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Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation
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Theriogenology 74 (2010) 563–568 www.theriojournal.com
Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation W.B. Feitosa, C.M. Mendes, M.P. Milazzotto, A.M. Rocha, L.F. Martins, R. Simões, F.F. Paula-Lopes, J.A. Visintin, M.E.O.A. Assumpção* Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil Received 7 September 2008; received in revised form 22 March 2010; accepted 22 March 2010
Abstract Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 ⫻ 106 cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 °C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P ⬍ 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. © 2010 Elsevier Inc. All rights reserved. Keywords: Exogenous DNA; DNA uptake; Sperm cells; Transgenic; Gene transfer
1. Introduction Landmark genetic manipulation studies in the 1980s demonstrated for the first time that genes could be transferred to pre-implantation embryos [1], resulting in birth of the first transgenic mammal [2]. During the last two decades, the ability to modify gene expression by trangenesis, particularly in mammals, has been an important advance in experimental and applied biology [3]. Sperm-mediated gene transfer has been described as a promising approach for transgenic animal production
* Corresponding author. Tel.: 55-11-30917665; fax: 55-11-30917412. E-mail address: [email protected] (M.E.O.A. Assumpção). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.03.016
[4]. The first evidence that spermatozoa were capable of taking up exogenous DNA was demonstrated in the early 1970s [5]. This was, however, not confirmed until the end of the 1980s, when it was reported that spermatozoa could behave as a vector, transferring exogenous DNA to the oocyte during in vitro fertilization, resulting in transgenic offspring [6,7]. These results were later repeated by several other groups [8 –11]. Although SMGT is a simple, fast, and low-cost method, it has not been widely used, as its efficiency ranges from 0 –100% [12], likely due to the low frequency of stable DNA integration in the genome and poor repeatability [12]. To increase SMGT efficiency, the kinetics of interactions between spermatozoa and exogenous DNA
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have been the focus of many studies [13]. Although prolonged incubation increased the amount of exogenous DNA associated with spermatozoa, it also decreased sperm viability [14]. Furthermore, large amounts of exogenous DNA associated with spermatozoa can trigger endonuclease activation, resulting in exogenous and endogenous DNA degradation in an apoptosis-like process [15]. This could explain, at least in part, differences in repeatability and efficiency of SMGT among various species, animals, and laboratories. Thus, the aims of this study were to evaluate the effects of exogenous DNA concentration and the incubation period on transfection rate and endogenous DNA integrity. 2. Materials and methods 2.1. Materials The plasmid pEYFP-Nuc (Clonetech, BD Biosciences, California, USA), which was 4.8 kb in size and linearized with Stu I, was used as exogenous DNA. Unless otherwise indicated, all materials were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Spermatozoa-DNA interaction One straw of frozen semen was thawed in a water bath at 37 °C for 30 s, and live spermatozoa were separated by centrifugation in a Percoll gradient (90 and 45%) for 5 min at 9000 ⫻ g. The sperm pellet was resuspended in fertilization medium [16] without heparin, centrifuged for 3 min at 9000 ⫻ g, resuspended in fertilization medium without heparin to a final concentration of 1 ⫻ 106 cells/mL, and incubated with 100, 300, or 500 ng/mL of pEYFP-NUC for 60 or 120 min at 39 °C and 5% (v/v) CO2 in air with high humidity. 2.3. DNA extraction After incubation with various concentrations of exogenous DNA for 60 or 120 min, spermatozoa were washed in PBS (Ca2⫹ and Mg free) for 3 min at 9000 ⫻ g and incubated with 0.1 mg DNase I for 30 min to remove exogenous DNA adsorbed on the sperm surface, but not internalized. After DNAse I treatment, spermatozoa were washed in PBS (Ca2⫹ and Mg free) for 3 min at 9000 ⫻ g. The sperm pellet was subjected to five freeze/thaw cycles to induce plasma membrane lysis, facilitating DNA retrieval, and then put in spermlysing buffer (50 L SDS 10%, 5 L Proteinase K 20 mg/mL, and 375 L sodium acetate 0.2 M) for 2 h at 56 °C. After incubation, spermatozoa were centrifuged
in a solution of phenol:chloroform:isoamyl alcohol (25: 24:1, v/v) at 16,000 ⫻ g for 2 min. The supernatant was removed and centrifuged in isopropanol at 16,000 ⫻ g for 2 min. The resultant pellet was centrifuged in ethanol (70%) at 16,000 ⫻ g for 2 min, dried, and resuspended in TE solution (Tris-Cl 10 mM, pH 8.0, and EDTA 1 mM) at 56 °C for 1 h. The recovered DNA was quantified with a spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), diluted to a final concentration of 1 ng/L in H2O, and stored at ⫺20 °C for later use as a template for real-time PCR. 2.4. Assessment of DNA uptake Quantitative DNA uptake analyses were performed by real time PCR [17] using the Mastercycler® ep reaplex (Eppendorf, Hamburg, Germany). Standard curves were generated using serial dilutions of pEYFP-NUC plasmid (2, 0.2, 0.02, 0.002, and 0.0002 ng). Quantitative PCR was performed in triplicate using a Platinum SYBR GreenER qPCR supermix (Invitrogen, Carlsbad, CA, USA) with a reaction solution containing 12.5 L of supermix, 300 nM of each primer, and 5 pg of DNA. Water was added to achieve a final volume of 25 L. Forward and reverse primers were 5=-ATGGCCGACAAGCAGAAGAAC-3= and 5=-TGCCGTCCTCGATGTTGTG-3=, respectively. The thermal cycler program was 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 64 °C for 1 min, and a melting curve. 2.5. Sperm chromatin structure assay A sperm chromatin structure assay (SCSA) assessed chromatin stability, using metachromatic staining with acridine orange (AO), as previously described [18]. Acridine orange emits green or red fluorescence when it intercalates with double- or single-stranded DNA, respectively. Briefly, sperm samples were diluted with TNE buffer (Tris 10 mM, pH 7.4, 0.15 M NaCl, and 1 mM EDTA) into a final concentration of approximately 1 ⫻ 106 cells/mL. An aliquot (200 L) was mixed with 400 L of a detergent/acid solution (0.1% Triton X-100, 0.15 M NaCl, and 0.08 N HCl, pH 1.4). After 30 s, spermatozoa were stained with 1.2 mL of AO (6 g/mL in 0.1 M citric acid, 0.2 M Na2HPO4, 1 mM EDTA, and 0.15 mM NaCl, pH 6.0). Within 3 min after AO staining, stained samples were analyzed by flow cytometry. 2.6. Assessment of sperm DNA integrity using a TUNEL assay Detection of DNA strand breaks (single- as well as double-stranded DNA) were done with a TUNEL as-
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say, using the Guava TUNEL Kit (Guava Technologies, Hayward, CA, USA), as previously described [19]. Briefly, spermatozoa were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 60 min at 4 °C and permeabilized with 1% Triton in PBS for 30 min at 4 °C. After fixation and permeabilization, spermatozoa were washed with washing buffer supplied with the kit, and then incubated in a DNA labeling mix (TdT reaction buffer, TdT enzyme, Brd-UTP, and distilled H2O) for 2 h at 37 °C. Thereafter, spermatozoa were centrifuged at 300 ⫻ g for 5 min in 200 L of rinsing buffer and incubated in Anti-BrdU staining mix (TRITC-conjugated anti-BrdU antibody) for 30 min at room temperature in the dark. Finally, 150 L of rinsing buffer was added, and spermatozoa were analyzed by flow cytometry. 2.7. Flow cytometry analysis Flow cytometry analyses (for TUNEL and SCSA) were performed using the Guava Easycyte Mini Flow Cytometry System (Guava Technologies, Hayward, CA, USA). This device contains a laser, which operates at 488 nm and emits a 20 mW visible laser radiation. A total of 10,000 events per sample were analyzed and data corresponding to yellow (PM1 photodetector— 583 nm), red (PM2 photodetector— 680 nm), and green fluorescence signals (PM3 photodetector—525 nm) were recorded after logarithmic amplification. Flow cytometry dot plots used were PM2/PM3 for the SCSA assay and PM1/FSC (Forward scatter) for the TUNEL assay. 2.8. Experimental design 2.8.1. Effects of exogenous DNA concentration and incubation period on exogenous DNA uptake by bovine spermatozoa The objective of this experiment was to evaluate the effects of exogenous DNA concentration and incubation period on exogenous DNA uptake. Spermatozoa were incubated with a pEYFP-Nuc plasmid at concentrations of 100, 300, or 500 ng, for 60 or 120 min, in sperm-TL at 39 °C in a humidified atmosphere of 5% (v/v) CO2. Exogenous DNA uptake was quantified by real-time PCR. This experiment was replicated three times using separate batches of frozen semen, and realtime PCR was performed in triplicate. 2.8.2. Effects of exogenous DNA concentration and incubation time on endogenous DNA integrity The aim of this experiment was to evaluate the effects of exogenous DNA concentration and incuba-
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tion period on endogenous DNA integrity. Spermatozoa were incubated with a pEYFP-Nuc plasmid at concentrations of 100, 300, or 500 ng for 60 or 120 min in sperm-TL at 39 °C in a humidified atmosphere of 5% (v/v) CO2. The percentages of endogenous DNA integrity were assessed by SCSA and TUNEL assays, coupled to flow cytometry. This experiment was replicated three times using different batches of frozen semen (10,000 spermatozoa/replicate). 2.9. Statistical analysis Data were analyzed by least-squares analysis of variance, using the General Linear Models (GLM) procedure of SAS (SAS Institute Inc., Cary, NC, USA). The mathematical model included the main effects of incubation period and DNA concentration, as well as their interaction. Main effects were considered fixed. Orthogonal contrasts and a mean separation procedure (pdiff) were performed when appropriate to located differences. Values are presented as mean ⫾ SEM, and P ⬍ 0.05 was considered significant. 3. Results 3.1. Effects of exogenous DNA concentration and incubation time on exogenous DNA uptake by bovine spermatozoa Incubation of frozen-thawed bovine spermatozoa with exogenous DNA resulted in spontaneous DNA uptake. It was noteworthy that exogenous DNA uptake by spermatozoa increased (P ⬍ 0.05) as incubation time increased from 60 to 120 min, but only for 500 ng of exogenous DNA (0.0063 ⫾ 0.009 and 0.0284 ⫾ 0.0277 ng, respectively). Similarly, the increase of exogenous DNA concentration from 100 to 500 ng only had an effect (P ⬍ 0.05) on exogenous DNA uptake by spermatozoa at 120 min of incubation (0.00226467 ⫾ 0.0012 and 0.0284 ⫾ 0.0277 ng, respectively; Fig. 1). 3.2. Effect of exogenous DNA on the DNA integrity of bovine spermatozoa assessed by flow cytometry Based on SCSA, there was no effect of exogenous DNA concentration or incubation period on the DNA integrity of spermatozoa incubated with 0 ng (96.56 ⫾ 0.7 and 91.56 ⫾ 3%), 100 ng (94.13 ⫾ 3.3 and 93.01 ⫾ 1.4%), 300 ng (94.48 ⫾ 3.8 and 93.66 ⫾ 2.9%), or 500 ng (94.96 ⫾ 1 and 93.74 ⫾ 1.9%) of exogenous DNA for 60 or 120 min, respectively. Similarly, based on the TUNEL assay, sperm DNA integrity was not affected when incubated with 0 ng (95.88 ⫾ 3.3 and 96.59 ⫾
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Fig. 1. Mean ⫹ SEM amounts of exogenous DNA internalized (assessed with real-time PCR) in bovine spermatozoa cells after 60 or 120 min of incubation with various concentrations of exogenous DNA. The uptake of DNA for 500 ng incubated for 60 min was greater (P ⬍ 0.05) than all other combinations (none of which were significantly different from each other).
2.8%), 100 ng (96.42 ⫾ 2.3 and 96.37 ⫾ 2.3%), 300 ng (96.61 ⫾ 2.9 and 96.4 ⫾ 1.8%), or 500 ng (96.36 ⫾ 2.4 and 95.49 ⫾ 3.2%) of exogenous DNA for 60 or 120 min. 4. Discussion Sperm-mediated gene transfer is a biochemically regulated process. Binding of exogenous DNA is mediated by a 30 –35 kDa plasma membrane protein, and the uptake and internalization of exogenous DNA is mediated by major histocompatibility complex-class II (MHC II) and CD4 [20]. In the present study, exogenous DNA uptake by frozen-thawed bovine spermatozoa was increased with the highest concentration of DNA and the longest incubation period. There are several techniques to determine the association between exogenous DNA and spermatozoa. For example, exogenous DNA can be localized in situ by autoradiography using radiolabeled plasmids or by fluorescence microscopy and flow cytometry using fluorescent-labeled plasmids. Although these methods localize the labeled DNA in situ, they have the inconvenience of being radioactive or failing to quantify the amount of exogenous DNA associated with spermatozoa. However, these limitations can be avoided with real-time PCR. In the current study, real-time PCR efficiently quantified exogenous plasmid DNA associated with spermatozoa. The duration of incubation of spermatozoa and exogenous DNA played a role in SMGT [7,9,21,22]. In the present work, there was a beneficial effect of an
extended incubation time on SMGT efficiency, but only with the highest concentration of exogenous DNA. Exogenous DNA starts to associate with bull spermatozoa after 15–20 min of incubation [9], with a maximal association at ⬃30 – 40 min [21–23]. Additionally, the prevalence of spermatozoa with exogenous DNA internalized in their nucleus peaked at 65%, after 60 min of incubation [24]. In the present study, internalization of exogenous DNA was greatest for the longest incubation period and highest concentration of DNA. However, it was noteworthy that the real-time PCR used to quantify internalization of exogenous DNA could not differentiate whether this increase was due to a greater amount of exogenous DNA internalized by each spermatozoa, or a greater proportion of spermatozoa which internalized the DNA. This critical question remains unresolved, but should yield important knowledge to understand and increase the efficiency of SMGT. Although SMGT efficiency was improved by a long incubation period and high concentration of exogenous DNA, these conditions could trigger endonuclease activity [15], presumably cleaving both exogenous and endogenous DNA in an apoptosis-like process [15]. Consequently fertilization rates [25] and the efficiency of exogenous DNA uptake were compromised [15], thereby reducing the rate of formation of viable transgenic embryos. In a previous study, exogenous DNA uptake had a negative effect on spermatozoa viability, manifested as reduced sperm motility [26]. In the present study, however, there was no apparent effect of incubation time or exogenous DNA concentration on endogenous DNA integrity which was evaluated using two methodologies (SCSA and TUNEL assay). The lack of exogenous DNA effect on DNA fragmentation could be attributed to the chromatin structure of bovine spermatozoa. In contrast to mouse spermatozoa, however, bovine spermatozoa lack protamine 2; they only have protamine 1 [27]. Since DNA fragmentation is more related to alterations of protamine 2 versus protamine 1 [28], this can confer greater resistance to apoptosis-like processes in bovine spermatozoa compared to murine spermatozoa. Furthermore, endonucleases from ejaculated spermatozoa are less reactive to exogenous DNA stimulation than endonucleases from epididymal spermatozoa [15]. Although exogenous DNA is extensively degraded during transfection of porcince spermatozoa [15], in bovine spermatozoa this DNA remains intact [29]. The endonuclease activity can also explain differences in DNA uptake by spermatozoa in the present
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study. We speculated that endonuclease activity was reduced over time following incubation with high DNA concentration reaching its saturation point. Perhaps endonuclease activity is time- and concentration-dependent of exogenous DNA. Besides the fact that bovine spermatozoa endonucleases activity is low [29], it is also possible that the result from DNA uptake by spermatozoa in the present study was due to low DNA/spermatozoa interaction and low uptake efficiency. Therefore, in order to optimize SMGT in bovine spermatozoa, it may be necessary to use a high exogenous DNA concentration and a long incubation period. In conclusion, the current findings were consistent with previous reports that bovine spermatozoa can spontaneously take up exogenous DNA. Internalization of exogenous DNA by spermatozoa was improved only when a high exogenous DNA concentration was associated with an extended incubation period. In addition, neither an increased concentration of exogenous DNA, nor a prolonged incubation period, nor both, induced endogenous DNA fragmentation as a consequence of an apoptosis-like process. Acknowledgements This study was supported by The State of São Paulo Research Foundation (FAPESP), fellowship 03/10234-7 and grant 03/07456-8. References [1] Brinster RL, Chen HY, Trumbauer ME, Avarbock MR. Translation of globin messenger RNA by the mouse ovum. Nature 1980;283:499 –501. [2] Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 1980;77:7380 – 4. [3] Celebi C, Guillaudeux T, Auvray P, Vallet-Erdtmann V, Jegou B. The making of ”transgenic spermatozoa”. Biol Reprod 2003; 68:1477– 83. [4] Smith K, Spadafora C. Sperm-mediated gene transfer: applications and implications. Bioessays 2005;27:551– 62. [5] Brackett BG, Baranska W, Sawicki W, Koprowski H. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Natl Acad Sci USA 1971; 68:353–7. [6] Arezzo F. Sea-urchin sperm as a vector of foreign genetic information. Cell Biol Int Rep 1989;13:391– 404. [7] Lavitrano M, Camaioni A, Fazio VM, Dolci S, Farace MG, Spadafora C. Sperm cells as vectors for introducing foreign DNA into eggs - genetic transformation of mice. Cell 1989; 57:717–23. [8] Atkinson PW, Hines ER, Beaton S, Matthaei KI, Reed KC, Bradley P. Association of exogenous DNA with cattle and insect spermatozoa in vitro, Mol Reprod Dev 1991;29:1–5.
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[9] Castro FO, Hernandez O, Uliver C, Solano R, Milanes C Aguilar A, Pérez A, de Armas R, Herrera L, de la Fuente J. Introduction of foreign DNA into the spermatozoa of farm animals.Theriogenology 1990;34:1099 –1110. [10] Gagne AM, Pothier F, Sirard MA. Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol Rep Dev 1991;29:6 –15. [11] Horan R, Powell R, Mcquaid S, Gannon F, Houghton JA. Association of foreign DNA with porcine spermatozoa. Arch Androl 1991;26:83–92. [12] Maione B, Lavitrano M, Spadafora C, Kiessling AA. Spermmediated gene transfer in mice. Mol Reprod Dev 1998;50: 406 –9. [13] Lavitrano M, Forni M, Bacci ML, Di Stefani C, Varzi V, Wang H, Seren E. Sperm mediated gene transfer in pig: Selection of donor boars and optimization of DNA uptake. Mol Reprod Dev 2003;64:284 –91. [14] Feitosa WB, Milazzotto MP, Simões R, Rovegno M, Nicacio AC, Nascimento AB, Gonçalves JSA, Visintin JA, Assumpção MEOA. Bovine sperm cells viability during incubation with or without exogenous DNA. Zygote 2009;17:315–20. [15] Maione B, Pittogi C, Achene L, Lorenzini R, Spadafora C. Activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA. DNA Cell Biol 1997;16:1087–97. [16] Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988;38:1171– 80. [17] Hoelker M, Mekchay S, Schneider H, Bracket BG, Tesfaye D, Jennen D, Tholen E, Gilles M, Rings F, Griese J, Schellander K. Quantification of DNA binding, uptake, transmission and expression in bovine sperm mediated gene transfer by RT-PCR: Effect of transfection reagent and DNA architecture. Theriogenology 2007;67:1097–1107. [18] Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P, Claussen OP. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic Hum Reprod 1999;14:1039 – 49. [19] Varum S, Bento C, Sousa APM, Gomes-Santos CSS, Henriques P, Almeida-Santos T, Teodósio C, Paiva A, Ramalho-Santos J. Characterization of human sperm populations using conventional parameters, surface ubquitination, and apoptotic markers. Fertil Steril 2007;87:572– 83. [20] Lavitrano M, Maione B, Forte E, Francolini M, Sperandio S, Testi R, et al. The interaction of sperm cells with exogenous DNA: a role of CD4 and major histocompatability complex class II molecules. Exp Cell Res 1997;233:56 – 62. [21] Lavitrano M, French D, Zani M, Frati L, Spadafora C. The interaction between exogenous DNA and sperm cells. Mol Reprod Dev 1992;31:161–9. [22] Sperandio S, Lulli V, Bacci M, Maione B, Spadafora C, Lavitrano M. Sperm-madiated DNA transfer in Bovine and Swine species. Anim Biotechnol 1996;7:59 –77. [23] Camaioni A, Russo MA, Odorisio T, Gandolfi F, Fazio V.M, Siracusa G. Uptake of exogenous DNA by mammalian spermatozoa specific localization of DNA on sperm heads. J Reprod Fertil 1992;96:203–12. [24] Francolini M, Lavitrano M, Lamia CL, French D, Frati L, Cotelli F, Spadafora C. Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells. Mol Reprod Dev 1993;34:133–9. [25] Tanghe S, Van Soon A, Sterckx V, Maes D, Kruif A. Assessment of different sperm quality parameters to predict
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in vitro fertility of bulls. Reprod Domest Anim 2002;37: 127–32. [26] Anzar M, Buhr MM. Spontaneous uptake of exogenous DNA by bull spermatozoa. Theriogenology 2006;65:683–90. [27] Maier WM, Nussbaum G, Domenjoud L, Klemm U, Engel W. The lack of protamine 2 (P2) in boar and bull spermatozoa is due to mutations within the P2 gene. Nucleic Acids Res 1990; 18:1249 –54.
[28] Carrel TC, Emery BR, Hammoud S. Altered protamine expression and diminished spermatogenesis: What is the link? Hum Reprod Update 2007;13:313–27. [29] Alderson J, Wilson B, Laible G, Pfeffer P, L’Huillier P. Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis. Anim Reprod Sci 2006;91: 23–30.
Theriogenology 74 (2010) 563–568 www.theriojournal.com
Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation W.B. Feitosa, C.M. Mendes, M.P. Milazzotto, A.M. Rocha, L.F. Martins, R. Simões, F.F. Paula-Lopes, J.A. Visintin, M.E.O.A. Assumpção* Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil Received 7 September 2008; received in revised form 22 March 2010; accepted 22 March 2010
Abstract Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 ⫻ 106 cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 °C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P ⬍ 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. © 2010 Elsevier Inc. All rights reserved. Keywords: Exogenous DNA; DNA uptake; Sperm cells; Transgenic; Gene transfer
1. Introduction Landmark genetic manipulation studies in the 1980s demonstrated for the first time that genes could be transferred to pre-implantation embryos [1], resulting in birth of the first transgenic mammal [2]. During the last two decades, the ability to modify gene expression by trangenesis, particularly in mammals, has been an important advance in experimental and applied biology [3]. Sperm-mediated gene transfer has been described as a promising approach for transgenic animal production
* Corresponding author. Tel.: 55-11-30917665; fax: 55-11-30917412. E-mail address: [email protected] (M.E.O.A. Assumpção). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.03.016
[4]. The first evidence that spermatozoa were capable of taking up exogenous DNA was demonstrated in the early 1970s [5]. This was, however, not confirmed until the end of the 1980s, when it was reported that spermatozoa could behave as a vector, transferring exogenous DNA to the oocyte during in vitro fertilization, resulting in transgenic offspring [6,7]. These results were later repeated by several other groups [8 –11]. Although SMGT is a simple, fast, and low-cost method, it has not been widely used, as its efficiency ranges from 0 –100% [12], likely due to the low frequency of stable DNA integration in the genome and poor repeatability [12]. To increase SMGT efficiency, the kinetics of interactions between spermatozoa and exogenous DNA
564
W.B. Feitosa et al. / Theriogenology 74 (2010) 563–568
have been the focus of many studies [13]. Although prolonged incubation increased the amount of exogenous DNA associated with spermatozoa, it also decreased sperm viability [14]. Furthermore, large amounts of exogenous DNA associated with spermatozoa can trigger endonuclease activation, resulting in exogenous and endogenous DNA degradation in an apoptosis-like process [15]. This could explain, at least in part, differences in repeatability and efficiency of SMGT among various species, animals, and laboratories. Thus, the aims of this study were to evaluate the effects of exogenous DNA concentration and the incubation period on transfection rate and endogenous DNA integrity. 2. Materials and methods 2.1. Materials The plasmid pEYFP-Nuc (Clonetech, BD Biosciences, California, USA), which was 4.8 kb in size and linearized with Stu I, was used as exogenous DNA. Unless otherwise indicated, all materials were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Spermatozoa-DNA interaction One straw of frozen semen was thawed in a water bath at 37 °C for 30 s, and live spermatozoa were separated by centrifugation in a Percoll gradient (90 and 45%) for 5 min at 9000 ⫻ g. The sperm pellet was resuspended in fertilization medium [16] without heparin, centrifuged for 3 min at 9000 ⫻ g, resuspended in fertilization medium without heparin to a final concentration of 1 ⫻ 106 cells/mL, and incubated with 100, 300, or 500 ng/mL of pEYFP-NUC for 60 or 120 min at 39 °C and 5% (v/v) CO2 in air with high humidity. 2.3. DNA extraction After incubation with various concentrations of exogenous DNA for 60 or 120 min, spermatozoa were washed in PBS (Ca2⫹ and Mg free) for 3 min at 9000 ⫻ g and incubated with 0.1 mg DNase I for 30 min to remove exogenous DNA adsorbed on the sperm surface, but not internalized. After DNAse I treatment, spermatozoa were washed in PBS (Ca2⫹ and Mg free) for 3 min at 9000 ⫻ g. The sperm pellet was subjected to five freeze/thaw cycles to induce plasma membrane lysis, facilitating DNA retrieval, and then put in spermlysing buffer (50 L SDS 10%, 5 L Proteinase K 20 mg/mL, and 375 L sodium acetate 0.2 M) for 2 h at 56 °C. After incubation, spermatozoa were centrifuged
in a solution of phenol:chloroform:isoamyl alcohol (25: 24:1, v/v) at 16,000 ⫻ g for 2 min. The supernatant was removed and centrifuged in isopropanol at 16,000 ⫻ g for 2 min. The resultant pellet was centrifuged in ethanol (70%) at 16,000 ⫻ g for 2 min, dried, and resuspended in TE solution (Tris-Cl 10 mM, pH 8.0, and EDTA 1 mM) at 56 °C for 1 h. The recovered DNA was quantified with a spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), diluted to a final concentration of 1 ng/L in H2O, and stored at ⫺20 °C for later use as a template for real-time PCR. 2.4. Assessment of DNA uptake Quantitative DNA uptake analyses were performed by real time PCR [17] using the Mastercycler® ep reaplex (Eppendorf, Hamburg, Germany). Standard curves were generated using serial dilutions of pEYFP-NUC plasmid (2, 0.2, 0.02, 0.002, and 0.0002 ng). Quantitative PCR was performed in triplicate using a Platinum SYBR GreenER qPCR supermix (Invitrogen, Carlsbad, CA, USA) with a reaction solution containing 12.5 L of supermix, 300 nM of each primer, and 5 pg of DNA. Water was added to achieve a final volume of 25 L. Forward and reverse primers were 5=-ATGGCCGACAAGCAGAAGAAC-3= and 5=-TGCCGTCCTCGATGTTGTG-3=, respectively. The thermal cycler program was 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 64 °C for 1 min, and a melting curve. 2.5. Sperm chromatin structure assay A sperm chromatin structure assay (SCSA) assessed chromatin stability, using metachromatic staining with acridine orange (AO), as previously described [18]. Acridine orange emits green or red fluorescence when it intercalates with double- or single-stranded DNA, respectively. Briefly, sperm samples were diluted with TNE buffer (Tris 10 mM, pH 7.4, 0.15 M NaCl, and 1 mM EDTA) into a final concentration of approximately 1 ⫻ 106 cells/mL. An aliquot (200 L) was mixed with 400 L of a detergent/acid solution (0.1% Triton X-100, 0.15 M NaCl, and 0.08 N HCl, pH 1.4). After 30 s, spermatozoa were stained with 1.2 mL of AO (6 g/mL in 0.1 M citric acid, 0.2 M Na2HPO4, 1 mM EDTA, and 0.15 mM NaCl, pH 6.0). Within 3 min after AO staining, stained samples were analyzed by flow cytometry. 2.6. Assessment of sperm DNA integrity using a TUNEL assay Detection of DNA strand breaks (single- as well as double-stranded DNA) were done with a TUNEL as-
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say, using the Guava TUNEL Kit (Guava Technologies, Hayward, CA, USA), as previously described [19]. Briefly, spermatozoa were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 60 min at 4 °C and permeabilized with 1% Triton in PBS for 30 min at 4 °C. After fixation and permeabilization, spermatozoa were washed with washing buffer supplied with the kit, and then incubated in a DNA labeling mix (TdT reaction buffer, TdT enzyme, Brd-UTP, and distilled H2O) for 2 h at 37 °C. Thereafter, spermatozoa were centrifuged at 300 ⫻ g for 5 min in 200 L of rinsing buffer and incubated in Anti-BrdU staining mix (TRITC-conjugated anti-BrdU antibody) for 30 min at room temperature in the dark. Finally, 150 L of rinsing buffer was added, and spermatozoa were analyzed by flow cytometry. 2.7. Flow cytometry analysis Flow cytometry analyses (for TUNEL and SCSA) were performed using the Guava Easycyte Mini Flow Cytometry System (Guava Technologies, Hayward, CA, USA). This device contains a laser, which operates at 488 nm and emits a 20 mW visible laser radiation. A total of 10,000 events per sample were analyzed and data corresponding to yellow (PM1 photodetector— 583 nm), red (PM2 photodetector— 680 nm), and green fluorescence signals (PM3 photodetector—525 nm) were recorded after logarithmic amplification. Flow cytometry dot plots used were PM2/PM3 for the SCSA assay and PM1/FSC (Forward scatter) for the TUNEL assay. 2.8. Experimental design 2.8.1. Effects of exogenous DNA concentration and incubation period on exogenous DNA uptake by bovine spermatozoa The objective of this experiment was to evaluate the effects of exogenous DNA concentration and incubation period on exogenous DNA uptake. Spermatozoa were incubated with a pEYFP-Nuc plasmid at concentrations of 100, 300, or 500 ng, for 60 or 120 min, in sperm-TL at 39 °C in a humidified atmosphere of 5% (v/v) CO2. Exogenous DNA uptake was quantified by real-time PCR. This experiment was replicated three times using separate batches of frozen semen, and realtime PCR was performed in triplicate. 2.8.2. Effects of exogenous DNA concentration and incubation time on endogenous DNA integrity The aim of this experiment was to evaluate the effects of exogenous DNA concentration and incuba-
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tion period on endogenous DNA integrity. Spermatozoa were incubated with a pEYFP-Nuc plasmid at concentrations of 100, 300, or 500 ng for 60 or 120 min in sperm-TL at 39 °C in a humidified atmosphere of 5% (v/v) CO2. The percentages of endogenous DNA integrity were assessed by SCSA and TUNEL assays, coupled to flow cytometry. This experiment was replicated three times using different batches of frozen semen (10,000 spermatozoa/replicate). 2.9. Statistical analysis Data were analyzed by least-squares analysis of variance, using the General Linear Models (GLM) procedure of SAS (SAS Institute Inc., Cary, NC, USA). The mathematical model included the main effects of incubation period and DNA concentration, as well as their interaction. Main effects were considered fixed. Orthogonal contrasts and a mean separation procedure (pdiff) were performed when appropriate to located differences. Values are presented as mean ⫾ SEM, and P ⬍ 0.05 was considered significant. 3. Results 3.1. Effects of exogenous DNA concentration and incubation time on exogenous DNA uptake by bovine spermatozoa Incubation of frozen-thawed bovine spermatozoa with exogenous DNA resulted in spontaneous DNA uptake. It was noteworthy that exogenous DNA uptake by spermatozoa increased (P ⬍ 0.05) as incubation time increased from 60 to 120 min, but only for 500 ng of exogenous DNA (0.0063 ⫾ 0.009 and 0.0284 ⫾ 0.0277 ng, respectively). Similarly, the increase of exogenous DNA concentration from 100 to 500 ng only had an effect (P ⬍ 0.05) on exogenous DNA uptake by spermatozoa at 120 min of incubation (0.00226467 ⫾ 0.0012 and 0.0284 ⫾ 0.0277 ng, respectively; Fig. 1). 3.2. Effect of exogenous DNA on the DNA integrity of bovine spermatozoa assessed by flow cytometry Based on SCSA, there was no effect of exogenous DNA concentration or incubation period on the DNA integrity of spermatozoa incubated with 0 ng (96.56 ⫾ 0.7 and 91.56 ⫾ 3%), 100 ng (94.13 ⫾ 3.3 and 93.01 ⫾ 1.4%), 300 ng (94.48 ⫾ 3.8 and 93.66 ⫾ 2.9%), or 500 ng (94.96 ⫾ 1 and 93.74 ⫾ 1.9%) of exogenous DNA for 60 or 120 min, respectively. Similarly, based on the TUNEL assay, sperm DNA integrity was not affected when incubated with 0 ng (95.88 ⫾ 3.3 and 96.59 ⫾
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Fig. 1. Mean ⫹ SEM amounts of exogenous DNA internalized (assessed with real-time PCR) in bovine spermatozoa cells after 60 or 120 min of incubation with various concentrations of exogenous DNA. The uptake of DNA for 500 ng incubated for 60 min was greater (P ⬍ 0.05) than all other combinations (none of which were significantly different from each other).
2.8%), 100 ng (96.42 ⫾ 2.3 and 96.37 ⫾ 2.3%), 300 ng (96.61 ⫾ 2.9 and 96.4 ⫾ 1.8%), or 500 ng (96.36 ⫾ 2.4 and 95.49 ⫾ 3.2%) of exogenous DNA for 60 or 120 min. 4. Discussion Sperm-mediated gene transfer is a biochemically regulated process. Binding of exogenous DNA is mediated by a 30 –35 kDa plasma membrane protein, and the uptake and internalization of exogenous DNA is mediated by major histocompatibility complex-class II (MHC II) and CD4 [20]. In the present study, exogenous DNA uptake by frozen-thawed bovine spermatozoa was increased with the highest concentration of DNA and the longest incubation period. There are several techniques to determine the association between exogenous DNA and spermatozoa. For example, exogenous DNA can be localized in situ by autoradiography using radiolabeled plasmids or by fluorescence microscopy and flow cytometry using fluorescent-labeled plasmids. Although these methods localize the labeled DNA in situ, they have the inconvenience of being radioactive or failing to quantify the amount of exogenous DNA associated with spermatozoa. However, these limitations can be avoided with real-time PCR. In the current study, real-time PCR efficiently quantified exogenous plasmid DNA associated with spermatozoa. The duration of incubation of spermatozoa and exogenous DNA played a role in SMGT [7,9,21,22]. In the present work, there was a beneficial effect of an
extended incubation time on SMGT efficiency, but only with the highest concentration of exogenous DNA. Exogenous DNA starts to associate with bull spermatozoa after 15–20 min of incubation [9], with a maximal association at ⬃30 – 40 min [21–23]. Additionally, the prevalence of spermatozoa with exogenous DNA internalized in their nucleus peaked at 65%, after 60 min of incubation [24]. In the present study, internalization of exogenous DNA was greatest for the longest incubation period and highest concentration of DNA. However, it was noteworthy that the real-time PCR used to quantify internalization of exogenous DNA could not differentiate whether this increase was due to a greater amount of exogenous DNA internalized by each spermatozoa, or a greater proportion of spermatozoa which internalized the DNA. This critical question remains unresolved, but should yield important knowledge to understand and increase the efficiency of SMGT. Although SMGT efficiency was improved by a long incubation period and high concentration of exogenous DNA, these conditions could trigger endonuclease activity [15], presumably cleaving both exogenous and endogenous DNA in an apoptosis-like process [15]. Consequently fertilization rates [25] and the efficiency of exogenous DNA uptake were compromised [15], thereby reducing the rate of formation of viable transgenic embryos. In a previous study, exogenous DNA uptake had a negative effect on spermatozoa viability, manifested as reduced sperm motility [26]. In the present study, however, there was no apparent effect of incubation time or exogenous DNA concentration on endogenous DNA integrity which was evaluated using two methodologies (SCSA and TUNEL assay). The lack of exogenous DNA effect on DNA fragmentation could be attributed to the chromatin structure of bovine spermatozoa. In contrast to mouse spermatozoa, however, bovine spermatozoa lack protamine 2; they only have protamine 1 [27]. Since DNA fragmentation is more related to alterations of protamine 2 versus protamine 1 [28], this can confer greater resistance to apoptosis-like processes in bovine spermatozoa compared to murine spermatozoa. Furthermore, endonucleases from ejaculated spermatozoa are less reactive to exogenous DNA stimulation than endonucleases from epididymal spermatozoa [15]. Although exogenous DNA is extensively degraded during transfection of porcince spermatozoa [15], in bovine spermatozoa this DNA remains intact [29]. The endonuclease activity can also explain differences in DNA uptake by spermatozoa in the present
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study. We speculated that endonuclease activity was reduced over time following incubation with high DNA concentration reaching its saturation point. Perhaps endonuclease activity is time- and concentration-dependent of exogenous DNA. Besides the fact that bovine spermatozoa endonucleases activity is low [29], it is also possible that the result from DNA uptake by spermatozoa in the present study was due to low DNA/spermatozoa interaction and low uptake efficiency. Therefore, in order to optimize SMGT in bovine spermatozoa, it may be necessary to use a high exogenous DNA concentration and a long incubation period. In conclusion, the current findings were consistent with previous reports that bovine spermatozoa can spontaneously take up exogenous DNA. Internalization of exogenous DNA by spermatozoa was improved only when a high exogenous DNA concentration was associated with an extended incubation period. In addition, neither an increased concentration of exogenous DNA, nor a prolonged incubation period, nor both, induced endogenous DNA fragmentation as a consequence of an apoptosis-like process. Acknowledgements This study was supported by The State of São Paulo Research Foundation (FAPESP), fellowship 03/10234-7 and grant 03/07456-8. References [1] Brinster RL, Chen HY, Trumbauer ME, Avarbock MR. Translation of globin messenger RNA by the mouse ovum. Nature 1980;283:499 –501. [2] Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 1980;77:7380 – 4. [3] Celebi C, Guillaudeux T, Auvray P, Vallet-Erdtmann V, Jegou B. The making of ”transgenic spermatozoa”. Biol Reprod 2003; 68:1477– 83. [4] Smith K, Spadafora C. Sperm-mediated gene transfer: applications and implications. Bioessays 2005;27:551– 62. [5] Brackett BG, Baranska W, Sawicki W, Koprowski H. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Natl Acad Sci USA 1971; 68:353–7. [6] Arezzo F. Sea-urchin sperm as a vector of foreign genetic information. Cell Biol Int Rep 1989;13:391– 404. [7] Lavitrano M, Camaioni A, Fazio VM, Dolci S, Farace MG, Spadafora C. Sperm cells as vectors for introducing foreign DNA into eggs - genetic transformation of mice. Cell 1989; 57:717–23. [8] Atkinson PW, Hines ER, Beaton S, Matthaei KI, Reed KC, Bradley P. Association of exogenous DNA with cattle and insect spermatozoa in vitro, Mol Reprod Dev 1991;29:1–5.
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