Hum. Reprod. Advance Access originally published online on September 9, 2005
Human Reproduction 2006 21(1):138-144; doi:10.1093/humrep/dei285
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Decreased mRNA transcripts of M-phase promoting factor and its regulators in the testes of infertile men
Y.M.Lin and Y.N.Teng contributed equally to this work. Departments of 1 Urology, 3 Obstetrics & Gynecology, and 4 Institute of Basic Medical Science, National Cheng Kung University, College of Medicine, Tainan, and 2 Department of Early Childhood Education and Nursery, Chia Nan University of Pharmacy and Science, Tainan, Taiwan
5 To whom correspondence should be addressed at: Division of Genetics, Department of Obstetrics and Gynecology, National Cheng Kung University Hospital, 138 Sheng Li Road, Tainan, Taiwan 704. E-mail: paolink{at}mail.ncku.edu.tw
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Abstract |
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BACKGROUND: M-phase promoting factor (MPF), which is comprised of Cyclin B and a catalytic subunit, Cdc2, is a key enzyme required for cells to enter M phase in both mitosis and meiosis. MPF activity is controlled by the stimulatory dephosphorylation of the Cdc25 family and the inhibitory phosphorylation of Wee1. We determined the levels of mRNA transcripts of MPF and its regulators in the testes of infertile men, and evaluated the relationship between the transcript levels and patients’ testicular phenotypes and sperm retrieval results. METHODS AND RESULTS: The mRNA transcript levels of CDC2, CCNB1, CCNB2, CDC25A, CDC25B, CDC25C and WEE1 in the testes of 37 azoospermic patients were examined by quantitative real-time polymerase chain reaction. Significant decreases in CDC2, CCNB1, CCNB2, CDC25A, CDC25C and WEE1 mRNA transcript levels were detected in patients with spermatogenic failure. CDC2 mRNA transcript levels correlated significantly with those of CCNB1 and CCNB2 mRNA. Significantly higher CDC2, CCNB1, CCNB2, CDC25C and WEE1 mRNA transcript levels were detected in 18 patients with successful sperm retrieval than in 11 patients with failed sperm retrieval. CONCLUSIONS: We suggest that the decreased mRNA transcripts of MPF and its regulators play important roles in human spermatogenesis.
Key words: male infertility/MPF/testis
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Introduction |
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Human spermatogenesis consists of stem cell mitotic proliferation, meiotic division and a metamorphic change to produce a haploid gamete. It is well known that both the mitotic and meiotic cycles in all eukaryotic organisms are initiated by activated M-phase promoting factor (MPF), which is composed of a regulatory subunit, Cyclin B, and a catalytic subunit, Cdc2 (cell division cycle 2) (Masui and Markert, 1971
). Activated MPF phosphorylates cellular machinery components involved in nuclear envelope breakdown, chromosome condensation, spindle assembly and cyclin degradation (Miake-Lye and Kirschner, 1985; Peter et al., 1990; Satterwhite et al., 1992), thus controlling both mitosis and meiosis.
MPF activation is controlled by the phosphorylation and dephosphorylation of three residues within the ATP binding cleft of Cdc2 (Gould and Nurse, 1989; Meijer et al., 1991; Wolgemuth et al., 2002). Phosphorylation of Thr161 is essential for Cdc2 activation. However, in the S and G2 phases, MPF activity is inhibited by the phosphorylation of Thr14 and Tyr15 residues, which is catalysed by Wee1 homologue (Wee1) and related protein kinases (Lee et al., 1991; Lundgren et al., 1991). On entry into M phase, this inhibition is removed by Cdc25 phosphatase activity. Maximal Cdc2 activity is achieved when it is fully dephosphorylated. Subsequently, rapid activation of MPF can lead to self-amplification as a result of the increased activity of dephosphorylated Cdc25 and the decreased activity of phosphorylated Wee1 (Figure 1) (Hoffmann et al., 1993; Tang et al., 1993; Parker et al., 1995; Watanabe et al., 1995).
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Two Cyclin B family members, Cyclin B1 (Ccnb1) and B2 (Ccnb2), have been identified in mammals, and both of them are expressed in testicular germ cells (Pines and Hunter, 1989; Chapman and Wolgemuth, 1992; 1993). In addition, three Cdc25 homologues have been identified and designated as Cdc25A, B and C (Sadhu et al., 1990; Nagata et al., 1991). All members of the Cdc25 family are involved in the M-phase regulation of mitosis and meiosis (Millar et al., 1991; Jinno et al., 1994; Nilsson and Hoffmann, 2000).
The role of MPF and its regulators in female reproduction has been well studied, and these proteins have been shown to be associated with the resumption of meiotic maturation, the inhibition of DNA replication between meiosis I and II, and the maintenance of the oocyte at metaphase II arrest until fertilization (Heikinheimo and Gibbons, 1998). To date, little is known about the role of MPF in human spermatogenesis. Considering the importance of MPF for driving cells into M phase for both mitosis and meiosis, it is tempting to speculate that the expression of MPF and its regulators may correlate with testicular phenotypes and haploid sperm production.
In this study we attempted to explore the importance of MPF and its regulators in human spermatogenesis, we determined the CDC2, CCNB1, CCNB2, CDC25A, CDC25B, CDC25C and WEE1 mRNA transcript levels in the testes of azoospermic patients using quantitative real time polymerase chain reaction (RT-PCR). We compared these mRNA transcript levels between patients with normal spermatogenesis and patients with spermatogenic failure, and evaluated the relationship between the mRNA transcript levels and the patients’ testicular phenotypes and sperm retrieval results.
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Materials and methods |
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Patients
This study recruited 37 patients presenting with azoospermia. The diagnosis of azoospermia was based on at least two separate semen analyses and two separate centrifuged semen sample analyses (3000 g, 15 min). All patients received a detailed physical examination, endocrinology profile testing, and a testicular biopsy and/or vaso-vesiculography. Patients with normal FSH levels, normal testicular volume, normal histology in the testicular biopsy and/or definite seminal tract obstruction in vaso-vesiculography were categorized as having obstructive azoospermia. Non-obstructive azoospermia was defined by the presence of spermatogenic defects (hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome) in the testicular biopsy, or by the presence of elevated serum FSH levels, a total testicular volume <30 ml and the absence of other applicable diagnoses. None of the patients possessed abnormal chromosome karyotypes or Y chromosome gene deletions. Informed consent was obtained from all patients enrolled in the study. The study was approved by The National Scientific Council of Taiwan and the Institutional Review Board of National Cheng Kung University Medical Centre.
Testicular samples
All patients underwent diagnostic testicular biopsy or sperm retrieval, and agreed to provide a small piece of testicular tissue (
5 mm in diameter) for further study. One-third of the tissue volume was immersed in Bouin’s solution and sent for histopathological diagnosis. This process allowed us to examine more than 100 cross-sections of seminiferous tubule. Given the presence of a mixture of components in the testicular samples, the testicular histopathology was categorized according to the most advanced pattern of spermatogenesis present. For example, some patients have the appearance of Sertoli cell-only in some tubules but with hypospermatogenesis in adjacent tubules, and such cases have been categorized as hypospermatogenesis. Two specialists reviewed all testicular slices. The remaining two-thirds of the tissue volume was cryopreserved for RNA extraction.
RNA extraction and complementary DNA synthesis
Total cellular RNA was extracted from the testicular tissue using standard methods (High PureTM RNA Tissue Kit; Boehringer Mannheim, Indianapolis, IN, USA), and quantified by measuring the absorbance at 260 nm. For complementary DNA (cDNA) synthesis, 12 ml aliquots of master mixture containing 2 ml of RNA, 1 ml of 500 ng/ml oligo(dT)12–18 primer (Gibco-BRL, Grand Island, NY, USA) and 9 ml of diethylpyrocarbonate-treated water were heated to 70°C for 10 min and put on ice. Reverse transcription reactions were performed in 20 ml volumes containing master mixture, 4 ml of 5x first strand synthesis buffer, 0.1 mol/l dithiothreitol, 10 mmol/l of each dNTP and 200 U of SuperscriptTM II RNase H– reverse transcriptase (Gibco-BRL). The temperature profile used was 42°C for 1 h, 75°C for 15 min, followed by cooling to 4°C. Aliquots of the cDNAs were stored at –20°C until use.
Primers and quantitative RT-PCR
The sequences of the forward and reverse primers used to amplify the human CDC2, CCNB1, CCNB2, CDC25A, CDC25B, CDC25C, WEE1 and hydroxymethylbilane synthase (HMBS) genes were designed using Light Cycler Probe Design Software v1.0 (Roche Applied Science, Penzberg, Germany). The HMBS gene was used as an endogenous control. The RT-PCR reactions were performed in a LightCycler® 3.5 Instrument (Roche Applied Science) using LightCycler capillaries and a master mix containing Taq DNA polymerase and SYBR Green I. The total volume of the PCR was 10 ml and contained 2 ml of cDNA template, 2 ml of primer mixture (final concentration 2 mmol/l), 4 mmol/l MgCl2 and 1 ml of SYBR Green I. The RT-PCR conditions began with denaturation at 95°C for 7 min, followed by 50 cycles consisting of denaturation at 95°C for 5 s, annealing at 60°C for 5 s and extension at 72°C for 10 s. All real-time experiments were run in triplicate, and the mean mRNA value was calculated. Negative controls without added template were included in each set of RT-PCR assays. The standard curve quantitation method was used in this study, and the amount of transcript in each sample was calculated by interpolation using the formula: (threshold cycle – y intercept)/S. The steady-state concentrations of mRNA for other genes in each testicular sample were normalized to the amount of HMBS mRNA.
Data analysis
Data was analysed using GraphPad Prism 4 statistical software (GraphPad Software, San Diego, CA, USA). The mRNA transcript levels of HMBS and transcript ratios of CDC2, CCNB1, CCNB2, CDC25A, CDC25B, CDC25C and WEE1 to HMBS in different histopathological groups were presented as the mean ± SEM. Transcript ratios in patients with varying degrees of spermatogenic failure were analysed using the Kruskal–Wallis test, and multiple pairwise comparisons were performed using Dunnett’s test. Pearson product moment correlation coefficients were calculated to determine the correlation between the transcript ratios for each gene. A P-value <0.05 was considered significant.
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Results |
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Clinical variables of the patients
Of the 37 patients with azoospermia, eight were diagnosed with obstructive azoospermia and normal spermatogenesis. Of these eight, there were two patients with failed vasectomy reversals, one patient with failed vaso-epididymostomy, two patients with bilateral epididymal obstructions and three patients with bilateral hypoplasia of the seminal vesicle. Sperm were successfully retrieved from all eight patients. Of the other 29 patients, with non-obstructive azoospermia, the histopathological data indicated hypospermatogenesis in 13 patients, maturation arrest in three patients, and Sertoli cell-only syndrome in 13 patients. Sperm were successfully retrieved from all 13 patients with hypospermatogenesis. Sperm were successfully retrieved from two of three patients with maturation arrest and from only three of 13 patients with Sertoli cell-only syndrome.
Comparison of mRNA transcript levels in patients with different testicular histopathologies
The HMBS mRNA transcript levels ranged from 1.73 to 9.16 x 103 copies/ng RNA (mean ± SEM, 3.91 ± 1.2 x 103 copies/ng RNA) for men with normal spermatogenesis, from 0.59 to 9.97 x 103 copies/ng RNA (mean ± SEM, 3.60 ± 0.8 x 103 copies/ng RNA) for men with hypospermatogenesis, from 2.23 to 6.44 x 103 copies/ng RNA (mean ± SEM, 4.48 ± 1.2 x 103 copies/ng RNA) for men with maturation arrest and from 1.29 to 5.55 x 103 copies/ng RNA (mean ± SEM, 2.86 ± 0.5 x 103 copies/ng RNA) for men with Sertoli cell-only syndrome. No significant difference was detected between these four groups (P = 0.703; Figure 2). The mRNA transcript ratios of each gene in patients with different testicular histopathologies are shown in Figure 2. Subdividing our patients into four groups according to the severity of testicular histopathology produced a progressive decrease in the mRNA transcript ratios for the CDC2, CCNB1, CCNB2, CDC25A, CDC25C and WEE1 genes, with P-values of 0.045, 0.0005, 0.018, 0.043, 0.0018 and 0.035, respectively. There was no significant differences in CDC25B mRNA transcript ratios between these groups (P = 0.1).
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Expression pattern of mRNA transcript levels reported as percent per quartile for each patient
Table I shows the patients’ characteristics and mRNA transcript ratios for all genes tested. The mRNA transcript ratios for each gene were displayed as percent per quartile from maximum to minimum levels. The transcript ratios in the 99th to 76th percentile were designated as fourth quartile (++++); ratios in the 75th to 51st percentile were designated as third quartile (+++); ratios in the 50th to 26th percentile were designated as second quartile (++); and ratios in the 25th to 1st percentile were designated as first quartile (+).
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Correlations between the mRNA transcript ratios for each gene
Table II shows the correlation coefficients that resulted from comparing the mRNA transcript ratios between the tested genes. The following mRNA transcript ratio correlations were low and calculated to be not significant: CDC25A compared with CDC2, CCNB1, CCNB2, CDC25B, CDC25C or WEE1; CDC25B compared with CDC2, CCNB1, CCNB2, CDC25A, CDC25C or WEE1; and CDC25C compared with CDC2, CCNB1, CCNB2, CDC25A, CDC25B or WEE1. In contrast, significant positive correlations were found for mRNA transcript ratio comparisons between CDC2 and CCNB1, CDC2 and CCNB2, CCNB1 and CCNB2, WEE1 and CDC2, WEE1 and CCNB1, and WEE1 and CCNB2.
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mRNA transcript ratios of MPF and its regulators and the results of sperm retrieval
We divided the 29 patients with spermatogenic failure into two groups based on the presence (18 patients) or absence (11 patients) of mature sperm recovered from microsurgical epididymal sperm aspiration or testicular sperm extraction (TESE). The mRNA transcript ratios for all genes tested were displayed with scattergrams (Figure 3). Significant differences were noted between these two groups for the CDC2, CCNB1, CCNB2, CDC25C and WEE1 genes with P-values of 0.004, 0.0005, <0.0001, 0.01 and 0.004, respectively (unpaired Student’s t-test). Conversely, no significant difference was noted for the HMBS, CDC25A and CDC25B genes (P = 0.279, 0.165 and 0.069, respectively).
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Discussion |
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Previous studies have shown that MPF subunits are expressed in mammalian testis and high levels of Cdc2 and Cyclin B expression as well as MPF activity have been demonstrated in rat and mouse testes (Chapman and Wolgemuth, 1992
; 1994; Rhee and Wolgemuth, 1995; Gromoll et al., 1997). Little is known about the mRNA or protein expression of MPF and its regulators in human testis. Only the protein expression of CDC25A is well described. In human testis, CDC25A protein is detectable in the cytoplasm of primary and secondary spermatocytes and then translocates from the cytoplasm to the nucleus of the round and elongating spermatids (Luetjens et al., 2004). In the present study, the CDC2, CCNB1 and CCNB2 mRNA transcript levels were decreased in patients with spermatogenic failure, indicating that MPF did indeed play a role in human spermatogenesis. In addition, the CDC25A, CDC25C and WEE1 mRNA transcript levels were significantly decreased in patients with spermatogenic failure, suggesting that the MPF’s regulators are also important for human spermatogenesis. Conversely, CDC25B mRNA transcript levels did not differ among these patients. These results may be derived from high levels of specific gene expression in the germ cells at specific stage of spermatogenesis. In adult mouse testes, Cdc2 transcripts are expressed in primary spermatocytes and Sertoli cells (Rhee and Wolgemuth, 1995), Ccnb1 transcripts are expressed in late meiotic cells, early round spermatids and Sertoli cells (Chapman and Wolgemuth, 1992; Gromoll et al., 1997), Ccnb2 transcripts are expressed in mid-to-late pachytene spermatocytes (Chapman and Wolgemuth, 1993), Cdc25A transcripts are expressed in spermatogonia and spermatocytes, Cdc25B transcripts are abundant in somatic cells, Cdc25C transcripts are localized in late pachytene-diplotene spermatocytes and spermatids (Wickramasinghe et al., 1995; Wu and Wolgemuth, 1995), and Wee1 transcripts are expressed in spermatogonia, primary and secondary spermatocytes (Wolgemuth et al., 2002). Therefore, reduction in the relative concentrations of germ cells in testis would certainly influence the relative gene expression levels, and the constant levels of CDC25B mRNA transcripts may have been due to their expression in somatic cells.
To date, most of the information regarding the biological functions of MPF and its regulators in mammals has been derived from genetically modified mice. Ccnb1-null mice die in utero, whereas Ccnb2-null mice develop normally and are fertile, indicating that Ccnb1 is essential for life and may compensate for the loss of Ccnb2 in the mutant mice (Brandeis et al., 1998). Another genetic ablation study showed that female Cdc25B-null mice were sterile and their oocytes remained arrested at prophase, whereas the male mice were fertile, suggesting that Cdc25B may have distinct functional differences between the sexes (Lincoln et al., 2002). Cdc25C-null mice were fertile and did not display any obvious abnormalities, indicating that Cdc25A and/or Cdc25B may compensate for the loss of Cdc25C (Chen et al., 2001). Cdc2-, Cdc25A- and Wee1-null mice have not yet been generated. Obviously, the functions of MPF and its regulators in mammalian spermatogenesis are not completely understood, owing in part to a complex series of upstream and downstream regulatory events (Wolgemuth, 2003). In the present study, the mRNA expression profiles of MPF and its regulators, as shown in Table I, may provide clues about which genes are important for human spermatogenesis. Among patients with normal spermatogenesis, two patients had lower mRNA transcript levels for CDC2, one patient had lower levels for CDC25A and three patients had lower levels for CDC25B (first quartile of the transcript levels of all patients), suggesting that CDC2, CDC25A and CDC25B may play less critical roles in human spermatogenesis. In patients with spermatogenic failure (hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome), successful sperm retrieval was almost always achieved in those patients whose mRNA transcript levels for CCNB1, CCNB2, CDC25C and WEE1 genes were moderate to high (second quartile or more of the transcript levels of all patients), suggesting that abundant expression of these four genes was critical for sperm production.
The use of quantitative RT-PCR has allowed more precise quantitation of the relative transcript levels of individual genes than in previous work. Thus, it provides a unique opportunity for understanding the relationships of MPF and its regulators in human testes. In this study, CDC2 mRNA transcript levels positively correlated with CCNB1 and CCNB2 mRNA transcript levels, implying that the both forms of Cyclin B may bind and activate CDC2 in human testes. Moreover, a good correlation was found between the mRNA transcript levels of CCNB1 and CCNB2, suggesting that their mRNA may be simultaneously expressed in human testes. This finding was consistent with previous studies showing that these two mammalian Cyclin Bs were co-expressed in most cultured cells (Wolgemuth et al., 2002).
It is also interesting to note that the WEE1 mRNA transcript levels positively correlated with CDK1, Cyclin B1 and Cyclin B2 mRNA transcript levels. Our data show coordinated expression patterns of WEE1 and MPF mRNA transcripts in human testes that suggests their functional interaction.
In patients with azoospermia caused by testicular failure, TESE plus ICSI is now considered the only way to achieve pregnancy. Currently, testicular samples for quantitative and/or qualitative evaluations are commonly used to predict the presence of sperm at TESE (Tournaye et al., 1997; Su et al., 1999; Seo and Ko, 2001). In this study, it is intriguing to note that two patients with maturation arrest (patients 22 and 23) and three patients with Sertoli cell-only syndrome (patients 25, 34 and 35) had successful TESE. An explanation for this is that the wet preparation specimens from those patients may contain some germ cell foci. There have been many reports of patients with diagnoses of maturation arrest or Sertoli cell-only who have undergone successful TESE (Silber et al., 1997; Tournaye et al., 1997). We believe that our histopathological diagnosis is convincing because there were generally more than 100 seminiferous tubule cross-sections available for examination in each patient, the advanced category system was applied, and the diagnoses were confirmed by two experienced specialists. Therefore, our results indicate the limited predictability of histopathological diagnosis at TESE.
Several marker genes, including protamine, cyclin A1 and BOULE, have been shown to correlate with the presence of testicular sperm in patients with spermatogenic failure (Steger et al., 2003; Wolgemuth et al., 2004; Schrader et al., 2002; Lin et al., 2005). Decrease in the mRNA transcripts of protamine 1 and the aberrant protamine 1 to protamine 2 mRNA ratio correlate significantly with efficiency of spermatogenesis (Steger et al., 2003). Quantitative determination of cyclin A1 mRNA expression appears to predict the presence of secondary spermatocytes/early spermatids or mature spermatids in patients with spermatogenic failure (Schrader et al., 2002). A significant decrease in BOULE transcript levels was detected in patients with spermatogenic failure, and BOULE transcripts progressively decreased with increasing severity of testicular failure (Lin et al., 2005). In the present study, higher levels of mRNA transcripts of CDC2, CCNB1, CCNB2, CDC25C and WEE1 genes significantly correlated with the success of TESE, implying that the measurement of mRNA transcripts of these genes may potentially be useful in predicting the presence of sperm in testis. However, it is also noted that the sample size is limited in the present study, and the possibility exists that more samples may change the predictive power reported. Therefore, the diagnostic value of this assay in patients with spermatogenic failure remains to be further investigated. At present, this molecular classification system may supplement histopathological evaluation of spermatogenic disorders.
In conclusion, we determined the mRNA transcript levels of MPF and its regulators in the testes of infertile men. Patients with spermatogenic failure were found to have decreased mRNA transcript levels of the CDC2, CCNB1, CCNB2, CDC25A, CDC25C and WEE1 genes. Positive correlations between the components of MPF and between MPF and WEE1 mRNA transcript levels were noted. Higher mRNA transcript levels of CDC2, CCNB1, CCNB2, CDC25C and WEE1 correlated with the success of sperm retrieval. Our results, for the first time, suggest that the mRNA transcripts of MPF and its regulators play important roles in human spermatogenesis.
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This study is supported by grants from the National Science Council of Taiwan (NSC 91–2314-B-006–149, NSC 91–3112-B-006–008, NSC 92–3112-B-006–002, NSC 93–3112-B-006–004, NSC 93–2314-B-006–69 and NSC 93–2314-B-006–078).
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Submitted on May 21, 2005; resubmitted on August 2, 2005; accepted on August 4, 2005.
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  Y. S. Cheng, P. L. Kuo, Y. N. Teng, T. Y. Kuo, C. L. Chung, Y. H. Lin, R. W. Liao, J. S. N. Lin, and Y. M. Lin Association of spermatogenic failure with decreased CDC25A expression in infertile men Hum. Reprod., September 1, 2006; 21(9): 2346 - 2352. [Abstract] [Full Text] [PDF]
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