Primordial Germ Cells In Embryos Research Articles

ZIF-1-mediated degradation of zinc finger proteins in the Caenorhabditis elegans germ line.

Rapid and conditional protein depletion is the gold standard genetic tool for deciphering the molecular basis of developmental processes. Previously, we showed that by conditionally expressing the E3 ligase substrate adaptor ZIF-1 in Caenorhabditis elegans somatic cells, proteins tagged with the first CCCH Zn finger 1 (ZF1) domain from the germline regulator PIE-1 degrade rapidly, resulting in loss-of-function phenotypes. The described role of ZIF-1 is to clear PIE-1 and several other CCCH Zn finger proteins from early somatic cells, helping to enrich them in germline precursor cells. Here, we show that proteins tagged with the PIE-1 ZF1 domain are subsequently cleared from primordial germ cells (PGCs) in embryos and from undifferentiated germ cells in larvae and adults by ZIF-1. We harness germline ZIF-1 activity to degrade a ZF1-tagged fusion protein from PGCs and show that its depletion produces phenotypes equivalent to those of a null mutation. Our findings reveal that ZIF-1 transitions from degrading CCCH Zn finger proteins in somatic cells to clearing them from undifferentiated germ cells, and that ZIF-1 activity can be harnessed as a new genetic tool to study the early germline.

Single-cell transcriptome analysis of the novel coronavirus (SARS-CoV-2) associated gene ACE2 expression in normal and non-obstructive azoospermia (NOA) human male testes.

Being infected by SARS-CoV-2 may cause damage to multiple organs in patients, such as the lung, liver and heart. Angiotensin-converting enzyme 2 (ACE2), reported as a SARS-CoV-2 receptor, is also expressed in human male testes. This suggests a potential risk in human male reproductive system. However, the characteristics of ACE2-positive cells and the expression of other SARS-CoV-2 process-related genes are still worthy of further investigation. Here, we performed singlecell RNA seq (scRNA-seq) analysis on 853 male embryo primordial germ cells (PGCs) and 2,854 normal testis cells to assess the effects of the SARS-CoV-2 virus on the male reproductive system from embryonic stage to adulthood. We also collected and constructed the scRNA-seq library on 228 Sertoli cells from three non-obstructive azoospermia (NOA) patients to assess the effects at disease state. We found that ACE2 expressing cells existed in almost all testis cell types and Sertoli cells had highest expression level and positive cells ratio. Moreover, ACE2 was also expressed in human male PGCs. In adulthood, the level of ACE2 expression decreased with the increase of age. We also found that ACE2 positive cells had high expressions of stress response and immune activation-related genes. Interestingly, some potential SARS-CoV-2 process-related genes such as TMPRSS2, BSG, CTSL and CTSB had different expression patterns in the same cell type. Furthermore, ACE2 expression level in NOA donors’ Sertoli cells was significantly decreased. Our work would help to assess the risk of SARS-CoV-2 infection in the male reproductive system.Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s11427-020-1705-0 and is accessible for authorized users.

Visualization and motility of primordial germ cells using green fluorescent protein fused to 3'UTR of common carp nanos-related gene

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. We previously visualized the PGCs of several teleostean embryos by injecting RNA synthesized from constructs encoding green fluorescent protein (GFP) fused to the 3'UTR of the zebrafish ( Danio rerio) nanos1 gene ( nos1). However, this technique was not always suitable for visualizing PGCs in embryos from all teleost species. In this study, we compared the visualization of PGCs in common carp ( Cyprinus carpio) embryos using two artificial constructs containing GFP fused to the 3'UTR of nanos from either common carp or zebrafish. Visualization was better using GFP fused to the 3'UTR of the nanos gene from common carp, compared with that from zebrafish. The visualized PGCs successfully migrated toward the gonadal ridge after transplantation into goldfish host embryos, suggesting that they maintained normal migratory motility. These techniques could be useful for the production of inter-specific germline chimeras using common carp donor PGCs.

Expression ofDazlandVasain turtle embryos and ovaries: evidence for inductive specification of germ cells

In bilaterian animals, germ cells are specified by the inductive/regulative mode or the predetermined (germ plasm) mode. Among tetrapods, mammals and urodeles use the inductive mode, whereas birds and anurans use the predetermined mode. From histological data it has been predicted that some reptiles including turtles use the inductive mode. Examining turtle oocytes, we find that Dazl RNA, Vasa RNA, and Vasa protein are not localized, suggesting that germ plasm is not present. In turtle embryos at somite stages, primordial germ cells (PGCs) expressing Dazl lie on a path from the lateral posterior extraembryonic endoderm through the gut to the gonad as previously described. In gastrulating embryos, cells expressing Dazl are found in the blastoporal plate and subsequently below the blastoporal plate, indicating that PGCs are generated at the equivalent of the early posterior primitive streak of mammals. Vasa RNA is expressed in somatic cells of gastrula to early somite stages, and Vasa RNA and protein are expressed in PGCs of later embryos. Taken together the evidence strongly suggests that turtles, and other reptiles (lacertoid lizards) with the same location of PGCs in embryos, use the inductive mode of germ cell specification. Phylogenetic analysis of the available evidence supports the following hypotheses: (1) the inductive mode is basal among reptiles, indicating that this mode was maintained as basal tetrapods evolved to amniotes, (2) the predetermined mode arose twice within reptiles, and (3) the induced mode may be used in several lepidosaurs whose PGCs are located in an unusual pattern distributed around the embryo.

The actin-binding protein Lasp promotes Oskar accumulation at the posterior pole of theDrosophilaembryo

During Drosophila oogenesis, Oskar mRNA is transported to the posterior pole of the oocyte, where it is locally translated and induces germ-plasm assembly. Oskar protein recruits all of the components necessary for the establishment of posterior embryonic structures and of the germline. Tight localization of Oskar is essential, as its ectopic expression causes severe patterning defects. Here, we show that the Drosophila homolog of mammalian Lasp1 protein, an actin-binding protein previously implicated in cell migration in vertebrate cell culture, contributes to the accumulation of Oskar protein at the posterior pole of the embryo. The reduced number of primordial germ cells in embryos derived from lasp mutant females can be rescued only with a form of Lasp that is capable of interacting with Oskar, revealing the physiological importance of the Lasp-Oskar interaction.

Who Stole the Chemokine?

Primordial germ cells (PGCs) in the zebrafish embryo face a long migration from the point at which they are specified during development to the location of the future gonad, and it takes some precise signaling to get them there. Major guidance cues are provided by the chemokine SDF-1a (stromal-derived factor-1 alpha), which attracts the PGCs by signaling through its receptor CXCR4b [chemokine (C-X-C motif) receptor 4b]. SDF-1a also binds to another receptor, CXCR7, but the function of this receptor in PGC cell migration has been uncertain. Boldajipour et al. ’s experiments indicate that CXCR7’s function is not to signal but rather to act as a sink that soaks up stray molecules of SDF-1a, thus adding to the precision of SDF-1a signaling through CXCR4b. Transplantation experiments taking PGCs from animals lacking CXCR7 into wild-type embryos showed that the critical function of CXCR7 was in somatic cells, not the PGCs. Microscopy of fluorescently tagged proteins showed that CXCR7 promoted internalization of SDF-1a, and human cells expressing CXCR7 depleted SDF-1a from the culture medium in which they were grown. In vivo, the inhibited motility of PGCs in embryos lacking CXCR7 was reproduced in embryos overexpressing SDF-1a, and depletion of CXCR4b suppressed the CXCR7 phenotype. Analysis of embryos in which SDF-1a was uniformly expressed but CXCR7 was expressed unevenly showed that PGCs moved to domains lacking CXCR7 (where concentrations of SDF-1a were presumably higher). The authors compare this decoy receptor strategy to a similar mechanism that functions in the resolution of inflammatory responses: In that case, interleukin-10 promotes expression of inflammatory chemokine receptors on mature dendritic cells, where they also appear not to signal but instead act to sequester proinflammatory chemokines. B. Boldajipour, H. Mahabaleshwar, E. Kardash, M. Reichman-Fried, H. Blaser, S. Minina, D. Wilson, Q. Xu, E. Raz, Control of chemokine-guided cell migration by ligand sequestration. Cell 132 , 463-473 (2008). [Online Journal]

Primordial germ cell formation in birds.

At the early somite stage, chicken embryo primordial germ cells comprise a population of about 200 dispersed, morphologically distinct cells, located in the extraembryonic germinal crescent. Tracing the origin of primordial germ cells at earlier stages is at present not possible by either cytological or immunohistochemical techniques. To resolve this difficulty we have microdissected embryos at early embryonic stages, isolated fragments from various positions and incubated them to the somite stage, when the primordial germ cells can be visualized accurately. From these studies a distinct developmental pattern has emerged. (1) At the uterine stage, when the area pellucida starts to thin out, there is already a defined area, confined to the most central part of the blastodisc, which constitutes the sole source of the future primordial germ cells. (2) As development proceeds, the area that gives rise to the primordial germ cells expands peripherally. (3) Primordial germ cells thereafter migrate as isolated cells, descending to the lower layer, the hypoblast. (4) This gradual process starts at the onset of blastulation and terminates towards the end of gastrulation. (5) The hypoblast, destined to become the yolk sac endoderm, is pushed anteriorly during gastrulation by morphogenetic movements to be replaced by the endoderm proper; the primordial germ cells are carried forwards with it and occupy an extraembryonic location.

The planarian nanos-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration

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

150 THE DISTRIBUTION PATTERN OF PRIMORDIAL GERM CELLS IN EARLY CHICK EMBRYOS

In all vertebrates, primordial germ cells (PGCs) appear during early stages of development in extragonadal sites, then they migrate to the gonad and give rise to ova or spermatozoa. Unlike in other species, however, in avian and reptile embryos, PGCs use the vascular system as a vehicle to transport them to the future gonadal region where they leave the blood vessels. The present study was carried out to know the details of this unique migration pathway and the proliferation of endogenous PGCs in chicken embryos. Whole of the chicken embryos during stages X [Roman numerals refer to the staging system of Eyal-Giladi and Kochav (1976 Dev. Biol. 49, 321–327) to 17 (Arabic numerals refer to the staging system of Hamburger and Hamilton (1951 J. Morphol. 88, 49-82))] or embryonic blood during stages 12 to 17 were immunohistochemical stained using specific antibody raised against chicken vasa homolog (CVH), which could be recognized as a marker for chicken PGCs. The distribution patterns and populations of PGCs in embryos were observed under a stereomicroscope. The numbers of PGCs were presented mean and standard deviation (mean � SD). Anti-CVH staining revealed the distribution and population of chicken PGCs in early chick embryos. PGCs existed mainly in the area pellucida and concentrated in the central zone at stage X. The mean number of PGCs per embryo at this stage was 130.4 � 31.9. With the formation of primitive streak, PGCs were carried anteriorly to the edge of the blastoderm. The PGCs scattered anteriorly began to concentrate to the anterior point of the head on the dorsal side of stage 10 embryos. The average number of PGCs per embryo at stage 10 was 439.3 � 93.6. The mean numbers of PGCs per embryo during stages X to 10 increased gradually as development progressed to stage 10. We found the entrance point of PGCs from anterior edge of the blastoderm to the vascular network during stages 10 to 11. In the blood, PGCs could be detected from all of the samples during stages 12 to 17. In contrast, no PGC was recognized in the future gonadal region before stage 14, and then they began to appear in the same region at stage 15. The mean numbers of PGCs that located in the future gonadal region during stages 15 to 17 increased intensively and were 97.3 � 57.3, 200.3 � 113.5, and 327.6 � 102.4, respectively. Interestingly, the numbers of PGCs within future gonadal region during stages 15 to 17 were consistently and significantly different (P < 0.05) between the left and right side of the region. The results suggest that chicken PGCs move from extraembryonic area to the vascular network during stages 10 to 11, circulate in the blood stream, and finally, they begin to leave the blood vessels actively and migrate to the future gonadal region at stage 15.

Altered primordial germ cell migration in the absence of transforming growth factor β signaling via ALK5

Transforming growth factor β (TGFβ) inhibits proliferation and promotes the migration of primordial germ cells (PGCs) towards explants of gonadal ridges in vitro. However, its effects in vivo are still unclear. Here, we analyzed the behavior of PGCs in embryos lacking TGFβ signaling via the type I receptor ALK5. TGFβ in vivo was neither a chemoattractant for PGCs, nor did it affect their proliferation during migration towards the gonadal ridges up to embryonic day (E)10. Unexpectedly, the absence of TGFβ signaling in fact resulted in significant facilitation of PGC migration out of the hindgut, due to the reduced deposition of collagen type I surrounding the gut of Alk5-deficient mutant embryos. Migratory PGCs adhere strongly to collagen; therefore, reduced collagen type I along the gut may result in reduced adhesion, facilitating migration into the dorsal mesenterium and gonadal ridges. Our results provide new evidence for the role of TGFβ signaling in migration of PGCs in vivo distinct from that described previously.

262. Towards derivation of primordial germ cells from murine embryonic stem cells

The process of primordial germ cell (PGC) specification begins at the earliest stages of murine embryogenesis. The mechanisms underlying this process are the strong instructive cues generated by the extra-embryonic ectoderm, which, via ligand-receptor signalling in the visceral endoderm, activate pathways in the proximal epiblast to induce the PGC phenotype. We have subjected murine embryonic stem (ES) cells to similar cues in order to drive PGC lineage specification in vitro. ES cells were differentiated as aggregates (embryoid bodies (EBs)), a process that is thought to recapitulate the early stages of embryogenesis by providing an environment conducive to the spontaneous emergence of multiple cell lineages. To date, we have shown that EBs can also support the spontaneous emergence of cells expressing PGC markers. Expression analysis was performed on EBs from 1 to 30 days in culture. PGC markers, including nanog, dazl, fragilis, stella and SSEA1, are expressed in undifferentiated ES cells, but rapidly become undetectable in EBs as the constituent ES cells undergo differentiation. The spontaneous emergence of cells expressing these markers occurred only following long-term EB culture. This indicates a lag in the signalling normally required for PGC specification. In vivo, the lack of BMP4, its receptor (ALK-2) or downstream signalling molecules (Smad 1 and 5), results in the absence of PGCs in embryos. Therefore, in order to enhance PGC specification in our in vitro system, we have added BMP4 into the culture media. Under these conditions, the emergence of cells expressing PGC markers occurs at both an apparently higher efficiency and in a shorter time period. This suggests that BMP4 response pathways are present within the EB context and, when activated, can direct PGC specification. Thus, by recapitulating an in vivo physical and biochemical environment, we are able to direct PGC lineage specification in vitro.

Effects of endosulfan and nonylphenol on the primordial germ cell population in pre-larval zebrafish embryos

A variety of chemicals released into the aquatic environment are capable of targeting the reproductive system in fish and other vertebrates. Some of the effects observed in exposed adults may arise by permanent organizational changes that occur during embryogenesis, including changes in gonad structure and function. Little work has addressed the effects of pesticides and industrial chemicals, many of which are recognized as endocrine disrupting chemicals, on early embryos. The recent cloning of the vasa gene in zebrafish, the mRNA of which is found in fertilized eggs and is later segregated into the primordial germ cells (PGCs), has provided a unique opportunity to examine PGC migration and positioning in early embryos. We utilized antisense RNA probes to vasa mRNA in whole mount in situ hybridization analysis in order to examine the early migration and distribution of PGCs in embryos exposed to endosulfan and nonylphenol. The data reveal that these chemicals cause alterations in the distribution of PGCs along the anterior–posterior axis in 24-h-old embryos. This suggests that the previously reported alterations in juvenile and adult gonad structure of various aquatic vertebrates following exposure to pesticides and industrial chemicals could be related in part to alterations in early PGC distribution.

Localization of oskar RNA regulates oskar translation and requires Oskar protein.

The site of oskar RNA and protein localization within the oocyte determines where in the embryo primordial germ cells form and where the abdomen develops. Initiation of oskar RNA localization requires the activity of several genes. We show that ovaries mutant for any of these genes lack Oskar protein. Using various transgenic constructs we have determined that sequences required for oskar RNA localization and translational repression map to the oskar 3'UTR, while sequences involved in the correct temporal activation of translation reside outside the oskar 3'UTR. Upon localization of oskar RNA and protein at the posterior pole, Oskar protein is required to maintain localization of oskar RNA throughout oogenesis. Stable anchoring of a transgenic reporter RNA at the posterior pole is disrupted by oskar nonsense mutations. We propose that initially localization of oskar RNA permits translation into Oskar protein and that subsequently Oskar protein regulates its own RNA localization through a positive feedback mechanism.

X-chromosome activity of the mouse primordial germ cells revealed by the expression of an X-linked lacZ transgene

We have determined the timing of the inactivation and reactivation of the X chromosome in the mouse primordial germ cells (PGCs) by monitoring the expression of an X-linked HMG-lacZ reporter gene. PGCs were identified by their distinct alkaline phosphatase activity and they were first localised in the primitive streak and allantoic bud of the 7.5-day gastrulating embryo. Although inactivation of the transgene was found in some PGCs at these sites, at least 85% of the population were still expressing the lacZ gene. This suggests that, although X-inactivation has commenced during gastrulation, the majority of PGCs still possess two active X chromosomes. Transgene activity remained unchanged during the relocation of PGCs to the hindgut endoderm, but decreased abruptly when PGCs left the hindgut and migrated through the mesentery. X-inactivation was completed during the migration of PGCs, but not simultaneously for the whole population. The first wave of PGCs entering the genital ridge at 9.5 days did not immediately re-activate the silent transgene until about 24 hours later. Re-activation of the transgene took place in over 80% of PGCs entering the genital ridge at 10.5-13.5 days p.c., preceding the entry into meiosis. About 90% of the meiotic germ cells in the 14.5-15.5 day fetal ovary expressed the transgene. Similar profiles of transgene activity were observed in PGCs of embryos that have inherited the lacZ transgene from different parents, showing unequivocally that X-inactivation in the germ cell lineage is not related to parental legacy.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of chick primordial germ cells from stages 4–8 embryos

Chick embryo primordial germ cells (PGCs) stages 4-8 were manually isolated for the first time from the late hypoblast layer. They were confirmed to be PGCs by periodic acid-Schiff (PAS) staining and examination by scanning electron microscopy (SEM). They were subsequently introduced onto a variety of artificial substrata. On two dimensional substrata, the cells change from a spherical shape covered with numerous microvilli to a rounded cell with a "skirt" of cytoplasm. Eventually a process projects from one side of the smooth cell. On a three dimensional substrate the PGCs change from a spherical shape covered with numerous microvilli to a smooth surfaced cell with a long single process. It is concluded that the PGCs which are originally spherical in situ in stage 4 alter their morphology both in vivo during their migration and in vitro studies.

Chapter 7 Cellular Interactions of Mouse Fetal Germ Cells In In Vitro Systems

This chapter discusses the germ cell differentiation in the mouse embryo and cellular interactions during early germ cell differentiation. The development of germ cells in the mouse embryo, as well as in all other mammals, can be schematically divided into four main periods—migratory, proliferative, sex differentiation, and cell death. In the mouse embryo primordial germ cells can be identified by their high alkaline phosphatase activity at the posterior end of the primitive streak about 8 days post coitum. In all mammalian embryos, waves of degeneration drastically reduce the number of germ cells before birth. Throughout their entire life history, germ cells do not exist as an independent tissue, but are always associated with other cells from which they may derive nutrients as well as developmental signals. It is likely that the sequential expression of the different genes that direct germ cell differentiation may be modulated by cellular interactions that involve cell contact and cell position as a function of time. There is evidence suggesting that during the developmental periods germ cells are involved in at least two different kinds of cellular interactions: germ cell– extracellular matrix and germ cell–somatic cell interactions.

Effects of γ-Ray Irradiation on Primordial Germ Cells in Embryos of Oryzias latipes

Effects of ..gamma.. rays on different stages of primordial germ cells were quantitatively studied in Oryzias latipes embryos. Stage 9, which is before the germ cell determination, exhibited the highest radiosensitivity to the elimination of primordial germ cells in embryos and hatching fry. Irradiation of embryos at this stage with 500-2000-rad ..gamma..-ray doses showed an exponential dose-effect relationship. A time-course study was also carried out on embryos and fry irradiated with 2000 rad at stage 9. A possibility was indicated that the onset of sex differentiation of germ cells may be independent of the population size of primordial germ cells at the genital ridges. Primordial germ cells became highly radioresistant from the stage 3 days after fertilization. In the time-course study of the embryos irradiated at 4 days after fertilization with 2000 rad, germ cells with enlarged nuclei were observed after 48 hr. Under the present experimental conditions, no ''immediate cell death'' was encountered.

Number of germ cells in known male and known female genotypes of vertebrate embryos (Oryzias latipes).

The number of primordial germ cells in embryos of known genotypic sex was the same in XY males and XX females until the gonad became recognizable as testis or ovary. It has been claimed that the heterogametic sex chromosome causes the gonad to differentiate as a testis in mammals and as an ovary in birds as a result of earlier and more mitoses. This claim was not supported in the present study where a sex difference in numbers of germ cells was first noted during differentiation of oocytes in the XX embryos.