Normal Embryonic and Germ Cell Development in Mice Lacking α1,3-Fucosyltransferase IX (Fut9) Which Show Disappearance of Stage-Specific Embryonic Antigen 1

ABSTRACT Stage-specific embryonic antigen 1 (SSEA-1), an antigenic epitope defined as a Lewis x carbohydrate structure, is expressed during the 8-cell to blastocyst stages in mouse embryos and in primordial germ cells, undifferentiated embryonic stem cells, and embryonic carcinoma cells. For many years, SSEA-1 has been implicated in the development of mouse embryos as a functional carbohydrate epitope in cell-to-cell interaction during morula compaction. In a previous study, α1,3-fucosyltransferase IX (Fut9) exhibited very strong activity for the synthesis of Lewis x compared to other α1,3-fucosyltransferases in an in vitro substrate specificity assay. Fut4 and Fut9 transcripts were expressed in mouse embryos. The Fut9 transcript was detected in embryonic-day-13.5 gonads containing primordial germ cells, but the Fut4 transcript was not. In order to identify the role of SSEA-1 and determine the key enzyme for SSEA-1 synthesis in vivo, we have generated Fut9-deficient (Fut9−/−) mice. Fut9−/− mice develop normally, with no gross phenotypic abnormalities, and are fertile. Immunohistochemical analysis revealed an absence of SSEA-1 expression in early embryos and primordial germ cells of Fut9−/− mice. Therefore, we conclude that expression of the SSEA-1 epitope in the developing mouse embryo is not essential for embryogenesis in vivo.

Download Full-text

In vivo and in vitro differentiation of male germ cells in the mouse

Reproduction ◽

10.1530/rep.1.00220 ◽

2004 ◽

Vol 128 (2) ◽

pp. 147-152 ◽

Cited By ~ 65

Author(s):

Orly Lacham-Kaplan

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Germ Cells ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Cellular Interactions ◽

Germ Cell Differentiation

Primordial germ cells appear in the embryo at about day 7 after coitum. They proliferate and migrate towards the genital ridge. Once there, they undergo differentiation into germ stem cells, known as ‘A spermatogonia’. These cells are the foundation of spermatogenesis. A spermatogonia commit to spermatogenesis, stay undifferentiated or degenerate. The differentiation of primordial germ cells to migratory, postmigratory and germ stem cells is dependent on gene expression and cellular interactions. Some of the genes that play a crucial role in germ cell differentiation are Steel, c-Kit, VASA, DAZL, fragilis, miwi, mili, mil1 and mil2. Their expression is stage specific, therefore allowing solid identification of germ cells at different developmental phases. In addition to the expression of these genes, other markers associated with germ cell development are nonspecific alkaline phosphatase activity, the stage specific embryonic antigen, the transcription factor Oct3/4 and β1- and α6-integrins. Commitment of cells to primordial germ cells and to A spermatogonia is also dependent on induction by the bone morphogenetic protein (BMP)-4. With this knowledge, researchers were able to isolate germ stem cells from embryonic stem cell-derived embryoid bodies, and drive these into gametes either in vivo or in vitro. Although no viable embryos were obtained from these gametes, the prospects are that this goal is not too far from being accomplished.

Download Full-text

Human Reproduction is Regulated by Retrotransposons derived from Ancient Hominin-Specific Viral Infections

10.21203/rs.3.rs-80780/v1 ◽

2020 ◽

Author(s):

Yu Tao ◽

Xiinyu Xiang ◽

Fei-Man Hsu ◽

Julien Pontis ◽

Didier Trono ◽

...

Keyword(s):

Germ Cells ◽

Viral Infections ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Dna Demethylation ◽

Human Reproduction ◽

Epigenetic Landscape

Abstract Germ cells are essential to pass DNA from one generation to the next. In human reproduction, germ cell development begins with the specification of primordial germ cells (PGCs) and a failure to specify PGCs leads to human infertility. Recent studies have revealed that the transcription factor network required for PGC specification has diverged in mammals, and this has a significant impact on our understanding of human reproduction. Here, we evaluated the emerging epigenetic landscape during hPGC specification using a combination of in vivo and in vitro analysis of hPGCs/hPGC-like cells (hPGCLCs) and human embryonic stem cells (hESCs). Our data reveals that hominid restricted Transposable Elements (TEs) partly derived from ancient viruses are pre-bound by the transcription factors TFAP2C and NANOG in undifferentiated hESCs, become transcriptionally induced during PGC specification and undergo dynamic epigenetic reprogramming leading to increased chromatin accessibility, localized DNA demethylation and establishment of broad peaks of H3K27ac. Using KRAB mediated CRISPRi we show that blocking this remodeling has a significant impact on hPGC specification. In summary, our data reveals that human reproduction requires the establishment of an epigenetic landscape during hPGC specification driven by the acquisition of hominid-specific TEs that were derived from ancient viral infections that entered the hominid germline less than 5 million years ago.

Download Full-text

Formative pluripotent stem cells show features of epiblast cells poised for gastrulation

Cell Research ◽

10.1038/s41422-021-00477-x ◽

2021 ◽

Author(s):

Xiaoxiao Wang ◽

Yunlong Xiang ◽

Yang Yu ◽

Ran Wang ◽

Yu Zhang ◽

...

Keyword(s):

Stem Cells ◽

Germ Cells ◽

Pluripotent Stem Cells ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Large Set ◽

Mouse Escs ◽

Epiblast Stem Cells ◽

Early Gastrula

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

Download Full-text

Expression of GFP Gene in Gonads of Chicken Embryos by Transfecting Primordial Germ Cells in vitro or in vivo using the PiggyBac Transposon Vector System

The Journal of Poultry Science ◽

10.2141/jpsa.0140197 ◽

2015 ◽

Vol 52 (3) ◽

pp. 227-231

Author(s):

Mitsuru Naito ◽

Takashi Harumi ◽

Takashi Kuwana

Keyword(s):

Germ Cells ◽

Primordial Germ Cells ◽

Vector System ◽

Piggybac Transposon ◽

Chicken Embryos ◽

Gfp Gene

Download Full-text

Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro

Development ◽

10.1242/dev.46.1.119 ◽

1978 ◽

Vol 46 (1) ◽

pp. 119-133

Author(s):

Janet Heasman ◽

C. C. Wylie

Keyword(s):

Xenopus Laevis ◽

Germ Cells ◽

Primordial Germ Cells ◽

Structural Features ◽

Culture Conditions ◽

Structural Basis ◽

Electron Microscopic ◽

Microscopic Studies

Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells. In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo. (b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filopodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo. (c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles. We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.

Download Full-text

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.

Download Full-text

Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells

Development ◽

10.1242/dev.122.4.1235 ◽

1996 ◽

Vol 122 (4) ◽

pp. 1235-1242 ◽

Cited By ~ 3

Author(s):

U. Koshimizu ◽

T. Taga ◽

M. Watanabe ◽

M. Saito ◽

Y. Shirayoshi ◽

...

Keyword(s):

Germ Cells ◽

Intracellular Signaling ◽

Primordial Germ Cells ◽

Cell Formation ◽

Embryonic Stem ◽

Oncostatin M ◽

Receptor Complexes ◽

Binding Subunit

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.

Download Full-text

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.

Download Full-text

Primary culture of porcine PGCs requires LIF and porcine membrane-bound stem cell factor

Zygote ◽

10.1017/s0967199498000215 ◽

1998 ◽

Vol 6 (3) ◽

pp. 271-275 ◽

Cited By ~ 17

Author(s):

Gabriela Durcova-Hills ◽

Katja Prelle ◽

Sigrid Müller ◽

Miodrag Stojkovic ◽

Jan Motlik ◽

...

Keyword(s):

Stem Cell ◽

Primary Culture ◽

Germ Cells ◽

Stem Cell Factor ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Leukaemia Inhibitory Factor ◽

Feeder Cells ◽

Membrane Bound

We studied the effect of murine leukaemia inhibitory factor (LIF), human basic fibroblast growth factor (bFGF) and porcine stem cell factor (SCF) on the survival and/or proliferation of porcine primordial germ cells (PGCs) obtained from 27-day-old embryos in vitro. PGCs were cultured in embryonic stem cell (ESC) medium supplemented with or without either LIF (1000 IU/ml) alone or LIF together with bFGF (10 ng/ml). They were seeded on mitotically inactivated feeder cells, either STO or transfected STO cells (STO#8), expressing the membrane-bound form of porcine SCF. PGCs were identified by their alkaline phosphatase (AP) activity and counted after 1, 3 and 5 days in culture. After 1 day of culture, PGCs cultured on STO#8 cells showed significantly higher survival than PGCs cultured on STO cells (p < 0.05). The combined effect of SCF and LIF caused a significant increase in PGC number by day 3 of culture when PGCs were cultured on either STO cells (p < 0.01) or STO#8 (p < 0.001). When SCF and LIF were used together with bFGF no increase in the PGC number was observed. Our results suggest that the membrane-bound form of porcine SCF plays a pivotal role in the primary culture of porcine PGCs and that bFGF is not required in vitro.

Download Full-text