Journal of Andrology Testis Workshop 2009
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Published-Ahead-of-Print August 1, 2007, DOI:10.2164/jandrol.107.002717
Journal of Andrology, Vol. 29, No. 1, January/February 2008
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.107.002717

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The Effect of Varicocele Repair on Experimental Varicocele-Induced Testicular Germ Cell Apoptosis

ADEM FAZLIOGLU*, INANC YILMAZ*, ÖZGüR METE{dagger}, FATIH KURTULUS*, OGUZHAN PARLAKKILIC*, ÖZGüR GüCTAS* AND METE CEK*

From the * Urology Department, Taksim Teaching Hospital, and the {dagger} Department of Pathology, Istanbul Medical School, Istanbul, Turkey.

Correspondence to: Mete Cek, Chief, Taksim Teaching Hospital Department of Urology (e-mail: cekmd{at}doruk.net.tr).
Received for publication March 14, 2007; accepted for publication July 30, 2007.

   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The purpose of this study was to evaluate the variance of apoptosis in rats in which experimental varicocele was induced and then treated by varicocelectomy. Forty adult male Wistar albino rats were used in this experimental study. Experimental varicocele was created in 30 rats. A total of 5 rats underwent a sham operation, and the remaining 5 rats were the control group. A total of 5 rats from the varicocele group were sacrificed on the 14th postoperative day, and 5 more were sacrificed on the 28th postoperative day to document the level of apoptosis due to varicocele. Varicocelectomy was performed on 20 rats with varicocele on the 14th postoperative day. These 20 rats were divided into 4 groups to evaluate the level of apoptosis in their testis after varicocelectomy. They were sacrificed on days 7, 14, 21, and 28 after varicocelectomy. The testes were fixated by perfusion with 10% formaldehyde and then placed in paraffin blocks. From each testis, 2 samples were stained with hematoxylin and eosin, and 2 samples were stained using the TUNEL method. In each specimen, apoptotic germ cells stained by TUNEL were counted in the cross section of 100 seminiferous tubules. The apoptotic index was defined by calculating the number of apoptotic cells per seminiferous tubule. Apoptotic index = total apoptotic germ cell count/100. In the adult rats on which experimental varicocele was performed, both in the second and fourth week, apoptosis in both left and right testes were significantly higher compared with the control group (with varicocele day 14: 0.25–0.26, with varicocele day 28: 0.28–0.32, control: 0.11–0.13). After varicocelectomy on the 7th and 14th days, the slight increase in the level of apoptosis continued (day 7 left testis: 0.30, day 7 right testis: 0.28; day 14 left testis: 0.25, day 14 right testis: 0.31). After varicocelectomy, apoptosis decreased significantly on day 21 (left testis: 0.16, right testis: 0,22), and on day 28 it was almost equal to the level of the control group (left testis: 0.14, right testis: 0.16). After the creation of unilateral varicocele, the level of apoptosis increased in both the left and right testes. Apoptosis in both testes decreased after surgical treatment.

     Key words: infertility, TUNEL


Varicocele is characterized by stasis and high pressure in the veins that constitute the pampiniform plexus (Raifer, 1998). Varicocele affects fertility and is the most common known cause of infertility, but it can be treated surgically (Pryor and Howards, 1987). Although there are many hypotheses to explain testicular failure due to varicocele, none of these has been proven. The latest factor, described in the pathophysiology of infertility, is apoptosis defined as programmed cell death.

The role of apoptosis in varicocele and how varicocele affects testicular tissue and spermatogenesis has not been clearly defined. As it is not ethical to obtain testicular biopsy specimens from normal or infertile males, it is essential to use animal testing in the study of varicocele. We performed experimental varicocele in adult rats and investigated the changes in the level of apoptosis in the testes before and after treatment by varicocelectomy. The changes in the level of apoptosis in their testes were investigated through the TUNEL method.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Forty adult male albino rats were used, their weight ranging between 240 and 320 g. The animals were fed in an environment of constant temperature, humidity and in a cycle of 12 hours of light and 12 hours of dark in standard laboratory conditions. We followed the antisepsis rules during our surgical procedures. For general anesthesia, inhalation of ether and intraperitoneal ketamine (100 mg) was administered. Experimental varicocele as defined by Saypol (Saypol et al, 1981) was performed in 30 of these rats.

A total of 5 rats were reserved as a control group and not operated on, while the remaining 5 rats underwent a sham operation. From the rats with varicocele, 5 were killed on day 14 and 5 on day 28 to investigate the changes in the level of apoptosis. The control group and the sham group also were sacrificed on day 14 for comparison. In 20 rats in which experimental varicocele was created, laparotomy was performed under general anesthesia on the 14th day, at which point significant apoptosis develops. Through the abdominal incision, the left spermatic vein was ligated with 3/0 silk and divided (varicocelectomy). These 20 rats were divided into 4 groups to investigate the changes in the level of apoptosis in their testes and sacrificed on days 7, 14, 21, and 28. Before performing orchidectomy, the intraoperative perfusion technique was used in order to fixate the testes. After fixation, bilateral orchidectomy was performed, and each testis was put in 10% formaldehyde solution. The tissues were processed in the laboratory and kept in paraffin blocks. Out of these blocks, slices of 5 µm thickness were taken and put on slides, which were covered with lysine. Two slides from each testis were stained with hematoxylin-eosin, and 2 were stained with TUNEL. The TUNEL technique was used to stain the apoptotic cells. Detection sets with catalogue number QIA 33 (Colorimetric; Oncogene Research Products, San Diego, Calif) were used.


Figure 1
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  		Figure 1. Apoptotic germ cells on day 14 after varicocelectomy, stained with TUNEL.

 
The slides were kept at 37°C for 12 hours, deparaffinated with xylene and ethanol, and then rinsed with 1x Tris-buffered saline (TBS); after completing this procedure, the specimens were dried. To increase the permeability of the specimens, 2 mg/mL proteinase K was diluted in 10 mM Tris, pH 8, 1:100. This specimen was covered with 100 µL of 20 µg/mL proteinase K. It was incubated at room temperature for 20 minutes and then rinsed with 1x TBS. The stage of endogenous peroxidase inactivation was started, and in the meantime, the solution for marking the reaction was prepared (57 µL TdT marking reaction solution and 3 µL TdT enzyme were transferred into the microspin tube in ice and stirred gently). The buffering solution on the specimen was taken out with drying paper. Care was taken not to touch the specimen. Then, the preprepared marking reaction solution (60 mL TdT) was applied to the specimen. The specimen was covered with paraffin film wider than the slides. The slides were put in a humid environment and incubated for 1.5 hours at 37°C. To finish the marking reaction, the paraffin film was lifted and the slide rinsed with 1x TBS. The specimen was covered with 100 µL stop solution, incubated for 5 minutes at room temperature, and rinsed with 1x TBS. At this stage of fixation, the specimen was covered with 100 µL blocking buffer solution and incubated for 10 minutes at room temperature. Diaminobenzydine (DAB) solution was prepared 5 minutes before the incubation was over (1 tablet DAB and 1 tablet H2O2/urea and 1 mL tap/faucet per 10 slides were dissolved in H2O. This was rinsed with 1x TBS.) The specimen was covered with 100 µL DAB solution. It was incubated at room temperature for 10 to 15 minutes. The slides were rinsed with dH2O. Methylene green contrast stain was applied immediately. In the cross section of each specimen, apoptotic germ cells stained with TUNEL were counted in 100 seminiferous tubules. Homogenously stained areas without any necrosis were taken into consideration during the counting. Apoptotic index was defined as apoptotic cell count per seminiferous tubule. (Apoptotic index = total apoptotic cell count/100). Figure 1 depicts apoptotic germ cells on day 14 after varicocelectomy, stained with TUNEL under a light microscope (x40).

SPSS 11.5 for Windows (SPSS Inc, Chicago, Ill) was used for statistical analysis. Descriptive analyses were applied in the analysis of the data.

The Kruskal-Wallis test was applied for variance analysis. The Mann-Whitney U test was used for multiple comparisons, and P < .05 was accepted as the level of statistical significance.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Apoptotic indices for the testis of each rat in each group were defined in Table 1. There was necrosis in the left testis of one rat in the control group that was left with varicocele and sacrificed on the 28th day and of another rat that was evaluated on the 7th day after varicocelectomy. The necrosis observed in three testes were thought to be related to technical errors during surgery. Three rats died during the study, and they also were excluded. Means (averages) and SDs were calculated. It was observed that left and right testes within each group showed comparable apoptotic indices. No statistically significant difference was found in any group, except the posttreatment day 21 group, (Table 2).


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  		Table 1. Apoptotic indices of the testes
 

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  		Table 2. Average apoptotic indices of the groups
 

No statistically significant difference was found between the sham operation group and the control group (P > .1). The apoptotic index, however, was found to be significantly higher in the group in which experimental varicocele was created and sacrificed on the 14th day (P < .005). Apoptosis slightly increased in the group in which experimental varicocele was created and sacrificed on the 28th day compared with those sacrificed on the 14th day, although this was not statistically significant. It was seen that apoptosis increases significantly on the 14th day after the creation of an experimental varicocelectomy (Figure 2).


Figure 2
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  		Figure 2. Increase of apoptosis after creation of varicocele.

 
There was no decrease in apoptosis in those sacrificed on days 7 and 14 after varicocelectomy compared with the group of rats with varicocele. On the other hand, the apoptotic index was significantly lower in both testes of the rats sacrificed 21 days after varicocelectomy. This decrease was statistically significant (P < .009) in the left testes, whereas it was not statistically significant in the right testes (P > .1). The decrease of apoptosis 28 days after treatment was significant in both testes (Figure 3).


Figure 3
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  		Figure 3. Chart of comparison of all groups.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Varicocele is characterized with dilatation, stasis, and high pressure in the veins of the pampiniform plexus (Raifer, 1998). It may develop secondary to the compression of the renal vein or internal spermatic vein due to tumors or other pathologies. However, infertile patients have primary varicocele most of the time. The diagnosis of varicocele before puberty is rather infrequent, and the youngest patient reported was 9 years old. The incidence of varicocele in male adolescents is similar to the incidence of varicocele in male adults and has been reported to be 16% (Sigman and Howards, 1998). No racial difference has been reported with regard to the incidence of varicocele (Lipschultz and Corriere, 1977).

The incidence of varicocele is around 35% to 40% among infertile men (Mostafa and Corriere, 2001). It has been shown that following varicocelectomy, semen parameters improve by 50% to 80%; testicular dimensions and testicular histology improve significantly (Kass and Belman, 1987). In the light of these data, it is believed that varicocele affects fertility and is the most important cause of infertility, which can be treated surgically (Saypol et al, 1981).

Various hypotheses have been suggested to explain the testicular damage occurring due to varicocele. Increase of temperature, increased or decreased testicular blood flow, reflux, and toxic effect of renal or adrenal metabolites, hypoxia, and hormonal disturbances are only some of the factors suggested to play a role in the pathophysiology of varicocele (Baker et al, 1985). McLeod has described the changes in testicular tissue seen in varicocele patients as "stress pattern" (McLeod, 1971). Stress pattern consists of immature, amorphous, and small cells. These cytomorphologic changes, although not typical, look like the changes seen in cells that undergo apoptosis (Takihara et al, 1991). Later studies have suggested that the degenerative changes in germ cells due to the increase in temperature are actually related to the development of apoptosis in testis. Apoptosis originates from the Greek word "apo-TOE-sis," which means, "falling of leaves in autumn" (Feng et al, 1999).

The constant equilibrium between apoptosis and mitosis regulates the required amount of cells in the tissue (Bellamy et al, 1995; Cummings et al, 1997). Genetically determined physiologic mechanisms regulate apoptosis necrosis, another type of cell death that is different than apoptosis. Nuclear picnosis, fragmentation of the DNA, and phagocytosis of the picnotic particles are the different properties of apoptosis (Majno and Torisl, 1995). Apoptosis, a form of cellular suicide, is an active procedure and requires high levels of ATP. While it is under control in physiologic conditions, it increases in various diseases that influence the testis (Fujisawa et al, 1999). Increased apoptosis is found in all of the following conditions: hormonal insufficiency, criptorchidism, and increase in blood flow to the testis, local temperature increase in testis, and hypoxia due to venous stasis (Fujisawa et al, 1999; Hikim et al, 2000). High apoptotic activity has been found in testicular dysfunction, and it has been reported that deterioration of spermatogenesis and hypospermatogenesis may be related to uncontrolled apoptosis (Fujisawa et al, 1999).

Lin and coworkers' discovery of increased apoptosis in the germ cells of testicular biopsies of patients with idiopathic testicular insufficiency suggested the hypothesis that the increase of apoptotic germ cells might be the cause of testicular dysfunction and infertility (Lin et al, 1997). The findings of Bacetti and coworkers that apoptotic sperm cell count is approximately a hundred times higher than it is in the patients with varicocele compared with those without varicocele imply that apoptosis may have a critical role in infertility due to varicocele (Baccetti et al, 1996).

In a study done by Simsek and coworkers, testicular biopsies were obtained from patients undergoing varicocelectomy and from healthy males. These investigators showed that apoptosis was seven times higher in the testicular tissue of patients with varicocele (Simsek et al, 1998). The only study suggesting that there is a decrease in the level of apoptosis in varicocele belongs to Fujisawa and coworkers. These investigators compared testicular biopsies obtained from subfertile patients with varicocele to those obtained from healthy individuals and found fewer apoptotic cells in the group with varicocele (Fujisawa et al, 1999). Various technical reasons (using formalin instead of Bouin solutions for fixation) were held responsible for the different results reported in this study. Fujisawa and coworkers compared the level of S-Fas in the semen of oligospermic patients with varicocele, oligospermic patients without varicocele, and healthy individuals. The level of S-Fas was low only in the group with varicocele. This shows that the Fas/Fas-L system, which is the major regulatory system on apoptosis in testes, is activated; in other words, apoptosis has increased. In the same study, S-Fas levels increased, which indicates that apoptosis had decreased (Cohen, 1993).

Animal studies have become important in exploring infertility, as ethical issues preclude biopsy on healthy males' testicles. Mobley was the first to create an experimental model of varicocele in dogs in 1976 (Mobley and Baumn Carleton, 1976). Saypol has developed a model of varicocele in rats by the partial ligation of the left renal vein (Saypol et al, 1981). All these studies have revealed an increase in testicular temperature, decrease in sperm density, abnormal spermatogenesis, and microscopic and macroscopic changes similar to those seen in men. However, the main pathophysiologic mechanism has not been discovered (Saypol et al, 1981; Weese et al, 1993). In many studies in which experimental varicocele has been created, increased levels of apoptosis in germ cells have been clarified. These studies showed that the level of testicular superoxide increased in varicocele and that vitamin E and melatonin, which decrease superoxide levels, decrease testicular germ cell apoptosis (Mostafa et al, 2001). Barqawi and coworkers have shown that significant apoptosis developed (double that of the control group) on day 14, and this reached a maximum level on day 28 (Barqawi et al, 2004). We have observed in our study that the apoptotic index in the testes of the rats following the creation of varicocele almost doubled on the 14th day (ie, from 0.15 to 0.25 compared with the control group) and continued to increase slowly until day 28, reaching 0.28. There was no increase of apoptosis in the sham group compared with the control group on day 14 or on day 28. Although Barqawi and coworkers did not detect a significant difference in contralateral (right) testis compared with the control group, we observed that the level of apoptotic germ cells of testes on both sides increased and decreased simultaneously. We observed that both testes are affected by the presence of varicocele not only by previous clinical experience, but also at the molecular level (Turner and Lopez, 1990; Tapanainen et al, 1993; Schlesinger et al, 1994; Mostafa et al, 2001). Turner and Lopez too have observed destruction and an increase of temperature in both testes of rats on day 30 after the induction of varicocele, whereas following treatment the temperature fell and healing occurred (Turner and Lopez, 1990). We performed high ligation to spermatic veins (varicocelectomy) on rats in which significant apoptosis developed and then evaluated the results of surgical treatment of varicocele with an interval of 1 week. There was no healing (reversal) of apoptosis of testicular germ cells on the postoperative first week; on the contrary, a slight increase could be detected. There was no difference with the control group on day 14. On day 21, it was seen that apoptosis significantly decreased, and on day 28 the results were similar to those of the control group.

Various methods can be used in the evaluation of apoptosis. Although each method has certain limitations, the TUNEL method has some advantages over others, such as being able to be applicable in tissue culture and paraffinated blocks and being more sensitive due to its ability to stain even preapoptotic cells. TUNEL is the standard method in evaluating apoptosis for these reasons (Kockx et al, 1998). However, it is necessary to note that cleaning the tissue of blood cells by perfusion before sampling the tissue, fixating it with 10% formalin, and application of the stain by an experienced team with care, are the factors that will increase the success rate of this sensitive method.

There is a need for investigation of the factors, mediators, and especially genes that influence apoptosis. A better understanding of apoptosis may bring a new insight into the treatment of infertility or the development of newer methods of contraception.


   Acknowledgments
 
The authors wish to thank Ms Kay Oosthuizen for her help in editing this manuscript.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Baccetti B, Collodel G, Piomboni P. Apoptosis in human ejaculated sperm cells (notulae seminologicae 9). J Submicrosc Cytol Pathol. 1996;28: 587 –596.[Medline]

Baker HGW, Burger HG, Dekretser DM. Testicular vein ligation and fertility in men with varicocele. Br Med J. 1985; 291: 1678 .[Abstract/Free Full Text]

Barqawi A, Caruso A, Meacham RB. Experimental varicocele induces testicular germ cell apoptosis in the rat. J Urol. 2004; 171: 501 –503.[CrossRef][Medline]

Bellamy CO, Malcomson RD, Harrison DJ, Wyllie AH. Cell death in health and disease: the biology and regulation of apoptosis. Cancer Biol. 1995;6: 3 –16.[CrossRef]

Cohen JJ. Apoptosis. Immunol Today. 1993; 14: 126 –130.[Medline]

Cummings MC, Winterford CM, Walker NI. Apoptosis. Am J Surg Pathol. 1997;21: 88 –101.[CrossRef][Medline]

Feng HL, Sandlow JI, Sparks AET, Sandra A, Zheng LJ. Decreased expression of the c-kit receptor is associated with increased apoptosis in subfertile human testes. Fertil Steril. 1999; 71: 85 –89.[CrossRef][Medline]

Fujisawa M, Hiramine C, Tanaka H, Okada H, Arakawa S, Kamidono S. Decrease in apoptosis of germ cells in the testis of infertile men with varicocele. World J Urol. 1999; 17: 296 –300.[CrossRef][Medline]

Hikim S, Amiya P, Wang C, Leung A, Swerdloff RS. Involvement of apoptosis in the induction of germ cell degeneration in adult rats after gonadotropin releasing hormone antogonist treatment. Endocrinology. 1995; 136: 2770 –2775.[Abstract]

Kass EJ, Belman AB. Reversal of testicular growth failure by varicocele ligation. J Urol. 1987; 79: 996 –998.

Kockx MM, Muhring J, Knaapen MWM, de Meyer GRY. RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis. Am J Pathol. 1998; 152: 885 –888.[Abstract]

Lin WW, Lamb DJ, Wheeler TM, Abrams J, Lipschultz LI, Kim ED. Apoptotic frequency is increased in spermatogenic maturation arrest and hypospermatogenic states. J Urol. 1997; 158: 1791 –1793.[CrossRef][Medline]

Lipschultz LI, Corriere JN Jr. Progressive testicular atrophy in the varicocele patient. J Urol. 1977; 117: 175 –180.[Medline]

Majno G, Torisl A. Apoptosis oncosis and necrosis. Am J Pathol. 1995;146: 3 –15.[Abstract]

McLeod J. Human male infertility. Obstet Gynecol Surg. 1971;26: 335 .

Mobley DF, Baumn Carleton CE. Studies in infertility. Experimental models in infertility. Experimental models of varicocele in the dog. Paper presented at: AUA meeting; 1976; Las Vegas, Nev.

Mostafa T, Anis TH, El-Nashar A, Imam H, Othman IA. Varicocelectomy reduces reactive oxygen species levels and increases antioxidant activity of seminal plasma from infertile men with varicocele. Int J Androl. 2001;24: 261 –265.[CrossRef][Medline]

Pryor JL, Howards SS. Varicocele. Urol Clin North Am. 1987;14: 499 –513.[Medline]

Raifer J. Varicocele. In: Walsh PC, Retik AB, Vaughan ED, Wein AJ, eds. Congenital Anomalies of the Scrotum and Testis. Campbell's Urology. Philadelphia: W.B. Saunders Company; 1998: 2186 .

Saypol DC, Howards SS, Turner TT. Influence of surgically induced varicocele in testicular blood flow, temperature, and histology in adult rats and dogs: J Clin Invest. 1981; 68: 39 –45.[Medline]

Schlesinger MH, Wilets IF, Nagler HM. Treatment outcome after varicocelectomy: a critical analysis. Urol Clin N Amer. 1994;21: 517 –529.[Medline]

Sigman M, Howards SS. Male infertility. In: Walsch PC, Retik A, Vaughan ED, Wein AJ, eds. Campbell's Urology. 7th edition. Philadelphia: W.B. Saunders. Company; 1998: 1287 –1330.

Simsek F, Türkeri L, Çevik I, Kamuran B, Akdas A. Role of apoptosis in testicular tissue damage caused by varicocele. Arch Esp Urol. 1998; 51: 947 –950.[Medline]

Takihara H, Sakatoku J, Cockett ATK. The pathophysiology of varicocele in male infertility. Fertil Steril. 1991; 55: 861 –868.[Medline]

Tapanainen JS, Tilly JL, Vihko KK, Hsueh AJW. Hormonal control of apoptotic cell death in the testis: gonadotropins and androgens as testicular cell survival factors. Mol Endocrinol. 1993; 7: 643 –650.[Abstract/Free Full Text]

Turner TT, Lopez TJ. Testicular blood flow in peripubertal and older rats with unilateral experimental varicocele and investigation into the mechanism of the bilateral response to the unilateral lesion. J Urol. 1990;144: 1018 –1021.[Medline]

Weese DL, Peaster ML, Hernandez RD, Leach GE, Lad PM, Zimmern PE. Chemoattractant agents and nerve growth factor stimulate human spermatozoal reactive oxygen species generation. Fertil Steril. 1993; 59: 869 .[Medline]





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