Evaluation of deoxyribonuclease activity in seminal plasma of ejaculated chicken semen

Keywords: deoxyribonucleases; chickens; seminal plasma; sperm; vectors; lipofection

Abstract

Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents. Methods: A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light. Results: The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L ~ 5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents. Conclusion: The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex

1 Introduction

The generation of transgenic animals in many species has been achieved by microinjection of exogenous genes into fertilized 1-cell embryo [1, 2]. However, this method requires special equipments and specific skill and is not useful for some species [3], specifically the avians, the fertilized 1-cell embryo of which is not accessible. For the generation of transgenic birds without fertilized 1-cell embryos, the following method has been developed: sperm-mediated gene transfer (SMGT) using spermatozoa as the vector for introducing exogenous genes into the zygote. The generation of transgenic rabbit [4], mouse [5] and porcine [6] has been reported using the method, in which spermatozoa are collected from the caudal epididymides or the ejaculated semen and incubated with exogenous genes. This method has been improved by using lipofection reagents and electroporation for introducing exogenous genes into spermatozoa [7]. More recently, testis-mediated gene transfer (TMGT) has been developed as an alternative method. In this method, exogenous genes are introduced into spermatozoa and spermatogonia in the testis by using lipofection reagents and spermatozoa ejaculated from this testis are used for artificial insemination. Generation of transgenic mammals was successfully achieved by the method of TMGT using lipofection reagents [8-11].

In contrast to the successful production of transgenic mammals by the sperm vector method, no transgenic bird has been efficiently produced by the method yet. In mice, spermatozoa for the SMGT method are usually collected from the caudal epididymides that do not contain seminal plasma mixed with accessory gland fluids. Accessory gland fluids are known to have deoxyribonuclease (DNase) activity that can digest exogenous genes introduced by the SMGT method [12]. On the other hand, chicken spermatozoa used for the sperm vector methods are collected from the ejaculated semen. Although chickens have no accessory glands, spermatozoa from the ejaculated semen contain seminal plasma. There is no literature concerning the stability of exogenous gene in the ejaculated chicken semen.

In the present communication, DNase activity was evaluated in the seminal plasma from the ejaculated chicken semen and the stability of DNA in the seminal plasma was tested using lipofection reagents.

2 Materials and methods

Semen was collected from mature White Leghorn chickens (8 month-old) by the conventional massage method. After collection, it was immediately centrifuged at 15 000 rpm for 3 min, and the resultant supernatant was used as the seminal plasma.

2.2 DNA substrate

A pEGFP-N1 vector (Clontech, Palo Alto, USA) was amplified by the polymerase chain reaction (PCR) using the forward primer 5'-ATTCTGCAGTCGACGGTACC-3' (position 631 to 650) and the reverse primer 5'-GTAGGTCAGGGTGGTCACGA-3' (position 879 to 859). The PCR reaction was performed in 10 µL of 1PCR buffer, 10 ng pEGFP-N1 vector, 0.2 mmol/L dNTPs, 0.25 U AmpliTaq Gold (Perkin Elmer Applied Biosystems, Foster, USA) and 0.2 µmol/L each of the synthetic primers. After an initial denaturation step (95 for 5 min), the amplification was performed in 40 cycles under a thermal profile of 95 for 1 min (denaturation), 56.8 for 1 min (annealing), 72 for 1 min (extension reaction) and the final extension at 72 for 5 min. A 249-bp fragment was used as the DNA substrate.

An aliquot of 10 µL DNA substrate (approx 300 ng), 5 µL seminal plasma and 15 µL pure water were mixed and incubated at 40 for 30 min. An aliquot of pure water instead of the seminal plasma was used as the control. Then, DNA was analyzed by electrophoresis on 2 % agarose gels containing ethidium bromide and the size was evaluated under UV light.

DNA substrate 20 µL (approx 600 ng) was mixed with 10 µL each of the lipofection reagents: SuperFect Transfection Reagent (QIAGEN, Hilden, Germany), DOTAP (Roche, Mannheim, Germany) and In Vivo GeneSHUTTLE (QUANTUM, Carlsbad, USA). Then, the lipofection-DNA solution was mixed with 10 µL seminal plasma and 20 µL pure water and incubated as above. The reaction solution was treated with an equivolume of phenol/chloroform, then centrifuged at 15,000 rpm for 7 min. The resultant supernatant was analyzed for DNA by agarose gel electrophoresis.

3 Results

The digestion of 249-bp DNA substrate was examined in the presence of seminal plasma. When DNA substrate was treated with seminal plasma at 40 for 30 min, it was completely diminished to be a new fragment with <100-bp size (Figure 1), suggesting the presence of the DNase activity in the ejaculated chicken seminal plasma.

Figure 1. Activity of heat-labile DNase in chicken seminal plasma DNA (approx 300 ng) was completely digested after incubation with seminal plasma (5 µL) at 40 for 30 min (Lane 5). The size of DNA did not change when DNA and seminal plasma were mixed just before application to electrophoresis (Lane 3); DNA was stable after incubation on ice for 30 min (Lane 4). DNA was stable after incubation with seminal plasma heated at 70 for 5 min (Lane 6). Size of DNA was decreased after incubation with seminal plasma heated at below 65 for 5 min (Lanes 7~10). Lane 1, 249-bp DNA substrate; Lane 2: seminal plasma alone; M: DNA mol wt marker.

After heat treatment of the seminal plasma at 70 for 5 min, the size of DNA substrate did not change, indicating that the ability of the seminal plasma to perform DNA digestion was diminished by 70 heat treatment. After heat treatment at 65 for 5 min, the size of DNA was decreased to approximately 200-bp. At below 60 , however, DNA substrate was completely digested to the <100-bp sized fragment. When DNA substrate was incubated on ice with the seminal plasma for 30 min or mixed with the seminal plasma just before application to electrophoresis, DNA remained intact.

3.3 Effect of divalent cations on DNase activity

DNA substrate was incubated with seminal plasma at 40 for 30 min in the presence of various concentrations (0.005 mmol/L~5 mmol/L) of EDTA. DNA remained intact after incubation with EDTA at concentrations above 0.5 mmol/L, but not below 0.25 mmol/L (Figure 2), indicating the blockage of the DNase activity by EDTA at concentrations above 0.5 mmol/L.

Figure 2. Effect of EDTA on DNase activity in seminal plasma DNA was incubated with seminal plasma at 40 for 30 min in the presence of various concentrations (0.005 mmol/L~5 mmol/L) of EDTA. The size of DNA did not change in the presence of 0.5 mmol/L~5 mmol/L EDTA (Lanes 3, 4), but did change in the presence of EDTA below 0.25 mmol/L (Lanes 5-7). Lane 1: 249-bp DNA substrate; Lane 2: DNA incubated with seminal plasma at 40 for 30 min; M: DNA mol wt marker.

DNA substrate was mixed with lipofection reagents and then incubated with seminal plasma. As shown in Figure 3, DNA remained intact, indicating that DNA is stable in the presence of lipofection reagents.

Figure 3. Effect of lipofection reagents on DNase activity in seminal plasma DNA mixed with each lipofection reagent, SuperFect Transfection Reagent (Lane 3), DOTAP (Lane 4) and In Vivo GeneSHUTTLE (Lane 5) was incubated with seminal plasma at 40 for 30 min, extracted with phenol/chloroform and subjected to electrophoresis. Lane 1: 249-bp DNA substrate; Lane 2: DNA incubated with seminal plasma without lipofection reagents; M: DNA mol wt marker.

The presence of DNase activity has been demonstrated in the semen of the bulls [13] and the humans [14] and in the seminal vesicle fluid of male mice [12]. In the present study, it was documented that the chicken seminal plasma also contains DNase activity, although there are no accessory glands in the roosters. We used a small DNA fragment for the measurement of DNase activity, because its fragmentation was easily identified on agarose electrophoresis as compared to plasmid DNA. The finding that the DNase activity was suppressed by heating at 70 for 5 min indicated the heat lability of the DNase, but it was relatively stable by heating at below 60 for 5 min. Since divalent cations, as calcium and magnesium, are generally required for DNase activity, it is reasonable that EDTA blocked the DNase activity in the seminal plasma [15]. At least 0.5 mmol/L EDTA was required for the blockage of the DNase activit.

The successful generation of transgenic animals by the sperm vector methods, SMGT and TMGT, was well documented. Several reports described that the spermatozoa have binding or incorporation sites of exogenous genes [10, 16-18]. It was proposed that MHC II molecules and the antigen CD4 present at the sperm head membrane were involved in the binding and internalization of exogenous genes [19]. However, these reports did not indicate that exogenous genes were bound or incorporated into spermatozoa as intact molecules. The DNase containing in the semen of mammals and birds may decrease the uptake of DNA as intact molecules and thus the efficient generation of transgenic animals by the sperm vector methods. In rodents, mature spermatozoa are usually collected from the epididymis, in which they are not mixed with seminal plasma. However, in avian species, spermatozoa from the ejaculated semen are used. It is strongly suggested that the DNase contained in the seminal plasma digests a part of the exogenous genes bound on the surface of spermatozoa. It was previously reported from our laboratory that the expression of exogenous genes was not observed but that exogenous genes were detected by PCR analysis of DNA isolated from the progeny [20]. The findings may result from digestion of the coding region for protein expression with no change in PCR amplifying region. Consequently, the generation of transgenic birds by the sperm vector methods may be inefficient with the ejaculated semen, if lipofection reagents are not used.

An interest fact that each lipofection reagent could protect the digestion of DNA by DNase was noted by several reports [21, 22]. Several lipofection reagents have been used for the efficient uptake of DNA into spermatozoa, although exogenous genes are not integrated to genomes [23]. In the present study, DNA remained intact after incubation of DNA substrate with seminal plasma in the presence of each lipofection reagent. The liposomal-DNA complex should be constructed by mixing DNA substrate with each lipofection reagent. The water-soluble DNase may not be able to interact with the complex rather than inhibition of the DNase by lipofection reagents, because the liposomal-DNA complex is formed as micelle [24, 25]. Actually, the complex could not migrate into agarose gels in electrophoretic analysis (data not shown).

In conclusion, although the male chicken has no accessory glands, the seminal plasma contains DNase activity. However, the enzyme does not digest DNA in the liposomal- DNA complex. The binding sites of exogenous genes on the chicken spermatozoa remain unknown, but the present study supports the validity of lipofection regents for the sperm vector methods.

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Correspondence to: Masa-aki Hattori, Ph.D., Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Fukuoka 812 8581, Japan.
Tel/Fax: +81-92-642 2938
E-mail: mhattori@agr.kyushu-u.ac.jp
Received 2002-12-16      Accepted 2003-07-03