Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis

Bastos, Henri; Lassalle, Bruno; Chicheportiche, Alexandra; Riou, Lydia; Testart, J; Allemand, Isabelle; Fouchet, Pierre

doi:10.1002/cyto.a.20129

Cited by 178 publications

(238 citation statements)

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“…This strategy enables the purification of (i) the side population (SP), which contains the spermatogonia and is highly enriched in germinal stem cells, and (ii) the more differentiated cell populations. 11,12 Caspase-9 is one of the effector caspases of the intrinsic pathway and in mice, its gene codes for two splice variants, proapoptotic caspase-9L mRNA and antiapoptotic caspase-9S mRNA. 13 Using primers encompassing the alternative splice site, caspase-9L mRNA was the only variant detected in testicular cells (Figure 2a).…”

Section: Caspase-3 Rnas Are Increased In Mtp53 Testicular Cell Subpopmentioning

confidence: 99%

Caspase-independent death of meiotic and postmeiotic cells overexpressing p53: calpain involvement

et al. 2006

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In a model of male sterility (MTp53) owing to enforced p53 expression in spermatocytes II and spermatids of transgenic mice, we focused on the role of caspases. Most of them are expressed in all differentiation stages, but only the transcriptional levels of caspase-2 and caspase-3 are modified in MTp53 germ cells. In normal testis, cleaved caspase-3 and caspase-9 are detected during the elongation of spermatids. Despite this constitutive presence of caspases during terminal differentiation, calpains are the main effectors of germ cell loss in MTp53 testes: calpain 1 RNA levels are increased, caspase-3-like activity is markedly decreased while calpain activity is higher and the calpain inhibitor E64d ((2S, 3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester) reduces TUNEL labeling in MTp53 testis, whereas pancaspase inhibitor zVADfmk (N-benzyloxycarbonyl-ValAla-Asp(OMe)-fluoromethylketone) has no effect. Our work suggests that despite the presence, and potent involvement, of caspases in male haploid cell maturation, calpains are the executioners of the death of terminally differentiating germ cells.

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Section: Caspase-3 Rnas Are Increased In Mtp53 Testicular Cell Subpopmentioning

confidence: 99%

Caspase-independent death of meiotic and postmeiotic cells overexpressing p53: calpain involvement

et al. 2006

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“…The Hoechst 33342 fluorescence of DNA in these cells depends on ploidy, accessibility of chromatin and Hoechst efflux out of the cells due to ATP-binding cassette transporter activity. As previously reported [9,10], the 'Side Population' phenotype of the male germ cells is based on efflux of Hoechst 33342 out of the cells determined by the expression of ABC transporter BCRP1. Given that there is no saturation of the Hoechst dye in spermatogonia, DNA in these cells is poorly stained and their ploidy cannot therefore be established [9,10].…”

Section: Resultsmentioning

confidence: 78%

“…As previously reported [9,10], the 'Side Population' phenotype of the male germ cells is based on efflux of Hoechst 33342 out of the cells determined by the expression of ABC transporter BCRP1. Given that there is no saturation of the Hoechst dye in spermatogonia, DNA in these cells is poorly stained and their ploidy cannot therefore be established [9,10]. 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.…”

Section: Resultsmentioning

confidence: 78%

“…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.…”

Section: Resultsmentioning

confidence: 86%

“…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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Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis

Cited by 178 publications

References 28 publications

Caspase-independent death of meiotic and postmeiotic cells overexpressing p53: calpain involvement

Caspase-independent death of meiotic and postmeiotic cells overexpressing p53: calpain involvement

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

Flow Cytometry of Murine Spermatocytes

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