Association of exogenous DNA with cattle and insect spermatozoa in vitro

Atkinson, Peter W.; Hines, Eric R.; Beaton, Sandra; Matthaei, Klaus I.; Reed, Ken C.; Bradley, Mark P.

doi:10.1002/mrd.1080290102

Cited by 77 publications

(54 citation statements)

References 16 publications

(14 reference statements)

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“…We therefore interpreted this pattern as indicative of dead sperm. Our finding is consistent with a previous study where similar postacrosomal staining accounted for O99% of labelling in coldshocked immotile sperm (Atkinson et al 1991). For each zona-free treatment, only rows with no common superscripts within a column differ significantly (P!0.05).…”

Section: Discussionsupporting

confidence: 92%

“…Following DNase treatment, less than half of these sperm retained their fluorescent label. This reduction is in agreement with the previous studies using radioactively (Atkinson et al 1991) or fluorescently labelled DNA (Carballada & Esponda 2001). In the label-retaining sperm fraction, exogenous DNA was protected from DNase digestion, either via association with the sperm membrane or via internalisation.…”

Section: Discussionsupporting

confidence: 92%

“…This may account for the lack of postacrosomal staining described in other studies. In our hands, preferential localisation of fluorescent signal in the postacrosomal region was only evident in immotile sperm, in accordance with previous reports (Brackett et al 1971, Atkinson et al 1991. This postacrosomal signal was uniform and DNase resistant, indicating that it was of a different quality from the randomly localised, DNase-sensitive puncta in live sperm.…”

Section: Discussionsupporting

confidence: 91%

“…While these assays demonstrated association between exogenous DNA and sperm, they provide no direct evidence for DNA uptake across the plasma membrane into the sperm cytoplasm or nucleus. Few studies have addressed DNA uptake into sperm using direct visualisation, such as autoradiography (Brackett et al 1971, Atkinson et al 1991, wide-field (Anzar & Buhr 2006) and confocal laser scanning epifluorescence microscopy of fluorescently labelled sperm (Bachiller et al 1991, Chan et al 2000 or transmission electron microscopy on thin sections (Kim et al 2010). These studies have shown DNA binding to the sperm surface, preferentially in the postacrosomal region, but not provided conclusive evidence for its uptake into sperm with an intact plasma membrane.…”

Section: Introductionmentioning

confidence: 99%

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Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Eghbalsaied¹,

Ghaedi²,

Laible³

et al. 2013

Reproduction

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Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (nZ96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (nZ751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68Z56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (nZ48) or GFP fluorescence (nZ94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Eghbalsaied¹,

Ghaedi²,

Laible³

et al. 2013

Reproduction

View full text Add to dashboard Cite

show abstract

“…Since the pioneering study by Brackett et al [1] showing that rabbit spermatozoa were able to incorporate SV40 DNA into their heads during incubation, the property of association of spermatozoa with heterologous DNA has been extensively studied [2][3][4][5]. These studies have facilitated the idea of using sperm as a vector to transfer exoge-actually carry the DNA into eggs, and the gene thus transferred is transcriptionally active [11][12][13][14][15][16][17].…”

mentioning

confidence: 99%

Possible Mechanisms for the Testis-Mediated Gene Transfer as a New Method for Producing Transgenic Animals.

Chang

Ikeda

Hayashi

et al. 1999

Journal of Reproduction and Development

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Abstract.We have recently shown that DNAs injected into the testis of either rats or mice can be transferred into eggs via sperm at fertilization and expressed and transmitted in the descendants (testis-mediated gene transfer). In the present study, we investigated the association of epididymal and ejaculated sperm with DNA injected into the testis of rats. In the first experiment, a chimeric gene of mouse metallothionein-I promoter (MT)/human growth hormone receptor (hGHR) labeled with FITC was prepared. This gene construct was mixed with cationic liposome and injected into rat testis, and 4 days later the cauda epididymis was dissected out and prepared for histological examination. A confocal microscopic observation showed that not only the head, but also the tail, of almost all spermatozoa in the epididymis was labeled with FITC. In the next experiment, 4 days after the injection of MT/hGHR gene encapsulated by liposome into the testis, the male was allowed to mate with females and spermatozoa were recovered from the uterus in the next morning. Spermatozoa were incubated in the presence or absence of deoxyribonuclease (DNase) I and then genomic DNA was extracted. The MT/hGHR gene was detected only in the spermatozoa incubated without DNase by means of PCR. These results support the notion that, in the case of our testis-mediated gene transfer, exogenous DNA injected with liposomes is not integrated into genome of the sperm and the integration occurs after fertilization.

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Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation

Patil

Khoo

1996

J. Exp. Zool.

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Mature sperm cells of zebrafish (Danio rerio) incubated with foreign DNA have the capacity to take up foreign DNA. Such uptake can be enhanced by electroporation. Mature spermatozoa of zebrafish were incubated and electroporated in the presence of either radiolabeled or unlabeled plasmid DNA at voltages of 500 or 1,000, or 1,500 V/cm. From the percentage of radiolabeled plasmids retained on the spermatozoa, some sperm showed an ability to spontaneously take up the plasmid DNA, and the ability was enhanced one‐ to twofold by electroporation. Fertilization of mature eggs with the treated sperm resulted in transmission of the plasmid DNA to the resulting offspring. Frequency of transgenic individuals, as monitored by polymerase chain reaction, increased marginally, more than doubled and nearly doubled in 500 V/cm, 1,000 V/cm, and 1,500 V/cm electroporated groups, respectively, when compared to the non‐electroporated group. These results indirectly implied that electroporation enhanced the capacity of spermatozoa to take up plasmid DNA. The increased field strength, however, had a deleterious effect on the motility of the sperm, causing clumping of sperms at high voltages. Light microscopic autoradiography of treated spermatozoa was able to show that the plasmid DNA was associated with the majority of sperm but was unable to differentiate whether it was present inside the nucleus or not. Ultrastructural in situ hybridization on thin sections of zebrafish spermatozoa, however, was able to show that the exogenous DNA was internalized into the nucleus and that electroporation enhanced this internalization. The results provide direct evidence for nuclear internalization of foreign DNA by non‐mammalian sperm as in mammalian sperm. © 1996 Wiley‐Liss, Inc.

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Association of exogenous DNA with cattle and insect spermatozoa in vitro

Cited by 77 publications

References 16 publications

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Possible Mechanisms for the Testis-Mediated Gene Transfer as a New Method for Producing Transgenic Animals.

Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation

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