scholarly journals 171 NORMAL REPROGRAMMING OF IMPRINTING IN PARTHENOGENETIC FEMALE GERM CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 236
Author(s):  
T. Horii ◽  
Y. Nagao ◽  
M. Kimura ◽  
I. Hatada
Keyword(s):  
Germ Cells ◽  
Restriction Analysis ◽  
Primordial Germ Cells ◽  
Fluorescent Microscopy ◽  
Embryonic Stem ◽  
Postnatal Growth ◽  
The Other ◽  
Imprinted Genes ◽  
Host Embryo ◽  
Parthenogenetic Embryos

Mammalian parthenotes cannot develop normally to term. Mouse parthenogenetic embryos die by Day 10 of gestation. On the other hand, viable parthenogenetic chimeras were produced by normal host embryos, although parthenogenetic cells were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. This can be explained by the aberrant expressions of imprinted genes in parthenogenetic cells. In female mice, erasure of imprints occurs around the time that primordial germ cells enter the gonad, and establishment of imprints occurs in the postnatal growth phase of oogenesis. In this study, we investigated whether aberrant imprints in parthenogenetic embryonic stem (PgES) cells can be erased through the germline. Diploid parthenogenetic embryos were produced by activation of (CBA × C57BL/6-EGFP) F1 mouse superovulated unfertilized oocytes by exposure to Sr2+ and cytochalasin B. Ten parthenogenetic blastocysts were plated and three PgES cell lines were isolated. Chimeras were made by injecting 10–15 PgES cells into ICR(CD-1) mouse blastocysts. Chimeras and chimeric tissues were detected by fluorescent microscopy. In all, 173 chimeric blastocysts were transferred to 9 recipient females, and 101 live pups containing 9 female and 21 male chimeras were born. No significant growth retardation was apparent in PgES chimeras, irrespective of their degree of chimerism. In 5 male chimeras killed at 1 day postpartum (dpp), PgES cells showed a restricted tissue contribution. The contribution to lung, liver, and intestine was considerably lower than in the other tissues such as brain, heart, spleen, and kidney. PgES derived or host embryo derived non-growing oocytes were isolated from dissociated ovaries of female chimeras at 1 dpp under fluorescent microscopy. Methylation imprints in non-growing oocytes were analyzed for maternally methylated imprinted genes Peg1, Snrpn, and Igf2r by the combined bisulfite restriction analysis (COBRA). In normal oocytes, imprints are expected to be erased and these genes are unmethylated at this stage. We observed that these genes were unmethylated in both PgES derived and host embryo derived non-growing oocytes. These results suggest that aberrant imprints in PgES cells can also be erased normally through the germline.

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

Cell Research ◽  
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.

Sex differentiation of germ cells in the teleost, Oryzias latipes, during normal embryonic development

Development ◽  
1972 ◽  
Vol 28 (2) ◽  
pp. 385-395
Author(s):  
Noriyuki Satoh ◽  
Nobuo Egami
Keyword(s):  
Germ Cells ◽  
Meiotic Prophase ◽  
Sex Differentiation ◽  
Primordial Germ Cells ◽  
Oryzias Latipes ◽  
The Other ◽  
Histological Structure ◽  
The Mean ◽  
Further Development ◽  
Normal Embryonic Development

Mitotic and meiotic activities of germ cells during early development in the medaka, Oryzias latipes, are dealt with in this report. Primordial germ cells were obviously distinguishable from somatic cells 3 days after fertilization and began to proliferate about 7 days after fertilization. The mean number of primordial germ cells increased during a period of 7–10 days after fertilization, reaching about 90 immediately before hatching. Newly hatched fry could be classified into two types according to the number and the nucleic activity of germ cells in the gonadal rudiment. One type consisted of fry containing about 100 germ cells and no cells in the meiotic prophase. In the other type of fry the number of germ cells increased by mitotic divisions and some of the cells began to enter into the meiotic prophase. During the course of further development the fry of the former type differentiated into males and the latter into females. Therefore it can be concluded that the morphological sex differentiation of germ cells occurs at the time of hatching. However, no sexual differences in the histological structure of somatic elements in the gonad are observable at that time.

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 ◽  
1996 ◽  
Vol 122 (4) ◽  
pp. 1235-1242 ◽  
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.

Collagen-alginate microspheres as a 3D culture system for mouse embryonic stem cells differentiation to primordial germ cells

Biologicals ◽  
2017 ◽  
Vol 48 ◽  
pp. 114-120 ◽  
Author(s):  
Vahid Mansouri ◽  
Mohammad Salehi ◽  
Mir davood Omrani ◽  
Zahra Niknam ◽  
Abdolreza Ardeshirylajimi
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Germ Cells ◽  
Culture System ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
3D Culture ◽  
Mouse Embryonic Stem Cells ◽  
3D Culture System

154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

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.

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

Zygote ◽  
1998 ◽  
Vol 6 (3) ◽  
pp. 271-275 ◽  
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.

scholarly journals Generation of developmentally competent oocytes and fertile mice from parthenogenetic embryonic stem cells

2021 ◽  
Author(s):  
Chenglei Tian ◽  
Linlin Liu ◽  
Ming Zeng ◽  
Xiaoyan Sheng ◽  
Dai Heng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Germ Cells ◽  
Genetic Manipulation ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Endocrine Function ◽  
Placental Development ◽  
Parthenogenetic Embryos

AbstractParthenogenetic embryos, created by activation and diploidization of oocytes, arrest at mid-gestation for defective paternal imprints, which impair placental development. Also, viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells (pESCs) derived from parthenogenetic embryos, presumably attributable to their aberrant imprinting. We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring. Moreover, normal expression of imprinted genes is found in the germ cells and the mice. pESCs exhibited imprinting consistent with exclusively maternal lineage, and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background. pESCs differentiated into primordial germ cell-like cells (PGCLCs) and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function. The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs, consistent with efficient reprogramming of methylation and genomic imprinting. These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting, offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function.

scholarly journals When Regenerative Medicine Faces the Challenges of Reproductive Medicine: A Review Study on Recent Advances in the Strategies for Derivation of Gametes from Stem Cells

2020 ◽  
pp. 42-52
Author(s):  
María Gil Juliá ◽  
José V. Medrano
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Developmental Stages ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Differentiation Strategy ◽  
Stem Cell Source ◽  
Potential Applications ◽  
Key Events

The murine model has allowed for the replication of all developmental stages of the mammalian germline in vitro, from embryonic stem cells to epiblast cells, primordial germ cells, and finally into functional haploid gametes. However, because of interspecies differences between mice and humans, these results are yet to be replicated in our species. Reports on the use of stem cells as a source of gametes, retrieved from public scientific databases, were analysed and classified according to the animal model used, the stem cell source and type, the differentiation strategy, and its potential application. This review offers a comprehensive compilation of recent publications of key events in the derivation of germ cells and gametogenesis in vitro, in both mice and human models. Additionally, studies intending to replicate the different stages in human cells in vitro, in order to obtain cells with a phenotype akin to functional human gametes, are also depicted. The authors present options for deriving gametes from stem cells in vitro and different reproductive options for specific groups of patients. Lastly, the potential applications of in vitro human gametogenesis are evaluated as well as the main limitations of the techniques employed. Even though it appears that we are far from being able to obtain gametes from pluripotent stem cells in vitro as a viable reproductive option, its current academic and clinical implications are extremely promising.

222 LOSS OF IMPRINTS OF PARTHENOGENETIC EMBRYONIC STEM CELLS IN MURINE CHIMERAS

2007 ◽  
Vol 19 (1) ◽  
pp. 228
Author(s):  
T. Horii ◽  
M. Kimura ◽  
S. Morita ◽  
Y. Nagao ◽  
I. Hatada
Keyword(s):  
Body Weight ◽  
Normal Level ◽  
Fluorescent Protein ◽  
Restriction Analysis ◽  
Methylation Status ◽  
Embryonic Stem ◽  
Fertility Treatment ◽  
Paternal Allele ◽  
Imprinted Genes ◽  
Parthenogenetic Embryo

Mammalian parthenotes with the 2 maternal genomes cannot develop to term. By contrast, chimeras produced by parthenogenetic and normal embryos can develop to term. However, parthenogenetic cells contribute to restricted cells and body weights of the chimeras are reduced. These effects are due to aberrant expressions of imprinted genes, with complete methylation of the maternally methylated genes and complete loss of the paternally methylated genes. On the other hand, parthenogenetic ES (PGES) chimeras show more normal tissue contribution of donor cells and body weight compared to parthenogenetic embryo (PG) chimeras. To elucidate the epigenetic mechanisms underlying this, we analyzed the epigenetic status of maternally methylated genes in murine PG and PGES chimeras. To make parthenogenetic chimeras, PG and PGES cells which express green fluorescent protein (GFP) were introduced into normal host embryos. Mouse embryonic fibroblasts (MEFs) from E13.5 chimeric fetuses were sorted by the fluorescence-activated cell sorter (FACS). Methylation status of parthenogenetic cells was analyzed by combined bisulfite restriction analysis (COBRA) and bisulfite sequencing. Methylation of maternally methylated genes, Peg1/Mest, Snrpn, and Igf 2r, was almost totally maintained in PG chimeras. Average methylatation percentages of PG-derived MEFs were 80% in Peg1/Mest, 84% in Snrpn, and 81% in Igf 2r (n = 6). In contrast, methylation in some PGES chimeras was partially reduced to normal level in all 3 genes (10–45%, n = 7). To clarify whether demethylation is correlated with expression of the imprinted genes, gene expression was analyzed by quantitative real-time RT-PCR. Among maternally imprinted genes, Peg1/Mest and Snrpn are expressed from the paternal allele, whereas Igf 2r is expressed from the maternal allele. Therefore, in parthenogenetic cells, loss of imprints is expected to up-regulate Peg1/Mest and Snrpn expression, and down-regulate Igf 2r expression. In fact, PGES-derived MEFs were up-regulated in Peg1/Mest and Snrpn expression, and down-regulated in Igf 2r expression. This study revealed that variations of imprint status were observed frequently in somatic cells of PGES cell origin. Demethylation could have occurred during establishment and/or maintenance of PGES cells. This demethylation that occurred in PGES cells could reprogram the maternally methylated imprinted genes and improve tissue contribution and body weight to normal level. The PGES cells with reprogramming ability might be utilized for fertility treatment and regenerative medicine.

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