Developmental stages, incubation temperature, and in vivo traceability of primordial germ cell in an important aquaculture species Piaractus mesopotamicus

Aquaculture ◽  
2021 ◽  
Vol 535 ◽  
pp. 736381
Author(s):  
Geovanna Carla Zacheo Coelho ◽  
Dilberto Ribeiro Arashiro ◽  
Tamiris Disselli ◽  
Matheus Pereira-Santos ◽  
Tatiana María Mira-López ◽  
...  
Keyword(s):  
Germ Cell ◽  
Developmental Stages ◽  
Incubation Temperature ◽  
Primordial Germ Cell ◽  
Piaractus Mesopotamicus

scholarly journals Preparation of a fish embryo for micromanipulation: staging of development, removal of the chorion and traceability of PGCs in Prochilodus lineatus

2019 ◽  
Vol 63 (1-2) ◽  
pp. 57-65
Author(s):  
Geovanna C. Z. Coelho ◽  
Isaac S. Yo ◽  
Tatiana M. Mira-López ◽  
Paulo S. Monzani ◽  
Dilberto R. Arashiro ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Incubation Temperature ◽  
Primordial Germ Cells ◽  
Survival Rates ◽  
Migration Route ◽  
Neotropical Fish ◽  
Fish Embryo ◽  
Prochilodus Lineatus ◽  
And Migration

The transplantation of primordial germ cells (PGCs) is a valuable tool for gene-banking and reconstitution by means of a germline chimera. For this technology, studies regarding developmental stages and traceability of PGCs are necessary. The objective of this study was to develop a micromanipulation procedure for the future establishment of cryobanks of PGCs in migratory characins. Incubation temperatures were evaluated at 22 ° C, 26 ° C, and 30 ° C in order to synchronize developmental stages. The highest hatching rates and the lowest abnormality rate arose at 26° C, which was considered to be the best incubation temperature. Enzymatic removal of the chorion was determined to be best using 0.05% pronase, in which the embryos presented better survival rates. In order to visualize PGCs in vivo, artificial GFP-nos1 3’UTR mRNA was injected and the migration route was observed in vivo as PGCs were visualized firstly at the segmentation stage (6 to 13 somites). The number of GFP positive cells ranged from 8 to 20 per embryo (mean of 13.8; n = 5). After hatching, GFP-positive cells increased to 14 to 27 embryos (mean of 19.8; n = 5). Visualization of the GFP-positive cells was possible at 10 days post hatching, and at this stage, the cells were positioned in the yolk extension region. This is the first report on PGC visualization in vivo in Neotropical fish; the obtained data provide information on the identification and migration of PGCs. The information presented in this work brings new insights in gene banking in Neotropical species and subsequent reconstitution through a germinal germline chimera.

scholarly journals Paracrine Insulin-Like Growth Factor Signaling Influences Primordial Germ Cell Migration: In Vivo Evidence from the Zebrafish Model

Endocrinology ◽  
2008 ◽  
Vol 149 (10) ◽  
pp. 5035-5042 ◽  
Author(s):  
Xianpeng Sang ◽  
Matthew S. Curran ◽  
Antony W. Wood
Keyword(s):  
Cell Migration ◽  
Germ Cell ◽  
Zebrafish Embryo ◽  
Expression Patterns ◽  
Primordial Germ Cell ◽  
Growth Factor Signaling ◽  
Zebrafish Model ◽  
Igf Signaling

IGF signaling has been shown to stimulate migration of multiple cell types in vitro, but few studies have confirmed an equivalent function for IGF signaling in vivo. We recently showed that suppression of IGF receptors in the zebrafish embryo disrupts primordial germ cell (PGC) migration, but the mechanism underlying these effects has not been elucidated. We hypothesized that PGCs are intrinsically dependent upon IGF signaling during the migratory phase of development. To test this hypothesis, we first examined the spatial expression patterns of IGF ligand genes (igf1, igf2a, and igf2b) in the zebrafish embryo. In situ analyses revealed distinct expression patterns for each IGF ligand gene, with igf2b mRNA expressed in a spatial pattern that correlates strongly with PGC migration. To determine whether PGC migration is responsive to IGF signaling in vivo, we synthesized gene hybrid expression constructs that permit conditional overexpression of IGF ligands by PGCs into the PGC microenvironment. Conditional overexpression of IGF ligands consistently disrupted PGC migration, confirming that PGC migration is sensitive to local aberrations in IGF signaling. Finally, we show that conditional suppression of IGF signaling, via PGC-specific overexpression of a mutant IGF-I receptor, disrupts PGC migration, confirming that zebrafish PGCs intrinsically require IGF signaling for directional migration in vivo. Collectively, these studies confirm an in vivo role for IGF signaling in cell migration and identify a candidate ligand gene (igf2b) regulating PGC migration in the zebrafish.

scholarly journals A transgenic DND1GFP fusion allele reports in vivo expression and RNA binding targets in undifferentiated mouse germ cells

2021 ◽  
Author(s):  
Victor A Ruthig ◽  
Tetsuhiro Yokonishi ◽  
Matthew B Friedersdorf ◽  
Sofia Batchvarova ◽  
Josiah Hardy ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Rna Binding ◽  
Cell Transformation ◽  
Primordial Germ Cell ◽  
Fetal Life ◽  
Mouse Line ◽  
Transgenic Mouse Line ◽  
Rna Immunoprecipitation

Abstract In vertebrates, the RNA binding protein (RBP) Dead End 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the pre-meiotic spermatogonial population. Utilizing the GFP-tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.

scholarly journals Single Cell Sequencing Reveals Early PGC-like Intermediates During Mouse Primed to Naïve Transition

2021 ◽  
Author(s):  
Jing Liu ◽  
Shengyong Yu ◽  
Chunhua Zhou ◽  
Jiangping He ◽  
Xingnan Huang ◽  
...  
Keyword(s):  
Germ Cell ◽  
Single Cell ◽  
Cell Fate ◽  
Single Cell Analysis ◽  
Primordial Germ Cell ◽  
Model System ◽  
Cell Analysis ◽  
Rna Seq ◽  
Single Cell Sequencing

Abstract Single cell analysis provides clarity unattainable with bulk approaches. Here we apply single cell RNA-seq to a newly established BMP4 induced mouse primed to naive transition (Bi-PNT) system and show that the reset is not a direct reversal of cell fate but through developmental intermediates. We first show that mEpiSCs bifurcate into c-Kit+ naïve and c-Kit- placenta-like cells, among which, the naive branch undergoes further transition through a primordial germ cell-like cells (PGCLCs) intermediate capable of spermatogenesis in vivo. Indeed, deficiency of Prdm1/Blimp1, the key regulator for PGC specification, blocks the induction of PGCLCs and naïve cells. Instead, Gata2 knockout arrests placenta-like fate, but facilitates the generation of PGCLCs. Our results not only reveal a newly cell fate dynamics between primed and naive states at single-cell resolution, but also provide a model system to explore mechanisms involved in regaining germline competence from primed pluripotency.

scholarly journals Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche

2017 ◽  
Author(s):  
Enrique Sosa ◽  
Di Chen ◽  
Ernesto J. Rojas ◽  
Jon D. Hennebold ◽  
Karen A. Peters ◽  
...  
Keyword(s):  
Germ Cell ◽  
Rhesus Macaque ◽  
Primordial Germ Cell ◽  
Stem Cell Differentiation ◽  
Cell Types ◽  
Major Bottleneck ◽  
Induced Pluripotent ◽  
Homologous Transplantation

AbstractA major challenge in stem cell differentiation validation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse model, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus macaque PGCLCs (rPGCLCs) from rhesus macaque induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation with the expression of VASA and MAGEA4, but not ENO2. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late PGCs and spermatogonia-like cells but do not complete the conversion into ENO2-positive spermatogonia.

scholarly journals Effects of Survival Motor Neuron Protein on Germ Cell Development in Mouse and Human

2021 ◽  
Vol 22 (2) ◽  
pp. 661
Author(s):  
Wei-Fang Chang ◽  
Min Peng ◽  
Jing Hsu ◽  
Jie Xu ◽  
Huan-Chieh Cho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Motor Neuron ◽  
Cell Biology ◽  
Muscular Atrophy ◽  
Primordial Germ Cell ◽  
Cell Types ◽  
Survival Motor Neuron

Survival motor neuron (SMN) is ubiquitously expressed in many cell types and its encoding gene, survival motor neuron 1 gene (SMN1), is highly conserved in various species. SMN is involved in the assembly of RNA spliceosomes, which are important for pre-mRNA splicing. A severe neurogenic disease, spinal muscular atrophy (SMA), is caused by the loss or mutation of SMN1 that specifically occurred in humans. We previously reported that SMN plays roles in stem cell biology in addition to its roles in neuron development. In this study, we investigated whether SMN can improve the propagation of spermatogonia stem cells (SSCs) and facilitate the spermatogenesis process. In in vitro culture, SSCs obtained from SMA model mice showed decreased growth rate accompanied by significantly reduced expression of spermatogonia marker promyelocytic leukemia zinc finger (PLZF) compared to those from heterozygous and wild-type littermates; whereas SMN overexpressed SSCs showed enhanced cell proliferation and improved potency. In vivo, the superior ability of homing and complete performance in differentiating progeny was shown in SMN overexpressed SSCs in host seminiferous tubule of transplant experiments compared to control groups. To gain insights into the roles of SMN in clinical infertility, we derived human induced pluripotent stem cells (hiPSCs) from azoospermia patients (AZ-hiPSCs) and from healthy control (ct-hiPSCs). Despite the otherwise comparable levels of hallmark iPCS markers, lower expression level of SMN1 was found in AZ-hiPSCs compared with control hiPSCs during in vitro primordial germ cell like cells (PGCLCs) differentiation. On the other hand, overexpressing hSMN1 in AZ-hiPSCs led to increased level of pluripotent markers such as OCT4 and KLF4 during PGCLC differentiation. Our work reveal novel roles of SMN in mammalian spermatogenesis and suggest new therapeutic targets for azoospermia treatment.

scholarly journals Association of culture of mouse urogenital complexes in media containing rodent sera with the appearance of primordial germ cell-like cells

Reproduction ◽  
2003 ◽  
pp. 519-526 ◽  
Author(s):  
T Mayanagi ◽  
K Ito ◽  
J Takahashi
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Calf Serum ◽  
Culture Method ◽  
Embryonic Germ Cells ◽  
Alkaline Phosphatase Staining

Primordial germ cells differentiate into germ cells and have the ability to reacquire totipotency. Mouse primordial germ cells are identified by alkaline phosphatase staining of the extraembryonic mesoderm, and they proliferate and migrate to reach the genital ridges. Mouse primordial germ cells have never been maintained in culture exclusively for longer than a week without differentiation or dedifferentiation. Moreover, primordial germ cells have not been proliferated with urogenital complexes in vitro, because gonad culture has never been successful. It was thought that primordial germ cells could proliferate in a culture of urogenital complex under modified medium conditions resembling those in vivo; however, organ culture of mouse gonad has been performed with fetal calf serum or equine serum, and those sera produce conditions different from those in vivo. Therefore, mouse urogenital complexes were cultured in media containing rodent sera. As a result, it was possible to proliferate primordial germ cell-like cells outside gonads, and these cells very closely resembled primordial germ cells. In addition, motile primordial germ cell-like cells could be obtained. The ability to maintain primordial germ cell-like cells in culture by this intra-species culture method is important in the study of gametogenesis. Furthermore, this method is useful as a source of stem cells such as embryonic germ cells.

scholarly journals DMRT1-mediated reprogramming drives development of cancer resembling human germ cell tumors with features of totipotency

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jumpei Taguchi ◽  
Hirofumi Shibata ◽  
Mio Kabata ◽  
Masaki Kato ◽  
Kei Fukuda ◽  
...  
Keyword(s):  
Germ Cell ◽  
Tumor Cells ◽  
Cell Fate ◽  
Primordial Germ Cell ◽  
Somatic Cells ◽  
Germ Cell Tumors ◽  
Differentiation Potential ◽  
Wide Range ◽  
The Impact

AbstractIn vivo reprogramming provokes a wide range of cell fate conversion. Here, we discover that in vivo induction of higher levels of OSKM in mouse somatic cells leads to increased expression of primordial germ cell (PGC)-related genes and provokes genome-wide erasure of genomic imprinting, which takes place exclusively in PGCs. Moreover, the in vivo OSKM reprogramming results in development of cancer that resembles human germ cell tumors. Like a subgroup of germ cell tumors, propagated tumor cells can differentiate into trophoblasts. Moreover, these tumor cells give rise to induced pluripotent stem cells (iPSCs) with expanded differentiation potential into trophoblasts. Remarkably, the tumor-derived iPSCs are able to contribute to non-neoplastic somatic cells in adult mice. Mechanistically, DMRT1, which is expressed in PGCs, drives the reprogramming and propagation of the tumor cells in vivo. Furthermore, the DMRT1-related epigenetic landscape is associated with trophoblast competence of the reprogrammed cells and provides a therapeutic target for germ cell tumors. These results reveal an unappreciated route for somatic cell reprogramming and underscore the impact of reprogramming in development of germ cell tumors.

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