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From the Department of Nephro-Urology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
| Correspondence to: Dr Yukihiro Umemoto, Department of Nephro-Urology, Nagoya City University Graduate School of Medical Sciences, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan (e-mail: cdn83230{at}par.odn.ne.jp). |
| Received for publication August 6, 2004; accepted for publication November 5, 2004. |
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Abstract |
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Key words: In vivo electroporation, lacZ, testis, apoptosis, gene transfection, ß-gal activity
At present, both virus-mediated and nonviral methods are used for gene transfer (GT) to the testes. Virus-mediated GT is popular because of its GT efficiency, but it might represent a high biohazard risk and can induce harmful effects such as uncontrolled infection or inflammation. Nonviral methods include lipofection and electroporation (EP), which are easier and safer. Several laboratories have performed GT to testicular cell and sperm in animals by EP (Muramatsu et al, 1996; Yamazaki et al, 1998; Sato et al, 2002), but in many cases, the purpose of GT is to make transgenic animals. The genetic risk of the procedure to offspring has not been investigated (Yomogida et al, 2002). When reproductive technology is applied to humans, not only transgene delivery efficacy and expression control but also its safety and ethics must be considered.
In this study, the effects of GT by EP on mouse testes were assessed by investigating histologic changes and the expression of apoptotic cells. The delivery method of plasmid was injection into the interstitial space of the testis. The fertility of GT-treated male mice was also evaluated by mating them with normal female mice and counting the number of offspring.
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Materials and Methods |
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Plasmid DNA
pCAGGS-lacZ, used as the adventitious gene, was constructed as a
cytomegalovirus enhancer/chicken ß-actin promoter connected with the
lacZ gene (Sawicki et al,
1998) and was donated by Professor Junichi Miyazaki (Osaka
University). This plasmid was amplified with a competent cell kit (Toyobo,
Osaka, Japan), purified with a Qiagen Maxi kit (Qiagen, Hilden, Germany), and
dissolved in 1x Tris-EDTA buffer at a concentration of 1 mg/mL with
0.04% trypan blue.
In Vivo Electroporation
Male mice were anesthetized with pentobarbital sodium (2 mg) injected
intraperitoneally, and the testes were abdominally exposed under a dissecting
microscope. The animals were divided into the following 5 groups
(Table): 1) 20 µL of plasmid
DNA solution was injected into the interstitial space of the testis with glass
pipettes (tip 30-40 µm diameter), followed by EP; 2) 20 µL of
phosphate-buffered saline (PBS) was injected instead of the lacZ
gene, followed by EP; 3) EP alone; 4) the plasmid DNA solution was injected
without EP; and 5) PBS was injected without EP. In groups 1 through 3, EP was
performed with a squarewave electroporator (ECM830; BTX, San Diego, Calif).
For this, the testis was held between a tweezers-type electrode, and square
electric pulses were applied (30 V, pulse length 100 ms, 6 pulses/s, interval
900 ms), after which the skin was stitched
(Muramatsu et al, 1996;
Yamazaki et al, 1998). In
groups 4 and 5, EP was not performed.
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The bilateral testes were removed at 1, 2, 4, 6, and 8 weeks after GT; 5 animals were used each week.
Staining of Testes for ß-Galactosidase Activity
X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside;
Sigma-Aldrich Japan, Tokyo, Japan) staining was used to detect LacZ expression
resulting from GT. The testes were fixed with 1% formaldehyde and 0.2%
glutaraldehyde solution in PBS containing 0.05% NP-40 and 1 mM
MgCl2 for 1 to 2 hours at 4°C, rinsed 3 times in PBS, and
stained for 2 hours at 37°C in PBS containing 0.04% X-gal, 5 mM
K3Fe(CN)6, 5 mM K4Fe(CN)6, and 1
mM MgCl2. The testes were embedded in paraffin, and 4-µm-thick
sections were cut and mounted on glass slides (Matsunami Glass, Osaka, Japan).
The sections were deparaffinized and hydrated by processing with xylene and a
graded series of alcohol (Yamazaki et al,
1998).
Detection of the lacZ Gene
The polymerase chain reaction (PCR) was used for detection of the
lacZ gene resulting from GT. DNA was extracted from the extraction
solution, which contained 50 mM Tris-HCl, 1 mM EDTA, 1% sodium dodecyl sulfate
(SDS), and proteinase K (1 mg/mL) from a part of the testes. The PCR reaction
was performed with 30 cycles at 94°C for 30 seconds, 63°C for 30
seconds, and 72°C for 30 seconds.
The primers used were lacZ-1 (5'-GCCGAAATCCCGAATCTCTA-3') and lacZ-2 (5'-GGCTTCATCCACCACATACA-3'). The enzyme used was Taq DNA polymerase (Takarashuzo, Shiga, Japan). The other components used in the PCR procedure were 5 µL of PCR buffer, 0.2 mM desoxynucleotide triphosphate (dNTP), 1.5 mM MgCl2, primer lacZ-1 at 1 µM, primer lacZ-2 at 1 µM, 1 µL of sample DNA, 0.5 µL of Taq DNA polymerase, and 34.5 µL of distilled water.
Evaluation of Spermatogenesis
The 4-µm-thick deparaffinized and hydrated sections were stained with
hematoxylin and eosin (Muto Pure Chemicals, Tokyo, Japan) and observed under a
light microscope. Disorder in the formation of seminiferous tubular sperm were
evaluated in each group on the basis of the Johnsen score (JS;
Johnsen, 1970), because the JS
is a simple and clear method and can be compared easily among groups.
Detection of Apoptosis
Apoptosis was examined by the TdT-mediated dUTP-biotin nick end-labeling
(TUNEL) method (Gavrieli et al,
1992). In situ end labeling was performed with an apoptosis in
situ detection kit (Wako, Osaka, Japan). For proteolytic treatment, the
deparaffinized and hydrated sections were treated with diluted protein
digestion enzyme solution at 37°C for 5 minutes. To label the 3' end
of DNA, TdT reaction solution was dripped on the sections, and they were
incubated in a moisturized box at 37°C for 10 minutes. Endogenous
peroxidase was inactivated by 3% H2O2 for 5 minutes at
room temperature. Diluted peroxidase-conjugated antibody solution was dripped,
and the sections were reacted for 10 minutes in a moisturized box at 37°C.
Then, diaminobenzidine tetrahydrochloride solution was dripped, and the
sections were incubated for 5 minutes at room temperature for the color
reaction.
For evaluation of apoptosis, the numbers of seminiferous tubules with TUNEL-positive cells were counted under a light microscope and are expressed here relative to all seminiferous tubules as the apoptosis index (AI) as we described previously (Umemoto et al, 2001). The frequency of apoptotic cells was compared between groups. Five male mice from each group (total of 10 testicles) were studied.
Fertility After In Vivo GT
The fertilization ability of 5 male mice from each group was determined by
mating each male with 3 normal adult females before surgery and at 1, 2, 4, 6,
and 8 weeks after the surgery. Newborn offspring were counted each week, and
the average number of offspring was compared between the 3 groups.
Newborn offspring derived from GT-treated males were examined for the expression of LacZ by PCR.
Statistical Analysis
The JS and AI are presented as means ± SD. Significance of
differences between groups was determined by ANOVA with the use of StatView
4.5 software (Abacus Concept Inc, Cary, NC) on a Power Macintosh (Apple Inc,
Cupertino, Calif) computer. P less than .05 was considered
significant.
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Results |
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The lacZ gene was detected by PCR at 1, 2, and 4 weeks in group 1 (Figure 2), but the gene was not detected in group 4 (data not shown). ß-Gal activity was absent, and the lacZ gene was not detected in groups 2, 3, or 5 because of the injection of PBS rather than the plasmid DNA (data not shown).
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Johnsen Score
The hematoxylin and eosin staining in group 1 is shown in
Figure 3. At 1 week after GT,
the number of disordered and hollow seminiferous tubules was greater than
before GT, and only a few spermatids were present in some of the tubules (JS =
5.5 ± 0.4). At 2 weeks after GT, spermatozoa and spermatids were absent
in many tubules (JS = 4.9 ± 0.3). At 4 weeks after GT, many
seminiferous tubules were severely damaged, and only a few spermatocytes were
present therein (JS = 4.4 ± 0.4). The seminiferous tubules recovered
gradually thereafter through 6 and 8 weeks after GT (JS = 6.3 ± 0.5 and
6.8 ± 0.7, respectively). At 1, 2, and 4 weeks after the procedure, the
JSs in groups 1 through 3 were significantly different from those in groups 4
and 5, although at 8 weeks, there were no significant differences between the
groups (in groups 2-5, data not shown). The results within groups 1 through 3
and within groups 4 and 5 were similar, indicating that the difference was
attributable to EP (Figure
4).
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Apoptosis Index
Figure 5 shows TUNEL
staining at 1 week after the procedure in groups 1 through 5. An average AI
for a nontreated control was 2.0 ± 0.2. In group 1, positive cells
appeared in many seminiferous tubules at 1, 2, and 4 weeks after GT, after
which the proportion of apoptotic cells decreased at 6 and 8 weeks (AI = 50.5%
± 3.1%, 35.6% ± 7.6%, 38.6% ± 4.4%, 13.5% ± 4.6%
and 11.3% ± 3.9% at 1, 2, 4, 6, and 8 weeks after GT, respectively). In
groups 2 and 3, positive cells also appeared in many seminiferous tubules, and
the AI values were similar to group 1
(Figure 5). In groups 4 and 5,
there were more apoptotic cells in seminiferous tubules than in the normal
testis, but the rates of positive cells were less than in groups 1 through 3.
At 1, 2, and 4 weeks after the surgery, the AIs in groups 1 through 3 were
significantly different from those in groups 4 and 5, but there were no
significant differences between the groups at 6 and 8 weeks
(Figure 6).
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Reproductive Ability
All the transfected males fathered normal offspring. The total numbers of
offspring before surgery and at 1, 2, 4, 6, and 8 weeks after GT were 40, 27,
23, 25, 23, and 39 animals in group 1, respectively; 38, 15, 23, 20, 34, and
43 in group 2; 35, 15, 17, 29, 32, and 29 animals in group 3; 36, 20, 19, 30,
29, and 45 animals in group 4; and 44, 34, 19, 29, 35, and 39 animals in group
5. There were no significant differences between the groups
(Figure 7). In all groups, the
number of offspring tended to decrease at 1, 2, and 4 weeks. However, it
recovered at 8 weeks.
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DNA was extracted from tail tissue of the offspring, and PCR was used to examine transmission of the transgene via transfected mature sperm. However, none of these offspring contained the lacZ gene (data not shown).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The presence of the blood-testis barrier renders it impossible to transfer materials to the testis by oral or intravenous injection routes. We therefore injected the adventitious gene into the interstitial space of the testis directly (group 1). This paper indicates that our methodology (Kojima et al, 2003) makes it possible to introduce a gene successfully into mouse testes by EP. This method is easy and requires little skill or special technique. Moreover, this delivery method provides the potential to transfect the somatic cells of the testis of an individual with spermatogenic failure because of a somatic cell defect. GT efficacy was evaluated by observing ß-gal activity and the lacZ gene, which disappeared after 4 weeks in group 1. This is attributed to the occurrence of ß-gal activity and the lacZ gene inside as well as outside the seminiferous tubules in group 1, thereby increasing apoptosis and phagocytosis, which caused the lacZ gene to disappear.
Currently, 2 types of GT are used in transgenics to a living body: virus-mediated and nonviral. Virus-mediated GT is the most widely used because of its high efficiency. However, it poses a high biohazard risk and is complicated to handle. It also might induce harmful effects such as uncontrolled infection or inflammation; hence, it was not chosen for the work with germ cells in this study. In contrast, nonviral vectors such as lipid-mediated systems are safe and easy to operate, but their transfection efficiencies are relatively low (Philip et al, 1993; Marshall, 1995). Another nonviral method, in vivo EP, has been shown to be efficient for transferring genes to the tissues of living animals (Heller et al, 1996; Muramatsu et al, 1997). This system indiscriminately delivers DNA molecules into all types of cells and has a markedly higher transfer efficiency than other nonviral transfer methods. In addition, EP is the easiest and most economic method for GT (Muramatsu et al, 1997). Another advantage is that it can be used for any type of tissue or cell, including germ cells. Recently, these methods have also been used for gene therapy (Nakashima et al, 2002).
The transfer of genes into testes is aimed at advancing fundamental reproductive technology (eg, the production of transgenic animals). However, few studies have examined obstacles to the use of GT in clinical applications.
In this study, cell damage in groups 1 through 3 persisted for 4 weeks after EP. Muramatsu et al (1998) reported that high-current EP produces excessive heat, causing irreversible tissue damage that is more evident at 1 month after EP than immediately after EP. However, in this study, damage to the testes had recovered slowly by 6 to 8 weeks after EP. In the mouse, 1 cycle of the spermatogenic process from spermatogonia to mature spermatozoa has been estimated to take about 35 days (Wolgemuth et al, 1991). Our results on cell damage and TUNEL staining suggest that the change in spermatogenesis is related to apoptosis. Because of the blood-testis barrier, Sertoli cells must supply many factors required for the maintenance and development of germ cells. Sertoli cells produce an apoptosis-promoting factor, and are known to regulate germ cell apoptosis via the production of the Fas ligand. The interaction of the Fas ligand with the Fas receptor in germ cells triggers cell death. Because injury to Sertoli cells induces the Fas ligand, the GT could indirectly activate Fas ligand expression by injuring Sertoli cells because of the repression of genes required for maintaining cellular processes (Scobey et al, 2001). Therefore, we speculate that the JSs decreased following apoptosis.
There are few reports of the effect of GT to testes on fertility (Yamazaki et al, 2000). Our results demonstrate that damage to spermatogenesis from GT by EP is only temporary. Moreover, because GT by EP can be used for germ cells and somatic cells, it could be an effective therapeutic strategy for idiopathic male infertility.
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Acknowledgments |
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Footnotes |
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