Chimaerism of primordial germ cells in the early postimplantation mouse embryo following microsurgical grafting of posterior primitive streak cells in vitro

A microsurgical grafting technique has been used to introduce primordial germ cell (PGC) precursors into intact primitive-streak-stage mouse embryos in vitro. Operated embryos were cultured for 36–40 h and then analysed by a combined histochemical and autoradiographic method. PGC chimaerism occurred in embryos that received grafts of caudal primitive streak cells but not adjacent embryonic endoderm or anterolateral ectoderm/mesoderm cells. Graftderived PGCs were found to be migrating through the gut endoderm alongside host-derived PGCs in approximately half of the chimaeric embryos whereas in the other 50% of cases PGCs remained at the site of grafting in association with graft-derived somatic cells. A similar pattern of somatic chimaerism was produced by primitive streak and anterolateral ectoderm/mesoderm grafts: the allantois was colonized predominantly, with, in addition, formation of amnion, surface ectoderm and caudal mesoderm in a few embryos. The majority of embryonic endoderm grafts failed to incorporate into host embryos and formed yolk-sac-like vesicles. The findings of this study indicate that (a) PGCs originate from the embryonic ectoderm via the primitive streak during development of the mouse embryo, and (b) anterolateral ectoderm and mesoderm cells are unable to form PGCs after heterotopic grafting to the posterior primitive streak site. The combined microsurgical and embryo culture methods provide an experimental system for the analysis of PGC development in intact mouse embryos.

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Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos

Development ◽

10.1242/dev.64.1.133 ◽

1981 ◽

Vol 64 (1) ◽

pp. 133-147

Author(s):

P. P. L. Tam ◽

M. H. L. Snow

Keyword(s):

Mitomycin C ◽

Germ Cells ◽

Compensatory Growth ◽

Primordial Germ Cells ◽

Primordial Germ Cell ◽

Primitive Streak ◽

Mouse Embryos ◽

Newborn Mice ◽

Embryonic Life ◽

And Migration

Primitive-streak-stage mouse embryos were treated with Mitomycin C injected intraperitoneally into pregnant females at 6·75–7·0 days post coitum. The newborn mice developed poorly and mortality was high during the suckling period. Many weaned survivors showed impaired fertility and poor breeding performance. Histological examination revealed a paucity of germ cells in the adult gonads. The deficiency was mainly caused by a severe reduction of the primordial germ cell population in early embryonic life, which was not fully compensated for during the compensatory growth phase of the Mitomycin C-treated embryo. Also contributing to such impaired fertility were retarded migration of the primordial germ cells into the genital ridges, poor development of the foetal gonad and secondary loss of the germ cells during gametogenesis in males.

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Protocol for controlled modeling of human epiblast and amnion development using stem cells

10.21203/rs.2.11520/v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Yi Zheng ◽

Jianping Fu

Keyword(s):

Stem Cells ◽

Primordial Germ Cells ◽

Primitive Streak ◽

Human Embryos ◽

Vitro Fertilization ◽

Human Embryology ◽

Post Implantation ◽

Advance Knowledge ◽

Early Human

Abstract Due to the inaccessibility of post-implantation human embryos and the restriction on in-vitro fertilization (IVF) embryos cultured beyond 14 days, the knowledge of early post-implantation human embryogenesis remains extremely limited. Recently, we have developed a microfluidic in-vitro platform, based on human pluripotent stem cells (hPSCs), which is capable of recapitulating several key developmental landmarks of early human post-implantation embryonic development, including lumenogenesis of the epiblast (EPI), amniogenesis, and specification of primordial germ cells (PGCs) and of primitive streak (PS) cells. Given its controllability and reproducibility, the microfluidic platform provides a powerful experimental platform to advance knowledge of human embryology and reproduction. This protocol describes the preparation of the microfluidic device and its implementation for modeling human post-implantation epiblast and amnion development using hPSCs.

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Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture

Development ◽

10.1242/dev.108.2.357 ◽

1990 ◽

Vol 108 (2) ◽

pp. 357-363 ◽

Cited By ~ 5

Author(s):

I. Godin ◽

C. Wylie ◽

J. Heasman

Keyword(s):

Long Range ◽

Primordial Germ Cells ◽

Primordial Germ Cell ◽

Great Majority ◽

Early Embryo ◽

Extrinsic Factors ◽

Alkaline Phosphatase Staining ◽

Hind Gut ◽

Feeder Cell Layers

The functional gametes of all vertebrates first arise in the early embryo as a migratory population of cells, the primordial germ cells (PGCs). These migrate to, and colonise, the genital ridges (GR) during the early organogenesis period, giving rise to the complete differentiating gonad. PGCs first become visible by alkaline phosphatase staining in the root of the developing allantois at 8.5 days post coitum (dpc). At 9.5 dpc they are found in the wall of the hind-gut and, during the following three days, they migrate along the hind-gut mesentery to the dorsal body wall, and then to the genital ridges. By 12.5 dpc, the great majority of PGCs have colonised the genital ridges. During this period the number of PGCs increases from less than 100 to approximately 4000. In a previous paper (Donovan et al. 1986), we showed that 10.5 dpc PGCs can be explanted from the hind-gut mesentery, and will spread and migrate on feeder cell layers. We showed also that the intrinsic ability of PGCs to spread and migrate changes as they colonise the genital ridges. In this paper, we examine extrinsic factors that control PGC behaviour in vitro. Using PGCs taken from 8.5 dpc embryos, at the beginning of their migratory phase, we show that culture medium conditioned by 10.5 dpc genital ridges causes an increase in the number of PGCs in these cultures. We also show that PGCs migrate towards 10.5 dpc genital ridges in preference to other explanted organs. These experiments show that genital ridges exert long-range effects on the migrating population of PGCs.(ABSTRACT TRUNCATED AT 250 WORDS)

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P–182 The mechanism of mouse embryo hatching and the impact of laser drilling the zona pellucida: an RNA sequencing study

Human Reproduction ◽

10.1093/humrep/deab130.181 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Y Liu ◽

C Jones ◽

K Coward

Keyword(s):

Treatment Group ◽

Mouse Embryo ◽

Mouse Embryos ◽

Z Score ◽

Rna Seq ◽

Metabolism Pathway ◽

Hatching Process ◽

The Impact

Abstract Study question What is the mechanism of embryo hatching? Will laser-assisted zona pellucida (ZP) drilling alter the embryonic transcriptome? Summary answer Hatching is an ATP-dependent process. Hatching is also associated with Rho-mediated signaling. Laser-assisted ZP drilling might cause alternation in embryo metabolism. What is known already Embryo hatching is a vital process for early embryo development and implantation. Animal data suggests that hatching is the result of multiple factors, such as mechanical pressure, protease activation, and the regulation of maternal secretions. However, little is known about the regulatory signaling mechanisms and the molecules involved. In addition, despite the extensive use of laser-assisted ZP drilling in the clinic, the safety profile of this technique at molecular level is very sparse. The impact of this technique on the embryonic transcriptome has not been studied systematically. Study design, size, duration Eighty mouse embryos were randomly divided into a laser ZP drilling group (n = 40) and an untreated group (n = 40). After treatment, embryos were cultured in vitro for two days. Then, hatching blastocyst (n = 8) and pre-hatching blastocyst (n = 8) from the untreated group, and the hatching blastocyst from the treatment group (n = 8) were processed for RNA sequencing (RNA-seq). Participants/materials, setting, methods Cryopreserved 8-cell stage mouse embryos (B6C3F1 × B6D2F1) were thawed, and a laser was used to drill the embryo ZP in the treatment group. Next, the treated and untreated embryos were individually cultured in vitro to the E4.5 blastocyst stage. The resulting blastocysts were lysed individually and used for subsequent cDNA library preparation and RNA-seq. Following data quality control and alignment, the RNA-seq data were processed for differentially expressed gene analysis and downstream functional analysis. Main results and the role of chance According to the RNA-seq data, 275 differentially expressed genes (DEGs) (230 up-regulated and 45 down-regulated, adjusted P < 0.05) were identified when comparing hatching and pre-hatching blastocysts in the control groups. Analysis suggested that the trophectoderm is the primary cell type involved in hatching, and revealed the potential molecules causing increased blastocyst hydrostatic pressure (Aqp3 and Cldn4). Functional enrichment analysis suggested that ATP metabolism and protein synthesis were activated in hatching blastocysts. DEGs were found to be significantly enriched in several gene ontology terms, particularly in terms of the organization of the cytoskeleton and actin polymerisation (P < 0.0001). Furthermore, according to QIAGEN ingenuity pathway analysis results, Rho signaling was implicated in blastocyst hatching (Actb, Arpc2, Cfl1, Myl6, Pfn1, Rnd3, Septin9, z-score=2.65, P < 0.0001). Moreover, the potential role of hormones (estrogen (z-score=2.24) and prolactin (z-score=2.4)) and growth factors (AGT (z-score=2.41) and FGF2 (z-score=2.213)) were implicated in the hatching process as indicated by the upstream regulator analysis. By comparing the transcriptome between laser-treated and untreated hatching blastocysts, 47 DEGs were identified (adjusted P < 0.05) following laser-assisted ZP drilling. These genes were enriched in metabolism-related pathways (P < 0.05), including the lipid metabolism pathway (Mvd, Mvk, Aacs, Gsk3a, Pik3c2a, Aldh9a1) and the xenobiotic metabolism pathway (Aldh18a1, Aldh9a1, Keap1, and Pik3c2a). Limitations, reasons for caution Findings in mouse embryos may not be fully representative of human embryos. Furthermore, the mechanism of hatching revealed here might only reflect the hatching process of embryos in vitro. Further studies are now necessary to confirm these findings in different conditions and species to determine their clinical significance. Wider implications of the findings: Our study profiled the mouse embryo transcriptome during in vitro hatching, identified potential key genes and mechanisms for future study. In addition, for the first time, we revealed the impact of laser-assisted ZP drilling on the transcriptome, this may help us to assess and improve the existing technique. Trial registration number Not applicable

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The allocation of cells in the presomitic mesoderm during somite segmentation in the mouse embryo

Development ◽

10.1242/dev.103.2.379 ◽

1988 ◽

Vol 103 (2) ◽

pp. 379-390 ◽

Cited By ~ 1

Author(s):

P.P. Tam

Keyword(s):

Mouse Embryo ◽

Colloidal Gold ◽

Wheat Germ ◽

Mouse Embryos ◽

Middle Region ◽

Presomitic Mesoderm ◽

Posterior Displacement ◽

Somite Formation ◽

Gold Conjugate

Orthotopic grafts of wheat germ agglutinin-colloidal gold conjugate (WGA-gold) labelled cells were used to demonstrate differences in the segmental fate of cells in the presomitic mesoderm of the early-somite-stage mouse embryos developing in vitro. Labelled cells in the anterior region of the presomitic mesoderm colonized the first three somites formed after grafting, while those grafted to the middle region of this tissue were found mostly in the 4th-7th newly formed somites. Labelled cells grafted to the posterior region were incorporated into somites whose somitomeres were not yet present in the presomitic mesoderm at the time of grafting. There was therefore an apparent posterior displacement of the grafted cells in the presomitic mesoderm. Colonization of somites by WGA-gold labelled cells was usually limited to two to three consecutive somites in the chimaera. The distribution of cells derived from a single graft to two somites was most likely due to the segregation of the labelled population when cells were allocated to adjacent meristic units during somite formation. Further spreading of the labelled cells to several somites in some cases was probably the result of a more extensive mixing of mesodermal cells among the somitomeres prior to somite segmentation.

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Epithelial—mesenchymal tissue interactions guiding otic capsule formation: the role of the otocyst

Development ◽

10.1242/dev.97.1.1 ◽

1986 ◽

Vol 97 (1) ◽

pp. 1-24

Author(s):

Joseph R. McPhee ◽

Thomas R. Van De Water

Keyword(s):

Mouse Embryo ◽

Mouse Embryos ◽

In Vitro Studies ◽

Otic Capsule ◽

Membranous Labyrinth ◽

Capsule Formation ◽

Tissue Interactions ◽

Mesenchymal Tissue

The otocyst is the epithelial anlage of the membranous labyrinth which interacts with surrounding cephalic mesenchyme to form an otic capsule. A series of in vitro studies was performed to gain a better understanding of the epithelial—mesenchymal interactions involved in this process. Parallel series of otocyst/mesenchyme (O/M) and isolated periotic mesenchyme (M) explants provided morphological and biochemical data to define the role of the otocyst in organizing and directing formation of its cartilaginous otic capsule. Explants were made from mouse embryos ranging in age from 10 to 14 days of gestation, and organ cultured under identical conditions until the chronological equivalent of 16 days of gestation. Expression of chrondrogenesis was determined by both histology and biochemistry. The in vitro behaviour of periotic mesenchyme explanted either with or without an otocyst supports several hypotheses that explain aspects of otic capsule development. The results indicate that (a) prior to embryonic day 12 the otocyst alone is not sufficient to stimulate chondrogenesis of the otic capsule within O/M explants; (b) the otocyst acts as an inductor of capsule chondrogenesis within O/M explants between embryonic days 12 to 13; (c) isolated mesenchyme within M explants taken from 13-day-old embryos are capable of initiating in vitro chondrogenesis, but without expressing capsule morphology in the absence of the otocyst; and (d) the isolated mesenchyme of M explants obtained from 14-day-old embryos expresses both chondrogenesis and otic capsule morphology in the absence of the otocyst. These findings suggest that the otocyst acts as an inductor of chondrogenesis of periotic mesenchyme tissue between embryonic days 11 to 13, and controls capsular morphogenesis between embryonic days 13 to 14 in the mouse embryo.

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The active migration of Drosophila primordial germ cells

Development ◽

10.1242/dev.121.11.3495 ◽

1995 ◽

Vol 121 (11) ◽

pp. 3495-3503 ◽

Cited By ~ 12

Author(s):

M.K. Jaglarz ◽

K.R. Howard

Keyword(s):

Primary Culture ◽

Germ Cells ◽

Actin Polymerization ◽

Primordial Germ Cells ◽

Primordial Germ Cell ◽

Cell Polarization ◽

Germ Cell Migration ◽

Oriented Movement

We describe our analysis of primordial germ cell migration in Drosophila wild-type and mutant embryos using high resolution microscopy and primary culture in vitro. During migratory events the germ cells form transient interactions with each other and surrounding somatic cells. Both in vivo and in vitro they extend pseudopodia and the accompanying changes in the cytoskeleton suggest that actin polymerization drives these movements. These cellular events occur from the end of the blastoderm stage and are regulated by environmental cues. We show that the vital transepithelial migration allowing exit from the gut primordium and passage into the interior of the embryo is facilitated by changes in the structure of this epithelium. Migrating germ cells extend processes in different directions. This phenomenon also occurs in primary culture where the cells move in an unoriented fashion at substratum concentration-dependent rates. In vivo this migration is oriented leading germ cells to the gonadal mesoderm. We suggest that this guidance involves stabilization of states of an intrinsic cellular oscillator resulting in cell polarization and oriented movement.

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154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab154 ◽

2016 ◽

Vol 28 (2) ◽

pp. 207

Author(s):

J. Galiguis ◽

C. E. Pope ◽

C. Dumas ◽

G. Wang ◽

R. A. MacLean ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Germ Cells ◽

Primordial Germ Cells ◽

Primordial Germ Cell ◽

Embryonic Stem ◽

Transcript Abundance ◽

Domestic Cat ◽

Sample Type

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

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Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients

Cells ◽

10.3390/cells10113099 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3099

Author(s):

Aline Fernanda de Souza ◽

Fabiana Fernandes Bressan ◽

Naira Caroline Godoy Pieri ◽

Ramon Cesar Botigelli ◽

Tamas Revay ◽

...

Keyword(s):

Germ Cell ◽

Turner Syndrome ◽

Genetic Disorder ◽

Primordial Germ Cells ◽

Primordial Germ Cell ◽

Cell Formation ◽

In Vitro Models ◽

Germline Development ◽

Germ Cell Specification

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.

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Neuroectodermal fate of epiblast cells in the distal region of the mouse egg cylinder: implication for body plan organization during early embryogenesis

Development ◽

10.1242/dev.121.1.87 ◽

1995 ◽

Vol 121 (1) ◽

pp. 87-98 ◽

Cited By ~ 13

Author(s):

G.A. Quinlan ◽

E.A. Williams ◽

S.S. Tan ◽

P.P. Tam

Keyword(s):

Cell Fate ◽

Neural Tube ◽

Primitive Streak ◽

Distal Region ◽

Small Population ◽

Surface Ectoderm ◽

Minor Contribution ◽

Stage Embryo ◽

A Minor

The developmental fate of cells in the distal region (distal cap) of the epiblast was analysed by fate mapping studies. The displacement and differentiation of cells labelled in situ with carbocyanine dyes and lacZ-expressing cells grafted to the distal cap were studied over a 48-hour period of in vitro development. The distal cap epiblast differentiates predominantly into neurectodermal cells. Cells at the anterior site of the distal cap colonise the fore-, mid- and hindbrain and contribute to non-neural ectoderm cells of the amnion and craniofacial surface ectoderm. Those cells in the most distal region of the epiblast contribute to all three brain compartments as well as the spinal cord and the posterior neuropore. Cells at the posterior site of the distal cap are mainly localised to the caudal parts of the neural tube. A minor contribution to the embryonic (paraxial and lateral) and extraembryonic (allantoic and yolk sac) mesoderm is also found. Epiblast cells located outside the distal cap give rise to surface ectoderm and other non-ectodermal derivatives, with only a minor contribution to the neuroectoderm. Results of this study provide compelling evidence that the precursor population of the neural tube is contained in the distal cap epiblast of the early-primitive-streak-stage embryo. Furthermore, the regionalisation of cell fate within this small population suggest that a preliminary craniocaudal patterning may have occurred in the neural primordium before neurulation.

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