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Keywords:

  • varicocele;
  • apoptosis;
  • infertility;
  • testis
Abbreviations
TUNEL

in situ terminal deoxynucleotidyl transferase-mediated dTUP nick-end labelling

ROS

reactive oxygen species

L-VDCC

l-type voltage-dependent calcium channel

AI

apoptotic index.

INTRODUCTION

  1. Top of page
  2. INTRODUCTION
  3. THE CHARACTERISTICS OF APOPTOSIS
  4. APOPTOSIS IN SPERMATOGENESIS
  5. APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE
  6. APOPTOSIS IN MEN WITH VARICOCELE
  7. ROS AND APOPTOSIS IN VARICOCELE
  8. CADMIUM AND APOPTOSIS IN VARICOCELE
  9. CONCLUSIONS
  10. CONFLICT OF INTEREST
  11. REFERENCES

Varicoceles have been recognized as a cause of male infertility for many years. However, it remains unclear as to why some men with varicoceles can father children and have normal semen, whereas others fail to improve, despite corrective surgery. Thus there is continuing controversy, as current concepts of the pathophysiology of varicoceles do not apply to all clinical situations [1]. Recently, molecular and genetic information was collected from men with varicoceles; this information may shed new light on the lower semen quality and poor sperm function associated with the condition.

Apoptosis is a process by which a specific and highly regulated cascade of intracellular events results in the involution and elimination of a cell from its environment [2]. Apoptosis is a normal physiological phenomenon in most tissues, and has a critical regulatory role. Apoptosis is also an important aspect of normal spermatogenesis. Therefore, alterations in the apoptosis of germ cells may be crucial in varicocele-related human infertility.

Varicoceles are associated both with oligospermia and abnormal spermatogenesis. However, it remains unclear how these sperm-related abnormalities are associated with varicoceles. Herein we review several studies in an attempt to determine the relationship between apoptosis and varicocele-related hypospermatogenesis.

THE CHARACTERISTICS OF APOPTOSIS

  1. Top of page
  2. INTRODUCTION
  3. THE CHARACTERISTICS OF APOPTOSIS
  4. APOPTOSIS IN SPERMATOGENESIS
  5. APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE
  6. APOPTOSIS IN MEN WITH VARICOCELE
  7. ROS AND APOPTOSIS IN VARICOCELE
  8. CADMIUM AND APOPTOSIS IN VARICOCELE
  9. CONCLUSIONS
  10. CONFLICT OF INTEREST
  11. REFERENCES

Apoptosis, also commonly referred to as programmed cell death, is the mechanism by which all cells die under normal conditions. Apoptotic cells are microscopically characterized by cell shrinkage, membrane blebbing, nuclear condensation, and fragmentation in the form of vesicles called ‘apoptotic bodies’[3]. Many pathways result in apoptosis, and these processes appear to be regulated on three levels [4]. In the cell membrane, specific membrane receptors mediate the death signals of the TNF receptor family, which consists of Fas and the Fas ligand. At the cytoplasmic level, signal transduction pathways involving cysteine proteases called caspases are also involved. Finally, at the nuclear level, specific apoptotic regulatory genes, including p53 and Bcl-2, also exert regulatory effects on apoptosis.

APOPTOSIS IN SPERMATOGENESIS

  1. Top of page
  2. INTRODUCTION
  3. THE CHARACTERISTICS OF APOPTOSIS
  4. APOPTOSIS IN SPERMATOGENESIS
  5. APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE
  6. APOPTOSIS IN MEN WITH VARICOCELE
  7. ROS AND APOPTOSIS IN VARICOCELE
  8. CADMIUM AND APOPTOSIS IN VARICOCELE
  9. CONCLUSIONS
  10. CONFLICT OF INTEREST
  11. REFERENCES

Spermatogenesis is an ongoing proliferative process, which results in the development of millions of spermatozoa per day. Apoptosis is a prerequisite for normal spermatogenesis in mammals, and is thought to ensure cellular homeostasis, and to facilitate the maintenance of the balance between germ cells and Sertoli cells. Germ cell apoptosis during normal spermatogenesis is estimated to result in the loss of 25–75% of potential mature sperm cells in the adult testes. Apoptosis appears to affect all three classes of germ cells, i.e. the spermatogonia, spermatocytes, and spermatids [5]. Several varicocele-associated factors, including heat stress, androgen deprivation, and exposure to toxic elements, may also induce pathways which result in apoptosis. In one study, the scrota of rats were exposed to a temperature of 43°C for 15 min; this heat stress culminated in a cell-specific and stage-specific apoptosis [6]. Similar apoptotic effects were reported as the results of heat stress in mice, using the cryptorchid model [7]. It was also reported that the pro-apoptotic Bax gene was expressed earlier and more abundantly than normal in susceptible germ cells after 30 min of scrotal heating. In this experiment, Bcl-2 expression was delayed, and this may have constituted an attempt to escape apoptosis [8]. Specific studies of apoptosis and androgen deprivation have also been reported, using rat models. Apoptotic induction was shown to occur in immature rats 4 days after hypophysectomy [9], whereas in mature rats, apoptosis could be induced by treatment with a GnRH antagonist [8]. Recent data also linked cadmium to apoptosis; the administration of cadmium chloride in a rat model induced the characteristic apoptotic ladder-like DNA patterns on agarose gel [10]. In humans, in testicular biopsy specimens obtained from men with varicocele, there was a correlation between cadmium deposition and the percentage of apoptotic cells found per round tubule [11].

APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE

  1. Top of page
  2. INTRODUCTION
  3. THE CHARACTERISTICS OF APOPTOSIS
  4. APOPTOSIS IN SPERMATOGENESIS
  5. APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE
  6. APOPTOSIS IN MEN WITH VARICOCELE
  7. ROS AND APOPTOSIS IN VARICOCELE
  8. CADMIUM AND APOPTOSIS IN VARICOCELE
  9. CONCLUSIONS
  10. CONFLICT OF INTEREST
  11. REFERENCES

Table 1 lists the results of animal models and human studies in which the relationship between apoptosis and varicoceles was investigated [12–29]. In a study using the in situ terminal deoxynucleotidyl transferase-mediated dTUP nick-end labelling (TUNEL) assay [12], the incidence of apoptosis was remarkably more frequent in the varicocele group, although the spermatogenic cells in the control group also had some degree of apoptosis. Testicular growth was slower in the varicocele than in the control group. In another study [15], there was greater apoptosis in the germ cells in a rat model 14 days after inducing a varicocele; there were 0.23 apoptotic cells per seminiferous tubule, compared with 0.05 cells in the controls (P < 0.002). However, at no point was the number of apoptotic germ cells in the contralateral testes of the varicocele group statistically different from those in the controls. In a similar experimental model, Cam et al.[17] also reported that the difference in apoptotic indices assessed using the in situ end-labelling method was also statistically significant between the sham and varicocele groups. In that study there were no statistically significant differences between the right and left testicles in both apoptosis and the levels of reactive oxygen species (ROS). This suggests that the varicocele functioned bilaterally, at least at the molecular level. In a study by Gurdal et al.[14], there was a significantly higher rate of apoptosis in the bilateral testicular tissue of rats with varicocele than in the sham-operated group. Furthermore, these authors reported a trend of increasing apoptosis with a longer duration of varicocele. In a study using flow cytometry [13], although there was no marked peak in apoptosis in the control group, there was such a peak in the varicocele group. The height of this peak increased directly with the duration of the experiment. Onur et al.[16] evaluated pro-apoptotic/anti-apoptotic markers in rats with an experimentally induced varicocele. There was a greater ratio of the pro-apoptotic Bax protein in the varicocele group, whereas there was no significant activity in the sham-operated group (P < 0.05).

Table 1.  Apoptosis in varicocele
RefSourcesMethods for apoptosisResults in the varicocele groupComments
  1. FC, flow cytometry; IHC, immunohistochemistry; MDA, malonedialdehyde; ISEL, in situ end-labelling.

Animal studies
[12]Testis tissueTUNELIncreased apoptosisSlower testicular growth in varicocele
[13]Testis tissueFCIncreased apoptosisIncreased the height of the peak with duration of varicocele
[14]Testis tissueISELIncreased apoptosis1) Increased apoptosis in bilateral testis 2) Trend of increased apoptosis with a longer duration
[15]Testis tissueTUNELIncreased apoptosisNo significant difference of contralateral testis between varicoceles and controls
[16]Testis tissueIHCIncreased Bax expressionIn the varicocele group: 1) Increased tissue MDA 2) Decreased antioxidant enzymes In the varicocele group exposed to melatonin: 1) Decreased Bax expression 2) Decreased tissue MDA 3) Increased antioxidant enzymes
[17]Testis tissueTUNELIncreased apoptosisIn the varicocele group: 1) Increased ROS In the varicocele group given vitamin E: 1) Decreased ROS 2) Decreased apoptosis (some degree)
[18]Testis tissueTUNELNo increased apoptosisIncreased p53 expression (determined by IHC)
Human studies
[19]Seminal plasmaTUNELIncreased apoptosis
[20]Testis tissueTUNELIncreased apoptosis
[21]Testis tissueTUNELDeceased apoptosis1) Decreased apoptosis of Sertoli cell in varicocele 2) No correlation between apoptosis and sperm concentration 3) No correlation between apoptosis and serum level of FSH, LH, testosterone or oestradiol
[11]Testis tissueTUNELIncreased apoptosisIncreased testicular cadmium level
[22]Seminal plasmaTUNELIncreased apoptosisIncreased Fas expression (determined by Western blot)
[23]Testis tissueIHCLower caspase-3 expressionCorrelation with caspase-3 and LH
[24]Testis tissueTUNELIncreased apoptosis1) Microdeletions of the L-VDCC in 40% 2) After varicocelectomy, lower sperm density in patients with microdeletions of the L-VDCC
[25]Seminal plasma (adolescents)Comet assayIncreased DNA fragmentation 
[26]Seminal plasmaELISADecreased soluble form FasAfter varicocelectomy, increased sFas
[27]Seminal plasmaFCIncreased DNA damageIncreased seminal ROS
[28]Testis tissueTUNELBilateral increased apoptosis In left varicoceleIn the varicocele group: 1) Bilateral increased cadmium in left varicocele After varicocelectomy: 1) Decreased apoptosis 2) Decreased cadmium level
[29]Seminal plasmaTUNELIncreased apoptosisNo correlation with apoptosis and semen quality

However, Kilinc et al.[18] detected no link between apoptosis and varicoceles, and documented p53 expression in an experimental varicocele model in rat testes. In that study, the sham-operated groups had a higher level of germ cell apoptosis than did rats with an experimental varicocele or the control groups. They suggested that the expression of mutant p53 in the testes of rats with varicocele might indicate that abnormal cells were not being normally eliminated during spermatogenesis. However, they also suspected that surgical manipulation of the renal vein in their model may have induced inflammation, which could also result in germ cell apoptosis.

Clearly, the current varicocele models differ from clinical varicoceles in humans in several respects. However, these results indicate that the varicocele model in the rat is associated with altered germ cell apoptosis, although some conflicting results related to varicocele and apoptosis are reported.

APOPTOSIS IN MEN WITH VARICOCELE

  1. Top of page
  2. INTRODUCTION
  3. THE CHARACTERISTICS OF APOPTOSIS
  4. APOPTOSIS IN SPERMATOGENESIS
  5. APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE
  6. APOPTOSIS IN MEN WITH VARICOCELE
  7. ROS AND APOPTOSIS IN VARICOCELE
  8. CADMIUM AND APOPTOSIS IN VARICOCELE
  9. CONCLUSIONS
  10. CONFLICT OF INTEREST
  11. REFERENCES

Apoptosis in the ejaculate

Up to 10% of sperm cells in the ejaculate of men with a varicocele were apoptotic, as compared with 0.1% in fertile controls [19]. That report concluded that varicocele induces apoptosis, which is initiated in the testicular tissue and is then expressed in the semen. Saleh et al.[27] showed that infertile men with varicoceles had significantly greater DNA damage in spermatozoa than had normal men. Bertolla et al.[25] also evaluated DNA fragmentation in adolescents with clinically diagnosed varicoceles, and determined that these boys had a higher percentage of cells with DNA fragmentation than did adolescents with no varicocele. Lin et al.[22] reported significantly greater apoptosis in patients with varicocele (29%) than in controls (9%) (P < 0.005). The expression of Fas protein was up-regulated in semen samples obtained from patients with varicocele when compared to a control group, whereas little or no changes in Fas ligand expression were detected in both groups. Fujisawa and Ishikawa [26] addressed the relationship between varicoceles and apoptosis by monitoring the concentrations of the soluble form of Fas (s-Fas) in seminal plasma, to characterize the Fas signalling system with regard to hypospermatogenesis as the result of varicocele. Recognizing that s-Fas could block Fas-dependent apoptosis, they screened the seminal plasma of oligospermic men with varicoceles, oligospermic men with no varicocele, and normal controls, for the levels of s-Fas and the s-Fas ligand. S-Fas was not detected in any of the cases, whereas s-Fas levels were specifically lower only in cases of varicocele. These reduced s-Fas levels were reversed by varicocelectomy. However, although higher temperatures may inhibit s-Fas production in patients with varicocele the reason for this decrease in s-Fas levels remains unknown. Brackin et al.[30] reported that, among men with varicoceles, there appears to be an inconsistent heat-shock protein response, underscoring the heterogeneity inherent in the mechanisms underlying the hypospermatogenesis which is often associated with varicoceles. These inconsistencies may explain, at least partly, why only two-thirds of the men tested by Fujisawa and Ishikawa [26] responded to varicocele surgery by generating greater levels of s-Fas in their seminal plasma.

In contrast to other studies, Chen et al.[29] identified no relationship between semen quality and apoptosis. Although the varicocele group had a significantly higher apoptotic index (AI) than in the fertile controls, semen quality and sperm motion characteristics were not significantly different between the groups. In summary, these data suggest that apoptosis may be significant in the spermatogenetic dysfunctions associated with varicoceles.

Apoptosis in the testicular tissue

Simsek et al.[20] also evaluated the presence of apoptosis in testicular tissue, using the TUNEL assay. Data obtained from 29 samples (varicocele, 24 patients; control, five) revealed that apoptosis was very rare in the testicular tissues of the control group. The mean percentage of apoptotic cells per total germ cell counted in a high-power field (×400) was 2% in the control and 14.7% in the varicocele group. It is clearly very important in case-control studies that the data collected from both cases and controls be of comparable accuracy. However, in the study by Simsek et al. the control testicular tissue was collected via orchidectomy for hormonal-ablation treatment in men with prostate cancer. This is probably attributable to the ethical restrictions inherent to acquiring testicular tissue from young fertile men. Hurley et al.[11] also reported that there were far more apoptotic nuclei in the seminiferous tubules of men with varicocele than in normal controls. Mayer et al.[24] assessed the number of apoptotic bodies in counted tubules, using the TUNEL assay, and detected microdeletions in the l-type voltage-dependent calcium channel (L-VDCC) using RT-PCR with primers. The results of that study suggested that microdeletions of the L-VDCC occurred in ≈ 40% of infertile men with varicoceles, and the mean count of apoptotic bodies/tubule was higher in men with varicocele with deleted IS6 (P < 0.04). After varicocelectomy the sperm densities were significantly lower in these patients (P < 0.01). Recently, Benoff et al.[28] reported that the percentage of apoptotic nuclei was noticeably higher in some men with varicoceles. These elevated values were consistent between testes in all the relevant individuals, despite the presence of varicocele only on the left side. This value was inversely related to increases in sperm concentration after varicocelectomy.

To our knowledge, only one group has reported findings which are contrary to those mentioned above [21,23]. Fujisawa et al.[21] reported fewer apoptotic germ cells in testicular biopsy material obtained from subfertile men with varicoceles than in biopsies of normal men (P < 0.05). There were also fewer apoptotic cells per Sertoli cell in the testes of men with varicocele than in those of normal men (P < 0.01). The evaluation of all specimens, including normal controls, revealed no significant correlations, either between the AI and sperm concentration on the seminal assay, or between the AI and Johnsen's mean scores. There was also no relationship between the AI and the serum levels of FSH, LH, testosterone or oestradiol. The same group subsequently assessed the expression of apoptosis-related proteins in infertile men with varicocele [23]. Although Bcl-2 was not expressed in the germ cells, these cells expressed low levels of Bax, with no significant differences between the testes of patients with varicoceles and the specimens from infertile men. Significantly fewer germ cells were detected in both testes from infertile patients with varicoceles stained for caspase-3, an enzyme which is activated when cells receive apoptosis-triggering signals, than in the testes of normal controls (P < 0.001). They suggested that apoptosis might be suppressed as the result of reduced expression of caspase-3, and that the mitochondrial pathway involving bcl-2 and Bax may not be involved in apoptotic regulation in germ cells.

Although various factors, including the clinical status of the patient, the methods used to quantify apoptosis, and the cell type assessed (ejaculatory sperm vs biopsy material), almost certainly have a role, such results appear to be contradictory.

ROS AND APOPTOSIS IN VARICOCELE

  1. Top of page
  2. INTRODUCTION
  3. THE CHARACTERISTICS OF APOPTOSIS
  4. APOPTOSIS IN SPERMATOGENESIS
  5. APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE
  6. APOPTOSIS IN MEN WITH VARICOCELE
  7. ROS AND APOPTOSIS IN VARICOCELE
  8. CADMIUM AND APOPTOSIS IN VARICOCELE
  9. CONCLUSIONS
  10. CONFLICT OF INTEREST
  11. REFERENCES

Seminal ROS may result in sperm DNA damage in patients with varicoceles. At the molecular level, ROS affect DNA directly, and alter the levels of intracellular Ca2+, which is known to be one of the more effective means of inducing apoptosis. Morphological alterations in testicular tissues have been reported as ‘stress patterns’ in patients with varicoceles. This stress pattern is reminiscent of, although not identical to, the cytomorphological changes in apoptosis. Hendin et al.[31] detected high levels of seminal ROS, and reduced total antioxidant capacity, in both fertile and infertile men with a clinical diagnosis of varicocele. Therefore, it was hypothesized that spermatozoal dysfunction in association with varicoceles may be related, at least in part, to elevated levels of sperm DNA damage induced by the high levels of ROS which are common in such patients.

In experimentally induced varicoceles [16] there was no significant difference between the sham-operated group and the melatonin-treated (10 mg/kg) varicocele group in the levels of the pro-apoptotic marker Bax. In addition, the melatonin–treated varicocele group had lower malonedialdehyde levels and high levels of antioxidant enzyme activity with the reduced Bax expression. Cam et al.[17] reported that ROS generation in a varicocele group treated with vitamin E was significantly lower than that in a varicocele group not treated with vitamin E. Although this difference was not significant, the AI values detected in the vitamin E group were lower than those in the varicocele group. These results suggest that ROS generation and apoptosis in the testicles can be induced by an experimentally created varicocele, and that the overproduction of ROS is crucial as an apoptosis-triggering mechanism.

In humans, Saleh et al.[27] also reported that infertile men with varicoceles had significant increases in spermatozoal DNA damage, which appeared to be associated with high ROS levels in the semen. This finding of high seminal ROS levels in patients with varicoceles might indicate that ROS plays a role in the pathogenesis of sperm DNA damage in such patients. However, further research will be required to determine the precise mechanisms responsible for the increase in DNA damage to spermatozoa from infertile men with varicoceles, and to determine whether repairing the varicocele can ameliorate such damage.

CADMIUM AND APOPTOSIS IN VARICOCELE

  1. Top of page
  2. INTRODUCTION
  3. THE CHARACTERISTICS OF APOPTOSIS
  4. APOPTOSIS IN SPERMATOGENESIS
  5. APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE
  6. APOPTOSIS IN MEN WITH VARICOCELE
  7. ROS AND APOPTOSIS IN VARICOCELE
  8. CADMIUM AND APOPTOSIS IN VARICOCELE
  9. CONCLUSIONS
  10. CONFLICT OF INTEREST
  11. REFERENCES

In one study [32] there were higher cadmium levels in the seminal plasma of infertile men with varicoceles than in those with no varicocele. Cadmium, a highly toxic substance, is one of the major toxicants in cigarette smoke, and has been implicated in apoptosis [33]. As the testes lack an active pump for removing cadmium, and resist the stress-induced expression of transportable cadmium-sequestering molecules, it is clear that, over time, testicular cadmium levels can accumulate. Increased cadmium levels on the testis side of the blood-testis barrier may exacerbate the effects of increased pressure, because cadmium itself alters the permeability of the barrier. This results in oedema, renders the blood-testis barrier more porous, and causes the testes to accumulate cadmium more rapidly [28].

Hurley et al.[11] correlated the number of apoptotic cells in the seminiferous tubules with the levels of cadmium in the testes. The testicular cadmium level was higher in patients with varicocele. The TUNEL assay showed apoptotic nuclei in the seminiferous tubules, increasing from <6% in the hypospermatogenic controls to ≈ 30% in patients with normal spermatogenesis, a varicocele and high cadmium level, and to >50% in the hypospermatogenic men with varicocele and a high cadmium level. These authors suggested that cadmium resulted in markedly high levels of apoptosis, ultimately culminating in oligospermia. Benoff et al.[28] also reported that both cadmium levels and the percentage of apoptotic nuclei tended to be high in some men with varicocele. These values were concordant in both testes, despite the presence of a varicocele only on the left side. The values were inversely related to increases in sperm concentration occurring after varicocelectomy. These data suggest that cadmium, a metal ion which is known to induce apoptosis, may contribute to this form of male infertility.

CONCLUSIONS

  1. Top of page
  2. INTRODUCTION
  3. THE CHARACTERISTICS OF APOPTOSIS
  4. APOPTOSIS IN SPERMATOGENESIS
  5. APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE
  6. APOPTOSIS IN MEN WITH VARICOCELE
  7. ROS AND APOPTOSIS IN VARICOCELE
  8. CADMIUM AND APOPTOSIS IN VARICOCELE
  9. CONCLUSIONS
  10. CONFLICT OF INTEREST
  11. REFERENCES

Varicoceles are a common cause of male infertility, but despite data obtained from animal models and human studies, the pathophysiology of varicocele-associated infertility remains unclear. As neither of these sources of data alone may fully explain the variable effects that a varicocele has on spermatogenesis and male fertility, the causes of infertility in varicoceles may be multifactorial. Although some controversy remains, new studies of the testicular tissues of men with varicoceles show elevated levels of apoptosis among developing germ cells, which may be the cause of oligospermia. In addition, as apoptotic levels tend to be higher in the spermatozoa ejaculated from men with varicocele than in those from healthy men, apoptosis is a potential cause of sperm DNA damage in such patients [34]. However, although varicoceles are fairly widely considered to induce apoptosis, current reports related to varicoceles and apoptosis are conflicting. Many of the pathogenic and physiopathological aspects of varicocele require further elucidation.

REFERENCES

  1. Top of page
  2. INTRODUCTION
  3. THE CHARACTERISTICS OF APOPTOSIS
  4. APOPTOSIS IN SPERMATOGENESIS
  5. APOPTOSIS IN EXPERIMENTALLY INDUCED VARICOCELE
  6. APOPTOSIS IN MEN WITH VARICOCELE
  7. ROS AND APOPTOSIS IN VARICOCELE
  8. CADMIUM AND APOPTOSIS IN VARICOCELE
  9. CONCLUSIONS
  10. CONFLICT OF INTEREST
  11. REFERENCES
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