`Side Population' cells in adult mouse testis express<i>Bcrp1</i>gene and are enriched in spermatogonia and germinal stem cells

Lassalle, Bruno; Bastos, Henri; Louis, J. P.; Riou, Lydia; Testart, J; Dutrillaux, Bernard; Fouchet, Pierre; Allemand, Isabelle

doi:10.1242/dev.00918

Cited by 144 publications

(189 citation statements)

References 46 publications

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“…In the mouse testis, putative stem cells have been selected on the basis of expression of c-Kit and the β1 and α6 integrins [134]. The expression of the transcription factors Oct-4 and Plfz has also been used to define murine testicular stem cells [135], and an SP population (dependent on ABCG2 activity) was found highly-enriched for in vivo cell repopulation ability, with these cells expressing α6 integrin and Stra8, a premeiotic germ cell-specific cytoplasmic protein [136]. In man, as in many primates, there are two types of A spermatogonia; A dark , likely to be facultative stem cells, and A pale , being progenitor cells [137].…”

Section: Male Gonadal and Accessory Sex Tissuementioning

confidence: 99%

Attributes of adult stem cells

Alison

Islam

2008

The Journal of Pathology

153

115

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While cultured embryonic stem (ES) cells can be harvested in abundance and appear to be the most versatile of cells for regenerative medicine, adult stem cells also hold promise, but the identity and subsequent isolation of these comparatively rare cells remains problematic in most tissues, perhaps with the notable exception of the bone marrow. The ability to continuously self-renew and produce the differentiated progeny of the tissue of their location are their defining properties. Identifying surface molecules (markers) that would aid in stem cell isolation is a major goal. Considerable overlap exists between different putative organspecific stem cells in their repertoire of gene expression, often related to self-renewal, cell survival and cell adhesion. More robust tests of 'stemness' are now being employed, using lineage-specific genetic marking and tracking to show production of long-lived clones and multipotentiality in vivo. Moreover, the characterization of normal stem cells in specific tissues may provide a dividend for the treatment of cancer. The successful treatment of neoplastic disease may well require the specific targeting of neoplastic stem cells, cells that may well have many of the characteristics of their normal counterparts.

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Section: Male Gonadal and Accessory Sex Tissuementioning

confidence: 99%

Attributes of adult stem cells

Alison

Islam

2008

The Journal of Pathology

153

115

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“…As a result, in this protocol the fraction of spermatogonia found in the 'Side Population' do not co-localise with the 4N DNA content plot. In contrast, as spermatocytes and spermatids do not exhibit an efflux of Hoechst, the concentration of Hoechst inside those cells is saturated [9,10] and they can effectively be separated by the technique used in our study. Using SM-PCR, the frequency of ESTR mutation at the Ms6-hm locus was evaluated in sorted spermatogonia, meiotic spermatocytes I as well as post-meiotic round and elongated spermatids.…”

mentioning

confidence: 85%

Section: Flow Cytometrymentioning

confidence: 99%

“…Cells were isolated from 3 month-old male mice using a two-step enzymatic digestion as previously described [9]. Magnetic-activated cell sorting (MACS) of α 6 -integrin positive cells was performed as we previously described [9] using rat anti-α 6 -integrin antibodies (GoH3, BD Biosciences) and mouse anti-rat Kappa antibody coupled to magnetic microbeads (Miltenyi Biotec, Paris, France). Testicular cell suspension, MACS α 6 -integrin-positive and negative cellular fractions were collected and suspended at 10 6 cells/ml for DNA staining with Hoechst 33342 (5 µg/ml, 1 hour at 32° C), and stained with propidium iodide (2 µg/ml) to exclude dead cells.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…Various fractions of germ cells were sorted with a purity of at least 97% on a dual-laser FACStarPlus flow cytometer (Becton Dickinson, Le Pont de Claix, France). Spermatogonia (SG) were sorted on MACS α 6 -integrin-positive cells fraction according to their ability to efflux the Hoechst 33342 (the 'Side Population' phenotype), while meiotic spermatocytes I (SC) and round spermatids (RS) were sorted from the α 6 -integrin-negative cells according to their DNA content [9]. Elongated spermatids (ES) were isolated according to forward scatter and their haploid DNA content on the testicular single-cell suspensions [10,11].…”

Section: Flow Cytometrymentioning

confidence: 99%

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Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline

Shanks¹,

Riou²,

Fouchet³

et al. 2008

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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Mouse expanded simple tandem repeat (ESTR) loci are the most unstable loci in the mouse genome. Despite the fact that over the last decade these loci have been extensively used for studying germline mutation induction in mice, to date little is known about the mechanisms underlying spontaneous and induced ESTR mutation. Here we used flow cytometry and single-molecule PCR to compare the frequency of ESTR mutation in four flow-sorted fractions of the mouse male germ cells -spermatogonia, spermatocytes I, round and elongated spermatids. The frequency and the spectrum of ESTR mutation did not significantly differ between different stages of mouse spermatogenesis. Considering these data and the results of other publications, we propose that spontaneous ESTR mutation is mostly attributed to replication slippage in spermatogonia and these loci may be regarded as a class of expanded microsatellites. IntroductionThe results of some recent publication show that mouse ESTR loci provide a sensitive technique for studying germline mutation induction following exposure to ionising radiation and chemical mutagens [1,2]. ESTR loci consist of long homogenous arrays of relatively short repeats (4-9 bp) and show a very high spontaneous mutation rate of length changes both in germline and somatic cells [3][4][5]. In this respect they clearly differ from another group of highly unstable tandem repeat DNA loci -the GC-rich human minisatellites, where the bulk of spontaneous mutations occur in the germline with very rare events taking place in the somatic cells [6]. Given this, ESTRs more closely resemble some microsatellite loci showing equally high instability in the germline and somatic tissues. In many publications it has been suggested, though not experimentally proven, that microsatellite mutation is most probably attributed to replication slippage [7]. However, numerous studies have also identified a unique class of the expanded disease-causing human microsatellites i.e., trinucleotide instability, which is clearly attributed to non-replication mechanisms [8]. It therefore remains unclear whether the very high spontaneous instability at ESTR loci is replication-driven or may be attributed to some other mechanisms. Here, to elucidate the still unknown mechanisms of ESTR spontaneous mutation, we have compared ESTR mutation frequencies in flow-sorted fractions of the male germ cells. Materials and methods Flow cytometryCBA/J male mice (Iffa Credo, Charles River, France) were used in this study. Cells were isolated from 3 month-old male mice using a two-step enzymatic digestion as previously described [9]. Magnetic-activated cell sorting (MACS) of α 6 -integrin positive cells was performed as we previously described [9] using rat anti-α 6 -integrin antibodies (GoH3, BD Biosciences) and mouse anti-rat Kappa antibody coupled to magnetic microbeads (Miltenyi Biotec, Paris, France). Testicular cell suspension, MACS α 6 -integrin-positive and negative cellular fractions were collected and suspended at 10 6 cells/ml for DNA stain...

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Flow Cytometry of Murine Spermatocytes

Gaysinskaya

Bortvin

2015

CP Cytometry

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Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument parameters, data display, and selection strategies. In addition, troubleshooting of flow cytometry experiments usually requires some fluency in technical principles and instrument specifications and settings. This unit describes setup and procedures for analysis and sorting of male meiotic prophase I (MPI) cells and other germ cells. Included are procedures that guide data acquisition, display, gating, and back‐gating critical for optimal data visualization and cell sorting. Additionally, a flow cytometry analysis of spermatogenesis‐defective testis is provided to illustrate the applicability of the technique to the characterization and purification of cells from mutant testis. © 2015 by John Wiley & Sons, Inc.

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`Side Population' cells in adult mouse testis expressBcrp1gene and are enriched in spermatogonia and germinal stem cells

Cited by 144 publications

References 46 publications

Attributes of adult stem cells

Attributes of adult stem cells

Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline

Flow Cytometry of Murine Spermatocytes

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