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a Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194-8511, Japan
b Laboratory of Cell Technology, Meiji Cell Technology Center, A Division of Meiji Milk Products Co., Ltd., Odawara 250-0862, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, in vivo gene transfer techniques have become popular as a tool for gene therapy and biological analysis at the whole-organ level, and several different methods have been developed thus far [14–16]. Virus-mediated gene transfer is the most widely used because of its high gene transfer efficiency; however, it is a high-risk biohazard. In contrast, nonviral vectors such as lipid-mediated systems are safe and easy, but the transfection efficiency is relatively low [16, 17]. Another nonviral method, in vivo electroporation (EP), has been shown to be an efficient method for transferring genes to the tissues of living animals [18–20]. This system indiscriminately delivers DNA molecules into any type of tissue cell and has a markedly higher transfer efficiency than other nonviral transfer systems [20].
In this study, as a possible alternative method for generating transgenic mice, we investigated an in vivo EP method using the testes of living mice to develop a simple assay system with which to analyze the regulatory elements of spermatogenic specific genes. A combination of foreign DNA injection into the seminiferous tubules and subsequent EP demonstrated an efficiency high enough for a transient expression assay to detect the activities of spermatogenic specific enhancer elements, which also enabled us to detect the histological distribution of cells expressing the transgenes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Crj:CD-1 strain male mice were purchased from Charles River Laboratories (Charles River Japan Inc., Kanagawa, Japan). Busulfan (Sigma, St. Louis, MO) was injected i.p. into 4- to 6-wk-old mice at a dose of 35 mg/kg to destroy the spermatogenic cells [21]. To study long-lasting expression of the transgene, mice were examined 3–4 wk after the treatment. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals (Mitsubisi Kasei Institute of Life Sciences Animal Care Committee, according to NIH #86–23).
Plasmid DNA
CMV-lacZ plasmid, which contains the Cytomegalovirus immediate early promoter/enhancer (CMV-IE) and the Escherichia coli lacZ gene, was purchased from Clontech (pCMVß; Palo Alto, CA). Prm-lacZ plasmid contains the lacZ gene driven by the enhancer/promoter region (-560 to +30, transcription initiation site as +1) of the mouse Protamine-1 (Prm-1) gene, and the sequence for the nuclear transporting signal was placed at the 5'-terminus of the ß-galactosidase coding sequence. Previous transgenic analyses revealed that a 113-base pair (bp) region between base pairs -150 and -37 of the Prm-1 gene is sufficient to confer postmeiotic spermatid-specific expression on a reporting gene [9]. A 5'-flanking sequence of Prm-1 containing the enhancer region was isolated by polymerase chain reaction (PCR) amplification using 5'-GTCTAGTAATGTCCAACACC and 5'-CCTGTGAGCAGGT GGAATTT as the primers and was used to construct Prm-lacZ plasmid.
Four plasmids containing the firefly luciferase (luc) gene were investigated for the luciferase reporting assay. Basic-luc contains only the luciferase gene without any promoter element (PGV-B; Toyo Ink Mfg. Co. Ltd., Tokyo, Japan). SV40 E/P-luc and SV40 P-luc contain the luciferase gene linked to both the enhancer and promoter regions of the SV40 early gene, and the SV40-promoter region alone, respectively (PGV-C, PGV-P; Toyo Ink Mfg. Co. Ltd.). Prm E/SV40 P-luc contains the luciferase gene driven by the SV40 early promoter region and the TATA-less prm-1 enhancer upstream sequence ranging from -560 to -33, which was isolated by PCR using two primers, 5'-GTCTAGTAATGTCCAACACC and 5'-GATACTAGTGGCCCCTAGGA. The Renilla luciferase gene under control of the CMV promoter (pRL-CMV; Promega, Madison, WI) was cotransfected in each assay to normalize the differences in transfection efficiency between the assays.
All the plasmid DNAs for injection were purified with Qiagen Maxi columns (Qiagen Inc., Chatworth, CA) and dissolved in HBS buffer (20 mM Hepes, 150 mM NaCl; pH 7.4) at concentrations of 1 µg/µl for CMV-lacZ and Prm-lacZ, and 0.5 µg/µl for the four luciferase plasmids, in which pRL-CMV was added at the concentration of 0.1 µg/µl and cotransfected.
In Vivo EP
Mice were anesthetized, and the testis was exposed under a dissecting microscope. A small (2–3-mm) incision was made in the tunica, and then 20 µl per testis of plasmid DNA solution, to which 0.04% Trypan blue dye had been added to monitor the accuracy of the injection, was injected into the seminiferous tubules (intratubular injection) or interstitial space of the testis (intratesticular injection) using injection glass pipettes (tip 30- to 40-µm in diameter) [21]. For the latter case, injections were made at three sites in each testis. After DNA injection, EP was performed with an electroporator (Electrosequare Porator T820; BTX, San Diego, CA). Testes were held between a tweezers-type electrode, and square electric pulses were applied eight times at 20–50 V with a constant time of 50 msec according to the procedure of Muramatsu et al. [20]. These treatments produced no noticeable damage on testes at histopathological observation. After EP treatment, the skin was stitched, and the mice were raised until analysis.
Histochemical Analysis
Histochemical staining of ß-galactosidase derived from transferred plasmid was performed as described previously [22]. The testes were fixed for 1–2 h in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3), rinsed three times in phosphate buffer containing 2 mM MgCl2 and 0.02% NP-40, and stained for 1–2 h at 37°C in the same buffer containing 0.1% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal; Sigma), 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN)6. For light microscopic observation, serial paraffin sections (8 or 15 µm) of the testes were prepared and counterstained with 0.5% eosin Y.
Dual Luciferase Assay
The dual luciferase reporter assay was performed according to the manufacturer's instructions (Promega). At 18 h after transfection, the whole testis was homogenized in 0.8 ml of ice-cold lysis buffer, and the crude lysates were clarified by centrifugation (12 000 rpm, 10 min) at 4°C. Ten microliters of the supernatant was first mixed with 100 µl of luciferase substrate to assay firefly luciferase reporter activity for 20 sec using a luminometer (Lumat LB 9501, Berthold, Wildbad, Germany), and then Renilla luciferase control activity was measured for 20 sec after addition of 100 µl of Stop&Glo buffer (Promega) to the reaction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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First, we compared the two types of injection methods with respect to the transgene expression efficiency in spermatogenic cells. Intratesticular injection is an injection of plasmid DNA into the interstitial space of the testis as described by Muramatsu et al. [20]. Intratubular injection is a DNA injection into the seminiferous tubules. CMV-lacZ was injected into mice (4 wk old) using each method, and the ß-gal expression patterns were compared 2 days later. As shown in Figure 1A, an intratesticular injection resulted in weak ß-gal expression throughout the entire testis, in which the interstitium as well as the seminiferous tubules were extensively stained (Fig. 1C). In contrast, an intratubular injection resulted in strong ß-gal expression along the seminiferous tubules (Fig. 1B), in which strong expression was observed in various stages of the spermatogenic cells and Sertoli cells in the seminiferous tubule, whereas positive cells were only slightly detected in the interstitial space outside the seminiferous tubules (Fig. 1D). These results indicated that the intratubular injection method was more suitable for gene transfer to spermatogenic germ cells as the target cells. Therefore, intratubular injection was used to inject DNA into the seminiferous tubules in subsequent experiments.
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Detection of Enhancer Activity of the Spermatogenic Gene
The enhancer activity of a spermatid-specific gene using a luciferase reporting system was studied to examine the possible application of an intratubular injection and in vivo EP as a new transient expression assay for spermatogenic specific genes. The Prm-1 gene encodes a sperm-specific chromosomal protein that replaces histone proteins during spermiogenesis [23–25]. The well-defined enhancer element Prm-1 was monitored as a typical indicator of spermatogenic stage-specific expression.
The four luciferase reporting plasmids were separately injected into adult mice (8 wk old), and the luciferase activities were determined 18 h after EP. The results are summarized in Table 1. By setting the luciferase activity of the promoterless vector (Basic-luc) injection at 1.0, the injections of the SV40-enhancer vector (SV40 E/P-luc) and the enhancerless vector (SV40P-luc) resulted in 44.3-fold and 5.2-fold increases in the luciferase activity, respectively, meaning that the SV40 enhancer itself showed about 8.5-fold enhancing activity. Transfection of PrmE/SV40P-luc driven by the Prm-1 enhancer resulted in a 10.0-fold increase, equivalent to about 1.9-fold compared with that of the enhancerless vector (SV40P-luc) and 22% of the enhancing activity of the SV40-enhancer vector. Considering that the population of round spermatid cells that specifically transcribe the Prm-1 gene is about 30% of all testicular cells [4, 26], the relatively lower activity of the Prm-1 enhancer appeared to be mainly due to the difference in the ratio of cells expressing each transgene. Moreover, when immature mouse testes (18–20 days after birth), which rarely have haploid cells, were used for the same experiments, the Prm-1 enhancer vector showed a small enhancing activity of about 0.9-fold compared with that of the SV40-enhancer vector. These results indicate that the transcriptional effect of the spermatogenic stage-specific Prm-1 enhancer is transiently detectable by this EP transfection method.
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Figure 2A shows the ß-gal staining of testes prepared two days after intratubular injection of Prm-lacZ plasmid. Although ß-gal-positive tubules were interspersed throughout the testis, sections of the positive tubules showed that the ß-gal-positive cells were detected only in the innermost layer of the seminiferous tubules, and the cells were in the elongated-spermatid stage (Fig. 2, B and C). Neither Sertoli cells nor spermatogenic germ cells of other stages exhibited any ß-gal staining. This expression pattern of the transfected Prm-lacZ was identical with the expression of the endogenous Prm-1 gene [27, 28].
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Long-Lasting Expression of the Transgene
One interesting aspect of this in vivo gene transfer technique is the possibility of tracing the developmental behavior of the transfected cells by detecting transgene expression as a long-lasting marker. When the testes at 4 wk after transfection of CMV-lacZ were examined with ß-gal staining, a small number of ß-gal-expressing cells were found to be scattered throughout the entire testis. A majority of the positive cells observed as dot-like localizations along the seminiferous tubules turned out to be Sertoli cells, which are nonproliferating somatic cells (Fig. 3A). Another pattern of staining was observed as a relatively long linear mass along the tubules, where ß-gal-expressing cells were found as a clump of spermatogenic germ cells present from the basal membrane to the inner lumen of the tubule (Fig. 3B), indicating that the CMV-lacZ transgene was incorporated into undifferentiated germ cells at the time of EP transfection.
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Busulfan-treated testes were used in an attempt to confirm that the transgene can be integrated into undifferentiated cells such as spermatogonia and the precursor stem cells. Adequate busulfan treatment turns off spermatogenesis transiently, after which a new spermatogenic cycle restarts from the surviving stem cells [21]. At 2 mo after EP transfection of CMV-lacZ into busulfan-treated testis, ß-gal staining was observed along the long axis of seminiferous tubules (Fig. 3C). In this tubule, ß-gal-expressing cells were detected in the reinitiated spermatogenic cell layers (Fig. 3D), whose image was in a pattern very similar to the pattern detected at 1 mo after gene transfer to the nontreated testis (Fig. 3B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EP is the easiest and most economical method for gene transfer. Another advantage is that it can be used for any type of tissue or cell [18, 20]. Recently, due to its efficient infection into numerous cell types, including nonproliferating tissue cells, adenovirus-mediated in vivo transfer has been regarded as the most attractive tool for treating a disease using gene therapy [15, 16, 29]. Blanchard and Boekelheide have carried out adenovirus-mediated gene transfer to adult rat testes [30]. They showed that expression of the SV40-lacZ transgene used in their study was detected only in Sertoli cells and Leydig cells but not in germ cells, indicating that adenovirus-mediated gene transfer to the testes has a strict cell-type preference and cannot deliver a transgene into all types of spermatogenic germ cells. In contrast, it has been reported that gene transfer by EP showed no preference for the target cell type. Muramatsu et al. [20] examined in vivo gene transfer by EP after DNA injection into the interstitial space of mouse testes. Transgene expression was detected in various types of testicular cells, both in the interstitium and seminiferous tubules. In our study using DNA injection into seminiferous tubules, significant expression of a transgene was restricted in germ cells and Sertoli cells within the tubules, but not in other somatic cells outside the tubules such as Leydig cells and peritubular cells (Fig. 1D). In short, transgenes retained in seminiferous tubules seem to be delivered equally to both germ and somatic cells residing within the tubules, and this has a positive effect on the efficiency of detecting a gene expressed in differentiating spermatogenic germ cells.
In the mouse, one spermatogenic cycle from spermatogonia to mature spermatozoa is estimated to take about 35 days [26]. Therefore, in about 1 mo all the differentiating germ cells in the premeiotic as well as postmeiotic stages finish maturing to spermatozoa and are released from the seminiferous epithelium to the epididymis. Nevertheless, our finding that ß-gal-positive cells were still detected in a clump of spermatogenic germ cells even after 1 mo of CMV-lacZ transfection (Fig. 3B) suggests that beyond the blood-testis barrier the foreign gene was transferred into the spermatogenic stem cells and the proliferating spermatogonia residing on the basal membrane.
This possibility was supported by the results obtained with transfection to busulfan-treated testes. The anti-cancer drug acts predominantly by killing proliferating active cells such as undifferentiated spermatogonia and transiently inhibiting the spermatogenic cycle. As a result, all developing spermatogenic cells disappear within 3 wk while some slowly dividing spermatogenic stem cells and nonproliferating somatic cells remain alive in busulfan-treated testes. After 2 mo of CMV-lacZ transfection to the busulfan-treated testis, we found a clump of ß-gal-positive cells in the repopulating spermatogenic cell layers (Fig. 3D), as well as characteristic localization of the transfectant cells along the axis of the seminiferous tubules (Fig. 3C). A previous study has revealed that undifferentiated progenitor type-A spermatogonia originating from a spermatogenic stem cell proliferate and extend horizontally onto the basement membrane [31]. Serial distribution along the tubules of spermatogenic stem cells has recently been reported in the testes of transgenic mice carrying a lacZ reporter driven by a specific promoter of a mouse retrotransposon gene that is specifically expressed in the spermatogenic stem cell [32]. Judging from these data, we believe that our EP transfection method is sufficiently efficient for introducing a foreign gene even into spermatogenic stem cells, and also that the transgene can be transmitted to the progenitor spermatogenic cells as development proceeds.
We have not examined whether the transgene was integrated into chromosomal DNA; however, it probably was since apparently stable expression of the transfected DNA was detected in stem germ cells and in their daughter cells. Further improvement of this method, in particular by raising the transfection efficiency into self-renewal stem cells, should allow us to perform in vivo functional analyses of a foreign gene during spermatogenesis in living testes, and ultimately this method could provide us with a new tool for producing transgenic animals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 427 24 6314; noce{at}libra.ls.m-kagaku.co.jp
3 Current address: Laboratory of Information Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444–8585, Japan.
4 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060–0810, Japan
5 Current address: Department of Biology, Faculty of Science, Konan University, Kobe 658–0072, Japan.
Accepted: July 28, 1998.
Received: March 26, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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