Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates adenylate cyclase and promotes proliferation of mouse primordial germ cells

Development ◽  
1996 ◽  
Vol 122 (1) ◽  
pp. 215-221 ◽  
Author(s):  
M. Pesce ◽  
R. Canipari ◽  
G.L. Ferri ◽  
G. Siracusa ◽  
M. De Felici
Keyword(s):  
Adenylate Cyclase ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Intracellular Camp ◽  
Type I ◽  
In Vitro Proliferation ◽  
Pituitary Adenylate Cyclase

During migration and for about 2 days after their arrival in the gonadal ridges, primordial germ cells (the embryonic precursors of gametes of the adult animal) proliferate actively. Certain growth factors, such as stem cell factor and leukemia inhibitory factor, seem to be essential for survival, proliferation and possibly differentiation of mouse primordial germ cell in vivo and/or in vitro. Similarly, increase in intracellular cAMP is followed by a marked enhancement of primordial germ cell proliferation, at least in culture. In the present study, we show that pituitary adenylate cyclase-activating polypeptides (PACAP-27 and PACAP-38), two neuropeptides of the secretin-glucagon-vasoactive intestinal polypeptide-GH-releasing hormone family, stimulate in vitro proliferation of mouse primordial germ cells, bind to primordial germ cells and gonadal somatic cells (possibly to type I PACAP receptor) and activate adenylate cyclase in the same cells. Moreover, PACAP-like immunoreactivity was found in gonadal ridges, mostly on germ cell surface. In conclusion, evidence is provided that PGC proliferation can be stimulated by certain bioactive polypeptides, thus suggesting a novel regulatory role for such compounds in early gonad development.

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.

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 Avian Primordial Germ Cells Are Bipotent for Male or Female Gametogenesis

2021 ◽  
Vol 9 ◽  
Author(s):  
Maeve Ballantyne ◽  
Lorna Taylor ◽  
Tuanjun Hu ◽  
Dominique Meunier ◽  
Sunil Nandi ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Bird Species ◽  
Male Sterile ◽  
Cell Competition ◽  
Germ Cell Differentiation ◽  
Heterogametic Sex

In birds, males are the homogametic sex (ZZ) and females are the heterogametic sex (ZW). Here, we investigate the role of chromosomal sex and germ cell competition on avian germ cell differentiation. We recently developed genetically sterile layer cockerels and hens for use as surrogate hosts for primordial germ cell (PGC) transplantation. Using in vitro propagated and cryopreserved PGCs from a pedigree Silkie broiler breed, we now demonstrate that sterile surrogate layer hosts injected with same sex PGCs have normal fertility and produced pure breed Silkie broiler offspring when directly mated to each other in Sire Dam Surrogate mating. We found that female sterile hosts carrying chromosomally male (ZZ) PGCs formed functional oocytes and eggs, which gave rise to 100% male offspring after fertilization. Unexpectedly, we also observed that chromosomally female (ZW) PGCs carried by male sterile hosts formed functional spermatozoa and produced viable offspring. These findings demonstrate that avian PGCs are not sexually restricted for functional gamete formation and provide new insights for the cryopreservation of poultry and other bird species using diploid stage germ cells.

scholarly journals Ligand–Receptor Interactions Elucidate Sex-Specific Pathways in the Trajectory From Primordial Germ Cells to Gonia During Human Development

2021 ◽  
Vol 9 ◽  
Author(s):  
Arend W. Overeem ◽  
Yolanda W. Chang ◽  
Jeroen Spruit ◽  
Celine M. Roelse ◽  
Susana M. Chuva De Sousa Lopes
Keyword(s):  
Germ Cell ◽  
Signaling Pathways ◽  
Germ Cells ◽  
Tyrosine Kinases ◽  
Intracellular Localization ◽  
Primordial Germ Cells ◽  
Cell Lineage ◽  
Systematic Analysis ◽  
Receptor Interactions

The human germ cell lineage originates from primordial germ cells (PGCs), which are specified at approximately the third week of development. Our understanding of the signaling pathways that control this event has significantly increased in recent years and that has enabled the generation of PGC-like cells (PGCLCs) from pluripotent stem cells in vitro. However, the signaling pathways that drive the transition of PGCs into gonia (prospermatogonia in males or premeiotic oogonia in females) remain unclear, and we are presently unable to mimic this step in vitro in the absence of gonadal tissue. Therefore, we have analyzed single-cell transcriptomics data of human fetal gonads to map the molecular interactions during the sex-specific transition from PGCs to gonia. The CellPhoneDB algorithm was used to identify significant ligand–receptor interactions between germ cells and their sex-specific neighboring gonadal somatic cells, focusing on four major signaling pathways WNT, NOTCH, TGFβ/BMP, and receptor tyrosine kinases (RTK). Subsequently, the expression and intracellular localization of key effectors for these pathways were validated in human fetal gonads by immunostaining. This approach provided a systematic analysis of the signaling environment in developing human gonads and revealed sex-specific signaling pathways during human premeiotic germ cell development. This work serves as a foundation to understand the transition from PGCs to premeiotic oogonia or prospermatogonia and identifies sex-specific signaling pathways that are of interest in the step-by-step reconstitution of human gametogenesis in vitro.

The active migration of Drosophila primordial germ cells

Development ◽  
1995 ◽  
Vol 121 (11) ◽  
pp. 3495-3503 ◽  
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.

Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens

2008 ◽  
Vol 20 (8) ◽  
pp. 900 ◽  
Author(s):  
Yoshiaki Nakamura ◽  
Yasuhiro Yamamoto ◽  
Fumitake Usui ◽  
Yusuke Atsumi ◽  
Yohei Ito ◽  
...  
Keyword(s):  
Sustained Release ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Sesame Oil ◽  
Chicken Embryos ◽  
Whole Mount ◽  
Phosphate Buffered Saline ◽  
Transfer Study

The aim of the present study was to improve the efficiency of endogenous primordial germ cell (PGC) depletion and to increase the ratio of donor PGCs in the gonads of recipient chicken embryos. A sustained-release emulsion was prepared by emulsifying equal amounts of Ca2+- and Mg2+-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 μg per 50 μL busulfan sustained-release emulsion was injected into the yolk. To determine the depletion and repopulation of PGCs in the gonads after 6 days incubation, whole-mount immunostaining was performed. The busulfan sustained-release emulsion significantly reduced the number of endogenous PGCs compared with control (P < 0.05). Moreover, the busulfan sustained-release emulsion significantly depleted endogenous PGCs compared with other previously reported busulfan delivery systems (P < 0.05), but with less variation, suggesting that the sustained-release emulsion delivered a consistent amount of busulfan to the developing chicken embryos. The PGC transfer study showed that the proportion of donor PGCs in the gonads of busulfan sustained-release emulsion-treated embryos after 6 days incubation increased 28-fold compared with control. In conclusion, the results demonstrate that exogenous PGCs are capable of migrating and settling in gonads from which endogenous PGCs have been removed using a busulfan sustained-release emulsion.

scholarly journals A Novel Role for Endogenous Pituitary Adenylate Cyclase Activating Polypeptide in the Magnocellular Neuroendocrine System

Endocrinology ◽  
2006 ◽  
Vol 147 (2) ◽  
pp. 791-803 ◽  
Author(s):  
E. R. Gillard ◽  
M. León-Olea ◽  
S. Mucio-Ramírez ◽  
C. G. Coburn ◽  
E. Sánchez-Islas ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Supraoptic Nucleus ◽  
Potential Role ◽  
Neuroendocrine System ◽  
Neuroendocrine Cells ◽  
Type I ◽  
Pituitary Adenylate Cyclase ◽  
Pacap Receptors ◽  
Magnocellular Neuroendocrine Cells

Central release of vasopressin (VP) by the magnocellular neuroendocrine cells (MNCs) responsible for systemic VP release is believed to be important in modulating the activity of these neurons during dehydration. Central VP release from MNC somata and dendrites is stimulated by both dehydration and pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP is expressed in MNCs, its potential role in the magnocellular response to dehydration is unexplored. The current study demonstrates that prolonged dehydration increases immunoreactivity for PACAP-27, PACAP-38, and the type I PACAP receptor in the supraoptic nucleus (SON) of the rat. In addition, PACAP stimulates local VP release in the euhydrated rat SON in vitro, and this effect is reduced by the PACAP receptor antagonist PAC6–27 (100 nm), suggesting the participation of PACAP receptors. Concomitant with its effects on local VP release, PACAP also reduces basal glutamate and aspartate release in the euhydrated rat SON. Furthermore, somatodendritic VP release elicited by acute dehydration is blocked by PAC6–27, suggesting that endogenous PACAP participates in this response. Consistent with this, RIA revealed that local PACAP-38 release within the SON is significantly elevated during acute dehydration. These results suggest that prolonged activation of hypothalamic MNCs is accompanied by up-regulation of PACAP and the type I PACAP receptor in these cells and that somatodendritic VP release in response to acute dehydration is mediated by activation of PACAP receptors by endogenous PACAP released within the SON. A potential role for PACAP in promoting efficient, but not exhaustive, systemic release of VP from MNCs during physiological challenge is discussed.

scholarly journals Purification of mouse primordial germ cells by Nycodenz

Reproduction ◽  
2003 ◽  
pp. 667-675 ◽  
Author(s):  
T Mayanagi ◽  
R Kurosawa ◽  
K Ohnuma ◽  
A Ueyama ◽  
K Ito ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Specific Antibody ◽  
Membrane Surface ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Specific Antigen ◽  
Cell Lineage ◽  
New Method ◽  
Purification Method

Primordial germ cells are important cells for the study of germ cell lineage. It has proved difficult to obtain highly purified primordial germ cells for preparation of a specific antibody. In the present study, a new method for purifying mouse primordial germ cells was developed using a Nycodenz gradient. Furthermore, the polyclonal anti-mouse primordial germ cells IgG derived from mouse primordial germ cells was prepared. As this IgG reacted only with primordial germ cells obtained at day 12.5 after mating, this antibody appeared to recognize the stage-specific antigen of primordial germ cells. One reason that a continuous primordial germ cell marker has not been obtained is because the purity of the primordial germ cells used has been too low to prepare the antibody. This new method represents a significant improvement in the purification of primordial germ cells; it is simpler than previous methods, and produced mouse primordial germ cells with a purity of more than 95%. In addition, the separation reagent Nycodenz is non-toxic and achieved separation of primordial germ cells without attachment of antibodies against the primordial germ cell membrane surface. This new purification method and stage-specific antibody will be useful for the analysis of the mechanisms of primordial germ cell migration.

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

Cells ◽  
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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