Editor-in-Chief
Prof. Yi-Fei WANG,
Asian J Androl 2006; 8 (1): 95-100 |
|
| ¡¡ | |
This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you. - Original Article - Testicular expression of survivin and human telomerase reverse transcriptase (hTERT) associated with spermatogenic function in infertile patients Steffen Weikert1, Frank Christoph1, Wolfgang Schulze2, Hans Krause1, Carsten Kempkensteffen1, Martin Schostak1,Kurt Miller1, Mark Schrader1 1Department of Urology, Charité-Universitätsmedizin Berlin, Campus B. Franklin, D-12200 Berlin, Germany Abstract Aim: To characterize the coexpression of survivin, an inhibitor of apoptosis (IAF), and human telomerase reverse transcriptase (hTERT) in human testes with varying spermatogenic function. Methods: Transcript levels of survivin mRNA and hTERT mRNA were determined in normal testes (n = 11) and testes with defective spermatogenesis (n = 28) using real-time reverse-transcription polymerase chain reaction (RT-PCR). The histological work-up was performed according to a modified Johnsen score. Results: Expressions of both survivin and hTERT were highest at median levels of 96.8 and 709 in normal spermatogenesis and dropped to 53.3 and 534 in testes with postmeiotic spermatogenic arrest (n = 10). In severe spermatogenic failure (n = 18), survivin expression was lacking in most specimens (n = 16), whereas at least low levels of testicular hTERT expression were largely detectable with a normalized expression of 73 in premeiotic spermatogenic arrest (n = 7) and 45 in patients with Sertoli cell-only syndrome (SCOS) (n = 3). Both survivin and hTERT expressions increased with a progressing Johnsen score (P for trend = 0.001). Conclusion: Although both survivin and hTERT are correlated with spermatogenic function, they show different expression patterns in testes of infertile patients. These findings substantiate results from studies in the rodent testis suggesting a predominant expression of survivin in meiotically dividing germ cells. (Asian J Androl 2006 Jan; 8: 95-100) Keywords: survivin; human telomerase reverse transcriptase; apoptosis; azoospermia; male infertility; spermatogenesis Correspondence to: Dr Steffen Weikert, M.D., Department of Urology, Charité-Universitätsmedizin Berlin, Campus B. Franklin,
Hindenburgdamm 30, D-12200 Berlin, Germany 1 Introduction
Normal production of germ cells is dependent on a precise balance of both proliferation and apoptotic cell death. The molecular mechanisms underlying the regulation of germ cell homeostasis in the testis are only partly understood. Genes involved in growth and proliferation as well as pro- and anti-apoptotic factors seem to play important roles in this context. Impaired proliferative capacity and dysregulated apoptosis can contribute to human spermatogenic disorders [1]. Genetic alterations, such as impaired expression of anti-apoptotic genes, are thought to be involved in idiopathic fertility defects [2-4]. Both survivin and telomerase are candidate regulatory genes that might affect proliferation and apoptosis in tissues with extensive cellular proliferation, such as the seminiferous epithelium. Telomerase activity has been linked to high proliferative capacity and correlates with the expression of the catalytic subunit of the telomerase enzyme, human telomerase reverse transcriptase (hTERT). In gonadal tissues, telomerase activity is restricted to specific germ cells, that is, spermatogonia, pachytene spermatocytes and round spermatids [5]. Evidence of a crucial role for telomerase in the maintenance of male fertility has come from animal models [6]. In humans, decreased telomerase activity [7-10] and impaired hTERT expression [9] have been linked to spermatogenic disorders. Thus, dysregulated expression of telomerase may be involved in spermatogenic failure. Among the mammalian apoptosis regulators, the inhibitor of apoptosis (IAP) protein family is just beginning to be elucidated for its role in spermatogenic function. Survivin is an IAP protein regulating apoptosis at cell division. Survivin has been studied extensively in cancer cells, but has only recently received serious attention in male germ cells. In the rodent testis, survivin expression has been localized to germ cells, especially mature spermatocytes [11, 12]. Relatively high levels of survivin expression have been demonstrated in normal human spermatogenesis and its downregulation associated with spermatogenic failure [13]. Taken together, these studies suggest that survivin is a candidate regulatory gene in male germ cell production. Recent evidences of telomerase [14] and survivin [12] involvement in the meiotic division of male rodent germ cells led us consider whether comparable expression patterns of these genes could be found at the mRNA level in human testes with varying spermatogenic function. The aim of this investigation was to determine the quantitative coexpression of survivin mRNA and hTERT mRNA in testicular biopsies from men with normal spermatogenesis and specific spermatogenic disorders.
2 Materials and methods
2.1 Patients All investigated tissue specimens were collected from patients presenting with azoospermia-related infertility at the Department of Urology, Charité-Universitätsmedizin Berlin, Campus B. Franklin, Berlin, Germany, and the Department of Andrology, University of Hamburg, Hamburg, Germany, between 1997 and 2002. The use of the specimens was approved by the Ethics Committee of the Free University of Berlin. All patients gave their informed consent prior to the biopsy. Two semen analyses per patient were carried out according to the World Health Organization (WHO) guidelines[15]. Morning baseline serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were determined prior to biopsy. Non-obstructive azoospermia was clinically diagnosed in the majority of patients investigated in this study.
2.2 Processing of the biopsy material For testicular biopsy, a 3-mm3 tissue sample was obtained through a small incision in the tunica albuginea and processed as previously described [13]. Briefly, the biopsy material was divided into several fractions. The largest part was reserved for a possible intracytoplasmic sperm injection and immediately placed in Sperm-freeze solution (Medicult, Hamburg, Germany). A small fraction of the specimen was intended for molecular investigation of gene expression by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). It was shock-frozen immediately after removal then stored at -80 ºC. Another small part of the specimen was placed in Stieve¡¯s solution and served as material for the histopathological investigation, which was performed according to a modified Johnsen score [16]. This score describes the preservation of spermatogenesis on a scale from 1 (no germ cells or Sertoli cells) to 10 (intact spermatogenesis).
2.3 RNA isolation and quantitative real-time RT-PCR Total RNA was extracted from the biopsy material using RNAzolB (WAK Chemie Medical, Bad Homburg, Germany) according to the manufacturer¡¯s instructions. The concentration and quality of RNA (28S/18S ratio) were determined using the Agilent Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany). Quantitative real-time fluorescence RT-PCR was performed using the LightCycler instrument (Roche Molecular Systems, Alameda, CA, USA) according to the manufacturer¡¯s instructions. In a one-step RT-PCR reaction, 250 ng total RNA were subjected to cDNA synthesis and subsequently amplified during 40 PCR cycles (0.5 s at 95 ºC; 10 s at 60 ºC; and10 s at 72 ºC). The mRNA encoding hTERT and the housekeeping porphobilinogen deaminase (PBGD) gene were processed in the same way. The final concentration of MgCl2 was 6 mmol/L for all PCR reactions. The respective primers for survivin (GenBank accession number AF077350) were sense 5¡¯ -AAA GAG CCA AGA ACA AAA TTG C-3¡¯ and antisense 5¡¯ -GAG AGA GAA GCA GCC ACT GTT AC-3¡¯ generating a 338-bp product. The hybridization probes used are specific for an internal segment of the amplified survivin fragment (FL probe: TGC TCT TGT TTT GTC TTG AAA GTG GC—FL and LC Red640 probe: CCA GAG GTG CTT CTG CCT GTG C—PH). The primers, probes and standards for hTERT and PBGD analyses were supplied with the LightCycler Telo TAGGG hTERT Quantification kit (Roche Diagnostics, Mannheim, Germany) and the h-PBGD Housekeeping Gene Set (Roche Diagnostics, Mannheim, Germany), respectively. To carry out a positive control and to establish an external standard curve, all measurements included the determination of standards for survivin, hTERT and PBGD. The survivin cRNA standard was generated using a cloned survivin cDNA fragment as described [17]. The LightCycler 3.3 (Roche Diagnostics, Mannheim, Germany) was used to analyze PCR kinetics and to calculate quantitative data. In each sample, copy numbers of survivin mRNA and hTERT mRNA were normalized by copy numbers of PBGD mRNA. Samples lacking housekeeping gene expression were excluded from analysis.
2.4 Statistical analysis To compare expression data of all histological groups, the Kruskal-Wallis test for non-parametric analysis of variance (anova) and the Jonckheere-Terpstra test for trend were performed using SPSS 10.5 (SPSS Inc., Chicago, IL, USA). Significant differences between two groups were assessed with the Mann-Whitney U-test or Fisher¡¯s exact test, as appropriate. Regression coefficients were computed to detail the correlation between gene expression and histopathological findings (Johnsen score).
3 Results
The histopathological work-up provided evidence of full spermatogenesis (Johnsen score 8-10, obstructive azoospermia) in 11 patients. Twenty testicular biopsies showed partial tubular atrophy with maturation arrest. Postmeiotic maturation arrest (MA) corresponding to a Johnsen score of 5-7 was seen in 10 of these cases. Histopathological findings in the remaining 10 samples were consistent with premeiotic spermatogenic arrest corresponding to a Johnsen score of 3-4. Histopathology demonstrated germ cell aplasia (Sertoli cell-only syndrome [SCOS], Johnsen score 2) in eight patients. Serum FSH, LH and testosterone levels were within the normal reference range in patients with normal spermatogenesis. Patients with spermogenic failure showed high variability of serum FSH with a mean ± SD of (17.3 ± 10.8) mIU/mL (range 4.0-34.2 mIU/mL). Neither the LH nor the testosterone levels differed significantly from those in the subgroup with normal spermatogenesis. PBGD expression levels did not differ significantly between the histological groups (Kruskal-Wallis ANOVA: P = 0.1). The median (range) PBGD expression was 10 486 copies (3 495-87 530) in tissue specimens with SCOS, 30 765 copies (7 609-81 770) in those with premeiotic MA, 56 120 copies (3 82 -107 300) in those with postmeiotic MA, and 47 270 copies (14 160- 201 800) in those with normal spermatogenesis. Comparisons of individual groups disclosed significant differences between SCOS and normal spermatogenesis (P = 0.016). This means that the criteria for an ideal internal standard was only partially met by PBGD. SCOS specimens may thus show a slight distortion towards higher normalized gene expression values. Survivin mRNA expression was measured in all of the biopsy specimens. Using serial dilutions of the survivin cRNA standard, the detection limit of the assay was determined to be 103 copies. Survivin mRNA expression was detectable in all specimens with normal spermatogenesis (n = 11) and in the majority (9 of 10) of those with postmeiotic MA (Johnsen score 5-7). In contrast, only 2 of the 10 specimens with premeiotic MA (Johnsen score 3-4) evidenced survivin mRNA expression. None of the samples from SCOS patients showed detectable survivin mRNA expression in the RT-PCR despite the presence of PBGD expression. Except in two specimens from patients with premeiotic spermatogenic failure, survivin mRNA expression was thus restricted to samples containing meiotically dividing or postmeiotic germ cells. Table 1 depicts the descriptive statistics of quantitative survivin mRNA expression in human testicular tissues with varying spermatogenic function. Survivin levels were highest in specimens with normal spermatogenesis and were decreased in those with a postmeiotic MA. Significant differences in testicular survivin levels were observed between the histological groups of testicular biopsies (Kruskal-Wallis anova, P < 0.001) with positive correlations between normalized survivin values and Johnsen scores (Figure 1, P for trend < 0.001). Comparison of individual groups disclosed differences between premeiotic arrest and postmeiotic arrest (Fisher¡¯s exact test, P = 0.013) as well as between postmeiotic MA and normal spermatogenesis (Mann-Whitney U-test, P = 0.031). Figure 1 illustrates the distribution of quantitative survivin expression by histology groups. Quantitative hTERT mRNA expression was analyzed in 31 specimens (SCOS, n = 3; premeiotic MA, n = 7; postmeiotic MA, n = 10; normal spermatogenesis, n = 11). The detection limit for hTERT analysis is 102 copies. hTERT mRNA expression was detected in all but one specimen with SCOS. Expression levels were highest in specimens with normal spermatogenesis and lowest in those with SCOS (Table 1). The Kruskal-Wallis anova showed significant differences in testicular hTERT values between the histological groups (P = 0.011). Normalized hTERT levels were positively correlated with the Johnsen score (Figure 2, P for trend = 0.001). However, differences were not statistically significant when individual histological groups were compared (Mann-Whitney U-test, P > 0.05). Testicular hTERT levels were lower in patients with spermatogenesis disorders than those in subjects with full spermatogenesis (P = 0.015). Owing to the relatively low housekeeping gene expression in SCOS samples, hTERT mRNA expression in these specimens may be lower than calculated. This should not interfere with the data on differences between hTERT and survivin expression patterns, but it may mask differences in hTERT levels between SCOS and other histological subgroups.
4 Discussion
The present study describes coexpression of the survivin and the telomerase subunit hTERT in human testicular tissue. Largely consistent with our findings, results of previous studies demonstrated decreased testicular telomerase activity in patients with spermatogenic failure [7, 9, 10]. However, telomerase activity appears to be present throughout the seminiferous epithelial cycle up to the round spermatid stage, at least in rat testis [5]. Thus, marked differences in testicular hTERT expression between specimens with MA and those with normal spermatogenesis seem counter-intuitive at first. However, they may be partly attributable to the fact that specific telomerase-positive cells like spermatogonia express hTERT at lower levels in testes with spermatogenic failure than in those with normal spermatogenesis. Moreover, given that the hTERT promoter is activated by estrogens [17], changes in the paracrine environment in patients with spermatogenic failure may also contribute to decreased testicular telomerase activity. These potential regulative mechanisms warrant further investi-gation. Corroborating previous preliminary data [13], we confirmed that the level of survivin mRNA expression is correlated with the severity of spermatogenic failure in infertile patients. In contrast to telomerase expression, however, that of survivin seems to be restricted to specimens containing meiotically dividing or haploid germ cells. These findings are in line with results in rodents demonstrating a preponderance of survivin protein and mRNA expression in meiotically dividing spermatocytes [11, 12]. Taken together, these studies raise the question of survivin involvement in the meiotic progression of germ cells. On the other hand, our results cannot exclude survivin expression by other cell types, for example, spermatogonia or even Sertoli cells, in the normal testis. Consistent with the role of survivin in the mitotic cell cycle, low levels of expression are found in spermatogonia in rats [12]. Thus, it remains to be elucidated which germ cell types actually express survivin in humans. Interestingly, we found coexpression of survivin and telomerase only in specimens characterized by the presence of haploid germ cells. In contrast, survivin expression was lacking despite the expression of hTERT in specimens showing severe spermatogenic failure. Telomerase expression thus seems to relate to the cellularity or pro liferative capacity of the seminiferous epithelium. On the other hand, recent evidence suggests additional roles of telomerase in male mammalian meiosis [6, 14]. Germ cells may require functional telomerase for both proliferation and meiotic division. Our study disclosed a relatively high variability of transcript levels within the histological subgroups, especially in the subgroup with normal spermatogenesis. This may indicate an influence of factors unrelated to the spermatogenic function on the expression levels of these genes. However, all patients with normal testicular histopathology had obstructive azoospermia. It is not yet clear whether even higher and less variable expression levels may occur in fertile men. It must also be considered that the observed differences between testicular survivin and telomerase expression could be due to artifacts. RT-PCR analysis of survivin mRNA may just lack the sensitivity to detect low expression levels of this IAP in premeiotic germ cells, such as spermatogonia, or even Sertoli cells. Indeed, low levels of survivin may be expressed in Leydig cells in the rodent testis [12]. Moreover, the described expression pattern of survivin mRNA remains to be confirmed at the protein level by other detection strategies such as immunohistochemistry. Low or no expression of anti-apoptotic genes such as survivin might play a role in the pathogenesis of spermatogenic disorders, as increased germ cell apoptosis has been observed in conjunction with impaired human spermatogenesis [1]. It remains to be elucidated whether altered survivin and/or telomerase expression are causative factors in the development of spermatogenic disorders. Overexpression of survivin in colon cancer cells has recently been shown to enhance telomerase activity by the upregulation of hTERT expression [18]. This opens up new perspectives on the question of germ cells having intimately linked survivin expression and telomerase activity to inhibit apoptosis and to prolong their cellular lifespan. Our findings indicate that testicular coexpression of survivin and hTERT is associated with normal spermatogenesis or postmeiotic maturation. Testes lacking haploid germ cells show hTERT, but not survivin mRNA expression, which supports the finding of predominant survivin expression during meiosis [11, 12]. References 1 Lin WW, Lamb DJ, Wheeler TM, Lipshultz LI, Kim ED.
In 2 Print CG, Loveland KL. Germ cell suicide: new insights into apoptosis during spermatogenesis. Bioessays 2000; 22: 423-30. 3 Yang B, Wang H, Gao XK, Chen BQ, Zhang YQ, Liu HL, et al. Expression and significance of Rap1A in testes of azoospermic subjects. Asian J Androl 2004; 6: 35-40. 4 Zhang XD, Yin LL, Zheng Y, Lu L, Zhou ZM, Sha JH. Expression of a novel beta adaptin subunit mRNA splice variant in human testes. Asian J Androl 2005; 7: 179-88. 5 Eisenhauer KM, Gerstein RM, Chiu CP, Conti M, Hsueh AJ. Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos. Biol Reprod 1997; 56: 1120-5. 6 Hemann MT, Rudolph KL, Strong MA, DePinho RA, Chin L, Greider CW. Telomere dysfunction triggers developmentally regulated germ cell apoptosis. Mol Biol Cell 2001; 12: 2023-30. 7 Fujisawa M, Tanaka H, Tatsumi N, Okada H, Arakawa S, Kamidono S. Telomerase activity in the testis of infertile patients with selected causes. Hum Reprod 1998; 13: 1476-9. 8 Schrader M, Muller M, Heicappell R, Krause H, Schulze W, Miller K. Telomerase activity and expression of telomerase subunits in the testicular tissue of infertile patients. Fertil Steril 2000; 73: 706-11. 9 Schrader M, Muller M, Heicappell R, Straub B, Miller K. Quantification of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA in testicular tissue of infertile patients. Asian J Androl 2001; 3: 263-70. 10 Yamamoto Y, Sofikitis N, Mio Y, Miyagawa I. Highly sensitive quantitative telomerase assay of diagnostic testicular biopsy material predicts the presence of haploid spermatogenic cells in therapeutic testicular biopsy in men with Sertoli cell-only syndrome. Hum Reprod 1999; 14: 3041-7. 11 Kobayashi K, Hatano M, Otaki M, Ogasawara T, Tokuhisa T. Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation. Proc Natl Acad Sci USA 1999; 96: 1457-62. 12 Wang Y, Suominen JS, Hakovirta H, Parvinen M, Martinand-Mari C, Toppari J, et al. Survivin expression in rat testis is upregulated by stem-cell factor. Mol Cell Endocrinol 2004; 218: 165-74. 13 Weikert S, Schrader M, Muller M, Krause H, Miller K. Expression of the apoptosis inhibitor survivin in testicular tissue of infertile patients. Int J Androl 2004; 27: 161-5. 14 Liu L, Franco S, Spyropoulos B, Moens PB, Blasco MA, Keefe DL. Irregular telomeres impair meiotic synapsis and recombination in mice. Proc Natl Acad Sci USA 2004; 101: 6496-501. 15 World Health Organization: Laboratory Manual for the Examination of Human Semen and Semen-cervical Mucus Interaction; 4th ed. New York: Cambridge University Press; 1999. 16 Holstein AF, Schulze W, Davidoff M. Understanding spermatogenesis is a prerequisite for treatment. Reprod Biol Endocrinol 2003; 1: 107. 17 Weikert S, Christoph F, Schrader M, Krause H, Miller K, Muller M. Quantitative analysis of survivin mRNA expression in urine and tumor tissue of bladder cancer patients and its potential relevance for disease detection and prognosis. Int J Cancer 2005; 116: 100-4. 18 Kyo S, Takakura M, Kanaya T, Zhuo W, Fujimoto K, Nishio Y, et al. Estrogen activates telomerase. Cancer Res 1999; 59: 5917-21. 19 Endoh T, Tsuji N, Asanuma K, Yagihashi A, Watanabe N. Survivin enhances telomerase activity via up-regulation of specificity protein 1- and c-Myc-mediated human telomerase reverse transcriptase gene transcription. Exp Cell Res 2005; 305: 300-11. |
| ¡¡ | |
