X and Y chromosome-bearing spermatozoa are equally able to uptake and internalize exogenous DNA by sperm-mediated gene transfer in swine

doi:10.1016/j.rvsc.2015.11.001

Research in Veterinary Science

Volume 104, February 2016, Pages 1-3

https://doi.org/10.1016/j.rvsc.2015.11.001 Get rights and content

Highlights

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Our group has produced genetically modified (GM) pigs since many years through SMGT.
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We have noticed a not significant positive trend in the birth of GM male piglets.
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We investigated if X and Y sorted sperms differentially internalized exogenous DNA.
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Data obtained from qPCR showed no significant differences among X and Y sperms.
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X and Y sperms equally internalize exogenous DNA during SMGT.

Abstract

Since proteomic differences between male X/Y chromosome-bearing gametes have recently been described, a question has been raised: could these differences be responsible for different behavior between X and Y chromosome-bearing spermatozoa during the binding and internalization of exogenous DNA in the swine species? In order to investigate this hypothesis, our group studied the process of the uptake and internalization of exogenous DNA in X and Y chromosome-bearing sperm sub-populations. No significant differences were found between sperm types in both the uptake and internalization of exogenous DNA. The quantity of internalized exogenous DNA was significantly lower than that of the uptaken DNA. In conclusion, our results showed that X and Y chromosomes-bearing spermatozoa have the same binding capacity and internalization of DNA, and the proteomic differences between them do not seem to interfere with these complex processes.

Section snippets

Acknowledgments

The authors thank Dr. Domenico Ventrella for the critical reading of the manuscript.

This study was supported by grants from: Ateneo Bologna, Research Funding Office 60% EF2012-EF2013 to MLB, MF, AZ, MS. Consorzio Interuniversitario per i Trapianti d'Organo, EF2008 to MF, AZ, MLB.

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Cited by (2)

Expression of fluorescent reporter protein was not obtained in ovine embryos produced through in vitro fertilization-sperm mediated gene transfer (IVF-SMGT)
2016, Small Ruminant Research
Citation Excerpt :
This was the method of choice for more than 20 years (Kues and Niemann, 2011), and transgenic mice, sheep, pigs, and cattle have been successfully produced using this technique (Garcia-Vazquez et al., 2009), but besides being expensive, this technique is still inefficient when used to generate transgenic farm animals (0.5–4%) (Garcia-Vazquez et al., 2011). More efficient protocols are now available (Kues and Niemann, 2011), including virus-mediated transgenesis, somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer (SMGT) (Zaniboni et al., 2016). These methods vary in efficacy and safety.
Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic animals on a mass scale using spermatozoa as vectors for exogenous DNA. In this study the possibility of using in vitro fertilization (IVF) −SMGT to produce transgenic ovine embryos was investigated for the first time. For this purpose, sperm were obtained from the epididymal testicle areas of 4 rams and different concentration of DNA (0.4, 0.8 and 1.6 μg) and TurboFect (0.25, 0.5, 1 and 2 μg) were used for sperm transfection (1 × 10⁶). Enhanced-GFP-expressing vector pEGFP-N1 was used as the carrier. In order to evaluate the performance of transgenic sperm, in vitro fertilization technology was used. After the preparing oocytes received from the ovaries of slaughterhouse origin, oocytes with more than three layers of granulosa and uniform cytoplasm were selected and matured in TCM-199 medium containing 10% fetal calf serum, follicle stimulating hormone (FSH) (5 μg/ml), β-17 estradiol (1 μg/ml) and sodium pyruvate 0.81 (Mm). Bracket and Oliphant’s (BO) medium and modified Charles Rosenkrans medium with amino acids (mCR2aa) were used for in vitro fertilization and culture, respectively. Results showed that transfected sperm with different concentrations of DNA and TurboFect carrier were unable to transfer the GFP gene to in vitro matured oocytes as the GFP gene was not expressed in neither zygotes nor morula stage embryos. Further optimization for SMGT improvement such as different transfection reagent and method like electroporation, using antioxidants in transfection medium to overcome the apoptosis and also separation of transfected sperm from untransfected ones before insemination have been suggested.
Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells
2017, Zygote

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X and Y chromosome-bearing spermatozoa are equally able to uptake and internalize exogenous DNA by sperm-mediated gene transfer in swine

Highlights

Abstract

Section snippets

Acknowledgments

Theriogenology

Theriogenology

J. Proteome

Regul. Pept.

Theriogenology

Theriogenology

Theriogenology

Exp. Cell Res.

Theriogenology

Theriogenology