Measurement of mRNA Poly(A) Tail Lengths in Drosophila Female Germ Cells and Germ-Line Stem Cells

2016 ◽  
pp. 93-102 ◽  
Author(s):  
Aymeric Chartier ◽  
Willy Joly ◽  
Martine Simonelig
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Germ Line

192 IDENTIFICATION AND CHARACTERIZATION OF Oct4-EGFP EXPRESSING CELLS IN TRANSGENIC PIG TESTIS

2014 ◽  
Vol 26 (1) ◽  
pp. 210
Author(s):  
M. Nowak-Imialek ◽  
N. Lachmann ◽  
D. Herrmann ◽  
F. Jacob ◽  
H. Niemann
Keyword(s):  
Stem Cells ◽  
Cell Population ◽  
Germ Cells ◽  
Germ Line ◽  
Cell Mass ◽  
Testicular Tissue ◽  
Egfp Expression ◽  
Cell Populations ◽  
Marker Genes ◽  
Transgenic Pigs

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.

Male germ line stem cells: from cell biology to cell therapy

2003 ◽  
Vol 15 (6) ◽  
pp. 323 ◽  
Author(s):  
David Pei-Cheng Lin ◽  
Ming-Yu Chang ◽  
Bo-Yie Chen ◽  
Han-Hsin Chang
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Germ Line ◽  
Current Status ◽  
Seminiferous Tubules ◽  
Male Germ Line ◽  
Male Germ Cells ◽  
Stem Cell Lines

Research using stem cells has several applications in basic biology and clinical medicine. Recent advances in the establishment of male germ line stem cells provided researchers with the ability to identify, isolate, maintain, expand and differentiate the spermatogonia, the primitive male germ cells, as cell lines under in vitro conditions. The ability to culture and manipulate stem cell lines from male germ cells has gradually facilitated research into spermatogenesis and male infertility, to an extent beyond that facilitated by the use of somatic stem cells. After the introduction of exogenous genes, the spermatogonial cells can be transplanted into the seminiferous tubules of recipients, where the transplanted cells can contribute to the offspring. The present review concentrates on the origin, life cycle and establishment of stem cell lines from male germ cells, as well as the current status of transplantation techniques and the application of spermatogonial stem cell lines.

The development of the testis of the Merino ram, with special reference to the orgin of the adult stem cells

1962 ◽  
Vol 13 (3) ◽  
pp. 487 ◽  
Author(s):  
CS Sapsford
Keyword(s):  
Stem Cells ◽  
Basement Membrane ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Adult Stem Cells ◽  
Germ Line ◽  
Primordial Germ Cells ◽  
Seminiferous Tubules ◽  
Stages Of Development ◽  
Striking Change

In the ram, as in other mammals, the sex cords are made up of two types of cell: indifferent cells (derivatives of the coelomic epithelium) and primordial germ cells. In the cords, each type pursues a separate and independent line of development to become respectively the Sertoli cells and the stem cells (type A spermatogonia) of the adult testis. The principal changes taking place in the primordial germ cells (gonocytes) are a reduction in the size and number of the Feulgen-positive particles in the nuclei, the appearance and subsequent fusion of the nucleoli, and, finally, an increase in the size of the nuclei. While these changes are taking place, the cytoplasm increases in volume and inclusions become more numerous. Cells which have undergone all these transformations have been called prospermatogonia. The cells of the germ line are at first more centrally placed in the sex cords than the indifferent cells. Just before spermatogenesis begins, they migrate to the basement membrane of the seminiferous tubules. All germ cells in tubules in which spermatogenesis has been initiated are seen as prospermatogonia. These cells become flattened against the basement membrane, and their nuclei become more oval in shape. They thus become identical with the stem cells of the adult. Little change is evident in the nuclei of the indifferent cells until puberty. Feulgen-positive material is found in the form of coarse granules at earlier stages of development. At puberty, these granules become dispersed to give a much more homogeneous nucleus. Concurrently, nuclei increase in size, and single or double true nucleoli can be identified. During development, increases in cytoplasmic volume take place. Although cell boundaries between indifferent cells cannot be seen in fixed material, phase contrast observations of fresh material have demonstrated that some forms exist as mononucleate units. It could not be determined whether the same was true in the case of Sertoli cells. No striking change in the relative numbers of glandular interstitial cells could be observed at different stages of development.

002. REGULATION OF PLURIPOTENCY AND CELL CYCLE IN FETAL GERM CELLS

2009 ◽  
Vol 21 (9) ◽  
pp. 2
Author(s):  
P. Western ◽  
J. Van Den Bergen ◽  
D. Miles ◽  
R. Ralli ◽  
A. Sinclair
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Transcriptional Regulation ◽  
Germ Cell ◽  
Germ Cells ◽  
Germ Line ◽  
Cell Lineage ◽  
Mitotic Arrest ◽  
Male Germ Cells ◽  
Developmental Potential

The germ cell lineage is unique in that it must ensure that the genome retains the complete developmental potential (totipotency) that supports development in the following generation. This is achieved through a number of mechanisms that prevent the early germ cell lineage from somatic differentiation and promote the capactity for functional totipotency. Part of this process involves the retained germ line expression of key genes that regulate pluripotency in embryonic stem cells, embryonic germ cells and some embryonal carcinoma cells, the stem cells of testicular tumours. Despite this, germ cells are not intrinsically pluripotent and must differentiate along the male or female pathways, a process which requires commitment of the bi-potential primordial germ cells to the spermatogenic (male) pathway and their entry into mitotic arrest, or to the oogenic pathway (females) and entry into meiosis. This involves robust regulation of regulatory networks controlling pluripotency, cell cycle and sex specific differentiation. Our work aims to further understand the mechanisms controlling differentiation, pluripotency and cell cycle in early male and female germ cells. Our data shows that mitotic arrest of male germ cells involves strict regulation of the G1-S phase check-point through the retinoblastoma protein. In addition, suppression of pluripotency in differentiating male germ cells involves post-transcriptional regulation of OCT4, transcriptional regulation of Sox2 and Nanog and methylation of the Sox2 and Nanog promoters. Further understanding of these processes promises to lead to a greater understanding of the molecular mechanisms underlying control of pluripotency, cell cycle and differentiation in the germ line and the initiation of germ cell derived testis tumours.

The germ line regulates somatic cyst cell proliferation and fate during Drosophila spermatogenesis

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2437-2447 ◽  
Author(s):  
P. Gonczy ◽  
S. DiNardo
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Cell Fate ◽  
Germ Cells ◽  
Germ Line ◽  
Lineage Tracing ◽  
Somatic Stem Cells ◽  
Daughter Cyst ◽  
Cyst Cell ◽  
Cyst Cells

Spermatogenesis relies on the function of germ-line stem cells, as a continuous supply of differentiated spermatids is produced throughout life. In Drosophila, there must also be somatic stem cells that produce the cyst cells that accompany germ cells throughout spermatogenesis. By lineage tracing, we demonstrate the existence of such somatic stem cells and confirm that of germ-line stem cells. The somatic stem cells likely correspond to the ultrastructurally described cyst progenitor cells. The stem cells for both the germ-line and cyst lineage are anchored around the hub of non-dividing somatic cells located at the testis tip. We then address whether germ cells regulate the behavior of somatic hub cells, cyst progenitors and their daughter cyst cells by analyzing cell proliferation and fate in testes in which the germ line has been genetically ablated. Daughter cyst cells, which normally withdraw from the cell cycle, continue to proliferate in the absence of germ cells. In addition, cells from the cyst lineage switch to the hub cell fate. Male-sterile alleles of chickadee and diaphanous, which are deficient in germ cells, exhibit similar cyst cell phenotypes. We conclude that signaling from germ cells regulates the proliferation and fate of cells in the somatic cyst lineage.

Germ cells of the mammalian female: A limited or renewable resource?

2021 ◽  
Author(s):  
Mathilde Hainaut ◽  
Hugh J Clarke
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Germ Cells ◽  
Germ Line ◽  
Marker Gene ◽  
Somatic Cells ◽  
Renewable Resource ◽  
Weight Of Evidence ◽  
Fetal Life

Abstract In many non-mammalian organisms, a population of germ-line stem cells supports continuing production of gametes during most or all the life of the individual, and germ-line stem cells are also present and functional in male mammals. Traditionally, however, they have been thought not to exist in female mammals, who instead generate all their germ cells during fetal life. Over the last several years, this dogma has been challenged by several reports, while supported by others. We describe and compare these conflicting studies with the aim of understanding how they came to opposing conclusions. We first consider studies that, by examining marker-gene expression, the fate of genetically marked cells, and consequences of depleting the oocyte population, addressed whether ovaries of post-natal females contain oogonial stem cells (OSC) that give rise to new oocytes. We next discuss whether ovaries contain cells that, even if inactive under physiological conditions, nonetheless possess OSC properties that can be revealed through cell-culture. We then examine studies of whether cells harvested after long-term culture of cells obtained from ovaries can, following transplantation into ovaries of recipient females, give rise to oocytes and offspring. Finally, we note studies where somatic cells have been re-programmed to acquire a female germ-cell fate. We conclude that the weight of evidence strongly supports the traditional interpretation that germ-line stem cells do not exist post-natally in female mammals. However, the ability to generate germ cells from somatic cells in vitro establishes a method to generate new gametes from cells of post-natal mammalian females.

scholarly journals Transcription factor AP2 controls cnidarian germ cell induction

Science ◽  
2020 ◽  
Vol 367 (6479) ◽  
pp. 757-762 ◽  
Author(s):  
Timothy Q. DuBuc ◽  
Christine E. Schnitzler ◽  
Eleni Chrysostomou ◽  
Emma T. McMahon ◽  
Febrimarsa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Germ Cell ◽  
Cell Fate ◽  
Germ Cells ◽  
Adult Stem Cells ◽  
Germ Line ◽  
Cell Induction ◽  
Cell Commitment ◽  
I Cells

Clonal animals do not sequester a germ line during embryogenesis. Instead, they have adult stem cells that contribute to somatic tissues or gametes. How germ fate is induced in these animals, and whether this process is related to bilaterian embryonic germline induction, is unknown. We show that transcription factor AP2 (Tfap2), a regulator of mammalian germ lines, acts to commit adult stem cells, known as i-cells, to the germ cell fate in the clonal cnidarian Hydractinia symbiolongicarpus. Tfap2 mutants lacked germ cells and gonads. Transplanted wild-type cells rescued gonad development but not germ cell induction in Tfap2 mutants. Forced expression of Tfap2 in i-cells converted them to germ cells. Therefore, Tfap2 is a regulator of germ cell commitment across germ line–sequestering and germ line–nonsequestering animals.

scholarly journals Cellular Analyses of the Mitotic Region in the Caenorhabditis elegans Adult Germ Line

2006 ◽  
Vol 17 (7) ◽  
pp. 3051-3061 ◽  
Author(s):  
Sarah L. Crittenden ◽  
Kimberly A. Leonhard ◽  
Dana T. Byrd ◽  
Judith Kimble
Keyword(s):  
Stem Cells ◽  
Caenorhabditis Elegans ◽  
Germ Cells ◽  
Germ Line ◽  
Germline Stem Cells ◽  
C Elegans ◽  
Brdu Label ◽  
Asymmetric Divisions ◽  
Brdu Labeling ◽  
Cell Niche

The Caenorhabditis elegans germ line provides a model for understanding how signaling from a stem cell niche promotes continued mitotic divisions at the expense of differentiation. Here we report cellular analyses designed to identify germline stem cells within the germline mitotic region of adult hermaphrodites. Our results support several conclusions. First, all germ cells within the mitotic region are actively cycling, as visualized by bromodeoxyuridine (BrdU) labeling. No quiescent cells were found. Second, germ cells in the mitotic region lose BrdU label uniformly, either by movement of labeled cells into the meiotic region or by dilution, probably due to replication. No label-retaining cells were found in the mitotic region. Third, the distal tip cell niche extends processes that nearly encircle adjacent germ cells, a phenomenon that is likely to anchor the distal-most germ cells within the niche. Fourth, germline mitoses are not oriented reproducibly, even within the immediate confines of the niche. We propose that germ cells in the distal-most rows of the mitotic region serve as stem cells and more proximal germ cells embark on the path to differentiation. We also propose that C. elegans adult germline stem cells are maintained by proximity to the niche rather than by programmed asymmetric divisions.

Establishment of mouse male germ line stem cells (GSCs) and in vitro differentiation to haploid germ cells

2003 ◽  
Vol 80 ◽  
pp. 1
Author(s):  
D.R. Lee ◽  
S.K. Kim ◽  
K.Y. Cha ◽  
Y.H. Yang ◽  
T.K. Yoon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cells ◽  
Germ Line ◽  
In Vitro Differentiation ◽  
Male Germ Line

scholarly journals Characterization of Alternative Splicing (AS) Events during Chicken (Gallus gallus) Male Germ-Line Stem Cell Differentiation with Single-Cell RNA-seq

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1469
Author(s):  
Changhua Sun ◽  
Kai Jin ◽  
Qisheng Zuo ◽  
Hongyan Sun ◽  
Jiuzhou Song ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Alternative Splicing ◽  
Cell Differentiation ◽  
Germ Cell ◽  
Germ Cells ◽  
Germ Line ◽  
Rna Seq ◽  
Germ Cell Differentiation ◽  
Genome Wide

Alternative splicing (AS) is a ubiquitous, co-transcriptional, and post-transcriptional regulation mechanism during certain developmental processes, such as germ cell differentiation. A thorough understanding of germ cell differentiation will help us to open new avenues for avian reproduction, stem cell biology, and advances in medicines for human consumption. Here, based on single-cell RNA-seq, we characterized genome-wide AS events in manifold chicken male germ cells: embryonic stem cells (ESCs), gonad primordial germ cells (gPGCs), and spermatogonia stem cells (SSCs). A total of 38,494 AS events from 15,338 genes were detected in ESCs, with a total of 48,955 events from 14,783 genes and 49,900 events from 15,089 genes observed in gPGCs and SSCs, respectively. Moreover, this distribution of AS events suggests the diverse splicing feature of ESCs, gPGCs, and SSCs. Finally, several crucial stage-specific genes, such as NANOG, POU5F3, LIN28B, BMP4, STRA8, and LHX9, were identified in AS events that were transmitted in ESCs, gPGCs, and SSCs. The gene expression results of the RNA-seq data were validated by qRT-PCR. In summary, we provided a comprehensive atlas of the genome-wide scale of the AS event landscape in male chicken germ-line cells and presented its distribution for the first time. This research may someday improve treatment options for men suffering from male infertility.

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