiosis, male germ cells undergo chromatin remodeling, including histone-to-protamine replacement. Male and female germ cells are also differentially marked by parental imprints, which contribute to sex determination in insects and mediate genomic imprinting in mammals. Here, we review epigenetic transitions during gametogenesis and discuss novel insights from animal and human studies.     

Extrinsic and intrinsic control of germ cell proliferation in Caenorhabditis elegans

The germ cells of Caenorhabditis elegans serve as a useful model to study the balance between proliferation and differentiation within the context of development and changing environmental signals experienced by the animal. Germ cells adjacent to a stem cell niche in the distal region of the gonad retain the capacity to divide during adulthood, making them unique from other cells in the organism. We will highlight recent advances in our understanding of mechanisms that control proliferation, as well as the signaling pathways involved in promoting mitosis at the expense of differentiation.     

Reprogramming mediated by stem cell fusion

Advances in mammalian cloning prove that somatic nuclei can be reprogrammed to a state of totipotency by transfer into oocytes. An alternative approach to reprogram the somatic genome involves the creation of hybrids between somatic cells and other cells that contain reprogramming activities. Potential fusion partners with reprogramming activities include embryonic stem cells, embryonic germ cells, embryonal carcinoma cells, and even differentiated cells. Recent advances in fusion-mediated reprogramming are discussed from the standpoints of the developmental potency of hybrid cells, genetic and epigenetic correlates of reprogramming, and other aspects involved in the reprogramming process. In addition, the utility of fusion-mediated reprogramming for future cell-based therapies is discussed.     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

Germ cell sex prior to meiosis in the rainbow trout

Germ cells make two major decisions when they move from an indeterminate state to their final stage of gamete production. One decision is sexual commitment for sperm or egg production, and the other is to maintain mitotic division or entry into meiosis. It is unclear whether the two decisions are made as a single event or separate events, because there has been no evidence for the presence of germ cell sex prior to meiosis. Here we report direct evidence in the fish rainbow trout that gonia have distinct sexuality. We show that dazl expression occurs in both male and female gonia but exhibits differential intracellular distribution. More strikingly, we show that boule is highly expressed in male gonia but absent in female gonia. Therefore, mitotic gonia possess sex, sperm/egg decision and mitosis/meiosis decision are two independent events, and sperm/egg decision precedes mitosis/meiosis decision in rainbow trout, making this organism a unique vertebrate model for mechanistic understanding of germ cell sex differentiation and relationship between the two decisions.     

Germ layer formation during Xenopus embryogenesis: the balance between pluripotency and differentiation

The African clawed frog, Xenopus laevis, has long been a model animal for the studies in the fields of animal cloning, developmental biology, biochemistry, cell biology, and physiology. With the aid of Xenopus, major molecular mechanisms that are involved in embryonic development have been understood. Germ layer formation is the first event of embryonic cellular differentiation, which is induced by a few key maternal factors and subsequently by zygotic signals. Meanwhile, another type of signals, the pluripotency factors in ES cells, which maintain the undifferentiated state, are also present during early embryonic cells. In this review, the functions of the pluripotency factors during Xenopus germ layer formation and the regulatory relationship between the signals that promote differentiation and pluripotency factors are discussed.     

The role of mitochondrial rRNAs and nanos protein in germline formation in Drosophila embryos

Germ cells, represented by male sperm and female eggs, are specialized cells that transmit genetic material from one generation to the next during sexual reproduction. The mechanism by which multicellular organisms achieve the proper separation of germ cells and somatic cells is one of the longest standing issues in developmental biology. In many animal groups, a specialized portion of the egg cytoplasm, or germ plasm, is inherited by the cell lineage that gives rise to the germ cells (germline). Germ plasm contains maternal factors that are sufficient for germline formation. In the fruit fly, Drosophila, germ plasm is referred to as polar plasm and is distinguished histologically by the presence of polar granules, which act as a repository for the maternal factors required for germline formation. Molecular screens have so far identified several of these factors that are enriched in the polar plasm. This article focuses on the molecular functions of two such factors in Drosophila, mitochondrial ribosomal RNAs and Nanos protein, which are required for the formation and differentiation of the germline progenitors, respectively.     

Moving towards the next generation.

In most organisms, primordial germ cells are set aside from the cells of the body early in development. To form an embryonic gonad, germ cells often have to migrate along complex routes through and along diverse tissues until they reach the somatic part of the gonad. Recent advances have been made in the genetic analysis of these early stages of germ line development. Here we review findings from Drosophila, zebrafish, and mouse; each organism provides unique insight into the mechanisms that determine germ cell fate and the cues that may guide their migration.     

生殖细胞形成的调控机制及体外培养体系的研究进展

生殖细胞是多细胞生物体遗传物质传递的载体,在发育生物学、临床医学及畜牧业生产等领域中具有广阔的应用前景。原始生殖细胞作为胚胎体内最早出现的生殖细胞,在发育过程中受多种信号因子的诱导,发生特化、迁移、分化及减数分裂,最终形成单倍体的配子,此过程在遗传学和表观遗传学方面受到严格的调控。另外,多能性干细胞向生殖细胞的分化以及生殖细胞的体外培养方面在最近均取得了较大的进展。该文将主要围绕原始生殖细胞,综述最近几年来关于生殖细胞形成中的转录调控及体外培养体系的进展。     

Turning germ cells into stem cells

Primordial germ cells (PGCs), the embryonic precursors of the gametes of the adult animal, can give rise to two types of pluripotent stem cells. In vivo, PGCs can give rise to embryonal carcinoma cells, the pluripotent stem cells of testicular tumors. Cultured PGCs exposed to a specific cocktail of growth factors give rise to embryonic germ cells, pluripotent stem cells that can contribute to all the lineages of chimeric embryos including the germline. The conversion of PGCs into pluripotent stem cells is a remarkably similar process to nuclear reprogramming in which a somatic nucleus is reprogrammed in the egg cytoplasm. Understanding the genetics of embryonal carcinoma cell formation and the growth factor signaling pathways controlling embryonic germ cell derivation could tell us much about the molecular controls on developmental potency in mammals.     

AKT signaling promotes derivation of embryonic germ cells from primordial germ cells

Primordial germ cells (PGCs) are embryonic germ cell precursors. Although the developmental potency of PGCs is restricted to the germ lineage, PGCs can acquire pluripotency, as verified by the in vitro establishment of embryonic germ (EG) cells and the in vivo production of testicular teratomas. PGC-specific inactivation of PTEN, which is a lipid phosphatase antagonizing phosphoinositide-3 kinase (PI3K), enhances both EG cell production and testicular teratoma formation. Here, we analyzed the effect of the serine/threonine kinase AKT, one of the major downstream effectors of PI3K, on the developmental potency of PGCs. We used transgenic mice that expressed an AKT-MER fusion protein, the kinase activity of which could be regulated by the ligand of modified estrogen receptor (MER), 4-hydroxytamoxifen. We found that hyperactivation of AKT signaling in PGCs at the proliferative phase dramatically augmented the efficiency of EG cell establishment. Furthermore, AKT signaling activation substituted to some extent for the effects of bFGF, an essential growth factor for EG cell establishment. By contrast, AKT activation had no effect on germ cells that were in mitotic arrest or that began meiosis at a later embryonic stage. In the transgenic PGCs, AKT activation induced phosphorylation of GSK3, which inhibits its kinase activity; enhanced the stability and nuclear localization of MDM2; and suppressed p53 phosphorylation, which is required for its activation. The p53 deficiency, but not GSK3 inhibition, recapitulated the effects of AKT hyperactivation on EG cell derivation, suggesting that p53 is one of the crucial downstream targets of the PI3K/AKT signal and that GSK3 is not.     

A germ cell-specific gene, Prmt5, works in somatic cell reprogramming

Germ cells possess the unique ability to acquire totipotency during development in vivo as well as give rise to pluripotent stem cells under the appropriate conditions in vitro. Recent studies in which somatic cells were experimentally converted into pluripotent stem cells revealed that genes expressed in primordial germ cells (PGCs), such as Oct3/4, Sox2, and Lin28, are involved in this reprogramming. These findings suggest that PGCs may be useful for identifying factors that successfully and efficiently reprogram somatic cells into toti- and/or pluripotent stem cells. Here, we show that Blimp-1, Prdm14, and Prmt5, each of which is crucial for PGC development, have the potential to reprogram somatic cells into pluripotent stem cells. Among them, Prmt5 exhibited remarkable reprogramming of mouse embryonic fibroblasts into which Prmt5, Klf4, and Oct3/4 were introduced. The resulting cells exhibited pluripotent gene expression, teratoma formation, and germline transmission in chimeric mice, all of which were indistinguishable from those induced with embryonic stem cells. These data indicate that some of the factors that play essential roles in germ cell development are also active in somatic cell reprogramming.     

Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads

The developmental fate of primordial germ cells in the mammalian gonad depends on their environment. In the XY gonad, Sry induces a cascade of molecular and cellular events leading to the organization of testis cords. Germ cells are sequestered inside testis cords by 12.5 dpc where they arrest in mitosis. If the testis pathway is not initiated, germ cells spontaneously enter meiosis by 13.5 dpc, and the gonad follows the ovarian fate. We have previously shown that some testis-specific events, such as mesonephric cell migration, can be experimentally induced into XX gonads prior to 12.5 dpc. However, after that time, XX gonads are resistant to the induction of cell migration. In current experiments, we provide evidence that this effect is dependent on XX germ cells rather than on XX somatic cells. We show that, although mesonephric cell migration cannot be induced into normal XX gonads at 14.5 dpc, it can be induced into XX gonads depleted of germ cells. We also show that when 14.5 dpc XX somatic cells are recombined with XY somatic cells, testis cord structures form normally; however, when XX germ cells are recombined with XY somatic cells, cord structures are disrupted. Sandwich culture experiments suggest that the inhibitory effect of XX germ cells is mediated through short-range interactions rather than through a long-range diffusible factor. The developmental stage at which XX germ cells show a disruptive effect on the male pathway is the stage at which meiosis is normally initiated, based on the immunodetection of meiotic markers. We suggest that at the stage when germ cells commit to meiosis, they reinforce ovarian fate by antagonizing the testis pathway.     

In vitro gamete derivation from pluripotent stem cells: progress and perspective

Germ cells constitute a highly specialized cell population that is indispensable for the continuation and evolution of the species. Recently, several research groups have shown that these unique cells can be produced in vitro from pluripotent stem cells. Furthermore, live births of offspring using induced germ cells have been reported in one study. These results suggest that it may be possible to investigate germ cell development ex vivo and to establish novel reproductive technologies. To this end, it is critical to assess if gamete induction processes in vitro faithfully recapitulate normal germ cell development in vivo. Here, this issue is discussed with a focus on the germ line specification and the sex-specific development of pre- and postnatal germ cells. The aim of this paper is to concisely summarize the past progress and to present some future issues for the investigation into in vitro gamete production from pluripotent stem cells.     

Sex Determination: Time for Meiosis? The Gonad Decides

Germ cell sex determination is directed by the gonad and is characterized by a difference in the timing of entry into meiosis. Recent data show that retinoic acid signalling is responsible for the induction of germ cell meiosis in the developing ovary. In the fetal testis, this process is inhibited by a retinoic acid metabolizing enzyme.     

Sexual dimorphism in the regulation of meiotic process in the rabbit

Meiosis, mitosis, and apoptosis during fetal and postnatal periods were investigated in order to explore mechanisms of sexual dimorphism in initiation of germ cell meiosis. Gonads were obtained from Japanese white rabbits from 23 to 51 days postcoitum (dpc). Gonadal thin sections were stained with hematoxylin and eosin. Germ cell alkaline phosphatase and apoptosis were detected with histochemical and immunohistochemical methods, respectively. In the ovary, meiotic germ cells were initially recognized at 29 dpc and arrested after enclosure within follicles. Similarly, meiotic germ cells were recognized outside seminiferous tubules at 29 dpc, but no meiotic figures were identified in intratubular spaces. Apoptotic germ cells were not recognized in the intratubular spaces before 35 dpc, and no apoptotic figures were recognized in the ovary during the period studied. In conclusion, the initiation of meiosis in testicular interstitial tissue at the time comparable to that in the ovary indicates that germ cells of both sexes have the ability to enter meiosis during the same stage of fetal development; and it appears most likely that delayed initiation of meiosis in the intratubular space is attributable to meiosis-inhibiting substance(s) present in seminiferous tubules.     

Small RNAs in the animal gonad: Guarding genomes and guiding development

Germ cells must safeguard, apportion, package, and deliver their genomes with exquisite precision to ensure proper reproduction and embryonic development. Classical genetic approaches have identified many genes controlling animal germ cell development, but only recently have some of these genes been linked to the RNA interference (RNAi) pathway, a gene silencing mechanism centered on small regulatory RNAs. Germ cells contain microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs); these are bound by members of the Piwi/Argonaute protein family. piwi genes were known to specify germ cell development, but we now understand that mutations disrupting germline development can also affect small RNA accumulation. Small RNA studies in germ cells have revealed a surprising diversity of regulatory mechanisms and a unifying function for germline genes in controlling the spread of transposable elements. Future challenges will be to understand the production of germline small RNAs and to identify the full breadth of gene regulation by these RNAs. Progress in this area will likely impact biomedical goals of manipulating stem cells and preventing diseases caused by the transposition of mobile DNA elements.