Spermatogonial proliferation and apoptosis in hypospermatogenesis associated with nonobstructive azoospermia

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1 FERTILITY AND STERILITY VOL. 76, NO. 5, NOVEMBER 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Spermatogonial proliferation and apoptosis in hypospermatogenesis associated with nonobstructive azoospermia Seiji Takagi, M.D., Naoki Itoh, M.D., Makoto Kimura, M.D., Takumi Sasao, M.D., and Taiji Tsukamoto, M.D. Department of Urology, Sapporo Medical University School of Medicine, Sapporo, Japan Objective: To provide evidence that a pathological process in spermatogonial proliferation and apoptosis may participate in developing hypospermatogenesis of infertile men. Design: Case-controlled retrospective analysis. Setting: University-based male infertility clinic. Patient(s): Thirty-four patients with idiopathic hypospermatogenesis. Intervention(s): Collecting blood samples for measurement of hormones and performing testicular biopsy for assessment of spermatogenesis. Main Outcome Measure(s): The expression of proliferating cell nuclear antigen (PCNA) of spermatogonia and the frequency of apoptosis of spermatogonia demonstrated by the in situ DNA 3 -end labeling method were investigated to determine the degree of cell degeneration. Result(s): We could classify 34 infertile patients into four subgroups according to spermatogonial proliferation and differentiation. No significant difference in the expression of PCNA was demonstrated between these four groups and the control group. In all groups, the balance of spermatogonial proliferation (PCNApositive rate) to apoptosis was significantly lower than that of the control group. Conclusion(s): It was demonstrated that accelerated apoptosis, rather than proliferative dysfunction in the mitotic phase, may induce the decreased number of spermatogonia in hypospermatogenesis. These findings suggest that disorders of the control and regulation of apoptosis may participate in the pathogenesis of idiopathic hypospermatogenesis. (Fertil Steril 2001;76: by American Society for Reproductive Medicine.) Key Words: Spermatogonia, cell proliferation, apoptosis, hypospermatogenesis Received January 23, 2001; revised and accepted May 4, Reprint requests: Naoki Itoh, M.D., Department of Urology, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, Sapporo , Japan (FAX: ; nitoh@sapmed.ac.jp) /01/$20.00 PII S (01) The etiology of idiopathic hypospermatogenesis has not been fully clarified. Many factors may affect this disorder so that clinically effective treatments are limited for infertile males with idiopathic hypospermatogenesis. Elucidating the causes and pathogenesis of hypospermatogenesis may be helpful in improving the outcome of the treatment for these patients. Spermatogenesis is one of the most elaborate processes of cell differentiation. The first process of spermatogenesis is spermatogonial proliferation for the maintenance of the cells as stem cells that can enter the cycle of the spermatogenetic process resulting in the production of spermatozoa. Throughout adult life, spermatogonial stem cells furnish cells for maturation in a cyclic pattern, at the same time renewing them to maintain a constant reservoir so as to produce spermatozoa continuously. The second process of spermatogenesis is differentiation of spermatogonia to spermatozoa with a meiotic phase and spermiogenesis. The role of spermatogonial proliferation in spermatogenesis has been extensively studied in a variety of experimental conditions, including during chemotherapy and irradiation therapy (1 3). These studies demonstrated that the recovery of spermatogenesis depends on the degree of stem cell killing. For the recovery of spermatogenesis from these conditions, a selfrenewing capacity of spermatogenesis is required to restore the cell populations and differentiating divisions to produce cells developing 901

2 into spermatozoa (1). However, the role of spermatogonial proliferation in infertile men has not yet been studied. The histological findings of testicular biopsy specimens of patients with idiopathic hypospermatogenesis demonstrate various patterns. It was suspected that some of the patients had a decrease of the spermatogonial population, suggested by a proliferative disorder during the first period of spermatogenesis. Therefore, in the current study, we hypothesized that a disorder of spermatogonial proliferation might be one of the causes of hypospermatogenesis in infertile males. To clarify this hypothesis, we investigated spermatogonial apoptosis as one of the causes of the decreased number of spermatogonia in patients with idiopathic hypospermatogenesis. Generally, a balance between cell proliferation and cell death determines the cell number (4). Not only an increment of spermatogonial death but also a decline of their proliferative activity may be involved in disturbance of their balance, resulting in hypospermatogenesis. Therefore, we investigated the expression of proliferating cell nuclear antigen (PCNA) of spermatogonia as an index of the proliferative activity (5), as well as the frequency of apoptosis of spermatogonia by in situ DNA 3 -end labeling to determine the degree of cell degeneration (6). By analyzing both proliferation and apoptosis of spermatogonia in infertile men, we found that an imbalance between cell proliferation and apoptosis of spermatogonia may partly cause impaired spermatogenesis. MATERIALS AND METHODS Subjects The subjects consisted of 61 Japanese patients with either azoospermia or severe oligozoospermia. Patients who had a medical history that included mumps orchitis, cryptorchism, receiving chemotherapy or irradiation therapy, genital tract infections, childhood inguinal herniorrhaphy, or pelvic surgery were excluded from this study. Patients with varicocele testis and hypogonadotropic hypogonadism were also excluded. All patients underwent testicular biopsies for assessment of spermatogenesis at our hospital after being informed of the benefits and risks of the procedure. These biopsies were performed from 1990 to At that time, we did not have an institutional review board in our hospital. We fully informed patients of all possibilities of the use of a part of these specimens for research. In addition, an initial overall histological evaluation allowed us to exclude from the study those specimens of 27 patients featuring seminiferous tubules with Sertoli cells only and maturation arrest, leaving 34 infertile men evaluable. Twenty-one additional patients who underwent a vasoreconstruction operation and testicular biopsy for obstructive azoospermia served as a control group in this study. Physical examination, semen analysis, and hormonal examination were performed in all patients. Seminal parameters of each subject were examined on two different occasions. Sperm concentration was estimated by using a Makler counting chamber (7). Serum was collected for the analyses of LH, FSH, and total T. Plasma LH and FSH levels were determined with an immunoradiometric assay system (LH: normal, 1.8 to 5.2 miu/ml; FSH: normal, 2.9 to 8.2 miu/ ml) (8). Intra-assay and interassay coefficients of variation in LH and FSH measurements were as follows: LH (intraassay), 1.54%; LH (interassay), 3.29%; FSH (intra-assay), 1.03%; FSH (interassay), 0.37%. The plasma total T concentration was determined by radioimmunoassay (normal, ng/ml). Intra-assay and interassay coefficients of variation in T were 3.2% and 5.5%, respectively. Blood was sampled on two different occasions between 10:00 and 12:00 AM. Mean values were used. Histological Examination of Spermatogenesis The biopsy specimens were fixed in Bouin s solution for 2 hours and in 10% neutral buffered formalin for 2 4 hours, embedded in paraffin, and stained with hematoxylin and eosin. We quantitatively analyzed 20 seminiferous tubules of a round cross-section selected at random per specimen by light microscopy. All germ cells and Sertoli cells were counted in each cross-section of the tubules. Germ cells were classified as spermatogonia (dark type A, pale type A, type B), primary spermatocytes (preleptotene, leptotene, zygotene, pachytene), round spermatids (secondary spermatocyte, Sa and Sb1 spermatid) and elongated spermatids (Sb2, Sc, and Sd spermatid). Immunohistochemical Staining of PCNA and In Situ TUNEL Staining Sections (5 m) were immunostained with an anti-pcna antibody (DAKO, Copenhagen, Denmark) by using the avidin-biotin method, followed by counterstaining with hematoxylin (5). The numbers of PCNA-positive spermatogonia and all spermatogonia were counted in 20 seminiferous tubules per specimen. The number of PCNA-positive spermatogonia per the total number of total spermatogonia was defined as the PCNA-positive rate. Terminal deoxy-nucleotidyl transferase-mediated digoxigenin-dutp nick-end labeling (TUNEL) was performed on formalin-fixed 5- m sections of specimens using an ApopTag Plus kit (Oncor, Gaithersburg, MD). In brief, digoxigenin-dutp end-labeled DNA was detected with an anti-digoxigenin-peroxidase antibody followed by peroxidase detection with 0.05% diaminobenzidine (DAB) and 0.02% H 2 O 2 (6). The numbers of apoptotic spermatogonia and all spermatogonia were counted in 20 seminiferous tubules per specimen. The number of apoptotic spermatogonia per weaning the total number spermatogonia was defined as the apoptotic rate. As the positive control for TUNEL assay, rat mammary glands obtained at the 4th day after weaning were used. Coefficients of variation of intra-assay and interassay for TUNEL assay were 6.3% and 11.9%, respectively. 902 Takagi et al. Spermatogonial proliferation and apoptosis Vol. 76, No. 5, November 2001

3 FIGURE 1 Four groups of infertile males classified by two criteria with quantitative analyses of spermatogenesis. The criteria consisted of the ratio of spermatogonia per Sertoli cell, used as an index of capability of spermatogonial proliferation, and the ratio of the number of spermatids to the number of spermatogonia, used as an index of capability of spermatogonial differentiation into spermatids. Statistical Analysis The StatView statistical analysis program (Abacus Concept, Inc., Berkeley, CA) was used to analyze all data. The Mann-Whitney U test and one-way analysis of variance (ANOVA) were used for statistical analyses. Statistically significant differences were confirmed when the P value was.05. For ANOVA, significant differences were evaluated by using the Turkey-Kramer honestly significant difference test for multiple comparisons. RESULTS Classifying Infertile Patients into Four Groups by Quantitative Analyses of Spermatogenesis We tried to classify infertile patients into four groups by quantitative analyses of spermatogenesis based on cell proliferation and differentiation. First, to determine which patients suffered from a disorder of proliferation of spermatogonia, we divided infertile patients into two major groups, depending on the ratio of spermatogonia per Sertoli cell (spermatogonia/sertoli cells/tubules) that was used as an index of capability for spermatogonial proliferation. In the control group, the mean 2SD of the ratio of spermatogonia to each Sertoli cell was 0.92, so that a ratio of 0.92 was defined as normal. Of 34 infertile patients, 17 patients had a decreased ratio of spermatogonia per Sertoli cell, compared with the control. Second, to distinguish which patients had a differentiation disturbance from spermatogonia to elongated spermatids, the two major groups were further classified into two groups each by the ratio of the number of elongated spermatids to the number of spermatogonia. The mean 2SD of this ratio was 0.61 in the control group, so that a ratio of 0.61 was defined as normal. Consequently, infertile males were classified into four groups using two parameters, the ratio of spermatogonia per Sertoli cell (cell proliferation) and the ratio of elongated spermatids to spermatogonia (cell differentiation; Fig. 1). Group A consisted of 9 patients with reduction in both the ratio of spermatogonia per Sertoli cell and the spermatid/ spermatogonia ratio. Group B included 8 patients, who had only a decreased ratio of spermatogonia per Sertoli cell. In group C, 7 patients had only a decreased spermatid/sper- FERTILITY & STERILITY 903

4 TABLE 1 The ratios of each germ cell number to Sertoli cell number in seminiferous tubules in the four groups and control. Group (No.) Spermatogonia Primary spermatocytes Round spermatids Elongated spermatids A (9) * * * * B (8) * * * C (7) * D (10) * * Control (21) * Note: All values are expressed as mean SD. Statistical analyses were performed by the Turkey-Kramer honestly significant difference test for multiple comparison. Statistical difference is indicated by different superscripts and was determined when the P value was.05. matogonia ratio. Group D consisted of 10 patients who had both a normal ratio of spermatogonia per Sertoli cell and spermatid/spermatogonium ratio. In brief, patients of group A had disorders in both spermatogonial proliferation and differentiation. Groups B and C had impairment in either spermatogonial differentiation (group B) or differentiation (group C). In patients of group D, spermatogonial proliferation and differentiation potential were maintained. The Degree of Spermatogenesis and Clinical Features of Infertile Men The ratios of each type of germ cell per Sertoli cell in the four groups are shown in Table 1. Infertile males were mostly classified equally into four groups according to our definitions. This finding indicated that the causes of hypospermatogenesis were heterogenous. Some patients had a disorder of spermatogonial proliferation or deterioration of spermatogonial differentiation, but others had both. For instance, in the patients of group B, the ratio of spermatogonia per Sertoli cell was significantly decreased according to this classification. However, both those of elongated and round spermatids were significantly higher in this group than in groups A and C (Table 1). In group C, numbers of spermatogonia were in the normal range; however, all germ cells after spermatogonia were significantly diminished, suggesting the impairment of spermatogonial differentiation. To compare clinical backgrounds among the four groups, several clinical findings, such as age, testicular size, parameters of semen analysis, and hormonal analysis are represented in Table 2. There was no significant difference in age distribution between any group and the control group. Testicular size of patients of group A was significantly smaller than that of the control group. Serum LH and FSH levels were elevated in the patients of group A. There was no significant difference in the hormonal analysis between group B and the control group. A significant elevation (P.05) of the serum FSH level was found in group C. In group D, serum LH and FSH levels were normal; however, the serum T level was significantly elevated compared with that of the control. Proliferating Cell Nuclear Antigen-Positive Rate and Apoptotic Rate in Hypospermatogenesis Spermatogonia and primary spermatocytes were stained positively with the anti-pcna antibody. The label was confined to the nucleus (Fig. 2). There was no significant difference in the PCNA-positive rate among infertile males and normal controls (Table 3). TABLE 2 Comparison of clinical characteristics of the four groups of infertile men. Group Age Testis volume (ml) Sperm density (per ml 10 6 ) Sperm motility (%) LH (IU/L) FSH (IU/L) T (ng/ml) A b b b B C a D a Control (azoospermia) Note: All values are expressed as mean SD. a P.05 vs. control group. b P.01 vs. control group. 904 Takagi et al. Spermatogonial proliferation and apoptosis Vol. 76, No. 5, November 2001

5 FIGURE 2 Immunostaining of PCNA in testicular biopsy specimens obtained from infertile patients. (Original magnification, 400). Spermatogonia and a few primary spermatocytes were stained positively. FIGURE 3 TUNEL-stained testicular biopsy specimens. (Original magnification, 400). A few spermatogonia within the basal compartment were stained positively. FERTILITY & STERILITY 905

6 TABLE 3 Comparison of PCNA-positive rate, apoptotic rate, and PCNA-positive rate per apoptotic rate in four infertile groups. Group PCNA-positive rate (%) Apoptotic rate (%) PCNA-positive rate/apoptotic rate A * * B C D * * Control * * Note: All values are expressed as mean SD. Statistical analyses were performed by the Turkey-Kramer honestly significant difference test for multiple comparison. Statistical difference is indicated by different superscripts and was determined when the P value was.05. The in situ end-labeling method detected apoptotic cells. The cells were identified as spermatogonia and, rarely, as primary spermatocytes (Fig. 3). The apoptotic rate was significantly elevated in group A compared with the cases of group D and the control (Table 2). The ratios of the PCNApositive rate to the apoptotic rate were analyzed as an index of the balance between cell proliferation and apoptosis of spermatogonia. The ratios in all four groups of infertile males were significantly higher than in normal controls. Group A had a significantly higher rate compared with group D. However, no other significant difference among groups was recognized (Table 3). DISCUSSION The causes of idiopathic hypospermatogenesis seem to be heterogeneous. The constant maintenance of the proliferation of spermatogonial stem cells during the first step of spermatogenesis is indispensable for maintaining the constant production of spermatozoa. However, the clinical implications of spermatogonial proliferation have not been fully studied in infertile men. Our study revealed that among 34 infertile patients with idiopathic hypospermatogenesis, there were 8 (23.5%) unique patients (group B) who had a decreased spermatogonial population suggesting a disorder of spermatogonial proliferation but who had almost normal differentiation in spermatogenesis subsequent to spermatogonial proliferation. The normal differentiation of spermatogenesis in those 8 was supported by evidence that they did not show any elevation of the serum FSH level. In contrast, 17 patients (groups A and C) who had a disturbance of differentiation as determined by their elongated spermatid/spermatogonium ratio showed abnormal elevation of serum FSH, indicating severe Sertoli cell dysfunction. Therefore, we investigated the cause of the decreased number of spermatogonia in patients with idiopathic hypospermatogenesis. Generally, a balance between cell proliferation and cell death determines the control of cell number (4). In patients with a decreased number of spermatogonia, this balance might be relatively inclined toward cell death. We speculated that proliferative dysfunction in the mitotic phase or accelerated germ-cell death might be one of the causes of this decrease. Therefore, we tried to assess both proliferative activity and the frequency of apoptosis of spermatogonia in infertile men, using immunohistochemistry. To our knowledge, there is no report on investigation of both proliferative activity and the frequency of apoptosis for spermatogenesis in humans. When we studied the proliferative dysfunction or accelerated apoptosis as a cause of the decreased number of spermatogonia, the results demonstrated a significant difference for the apoptotic index only between group A and the control group. Moreover, when we focused on the balance between cell proliferation and apoptosis of spermatogonia, this balance in all four groups was strongly inclined toward apoptosis. On the basis of these observations, it was suggested that an elevated apoptotic rate of spermatogonia may have a more crucial role than a decrease of spermatogonial proliferation as one of causes of hypospermatogenesis. Of course, not only spermatogonial apoptosis but also other factors may be involved in defective spermatogenesis. Moreover, the accelerated spermatogonial apoptosis might be a result, not a cause, of defective testicular tissue. Recently, using molecular cell biology techniques, many genes and proteins were found to be expressed in each process of spermatogenesis. Among them, the c-kit protoncogene that encodes for transmembrane protein receptors has been demonstrated to be important for germ cell migration, development, and maturation (9). Yoshinaga et al. (10) demonstrated that blocking the c-kit receptor in mice would cause sterility by preventing the proliferation of the stem cells necessary for spermatogenesis. Although group B patients in this study had normal expression of PCNA as assessed for proliferative activity, other factors such as c-kit and stem cell factor, which may participate in the regulation 906 Takagi et al. Spermatogonial proliferation and apoptosis Vol. 76, No. 5, November 2001

7 of proliferation of spermatogonia themselves, may affect the number of spermatogonia. Recent evidence suggests that alterations in apoptosis contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, and neurodegenerative disorders (11). In the testis, germ cell apoptosis occurs during normal spermatogenesis and continuously throughout life (12). The regulation of apoptosis appears to play an important role during normal spermatogenesis. Accelerated germ cell apoptosis has been observed in a variety of experimental conditions, such as those after irradiation (13), toxicant exposures (14), gonadotropin-releasing hormone antagonist treatment (15), heat stress (16), and mild hypothermia (17). More recently, Lin et al. (18) demonstrated that increased apoptosis occurred in maturation arrest and hypospermatogenesis in infertile men. These histological findings resemble those of group A in our study. These results suggested that accelerated apoptosis may participate in the pathogenesis of idiopathic hypospermatogenesis in some infertile patients. However, the control and mechanisms of apoptosis in spermatogenesis have not been elucidated. Experimental models of disorders of regulation of apoptosis have been reported. Several of them used transgenic mice overexpressing bcl-2 and others that were bax-deficient and which developed male infertility as a result of disordered seminiferous tubules with an accumulation of atypical premeiotic germ cells but no mature spermatozoa (19, 20). To investigate whether these genes and proteins associated with apoptosis participate in spermatogenesis may help to elucidate the pathogenesis of idiopathic hypospermatogenesis. Although clinically effective treatments for males with idiopathic hypospermatogenesis are limited, Foresta et al. (21) reported that FSH treatment increases the spermatogonial population in men who have normal FSH levels and a testicular histology characterized by hypospermatogenesis without maturational disturbance. The responders to FSH treatment in their report appear to resemble group B patients in our study, who had normal FSH levels and a decreased spermatogonial population without maturational disturbance. Experimentally, the number of pale type A spermatogonia could be increased by FSH treatment in the monkey (22). Appropriate patient selection may lead to a satisfactory outcome with FSH treatment. In conclusion, we demonstrated for the first time that some infertile males have a disorder of differentiation of spermatogonial stem cells. Accelerated apoptosis, rather than proliferative dysfunction in the mitotic phase, may induce the decreased number of spermatogonia in these patients. These findings suggest that disorders of the control and regulation of apoptosis may participate in the pathogenesis of idiopathic hypospermatogenesis. Elucidating the mechanism and regulation of apoptosis in spermatogenesis may help to resolve at least some problems in the pathogenesis of male infertility. References 1. Meistrich ML. Effects of chemotherapy and radiotherapy on spermatogenesis. Eur Urol 1993;23: Bustos-Obregon E, Rodriguez H. Testicular x-ray irradiation in adult mice as a model to study spermatogonial proliferation. Andrologia 1991;23: Judas L, Bentzen SM, Hansen PV, Overgaard J. Proliferative response of mouse spermatogonial stem cells after irradiation: a quantitative model analysis of experimental data. Cell Prolif 1996;29: Raff MC. Social controls on cell survival and cell death. Nature 1992;356: Hall PA, Levison DA. Review: assessment of cell proliferation in histological material. J Clin Pathol 1990;43: Gavrieli Y, Sherman Y, Ben-Sasson SA. 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