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Corrado Spadafora - One of the best experts on this subject based on the ideXlab platform.
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MINI-REVIEW: DEVELOPMENTS IN REPRODUCTIVE BIOLOGY AND MEDICINE Sperm-mediated ‘reverse ’ gene transfer: a role of reverse transcriptase in the generation of new genetic information
2015Co-Authors: Corrado SpadaforaAbstract:Sperm-mediated gene transfer (SMGT) is a procedure through which new genetic traits are introduced in animals by exploiting the ability of spermatozoa to take up Exogenous DNA molecules and deliver them to oocytes at fertilization. The interaction of Exogenous DNA with sperm cells is a regulated process mediated by specific factors; among those, a reverse transcriptase (RT) activity plays a central role in SMGT. ‘Retro-genes ’ are generated either through reverse transcription of Exogenous RNA internalized in spermatozoa, or through sequential transcription, splicing and reverse transcription of Exogenous DNA. The resulting retro-genes are delivered to oocytes and transmitted to embryos and born animals as low-copy, transcriptionally competent, extrachromosomal structures capable of deter-mining new phenotypic traits. Retro-genes can be further transmitted through sexual reproduction from founders to their F1 progeny: new genetic and phenotypic features, unlinked to chromosomes, can thus be generated and inherited in a non-Mendelian ratio. We have called this phenomenon sperm-mediated ‘reverse ’ gene transfer (SMRGT). Thus, a RT-mediated machinery operates in sperm cells and is responsible for the genesis and non-Mendelian propagation of new genetic information. The features of RT-generated traits elicited in SMRGT resemble those characterized in recent studies of RNA-mediated inheritance of extra-genomic information
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activation of endogenous nucleases in mature sperm cells upon interaction with Exogenous DNA
DNA and Cell Biology, 1997Co-Authors: B Maione, C Pittoggi, L Achene, Rodolfo Lorenzini, Corrado SpadaforaAbstract:Mature sperm cells, either of epididymal origin or ejaculated and depleted of seminal fluid, are spontaneously able to bind Exogenous DNA molecules which are subsequently internalized into sperm nuclei. Southern blot analysis showed that the internalized DNA was specifically cleaved by sperm endonucleases and showed typical fragmentation patterns of localized hypersensitivity. Nucleases were activated in response to the internalization of Exogenous DNA by sperm cells and their activity increased with the DNA concentration. Nuclease activation was efficient in epididymal sperm cells, while being drastically reduced in ejaculated washed spermatozoa. Nucleases were Ca++ dependent, and were, respectively, inhibited and activated by preincubating sperm cells with Aurintricarboxylic Acid (ATA) and Ca++ Ionophore A23187, which are known to, respectively, inhibit and activate apoptosis in somatic cells. Moreover, nuclease activation also caused a partial degradation of the sperm endogenous chromosomal DNA; cleave...
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the interaction of sperm cells with Exogenous DNA a role of cd4 and major histocompatibility complex class ii molecules
Experimental Cell Research, 1997Co-Authors: Marialuisa Lavitrano, Maura Francolini, Barbara Maione, E Forte, S Sperandio, R Testi, Corrado SpadaforaAbstract:Mouse epidydimal sperm cells have the spontaneous ability to take up Exogenous DNA, a part of which is further internalized into nuclei. We report here that sperm cells from MHC class II knockout mice have a reduced ability to bind DNA compared to sperm cells from wild-type animals. Spermatozoa from CD4 knockout mice are instead fully capable of binding Exogenous DNA, yet lose the ability to further internalize it. MHC class II expression was not detected on sperm heads using monoclonal antibodies. In contrast, CD4 molecules were found on sperm heads by both immunofluorescence and Western blot analysis. Moreover, we show that nuclear internalization of Exogenous DNA was prevented in wild-type sperm cells preincubated with anti-CD4 mAbs. These results support the conclusion that CD4 and MHC class II molecules play distinct roles in the process of sperm/DNA interaction: though not present in mature sperm cells, MHC class II expression appears to be required during spermatogenesis to produce sperm cells capable of taking up foreign DNA, while CD4 molecules present on sperm cells mediate the nuclear internalization of sperm-bound DNA.
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the mechanism of binding of Exogenous DNA to sperm cells factors controlling the DNA uptake
Experimental Cell Research, 1995Co-Authors: Massimo Zani, Marialuisa Lavitrano, Deborah French, Valentina Lulli, Barbara Maione, Sabina Sperandio, Corrado SpadaforaAbstract:Abstract Mature sperm cells have the spontaneous capability of taking up Exogenous DNA. Potential substrates for the interaction of the DNA with the sperm heads are specific classes of DNA-binding proteins. In the present work three major classes of DNA-binding proteins were identified by Southwestern analysis of sperm head protein extracts: a first class of about 50 kDa in molecular weight, a second one of 30-35 kDa, and finally a third one below 20 kDa. The latter group most probably contains sperm protamines. Our attention was particularly focused on the 30- to 35-kDa proteins as a substrate for DNA binding, as they represented the only group whose electrophoretic mobility was conserved among mammalian species. In addition they were the only class of DNA-binding proteins accessible to Exogenous DNA in intact sperm cells. The purified 30- to 35-kDa proteins interacted in vitro with Exogenous DNA and generated discrete protein/DNA complexes as determined by band shift assay. A factor blocking the binding of Exogenous DNA to sperm cells was also identified in the seminal fluid of mammals and in echinoid spermatozoa. The factor also exerted a powerful inhibitory effect on DNA uptake in sperm cells of heterologous species. The 30- to 35-kDa DNA-binding proteins appeared to be the specific target through which the inhibition was mediated. In the presence of the inhibitory factor, the 30- to 35-kDa lost the ability to bind Exogenous DNA. Thus, the interaction of Exogenous DNA with sperm cells does not appear to be a casual event but, on the contrary, relies on a molecular mechanism based on the cooperation of specific protein factors.
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evidence for nuclear internalization of Exogenous DNA into mammalian sperm cells
Molecular Reproduction and Development, 1993Co-Authors: Maura Francolini, Marialuisa Lavitrano, Carla Lora Lamia, Deborah French, Luigi Frati, Franco Cotelli, Corrado SpadaforaAbstract:Mature sperm cells have the spontaneous capacity to take up Exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to Exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the Exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.
Marialuisa Lavitrano - One of the best experts on this subject based on the ideXlab platform.
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sperm mediated gene transfer in pig selection of donor boars and optimization of DNA uptake
Molecular Reproduction and Development, 2003Co-Authors: Marialuisa Lavitrano, Monica Forni, Maria Laura Bacci, Carla Di Stefano, Vincenzo Varzi, Hongjun Wang, E SerenAbstract:Transgenic animals are produced primarily by microinjecting Exogenous DNA into the male pronuclei of a zygote. Microinjection is successful in mice but not efficient in farm animals, limiting its general utility. We have pursued an alternative technology for producing transgenic animals: Sperm Mediated Gene Transfer (SMGT). Based on our finding that sperm cells bind and internalize Exogenous DNA, we used sperm as a vector for transmitting, not only their own DNA, but also, the Exogenously-introduced gene of interest to the zygote. SMGT is highly efficient (up to greater than 80%) and relatively inexpensive; it can be used in species refractory to microinjection, whenever reproduction is mediated by gametes. In this report, we describe the procedure for selection of sperm donors and optimization of DNA uptake that are the key steps for the successful outcome of SMGT. We found that the nominal parameters that boar sperm should possess to serve as a good vector for Exogenous DNA are the quality of semen based on standard parameters used in conventional animal breeding programs (volume, concentration, presence of abnormal sperm cells, motility at time of collection, and high progressive motility after 2 hr) and the ability of the sperm cells to take up and internalize Exogenous DNA. The results described provide significant advances in SMGT technology applied to pigs, so that transgenic pigs can be efficiently obtained. Mol. Reprod. Dev. 64: 284–291, 2003. © 2003 Wiley-Liss, Inc.
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the interaction of sperm cells with Exogenous DNA a role of cd4 and major histocompatibility complex class ii molecules
Experimental Cell Research, 1997Co-Authors: Marialuisa Lavitrano, Maura Francolini, Barbara Maione, E Forte, S Sperandio, R Testi, Corrado SpadaforaAbstract:Mouse epidydimal sperm cells have the spontaneous ability to take up Exogenous DNA, a part of which is further internalized into nuclei. We report here that sperm cells from MHC class II knockout mice have a reduced ability to bind DNA compared to sperm cells from wild-type animals. Spermatozoa from CD4 knockout mice are instead fully capable of binding Exogenous DNA, yet lose the ability to further internalize it. MHC class II expression was not detected on sperm heads using monoclonal antibodies. In contrast, CD4 molecules were found on sperm heads by both immunofluorescence and Western blot analysis. Moreover, we show that nuclear internalization of Exogenous DNA was prevented in wild-type sperm cells preincubated with anti-CD4 mAbs. These results support the conclusion that CD4 and MHC class II molecules play distinct roles in the process of sperm/DNA interaction: though not present in mature sperm cells, MHC class II expression appears to be required during spermatogenesis to produce sperm cells capable of taking up foreign DNA, while CD4 molecules present on sperm cells mediate the nuclear internalization of sperm-bound DNA.
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the mechanism of binding of Exogenous DNA to sperm cells factors controlling the DNA uptake
Experimental Cell Research, 1995Co-Authors: Massimo Zani, Marialuisa Lavitrano, Deborah French, Valentina Lulli, Barbara Maione, Sabina Sperandio, Corrado SpadaforaAbstract:Abstract Mature sperm cells have the spontaneous capability of taking up Exogenous DNA. Potential substrates for the interaction of the DNA with the sperm heads are specific classes of DNA-binding proteins. In the present work three major classes of DNA-binding proteins were identified by Southwestern analysis of sperm head protein extracts: a first class of about 50 kDa in molecular weight, a second one of 30-35 kDa, and finally a third one below 20 kDa. The latter group most probably contains sperm protamines. Our attention was particularly focused on the 30- to 35-kDa proteins as a substrate for DNA binding, as they represented the only group whose electrophoretic mobility was conserved among mammalian species. In addition they were the only class of DNA-binding proteins accessible to Exogenous DNA in intact sperm cells. The purified 30- to 35-kDa proteins interacted in vitro with Exogenous DNA and generated discrete protein/DNA complexes as determined by band shift assay. A factor blocking the binding of Exogenous DNA to sperm cells was also identified in the seminal fluid of mammals and in echinoid spermatozoa. The factor also exerted a powerful inhibitory effect on DNA uptake in sperm cells of heterologous species. The 30- to 35-kDa DNA-binding proteins appeared to be the specific target through which the inhibition was mediated. In the presence of the inhibitory factor, the 30- to 35-kDa lost the ability to bind Exogenous DNA. Thus, the interaction of Exogenous DNA with sperm cells does not appear to be a casual event but, on the contrary, relies on a molecular mechanism based on the cooperation of specific protein factors.
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evidence for nuclear internalization of Exogenous DNA into mammalian sperm cells
Molecular Reproduction and Development, 1993Co-Authors: Maura Francolini, Marialuisa Lavitrano, Carla Lora Lamia, Deborah French, Luigi Frati, Franco Cotelli, Corrado SpadaforaAbstract:Mature sperm cells have the spontaneous capacity to take up Exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to Exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the Exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.
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the interaction between Exogenous DNA and sperm cells
Molecular Reproduction and Development, 1992Co-Authors: Marialuisa Lavitrano, Deborah French, Luigi Frati, Massimo Zani, Corrado SpadaforaAbstract:Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the Exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for Exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.
Mark E Davis - One of the best experts on this subject based on the ideXlab platform.
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pegylation significantly affects cellular uptake and intracellular trafficking of non viral gene delivery particles
European Journal of Cell Biology, 2004Co-Authors: Swaroop Mishra, Paul Webster, Mark E DavisAbstract:In vitro studies of non-viral gene delivery vectors are typically not performed at physiological conditions, and thus may not provide meaningful results for in vivo investigations. We determine if polycation-plasmid DNA complexes (polyplexes) exploited for in vitro studies behave similarly to variants more applicable to in vivo use by examining their cellular uptake and trafficking. Branched polyethylenimine (25 kDa) or a linear beta-cyclodextrin-containing polymer are each used to formulate polyplexes, which can be PEGylated (PEG: poly(ethylene glycol)) to create particles stable in physiological salt concentrations. Particle size, cellular uptake, intracellular trafficking, and reporter gene expression are reported for polyplexes and for their PEGylated variants. PEGylation confers salt stability to particles but produced a reduction in luciferase expression. Examination of in vitro particle internalization by transmission electron microscopy shows unmodified polyplexes entering cells as large aggregates while PEGylated particles remain small and discrete, both outside and within cells. Unmodified and PEGylated particles enter cells through the endocytic pathway and accumulate in a perinuclear region. Immunolabeling reveals unpackaged Exogenous DNA in the cytoplasm and nuclei. It appears all particle types traffic towards the nucleus within vesicles and undergo degradation in vesicles and/or cytoplasm, and eventually some Exogenous DNA enters the nucleus, where it is transcribed. In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.
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pegylation significantly affects cellular uptake and intracellular trafficking of non viral gene delivery particles
European Journal of Cell Biology, 2004Co-Authors: Swaroop Mishra, Paul Webster, Mark E DavisAbstract:Summary In vitro studies of non-viral gene delivery vectors are typically not performed at physiological conditions, and thus may not provide meaningful results for in vivo investigations. We determine if polycation-plasmid DNA complexes (polyplexes) exploited for in vitro studies behave similarly to variants more applicable to in vivo use by examining their cellular uptake and trafficking. Branched polyethylenimine (25 kDa) or a linear β-cyclodextrin-containing polymer are each used to formulate polyplexes, which can be PEGylated (PEG: poly(ethylene glycol)) to create particles stable in physiological salt concentrations. Particle size, cellular uptake, intracellular trafficking, and reporter gene expression are reported for polyplexes and for their PEGylated variants. PEGylation confers salt stability to particles but produced a reduction in luciferase expression. Examination of in vitro particle internalization by transmission electron microscopy shows unmodified polyplexes entering cells as large aggregates while PEGylated particles remain small and discrete, both outside and within cells. Unmodified and PEGylated particles enter cells through the endocytic pathway and accumulate in a perinuclear region. Immunolabeling reveals unpackaged Exogenous DNA in the cytoplasm and nuclei. It appears all particle types traffic towards the nucleus within vesicles and undergo degradation in vesicles and/or cytoplasm, and eventually some Exogenous DNA enters the nucleus, where it is transcribed. In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.
H Harashima - One of the best experts on this subject based on the ideXlab platform.
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localization of Exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein injection
Journal of Controlled Release, 2013Co-Authors: Yukari Yasuzaki, Yuma Yamada, Tsutomu Kanefuji, H HarashimaAbstract:Mitochondrial genetic disorders are a major cause of mitochondrial diseases. It is therefore likely that mitochondrial gene therapy will be useful for the treatment of such diseases. Here, we report on the possibility of mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. The HLV injection procedure, a useful method for transgene expression in skeletal muscle, involves the rapid injection of a large volume of naked plasmid DNA (pDNA) into the distal vein of a limb. We hypothesized that the technique could be used to deliver pDNA not only to nuclei but also to mitochondria, since cytosolic pDNA that is internalized by the method may be able to overcome mitochondrial membrane. We determined if pDNA could be delivered to myofibrillar mitochondria by HLV injection by PCR analysis. Mitochondrial toxicity assays showed that the HLV injection had no influence on mitochondrial function. These findings indicate that HLV injection promises to be a useful technique for in vivo mitochondrial gene delivery.
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the location of the left handedly curved DNA sequence affects Exogenous DNA expression in vivo
Archives of Biochemistry and Biophysics, 2007Co-Authors: H Kamiya, Takashi Ohyama, S Fukunaga, H HarashimaAbstract:The intranuclear disposition of a plasmid is extremely important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on plasmid expression were examined in vivo. A naked luciferase-plasmid was delivered into mouse liver by a hydrodynamics-based injection, and the luciferase activities were quantitated at various time points. The location of the left-handedly curved sequence determined the transgene expression, without affecting the amount of intranuclear Exogenous DNA. The plasmid containing the curved sequence at the location that results in the exposure of the TATA box out of the nucleosome core showed the highest expression. These results suggest that sequences with high histone affinity could control transgene expression from plasmids in vivo.
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intranuclear disposition of Exogenous DNA in vivo silencing methylation and fragmentation
FEBS Letters, 2006Co-Authors: Hiroshi Ochiai, H Harashima, H KamiyaAbstract:The intranuclear disposition of Exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the Exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of Exogenous DNA, and its 'silencing', occurred in parallel in the nucleus.
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visualization of intracellular trafficking of Exogenous DNA delivered by cationic liposomes
Biochemical and Biophysical Research Communications, 2002Co-Authors: H Kamiya, Yuka Fujimura, Ichiro Matsuoka, H HarashimaAbstract:To visualize the intracellular trafficking of Exogenous DNAs delivered by cationic liposomes, rhodamine-labeled DNAs were transfected into NIH3T3 cells and observed by confocal laser microscopy. After 0.5- to 1-h incubations, the DNAs reached the nucleus with a much higher frequency than that expected from the cell division rate. This result suggests that DNAs can enter the nucleus in the presence of the nuclear membrane. Interestingly, some DNAs appeared to extend through the nuclear membrane in the aggregated form which were much larger than the nuclear pore complex. The DNAs which have passed through the nuclear membrane were stained with SYTO 24, a DNA labeling reagent. The stained part may be "naked" DNA that is free of lipids or proteins. This observation indicates that a complex containing DNA fuses with the nuclear membrane and then naked DNA is released into the nucleus.
Deborah French - One of the best experts on this subject based on the ideXlab platform.
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the mechanism of binding of Exogenous DNA to sperm cells factors controlling the DNA uptake
Experimental Cell Research, 1995Co-Authors: Massimo Zani, Marialuisa Lavitrano, Deborah French, Valentina Lulli, Barbara Maione, Sabina Sperandio, Corrado SpadaforaAbstract:Abstract Mature sperm cells have the spontaneous capability of taking up Exogenous DNA. Potential substrates for the interaction of the DNA with the sperm heads are specific classes of DNA-binding proteins. In the present work three major classes of DNA-binding proteins were identified by Southwestern analysis of sperm head protein extracts: a first class of about 50 kDa in molecular weight, a second one of 30-35 kDa, and finally a third one below 20 kDa. The latter group most probably contains sperm protamines. Our attention was particularly focused on the 30- to 35-kDa proteins as a substrate for DNA binding, as they represented the only group whose electrophoretic mobility was conserved among mammalian species. In addition they were the only class of DNA-binding proteins accessible to Exogenous DNA in intact sperm cells. The purified 30- to 35-kDa proteins interacted in vitro with Exogenous DNA and generated discrete protein/DNA complexes as determined by band shift assay. A factor blocking the binding of Exogenous DNA to sperm cells was also identified in the seminal fluid of mammals and in echinoid spermatozoa. The factor also exerted a powerful inhibitory effect on DNA uptake in sperm cells of heterologous species. The 30- to 35-kDa DNA-binding proteins appeared to be the specific target through which the inhibition was mediated. In the presence of the inhibitory factor, the 30- to 35-kDa lost the ability to bind Exogenous DNA. Thus, the interaction of Exogenous DNA with sperm cells does not appear to be a casual event but, on the contrary, relies on a molecular mechanism based on the cooperation of specific protein factors.
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evidence for nuclear internalization of Exogenous DNA into mammalian sperm cells
Molecular Reproduction and Development, 1993Co-Authors: Maura Francolini, Marialuisa Lavitrano, Carla Lora Lamia, Deborah French, Luigi Frati, Franco Cotelli, Corrado SpadaforaAbstract:Mature sperm cells have the spontaneous capacity to take up Exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to Exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the Exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.
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the interaction between Exogenous DNA and sperm cells
Molecular Reproduction and Development, 1992Co-Authors: Marialuisa Lavitrano, Deborah French, Luigi Frati, Massimo Zani, Corrado SpadaforaAbstract:Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the Exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for Exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.