Number Of Primordial Germ Cells Research Articles

Esrrb Complementation Rescues Development of Nanog-Null Germ Cells

SummaryThe transcription factors (TFs) Nanog and Esrrb play important roles in embryonic stem cells (ESCs) and during primordial germ-cell (PGC) development. Esrrb is a positively regulated direct target of NANOG in ESCs that can substitute qualitatively for Nanog function in ESCs. Whether this functional substitution extends to the germline is unknown. Here, we show that germline deletion of Nanog reduces PGC numbers 5-fold at midgestation. Despite this quantitative depletion, Nanog-null PGCs can complete germline development in contrast to previous findings. PGC-like cell (PGCLC) differentiation of Nanog-null ESCs is also impaired, with Nanog-null PGCLCs showing decreased proliferation and increased apoptosis. However, induced expression of Esrrb restores PGCLC numbers as efficiently as Nanog. These effects are recapitulated in vivo: knockin of Esrrb to Nanog restores PGC numbers to wild-type levels and results in fertile adult mice. These findings demonstrate that Esrrb can replace Nanog function in germ cells.

Germline competency of human embryonic stem cells depends on eomesodermin.

In humans, germline competency and the specification of primordial germ cells (PGCs) are thought to occur in a restricted developmental window during early embryogenesis. Despite the importance of specifying the appropriate number of PGCs for human reproduction, the molecular mechanisms governing PGC formation remain largely unexplored. Here, we compared PGC-like cell (PGCLC) differentiation from 18 independently derived human embryonic stem cell (hESC) lines, and discovered that the expression of primitive streak genes were positively associated with hESC germline competency. Furthermore, we show that chemical inhibition of TGFβ and WNT signaling, which are required for primitive streak formation and CRISPR/Cas9 deletion of Eomesodermin (EOMES), significantly impacts PGCLC differentiation from hESCs. Taken together, our results suggest that human PGC formation involves signaling and transcriptional programs associated with somatic germ layer induction and expression of EOMES.

Role of maternal Xenopus syntabulin in germ plasm aggregation and primordial germ cell specification

The localization and organization of mitochondria- and ribonucleoprotein granule-rich germ plasm is essential for many aspects of germ cell development. In Xenopus, germ plasm is maternally inherited and is required for the specification of primordial germ cells (PGCs). Germ plasm is aggregated into larger patches during egg activation and cleavage and is ultimately translocated perinuclearly during gastrulation. Although microtubule dynamics and a kinesin (Kif4a) have been implicated in Xenopus germ plasm localization, little is known about how germ plasm distribution is regulated. Here, we identify a role for maternal Xenopus Syntabulin in the aggregation of germ plasm following fertilization. We show that depletion of sybu mRNA using antisense oligonucleotides injected into oocytes results in defects in the aggregation and perinuclear transport of germ plasm and subsequently in reduced PGC numbers. Using live imaging analysis, we also characterize a novel role for Sybu in the collection of germ plasm in vegetal cleavage furrows by surface contraction waves. Additionally, we show that a localized kinesin-like protein, Kif3b, is also required for germ plasm aggregation and that Sybu functionally interacts with Kif3b and Kif4a in germ plasm aggregation. Overall, these data suggest multiple coordinate roles for kinesins and adaptor proteins in controlling the localization and distribution of a cytoplasmic determinant in early development.

Development and gonadal sex differentiation in the neotenic urodele: Ambystoma mexicanum

Ambystoma mexicanum is an endemic neotenic urodele amphibian from Mexico; although it belongs to the Salamandridae family, it is characterized by retaining larval structures and hence has been widely used as an experimental model. In the present study, we describe the main events of gonadal morphogenesis in A. mexicanum and correlate these with stages in embryonic and larval development. In this way, it was established that during stage 41 (St41), the gonadal primordium is formed, consisting of primordial germ cells (PGC) and somatic cells. During St45, the undifferentiated gonad is formed from a larger number of PGC interacting with somatic cells. During St53, the germ and somatic cells arrange into the cortical and medullary region. As development proceeds between St55 and 57, morphological differentiation of the gonadal sex takes place, primarily manifested in ovarian differentiation. Our observations and the way these correlate with other urodeles suggest that gonadal morphogenesis in A. mexicanum does not depend on larval age. Besides, onset of gonadal sexual differentiation takes place from St53 onward, evidenced by ovarian structural changes, thus neotenic condition does not influence gonadal differentiation events. Finally, it has been established that gonadal development is controlled by chronological regulation that differs from that of somatic development which in the case of A. mexicanum suggests that gonadal development is completely independent of metamorphosis, thus implying a process of heterochrony.

Sexual dimorphic expression of dnd in germ cells during sex reversal and its requirement for primordial germ cell survival in protogynous hermaphroditic grouper

Dead end (dnd), vertebrate-specific germ cell marker, had been demonstrated to be essential for primordial germ cell (PGC) migration and survival, and the link between PGC number and sex change had been revealed in some teleost species, but little is known about dnd in hermaphroditic vertebrates. In the present study, a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides) dnd homologue (Ecdnd) was identified and characterized. Quantitative real-time PCR and in situ hybridization analysis revealed a dynamic and sexually dimorphic expression pattern in PGCs and germ cells of gonads. During sex changing, the Ecdnd transcript sharply increased in early transitional gonad, reached the highest level at late transitional gonad stage, and decreased after testis maturation. Visualization of zebrafish PGCs by injecting with RFP-Ecdnd-3'UTR RNA and GFP-zfnanos3-3'UTR RNA confirmed importance of Ecdnd 3'UTR for the PGC distribution. In addition, knockdown of EcDnd by using antisense morpholinos (MO) caused the ablation of PGCs in orange-spotted grouper. Therefore, the current data indicate that Ecdnd is essential for PGCs survival and may serve as a useful germ cell marker during gametogenesis in hermaphroditic grouper.

Isolation and Number of Gonadal Primordial Germ Cells (Gonadal PGCs) on the Stages of Early Embryonic Development of KUB Chicken

Primordial germ cells (PGCs) are cells that will differentiate themselves into spermatogonia in the testis or oogonia in the ovary. Primordial germ cells arise from epiblast and circulate through the bloodstream and finally entering gonadal anlage. The aim of this study was to determine the number of gonadal PGCs of KUB chicken at different development stages. Sixty KUB chicken fertile eggs were divided into four groups (6, 7, 8, and 9 days incubation periods), and incubated at 38 oC with a humidity of 60%. Harvesting was synchronized to the embryonic development at 6-9 d. Gonads were collected using sharp tweezers, and were placed in Eppendorf tube 1.5 mL containing 500 µL PBS [-]. Gonadal PGCs were purified using PBS [-]. The results showed that the average number of gonadal PGCs at 6, 7, 8, and 9 d were 113.7; 143.5; 92.9; and 85.7 cells per embryo, respectively. Number of gonadal PGCs per embryo of KUB chicken were significantly affected by stage of embryonic development (P<0.05), which reached a peak at day 7 of incubation, so that the isolation and collection of PGCs from the gonads were recommended at day 7 of incubation. This information is useful in production of germline chimera of other Indonesian local chickens.

Xenopus Vasa Homolog XVLG1 is Essential for Migration and Survival of Primordial Germ Cells.

Xenopus vasa-like gene 1 (XVLG1), a DEAD-Box Helicase 4 (DDX4) gene identified as a vertebrate vasa homologue, is required for the formation of primordial germ cells (PGCs). However, it remains to be clarified when and how XVLG1 functions in the formation of the germ cells. To gain a better understanding of the molecular mechanisms underlying XVLG1 during PGC development, we injected XVLG1 morpholino oligos into germ-plasm containing blastomeres of 32-cell stage of Xenopus embryos, and traced cell fates of the injected blastomere-derived PGCs. As a result of this procedure, migration of the PGCs was impaired and the number of PGCs derived from the blastomeres was significantly decreased. In addition, TUNEL staining in combination with in situ hybridization revealed that the loss of PGCs peaked at stage 27 was caused by apoptosis. This data strongly suggests an essential role for XVLG1 in migration and survival of the germ cells.

Development and Conservation of Gonadal Primordial Germ Cells for Preservation of Local Chicken in Indonesia

One of the &lt;em&gt;ex situ&lt;/em&gt; conservation techniques for poultry that recently developed was to collect primordial germ cell (PGC) or gonadal primordial germ cell (gPGC) that isolated from embryo development. Primordial germ cells (PGC) are embryonic cells that migrate to the gonads and form the precursors of gametes. The unique nature and accessibility of PGC during the early development provides an opportunity to manipulate the poultry germplasm, for example by forming germline chimeras. There are some stages that must be done through isolation and collection of PGC from its resources i.e. blastoderm, embryonic circulation blood and gonad. PGC collection originating from the gonads is one of existing PGC resources and technologies. gonadal PGC have advantages compared with other sources, namely (1) A large number of gonadal PGC can be taken from an embryo; and (2) A collection of gonadal PGC can be used in developing management systems of local avian germplasm conservation. This review is intended to describe the usefulness of isolation and collection technology of gonadal PGC as the local poultry germplasm conservation in Indonesia.

Conserved role of Ovo in germline development in mouse and Drosophila

Ovo, which encodes a transcription factor with Zn-finger domains, is evolutionarily conserved among animals. In Drosophila, in addition to its zygotic function for egg production, maternal ovo activity is required in primordial germ cells (PGCs) for expression of germline genes such as vasa and nanos. In this study, we found that maternal Ovo accumulates in PGC nuclei during embryogenesis. In these cells, ovo serves a dual function: activation of genes expressed predominantly in PGCs, and conversely suppression of somatic genes. Reduction of ovo activity in PGCs makes them unable to develop normally into germ cells of both sexes. In mice, knockout of the ovo ortholog, Ovol2, which is expressed in PGCs, decreases the number of PGCs during early embryogenesis. These data strongly suggest that ovo acts as part of an evolutionarily conserved mechanism that regulates germline development in animals.

Gonadogenesis analysis and sex differentiation in cultured turbot (Scophthalmus maximus).

As a flatfish, the turbot (Scophthalmus maximus) is one of the most important farmed fish species with great commercial value, which has a strong sexual dimorphism on growth rate and sexual maturity. In this study, using histology, the basic information on proliferation and migration of germ cells and early gonadal development during sex differentiation in turbot were described in detail. There were six to nine individual primordial germ cells (PGCs) with large nuclei until 15days post-hatching (dph). The PGCs located under the mesonephric ducts undergo migration along the dorsal mesentery toward the region of the genital ridge from 0 to 15dph. During migration, the number of PGCs was constant, and the expression of vasa had no significant changes. At 20dph, the aggregation of somatic cells at genital ridge indicated the formation of primary gonad. Furthermore, the number of PGCs was increased to 60 and the expression of vasa was upregulated for the first time. The undifferentiated gonads with no morphological indications of sex differentiation grew larger with the increase in germ cells and somatic cells number/size from 20 to 35dph. During 36-52dph, cytological gonadal differentiation was observed. In presumptive testes of type I gonadal tissue (with a lance shape), the number of germ cells increased steadily and the germ cells had the same characteristics as before. Meanwhile, in presumptive ovaries of type II gonadal tissue (with a club-like shape), the germ cells proliferated and induced in two different populations of germ cells. One type had the morphological characteristics as undifferentiated germ cells, while the other type of germ cells underwent mitosis exhibiting smaller size and mottled nuclei. At 60dph, ovarian cavity was present in the gonad of type II, which would develop into ovaries. However, spermatogonial cysts were not noticed in the gonad of type I until 90dph, which indicated the formation of the testes.

Mitochondrial trafficking through Rhot1 is involved in the aggregation of germinal granule components during primordial germ cell formation in Xenopus embryos.

In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline-Mt). However, the role of germline-Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline-Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C-terminal mitochondrial transmembrane domain, inhibited the aggregation of germline-Mt during early development. In Rhot1-inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail-bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.

Msx1 and Msx2 function together in the regulation of primordial germ cell migration in the mouse

Primordial germ cells (PGCs) are a highly migratory cell population that gives rise to eggs and sperm. Much is known about PGC specification, but less about the processes that control PGC migration. In this study, we document a deficiency in PGC development in embryos carrying global homozygous null mutations in Msx1 and Msx2, both immediate downstream effectors of Bmp signaling pathway. We show that Msx1−/−;Msx2−/− mutant embryos have defects in PGC migration as well as a reduced number of PGCs. These phenotypes are also evident in a Mesp1-Cre-mediated mesoderm-specific mutant line of Msx1 and Msx2. Since PGCs are not marked in Mesp1-lineage tracing, our results suggest that Msx1 and Msx2 function cell non-autonomously in directing PGC migration. Consistent with this hypothesis, we noted an upregulation of fibronectin, well known as a mediator of cell migration, in tissues through which PGCs migrate. We also noted a reduction in the expression of Wnt5a and an increase in the expression in Bmp4 in such tissues in Msx1−/−;Msx2−/− mutants, both known effectors of PGC development.

Discrete somatic niches coordinate proliferation and migration of primordial germ cells via Wnt signaling.

Inheritance depends on the expansion of a small number of primordial germ cells (PGCs) in the early embryo. Proliferation of mammalian PGCs is concurrent with their movement through changing microenvironments; however, mechanisms coordinating these conflicting processes remain unclear. Here, we find that PGC proliferation varies by location rather than embryonic age. Ror2 and Wnt5a mutants with mislocalized PGCs corroborate the microenvironmental regulation of the cell cycle, except in the hindgut, where Wnt5a is highly expressed. Molecular and genetic evidence suggests that Wnt5a acts via Ror2 to suppress β-catenin-dependent Wnt signaling in PGCs and limit their proliferation in specific locations, which we validate by overactivating β-catenin in PGCs. Our results suggest that the balance between expansion and movement of migratory PGCs is fine-tuned in different niches by the opposing β-catenin-dependent and Ror2-mediated pathways through Wnt5a This could serve as a selective mechanism to favor early and efficient migrators with clonal dominance in the ensuing germ cell pool while penalizing stragglers.

Perchlorate Exposure Reduces Primordial Germ Cell Number in Female Threespine Stickleback.

Perchlorate is a common aquatic contaminant that has long been known to affect thyroid function in vertebrates, including humans. More recently perchlorate has been shown to affect primordial sexual differentiation in the aquatic model fishes zebrafish and threespine stickleback, but the mechanism has been unclear. Stickleback exposed to perchlorate from fertilization have increased androgen levels in the embryo and disrupted reproductive morphologies as adults, suggesting that perchlorate could disrupt the earliest stages of primordial sexual differentiation when primordial germ cells (PGCs) begin to form the gonad. Female stickleback have three to four times the number of PGCs as males during the first weeks of development. We hypothesized that perchlorate exposure affects primordial sexual differentiation by reducing the number of germ cells in the gonad during an important window of stickleback sex determination at 14–18 days post fertilization (dpf). We tested this hypothesis by quantifying the number of PGCs at 16 dpf in control and 100 mg/L perchlorate-treated male and female stickleback. Perchlorate exposure from the time of fertilization resulted in significantly reduced PGC number only in genotypic females, suggesting that the masculinizing effects of perchlorate observed in adult stickleback may result from early changes to the number of PGCs at a time critical for sex determination. To our knowledge, this is the first evidence of a connection between an endocrine disruptor and reduction in PGC number prior to the first meiosis during sex determination. These findings suggest that a mode of action of perchlorate on adult reproductive phenotypes in vertebrates, including humans, such as altered fecundity and sex reversal or intersex gonads, may stem from early changes to germ cell development.

Dazl is a critical player for primordial germ cell formation in medaka.

The DAZ family genes boule, daz and dazl have conserved functions in primordial germ cell (PGC) migration, germ stem cell proliferation, differentiation and meiosis progression. It has remained unknown whether this family is required for PGC formation in developing embryos. Our recent study in the fish medaka (Oryzias latipes) has defined dnd as the critical PGC specifier and predicted the presence of additional factors essential for PGC formation. Here we report that dazl is a second key player for medaka PGC formation. Dazl knockdown did not prevent PGC formation even in the absence of normal somatic structures. It turned out that a high level of Dazl protein was maternally supplied and persisted until gastrulation, and hardly affected by two antisense morpholino oligos targeting the dazl RNA translation. Importantly, microinjection of a Dazl antibody remarkably reduced the number of PGCs and even completely abolished PGC formation without causing detectable somatic abnormality. Therefore, medaka PGC formation requires the Dazl protein as maternal germ plasm component, offering first evidence that dazl is a critical player in PGC formation in vivo. Our results demonstrate that antibody neutralization is a powerful tool to study the roles of maternal protein factors in PGC development in vivo.

Dnd Is a Critical Specifier of Primordial Germ Cells in the Medaka Fish.

SummaryPrimordial germ cell (PGC) specification occurs early in development. PGC specifiers have been identified in Drosophila, mouse, and human but remained elusive in most animals. Here we identify the RNA-binding protein Dnd as a critical PGC specifier in the medaka fish (Oryzias latipes). Dnd depletion specifically abolished PGCs, and its overexpression boosted PGCs. We established a single-cell culture procedure enabling lineage tracing in vitro. We show that individual blastomeres from cleavage embryos at the 32- and 64-cell stages are capable of PGC production in culture. Importantly, Dnd overexpression increases PGCs via increasing PGC precursors. Strikingly, dnd RNA forms prominent particles that segregate asymmetrically. Dnd concentrates in germ plasm and stabilizes germ plasm RNA. Therefore, Dnd is a critical specifier of fish PGCs and utilizes particle partition as a previously unidentified mechanism for asymmetric segregation. These findings offer insights into PGC specification and manipulation in medaka as a lower vertebrate model.

P elements and the determinants of hybrid dysgenesis have different dynamics of propagation in Drosophila melanogaster populations.

Intraspecific hybrid dysgenesis (HD) appears after some strains of D. melanogaster are crossed. The predominant idea is that the movement of transposable P elements causes HD. It is believed that P elements appeared in the D. melanogaster genome in the middle of the last century by horizontal transfer, simultaneously with the appearance of HD determinants. A subsequent simultaneous expansion of HD determinants and P elements occurred. We analyzed the current distribution of HD determinants in natural populations of D. melanogaster and found no evidence of their further spread. However, full-sized P elements were identified in the genomes of all analyzed natural D. melanogaster strains independent of their cytotypes. Thus, the expansion of P elements does not correlate with the expansion of HD determinants. We found that the ovaries of dysgenic females did not contain germ cells despite the equal number of primordial germ cells in early stages in dysgenic and non-dysgenic embryos. We propose that HD does not result from DNA damage caused by P element transposition, but it would be the disruption in the regulation of dysgenic ovarian formation that causes the dysgenic phenotypes.

MCM9 deficiency delays primordial germ cell proliferation independent of the ATM pathway.

Maintenance of genome integrity is crucial for the germline, and this is reflected by lower mutation rates in gametes than somatic cells. Germ cells at different stages employ different DNA damage response (DDR) mechanisms. In response to certain DNA repair defects, primordial germ cells (PGCs) either undergo apoptosis or delayed proliferation, although little is known about the underlying mechanisms that govern these outcomes. Here, we report genetic studies of DDR pathways that underlie germ cell depletion in mice mutant for minichromosome maintenance 9 (Mcm9), a gene that plays a role in homologous recombination repair (HRR). Germ cell depletion in these mice is a result of reduced PGC numbers both before and after they arrive in the primitive gonads. This reduction was attributable to reduced proliferation, not apoptosis, and this response was independent of ATM-CHK2-TRP53-P21 signaling. This mechanism of PGC depletion differs from that in Fancm mutants, which also display reduced PGC depletion that is partially orchestrated by the ATM-TRP53-P21 pathway. Germ cell depletion in mice doubly deficient for FANCM and MCM9 was additive, indicating that the damage caused by each mutation triggers different DDR pathways to slow the cell cycle as a means to preserve genomic integrity. genesis 53:678-684, 2015. © 2015 Wiley Periodicals, Inc.

How Is the Number of Primordial Follicles in the Ovarian Reserve Established?

The number of primordial follicles in the ovarian reserve is an important determinant of the length of the ovarian lifespan, and therefore the fertility of an individual. This reserve contains all of the oocytes potentially available for fertilization throughout the fertile lifespan. The maximum number is set during pregnancy or just after birth in most mammalian species; current evidence does not support neofolliculogenesis after the ovarian reserve is established, although this is increasingly being reexamined. Under physiological circumstances, this number will be influenced by the number of primordial germ cells initially specified in the epiblast of the developing embryo, their proliferation during and after migration to the developing gonads, and their death during oogenesis and formation of primordial follicles at nest breakdown. Death of germ cells during the establishment of the ovarian reserve occurs principally by autophagy or apoptosis, although the triggers that initiate these remain elusive. This review outlines the regulatory steps that determine the number of primordial follicles and thus the number of oocytes in the ovarian reserve at birth, using the mouse as the model, interspersed with human data where available. This information has application for understanding the variability in duration of fertility that occurs between normal individuals and with age, in premature ovarian insufficiency, and after chemotherapy or radiotherapy.

A perspective on the evolution of germ-cell development and germinal mosaics of deleterious mutations

In many animals a small number of primordial germ cells (PGCs) are set aside early in development, mitosis and mitochondrial DNA syntheses are arrested, transcription is stopped or reduced, and the PGCs migrate later to the emerging gonads and become germ cells. What could be the evolutionary advantage of sequestering non-dividing PGCs early in development? A commonly cited advantage is a reduction in the number of new deleterious mutations that would occur if there were additional divisions in PGCs early in development. We would like to add to this advantage the fact that these additional mutations in PGCs give rise to germinal mosaics (i.e., premeiotic clusters of mutation) in multiple progeny of the same individual, thus having a larger detrimental effect on the evolutionary fitness of their carriers. Here, we reviewed published studies providing evidence that germinal mosaics of deleterious mutant alleles are not rare, occur for all types of genetic damage, and have been observed in all tested organisms and in nature. We propose the hypothesis that PGC sequestration during early animal development may have evolved in part in response to selection for preventing the occurrence of premeiotic clusters of deleterious mutant alleles, and describe a series of predictions that would allow the assessment of the potential role of germinal mosaics on the evolution of PGC sequestration.