Editor-in-Chief
Prof. Yi-Fei WANG,
Asian J Androl 2005; 7 (4): 369-373 |
|
| กก | |
This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you. - Original Article - Erythropoietin gene transfer into rat testes by in vivo electropo-ration may reduce the risk of germ cell loss caused by cryptorchidism Masaki Dobashi1, Kazumasa Goda1, Hiroki Maruyama2, Masato Fujisawa1 1Division of Urology, Department of Organs Therapeutics, Faculty of Medicine, Kobe University Graduate School of
Medicine, Kobe 650-0017, Japan
Abstract Aim: To investigate the effects of rat Erythropoietin (Epo) on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation. Methods: Sprague-Dawley rats with surgically-induced unilateral cryptorchidism were divided into three groups: the first group was given intratesticular injections of pCAGGS-Epo (pCAGGS-Epo group), the second group was given intratesticular injections of pCAGGS (pCAGGS group), and the third group were given intratesticular injections of phosphate-buffered saline (PBS group). At the same time, square electric pulses of 30 V were applied six times with a time constant of 100 ms. One or two weeks after injection, each testis was weighed and the ratio of the total number of germ cells to that of Sertoli cells (G/S ratio) was calculated to evaluate the impairment of spermatogenesis. Ten testes taken from each of the three groups were examined at each time point. Results: The testicular weight after the injection of pCAGGS-Epo or pCAGGS control plasmid was (0.85 ± 0.08) g and (0.83 ± 0.03) g, respectively, at week 1 (P = 0.788) and (0.62 ± 0.06) g and (0.52 ± 0.02) g, respectively, at week 2 (P = 0.047). At week 1, spermatids and sperm were more abundant in testes with pCAGGS-Epo than those in the control testes. At week 2, spermatids and sperm were hardly detected in either group. The G/S ratio was 23.27 ± 6.80 vs. 18.63 ± 5.30 at week 1 (P = 0.0078) and 7.16 ± 3.06 vs. 6.05 ± 1.58 at week 2 (P = 0.1471), respectively. Conclusion: The transfer of Epo to rat testes by in vivo electroporation may reduce the risk of the germ cell loss caused by cryptorchidism. (Asian J Androl 2005 Dec; 7: 369-373) Keywords: electroporation; gene transfer techniques; erythropoietin; spermatogenesis; Epo Correspondence to: Dr Masato Fujisawa, M.D., Division of
Urology, Department of Medicine, Kawasaki Medical School, 577
Matsushima, Kurashiki, Okayama 701-0192, Japan.
1 Introduction
Impaired spermatogenesis is an adverse effect of cryptorchidism in many mammals; however, the impairment can be reversed by orchidopexy soon after birth [1]. One of the models widely used to investigate testicular function was the experimental cryptorchid model. In rats, testicular weight has been found to decrease in 4 days to 2 weeks after an experimental induction of cryptorchidism [2]. Histopathologically, seminiferous tubules were reduced in diameter, and only a few tubules showed active spermatogenesis [3]. Erythropoietin (Epo), a hematopoietic cytokine, regulates erythrocyte production by acting on the proliferation, differentiation, and apoptosis of erythroid progenitor cells [4]. It has been demonstrated that Epo stimulated steroidogenesis in the testis. It influenced Leydig cell steroidogenesis of rat in vitro by stimulating testosterone production [5], and intravenous Epo administration increases testosterone production [6], although the mechanism by which this occured has not been completely clarified. In vivo gene transfer techniques have become popular tools in gene therapy and biological analysis to the various organs of rats, mice and others and several different methods have been developed thus far [7]. Virus-mediated gene transfer was the most widely used as it has a high gene transfer rate; however, it was also a high-risk biohazard. Although non-viral vectors such as lipid-mediated systems were safer and easier, the transfection efficiency rate was relatively low [8]. In vivo electroporation has been shown to be an efficient method of transferring genes to the tissues of living animals [9]. This system indiscriminately delivered DNA segments to any type of tissue cell and had markedly higher transfer efficiency rates than other non-viral transfer systems. We investigated the effects of rat Epo on spermatogenesis by transferring rat Epo genes into cryptorchid testes by means of in vivo electroporation.
2 Materials and methods
2.1 Plasmid vectors Plasmid pCAGGS-Epo [10] was constructed by inserting rat Epo cDNA into a unique XhoI site between the CAG promoter and the 3กฏ-flanking sequence of rabbit b-globin genes in the pCAGGS expression vector [11]. The empty pCAGGS was used as a control. DNA was diluted in phosphate-buffered saline (PBS) to 1.0 μg/μL immediately before injection.
2.2 Experimental procedures All experimental procedures were conducted in strict accordance with the Kobe University Guide for the Care and Use of Laboratory Animals. Seven-week-old male Sprague-Dawley rats had cryptorchidism surgically induced [12]. Under general anesthesia (sodium pento-barbital), the right testis of each rat was briefly surgically exposed when a mid-line incision was made, gubernaculum was cut and then the testis was replaced in the abdominal cavity. At the same time, each plasmid DNA (50 μg) diluted in 1.0 μg/μL of PBS was injected into the right testis with a 27-gauge needle connected to a 1-mL capacity syringe. Immediately after injection of the plasmid DNA, we applied square electric pulses of 30 V six times per 100 ms with a disk-type electrodes of an electro-square porator T820 (BTX, San Diego, CA, USA). Later, to evaluate the likelihood of testicular damage, we analyzed the testis sections that had been injected with the 50 μL of PBS solution containing no DNA. The testes were removed so that we could evaluate spermatogenesis and testicular weight at weeks 1 and 2. Ten testes taken from each of the three groups were examined at each time point.
2.3 X-gal staining Plasmid pCAGGS-lacZ expresses Escherichia coli b-galactosidase in the cytoplasm [11]. Rats were killed 2 days after the injection of 50 μg of pCAGGS-lacZ, and then the testes were rapidly dissected. The testes were fixed in 0.25 % glutaraldehyde and then stained with a 5-brome-4-chloro-3-indolyl-b-d-galactopyranoside (X-gal) solution mixture prepared as follows: 5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6·3H2 O, 0.2 mmol/L MgCl2, 1 mg/mL X-gal. Sections 4 μm in thickness were mounted on silanized slides, and were then counterstained with eosin.
2.4 Total tissue RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) At weeks 1 and 2 after gene transfer, the rats received either pCAGGS-Epo or pCAGGS and then were killed. The total RNA was extracted using a TRIzol reagent (Life Technologies, New York, NY, USA) and treated with RNase-free DNase. Complementary (c)DNA was prepared from 1 μg of total RNA by random priming, using a RNA PCR kit (TaKaRa, Kyoto, Japan) and PCR reactions were performed with a thermocycler (Program Temp Control System PC-800, ASTEC, Fukuoka, Japan). The lengths of the products were 323 bp for Epo mRNA and 207 bp for GAPDH mRNA. The primer sequences were as follows: Epo (sense: 5กฏ-TCTGACTGACCGCGTTACTC-3กฏ, antisense: 5กฏ-GACTTTGGTATCTGGGACGG-3กฏ), GAPDH (sense: 5กฏ-CTGGCGTCTTCACCACCA-3กฏ, antisense: 5กฏ-AGTTGTCATGGATGACCT-3กฏ).
2.5 Evaluation of spermatogenesis The testes were fixed in 10 % buffered formaldehyde, embedded in paraffin for sectioning and stained with hematoxylin and eosin. The numbers of germ cells and Sertoli cells were counted and the ratio of the total number of germ cells to that of Sertoli cells (G/S ratio) was calculated. Although the impairment of maturation or the decrease of numbers of germ cells in the testes with hypospermatogenesis was observed, the number of Sertoli cells was stable. The impairment of spermatogenesis was reflected in the G/S ratio.
2.6 Statistical analysis The statistical significance of difference was evaluated by unpaired t-test. P < 0.05 was considered statistically significant.
3 Results
3.1 Localization of pCAGGS-lacZ gene expression X-gal staining was positive in the interstitial cells and the spermatogonium-like cells located close to the basement membrane of the seminiferous tubule in the pCAGGS-lacZ-injected testes (Figure 1). No instances of pathological damage were noted in this treated area.
3.2 RT-PCR detection of Epo mRNA in testes injected with pCAGGS-Epo Epo mRNA was detected only in the testes injected with pCAGGS-Epo and not in those injected with pCAGGS; while control GAPDH mRNA was detected in both of the groups (Figure 2). We were also able to confirm the gene transfer into the testes by in vivo electroporation.
3.3 Testicular weight One week after the injection, there was no difference found in the testicular weight between pCAGGS-Epo-injected rats (n = 10) and pCAGGS-injected rats (n = 10): (0.85 ± 0.08) g vs.(0.83 ± 0.03) g, respec-tively (P = 0.788). The testicular weight 2 weeks after the injection was significantly heavier in pCAGGS-Epo-injected rats (n = 10) than that in pCAGGS-injected rats (n = 10): (0.62 ± 0.06) g vs. (0.52 ± 0.02) g, respectively (P = 0.047) (Table 1). The testicular weight where there had been no gene transfer (PBS) was (0.89 ± 0.05) g at week 1 (n = 10) and (0.55 ± 0.07) g at week 2 (n = 10) after the operation. No difference between the PBS group and pCAGGS group was noted. 3.4 Histochemical findings One week after the pCAGGS injection, spermatogonia, spermatocytes, spermatids and a few sperm were detec-ted in the testes (Figure 3A). At week 1, spermatids and sperm were more abundant in the testes with pCAGGS-Epo than that in the testes with pCAGGS (Figure 3C). The G/S ratios 1 week after the injections with pCAGGS-Epo and pCAGGS were 23.27 ± 6.80 and 18.63 ± 5.30, respectively (P = 0.0078) (Table 1). At week 2, spermatids and sperm were hardly detected in either group (Figure 3B, D). The G/S ratios 2 weeks after the injections with pCAGGS-Epo and pCAGGS were 7.16 ± 3.06 and 6.05 ± 1.58, respectively (P = 0.1471) (Table 1). These results demonstrated that the G/S ratio in the pCAGGS-Epo group was larger than that in the pCAGGS group at week 1 after the induction of cryptorchidism, but not after 2 weeks. The G/S ratios in the group where there were no gene transfer (PBS) was 19.98 ± 5.37 at week 1 and 6.36 ± 3.14 at week 2, repectively. No difference was seen between the PBS group and pCAGGS group.
4 Discussion
The present study was the first report to demonstrate that the Epo gene can be transferred into rat testes by in vivo electroporation and that germ cell loss due to cryptorchidism might be reduced to some extent as a result of the transfer. In a previously published report [13], the testicular weight of rats decreased 6 days after the induction of cryptorchidism. Similarly, in this study, the weight of the right testes that had been injected with pCAGGS had decreased by the 7th day after the induction of cryptorchidism. Some reports demonstrated that Epo stimulates spermatogenesis or steroidogenesis, for example, Epo might reduce the risk of germ cell loss in boys with cryptorchidism [14]; Epo influenced rat Leydig cell steroidogenesis in vitro by stimulating testosterone production [5], and in humans, intravenous Epo administration increased testosterone production [6]. Moreover, a recent report described how Epo receptors were detected in rat Leydig cells [15]. We suggested that in the present study, the reversal of cryptorchidism-induced germ cell loss was the result of the transfection of Epo genes into Leydig cells. As described previously, the lacZ gene was transfected by in vivo electroporation and was expressed in the interstitial cells or spermatogonium-like cells that were located close to the basement membrane of the seminiferous tubule. In the testis, interactions among Sertoli cells, peritubular myoid cells, Leydig cells and germ cells were thought to be essential for spermatogenesis to occur [16]. We suggested that the reversal of cryptorchidism-induced germ cell loss was a result of the interactions of the cells into which the Epo gene was transfected. Quantitative analysis of germ cell numbers has been used to objectively assess spermatogenesis. Prior studies have demonstrated a significant correlation between sperm concentration in the ejaculate and the total spermatogenesis (sum of all germ cells), total spermatid count (sum of early and late spermatids) and the ratio of late spermatids per Sertoli cell [17]. Such studies have also proposed a simplified and rapid technique, which quantified spermatogenesis by determining the mean number of late spermatids per seminiferous tubule in the human testis [18]. The quantification of germ cell numbers and its relation to Sertoli cell numbers has also been developed and used extensively in rat studies to evaluate spermatogenesis or estimate daily sperm production [19, 20]. The present study demonstrated how Epo gene transduction significantly increased the total number of germ cells per Sertoli cell (G/S ratio). In the present study, we showed that the G/S ratio in rats injected with pCAGGS-Epo was larger than that in rats injected with pCAGGS at week 1 after the induction of cryptorchidism, but not at week 2. The testes of the rats in the pCAGGS group weighed less than those of the rats in the pCAGGS-Epo group at week 2 after induction of cryptorchidism, but not at week 1. We had thought that Epo could reduce the deleterious effect of surgical cryptorchidism on spermatogenesis, but the impaired spermatogenesis did not show any signs of reco-very. At week 1 there was no significant difference in the destruction of seminiferous tubules between the two groups, but the reduction of the number of germ cell in the pCAGGS group was observed more than in the pCAGGS-Epo group. It was considered that the difference in the testicular weight would be primarily caused by the loss of seminiferous tubules rather than the pathological changes in the tubules such as the G/S ratio. Therefore, no significant difference was observed in the testicular weight at week 1. At week 2 after the induction of cryptorchidism, the more progressive destruction and loss of seminiferous tubules were observed in the control testes more than that in the Epo-transfected testes. Therefore, testicular weight decreased significantly in the control, compared with the Epo-transfected testes. In contrast, germ cells themselves in the remaining tubules were more severely impaired by cryptorchidism at week 2 than they were at week 1, resulting in the two groups displaying the same G/S ratio. In conclusion, we demonstrated that Epo gene transfer in rat testes by in vivo electroporation was efficient and it might to some extent reduce the loss of germ cells caused by cryptorchidism.
Acknowledgment
The authors are grateful to Dr J. Miyazaki of the Division of Stem Cell Regulation Research, G6, Osaka University Medical School, for his kind gift of pCAGGS. This work was supported by a Grant-in-Aid for Scientific Research (No. 14571500) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References 1 Alpert PF, Klein RS. Spermatogenesis in the unilateral cryptorchid testis after orchiopexy. J Urol 1983; 129: 301-2. 2 Jegou B, Risbridger GP, de Kretser DM. Effects of experimental cryptorchidism on testicular function in adult rats. J Androl 1983; 4: 88-94. 3 Jegou B, Laws AO, de Kretser DM. The effect of cryptorchidism and subsequent orchidopexy on testicular function in adult rats. J Reprod Fertil 1983; 69: 137-45. 4 Krantz SB. Erythropoietin. Blood 1991; 77: 419-34. 5 Foresta C, Mioni R, Bordon P, Gottardello F, Nogara A, Rossato M. Erythropoietin and testicular steroidogenesis: the role of second messengers. Eur J Endocrinol 1995; 132: 103-8. 6 Foresta C, Mioni R, Bordon P, Miotto D, Montini G, Varotto A. Erythropoietin stimulates testosterone production in man. J Clin Endocrinol Metab 1994; 78: 753-6. 7 Thierry AR, Lunardi-Iskandar Y, Bryant JL, Rabinovich P, Gallo RC, Mahan LC. Systemic gene therapy: biodistribution and long-term expression of a transgene in mice. Proc Natl Acad Sci USA 1995; 92: 9742-6. 8 Philip R, Liggitt D, Philip M, Dazin P, Debs R. In vivo gene delivery. J Biol Chem 1993; 268: 16087-90. 9 Muramatsu T, Shibata O, Ryoki S, Ohmori Y, Okumura J. Foreign gene expression in the mouse testis by localized in vivo gene transfer. Biochem Biophys Res Commun 1997; 233: 45-9. 10 Maruyama H, Sugawa M, Moriguchi Y, Imazeki I, Ishikawa Y, Ataka K, et al. Continuous erythropoietin delivery by muscle-targeted gene transfer using in vivo electroporation. Hum Gene Ther 2000; 11: 429-37. 11 Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 1991; 108: 193-9. 12 Jones TM, Anderson W, Fang VS, Landau RL, Rosenfield RL. Experimental cryptorchidism in adult male rats: histological and hormonal sequelae. Anat Rec 1977; 189: 1-27. 13 Nishimune Y, Maekawa M, Sakamaki L. Effect of duration of cryptorchidism on ability of mouse germ cells to regenerate and differentiate. Arch Androl 1986; 16: 89-96. 14 Cortes D, Visfeldt J, Thorup JM. Erythropoietin may reduce the risk of germ cell loss in boys with cryptorchidism. Horm Res 2001; 55: 41-5. 15 Mioni R, Gottardello F, Bordon P, Montini G, Foresta C. Evidence for specific binding and stimulatory effects of recombinant human erythropoietin on isolated adult rat Leydig cells. Acta Endocrinol (Copenh) 1992; 127: 459-65. 16 Hadrey MA, Dym M. Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization. Biol Reprod 1987; 37: 1283-9. 17 Zukerman Z, Rodriguez-Rigau LJ, Weiss DB, Chowdhury AK, Smith KD, Steinberger E. Quantitative analysis of the seminiferous epithelium in human testicular biopsies, and the relation of spermatogenesis to sperm density. Fertil Steril 1978; 30: 448-55. 18 Silber SJ, Rodriguez-Rigau LJ. Quantitative analysis of testicle biopsy: determination of partial obstruction and prediction of sperm count after surgery for obstruction. Fertil Steril 1981; 36: 480-5. 19 Roosen-Runge EC. Untersuchungen uber die Degeneration Samenbildender Zellen in der Nomalen Spermatogenese der Ratte. Z Zellforsch Mikrosk Anat 1955; 41: 221-35. 20 Russell LD, Peterson RN. Determination of the elongate spermatid-Sertoli cell ratio in various mammals. J Reprod Fertil 1984; 70: 635-41. |
| กก | |
