The differentiation of mouse spermatids is one critical process for the production of a functiona... more The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.
A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ... more A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ultraviolet damage endonuclease (UVDE) generates 3'-OH ends at the two major UV induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone dimers (6,4 PPs), allowing for their capture after biotin end-labeling. qPCR amplification of biotinylated DNA enables parallel measuring of DNA damage in several loci, which can then be combined with high-throughput screening of cell survival to test genotoxic reagents. Alternatively, a library of captured sequences could be generated for a genome wide study of damage sites and large-scale assessment of repair kinetics in different regions of the genome, using next-generation sequencing. The assay is suitable to study any DNA lesion that can be converted into 3'-OH by UVDE, or other enzymes. Toward these goals, we compared UVDE with the classical T4 endonuclease V (T4V) assay. We showed that there is a linear correlation between UV dose, 3'-OH formation and capture by immunoprecipitation, together with its potential application for in vivo studies.
Transient DNA breaks and evidence of DNA damage response have recently been reported during the c... more Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.
... as one could have expected, the perturbation of the poly(ADP-ribose) metabolism clearly impac... more ... as one could have expected, the perturbation of the poly(ADP-ribose) metabolism clearly impacts the differen ... as seen in untreated controls and reported by some studies monitoring the effects of some ... Impact of This Transient Window of Genetic Instability on Clinical Practices ...
This paper reviews the possible origin of sperm DNA fragmentation and focuses on the nuclear even... more This paper reviews the possible origin of sperm DNA fragmentation and focuses on the nuclear events associated with spermiogenesis as a potential source of genetic instability and reduced fertilizing potential of the mature male gamete. Recent findings suggest a programmed DNA fragmentation and DNA damage response during the chromatin remodeling steps in spermatids. We also discuss the spermatid DNA repair mechanisms and the possible involvement of condensing proteins, such as transition proteins and protamines, in the process, as this DNA fragmentation is normally not found in late spermatids. We propose that alterations in the chromatin remodeling steps or DNA repair in elongating spermatids may lead to persistent DNA breaks. This vulnerable step of spermiogenesis may provide a clue to the etiology of sperm DNA fragmentation associated with infertility in humans. This vulnerability is further emphasized given the haploid character of spermatids that must resolve programmed double-stranded breaks by an error-prone DNA repair mechanism. Therefore, spermiogenesis has probably been overlooked as an important source of genetic instability.
Determination of cellular DNA damage has so far been limited to global assessment of genome integ... more Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.
At the sequence level, genetic diversity is provided by de novo transmittable mutations that may ... more At the sequence level, genetic diversity is provided by de novo transmittable mutations that may act as a substrate for natural selection. The gametogenesis process itself is considered more likely to induce endogenous mutations and a clear male bias has been demonstrated from recent next-generation sequencing analyses. As new experimental evidence accumulates, the post-meiotic events of the male gametogenesis (spermiogenesis) appear as an ideal context to induce de novo genetic polymorphism transmittable to the next generation. It may prove to be a major component of the observed male mutation bias. As spermatids undergo chromatin remodeling, transient endogenous DNA double-stranded breaks are produced and trigger a DNA damage response. In these haploid cells, one would expect that the non-templated, DNA endjoining repair processes may generate a repertoire of sequence alterations in every sperm cell potentially transmittable to the next generation. This may therefore represent a novel physiological mechanism contributing to genetic diversity and evolution.
Wood preserved with chromated copper arsenate (CCA) and alkaline copper quaternary (ACQ) was mixe... more Wood preserved with chromated copper arsenate (CCA) and alkaline copper quaternary (ACQ) was mixed with artificial rainwater, to generate leachates containing As, Cr and Cu. Then, leachates were applied to two soils at rates of 13-169 mg As kg(-1) soil (dry weight basis), 12-151 mg Cr kg(-1) and 10-216 mg Cu kg(-1). Metal bioavailability was evaluated after 28 days using the earthworm Eisenia fetida (Savigny). Metal concentrations in earthworm tissue ranged from negligible to 80 mg As kg(-1) (dry weight basis), 89 mg Cr kg(-1) and 90 mg Cu kg(-1), which appeared to be non-lethal to E. fetida. There was less Cu available to earthworms in the Courval soil (pH 7.8) than the Châteauguay soil (pH 6.8), but earthworm growth and reproduction were not affected by exposure to Cu from ACQ-treated wood. In contrast, earthworms exposed to As, Cr and Cu from the CCA-treated wood gained weight more quickly in the Courval soil (1.3-21 mg g(-1) initial biomass days) than in the Châteauguay soil (0.2-7.8 mg g(-1) day(-1)), but fewer than 20% of the cocoons deposited by the faster-growing earthworms hatched by the end of the 56 days ecotoxicology test. It appeared that E. fetida can allocate more energy to growth than reproduction, delaying cocoon development and hatching in some situations. Further information is needed on the soil factors that may induce such behavior, as it can affect the interpretation of results from the earthworm ecotoxicology test.
A precise packaging of the paternal genome during spermiogenesis is essential for fertilization a... more A precise packaging of the paternal genome during spermiogenesis is essential for fertilization and embryogenesis. Most of the nucleosomal DNA supercoiling must be eliminated in elongating spermatids (ES), and transient DNA strand breaks are observed that facilitate the process. Topoisomerases have been considered as ideal candidates for the removal of DNA supercoiling, but their catalytic activity, in the context of such a major chromatin remodeling, entails genetic risks. Using immunofluorescence, we confirmed that topoisomerase II beta (TOP2B) is the type II topoisomerase present in ES between steps 9 and 13. Interestingly, the detection of TOP2B was found coincident with detection of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme known to resolve topoisomerase-mediated DNA damage. The presence of gamma-H2AX (also known as H2AFX) coincident with DNA strand breakage was also confirmed at these steps and indicates that a DNA damage response is triggered. Active DNA repair in ES was demonstrated using a fluorescent in situ DNA polymerase activity assay on squash preparations of staged tubules. In the context of haploid spermatids, any unresolved double-strand breaks, resulting from a failure in the rejoining process of TOP2B, must likely rely on the error-prone nonhomologous end joining, because homologous recombination cannot proceed in the absence of a sister chromatid. Because this process is part of the normal developmental program of the spermatids, dramatic consequences for the genomic integrity of the developing male gamete may arise should any alteration in the process occur.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2009
The observation that PrP is present in the cytosol of some neurons and non-neuronal cells and tha... more The observation that PrP is present in the cytosol of some neurons and non-neuronal cells and that the Nterminal signal peptide is slightly inefficient has brought speculations concerning a possible function of the protein in the cytosol. Here, we show that cells expressing a cytosolic form of PrP termed cyPrP display a large juxtanuclear cytoplasmic RNA organelle. Although cyPrP spontaneously forms aggresomes, we used several mutants to demonstrate that the assembly of this RNA organelle is independent from cyPrP aggregation. Components of the organelle fall into three classes: mRNAs; proteins, including the RNAseIII family polymerase Dicer, the decapping enzyme Dcp1a, the DEAD-box RNA helicase DDX6, and the small nuclear ribonucleoprotein-associated proteins SmB/B′/N; and non-coding RNAs, including rRNA 5S, tRNAs, U1 small nuclear RNA, and microRNAs. This composition is similar to RNA granules or chromatoid bodies from germ cells, or planarian stem cells and neurons, which are large ribonucleoprotein complexes predicted to function in RNA processing and posttranscriptional gene regulation. The domain of PrP encompassing residues 30 to 49 is essential for the formation of the RNA particle. Our findings confirm the intriguing relation between PrP and RNA in cells, and underscore an unexpected function for cytosolic PrP: assembling a large RNA processing center which we have termed PrP-RNP for PrP-induced ribonucleoprotein particle.
The differentiation of mouse spermatids is one critical process for the production of a functiona... more The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.
A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ... more A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ultraviolet damage endonuclease (UVDE) generates 3'-OH ends at the two major UV induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone dimers (6,4 PPs), allowing for their capture after biotin end-labeling. qPCR amplification of biotinylated DNA enables parallel measuring of DNA damage in several loci, which can then be combined with high-throughput screening of cell survival to test genotoxic reagents. Alternatively, a library of captured sequences could be generated for a genome wide study of damage sites and large-scale assessment of repair kinetics in different regions of the genome, using next-generation sequencing. The assay is suitable to study any DNA lesion that can be converted into 3'-OH by UVDE, or other enzymes. Toward these goals, we compared UVDE with the classical T4 endonuclease V (T4V) assay. We showed that there is a linear correlation between UV dose, 3'-OH formation and capture by immunoprecipitation, together with its potential application for in vivo studies.
Transient DNA breaks and evidence of DNA damage response have recently been reported during the c... more Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.
... as one could have expected, the perturbation of the poly(ADP-ribose) metabolism clearly impac... more ... as one could have expected, the perturbation of the poly(ADP-ribose) metabolism clearly impacts the differen ... as seen in untreated controls and reported by some studies monitoring the effects of some ... Impact of This Transient Window of Genetic Instability on Clinical Practices ...
This paper reviews the possible origin of sperm DNA fragmentation and focuses on the nuclear even... more This paper reviews the possible origin of sperm DNA fragmentation and focuses on the nuclear events associated with spermiogenesis as a potential source of genetic instability and reduced fertilizing potential of the mature male gamete. Recent findings suggest a programmed DNA fragmentation and DNA damage response during the chromatin remodeling steps in spermatids. We also discuss the spermatid DNA repair mechanisms and the possible involvement of condensing proteins, such as transition proteins and protamines, in the process, as this DNA fragmentation is normally not found in late spermatids. We propose that alterations in the chromatin remodeling steps or DNA repair in elongating spermatids may lead to persistent DNA breaks. This vulnerable step of spermiogenesis may provide a clue to the etiology of sperm DNA fragmentation associated with infertility in humans. This vulnerability is further emphasized given the haploid character of spermatids that must resolve programmed double-stranded breaks by an error-prone DNA repair mechanism. Therefore, spermiogenesis has probably been overlooked as an important source of genetic instability.
Determination of cellular DNA damage has so far been limited to global assessment of genome integ... more Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.
At the sequence level, genetic diversity is provided by de novo transmittable mutations that may ... more At the sequence level, genetic diversity is provided by de novo transmittable mutations that may act as a substrate for natural selection. The gametogenesis process itself is considered more likely to induce endogenous mutations and a clear male bias has been demonstrated from recent next-generation sequencing analyses. As new experimental evidence accumulates, the post-meiotic events of the male gametogenesis (spermiogenesis) appear as an ideal context to induce de novo genetic polymorphism transmittable to the next generation. It may prove to be a major component of the observed male mutation bias. As spermatids undergo chromatin remodeling, transient endogenous DNA double-stranded breaks are produced and trigger a DNA damage response. In these haploid cells, one would expect that the non-templated, DNA endjoining repair processes may generate a repertoire of sequence alterations in every sperm cell potentially transmittable to the next generation. This may therefore represent a novel physiological mechanism contributing to genetic diversity and evolution.
Wood preserved with chromated copper arsenate (CCA) and alkaline copper quaternary (ACQ) was mixe... more Wood preserved with chromated copper arsenate (CCA) and alkaline copper quaternary (ACQ) was mixed with artificial rainwater, to generate leachates containing As, Cr and Cu. Then, leachates were applied to two soils at rates of 13-169 mg As kg(-1) soil (dry weight basis), 12-151 mg Cr kg(-1) and 10-216 mg Cu kg(-1). Metal bioavailability was evaluated after 28 days using the earthworm Eisenia fetida (Savigny). Metal concentrations in earthworm tissue ranged from negligible to 80 mg As kg(-1) (dry weight basis), 89 mg Cr kg(-1) and 90 mg Cu kg(-1), which appeared to be non-lethal to E. fetida. There was less Cu available to earthworms in the Courval soil (pH 7.8) than the Châteauguay soil (pH 6.8), but earthworm growth and reproduction were not affected by exposure to Cu from ACQ-treated wood. In contrast, earthworms exposed to As, Cr and Cu from the CCA-treated wood gained weight more quickly in the Courval soil (1.3-21 mg g(-1) initial biomass days) than in the Châteauguay soil (0.2-7.8 mg g(-1) day(-1)), but fewer than 20% of the cocoons deposited by the faster-growing earthworms hatched by the end of the 56 days ecotoxicology test. It appeared that E. fetida can allocate more energy to growth than reproduction, delaying cocoon development and hatching in some situations. Further information is needed on the soil factors that may induce such behavior, as it can affect the interpretation of results from the earthworm ecotoxicology test.
A precise packaging of the paternal genome during spermiogenesis is essential for fertilization a... more A precise packaging of the paternal genome during spermiogenesis is essential for fertilization and embryogenesis. Most of the nucleosomal DNA supercoiling must be eliminated in elongating spermatids (ES), and transient DNA strand breaks are observed that facilitate the process. Topoisomerases have been considered as ideal candidates for the removal of DNA supercoiling, but their catalytic activity, in the context of such a major chromatin remodeling, entails genetic risks. Using immunofluorescence, we confirmed that topoisomerase II beta (TOP2B) is the type II topoisomerase present in ES between steps 9 and 13. Interestingly, the detection of TOP2B was found coincident with detection of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme known to resolve topoisomerase-mediated DNA damage. The presence of gamma-H2AX (also known as H2AFX) coincident with DNA strand breakage was also confirmed at these steps and indicates that a DNA damage response is triggered. Active DNA repair in ES was demonstrated using a fluorescent in situ DNA polymerase activity assay on squash preparations of staged tubules. In the context of haploid spermatids, any unresolved double-strand breaks, resulting from a failure in the rejoining process of TOP2B, must likely rely on the error-prone nonhomologous end joining, because homologous recombination cannot proceed in the absence of a sister chromatid. Because this process is part of the normal developmental program of the spermatids, dramatic consequences for the genomic integrity of the developing male gamete may arise should any alteration in the process occur.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2009
The observation that PrP is present in the cytosol of some neurons and non-neuronal cells and tha... more The observation that PrP is present in the cytosol of some neurons and non-neuronal cells and that the Nterminal signal peptide is slightly inefficient has brought speculations concerning a possible function of the protein in the cytosol. Here, we show that cells expressing a cytosolic form of PrP termed cyPrP display a large juxtanuclear cytoplasmic RNA organelle. Although cyPrP spontaneously forms aggresomes, we used several mutants to demonstrate that the assembly of this RNA organelle is independent from cyPrP aggregation. Components of the organelle fall into three classes: mRNAs; proteins, including the RNAseIII family polymerase Dicer, the decapping enzyme Dcp1a, the DEAD-box RNA helicase DDX6, and the small nuclear ribonucleoprotein-associated proteins SmB/B′/N; and non-coding RNAs, including rRNA 5S, tRNAs, U1 small nuclear RNA, and microRNAs. This composition is similar to RNA granules or chromatoid bodies from germ cells, or planarian stem cells and neurons, which are large ribonucleoprotein complexes predicted to function in RNA processing and posttranscriptional gene regulation. The domain of PrP encompassing residues 30 to 49 is essential for the formation of the RNA particle. Our findings confirm the intriguing relation between PrP and RNA in cells, and underscore an unexpected function for cytosolic PrP: assembling a large RNA processing center which we have termed PrP-RNP for PrP-induced ribonucleoprotein particle.
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Papers by Frederic Leduc