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The Fas system may have a role in male reproduction
The Fas system may have a role in male reproduction Ciler Celik-Ozenci, D.D.S., Ph.D.,a Zeliha Sahin, M.Sc.,a Ismail Ustunel, Ph.D.,a Gokhan Akkoyunlu, Ph.D.,a Tibet Erdogru, M.D.,b Emin Turkay Korgun, Ph.D.,a Mehmet Baykara, M.D.,b and Ramazan Demir, Ph.D.a a Department of Histology and Embryology and b Department of Urology, Akdeniz University, Faculty of Medicine, Antalya, Turkey
Objective: To assess what the distributions of Fas system proteins are in normal rat testicular tissue; to assess whether there is a change in these distributions and in expression levels with experimentally-induced varicocele of 9, 11, and 13 weeks; and to assess whether there is a relationship between apoptosis and the Fas system in varicocele-induced rat testis. Design: Comparative and controlled study. Setting: University animal care and operation unit. Animal(s): Wistar male rats for experimental and control groups. Intervention(s): The control group underwent sham operation (n ⫽ 6). Rats in experimental groups underwent partial ligation of the renal vein to induce an experimental varicocele and then were killed at 9 (n ⫽ 6), 11 (n ⫽ 6), and 13 (n ⫽ 6) weeks after induction of varicocele. Main Outcome Measure(s): Tissues were fixed and processed for paraffin and Araldite embedding, and subsequently immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, and transmission electron microscopy were performed. In addition, Western blotting was applied. Result(s): In control testis, we detected the expression of FasL in spermatids, interestingly at the progressing stages of acrosome formation and in the heads of the spermatozoa being released to lumen. Varicocele induction revealed a significant down-regulation of this protein, especially 11 weeks after the operation, without altering its distribution. Fas protein was present in cytoplasmic extrusions of the elongated spermatids and evidently in Leydig cells of the interstitial tissue. The expression of Fas protein was diminished after 11 weeks of varicocele induction, both in Leydig cells and in cytoplasmic extrusions. The decrease of Fas was significant in the 13-weekold varicocele group, whereas that of FasL was significant in the 11-week-old varicocele group. Compared with sham-operated animals, a minor increase in the number of apoptotic germ cells in varicocele groups was detected. Conclusion(s): Our results exposed other possible important roles of the Fas system in addition to than apoptosis in male reproduction. We suggest that the role of the Fas system needs further investigation both in animal models and in human male infertility. (Fertil Steril威 2006;85(Suppl 1):1168 –78. ©2006 by American Society for Reproductive Medicine.) Key Words: Testis, varicocele, Fas, FasL, rat, immunohistochemistry, electron microscopy, Western blot, TUNEL
Apoptosis of select germ cells occurs normally in the testis and is essential for the normal maintenance of spermatogenesis (1–3). Regular apoptosis of spermatogenic cells is required to maintain proper testicular homeostasis, although increased cell death can result in defective spermatogenesis leading to infertility (4). Increases in the incidence of germ cell apoptosis often are observed as a result of various forms of physical or chemical injury to the testis (5). The Fas signaling system is a widely recognized apoptosisinitiating pathway (6 –9). In the testis, ligation of Fas on select germ cells by Sertoli cell– expressed FasL is proposed Received November 25, 2004; revised and accepted August 17, 2005. Supported by Akdeniz University Research Foundation, Antalya, Turkey (project number 2002.02.0122.003). Reprint requests: Ciler Celik-Ozenci, D.D.S., Ph.D., Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, 07070, Antalya, Turkey (FAX: ⫹90-242-2274486; E-mail: cilercelik@ yahoo.com).
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as a paracrine signaling system by which Sertoli cells initiate apoptosis of Fas-bearing germ cells (10 –13). However, recently, in several reports, it has been shown that FasL is expressed in spermatogenic cells, not in Sertoli cells (14 – 16). Therefore, a discussion arose in the literature regarding the necessity of reconsideration of the roles so far proposed for this molecule in the testis, such as maintenance of immunoprivilege and regulation of physiological germ cell apoptosis. Moreover, there still are various results related to the localization of Fas protein in the literature (11, 13, 17–19). Varicoceles are pathological dilations of the venous pampiniform plexus of the spermatic cord and occur more frequently on the left side (20). Although varicocele appears to be associated with male infertility, it has been well documented that most men with varicocele are fertile (21). Moreover, subclinical varicoceles do not play a major role in male infertility (22). Clinical varicocele is observed in 10%–20% of the general male population, whereas it is demonstrated in
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19% to 41% of men presenting for infertility investigations (20, 23–25). The pathophysiologies of testicular damage in varicocele are not completely understood; however, gross testicular alterations associated with varicocele are well documented. The effect of the varicocele varies but often may result in a generalized impairment of sperm production, characterized by abnormal sperm quality, ranging from oligozoospermia to complete nonobstructive azoospermia. Furthermore, varicocele influences not only the physiology and the reproductive potential of the sperm but also the fertilizing capacity of the haploid gamete (26).
mm diameter. The wire was removed from the ligature, and the vessel expanded to an external diameter of approximately 1 mm. After instillation of 10 mg/mL of gentamicin sulfate into the abdominal cavity, midline incision of abdominal wall and anterior abdominal muscles were repaired separately. After identification of left varicocele by observation of the dilatation of the internal spermatic veins (36), both testicles then were harvested from the same incision. All surgical procedures were performed under ⫻3.5 magnification. Sham-operated rats underwent a similar procedure: the left renal vein was dissected free but was not ligated.
There are many studies in the literature indicating an increased germ cell apoptosis as a result of varicocele formation (27–34). However, how the varicocele pathology affects the expression of FasL and Fas proteins has not yet been studied. The present study was organized to explain three important open questions: first, what are the distributions of the Fas system proteins in normal rat testicular tissue? Second, are their distribution and expression levels altered with the time progression of experimentally induced varicocele? Third, is there a relationship between apoptosis and Fas system in varicocele-induced rat testis?
The experimental protocol was approved by the animal core and usage comittee of Akdeniz University and was in accordance with the Declaration of Helsinki and International Association for the Study of Pain guidelines.
MATERIALS AND METHODS Animals A total of 24 male Rattus norvegicus (Wistar) rats obtained from Akdeniz University Laboratories were included in the study. The establishment of the control and experimental groups were described elsewhere (35). Briefly, the adult animals (7 weeks old) were used for control group and underwent sham operation when they were 18 weeks old (n ⫽ 6). Seven-week-old animals were used for experimental groups and underwent partial ligation of renal vein (n ⫽ 18). A minimum of six animals was sampled at each of the following time points after the varicocele operation: 9 weeks (7-week-old rats, 9 weeks later), 11 weeks (7-week-old rats, 11 weeks later), and 13 weeks (7-week-old rats, 13 weeks later). After anesthesia was performed by using Xylasine (Rompun, 15 mg/kg IM; Bayer AG, Leverkusen, Germany) and ketamine (Ketalar, 75 mg/kg, IM; Parke Davis, Istanbul, Turkey), experimental varicocele was created in 18 rats as described elsewhere (35, 36). The upper left abdominal quadrant was approached through a midline laparotomy incision. The abdominal contents were packed to the right to visualize the left kidney, adrenal and renal veins, and the left spermatic vein as it inserts into the left renal vein. By careful blunt dissection, a tunnel was made in the fat and connective tissues surrounding the left renal vein, and the vein is cleared of adhering tissue in a position medial to the insertion of the spermatic and adrenal veins. A 4 – 0 silk suture was tied to partially occlude the left renal vein at the point that the vessel was cleared of other tissue. The ligature was made around a metal wire of 0.85 Fertility and Sterility姞
Tissue Processing Testes were obtained from sham-operated (control) and varicocele-induced adult Wistar rats weighing 200 –250 g. Animals were killed at 9, 11, and 13 weeks after creation of varicocele, and testes were delivered into the abdomen. Tissues were fixed by immersion in Bouin’s (picric acid, formalin, and glacial acetic acid) fixative and were processed routinely for paraffin embedding. Immunohistochemistry For Fas and FasL immunostaining, fixed sections were deparaffinized and blocked for endogenous peroxidase activity with methanol containing 3% H2O2 for 10 minutes and for nonspecific binding with universal blocking reagent (BioGenex, San Ramon, CA) for 10 minutes at room temperature. Rabbit polyclonal Fas and rabbit polyclonal FasL primary antibodies (Santa Cruz Biotechnology Inc, Santa Cruz, CA) were applied in a dilution of 1 g of IgG per milliliter for 1.5 hours at room temperature. Negative control stainings were performed by replacing the primary antibodies with their nonimmune isotypes at the same concentration. The antibodies were detected with an avidin– biotin horseradish peroxidase complex with a Universal LSAB Kit (Dako, Glostrup, Denmark). Antibody complexes were visualized by incubation with diaminobenzidine (DAB) chromogen (BioGenex). Sections were counterstained with Mayer’s hematoxylin (Dako) for 30 seconds, dehydrated, mounted, and examined by light microscopy. Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick End-Labeling Apoptosis in testicular tissue was detected by enzymatic labeling of DNA strand breaks by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). Paraffin sections of 5-m thickness from the testicular tissues were cut and placed on slides covered with poly-L-lysine, and after drying, the slides were left in the incubator at 45°C overnight and at 60°C for 1169
1 hour. After deparaffinization and rehydration, slides were washed twice in phosphate-buffered saline for 5 minutes. After incubation of slides with the permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 8 minutes at 4°C and washing twice with phosphate-buffered saline for 5 minutes, the labeling reaction was performed by using 50-L of TUNEL reagent for each sample, except negative control, in which reagent without enzyme was added and incubated for 1 hour at 37°C. After phosphatebuffered saline washings, slides were incubated with converter reagent for 30 minutes at 37°C. After washing, color development for localization of cells containing labeled DNA strand breaks was performed by incubating the slides with Fast Red substrate solution for 10 minutes. Labeling with TUNEL was conducted with a Cell Death Detection kit (Roche, Mannheim, Germany) and was performed according to the manufacturer’s instructions. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Blotting Protein extraction and immunoblot analysis were performed as described elsewhere (37). In brief, tissues were weighed and put into homogenationhomogenation buffer (10 mM Tris-HCL, 1 mM ethylenediaminetetraacetic acid, 2.5% sodium dodecyl sulfate, 1 mM phenylmethylsulfonylfluroide, and 1 g/mL of leupeptin) supplemented with CompleteR protease inhibitor cocktail (Boehringer, Mannheim, Germany). After homogenization, samples were processed by centrifuge at 10,000 ⫻ g for 10 minutes. Supernatants were collected and stored at ⫺70°C. The protein concentration was determined by Lowry assay (38), and 50 g of protein was applied per lane. Before electrophoresis, samples were boiled for 5 minutes at 95°C. Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then were transferred onto nitrocellulose membranes (Pharmacia) to remain overnight in a buffer containing 0.2 mol/L glycine, 25 mM Tris, and 20% methanol. Successful transfer was confirmed by Ponceau S (Sigma) staining of the blots. The membranes were blocked for 1 hour with 5% nonfat dry milk (BioRad, Hercules, CA) and 0.1% Tween 20 (Sigma) in 0.14 mol/L Tris-buffered saline (TBS; pH 7.2–7.4) at 4°C. Blotting membranes were incubated for 1 hour at room temperature with rabbit antisera against Fas and FasL, both diluted as 2 g/mL (Santa Cruz). After washing steps, the membranes were further incubated with goat anti-rabbit IgG horseradish peroxidase conjugate (BioRad) diluted 1:5,000 for 1 hour at room temperature. Immunolabeling was visualized by using the chemiluminescence-based SuperSignal CL HRP Substrate System (Pierce, Rockford, IL), and the membranes were exposed to Hyperfilm (Amersham), which subsequently was quantified with an Alpha Digi Doc 1000 gel documentation unit (Alpha Innotech Corporation, CA). Control blots were 1170
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incubated with antibody diluent only instead of the primary antiserum. -Actin antibody (1:5,000 dilution) was used as an internal control for each blotting to confirm the equal loading of the samples. Electron Microscopy Morphological studies were performed as described elsewhere (39). Samples of rat testicular tissues were fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 4 hours and were postfixed in 1% phosphate-buffered osmium tetroxide for 2 hours. The specimens were dehydrated in ethanol and embedded in Araldite epoxy resin (SPI-CHEM, Structure Probe, West Chester, PA). Semi-thin sections were stained with toluidine blue. Ultra-thin sections were double stained with uranyl acetate and lead citrate and examined with an electron microscope (Leo 906 E, Zeiss, Oberkochen, Germany). Statistical Analysis Statistical analyses of FasL and Fas Western blot quantifications were compared by one-way analysis of variance tests followed by post hoc Tukey tests for parametric data. Comparisons were made vs. control group. Probability values of ⬍.05 were considered significant, and values are presented as mean ⫾ SEM. RESULTS Histological Evaluation of the Varicocele Model Histological evaluation of our experimental model was determined according to our previously published study (35). Briefly, testis tissue specimens harvested from sham and from the 9-, 11-, and 13-week varicocele groups were stained with hematoxylin and eosin. In varicocele groups, disintegration of the germinal epithelium and degenerative appearance was observed visibly by light microscopy. In our recently published study, we found that degenerative tubules were evident, especially in 11-week and 13-week varicocele groups, when compared with the sham-operated rat testis (35). In the present study, we used the same tissue blocks for sham-operated and experimental groups. Localization of FasL and Fas in Rat Testis To clarify the cell types that express FasL and Fas proteins, immunohistochemical studies were performed with rat testicular tissue. In control tissue (sham-operated rats), FasL immunostaining was clearly visible only in spermatogenic cells but not in Sertoli cells. In the seminiferous tubule sections, FasL immunostaining mainly was observed in Golgi structures (acrosomal vesicle) of round spermatids (Fig. 1a), with a progressive expression in the cap phase of the sperm acrosome (acrosomal cap) resembling a semi-ring (Fig. 1b). Finally, FasL was present in the heads of elongated spermatids, most
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FIGURE 1 Localization of FasL protein in sham-operated (a– c) and varicocele-induced (d– h) rat testis. (a) FasL first appears in Golgi vesicles of round spermatids (arrowheads and inset), (b) then in acrosomal caps of round spermatids (arrowheads and inset), and (c) finally in heads of elongating spermatids (arrowheads and inset). (d) In the 9-week-old varicocele group, the expression of FasL was similar to that of control, as each phase of the expression pattern (arrows) was seen in tubuli sections. (e, f) In the 11-week-old varicocele group, there were cells still expressing FasL in their heads (arrows), whereas some cells did not express this protein accurately (arrowheads and insets). (g, h) In the 13-week-old varicocele group, some cells were negative in the same tubule (arrowheads), whereas some cells were positive for FasL (arrows). (i) Negative-control section; note the absence of FasL immunostaining. Scale bars represent 100 m (panel i) or 10m (panels a– h).
Celik-Ozenci. Fas system in varicocele-induced rat testis. Fertil Steril 2006.
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FIGURE 2 Localization of Fas protein in sham-operated (a) and varicocele-induced (b– d) rat testis. (a) Fas was present in cytoplasmic extrusions of elongated spermatids (arrow) and also in Leydig cells of the interstitial tissue (inset, arrowhead). (b) Similar to control, Fas was present in cytoplasmic extrusions (arrow) and in Leydig cells (arrowhead and inset) in the 9-week-old varicocele group. (c) Diminished intensity for Fas in Leydig cells can be seen (arrowhead and inset) in the 11-week-old varicocele group. (d) There were still a few cells immunostained with Fas in the 13-week-old varicocele group (arrowhead and inset). Scale bars represent 10 m.
Celik-Ozenci. Fas system in varicocele-induced rat testis. Fertil Steril 2006.
likely localized in their acrosomes (Fig. 1c). An intense immunostaining of FasL protein was detectable in both round and elongated spermatids. In the sham-operated rats, Fas immunostaining was essentially localized in cytoplasmic extrusions of the elongated spermatids (Fig. 2a) and in Leydig cells (Fig. 2A, inset). Down-Regulation of Fasl and Fas in Varicocele-Induced Rat Testis In the 9-week-old varicocele group, the distributions and the intensities of the FasL were similar to the sham-operated group. The protein was localized in Golgi vesicles of round spermatids, in the cap phase of developing sperm acrosome, and in the heads of elongated spermatids (Fig. 1d). In the 11-week-old varicocele group, positive immunolabeling still was present in round and elongated spermatids, but at a reduced intensity when compared with the control testis. We observed that in some tubules, round spermatids expressed FasL precisely (Fig. 1f), whereas, in some tubules, the expression of this protein was not seen accurately (Fig. 1f, inset). Moreover, FasL was present in elongated sperm, whereas its expression in elongated sperm was less in an adjacent tubule (Fig. 1e and inset).
the intensity of Fas protein were parallel to that in the sham-operated group (Fig. 2b and inset). Furthermore, starting from 11 weeks, the intensity of Fas immunostaining reduced gradually (Fig. 2c and inset). In the 13-week-old varicocele group, localization of this protein was limited to Leydig cells but with diminished intensity (Fig. 2d, inset). Evaluation of TUNEL Staining We observed TUNEL-positive cells in both sham-operated and varicocele-induced testes. Because the numbers of apoptotic cells were quite few in study groups, we did not perform an apoptotic index; thus, we gave representative figures for each of the study groups (Fig. 3a– d). Although we detected that apoptotic cells in the varicocele groups were slightly higher than in sham, this difference was not considerable. Additionally, we did not observe any significant valuable differences in the number of TUNEL-positive cells between the experimental groups.
Finally, in the 13-week-old varicocele group, immunolabeling of FasL still existed in some of the seminiferous tubules; however, there was a distinct decrease in its immunostaining intensity in both round and elongated spermatids (Fig. 1g and h). It was observed that not every single spermatid contained this protein regularly (Fig. 1h).
Confirmation of FasL and Fas Down-Regulation by Western Blot The presence of rat FasL and Fas proteins were also observed by Western blots using lysates of testis (Fig. 4). We detected FasL at 38 kDa and Fas at 48 kDa, as suggested by the manufacturer. Our results revealed that the expression of Fas decreased statistically significantly (P⬍.05) after the 13-week-old varicocele group (Fig. 5a), whereas the decrease of FasL was earlier at the 11-week-old varicocele group when compared with the case of sham-operated controls (Fig. 5b).
Fas immunoreactivity was present in cytoplasmic extrusions of elongated spermatids and in Leydig cells of the 9-, 11-, and 13-week-old varicocele groups at various densities. In the 9-week-old varicocele group, both the localization and
Evaluation of Electron-Microscopic Findings Ultrastructural analysis confirmed the effect of experimental varicocele in rat testis. Varicocele resulted in injuries of
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FIGURE 3 Detection of TUNEL-positive apoptotic germ cell nuclei (arrows) in seminiferous tubular cross section. (a) Sham, (b) 9-week-old varicocele, (c) 11-week-old varicocele, and (⬎d) 13-week-old varicocele.
Celik-Ozenci. Fas system in varicocele-induced rat testis. Fertil Steril 2006.
Sertoli–Sertoli cell and Sertoli–spermatogenic cell contacts (Fig. 6A and B). DISCUSSION We examined the expression of the Fas–FasL system in sham-operated and in varicocele-induced rat testis by immunohistochemistry and Western blot, as well as, by ultrastructural analysis. Moreover, to assess whether there is any relevance to the expression of the Fas–FasL system, apoptotic cells were evaluated by TUNEL method. Our results revealed that in control testis, FasL was visibly localized to round and elongated spermatids. This finding is contrary to most of the extensive statements in the literature, which argue that FasL is present in Sertoli cells rather than in germ cells of the seminiferous tubules (10 –13). However, recently, the findings of D’Alessio et al. (14) for the first time provided direct evidence based on Northern blot analysis Fertility and Sterility姞
that the attribution of testicular expression of FasL to Sertoli cells is erroneous. Elsewhere, it has been pointed out that there is continued increase of erroneous ideas about FasL as a result of technical problems related to specificity of the antibodies or as a result of the paradigm, already not accepted by several reports (40 – 43), that this protein has a role in immunoprivilege and in tumor escape. It is generally accepted that in the testis, FasL is expressed in Sertoli cells, and spermatogenic cells that contain its receptor would go to apoptosis (44, 45). Conversely, D’Alessio et al. (14) have found the transcription of this protein predominantly in round spermatids and, to lesser extent, in pachytene spermatocytes of mouse and rat testis. When they used isolated germ cells of the seminiferous tubules, they found the mRNA signal for FasL only in postmeiotic germ cells, particularly in elongated spermatids, whereas Sertoli cells were negative (14). The protein expression of FasL was found at a significant level 1173
FIGURE 4 Expression of Fas and FasL proteins in sham and different varicocele weeks determined by Western blot analysis using -actin as an internal standard.
found in germ cells and protein displayed on the surface of mouse spermatozoa may represent a self-defense mechanism against lymphocytes that are present in the female genital tract. Moreover, there are other studies in the literature that support this idea. Takeda et al. (49) investigated whether a graft can be protected from alloimmune responses by manipulating the Fas–FasL-system; thus, they transplanted allogeneic islets under the kidney capsule of diabetic mice together with testicular tissue. They found that FasL-positive testicular allografts protected composite islet allografts and concluded that manipulation of Fas–FasL mediated apoptosis is a suitable strategy for controlling rejection of islet allografts (49). Braendstrup et al. (50) studied the expressions of Fas and FasL in seminomas with surrounding normal and precancerous seminiferous tubule tissue and found that FasL was expressed in virtually all normal seminiferous tubules, whereas in precancerous tubules, the expression was discontinuous and less pronounced. Thus, the investigators discussed the concept of the testis being an immunologically privileged area and immune reaction to seminomas (50). Formerly, the involvement of immunological mechanisms in the development of pathological changes in the testes affected with varicocele was suggested (51).
Celik-Ozenci. Fas system in varicocele-induced rat testis. Fertil Steril 2006.
on spermatid after applying permeabilization (14) because there are several findings suggesting that FasL may be stored in specialized cytoplasmic vesicles (46, 47). In another recent study, FasL was found to be present intensely in elongated spermatid cytoplasm, especially after estrogen induction, and those investigators concluded that spermatogenic cells’ ability to control germ cell apoptosis is independent of Sertoli cells (15). Our FasL immunostaining findings are in accordance with D’Alessio et al. (14). Moreover, we found that the protein expression of FasL first was seen in acrosomal vesicle, continued with acrosomal cap, and finally became localized in heads (most likely in acrosomes) of the developing spermatids. Additionally, D’Alessio et al. (14) found that murine spermatozoa display on their surface a high concentration of FasL. Those investigators have stated that the roles so far proposed for the Fas system in the testis, such as maintenance of immunoprivilege (48) and regulation of physiological germ cell apoptosis (11), need reconsideration because both hypotheses are based on an erroneous cellular localization of Fas and FasL in the seminiferous epithelium. Because of our immunohistochemical results, we believe and also agree with the proposition that the presence of FasL on the sperm head, most likely on the sperm acrosome, is necessary for male gametes to escape both the autoimmune reaction during their journey through the male genital tract and the allogenic reaction that may take place in the female’s body. Recently, Riccioli et al. (16) stated that FasL mRNA 1174
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We have immunolocalized Fas protein essentially in Leydig cells and also in the cytoplasmic extrusions of elongated spermatids. The presence of Fas in cytoplasmic extrusions of elongated spermatids is first reported in our study, and with this limited amount of data we might speculate that the protein is necessary for accurate cytoplasmic extrusion. Previously, it was found that Fas was predominantly localized to Leydig cells, whereas FasL was localized to Sertoli cells in normal mouse testis, and there was no obvious association between the Fas system and occurrence of apoptosis (13). As an immune-privileged organ (48), the testis is known to express FasL at the highest level among tissues (52), whereas the level of Fas expression is very low. Moreover, FasL expression is exclusively restricted to Sertoli cells (53), and that of Fas is largely found in Leydig cells, in addition to a few germ cells (54). Francavilla et al. (17) found a coexpression pattern of Fas and FasL in Leydig cells of adult testis. Immunostaining for Fas was reported in Leydig cells of human testis (19), but for the first time, coexpression of Fas and FasL at both mRNA and protein levels in the Leydig cell was documented (17) and suggested that this colocalization was not associated with the evidence of apoptotic degeneration of Leydig cell, suggesting that the coupling of Fas by its ligand does not transduce a death signal in this cell type. In testis sections from control animals, most, but not all, Leydig cells express Fas and FasL proteins (18). There are some reports that support FasL expression in Leydig cells (54, 55), whereas there are some that do not show FasL expression in the interstitium (11). Another important aspect of our study is that we studied the role of Fas–FasL system in varicocele-induced rat testis
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FIGURE 5 Quantitative analysis of Western blot results (per-group n ⫽ 6). The values in the bar graphs were obtained by dividing the protein intensity value to its corresponding -actin intensity value. (a) Fas protein was significantly reduced in the 13-week-old varicocele group when compared with sham. (b) FasL expression was significantly less in the 11-week and 13-week-old varicocele groups when compared with sham. *P⬍.05.
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for the first time. Studies state that unilateral varicocele has been associated with bilateral physiologic and histological changes in the testis of humans and laboratory animals (56). However, a recent study by Barqawi et al. (27) demonstrated that the creation of experimental varicocele in rats generated a significant increase in germ cell apoptosis in the ipsilateral testis. We studied the effects of varicocele in the ipsilateral Fertility and Sterility姞
testes; thus, contralateral testes were not evaluated in the present study. It is accepted in the literature that apoptosis is increased in ejaculated spermatozoa in patients with varicocele compared with normal fertile men (30, 31) and is increased in testes of human with varicocele (27, 28, 57). Moreover, experimental 1175
FIGURE 6 Ultrastructural features of varicocele-induced rat testicular tissue. (a) General appearance of germinal epithelium in the seminiferous tubule wall of the 13-week-old varicocele group. (b) Arrows indicate the disturbance of contact points between spermatogonium (SG) and Sertoli cell (ST) in the 13-week-old varicocele group. L ⫽ lumen. Scale bars represent 10 m (a) and 2 m (b).
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models have supported this idea that the creation of experimental varicocele generated an increase in germ cell apoptosis in rats when compared with controls (27, 32, 33). There are also contradictory statements in the literature indicating that apoptosis is decreased in germ cells in the testis of infertile men with varicocele as compared with normal men (58). In addition, Tanaka et al. (59) suggested that apoptosis in testes of infertile men with varicocele might be suppressed as a result of decreased caspase-3 expression. A recent study by Kilinc et al. (60) suggested no relationship between apoptosis and varicocele in rats. Another recent study revealed that busulfan-induced spermatogenic germ cell apoptosis pathway is independent of p53 or Fas–FasL; the investigators found that although the number of TUNELpositive cells increased, the expression of FasL decreased by progressing time of busulfan treatment (61). It is clear from the studies mentioned that the current literature related to varicocele and apoptosis still contains conflicting results, both in human subjects and animal models. Additionally, using the FasL-defective gld mice, Hikim et al. (62) have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system may be dispensable for heat-induced germ cell apoptosis in the testis. Even though there is an increase in the number of apoptotic cells, this was not related to Fas–FasL system (62). There are limited data on the role of the Fas–FasL system in the apoptotic mechanism in varicocele-associated testis. We performed the current study in an effort to shed addi1176
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tional light on this subject. To our knowledge, until the present study, nobody has searched for the role of the Fas– FasL system in experimental varicocele of rat testis. We observed increased numbers of degenerative tubules and decreased immunostaining of the Fas–FasL system with progression of varicocele over time. In contrast, the presence of TUNEL-positive cells slightly increased in varicocele-induced testes. Germ cell death clearly was seen among spermatogonia and spermatocytes but was scarce. Therefore, we claim that the Fas–FasL system may not only be associated with apoptotic mechanisms but also may be related to the survival of sperm from immunological attack. There are many studies in the literature concerning the ultrastructural effects of varicocele to the seminiferous epithelium. Spermatogenic arrest, defects of the basal lamina, and injuries of the junctional complexes are the most frequent anomalies observed by the researchers (63– 65). Our results also confirmed the disintegration of the seminiferous epithelium in varicocele-induced testicular tissue. Our results have led us to comment on two issues. First, the presence of FasL in normal testicular tissue appears to be related to the survival of spermatozoa from a possible immunological attack that may occur in either the male or female genital tract. Consequently, the relative decrease of this protein in the varicocele model might increase the number of cells that could not survive these possible attacks, therefore leading to varicocele-associated male infertility.
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Second, the presence of Fas in cytoplasmic extrusions of elongated spermatids might be necessary for normal spermiogenesis, especially for normal sperm head morphology. Thus, the decrease of this protein in varicocele-induced testicular tissue might result with defective cytoplasmic extrusion of cells. However, we do believe that this hypothesis still needs further investigation. On the other hand, from human studies, it is known that varicocele-related male infertility is associated with impaired disposal of residual sperm cytoplasm by the testis (66). Moreover, the decrease of Fas protein in Leydig cells of varicocele-induced testis might be a kind of resistance of these androgen-producing cells to the pathology, to compensate for the T levels. In conclusion, this is the first study related to the relationship between the Fas–FasL system and varicocele-induced rat testis. According to our results, we think that FasL is necessary for sperm survival from possible immunological attack in either gender’s genital tract, and the presence of varicocele may reduce these cells’ survival chance by affecting the FasL expression in their heads, most probably in their acrosomes. Moreover, the existence of varicocele diminishes the expression of Fas in Leydig cells and in cytoplasmic extrusions of elongated spermatids, suggesting that Fas protein might have another unknown role instead of, or in addition to, apoptosis in testicular tissue. The topic is still under discussion and needs further investigation.
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20. 21. 22.
Acknowledgments: The authors thank Arife Demı˙rtop and Hakan Er (Transmission Electron Microscopy Imaging and Analysis [TEMGA] Unit) for their excellent technical assistance of transmission electron microscopy studies.
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35. Sahin Z, Bayram Z, Celik-Ozenci C, Akkoyunlu G, Seval Y, Erdogru T, et al. Effect of experimental varicocele on the expressions of notch 1, 2 and 3 in rat testes: an immunohistochemical study. Fertil Steril 2005;83:86 –94. 36. Turner TT. The study of varicocele through the use of animal models. Hum Reprod Update 2001;7:78 – 84. 37. Hahn D, Blaschitz A, Korgun ET, Lang I, Desoye G, Skofitsch G, et al. From maternal glucose to fetal glycogen: expression of key regulators in the human placenta. Mol Hum Reprod 2001;7:1173– 8. 38. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. 39. Demir R, Kayisli UA, Seval Y, Celik-Ozenci C, Korgun ET, DemirWeusten AY, et al. Sequential expression of VEGF and its receptors in human placental villi during very early pregnancy: differences between placental vasculogenesis and angiogenesis. Placenta 2004; 25:560 –72. 40. Restifo NP. Not so Fas: re-evaluating the mechanisms of immune privilege and tumor escape. Nat Med 2000;6:493–5. 41. Allison J, Georgiou HM, Strasser A, Vaux DL. Transgenic expression of CD95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts. Proc Natl Acad Sci USA 1997;94:3943–7. 42. Arai H, Gordon D, Nabel EG, Nabel GJ. Gene transfer of Fas ligand induces tumor regression in vivo. Proc Natl Acad Sci USA 1997;94: 13862–7. 43. Kang SM, Schneider DB, Lin Z, Hanahan D, Dichek DA, Stock PG, et al. Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction. Nat Med 1997;3:738 – 43. 44. Akingbemi BT, Hardy MP. Oestrogenic and antiandrogenic chemicals in the environment: effects on male reproductive health. Ann Med 2001;33:391– 403. 45. Williams K, Saunders PT, Atanassova N, Fisher JS, Turner KJ, Millar MR, et al. Induction of progesterone receptor immunoexpression in stromal tissue throughout the male reproductive tract after neonatal oestrogen treatment of rats. Mol Cell Endocrinol 2000;164:117–31. 46. Albanese J, Meterissian S, Kontogiannea M, Dubreuil C, Hand A, Sorba S, et al. Biologically active Fas antigen and its cognate ligand are expressed on plasma membrane-derived extracellular vesicles. Blood 1998;91:3862–74. 47. Martinez-Lorenzo MJ, Anel A, Gamen S, Monleon I, Lasierra P, Larrad L, et al. Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles. J Immunol 1999;163:1274 – 81. 48. Bellgrau D, Gold D, Selawry H, Moore J, Franzusoff A, Duke RC. A role for CD95 ligand in preventing graft rejection. Nature 1995;377: 630 –2. 49. Takeda Y, Gotoh M, Dono K, Nishihara M, Grochowiecki T, Kimura F, et al. Protection of islet allografts transplanted together with Fas ligand expressing testicular allografts. Diabetologia 1998;41:315–21. 50. Braendstrup O, Bols B, Jensen L. Fas and Fas-ligand expression in seminomatous testes. APMIS 1999;107:431– 6.
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51. Raitsina SS, Kurnosov AV, Iarovaia IM, Gladkova NS, Davydova AI. Autoimmune nature of spermatogenesis disorders in varicocele models in rats. Biull Eksp Biol Med 1979;87:44 – 8. 52. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993;75:1169 –78. 53. French LE, Hahne M, Viard I, Radlgruber G, Zanone R, Becker K, et al. Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover. J Cell Biol 1996;133:335– 43. 54. Li H, Ren J, Dhabuwala CB, Shichi H. Immunotolerance induced by intratesticular antigen priming: expression of TGF-beta, Fas and Fas ligand. Ocul Immunol Inflamm 1997;5:75– 84. 55. Sugihara A, Saiki S, Tsuji M, Tsujimura T, Nakata Y, Kubota A, et al. Expression of Fas and Fas ligand in the testes and testicular germ cell tumors: an immunohistochemical study. Anticancer Res 1997;17: 3861–5. 56. Hurt GS, Howards SS, Turner TT. The effects of unilateral, experimental varicocele are not mediated through the ipsilateral testis. J Androl 1987;8:403– 8. 57. Fujisawa M, Ishikawa T. Soluble forms of Fas and Fas ligand concentrations in the seminal plasma of infertile men with varicocele. J Urol 2003;170:2363–5. 58. Fujisawa M, Hiramine C, Tanaka H, Okada H, Arakawa S, Kamidono S. Decrease in apoptosis of germ cells in the testes of infertile men with varicocele. World J Urol 1999;17:296 –300. 59. Tanaka H, Fujisawa M, Okada H, Kamidono S. Apoptosis-related proteins in the testes of infertile men with varicocele. BJU Int 2002; 89:905–9. 60. Kilinc F, Guvel S, Kayaselcuk F, Aygun C, Egilmez T, Ozkardes H. p53 expression and apoptosis in varicocele in the rat testis. J Urol 2004;172:2475– 8. 61. Choi YJ, Ok DW, Kwon DN, Chung JI, Kim HC, Yeo SM, et al. Murine male germ cell apoptosis induced by busulfan treatment correlates with loss of c-kit-expression in a Fas/FasL- and p53-independent manner. FEBS Lett 2004;575:41–51. 62. Hikim AP, Lue Y, Yamamoto CM, Vera Y, Rodriguez S, Yen PH, et al. Key apoptotic pathways for heat-induced programmed germ cell death in the testis. Endocrinology 2003;144:3167–75. 63. Tian L. Influence of experimental varicocele on the junctions of seminiferous epithelium. Zhonghua Bing Li Xue Za Zhi 1991;20:211–3. 64. Wang R, Chang JS, Zhou XM, Chen DY. Varicocele in the rat: a new experimental model. Effect on histology, ultrastructure and temperature of the testis and the epididymis. Urol Res 1991;19:319 –22. 65. Santoro G, Romeo C, Impellizzeri P, Gentile C, Anastasi G, Santoro A. Ultrastructural and immunohistochemical study of basal lamina of the testis in adolescent varicocele. Fertil Steril 2000;73:699 –705. 66. Zini A, Defreitas G, Freeman M, Hechter S, Jarvi K. Varicocele is associated with abnormal retention of cytoplasmic droplets by human spermatozoa. Fertil Steril 2000;74:461– 4.
Fas system in varicocele-induced rat testis
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Objective: To assess what the distributions of Fas system proteins are in normal rat testicular tissue; to assess whether there is a change in these distributions and in expression levels with experimentally-induced varicocele of 9, 11, and 13 weeks; and to assess whether there is a relationship between apoptosis and the Fas system in varicocele-induced rat testis. Design: Comparative and controlled study. Setting: University animal care and operation unit. Animal(s): Wistar male rats for experimental and control groups. Intervention(s): The control group underwent sham operation (n ⫽ 6). Rats in experimental groups underwent partial ligation of the renal vein to induce an experimental varicocele and then were killed at 9 (n ⫽ 6), 11 (n ⫽ 6), and 13 (n ⫽ 6) weeks after induction of varicocele. Main Outcome Measure(s): Tissues were fixed and processed for paraffin and Araldite embedding, and subsequently immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, and transmission electron microscopy were performed. In addition, Western blotting was applied. Result(s): In control testis, we detected the expression of FasL in spermatids, interestingly at the progressing stages of acrosome formation and in the heads of the spermatozoa being released to lumen. Varicocele induction revealed a significant down-regulation of this protein, especially 11 weeks after the operation, without altering its distribution. Fas protein was present in cytoplasmic extrusions of the elongated spermatids and evidently in Leydig cells of the interstitial tissue. The expression of Fas protein was diminished after 11 weeks of varicocele induction, both in Leydig cells and in cytoplasmic extrusions. The decrease of Fas was significant in the 13-weekold varicocele group, whereas that of FasL was significant in the 11-week-old varicocele group. Compared with sham-operated animals, a minor increase in the number of apoptotic germ cells in varicocele groups was detected. Conclusion(s): Our results exposed other possible important roles of the Fas system in addition to than apoptosis in male reproduction. We suggest that the role of the Fas system needs further investigation both in animal models and in human male infertility. (Fertil Steril威 2006;85(Suppl 1):1168 –78. ©2006 by American Society for Reproductive Medicine.) Key Words: Testis, varicocele, Fas, FasL, rat, immunohistochemistry, electron microscopy, Western blot, TUNEL
Apoptosis of select germ cells occurs normally in the testis and is essential for the normal maintenance of spermatogenesis (1–3). Regular apoptosis of spermatogenic cells is required to maintain proper testicular homeostasis, although increased cell death can result in defective spermatogenesis leading to infertility (4). Increases in the incidence of germ cell apoptosis often are observed as a result of various forms of physical or chemical injury to the testis (5). The Fas signaling system is a widely recognized apoptosisinitiating pathway (6 –9). In the testis, ligation of Fas on select germ cells by Sertoli cell– expressed FasL is proposed Received November 25, 2004; revised and accepted August 17, 2005. Supported by Akdeniz University Research Foundation, Antalya, Turkey (project number 2002.02.0122.003). Reprint requests: Ciler Celik-Ozenci, D.D.S., Ph.D., Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, 07070, Antalya, Turkey (FAX: ⫹90-242-2274486; E-mail: cilercelik@ yahoo.com).
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as a paracrine signaling system by which Sertoli cells initiate apoptosis of Fas-bearing germ cells (10 –13). However, recently, in several reports, it has been shown that FasL is expressed in spermatogenic cells, not in Sertoli cells (14 – 16). Therefore, a discussion arose in the literature regarding the necessity of reconsideration of the roles so far proposed for this molecule in the testis, such as maintenance of immunoprivilege and regulation of physiological germ cell apoptosis. Moreover, there still are various results related to the localization of Fas protein in the literature (11, 13, 17–19). Varicoceles are pathological dilations of the venous pampiniform plexus of the spermatic cord and occur more frequently on the left side (20). Although varicocele appears to be associated with male infertility, it has been well documented that most men with varicocele are fertile (21). Moreover, subclinical varicoceles do not play a major role in male infertility (22). Clinical varicocele is observed in 10%–20% of the general male population, whereas it is demonstrated in
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19% to 41% of men presenting for infertility investigations (20, 23–25). The pathophysiologies of testicular damage in varicocele are not completely understood; however, gross testicular alterations associated with varicocele are well documented. The effect of the varicocele varies but often may result in a generalized impairment of sperm production, characterized by abnormal sperm quality, ranging from oligozoospermia to complete nonobstructive azoospermia. Furthermore, varicocele influences not only the physiology and the reproductive potential of the sperm but also the fertilizing capacity of the haploid gamete (26).
mm diameter. The wire was removed from the ligature, and the vessel expanded to an external diameter of approximately 1 mm. After instillation of 10 mg/mL of gentamicin sulfate into the abdominal cavity, midline incision of abdominal wall and anterior abdominal muscles were repaired separately. After identification of left varicocele by observation of the dilatation of the internal spermatic veins (36), both testicles then were harvested from the same incision. All surgical procedures were performed under ⫻3.5 magnification. Sham-operated rats underwent a similar procedure: the left renal vein was dissected free but was not ligated.
There are many studies in the literature indicating an increased germ cell apoptosis as a result of varicocele formation (27–34). However, how the varicocele pathology affects the expression of FasL and Fas proteins has not yet been studied. The present study was organized to explain three important open questions: first, what are the distributions of the Fas system proteins in normal rat testicular tissue? Second, are their distribution and expression levels altered with the time progression of experimentally induced varicocele? Third, is there a relationship between apoptosis and Fas system in varicocele-induced rat testis?
The experimental protocol was approved by the animal core and usage comittee of Akdeniz University and was in accordance with the Declaration of Helsinki and International Association for the Study of Pain guidelines.
MATERIALS AND METHODS Animals A total of 24 male Rattus norvegicus (Wistar) rats obtained from Akdeniz University Laboratories were included in the study. The establishment of the control and experimental groups were described elsewhere (35). Briefly, the adult animals (7 weeks old) were used for control group and underwent sham operation when they were 18 weeks old (n ⫽ 6). Seven-week-old animals were used for experimental groups and underwent partial ligation of renal vein (n ⫽ 18). A minimum of six animals was sampled at each of the following time points after the varicocele operation: 9 weeks (7-week-old rats, 9 weeks later), 11 weeks (7-week-old rats, 11 weeks later), and 13 weeks (7-week-old rats, 13 weeks later). After anesthesia was performed by using Xylasine (Rompun, 15 mg/kg IM; Bayer AG, Leverkusen, Germany) and ketamine (Ketalar, 75 mg/kg, IM; Parke Davis, Istanbul, Turkey), experimental varicocele was created in 18 rats as described elsewhere (35, 36). The upper left abdominal quadrant was approached through a midline laparotomy incision. The abdominal contents were packed to the right to visualize the left kidney, adrenal and renal veins, and the left spermatic vein as it inserts into the left renal vein. By careful blunt dissection, a tunnel was made in the fat and connective tissues surrounding the left renal vein, and the vein is cleared of adhering tissue in a position medial to the insertion of the spermatic and adrenal veins. A 4 – 0 silk suture was tied to partially occlude the left renal vein at the point that the vessel was cleared of other tissue. The ligature was made around a metal wire of 0.85 Fertility and Sterility姞
Tissue Processing Testes were obtained from sham-operated (control) and varicocele-induced adult Wistar rats weighing 200 –250 g. Animals were killed at 9, 11, and 13 weeks after creation of varicocele, and testes were delivered into the abdomen. Tissues were fixed by immersion in Bouin’s (picric acid, formalin, and glacial acetic acid) fixative and were processed routinely for paraffin embedding. Immunohistochemistry For Fas and FasL immunostaining, fixed sections were deparaffinized and blocked for endogenous peroxidase activity with methanol containing 3% H2O2 for 10 minutes and for nonspecific binding with universal blocking reagent (BioGenex, San Ramon, CA) for 10 minutes at room temperature. Rabbit polyclonal Fas and rabbit polyclonal FasL primary antibodies (Santa Cruz Biotechnology Inc, Santa Cruz, CA) were applied in a dilution of 1 g of IgG per milliliter for 1.5 hours at room temperature. Negative control stainings were performed by replacing the primary antibodies with their nonimmune isotypes at the same concentration. The antibodies were detected with an avidin– biotin horseradish peroxidase complex with a Universal LSAB Kit (Dako, Glostrup, Denmark). Antibody complexes were visualized by incubation with diaminobenzidine (DAB) chromogen (BioGenex). Sections were counterstained with Mayer’s hematoxylin (Dako) for 30 seconds, dehydrated, mounted, and examined by light microscopy. Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick End-Labeling Apoptosis in testicular tissue was detected by enzymatic labeling of DNA strand breaks by using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). Paraffin sections of 5-m thickness from the testicular tissues were cut and placed on slides covered with poly-L-lysine, and after drying, the slides were left in the incubator at 45°C overnight and at 60°C for 1169
1 hour. After deparaffinization and rehydration, slides were washed twice in phosphate-buffered saline for 5 minutes. After incubation of slides with the permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 8 minutes at 4°C and washing twice with phosphate-buffered saline for 5 minutes, the labeling reaction was performed by using 50-L of TUNEL reagent for each sample, except negative control, in which reagent without enzyme was added and incubated for 1 hour at 37°C. After phosphatebuffered saline washings, slides were incubated with converter reagent for 30 minutes at 37°C. After washing, color development for localization of cells containing labeled DNA strand breaks was performed by incubating the slides with Fast Red substrate solution for 10 minutes. Labeling with TUNEL was conducted with a Cell Death Detection kit (Roche, Mannheim, Germany) and was performed according to the manufacturer’s instructions. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Blotting Protein extraction and immunoblot analysis were performed as described elsewhere (37). In brief, tissues were weighed and put into homogenationhomogenation buffer (10 mM Tris-HCL, 1 mM ethylenediaminetetraacetic acid, 2.5% sodium dodecyl sulfate, 1 mM phenylmethylsulfonylfluroide, and 1 g/mL of leupeptin) supplemented with CompleteR protease inhibitor cocktail (Boehringer, Mannheim, Germany). After homogenization, samples were processed by centrifuge at 10,000 ⫻ g for 10 minutes. Supernatants were collected and stored at ⫺70°C. The protein concentration was determined by Lowry assay (38), and 50 g of protein was applied per lane. Before electrophoresis, samples were boiled for 5 minutes at 95°C. Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then were transferred onto nitrocellulose membranes (Pharmacia) to remain overnight in a buffer containing 0.2 mol/L glycine, 25 mM Tris, and 20% methanol. Successful transfer was confirmed by Ponceau S (Sigma) staining of the blots. The membranes were blocked for 1 hour with 5% nonfat dry milk (BioRad, Hercules, CA) and 0.1% Tween 20 (Sigma) in 0.14 mol/L Tris-buffered saline (TBS; pH 7.2–7.4) at 4°C. Blotting membranes were incubated for 1 hour at room temperature with rabbit antisera against Fas and FasL, both diluted as 2 g/mL (Santa Cruz). After washing steps, the membranes were further incubated with goat anti-rabbit IgG horseradish peroxidase conjugate (BioRad) diluted 1:5,000 for 1 hour at room temperature. Immunolabeling was visualized by using the chemiluminescence-based SuperSignal CL HRP Substrate System (Pierce, Rockford, IL), and the membranes were exposed to Hyperfilm (Amersham), which subsequently was quantified with an Alpha Digi Doc 1000 gel documentation unit (Alpha Innotech Corporation, CA). Control blots were 1170
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incubated with antibody diluent only instead of the primary antiserum. -Actin antibody (1:5,000 dilution) was used as an internal control for each blotting to confirm the equal loading of the samples. Electron Microscopy Morphological studies were performed as described elsewhere (39). Samples of rat testicular tissues were fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 4 hours and were postfixed in 1% phosphate-buffered osmium tetroxide for 2 hours. The specimens were dehydrated in ethanol and embedded in Araldite epoxy resin (SPI-CHEM, Structure Probe, West Chester, PA). Semi-thin sections were stained with toluidine blue. Ultra-thin sections were double stained with uranyl acetate and lead citrate and examined with an electron microscope (Leo 906 E, Zeiss, Oberkochen, Germany). Statistical Analysis Statistical analyses of FasL and Fas Western blot quantifications were compared by one-way analysis of variance tests followed by post hoc Tukey tests for parametric data. Comparisons were made vs. control group. Probability values of ⬍.05 were considered significant, and values are presented as mean ⫾ SEM. RESULTS Histological Evaluation of the Varicocele Model Histological evaluation of our experimental model was determined according to our previously published study (35). Briefly, testis tissue specimens harvested from sham and from the 9-, 11-, and 13-week varicocele groups were stained with hematoxylin and eosin. In varicocele groups, disintegration of the germinal epithelium and degenerative appearance was observed visibly by light microscopy. In our recently published study, we found that degenerative tubules were evident, especially in 11-week and 13-week varicocele groups, when compared with the sham-operated rat testis (35). In the present study, we used the same tissue blocks for sham-operated and experimental groups. Localization of FasL and Fas in Rat Testis To clarify the cell types that express FasL and Fas proteins, immunohistochemical studies were performed with rat testicular tissue. In control tissue (sham-operated rats), FasL immunostaining was clearly visible only in spermatogenic cells but not in Sertoli cells. In the seminiferous tubule sections, FasL immunostaining mainly was observed in Golgi structures (acrosomal vesicle) of round spermatids (Fig. 1a), with a progressive expression in the cap phase of the sperm acrosome (acrosomal cap) resembling a semi-ring (Fig. 1b). Finally, FasL was present in the heads of elongated spermatids, most
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FIGURE 1 Localization of FasL protein in sham-operated (a– c) and varicocele-induced (d– h) rat testis. (a) FasL first appears in Golgi vesicles of round spermatids (arrowheads and inset), (b) then in acrosomal caps of round spermatids (arrowheads and inset), and (c) finally in heads of elongating spermatids (arrowheads and inset). (d) In the 9-week-old varicocele group, the expression of FasL was similar to that of control, as each phase of the expression pattern (arrows) was seen in tubuli sections. (e, f) In the 11-week-old varicocele group, there were cells still expressing FasL in their heads (arrows), whereas some cells did not express this protein accurately (arrowheads and insets). (g, h) In the 13-week-old varicocele group, some cells were negative in the same tubule (arrowheads), whereas some cells were positive for FasL (arrows). (i) Negative-control section; note the absence of FasL immunostaining. Scale bars represent 100 m (panel i) or 10m (panels a– h).
Celik-Ozenci. Fas system in varicocele-induced rat testis. Fertil Steril 2006.
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FIGURE 2 Localization of Fas protein in sham-operated (a) and varicocele-induced (b– d) rat testis. (a) Fas was present in cytoplasmic extrusions of elongated spermatids (arrow) and also in Leydig cells of the interstitial tissue (inset, arrowhead). (b) Similar to control, Fas was present in cytoplasmic extrusions (arrow) and in Leydig cells (arrowhead and inset) in the 9-week-old varicocele group. (c) Diminished intensity for Fas in Leydig cells can be seen (arrowhead and inset) in the 11-week-old varicocele group. (d) There were still a few cells immunostained with Fas in the 13-week-old varicocele group (arrowhead and inset). Scale bars represent 10 m.
Celik-Ozenci. Fas system in varicocele-induced rat testis. Fertil Steril 2006.
likely localized in their acrosomes (Fig. 1c). An intense immunostaining of FasL protein was detectable in both round and elongated spermatids. In the sham-operated rats, Fas immunostaining was essentially localized in cytoplasmic extrusions of the elongated spermatids (Fig. 2a) and in Leydig cells (Fig. 2A, inset). Down-Regulation of Fasl and Fas in Varicocele-Induced Rat Testis In the 9-week-old varicocele group, the distributions and the intensities of the FasL were similar to the sham-operated group. The protein was localized in Golgi vesicles of round spermatids, in the cap phase of developing sperm acrosome, and in the heads of elongated spermatids (Fig. 1d). In the 11-week-old varicocele group, positive immunolabeling still was present in round and elongated spermatids, but at a reduced intensity when compared with the control testis. We observed that in some tubules, round spermatids expressed FasL precisely (Fig. 1f), whereas, in some tubules, the expression of this protein was not seen accurately (Fig. 1f, inset). Moreover, FasL was present in elongated sperm, whereas its expression in elongated sperm was less in an adjacent tubule (Fig. 1e and inset).
the intensity of Fas protein were parallel to that in the sham-operated group (Fig. 2b and inset). Furthermore, starting from 11 weeks, the intensity of Fas immunostaining reduced gradually (Fig. 2c and inset). In the 13-week-old varicocele group, localization of this protein was limited to Leydig cells but with diminished intensity (Fig. 2d, inset). Evaluation of TUNEL Staining We observed TUNEL-positive cells in both sham-operated and varicocele-induced testes. Because the numbers of apoptotic cells were quite few in study groups, we did not perform an apoptotic index; thus, we gave representative figures for each of the study groups (Fig. 3a– d). Although we detected that apoptotic cells in the varicocele groups were slightly higher than in sham, this difference was not considerable. Additionally, we did not observe any significant valuable differences in the number of TUNEL-positive cells between the experimental groups.
Finally, in the 13-week-old varicocele group, immunolabeling of FasL still existed in some of the seminiferous tubules; however, there was a distinct decrease in its immunostaining intensity in both round and elongated spermatids (Fig. 1g and h). It was observed that not every single spermatid contained this protein regularly (Fig. 1h).
Confirmation of FasL and Fas Down-Regulation by Western Blot The presence of rat FasL and Fas proteins were also observed by Western blots using lysates of testis (Fig. 4). We detected FasL at 38 kDa and Fas at 48 kDa, as suggested by the manufacturer. Our results revealed that the expression of Fas decreased statistically significantly (P⬍.05) after the 13-week-old varicocele group (Fig. 5a), whereas the decrease of FasL was earlier at the 11-week-old varicocele group when compared with the case of sham-operated controls (Fig. 5b).
Fas immunoreactivity was present in cytoplasmic extrusions of elongated spermatids and in Leydig cells of the 9-, 11-, and 13-week-old varicocele groups at various densities. In the 9-week-old varicocele group, both the localization and
Evaluation of Electron-Microscopic Findings Ultrastructural analysis confirmed the effect of experimental varicocele in rat testis. Varicocele resulted in injuries of
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FIGURE 3 Detection of TUNEL-positive apoptotic germ cell nuclei (arrows) in seminiferous tubular cross section. (a) Sham, (b) 9-week-old varicocele, (c) 11-week-old varicocele, and (⬎d) 13-week-old varicocele.
Celik-Ozenci. Fas system in varicocele-induced rat testis. Fertil Steril 2006.
Sertoli–Sertoli cell and Sertoli–spermatogenic cell contacts (Fig. 6A and B). DISCUSSION We examined the expression of the Fas–FasL system in sham-operated and in varicocele-induced rat testis by immunohistochemistry and Western blot, as well as, by ultrastructural analysis. Moreover, to assess whether there is any relevance to the expression of the Fas–FasL system, apoptotic cells were evaluated by TUNEL method. Our results revealed that in control testis, FasL was visibly localized to round and elongated spermatids. This finding is contrary to most of the extensive statements in the literature, which argue that FasL is present in Sertoli cells rather than in germ cells of the seminiferous tubules (10 –13). However, recently, the findings of D’Alessio et al. (14) for the first time provided direct evidence based on Northern blot analysis Fertility and Sterility姞
that the attribution of testicular expression of FasL to Sertoli cells is erroneous. Elsewhere, it has been pointed out that there is continued increase of erroneous ideas about FasL as a result of technical problems related to specificity of the antibodies or as a result of the paradigm, already not accepted by several reports (40 – 43), that this protein has a role in immunoprivilege and in tumor escape. It is generally accepted that in the testis, FasL is expressed in Sertoli cells, and spermatogenic cells that contain its receptor would go to apoptosis (44, 45). Conversely, D’Alessio et al. (14) have found the transcription of this protein predominantly in round spermatids and, to lesser extent, in pachytene spermatocytes of mouse and rat testis. When they used isolated germ cells of the seminiferous tubules, they found the mRNA signal for FasL only in postmeiotic germ cells, particularly in elongated spermatids, whereas Sertoli cells were negative (14). The protein expression of FasL was found at a significant level 1173
FIGURE 4 Expression of Fas and FasL proteins in sham and different varicocele weeks determined by Western blot analysis using -actin as an internal standard.
found in germ cells and protein displayed on the surface of mouse spermatozoa may represent a self-defense mechanism against lymphocytes that are present in the female genital tract. Moreover, there are other studies in the literature that support this idea. Takeda et al. (49) investigated whether a graft can be protected from alloimmune responses by manipulating the Fas–FasL-system; thus, they transplanted allogeneic islets under the kidney capsule of diabetic mice together with testicular tissue. They found that FasL-positive testicular allografts protected composite islet allografts and concluded that manipulation of Fas–FasL mediated apoptosis is a suitable strategy for controlling rejection of islet allografts (49). Braendstrup et al. (50) studied the expressions of Fas and FasL in seminomas with surrounding normal and precancerous seminiferous tubule tissue and found that FasL was expressed in virtually all normal seminiferous tubules, whereas in precancerous tubules, the expression was discontinuous and less pronounced. Thus, the investigators discussed the concept of the testis being an immunologically privileged area and immune reaction to seminomas (50). Formerly, the involvement of immunological mechanisms in the development of pathological changes in the testes affected with varicocele was suggested (51).
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on spermatid after applying permeabilization (14) because there are several findings suggesting that FasL may be stored in specialized cytoplasmic vesicles (46, 47). In another recent study, FasL was found to be present intensely in elongated spermatid cytoplasm, especially after estrogen induction, and those investigators concluded that spermatogenic cells’ ability to control germ cell apoptosis is independent of Sertoli cells (15). Our FasL immunostaining findings are in accordance with D’Alessio et al. (14). Moreover, we found that the protein expression of FasL first was seen in acrosomal vesicle, continued with acrosomal cap, and finally became localized in heads (most likely in acrosomes) of the developing spermatids. Additionally, D’Alessio et al. (14) found that murine spermatozoa display on their surface a high concentration of FasL. Those investigators have stated that the roles so far proposed for the Fas system in the testis, such as maintenance of immunoprivilege (48) and regulation of physiological germ cell apoptosis (11), need reconsideration because both hypotheses are based on an erroneous cellular localization of Fas and FasL in the seminiferous epithelium. Because of our immunohistochemical results, we believe and also agree with the proposition that the presence of FasL on the sperm head, most likely on the sperm acrosome, is necessary for male gametes to escape both the autoimmune reaction during their journey through the male genital tract and the allogenic reaction that may take place in the female’s body. Recently, Riccioli et al. (16) stated that FasL mRNA 1174
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We have immunolocalized Fas protein essentially in Leydig cells and also in the cytoplasmic extrusions of elongated spermatids. The presence of Fas in cytoplasmic extrusions of elongated spermatids is first reported in our study, and with this limited amount of data we might speculate that the protein is necessary for accurate cytoplasmic extrusion. Previously, it was found that Fas was predominantly localized to Leydig cells, whereas FasL was localized to Sertoli cells in normal mouse testis, and there was no obvious association between the Fas system and occurrence of apoptosis (13). As an immune-privileged organ (48), the testis is known to express FasL at the highest level among tissues (52), whereas the level of Fas expression is very low. Moreover, FasL expression is exclusively restricted to Sertoli cells (53), and that of Fas is largely found in Leydig cells, in addition to a few germ cells (54). Francavilla et al. (17) found a coexpression pattern of Fas and FasL in Leydig cells of adult testis. Immunostaining for Fas was reported in Leydig cells of human testis (19), but for the first time, coexpression of Fas and FasL at both mRNA and protein levels in the Leydig cell was documented (17) and suggested that this colocalization was not associated with the evidence of apoptotic degeneration of Leydig cell, suggesting that the coupling of Fas by its ligand does not transduce a death signal in this cell type. In testis sections from control animals, most, but not all, Leydig cells express Fas and FasL proteins (18). There are some reports that support FasL expression in Leydig cells (54, 55), whereas there are some that do not show FasL expression in the interstitium (11). Another important aspect of our study is that we studied the role of Fas–FasL system in varicocele-induced rat testis
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FIGURE 5 Quantitative analysis of Western blot results (per-group n ⫽ 6). The values in the bar graphs were obtained by dividing the protein intensity value to its corresponding -actin intensity value. (a) Fas protein was significantly reduced in the 13-week-old varicocele group when compared with sham. (b) FasL expression was significantly less in the 11-week and 13-week-old varicocele groups when compared with sham. *P⬍.05.
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for the first time. Studies state that unilateral varicocele has been associated with bilateral physiologic and histological changes in the testis of humans and laboratory animals (56). However, a recent study by Barqawi et al. (27) demonstrated that the creation of experimental varicocele in rats generated a significant increase in germ cell apoptosis in the ipsilateral testis. We studied the effects of varicocele in the ipsilateral Fertility and Sterility姞
testes; thus, contralateral testes were not evaluated in the present study. It is accepted in the literature that apoptosis is increased in ejaculated spermatozoa in patients with varicocele compared with normal fertile men (30, 31) and is increased in testes of human with varicocele (27, 28, 57). Moreover, experimental 1175
FIGURE 6 Ultrastructural features of varicocele-induced rat testicular tissue. (a) General appearance of germinal epithelium in the seminiferous tubule wall of the 13-week-old varicocele group. (b) Arrows indicate the disturbance of contact points between spermatogonium (SG) and Sertoli cell (ST) in the 13-week-old varicocele group. L ⫽ lumen. Scale bars represent 10 m (a) and 2 m (b).
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models have supported this idea that the creation of experimental varicocele generated an increase in germ cell apoptosis in rats when compared with controls (27, 32, 33). There are also contradictory statements in the literature indicating that apoptosis is decreased in germ cells in the testis of infertile men with varicocele as compared with normal men (58). In addition, Tanaka et al. (59) suggested that apoptosis in testes of infertile men with varicocele might be suppressed as a result of decreased caspase-3 expression. A recent study by Kilinc et al. (60) suggested no relationship between apoptosis and varicocele in rats. Another recent study revealed that busulfan-induced spermatogenic germ cell apoptosis pathway is independent of p53 or Fas–FasL; the investigators found that although the number of TUNELpositive cells increased, the expression of FasL decreased by progressing time of busulfan treatment (61). It is clear from the studies mentioned that the current literature related to varicocele and apoptosis still contains conflicting results, both in human subjects and animal models. Additionally, using the FasL-defective gld mice, Hikim et al. (62) have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system may be dispensable for heat-induced germ cell apoptosis in the testis. Even though there is an increase in the number of apoptotic cells, this was not related to Fas–FasL system (62). There are limited data on the role of the Fas–FasL system in the apoptotic mechanism in varicocele-associated testis. We performed the current study in an effort to shed addi1176
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tional light on this subject. To our knowledge, until the present study, nobody has searched for the role of the Fas– FasL system in experimental varicocele of rat testis. We observed increased numbers of degenerative tubules and decreased immunostaining of the Fas–FasL system with progression of varicocele over time. In contrast, the presence of TUNEL-positive cells slightly increased in varicocele-induced testes. Germ cell death clearly was seen among spermatogonia and spermatocytes but was scarce. Therefore, we claim that the Fas–FasL system may not only be associated with apoptotic mechanisms but also may be related to the survival of sperm from immunological attack. There are many studies in the literature concerning the ultrastructural effects of varicocele to the seminiferous epithelium. Spermatogenic arrest, defects of the basal lamina, and injuries of the junctional complexes are the most frequent anomalies observed by the researchers (63– 65). Our results also confirmed the disintegration of the seminiferous epithelium in varicocele-induced testicular tissue. Our results have led us to comment on two issues. First, the presence of FasL in normal testicular tissue appears to be related to the survival of spermatozoa from a possible immunological attack that may occur in either the male or female genital tract. Consequently, the relative decrease of this protein in the varicocele model might increase the number of cells that could not survive these possible attacks, therefore leading to varicocele-associated male infertility.
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Second, the presence of Fas in cytoplasmic extrusions of elongated spermatids might be necessary for normal spermiogenesis, especially for normal sperm head morphology. Thus, the decrease of this protein in varicocele-induced testicular tissue might result with defective cytoplasmic extrusion of cells. However, we do believe that this hypothesis still needs further investigation. On the other hand, from human studies, it is known that varicocele-related male infertility is associated with impaired disposal of residual sperm cytoplasm by the testis (66). Moreover, the decrease of Fas protein in Leydig cells of varicocele-induced testis might be a kind of resistance of these androgen-producing cells to the pathology, to compensate for the T levels. In conclusion, this is the first study related to the relationship between the Fas–FasL system and varicocele-induced rat testis. According to our results, we think that FasL is necessary for sperm survival from possible immunological attack in either gender’s genital tract, and the presence of varicocele may reduce these cells’ survival chance by affecting the FasL expression in their heads, most probably in their acrosomes. Moreover, the existence of varicocele diminishes the expression of Fas in Leydig cells and in cytoplasmic extrusions of elongated spermatids, suggesting that Fas protein might have another unknown role instead of, or in addition to, apoptosis in testicular tissue. The topic is still under discussion and needs further investigation.
13.
14.
15.
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17.
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20. 21. 22.
Acknowledgments: The authors thank Arife Demı˙rtop and Hakan Er (Transmission Electron Microscopy Imaging and Analysis [TEMGA] Unit) for their excellent technical assistance of transmission electron microscopy studies.
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