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Electroporated Transgene-Rescued Spermatogenesis in Infertile Mutant Mice with a Sertoli Cell Defect¹

^a Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan

	ABSTRACT

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The molecular basis of most human male infertility arising from spermatogenesis disruption is poorly understood because of a lack of useful investigation systems. To study the roles of the supporting Sertoli cells in mammalian spermatogenesis, we improved an electroporation technique for seminiferous tubules in vivo. Because Sertoli cells barely proliferate in mature testis, linear transgenes are not incorporated into the genome and quickly degrade. However, circular expression vector is stably expressed in Sertoli cells for a long period. By electrotransformation of a complete cDNA, we rescued defective spermatogenesis in infertile Sl^17H/Sl^17H mutant mice with partial dysfunction of stem cell factor in Sertoli cells. Application of this gene transfer system will facilitate both the understanding of spermatogenesis and the development of new gene therapies for human male infertility.

gene regulation, Sertoli cells, spermatogenesis, testis

	INTRODUCTION

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More than one tenth of human couples are known to suffer from infertility, and half of these cases are attributable to deficient spermatogenesis in males [1]. Mammalian spermatogenesis is composed of three biologically significant processes: self-renewal and differentiation of germinal stem cells, meiotic recombination in spermatocytes after spermatogonial mitotic proliferation, and a dramatic morphological change in haploid germ cells to mature sperm. These serial processes are regulated strictly and cyclically to produce a constant and sufficient supply of sperm [2–4]. The supporting somatic Sertoli cells provide the microenvironment essential for germ cell proliferation and differentiation [1, 5, 6]. Although the molecular basis of almost all disorders of human spermatogenesis remains unclear [7], some disorders are caused by Sertoli cell dysfunction rather than germ cell dysfunction [8]. Understanding the roles of Sertoli cells will help elucidate the regulatory mechanism of spermatogenesis.

In vivo analyses using transgenic or gene-disrupted animals are effective and informative, especially in testicular germ cell studies [9–11], where no suitable culture system currently exists. Although in vivo transfection of DNA into testicular cells may be an efficient way to study gene function in spermatogenesis and to produce transgenic animals, an efficient gene transfer system has yet to be established [12–16]. Virus-mediated gene delivery systems are useful because of their high gene transfer efficiency [15–17]. However, they have a high biohazard risk, are complicated to handle, and can induce harmful effects as uncontrolled infection or inflammation [18]. Nonviral systems are easy and safe to operate but cannot incorporate transgenes efficiently and stably into the genome of germinal stem cells or supporting somatic cells in testis [12–14].

To improve in vivo electroporation as a tool for investigating the roles of Sertoli cells in mammalian spermatogenesis, this study examined conditions for the introduction and stable expression of transgenes in Sertoli cells. Furthermore, we investigated the rescue of spermatogenesis in Sl^17H/Sl^17H mutant mice, which are infertile because of an altered stem cell factor (SCF) cytoplasmic domain resulting from a splicing defect [19, 20]. Although a few undifferentiated spermatogonia remain in the mutant testes, they fail to continue spermatogonial differentiation after the initial wave of spermatogenesis [6, 19].

	MATERIALS AND METHODS

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Animals

Postnatal Day 12 C57BL/6 male mice were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan), and Sl^17H/Sl^17H mutant mice were raised in our animal facilities. All animal experiments conformed to the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Research Institute for Microbial Diseases, Osaka University).

Expression Vectors

As circular DNAs, we used noncut pCXN-EGFP plasmid containing the cytomegalovirus-enhanced chicken beta-actin promoter and enhanced green fluorescence protein (EGFP) [21]. As a linear DNA, we prepared one-cut pCXN-EGFP at the PvuI site. For the rescue of Sl mutant mice, we used the KL1 expression vector carrying CMV-1E and complete SCF cDNA, KL-1 [22]. All the DNAs for injection were dissolved in Tris-EDTA (TE) buffer at a concentration of 1 mg/ml with 0.04% Trypan blue.

DNA Injection and Electroporation

Mice were anesthetized, and testes were exposed under a dissecting microscope. A glass micropipette was inserted into the rete testis for injection into seminiferous tubules. About 3–5 µl of DNA solution was injected into each testis as monitored by the blue dye. Electric pulses were charged with an electric pulse generator (Electroporator CUY-21, Tokiwa-Science, Fukuoka, Japan). Testes were held between a pair of tweezer-type electrodes (CUY650, Tokiwa-Science) and square electric pulses were applied eight times in four different directions at 10–50 V and 50 msec duration.

Fluorescent Stereomicroscopic Observation under UV Light

Transfected testes were observed using a fluorescent stereomicroscope under UV excitation light and were photographed with a Leica DC200 (Leica Microscopy System Ltd., Wetzlar, Germany) set to the stereomicroscope.

Histological Analysis

The testes were fixed with 4% paraformaldehyde in PBS for 8 h, dehydrated with acetone, and embedded in glycol methacrylate (Technovit 8100; Heraeus Kulzer GmbH, Wehrheim, Germany), then cut into 5-µm-thick sections and observed under a fluorescent microscope. After GFP fluorescence was photographed, the same section was stained with hematoxylin and observed under a photomicroscope.

Northern Blot Analysis

Total RNAs were isolated with a Sepasol RNA-I Reagent (NACALAI TESQE, Kyoto, Japan) and aliquots (15 µg) were electrophoresed on a 1% agarose/formaldehyde gel. After transfer to a Zeta-Probe Blotting Membrane (Bio-Rad Laboratories, Hercules, CA), the RNA was hybridized to ³²P-labeled EGFP cDNA probe.

	RESULTS

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Specific Expression of Transgene in Sertoli Cells

To improve the efficiency of gene transfer into Sertoli cells, we used the testes of 12-day-old mice. These mice have a limited number of differentiated germ cells in their seminiferous tubules. We electroporated vector DNAs, carrying EGFP as a maker in linear or circular form, into the seminiferous tubules (Fig. 1). In transfected cells, EGFP was very strong in whole testes on Day 1 after transfection with both circular and linear vector DNA (Fig. 1A, Day 1). At this time, we were able to easily detect many fluorescence-positive Sertoli cells and a few green germ cells in the transfected testes, independent of the transgene structures (Fig. 1B, Day 1, and Table 1). Only 3 days after electroporation, the fluorescence of whole testis transfected with linear DNA was greatly reduced due to the great decrease of the fluorescence-positive cell population (Fig. 1A, Day 3, and Table 1). In the testicular cross-sections, only a few germ cells and a few Sertoli cells could be detected as fluorescence-positive cells (Fig. 1B, Day 3, and Table 1). At 7 days, almost no fluorescence-positive cells were observed in transfected testes with the linear form of DNA (Table 1). In contrast, circular transgene was stably expressed in the Sertoli cells, even after 35 days (Fig. 1 and Table 1). Transfected testes had qualitatively normal spermatogenesis, and we could trace the detailed microscopic structure of Sertoli cells by the fluorescence of EGFP expressed in the seminiferous tubules (Fig. 1B, Day 35, right panel).

View larger version (90K): [in this window] [in a new window]   FIG. 1. Low-magnification fluorescence stereomicroscope views of transfected testes with circular DNA (cir) or linear DNA (lin) at 1, 3, 7, and 35 days after electroporation (A). Cross-sections of transfected testes under excitation light followed by counterstaining with hematoxylin (B). Transfected cells were detected as fluorescence-positive cells. The shape of labeled germ cells was round (arrows). The transfected Sertoli cells were easily identified by their unique tree-shaped fluorescence (arrowheads) with normal spermatogenesis, particularly at 35 days after electroporation (right panel: higher magnification). Bars = 1 mm (A) or 100 µm (B)

Efficiency and Damage of In Vivo Electroporation

Because various kinds of stress are known to induce damage in testicular germ cells during differentiation [23, 24], we examined the effect of electroporation on spermatogenesis and testicular growth using 12-day-old testes. Testicular damage is roughly estimated by weight loss after 5 wk. The decrease in testicular size and weight was associated with the voltage used for electroporation; as voltage increased, testes became smaller (Fig. 2A) and lost more weight (Fig. 2C). In these testes, we detected the luminal enlargement of seminiferous tubules (Fig. 2D) and the degeneration of some peripheral seminiferous tubules under the capsule (Fig. 2E). At the same time, transformation efficiency appeared to increase with increasing voltage (Fig. 2B), although we could not access it precisely because of the damage. Thus, we selected 20 V as the optimal electroporation voltage because this achieved relatively high transfection efficiency without producing detectable qualitative histologic damage to spermatogenesis (Fig. 1B, Day 35).

View larger version (65K): [in this window] [in a new window]   FIG. 2. Stereomicroscopic views of transfected testes charged with various voltages and observed under visible (A) or excitation (B) light after 5 wk. Voltage is indicated on each testis (A). Loss of testicular weight 5 wk after electric charge in 12-day-old testes (C). All values are means ± SD (measured number of testes charged with 0, 10, 15, 20, 25, 30, 40, and 50 V are 4, 4, 4, 12, 12, 15, 10, and 12, respectively). Cross-sections of a testis at 5 wk after being charged with 50 V, observed with fluorescence microscope under excitation light (D, left), followed by counterstaining of the same section with hematoxylin (D, right) or another section just stained with hematoxylin (E). In higher voltage charged testes, luminal enlargement of seminiferous tubules (D) or complete degeneration of seminiferous tubules under the capsule (D, left) was observed. In these testes, many fluorescence-positive Sertoli cells were identified easily (E, right), like in 20 V charged testes (Fig. 1B, Day 35). Bars = 2 mm (A, B) or 100 µm (D, E)

Stability of Transgene Expression in Sertoli Cells

Next, we confirmed that the circular transgene electroporated into Sertoli cells was stably expressed for a long period. EGFP fluorescence was detected in all transfected testes (six testes tested) even 11 mo after electroporation (Fig. 3A). In addition, transcription of the transgene was at the same level at 11 mo as it was at 5 wk after treatment (Fig. 3B). The fluorescence of the cross-section of the testes was also very similar to that of 35 days after electroporation (data not shown).

View larger version (34K): [in this window] [in a new window]   FIG. 3. The EGFP expression in the testis well after electroporation. A) Fluorescence stereomicroscopic view of the testis 11 mo after electroporation. Bar = 2 mm. B) Northern blot analysis of the RNA extracted from the testes 5 wk and 11 mo after transfection

Rescue of Spermatogenesis in Sl^17H/Sl^17H Infertile Mutant Mice

To prove the benefit of permanent transgene expression, we attempted to rescue spermatogenesis in Sl^17H/Sl^17H infertile mutant mice using this gene transfer system. We electroporated expression vector DNA having complete SCF cDNA together with the DNA of EGFP reporter genes to 12-day-old testes of the mutant mice. As a control, some mutant testes were treated with only the EGFP reporter gene DNA. At 2.5 mo after electroporation, we could easily detect the transfected Sertoli cells by EGFP reporter-gene fluorescence (Fig. 4, A and D). As expected, only Sertoli cells transfected with complete SCF cDNA were able to support normal spermatogenesis (Fig. 4, B and C, and Table 2). Thus, the complete membranous type SCF produced by Sertoli cells was sufficient to support normal germ cell differentiation in Sl^17H/Sl^17H infertile mutant testes. Because the mutant testis does not have sufficient numbers of spermatogonia to recover fertility [6], we have not yet gotten any pups from these mice under normal mating conditions. To get pups from these rescued mice, we would have to perform intracytoplasmic sperm injection (ICSI) using their immature sperm.

View larger version (143K): [in this window] [in a new window]   FIG. 4. Cross-sections of transfected Sl17H/Sl17H testis 2.5 mo after electroporation. EGFP expression vector was electroporated with (A–C) or without (D–F) complete SCF cDNA. Spermatogenesis was recovered only in the seminiferous tubules containing Sertoli cells transfected with the complete SCF cDNA; under excitation light (A; control is D) and the same section stained with hematoxylin (B, E and C, F in higher magnification; controls are E and F). B) No spermatogenesis was observed in the seminiferous tubules with any fluorescence. Arrowheads indicate undifferentiated spermatogonia. Bars = 100 µm

	DISCUSSION

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As reported previously, in vivo electroporation is an effective method for the transfer of DNA into testicular cells. However, in spite of its potential, the low viability of electroporated germ cells and the poor stability of transfected genes have prevented this form of DNA transfer from becoming a major tool in infertility treatment [12–14]. In this study, we focused on the supporting Sertoli cells. Using 12-day-old mouse testes as target organs, we demonstrated the high efficiency of gene transfer and also the long-time stability of transgenes in Sertoli cells. The circular transgene was stably expressed in Sertoli cells, and the encoded protein was expressed for a long period. In contrast, the linear transgene disappeared immediately. It is difficult to incorporate linear DNA vectors into the genome of nonproliferating cells like mature Sertoli cells, and they are inactivated soon after transfection (Fig. 1). Furthermore, these linear transgenes might be degraded easily by nucleases in Sertoli cells. In contrast, the majority of fluorescent-positive germ cells disappeared soon, and all of them were lost in a month (Table 1). Although the promoter activity used here became weaker following the haploid germ cell differentiation, especially at the late stage of elongated spermatids [6, 21], it suggests that the transgenes were not incorporated into the genome of germinal stem cells.

The permanent expression of circular transgenes in Sertoli cells will facilitate the investigation of specific gene function in spermatogenesis. Furthermore, specifically controlled or hyperexpressed transgenes in Sertoli cells will provide a new method of functional assay of gene products and a promoter assay of Sertoli cell-specific genes in vivo without producing transgenic animals. Use of this technique with knockout or mutant mice that have defective Sertoli cell genes may be particularly effective and may provide useful information about gene function and the molecular basis of spermatogenesis.

The application of gene therapy to gonads will provoke ethical questions if transgene are incorporated into germ cells. Here, we demonstrate that a circular transgene was stably incorporated into mature Sertoli cells and was expressed for a long period but was minimally incorporate into germ cells. The stability of circular transgenes does not necessarily indicate that the vector DNA is integrated into the genome; it may become stably established as circular DNA molecules, like plasmid or viral DNA, and be effectively expressed for a long time. In any case, the stable expression of vector DNA into nonproliferating cells suggests that this method could be applied to other terminally differentiated cells, such as neurons, hepatocytes, and myocytes [25]. At present, these nondividing differentiated cells rarely incorporate transgenes. Recently, lentiviral vectors such as HIV vector have been developed for gene transfer, but they have a high biohazard risk [26, 27]. The accumulation of knowledge about the molecular basis of human male infertility will facilitate the application of this method as a gene therapy for patients with Sertoli cell dysfunction causing spermatogenic maturation arrest similar to the Sl mutant in mice.

	FOOTNOTES

 
First decision: 19 December 2001.

¹ This work was supported by H13-genom-009 from the Ministry of Health, Labor, and Welfare.

² Correspondence: Yoshitake Nishimune, Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp

Accepted: March 20, 2002.

Received: November 21, 2001.

	REFERENCES

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1. McLachlan RI, Mallidis C, Ma K, Bhasin S, deKretser DM. Genetic disorders and spermatogenesis. Reprod Fertil Dev 1998 10:97-104[CrossRef][Medline]
2. Russell LD, Ettlin RA, SinhaHikim AP, Clegg ED. Histological and Histopathological Evaluation of the Testis. Clearwater, FL: Cache River Press; 1990: 1–58
3. Parvinen P. Cyclic function of Sertoli cells. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993: 331–347
4. Yomogida K, Ohtani H, Harigae H, Ito E, Nishimune Y, Engel JD, Yamamoto M. Developmental stage- and spermatogenic cycle-specific expression of transcription factor GATA-1 in mouse Sertoli cells. Development 1994 120:1759-1766[Abstract]
5. Sharpe R. Experimental evidence for Sertoli-germ cell and Sertoli-Leydig cell interaction. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993: 391–418
6. Ohta H, Yomogida K, Dohmae K, Nishimune Y. Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF. Development 2000 127:2125-2131[Abstract]
7. Johannes HP. Towards an understanding of the genetics of human male infertility: lessons from files. Trends Genet 2000 16:565-572[CrossRef][Medline]
8. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nature Med 2000 6:29-34[CrossRef][Medline]
9. Escalier D. Impact of genetic engineering on the understanding of spermatogenesis. Hum Reprod Update 2001 7:191-210[Abstract/Free Full Text]
10. Meng X, Lindahl M, Hyvonen ME, Parvinen M, deRooij DG, Hess MW, Raatikainen AA, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000 287:1489-1493[Abstract/Free Full Text]
11. Furuchi T, Masuko K, Nishimune Y, Obinata M, Matsui Y. Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia. Development 1996 122:1703-1709[Abstract]
12. Yamazaki Y, Fujimoto H, Ando H, Ohyama T, Hirota Y, Noce T. In vivo gene transfer to mouse spermatogenic cells by deoxyribonucleic acid injection into seminiferous tubules and subsequent electroporation. Biol Reprod 1998 59:1439-1444[Abstract/Free Full Text]
13. Huang Z, Tamura M, Sakurai T, Chuma S, Saito T, Nakatsuji N. In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene. FEBS Lett 2000 487:248-251[CrossRef][Medline]
14. Yamazaki Y, Yagi T, Ozaki T, Imoto K. In vivo transfer to mouse spermatogenic cells using green fluorescent protein as a marker. J Exp Zool 2000 286:212-218[CrossRef][Medline]
15. Blanchard KT, Boekelheide K. Adenovirus-mediated gene transfer to rat testis in vivo. Biol Reprod 1997 56:495-500[Abstract]
16. Hall SJ, Bar-Chama N, Ta S, Gorden JW. Direct exposure of mouse spermatogenic cells to high doses of adenovirus gene therapy vector does not result in germ cell transduction. Hum Gene Ther 2000 11::1705-1712[CrossRef][Medline]
17. Nagano M, Shinohara T, Avarbock MR, Brinster RL. Retrovirus gene delivery into male germ line stem cells. FEBS Lett 2000 475:7-10[CrossRef][Medline]
18. Scobey MJ, Bertera S, Somers JP, Watkins SC, Zeleznik AJ, Walker WH. Delivery of a cyclic adenosine 3',5'-monophosphate response element-binding protein (CREB) mutant to seminiferous tubules results in impaired spermatogenesis. Endocrinology 2001 142:948-954[Abstract/Free Full Text]
19. Brannan CI, Bedell MA, Resnick JL, Epping JJ, Handel MA, Williams DE, Lyman SD, Donovan PJ, Jenkins NA, Copeland NG. Developmental abnormalities in Steel^17H mice result from a splicing defect in the steel factor cytoplasmic tail. Gene Dev 1992 6:1832-1842[Abstract/Free Full Text]
20. Wehrle-Haller B, Weston JA. Altered cell-surface targeting of stem cell factor causes loss of melanocyte precursors in Steel^17H mutant mice. Dev Biol 1999 210:71-86[CrossRef][Medline]
21. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. Green mice as a source of ubiquitous green cells. FEBS Lett 1997 407:313-319[CrossRef][Medline]
22. Tajima Y, Huang EJ, Vosseler K, Ono M, Moore MAS, Besmer P. Role of dimerization of the membrane-associated growth factor kit ligand in juxtacrine signaling: stability-phenotypes in hematopoiesis. J Exp Med 1998 187:1415-1461
23. Gasinska A, Hill S. The effect of hyperthermia on the mouse testis. Neoplasma 1990 37:357-366[Medline]
24. Meistrich ML. Effects of chemotherapy and radiotherapy on spermatogenesis. Eur Urol 1993 23:136-142[Medline]
25. Mir LM, Bureau MF, Gehl J, Ranfara R, Rouy D, Callaud JM, Delare P, Branellec D, Shwartz D, Sherman D. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci U S A 1999 96:4262-4267[Abstract/Free Full Text]
26. Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nature Med 2001 7:33-40[CrossRef][Medline]
27. Kafri T, Blomer U, Peterson DA, Gage FH, Verma IM. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 1997 17:314-317[Medline]

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