Stesura Seveso Archivio Italiano di Urologia e Andrologia 2015; 87, 3198 ORIGINAL PAPER The histology and the proapoptotic control in the ipsilateral and the contralateral testes following unilateral vasectomy Aydin Ismet Hazar 1, Basri Cakiroglu 2*, Ertan Sakalli 3, Mustafa Bahadir Can Balci 1, Erkan Eyyupoglu 4, Tuncay Tas 1, Orhun Sinanoglu 5, Pinar Tuzlali 6, Nusret Can Cilesiz 1 1 Taksim Training and Research Hospital, Department of Urology Istanbul, Turkey; 2 Hisar Intercontinental Hospital, Department of Urology Istanbul, Turkey; 3 Zonguldak State Hospital, Department of Urology, Zonguldak, Turkey; 4 Amasya Training and Research Hospital, Department of Urology, Amasya, Turkey; 5 Maltepe University Medical School, Department of Urology, Istanbul, Turkey; 6 Taksim Training and Research Hospital, Department of Pathology, Istanbul, Turkey. Objective: The aim of this study was to enlighten both the testicular histology and the genetic aspects of the apoptotic process. Thus an experi- mental study was designed with a model of unilateral vasec- tomy. Methods: Twenty-two adult male rats were used and 4 main groups were formed. The first (A), the second (B), the third (C), and the fourth group (D) consisted of 4, 4, 4 and 10 rats respectively. Rats in group A had sham operation while rats in other groups (B, C, D) underwent left vasectomy opera- tion including binding of ductus deferens with a 3/0 silk and cutting a minimum of 1 cm part while preserving the vascu- lar structure under 9x magnification. Rats undergoing uni- lateral vasectomy were sacrificed at the 1st, 2nd and 8th weeks and their testicular structure and proapoptotic gene proteins were compared with that of the control group undergoing sham operation. Results: We found that vasectomy gradually caused destruc- tion and both ipsilateral and contralateral testicles were affected showing initial apoptosis. Conclusion: The procedure causes destruction in the testicu- lar structure by causing bilateral intratubular germ cell necrosis, unilateral obstruction, increase in the tubular pres- sure and processes that are aggravated by some probable autoimmune reactions. KEY WORDS: Testis; Spermatogenesis; Vasectomy; Apoptosis; BCL-2 protein. Submitted 18 December 2014; Accepted 31 March 2015 Summary No conflict of interest declared. gene’s pro regulation, and they have great importance in the modulation of this process (2-4). Since it was first detected in B cell lymphoma-2 cells it was named as BCL-2. Some proteins of the BCL-2 gene family such as “bcl-2, bcl-xL and bcl-w” support cell growth and play an apoptotic role while others such as “bax, bam, bad, bok, bid and bim” antagonise the effect of others and play an apoptotic role (5). The competition between these two cells and their being a homodimer such as bcl/bcl, bax/bax or a heterodimer such as bax/bcl assign the cell either to die or to live (6, 7). The naming “bax” is formed as bcl-2 associated x protein and is based on the rala- tionship between these two immunohistochemical (IHC) measurements of the proapoptotic and antiapoptotic proteins are used in determining the apoptosis as well as the detection of the DNA fragmantation (8). Bax overex- pression has been shown with drug-induced apoptosis in human germ cells (9). The same finding was found in animal experiments after vasectomy (10, 11). Although vasectomy is a popular male contraception method and its reversals have been widely studied, the effect of vasectomy on testicular histology is still contro- versial (12, 13). However, even apoptosis in the ejaculate has been studied in diabetes, schizophrenia and in immunopathologies (14) the relationship between the post vasectomial testicular histology and apoptosis and its clinical effects haven’t been studied widely (8, 15-18). In this study adult rat unilateral vasectomy was com- pared with the control group and the answer to 4 ques- tions were required: 1) the role of vasectomy on testicu- lar damage 2) its genetic effect based on bax in apopto- sis in normal spermatogenesis 3) the effects of vasectomy on the contralateral testicle 4) the possible effects of the unilateral vas deferens damage due to inguinal surgery on testicular histopathology. MATERIALS AND METHODS In this experimental study, 22 adult male Sprague Dawley 16 weeks old rats weighing 250 grams were used and studied in 4 main groups. The first group (A) consisted of 4 rats, the second (B) 4, the third (C) 4 and the fourth DOI: 10.4081/aiua.2015.3.198 INTRODUCTION Spermatogenesis is a process in which spermatogonial stem cells form mature sperm cells (spermatozoa) by cell differentiation which is characterized by mitotic and meiotic cell divisions. Germ cell deaths occur in addition to cell growth and proliferation during normal sper- matogenesis which is of critical importance in the for- mation of the mature sperm cells (1). Apoptosis is the process that destructs the cells with a damaged DNA after receiving internal or external induction signals. The effectors of this death process are the caspases and the Bcl-2 protein family which is responsible for 17 p53 Hazar corr_Stesura Seveso 30/09/15 09:28 Pagina 198 199Archivio Italiano di Urologia e Andrologia 2015; 87, 3 Proapoptotic control in unilateral vasectomy group (D) 10 rats. All the rats had anesthesia with intraperitoneal 25 mg/kg pentothal injection. While rats in group A had sham operation in an aseptic environ- ment, rats in group B, C, D had a left vasectomy opera- tion in which ductus deferens was tied with a 3/0 silk and a minimum of 1 cm part was excised with an inguinal incision preserving the vascular structure. Animals were sacrificed with 75 mg/kg pentothal injec- tion and standard orchiectomy was applied and the tes- ticular histopathology was examined at the 1st (B), the 2nd (C) and the 8th (D) week. Histopathology The testes were fixed in Bouin solution and buried in paraffin and sections were prepared from tissue blocks. All the samples were examined for inflamatory infiltrate, edema, fibrosis, sperm granuloma, intratubular spermat- ic necrosis. IHC Coloring Bax B-9 rat monoclonal IgG2b antibody which is essen- tial for coloring was used. Bax protein eliminates the inhibitory effects of BCL-2 gene and speeds up apoptot- ic deaths, thus Bax antibody binds to the Bax protein in the cell in which the apoptotic death is increased. In the beginning of the procedure, tissue sections were incubated in hydrogen peroxidase solution for 15 min- utes then washed with Phosphate Buffer Saline (PBS) twice, citrate tamponade was made in microwave and then cooled in room temperature for 20 minutes, after- wards washed with PBS for four times. In order to avoid non specific arka plân coloring, ultra V Block application (Protein blockage) was made in room temperature for 5 minutes. It was not washed, but Bax antibody was used to cover the tissue by dropping the Ultra V Block onto the microscope slide and incubated for an hour. Dilution was made at 1/100. Then it was washed with PBS for 4 times. Afterwards “Biotinylated Goat Anti polyvalent Solution” (link saru) was dropped and after 20 minutes was washed with PBS for 4 times and incubated with AEC Chromogen until it was colored (AEC preparation: 1 drop of chromogen was added to 1 ml of substrate and washed with distilled water). The contrast coloring was made with Mayer’s Haematoxylin that was kept there for 1 minute maxi- mum before being washed with distiled water and closed with ultramount. Preparates were examined with a light microscope and Bax (+) coloring was defined as granular type coloring of the cytoplasm. Increased intensity of coloring (+), excessive increase in coloring (++) and nor- mal coloring were recorded. Randomized 10 tubules diameters were measured in all the samples under the ocular micrometer of the Olympus CH30 light micro- scope. Necrosis level of intratubular germ cells were clas- sified according to their cell level. Statistical analysis Kruskal Wallis test was used to compare groups, Mann- Whitney U test was used for dual comparisons and Fisher (exact) test was used to compare the qualitative data. All the results were evaluated in a CI of 95% and p < 0.05 was defined as statistical significance. RESULTS After the IHC, all four groups were evaluated for apopto- sis and morphology and these results were found: all 4 cases in the control group had spontaneous apoptosis and apoptotic coloring was dominant (+) in the spermatocyte level in 3 cases and in the spermatid level in 1 case. At the 1st week of the unilateral vasectomy bax coloring level increased to (++) while it remained the same (+) in one case. Dominant level was the spermatocyte level, the same as the control group. On the contralateral side bax coloring was (+) but in 3 cases bax dominant cell level had increased to spermatids while in one case it remained in the spermatocyte level. At the 2nd (C) and the 8th weeks (D) bax colorings were negative both in the vasectomy operated side and the contralateral side. The difference in the bax positivity in the vasectomy operated testes were statistically significant when compared with the 2nd and the 8th weeks. Likewise 1st week’s bax positivity of the contralateral side was statistically significant when com- pared to that of the 2nd and the 8 weeks (Table 1). During the first week, neither the ipsilateral nor the con- tralateral testicles had any inflammation. At the second week 3 of the ipsilateral testicles had inflamation of the tunica albuginea while there were no such inflammation in the contralateral testis. At the 8th week inflammation was detected in 4 vasectomy operated testes and in 2 con- tralateral testes. When the inflammation rate of the first Control Bax Bax cell (dominant) Case 1 Left testis (+) Spermatocyte Right testis (+) Spermatocyte Case 2 Left testis (+) Spermatocyte Right testis (+) Spermatocyte Case 3 Left testis (+) Spermatocyte Right testis (+) Spermatocyte Case 4 Left testis (+) Spermatid Right testis (+) Spermatocyte 1st Week Case 1 V1 (++) Spermatocyte C1 (+) Spermatid Case 2 V2 (++) Spermatocyte C2 (+) Spermatocyte Case 3 V3 (++) Spermatocyte C3 (+) Spermatid Case 4 V4 (+) Spermatocyte C4 (+) Spermatid 2nd Week Case 1 V1 (-) (-) C1 (-) (-) Case 2 V2 (-) (-) C2 (-) (-) Case 3 V3 (-) (-) C3 (-) (-) Case 4 V4 (-) (-) C4 (-) (-) V = vasectomy = left testis, C = Contralateral testis = right testis, the staining type: None( -), Low (+), Moderate (++) Table 1. Weekly Bax staining results. Hazar corr_Stesura Seveso 30/09/15 09:28 Pagina 199 Archivio Italiano di Urologia e Andrologia 2015; 87, 3 A. Ismet Hazar, B. Cakiroglu, E. Sakalli, M. Bahadir Can Balci, E. Eyyupoglu, T.Tas, O. Sinanoglu, P. Tuzlali, N. Can Cilesiz 200 week was compared with the patients 2nd (p < 0.0001) and the 8th week (p < 0.001) statistically significant increase was detected. When the contralateral testicles were evalu- ated, no such increase according to weeks was detected (p > 0.05). When the ipsilateral and the contralateral testes were compared there were no statistically significant dif- ference at the 1st week (p > 0.05) but at the end of the 2nd and the 8th week, inflammation rate was higher in the vasectomy operated group (p < 0.001) (Table 2). Peritubular fibrosis was seen in the vasectomy operated group at the first week and 2 cases at the 2nd and 8th week while it wasn’t seen in any in the contralateral group. While there was a significant rise in peritubular fibrosis at 2nd weeks when compared with the 1st week (p < 0.01), there was no significant increase at 8th week when compared to the 2nd (p > 0.05). In the vasectomy group peritubular fibrosis was significantly higher at the 1st, 2nd and 8th week when compared to the contralateral testicles (p < 0.0001). Intratubular germ cell necrosis was not detected during the 1st week. At the 2nd week, 4 cases had moderate and 1 case had mild intratubular germ cell necrosis while it was not seen in the contralateral side. Dominant level was the spermatocyte level in 2 cases and the spermatid level in 2 cases. At the 8th week 9 out of 10 testes with vasectomy had intratubular germ cell necrosis. Intratubular germ cell necrosis was mild in 2 cases, mod- erate in 5 cases and severe in 2 cases. Dominant level was the 1° spermatocyte level in 1 case, the 2° spermatocyte level in 7 cases and the spermatid level in 1 case. When the contralateral testes were examined intratubu- lar germ cell necrosis was found in 9 out of 10 cases. In 6 cases it was moderate and in 3 cases it was mild. Dominant level was the spermatid level in 5 cases, the 1° spermato- cyte level in 3 cases and 2° spermatocyte level in 1 case (Table 2). Statistical analyses revealed that germ cell necrosis mainly in the spermatid area was statistically significant at the 2nd week when compared with that of the 8th week (p < 0.0001). There was statistically signifi- cant increase in intratubular germ necrosis at the 2nd and the 8th week when compared with the 1st week (p < 0.001). When the contralateral testicles were evaluated inter- nally, increase in germ cell necrosis in sper- matids at the 8th week was statistically sig- nificant (p < 0.0001). In the preoperative operation no sperm granuloma was seen while it was encoun- tered in one case at the 2nd week and in 3 cases at the 8th week. When compared with the 1st week, the possibilty of granuloma detection rate was higher at the 2nd and the 8th week and this difference was statistically significant (p < 0.0001). No testicular necro- sis or atrophy were shown. The morphology of the epididymis was pre- served at the 8th week. The diameter of the tubules was 235.3 ± 12.9 μ in the vasectomy operated side and 248 ± 12.9 μ in the con- tralateral side at the 1st week. In the second week the mean diameter of the tubule in the vasectomy operated testicle was 234.5 ± 3.87 μ and the mean diameter of the contralateral side was 249.3 ± 3.20 μ. At the 8th week this diameter was measured as 230.1 ± 7.66 μ in the ipsi- lateral side and 250.4 ± 2.5 μ in the contralateral side. When the statistical analyses were made for the mean tubule diameters and weekly change, the difference was not statistically significant at the 1st week (p > 0.05) while it was statistically significant at the 2nd (p < 0.05) and the 8th week (p < 0.001). There was no statistically significant difference when the vasectomy operated testes were compared with each other (p > 0.05) (Table 3). 1st Week Inflammation Pt fibrosis Intratubular necrosis Sperm granuloma V1 (-) (+) (-) (-) C1 (-) (-) (-) (-) V2 (-) (-) (-) (-) C2 (-) (-) (-) (-) V3 (-) (-) (-) (-) C3 (-) (-) (-) (-) V4 (-) (-) (-) (-) C4 (-) (-) (-) (-) 1st Week Inflammation Pt fibrosis Intratubular necrosis Sperm granuloma Degree Dominant V1 (-) (-) (++) 2°spermatocyte (-) C1 (-) (-) (-) (-) (-) V2 (+)T.Albuginea (+) (++) Spermatid (+) C2 (-) (-) (-) (-) (-) V3 (+)T.Albuginea (+) (+) 2°spermatocyte (-) C3 (-) (-) (-) (-) (-) V4 (+)T.Albuginea (-) (++) Spermatid (-) C4 (-) (-) (-) (-) (-) 8th Week Intratubular necrosis Pt fibrosis Sperm granuloma Inflammation Degree Dominant Case 1 V1 (+) 1°spermatocyte (-) (-) (+) C1 (++) Spermatid (-) (-) (-) Case 2 V2 (++) 2°spermatocyte (+) (+) (-) K2 (++) Sperma (-) (-) (-) Case 3 V3 (++) 2°Spermatosit (-) (+) (+) C3 (++) Spermatid (-) (-) (-) Case 4 V4 (+++) 2°spermatocyte (-) (-) (-) C4 (+) Spermatid (-) (+) (-) Case 5 V5 (++) 2°spermatocyte (-) (-) (-) C5 (++) Sperma (-) (-) (-) Case 6 V6 (+) 2°spermatocyte (+) (+) (+) C6 (++) Sperma (-) (-) (+) Case 7 V7 (++) Spermatid (-) (-) (-) C7 (++) Spermatid (-) (-) (-) Case 8 V8 (+++) 2°spermatocyte (-) (-) (-) C8 (+) Spermatid (-) (-) (-) Case 9 V9 (++) 2°spermatocyte (-) (-) (+) C9 (-) (-) (-) (-) (+) Case 10 V10 (-) (-) (-) (-) (-) C10 (+) 2°spermatocyte (-) (-) (-) Table 2. Weekly histopathology results. Hazar corr_Stesura Seveso 30/09/15 09:28 Pagina 200 201Archivio Italiano di Urologia e Andrologia 2015; 87, 3 Proapoptotic control in unilateral vasectomy DISCUSSION The effect of vasectomy on testicular histology is still controversial. Testicular stem cells and spermatids which are formed by differentiated stem cells are in close rela- tionship with the seminifer tubules’ luminar cells and Sertoli cells of the testicular interstitium. In order to maturize to sperm cells they need the epididymis and its functions as well. The high rate of fertilisation in con- ductive fertilisation techniques after vasectomy and its reversals suggest that vasectomy does not cause an important disorder in testicular functions. Althrough the studies in the literature are controversial, there is more data supporting that there is a worsening of the sperm functions and dilatation in the seminiferous tubules in the early stages and inflammation and per- itubular fibrosis with a decrease in germ cell levels in the later stages (19). Spermatic cysts behave as shock absorbers and help in preserving the functions and give physiological support (20). Lower fertilization rates in patients with short-term vasectomy than in patients with long-term vasectomy support the late stage findings of testicular histology. Recent studies have shown that apoptosis is the most important mechanism for sper- matogenesis in germ cells. Apoptosis has been widely studied in the spermatogonia, the spermatocytes and spermatids and, many apoptotic factors have been described. Testicular germ cell apoptosis is a physiologi- cal event that occurs throughout life (21). In the end the number of potential spermatozoa decrease by 75%. Apoptosis has 2 functions in spermatogenesis. The first is to limit the germ cell population which Sertoli cells can support, the second is to be selective in decreasing the abnormal spermatozoa (21). The relationship between the fertility and apoptosis was shown by the increased apoptosis in patients with oligospermia. On the other hand there are few studies regarding the effect of vasectomy on this process and its genetic control. There’s almost no studies on the apoptotic process in normal testicles. Its clinical importance is the potential effect on fertility in vas deferens injuries that does not last with orchiectomy. We did not detect any testicular necrosis or atrophy in our experimental study. But there were increase in sperm granuloma formations and significant increase in tubular diameters and decrease in the wall thickness. The increase of the granuloma is more significant at the 8th week. Since spermatogenesis continues after vasectomy, there can be leaks from the slimming tubules due to the widening of the epididymis and ductus deferens and this stimulates the granuloma formation. It is possible that the granulomas relieve the inflamatory pres- sure. It may be related to intraluminar phagocytosis. Phagocytosis causes stimula- tion of the immune system which cause anti- sperm antibodies formation. On the other hand it is also possible that extravasation may induce autoimmune responses. There are many articles about the autoimmune response to the testicular tissue. T lympho- cytic cells and monocytes are responsible from this immunity and phagocytosis. After detecting the antigen, T lymphocytic cells stimulate B lymphocytic cells to produce antibodies. Monocyte-monocyte interaction leads the phagocytosis pathway to activate and this formation causes inflamma- tion and fibrosis in testicular germ cells (22). In our study in the later stages at the 8th week in 9 out of 10 cases intratubular germ cell necrosis was present in vasectomy operated side, in addition to inflammation and fibrosis, 2 cases had mild, 5 cases had moderate and 2 cases had severe intratubular germ cell necrosis. Dominant stage was the 1° spermatocyte in one case, the 2° spermatocyte in 7 cases and the spermatid in one case. Studies that explain the impairment in spermatogenesis after iatrogenic damage to vas deferens are classified as: 1) immunologic response to the testicular tissue 2) reac- tive oxygen species 3) apoptotic changes in germ cells (23-26). However, it is also suggested that adult vas def- erens obstruction does not affect apoptosis (27). Abnormal spermatogenesis was shown to occur due to dysfunctional expression of the genes (28-34). In our study we observed that increased apoptosis in the testis undergoing vasectomy This is compatible with previous literature findings (35). Thus, with our vasectomy exper- iment protocol, we tried to find an answer to 2 main questions which include the changes in the ipsilateral histology and apoptosis. Bax staining increased in the contralateral testicle during the initial period in our vasectomy model. The affected cell level was more mature in contrast to the ipsilateral testicle. It is known that apoptosis in stem cells affects the physiological regulation. Despite the fact, this high apoptosis level in the low-resistant spermatids which are under high exposure to external factors in the contralat- eral testicle suggest a possible systemic effect after vasec- tomy (36, 37). This early stage of apoptosis occurs without intratubular and peritubular changes and the granuloma formation may not be dependent on the immunity. We found find- ings suggesting that this effect supports the late stage inflammation. We believe that Bax proteins has a role in this modulation. We did not detect any tubular or per- itubular pathologies in the contralateral testis since the increase of pressure does not affect the contralateral one. If these findings with common avascular fibrosis which is seen during the complete mobilization of vas deferens in inguinal surgery was further evaluated, it may enlight- en the underlying factors of this situation which causes sterility with a range of 78%. Furthermore, the excessive mobilization of the cord and the operation technique are highly important in the inguinal surgery in order to sus- tain fertility (38). Tubule diameters 1st Week 2nd Week 8th Week KW pV1 Vasectomy 235.3 ± 12.09 234.5 ± 3.87 230.1 ± 7.66 1.38 > 0.05 Contralateral testes 248 ± 3.91 249.3 ± 3.20 250.4 ± 2.5 2.54 > 0.05 MW 2 0 0 p > 0.05 < 0.05 < 0.001 Table 3. The testicular tubular diameter change per weeks in the ipsilateral and contralateral testi. Hazar corr_Stesura Seveso 30/09/15 09:28 Pagina 201 Archivio Italiano di Urologia e Andrologia 2015; 87, 3 A. Ismet Hazar, B. Cakiroglu, E. Sakalli, M. Bahadir Can Balci, E. Eyyupoglu, T.Tas, O. Sinanoglu, P. Tuzlali, N. Can Cilesiz 202 CONCLUSIONS The damage in testicular histology following the obstruc- tion of ductus deferens depends by complex mecha- nisms rather than a single mechanism. Apoptosis is responsible for the initial germinal cell damage. Unilateral ductal obstruction causes damage on the tes- ticular structure by causing bilateral germ cell necrosis due to the increase in pressure and processes that are triggered by the autoimmune reactions. We have shown that it is not essential to initiate the immune reaction for the sperm granuloma formation and vasectomy alone can trigger the immune response and impair the sper- matogenesis. AUTHORS’ CONTRIBUTIONS AIH, BC and MBCB and conducted the experiments and participated in acquisition, analysis, and interpretation of data. 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Billig H, Furuta I, Rivier C, et al. Apoptosis in testis germ cells: developmental changes in gonadotropin dependence and localization to selective tubule stages.Endocrinology. 1995; 136:5-12. 38. Matsuda T, Muguruma K, Horii Y, et al. Serum antisperm anti- bodies in men with vas deferens obstruction caused by childhood inguinal herniorhaphy.Fertil Steril 1993; 59:1095. Correspondence Aydin Ismet Hazar, MD Mustafa Bahadir Can Balci, MD Tuncay Tas, MD Nusret Can Cilesiz, MD Taksim Training and Research Hospital, Department of Urology, Istanbul, Turkey Basri Cakiroglu, MD (Corresponding Autor) drbasri@hotmail.com Hisar Intercontinental Hospital Department of Urology Saray Mah. Siteyolu Cad., No:7 34768 Umraniye, Istanbul, Turkey Ertan Sakalli, MD Zonguldak State Hospital, Department of Urology, Zonguldak, Turkey Erkan Eyyupoglu, MD Amasya Training and Research Hospital, Department of Urology, Amasya, Turkey Orhun Sinanoglu, MD Maltepe University Medical School, Department of Urology, Istanbul, Turkey Pinar Tuzlali, MD Taksim Training and Research Hospital, Department of Pathology, Istanbul, Turkey Hazar corr_Stesura Seveso 30/09/15 09:28 Pagina 203