Introduction
In the past decades, the ability to modify gene expression through transgenesis, particularly in mammals, has become one of the most important advances in the field of applied and experimental biology (Celebi et al., Reference Celebi, Guillaudeux, Auvray, Vallet-Erdtmann and Jégou2003). Sperm-mediated gene transfer (SMGT), a simple, low-cost technique, which, in theory, can be used in all species that reproduce through gametes, is drawing researchers’ attentions. Nevertheless SMGT presents inconsistent results and is difficult to reproduce (Maione et al., Reference Maione, Pittogi, Achene, Lorenzini and Spadafora1998).
A better understanding of how exogenous DNA molecules interact with sperm cells is crucial to optimize SMGT. Longer incubation time results in a higher binding rate of exogenous DNA and sperm cells followed by the internalization of the nucleus (Castro et al., Reference Castro, Hernandez, Uliver, Solano, Milanés, Aguilar, Pérez, De Armas, Herrwera and De La Fuente1990; Francoline et al., Reference Francolini, Lavitrano, Lamia, French, Frati, Cotelli and Spadafora1993). However, longer incubation time can affect sperm viability resulting in lower fertilization rates and consequently fewer transgenic embryos. Understanding how incubation time and exogenous DNA affect sperm viability might reduce the inconsistency of results and improve cell transfection rates.
In order to maximize the production of transgenic animals it is necessary to determine when, for how long, what type and what amount of exogenous DNA should be added to the sperm, so that exogenous DNA is efficiently bound and internalized by the majority of sperm cells. Therefore, understanding the relationship between exogenous DNA and sperm is crucial. Sperm viability influences the efficiency of exogenous DNA binding and internalization. However, SMGT protocols reveal that little is known about the effect of exogenous DNA on sperm viability.
The aim of this study was to assess the effect of exogenous DNA addition and incubation time on sperm motility, plasma membrane integrity, acrosome membrane integrity and mitochondrial membrane potential.
Material and methods
Preparation of sperm and incubation with exogenous DNA
Bovine semen was thawed in water at 37 °C for 30 s and subjected to Percoll gradient separation (45 and 90%) at 600 g for 30 min. To remove excess Percoll, the sediment was resuspended and washed in Sperm-TALP medium at 200 g for 5 min. Sperm was suspended in fertilization medium (Parrish et al., Reference Parrish, Susko-Parrish, Winer and First1988) without heparin at a concentration of 5 × 106/ml and incubated at 39 °C and in 5% CO2 (v/v) in air and under high humidity for 0 (incubation time control), 1, 2, 3 and 4 h with or without (exogenous DNA control) 500 ng/ml of plasmid pEYFP–NUC (Clontech, BD Biosciences), linearized with StuI restriction enzyme.
Assessment of motility, plasma and acrosome membrane integrity and mitochondrial membrane potential
Sperm motility was assessed by evaluating sperm placed on a slide, covered with a coverslip and examined under a light microscope. Fluorescent probes, propidium iodide (PI) and Hoechst 33342 (H342), were used to assess plasma membrane integrity. Fluorescently labelled Pisum sativum lectin (PNA–FITC) was used to assess acrosome integrity. Mitochondrial function was assessed with the JC-1 probe, which marks mitochondria that have high membrane potentials as fluorescencent red and mitochondria with low membrane potentials as fluorescencent green.
A volume (2 μl) of Hoechst 33342 (40 μg/ml in DMSO) was added to 150 μl of semen that had been diluted in TALP (5 × 106 sperm/ml) and incubated for 10 min at 37 °C. Then 3 μl propidium iodide (0.5 mg/ml in Dulbecco's phosphate-buffered saline solution – DPBS), 2 μl JC-1 (76.5 μM in DMSO) and 50 μl PNA–FITC (100 μg/ml added 10% of sodium azide solution at 10%) were added and incubated for an additional 10 min at 25 °C. Following incubation, 10 μl of the solution was spread on a slide and covered with a glass coverslip. At least 200 cells were examined under an epifluorescence microscope (Olympus) using 355, 490 and 520 nm excitation filters and 465, 252 and 610 nm emission filters, respectively. The association of the four probes allowed us to classify sperm into eight different groups (Celeghini et al., Reference Celeghini, Arruda, Andrade, Nascimento and Raphael2007), described in Table 1.
Classification of sperm according to fluorescence of propidium iodide, Hoechst, FITC–PSA and JC-1 probes.
Statistical analysis
For the purpose of this study we used semen samples from three bulls, in a total of 10 repetitions per treatment in a 5 × 2 factorial analysis. In all evaluations 200 cells were counted in nine quadrants of the slide. Experiment data were analysed using statistical software MINITAB Release 14. Prior to software analysis the homogeneity and distributions of variables was verified and data were subjected to analysis of variance and comparison using the Turkey test with a 5% significance level.
Results
After 4 h of incubation, exogenous DNA did not affect sperm motility (Table 2), plasma and acrosome membrane integrity and mitochondrial membrane potential. However, increased incubation time reduced sperm viability. In the group that was incubated with exogenous DNA and in the group incubated without exogenous DNA, sperm motility was high after 1 h of incubation (p < 0.05). Nevertheless, in both groups, sperm motility was reduced when compared with the control group.
Average motility of sperm incubated with (+) or without (−) exogenous DNA.
Different letters represent significant differences (p < 0.05). AThe effect of exogenous DNA; comparison between lines in the same column. a–dThe effect of incubation time; comparison between columns in the same line.
Class 1 sperm were more frequent after 1 h of incubation with and without exogenous DNA, but these results were still lower than results from the control group (0 h) (p < 0.05). Regarding classes 2, 3, 4 and 7, no variation was noticed throughout the incubation period. Class 5 sperm were more frequent at 0, 1 and 2 h of incubation without exogenous DNA and at 1 h incubation with exogenous DNA (p < 0.05). Class 6 sperm were more frequently observed at 2, 3 and 4 h incubation without exogenous DNA and at 3 and 4 h incubation with exogenous DNA (p < 0.05). Class 8 sperm were more frequently seen after 3 and 4 h of incubation (p < 0.05) with and without exogenous DNA (Table 3).
Percentage of sperm incubated with (+) or without (−) exogenous DNA at different time points and distributed in eight classes according to fluorescent probes: propidium iodide, Hoechst, FITC–PSA and JC-1.
a–dDifferent letters represent significant differences (p < 0.05) between the effect of incubation time, comparison between columns in each class.
Sperm that presented plasma membrane integrity (PMI) (Σ classes 1, 2, 3 and 4), acrosome membrane integrity (AMI) (Σ classes 1, 2, 5 and 6) and high mitochondrial membrane potential (HMMP) (Σ classes 1, 3, 5 and 7) were evaluated separately (Fig. 1). Sperm that presented plasma membrane integrity were more frequently observed at 0 and 1 h incubation with and without exogenous DNA (p < 0.05). A greater number of sperm with acrosome membrane integrity was observed at 0 and 1 h of incubation with and without exogenous DNA and at 2 h incubation without exogenous DNA (p < 0.05). The number of sperm with high mitochondrial membrane potential was also higher at 0 and 1 h incubation (p < 0.05).
Percentage of bovine sperm with: (a) acrosome membrane integrity; (b) plasma membrane integrity; and (c) high mitochondrial membrane potential after different incubation times without or with exogenous DNA. Different letters represent significant difference (p < 0.05). Uppercase letters represent the effect of incubation time on sperm cell viability after incubation without exogenous DNA; lowercase letters represent the effect of incubation time on sperm cell viability after incubation with exogenous DNA.
Discussion
This study assessed the effect of incubation time and exogenous DNA addition on sperm motility, plasma membrane integrity, acrosome membrane integrity and mitochondrial membrane potential. According to our results, exogenous DNA has no influence on sperm viability (exogenous DNA uptake by sperm cells was confirmed by PCR in all incubation times, data not shown), which, on the other hand, is affected by the incubation time.
In SMGT, exogenous DNA interacts with sperm cells after 15 min of incubation and reaches a maximum level of binding at 40–45 min, followed by a plateau (Castro et al., Reference Castro, Hernandez, Uliver, Solano, Milanés, Aguilar, Pérez, De Armas, Herrwera and De La Fuente1990; Camaioni et al., Reference Camaioni, Russo, Odorisio, Gandolfi, Fazio and Siracusa1992). Results of the present study agree with data presented in other studies (Castro et al., Reference Castro, Hernandez, Uliver, Solano, Milanés, Aguilar, Pérez, De Armas, Herrwera and De La Fuente1990; Camaioni et al., Reference Camaioni, Russo, Odorisio, Gandolfi, Fazio and Siracusa1992) and might explain the existence of a plateau phase in SMGT. According to these studies, following 1 h of incubation sperm presented reduced motility, plasma and acrosome membrane disruption and a reduction in mitochondrial membrane potential.
In the present study, when compared with sperm incubated without exogenous DNA, the addition of exogenous DNA to the incubation medium did not influence plasma and acrosome membrane integrity or mitochondrial membrane potential. These results conflict with results presented by Anzar & Buhr (Reference Anzur and Buhr2006), who reported that DNA uptake reduces plasma membrane integrity. However, our results confirm data presented by Lavitrano et al. (Reference Lavitrano, Camaioni, Fazio, Dolci, Farace and Spadafora1989) who reported that incubation with exogenous DNA did not cause morphologic changes in sperm. Despite the finding that exogenous DNA uptake did not influence sperm viability, other studies reported that sperm viability influences fertilization and binding of exogenous DNA molecules within sperm cells (Lavitrano et al., Reference Lavitrano, Camaioni, Fazio, Dolci, Farace and Spadafora1989; Castro et al., Reference Castro, Hernandez, Uliver, Solano, Milanés, Aguilar, Pérez, De Armas, Herrwera and De La Fuente1990; Horan et al., Reference Horan, Powell, Mcquaid, Gannon and Houghton1991; Anzar & Buhr, Reference Anzur and Buhr2006).
Increased incubation time reduced average motility of sperm incubated with and without exogenous DNA. Motility reduction during incubation might play an important role in the transfection of sperm cells. Furthermore, sperm with good motility showed higher exogenous DNA binding rates than sperm with no motility (Horan et al., Reference Horan, Powell, Mcquaid, Gannon and Houghton1991). However, it is not known whether this increased binding rate is the consequence of more efficient DNA interaction and internalization mechanisms or of greater exposure of motile sperm to exogenous DNA molecules, which would favour the interaction of these molecules with the sperm cells.
In vivo and in vitro studies reported that reduced motility also affects fertilization (Kjaestad et al., Reference Kjaestad, Ropstad and Berg1993; Holt et al., Reference Holt, Holt, Moore, Reed and Curnock1997; Tanghe et al., Reference Tanghe, Van Soon, Sterckx, Maes and Kruif2002). Thus, an incubation time with reduced effect on sperm motility not only improves transfection efficiency but also prevents the reduction of fertilization rates.
The interaction, internalization and integration of exogenous DNA within sperm cells is not merely the consequence of a passive and uncontrolled process, it is regulated by factor-specific mechanisms that metabolically activate the cells (Smith & Spadafora, Reference Smith and Spadafora2005). Thus, SMGT might involve energy consumption. Mitochondrial membrane potential of sperm was not affected by exogenous DNA addition. Initially these results suggested that the mechanism that regulates SMGT is an isolated event that does not consume mitochondrial energy, or that energy consumption is extremely low and is not detected by the fluorescent probe JC-1.
However, flagellum motility does not depend exclusively on ATP derived from mitochondrial oxidative phosphorylation. Several studies have shown that glucolysis throughout the main piece is the most important ATP source for the flagellum (Bradley et al., Reference Bradley, Geelan, Leitch and Goldberg1996; Westhoff & Kamp, Reference Westhoff and Kamp1997; Bunch et al., Reference Bunch, Welch, Magyar, Eddy and O'Brien1998; Mori et al., Reference Mori, Nakamura, Welch, Gotoh, Goulding, Fujioka and Eddy1998; Travis et al., Reference Travis, Foster, Rosenbaum, Visconti, Gerton, Kopf and Moss1998). Therefore, considering that SMGT is an energy-demanding process, energy might be derived from glucolysis instead of from the mitochondria.
The plasma membrane also plays an important role in sperm and exogenous DNA interaction. Atikinson et al. (Reference Atkinson, Hines, Beaton, Matthaei, Reed and Bradley1991) did not observe any difference in the percentage of intact sperm with exogenous DNA molecules (18.54%) and disrupted sperm (19.2%). However, they observed a difference in the exogenous DNA-binding site, which suggests that plasma membrane integrity might influence SMGT. Even though the plasma membrane affects exogenous DNA uptake, our results showed that exogenous DNA not affect plasma membrane integrity. On the other hand, Lavitrano et al. (Reference Lavitrano, Camaioni, Fazio, Dolci, Farace and Spadafora1989) observed that only live cells bind to exogenous DNA molecules. This information was later confirmed by Castro et al. (Reference Castro, Hernandez, Uliver, Solano, Milanés, Aguilar, Pérez, De Armas, Herrwera and De La Fuente1990) and Anzar & Buhr (Reference Anzur and Buhr2006). Furthermore, a sperm with functional disruption of the plasma membrane is considered deteriorated (dead) and is incapable of in vivo or in vitro fertilization (Silva & Gadella, Reference Silva and Gadella2006).
In this study exogenous DNA did not affect acrosome membrane integrity. However, incubation time played a role, causing acrosome membrane disruption during increased incubation periods. Acrosome membrane integrity does not influence exogenous DNA binding to sperm cells without a difference in exogenous DNA uptake by sperm with and without acrosome reaction (Horan et al., Reference Horan, Powell, Mcquaid, Gannon and Houghton1991).
Nevertheless, despite being no correlation between acrosome membrane integrity and exogenous DNA uptake rates, acrosome membrane integrity is crucial for fertilization. The acrosome must remain intact before and during the passage of the sperm through the isthmus of the uterine tube and until it reaches the zona pellucida. Early acrosome reaction (Silva & Gadella, Reference Silva and Gadella2006) or absence thereof (Bielfeld et al., Reference Bielfeld, Faredi, Zaneveld and Dejomge1994) hinders zona pellucida penetration and consequently reduces fertilization capacity.
Our study shows that whereas exogenous DNA does not influence sperm viability, longer incubation times reduce sperm viability. According to the literature, reduced sperm viability affects binding of exogenous DNA to sperm cells (Lavitrano et al., Reference Lavitrano, Camaioni, Fazio, Dolci, Farace and Spadafora1989; Castro et al., Reference Castro, Hernandez, Uliver, Solano, Milanés, Aguilar, Pérez, De Armas, Herrwera and De La Fuente1990; Atkinson et al., Reference Atkinson, Hines, Beaton, Matthaei, Reed and Bradley1991; Horan et al., Reference Horan, Powell, Mcquaid, Gannon and Houghton1991; Anzar & Buhr, Reference Anzur and Buhr2006). Thus, increasing the incubation time and reducing sperm viability might reduce SMGT efficiency and fertilization.
Acknowledgements
The authors thank the State of Sao Paulo Research Foundation (FAPESP) for financial support (Fellowship: 03/10234–7 and Grant: 03/07456–8) and Melanie Klemm for the translation and revision of the text.
