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The impact of exogenous DNA on the structure of sperm of olive flounder (Paralichthys olivaceus)
Animal Reproduction Science 149 (2014) 305–310
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
The impact of exogenous DNA on the structure of sperm of olive flounder (Paralichthys olivaceus) Nian Xin, Tiantian Liu, Haitao Zhao, Zhenwei Wang, Jinxiang Liu, Quanqi Zhang, Jie Qi ∗ Key Laboratory of Marine Genetics and Breeding, College of Marine Life Science, Ocean University of China, Qingdao, Shandong, 266003, China
a r t i c l e
i n f o
Article history: Received 13 February 2014 Received in revised form 7 June 2014 Accepted 23 June 2014 Available online 3 July 2014 Keywords: DNase Liposome-DNA complexes Naked-DNA Paralichthys olivaceus Sperm-mediated gene transfer (SMGT)
a b s t r a c t Sperm-mediated gene transfer (SMGT) is a promising transgenic technology that relies on the capability of sperm to internalize exogenous DNA. In marine fish, however, the interaction between sperm and exogenous DNA appears to be deficient. Here, we demonstrated significant DNase activity in the seminal plasma of the olive flounder. When incubated with naked-DNA, the spermatozoa lost their structural integrity, including the head, mitochondria and flagellum, in an incubation time-dependent manner. However, internalization of a liposome-DNA complex resulted in the structural integrity of the spermatozoa being maintained, even when using incubation times of up to 50 min. We concluded that in the olive flounder, SMGT is possible by integrating liposome-DNA complexes, rather than naked-DNA alone, into the sperm. In brief, removal of the seminal plasma and packaging the exogenous DNA were necessary for successful SMGT in the olive flounder. © 2014 Published by Elsevier B.V.
1. Introduction Sperm-mediated gene transfer (SMGT) has been used extensively, because of its efficiency and cost-effectiveness, for generating transgenic animals. The first evidence that sperm could internalize exogenous DNA was reported in the early 1970s (Brackett et al., 1971). Significant progress was subsequently made using SMGT to generate transgenic embryos and animals in many species, including amphibians (Habrova et al., 1996), birds (Nakanishi & Iritani, 1993; Collares et al., 2011) and mammals (Lavitrano et al., 1989; Hoelker et al., 2007) as well as other species, such as the sea urchin and silkworm (Arezzo, 1989; Zhou et al., 2012). The efficiency of SMGT was limited by the effectiveness of
∗ Corresponding author. Tel.: +86 532 82031986; fax: +86 532 82031986. E-mail address: [email protected] (J. Qi). http://dx.doi.org/10.1016/j.anireprosci.2014.06.029 0378-4320/© 2014 Published by Elsevier B.V.
the internalization of the exogenous DNA by the sperm as well as by the frequency of stable integration of the exogenous DNA in the genome (Maione et al., 1998). To increase the efficiency of SMGT, the interaction between exogenous DNA and sperm was the focus of many studies. In mammals, for example, it was shown that in addition to the existence of positive factors, which contributed to the internalization of exogenous DNA by the sperm nucleus (Zani et al., 1995), there were also negative regulatory elements, such as inhibitory factor 1 (IF-1) and DNase in the seminal plasma (Carballada & Esponda, 2001). Once the negative regulatory elements were triggered, the exogenous nakedDNA was subsequently degraded (Maione et al., 1997). To avoid this degradation effect, the naked-DNA was coated with materials such as liposomes, nanopolymer and halloysite clay nanotubes to form complexes (Lai et al., 2001; Campos et al., 2011b), to prevent contact directly between the sperm and the exogenous DNA. In addition, several other factors such as incubation time of the sperm with
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the exogenous DNA, the amount of exogenous DNA used per sperm cell, DNA architecture and semen quality were also shown to affect the uptake of exogenous DNA by the sperm (Lavitrano et al., 2003; Lavitrano et al., 2006; Hoelker et al., 2007). Although several specific methodologies have been developed (Horan et al., 1992; Chang et al., 2002), the transgenic efficiency of SMGT was never very successful in marine fish because the interaction between the sperm and the exogenous DNA was not fully understood. The aim of this study was therefore to determine: (1) the existence of DNase activity in the seminal plasma and (2) the impact of naked-DNA and liposome-DNA complexes on sperm viability in the olive flounder. These data provide important information for developing an SMGT protocol in marine fish such as the olive founder.
the jetPEItm reagent and ‘#’ is the number of nmoles of phosphate (P) per g of DNA. To confirm the N/P ratio for optimal sperm fertility (i.e., the survival rate of embryos to the blastula stage, based on 500 embryos at the 1-cell stage), a series of solutions with various N/P ratios (i.e., N/P = 3, 4, 5, 6, 7, 8, 9, and 10) were designed according to the jetPEItm kit (Promega) manufacturer’s instructions. The oocytes were collected from healthy female fish by gentle abdominal massage and then fertilized with the sperm-free seminal plasma and treated by exogenous naked-DNA or liposome-DNA complexes (described in Section 2.2) at different N/P ratios. Finally, the optimal N/P ratio was selected according to the highest fertility rate.
2. Materials and methods
2.4. Preparation of the Liposome-DNA complexes
2.1. Animals and sperm collection Adult olive flounder were maintained in a culture pond at Hai Yang, Shandong Province, China. The temperature and salinity of the seawater was maintained at 15 ± 2 ◦ C, and 30%, respectively, and the fish were kept on a 14 h:10 h light/dark cycle in standard fish facility conditions. The spermatozoa of three males were collected during March to May. Fish were carefully turned onto their ocular side and then the abdomen and genital pore were dried with sterile filter paper to prevent the sperm from being activated by seawater and/or urine. The sperm were artificially ejected by massage using gloved hand; a 5 mL straw was then used to collect the sperm ejected from the genital pore, and subsequently transfer them into a 1.5 mL microtube. 2.2. Removal of the seminal plasma from the semen Immediately after sperm collection, the seminal plasma was removed by centrifugation at 12,000 rpm for 15 min after which the supernatant (containing the seminal plasma) was discarded carefully. The sperm were diluted to a final concentration of 106 cells/l in Ringer’s solution (13.5 g/L NaCl, 0.6 g/L KCl, 0.02 g/L NaHCO3 ) with Ca2+ and Mg2+ free to avoid the activation of sperm. The centrifuged and diluted sperm were then transferred to a clean 1.5 mL microtube and kept at room temperature for immediate use in experiments. 2.3. Appropriate N/P ratio selection The N/P ratio represents the ionic balance within the liposome–DNA complexes; it refers to the number of nitrogen residues (N) in the transfection reagent per phosphate (P) of DNA. The overall charge of the liposome-DNA complexes is a crucial factor in transfection experiments, and is determined by the N/P ratio, such that a positive charge is necessary for successful complex integration by promoting interaction with anionic proteoglycans on the cell surface. The N/P ratio is calculated using the following formula: N/P ratio = (7.5* × l of jetPEItm )/(3# × g of DNA), where ‘*’ is the concentration of nitrogen (N) residues in
To form the liposome-DNA complexes, 1 g of the circular pEGFP-C1 DNA plasmid (Clontech) was incubated with 2 l transfection reagent to a final volume of 50 l in reagent buffer from the jetPEItm kit, according to the manufacturer’s standard protocol. The mixture was then incubated for 15–20 min at room temperature after which it was stored for either 2 h at room temperature or 24 h at 4 ◦ C.
2.5. Detection of DNase activity in the seminal plasma The exogenous naked-DNA plasmid or liposome-DNA complexes (1 g) were incubated with 20 l seminal plasma or DNaseI (New England Biolabs) for 5 min, 15 min or 30 min at 14 ◦ C or 23 ◦ C, which is the temperature range for growth and breeding of the olive flounder, as well as at 37 ◦ C, which is the optimum reaction temperature for DNaseI. Subsequently, the mixture was analyzed by electrophoresis on a 1% agarose gel containing ethidium bromide (0.5 g/mL) and visualized under a UV light.
2.6. Preparation of naked-DNA or liposome-DNA and sperm mixtures Sperm were mixed with either the naked-DNA or with the liposome-DNA complexes. 1 g naked-DNApEGFP-C1 or 100 l liposome-DNA containing 1 g pEGFP-C1 plasmid DNA was incubated with 10 l seminal plasma-free sperm for 10 min, 15 min, 30 min, or 50 min at room temperature.
2.7. Analysis of sperm structure by scanning electron microscopy The sperm that were treated with either naked-DNA or liposome-DNA were fixed using 2.5% glutaraldehyde for subsequent scanning electron microscopy (JSM-840, JEOL, and Japan) to investigate the effect of naked-DNA and liposome-DNA on the structure of the sperm following treatment for 10 min, 15 min, 30 min or 50 min.
N. Xin et al. / Animal Reproduction Science 149 (2014) 305–310
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3.3. Effect of naked-DNA and liposome-DNA on sperm
Fig. 1. Detection of DNase activity in Paralichthys olivaceus seminal plasma. DNase activity was analyzed by electrophoresis on 1% agarose gel. M, 1 kb DNA molecular marker; lanes 1–3, mixture of seminal plasma and plasmid for 5 min, 15 min or 30 min at 14 ◦ C; lanes 4–6, mixture of seminal plasma and plasmid for 5 min, 15 min or 30 min at 23 ◦ C; lanes 7 pEGFP-C1 alone; lanes 8-10, mixture of DNase I and naked-DNA plasmid for 5 min, 15 min or 30 min at 37 ◦ C. The pEGFP-C1 plasmid was digested when it was incubated with seminal plasma at both 14 ◦ C and 23 ◦ C.
2.8. Data analysis The fertility rate among the controls and at different N/P ratios was compared using the ANOVA method. Data were considered to be significantly different at P < 0.05. 3. Results 3.1. Detection of DNase activity in the seminal plasma To detect if the seminal plasma exhibited any DNase activity, the pEGFP-C1 plasmid was mixed with seminal plasma, for varying lengths of time and at different temperatures. The results showed that the pEGFP-C1 plasmid was digested when it was incubated with seminal plasma; this is somewhat similar to the positive control when pEGFP-C1 plasmid was digested with DNaseI at 37 ◦ C. However in the latter case, the DNAase I digested the exogenous DNA completely (Fig. 1). The pEGFP-C1 plasmid was digested when it was incubated in seminal plasma at both 14 ◦ C and 23 ◦ C. 3.2. Appropriate N/P ratio selection based on the fertility rate As an N/P ratio greater than 3 is required to obtain a positive charge, we tested N/P ratios of between 3 and 10. Liposome-DNA complexes at different N/P ratios were prepared and incubated with seminal plasma-free sperm for 30 min according to the jetPEItm kit instructions, in order to determine the fertility rate (Fig. 2). Compared with the untreated sperm, the fertility rate was not significantly different when the N/P ratio was 3, 4, 5 or 6 (i.e., in all cases P > 0.05). On the other hand, the fertility rate was significantly lower than the control when the N/P ratio was between 7 and 10 (i.e., P < 0.01). According to the P values calculated, N/P ratios of 5 and 6 were selected as being most suitable for the sperm incubation experiments. Furthermore, a comparison of the average fertility rate with N/P ratios of 5 and 6 showed that the former was higher than the latter. Thus, an N/P ratio of 5 was determined to be optimal for the preparation of the liposome-DNA complexes.
When sperm were incubated with liposome-DNA complexes at 10 ng/106 sperm cells, for 5 min, 10 min, 15 min, 30 min or 50 min, the subsequent fertilization rate was not significantly different than when they were incubated with untreated sperm for the same incubation times (Table 1). On the other hand, when sperm were incubated with naked-DNA, the fertilization rate decreased to 50.1% and 33.4% following incubation times of 10 min and 15 min, respectively; and dropped to approximately zero after an incubation time of 30 min (Table 1). The quality of the sperm following treatment with naked-DNA or liposome-DNA complexes was investigated using scanning electron microscopy. Untreated sperm maintained a normal morphology, including the head, mitochondria and flagellum, after incubation times of 10 min, 15 min, 30 min or 50 min at 14 ◦ C (Fig. 3A–D). In addition, sperm incubated with naked-DNA, also exhibited a relatively normal morphology at 10 min (Fig. 3E); but the mitochondria and flagellum showed obvious degradation after 15 min (Fig. 3F, more severe deterioration by 30 min (Fig. 3G) and they had decomposed completely by 50 min (Fig. 3H). On the other hand, sperm treated with liposomeDNA complexes exhibited an intact morphology at all the time points tested (Fig. 3I–M). These results indicate that the sperm were well protected when using liposome-DNA complexes by isolating the exogenous DNA from the sperm, and in this way, the structural integrity of the sperm were maintained. 4. Discussion It is well known that sperm are capable of combining and internalizing exogenous DNA in some species. In fish, SMGT has been previously applied successfully to several species of transgenic fish, such as the silver sea bream (Lu et al., 2002) and the South American catfish (Campos et al., 2011a). In the case of the catfish, a transgene efficiency of exogenous DNA into larvae of 25% and 38% was identified by PCR and EGFP expression, respectively (Collares et al., 2010). Indeed, the principle of SMGT is based on the ability of sperm to combine and internalize exogenous DNA. However, the interaction between exogenous naked-DNA and the sperm plasma membrane is not random, but is known to be dependent on a specific region of the sperm head called the post-acrosomal region. (Lavitrano et al., 1992). In addition, it has been reported that the sperm plasma membrane plays a critical role in its interaction with DNA (Perry et al., 1999; Anzar & Buhr, 2006; Kurome et al., 2007). More recently, however, Lanes et al. (2009) reported that sperm are effected by endonuclease activity following the degradation of exogenous and endogenous DNA, and they concluded that there is strong DNase activity in the seminal plasma of the Brazilian flounder (Lanes et al., 2009). Therefore, in some species the sperm needs to be incubated with exogenous DNA indirectly to product transgenic embryos (Khoo et al., 1992), whereas in others, the sperm may be incubated directly with the DNA. This is therefore an important detail that needs to be determined before SMGT is developed for a particular species.
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Fig. 2. The effect of N/P ratio on the fertility rate of eggs fertilized with spermatozoa following internalization of a liposome-DNA complex. At N/P ratios of 3, 4, 5 and 6, the fertility rates were 89.3%, 87%, 84.6%, and 81%, respectively, which were similar (i.e., with P > 0.05) to the untreated sperm control group with a fertility rate of 89.3%. On the other hand, at N/P ratios of 7, 8, 9 and 10, the fertility rates were significantly lower (at P < 0.01) than the control, with values of 69.7%, 63.7%, 50% and 48.3%, respectively. Groups marked with the same letter means no statistically difference. Table 1 The effect of incubation time on the fertility rate among different combination groups at an N/P ratio of 5. Combination of plasmid and sperm
Untreated sperm pEGFP-C1plasmid/sperm Lipo-Complex-pEGFP-C1 plasmid/sperm
Fertility rate (%) Incubation time (min) 0
10
15
30
50
90.3 90.1 91.4
90.0 50.1 85.7
87.2 33.4 82.8
83.5 0 78.2
80.2 0 77.8
Fig. 3. Comparison of the morphology of sperm following treatment with either naked-DNA or liposome-DNA complexes at various incubation times. The morphology of untreated sperm in (A–D) the control group; and following treatment with either (E–H) naked-DNA or (I–L) liposome- DNA complexes. Sperm were treated for (A, E, I) 10 min, (B, F, J) 15 min, (C, G, K) 30 min; or (D, H, L) 50 min. The white arrows indicate the mitochondria of the sperm. When sperm were treated with naked-DNA, the mitochondria disappeared gradually over time until, by 50 min, the sperm were completely decomposed. On the other hand, in the untreated sperm and in those treated with liposome-DNA complexes, the mitochondria maintained a normal structural integrity at all the incubation times tested.
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Indeed, our results indicated that the seminal plasma of the olive flounder exhibited obvious DNase activity such that it completely degraded the pEGFP-C1 plasmid. This finding is similar to that of previous studies in mammals (Carballada & Esponda, 2001) and in the Brazilian flounder (Lanes et al., 2009). To our knowledge, this is the first report describing that there were a DNase activity in the seminal plasma of the olive flounder. These experiments suggest that a mechanism similar to that in mammals likely exists in fish. Sperm motility is important for sperm to attach, incorporate, and internalize exogenous DNA, and it is also critical for maintaining successful fertilization with an occyte. Several views support the idea that mitochondrial function is related to sperm motility as the mitochondria generate ATP (cellular energy) by oxidative phosphorylation. ATP is required for sperm motility and hyperactivation, which suggests that sperm mitochondrial function may be important for flagella propulsion and sperm fertilization capacity (Rajender et al., 2010; Ramalho-Santos & Amaral, 2013). Consequently, the physical condition of the mitochondria is thought to be a good determinant of sperm motility and thus quality. In this study, the naked-DNA indeed leads to a degradation of the sperm mitochondria, resulting in a shortened flagellum. In the liposome-DNA complexestreated groups, the change of morphological specificity was not obvious during even the longest incubation periods (i.e., 50 min), but in the exogenous naked-DNA group, the sperm mitochondria were degraded completely after incubated 50 min. However, a previous report on the South American catfish demonstrated that the pEGFP vector was internalized by sperm cells at even lower concentrations without motility loss after seminal plasma removal (Campos et al., 2011a). Furthermore, Collares et al. demonstrated the transgene transmission efficiency of exogenous DNA into silver catfish larvae through SMGT technology was 25% and 38%, respectively, identified by PCR and EGFP expression (Collares et al., 2010), and also showed that removal of the biological constituents of seminal plasma could lead to an increase in the incorporation of foreign DNA. 5. Conclusion Our results demonstrate that: (1) the spermatozoa of Paralichthys olivaceus exhibit DNase activity, and for successful SMGT, it was necessary to remove the seminal plasma. In addition, (2) naked-DNA induced sperm damage; but (3) liposomes coated on the DNA surface could protect the sperm from the harmful effects of exogenous DNA. In summary, the use of liposome-DNA complexes in transfection is a promising approach for the establishment of SMGT in the olive flounder. Acknowledgments We appreciate the help of Dr. Sarah E Webb, The Hong Kong University of Science & Technology (HKUST), for helpful comments on this manuscript. This study was supported by grants from the National High Technology Research and Development Program of China (No. 2012AA10A401).
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The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
The impact of exogenous DNA on the structure of sperm of olive flounder (Paralichthys olivaceus) Nian Xin, Tiantian Liu, Haitao Zhao, Zhenwei Wang, Jinxiang Liu, Quanqi Zhang, Jie Qi ∗ Key Laboratory of Marine Genetics and Breeding, College of Marine Life Science, Ocean University of China, Qingdao, Shandong, 266003, China
a r t i c l e
i n f o
Article history: Received 13 February 2014 Received in revised form 7 June 2014 Accepted 23 June 2014 Available online 3 July 2014 Keywords: DNase Liposome-DNA complexes Naked-DNA Paralichthys olivaceus Sperm-mediated gene transfer (SMGT)
a b s t r a c t Sperm-mediated gene transfer (SMGT) is a promising transgenic technology that relies on the capability of sperm to internalize exogenous DNA. In marine fish, however, the interaction between sperm and exogenous DNA appears to be deficient. Here, we demonstrated significant DNase activity in the seminal plasma of the olive flounder. When incubated with naked-DNA, the spermatozoa lost their structural integrity, including the head, mitochondria and flagellum, in an incubation time-dependent manner. However, internalization of a liposome-DNA complex resulted in the structural integrity of the spermatozoa being maintained, even when using incubation times of up to 50 min. We concluded that in the olive flounder, SMGT is possible by integrating liposome-DNA complexes, rather than naked-DNA alone, into the sperm. In brief, removal of the seminal plasma and packaging the exogenous DNA were necessary for successful SMGT in the olive flounder. © 2014 Published by Elsevier B.V.
1. Introduction Sperm-mediated gene transfer (SMGT) has been used extensively, because of its efficiency and cost-effectiveness, for generating transgenic animals. The first evidence that sperm could internalize exogenous DNA was reported in the early 1970s (Brackett et al., 1971). Significant progress was subsequently made using SMGT to generate transgenic embryos and animals in many species, including amphibians (Habrova et al., 1996), birds (Nakanishi & Iritani, 1993; Collares et al., 2011) and mammals (Lavitrano et al., 1989; Hoelker et al., 2007) as well as other species, such as the sea urchin and silkworm (Arezzo, 1989; Zhou et al., 2012). The efficiency of SMGT was limited by the effectiveness of
∗ Corresponding author. Tel.: +86 532 82031986; fax: +86 532 82031986. E-mail address: [email protected] (J. Qi). http://dx.doi.org/10.1016/j.anireprosci.2014.06.029 0378-4320/© 2014 Published by Elsevier B.V.
the internalization of the exogenous DNA by the sperm as well as by the frequency of stable integration of the exogenous DNA in the genome (Maione et al., 1998). To increase the efficiency of SMGT, the interaction between exogenous DNA and sperm was the focus of many studies. In mammals, for example, it was shown that in addition to the existence of positive factors, which contributed to the internalization of exogenous DNA by the sperm nucleus (Zani et al., 1995), there were also negative regulatory elements, such as inhibitory factor 1 (IF-1) and DNase in the seminal plasma (Carballada & Esponda, 2001). Once the negative regulatory elements were triggered, the exogenous nakedDNA was subsequently degraded (Maione et al., 1997). To avoid this degradation effect, the naked-DNA was coated with materials such as liposomes, nanopolymer and halloysite clay nanotubes to form complexes (Lai et al., 2001; Campos et al., 2011b), to prevent contact directly between the sperm and the exogenous DNA. In addition, several other factors such as incubation time of the sperm with
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N. Xin et al. / Animal Reproduction Science 149 (2014) 305–310
the exogenous DNA, the amount of exogenous DNA used per sperm cell, DNA architecture and semen quality were also shown to affect the uptake of exogenous DNA by the sperm (Lavitrano et al., 2003; Lavitrano et al., 2006; Hoelker et al., 2007). Although several specific methodologies have been developed (Horan et al., 1992; Chang et al., 2002), the transgenic efficiency of SMGT was never very successful in marine fish because the interaction between the sperm and the exogenous DNA was not fully understood. The aim of this study was therefore to determine: (1) the existence of DNase activity in the seminal plasma and (2) the impact of naked-DNA and liposome-DNA complexes on sperm viability in the olive flounder. These data provide important information for developing an SMGT protocol in marine fish such as the olive founder.
the jetPEItm reagent and ‘#’ is the number of nmoles of phosphate (P) per g of DNA. To confirm the N/P ratio for optimal sperm fertility (i.e., the survival rate of embryos to the blastula stage, based on 500 embryos at the 1-cell stage), a series of solutions with various N/P ratios (i.e., N/P = 3, 4, 5, 6, 7, 8, 9, and 10) were designed according to the jetPEItm kit (Promega) manufacturer’s instructions. The oocytes were collected from healthy female fish by gentle abdominal massage and then fertilized with the sperm-free seminal plasma and treated by exogenous naked-DNA or liposome-DNA complexes (described in Section 2.2) at different N/P ratios. Finally, the optimal N/P ratio was selected according to the highest fertility rate.
2. Materials and methods
2.4. Preparation of the Liposome-DNA complexes
2.1. Animals and sperm collection Adult olive flounder were maintained in a culture pond at Hai Yang, Shandong Province, China. The temperature and salinity of the seawater was maintained at 15 ± 2 ◦ C, and 30%, respectively, and the fish were kept on a 14 h:10 h light/dark cycle in standard fish facility conditions. The spermatozoa of three males were collected during March to May. Fish were carefully turned onto their ocular side and then the abdomen and genital pore were dried with sterile filter paper to prevent the sperm from being activated by seawater and/or urine. The sperm were artificially ejected by massage using gloved hand; a 5 mL straw was then used to collect the sperm ejected from the genital pore, and subsequently transfer them into a 1.5 mL microtube. 2.2. Removal of the seminal plasma from the semen Immediately after sperm collection, the seminal plasma was removed by centrifugation at 12,000 rpm for 15 min after which the supernatant (containing the seminal plasma) was discarded carefully. The sperm were diluted to a final concentration of 106 cells/l in Ringer’s solution (13.5 g/L NaCl, 0.6 g/L KCl, 0.02 g/L NaHCO3 ) with Ca2+ and Mg2+ free to avoid the activation of sperm. The centrifuged and diluted sperm were then transferred to a clean 1.5 mL microtube and kept at room temperature for immediate use in experiments. 2.3. Appropriate N/P ratio selection The N/P ratio represents the ionic balance within the liposome–DNA complexes; it refers to the number of nitrogen residues (N) in the transfection reagent per phosphate (P) of DNA. The overall charge of the liposome-DNA complexes is a crucial factor in transfection experiments, and is determined by the N/P ratio, such that a positive charge is necessary for successful complex integration by promoting interaction with anionic proteoglycans on the cell surface. The N/P ratio is calculated using the following formula: N/P ratio = (7.5* × l of jetPEItm )/(3# × g of DNA), where ‘*’ is the concentration of nitrogen (N) residues in
To form the liposome-DNA complexes, 1 g of the circular pEGFP-C1 DNA plasmid (Clontech) was incubated with 2 l transfection reagent to a final volume of 50 l in reagent buffer from the jetPEItm kit, according to the manufacturer’s standard protocol. The mixture was then incubated for 15–20 min at room temperature after which it was stored for either 2 h at room temperature or 24 h at 4 ◦ C.
2.5. Detection of DNase activity in the seminal plasma The exogenous naked-DNA plasmid or liposome-DNA complexes (1 g) were incubated with 20 l seminal plasma or DNaseI (New England Biolabs) for 5 min, 15 min or 30 min at 14 ◦ C or 23 ◦ C, which is the temperature range for growth and breeding of the olive flounder, as well as at 37 ◦ C, which is the optimum reaction temperature for DNaseI. Subsequently, the mixture was analyzed by electrophoresis on a 1% agarose gel containing ethidium bromide (0.5 g/mL) and visualized under a UV light.
2.6. Preparation of naked-DNA or liposome-DNA and sperm mixtures Sperm were mixed with either the naked-DNA or with the liposome-DNA complexes. 1 g naked-DNApEGFP-C1 or 100 l liposome-DNA containing 1 g pEGFP-C1 plasmid DNA was incubated with 10 l seminal plasma-free sperm for 10 min, 15 min, 30 min, or 50 min at room temperature.
2.7. Analysis of sperm structure by scanning electron microscopy The sperm that were treated with either naked-DNA or liposome-DNA were fixed using 2.5% glutaraldehyde for subsequent scanning electron microscopy (JSM-840, JEOL, and Japan) to investigate the effect of naked-DNA and liposome-DNA on the structure of the sperm following treatment for 10 min, 15 min, 30 min or 50 min.
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3.3. Effect of naked-DNA and liposome-DNA on sperm
Fig. 1. Detection of DNase activity in Paralichthys olivaceus seminal plasma. DNase activity was analyzed by electrophoresis on 1% agarose gel. M, 1 kb DNA molecular marker; lanes 1–3, mixture of seminal plasma and plasmid for 5 min, 15 min or 30 min at 14 ◦ C; lanes 4–6, mixture of seminal plasma and plasmid for 5 min, 15 min or 30 min at 23 ◦ C; lanes 7 pEGFP-C1 alone; lanes 8-10, mixture of DNase I and naked-DNA plasmid for 5 min, 15 min or 30 min at 37 ◦ C. The pEGFP-C1 plasmid was digested when it was incubated with seminal plasma at both 14 ◦ C and 23 ◦ C.
2.8. Data analysis The fertility rate among the controls and at different N/P ratios was compared using the ANOVA method. Data were considered to be significantly different at P < 0.05. 3. Results 3.1. Detection of DNase activity in the seminal plasma To detect if the seminal plasma exhibited any DNase activity, the pEGFP-C1 plasmid was mixed with seminal plasma, for varying lengths of time and at different temperatures. The results showed that the pEGFP-C1 plasmid was digested when it was incubated with seminal plasma; this is somewhat similar to the positive control when pEGFP-C1 plasmid was digested with DNaseI at 37 ◦ C. However in the latter case, the DNAase I digested the exogenous DNA completely (Fig. 1). The pEGFP-C1 plasmid was digested when it was incubated in seminal plasma at both 14 ◦ C and 23 ◦ C. 3.2. Appropriate N/P ratio selection based on the fertility rate As an N/P ratio greater than 3 is required to obtain a positive charge, we tested N/P ratios of between 3 and 10. Liposome-DNA complexes at different N/P ratios were prepared and incubated with seminal plasma-free sperm for 30 min according to the jetPEItm kit instructions, in order to determine the fertility rate (Fig. 2). Compared with the untreated sperm, the fertility rate was not significantly different when the N/P ratio was 3, 4, 5 or 6 (i.e., in all cases P > 0.05). On the other hand, the fertility rate was significantly lower than the control when the N/P ratio was between 7 and 10 (i.e., P < 0.01). According to the P values calculated, N/P ratios of 5 and 6 were selected as being most suitable for the sperm incubation experiments. Furthermore, a comparison of the average fertility rate with N/P ratios of 5 and 6 showed that the former was higher than the latter. Thus, an N/P ratio of 5 was determined to be optimal for the preparation of the liposome-DNA complexes.
When sperm were incubated with liposome-DNA complexes at 10 ng/106 sperm cells, for 5 min, 10 min, 15 min, 30 min or 50 min, the subsequent fertilization rate was not significantly different than when they were incubated with untreated sperm for the same incubation times (Table 1). On the other hand, when sperm were incubated with naked-DNA, the fertilization rate decreased to 50.1% and 33.4% following incubation times of 10 min and 15 min, respectively; and dropped to approximately zero after an incubation time of 30 min (Table 1). The quality of the sperm following treatment with naked-DNA or liposome-DNA complexes was investigated using scanning electron microscopy. Untreated sperm maintained a normal morphology, including the head, mitochondria and flagellum, after incubation times of 10 min, 15 min, 30 min or 50 min at 14 ◦ C (Fig. 3A–D). In addition, sperm incubated with naked-DNA, also exhibited a relatively normal morphology at 10 min (Fig. 3E); but the mitochondria and flagellum showed obvious degradation after 15 min (Fig. 3F, more severe deterioration by 30 min (Fig. 3G) and they had decomposed completely by 50 min (Fig. 3H). On the other hand, sperm treated with liposomeDNA complexes exhibited an intact morphology at all the time points tested (Fig. 3I–M). These results indicate that the sperm were well protected when using liposome-DNA complexes by isolating the exogenous DNA from the sperm, and in this way, the structural integrity of the sperm were maintained. 4. Discussion It is well known that sperm are capable of combining and internalizing exogenous DNA in some species. In fish, SMGT has been previously applied successfully to several species of transgenic fish, such as the silver sea bream (Lu et al., 2002) and the South American catfish (Campos et al., 2011a). In the case of the catfish, a transgene efficiency of exogenous DNA into larvae of 25% and 38% was identified by PCR and EGFP expression, respectively (Collares et al., 2010). Indeed, the principle of SMGT is based on the ability of sperm to combine and internalize exogenous DNA. However, the interaction between exogenous naked-DNA and the sperm plasma membrane is not random, but is known to be dependent on a specific region of the sperm head called the post-acrosomal region. (Lavitrano et al., 1992). In addition, it has been reported that the sperm plasma membrane plays a critical role in its interaction with DNA (Perry et al., 1999; Anzar & Buhr, 2006; Kurome et al., 2007). More recently, however, Lanes et al. (2009) reported that sperm are effected by endonuclease activity following the degradation of exogenous and endogenous DNA, and they concluded that there is strong DNase activity in the seminal plasma of the Brazilian flounder (Lanes et al., 2009). Therefore, in some species the sperm needs to be incubated with exogenous DNA indirectly to product transgenic embryos (Khoo et al., 1992), whereas in others, the sperm may be incubated directly with the DNA. This is therefore an important detail that needs to be determined before SMGT is developed for a particular species.
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Fig. 2. The effect of N/P ratio on the fertility rate of eggs fertilized with spermatozoa following internalization of a liposome-DNA complex. At N/P ratios of 3, 4, 5 and 6, the fertility rates were 89.3%, 87%, 84.6%, and 81%, respectively, which were similar (i.e., with P > 0.05) to the untreated sperm control group with a fertility rate of 89.3%. On the other hand, at N/P ratios of 7, 8, 9 and 10, the fertility rates were significantly lower (at P < 0.01) than the control, with values of 69.7%, 63.7%, 50% and 48.3%, respectively. Groups marked with the same letter means no statistically difference. Table 1 The effect of incubation time on the fertility rate among different combination groups at an N/P ratio of 5. Combination of plasmid and sperm
Untreated sperm pEGFP-C1plasmid/sperm Lipo-Complex-pEGFP-C1 plasmid/sperm
Fertility rate (%) Incubation time (min) 0
10
15
30
50
90.3 90.1 91.4
90.0 50.1 85.7
87.2 33.4 82.8
83.5 0 78.2
80.2 0 77.8
Fig. 3. Comparison of the morphology of sperm following treatment with either naked-DNA or liposome-DNA complexes at various incubation times. The morphology of untreated sperm in (A–D) the control group; and following treatment with either (E–H) naked-DNA or (I–L) liposome- DNA complexes. Sperm were treated for (A, E, I) 10 min, (B, F, J) 15 min, (C, G, K) 30 min; or (D, H, L) 50 min. The white arrows indicate the mitochondria of the sperm. When sperm were treated with naked-DNA, the mitochondria disappeared gradually over time until, by 50 min, the sperm were completely decomposed. On the other hand, in the untreated sperm and in those treated with liposome-DNA complexes, the mitochondria maintained a normal structural integrity at all the incubation times tested.
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Indeed, our results indicated that the seminal plasma of the olive flounder exhibited obvious DNase activity such that it completely degraded the pEGFP-C1 plasmid. This finding is similar to that of previous studies in mammals (Carballada & Esponda, 2001) and in the Brazilian flounder (Lanes et al., 2009). To our knowledge, this is the first report describing that there were a DNase activity in the seminal plasma of the olive flounder. These experiments suggest that a mechanism similar to that in mammals likely exists in fish. Sperm motility is important for sperm to attach, incorporate, and internalize exogenous DNA, and it is also critical for maintaining successful fertilization with an occyte. Several views support the idea that mitochondrial function is related to sperm motility as the mitochondria generate ATP (cellular energy) by oxidative phosphorylation. ATP is required for sperm motility and hyperactivation, which suggests that sperm mitochondrial function may be important for flagella propulsion and sperm fertilization capacity (Rajender et al., 2010; Ramalho-Santos & Amaral, 2013). Consequently, the physical condition of the mitochondria is thought to be a good determinant of sperm motility and thus quality. In this study, the naked-DNA indeed leads to a degradation of the sperm mitochondria, resulting in a shortened flagellum. In the liposome-DNA complexestreated groups, the change of morphological specificity was not obvious during even the longest incubation periods (i.e., 50 min), but in the exogenous naked-DNA group, the sperm mitochondria were degraded completely after incubated 50 min. However, a previous report on the South American catfish demonstrated that the pEGFP vector was internalized by sperm cells at even lower concentrations without motility loss after seminal plasma removal (Campos et al., 2011a). Furthermore, Collares et al. demonstrated the transgene transmission efficiency of exogenous DNA into silver catfish larvae through SMGT technology was 25% and 38%, respectively, identified by PCR and EGFP expression (Collares et al., 2010), and also showed that removal of the biological constituents of seminal plasma could lead to an increase in the incorporation of foreign DNA. 5. Conclusion Our results demonstrate that: (1) the spermatozoa of Paralichthys olivaceus exhibit DNase activity, and for successful SMGT, it was necessary to remove the seminal plasma. In addition, (2) naked-DNA induced sperm damage; but (3) liposomes coated on the DNA surface could protect the sperm from the harmful effects of exogenous DNA. In summary, the use of liposome-DNA complexes in transfection is a promising approach for the establishment of SMGT in the olive flounder. Acknowledgments We appreciate the help of Dr. Sarah E Webb, The Hong Kong University of Science & Technology (HKUST), for helpful comments on this manuscript. This study was supported by grants from the National High Technology Research and Development Program of China (No. 2012AA10A401).
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The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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