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Hum. Reprod. Advance Access originally published online on May 23, 2006
Human Reproduction 2006 21(9):2346-2352; doi:10.1093/humrep/del163
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Association of spermatogenic failure with decreased CDC25A expression in infertile men

Yu Sheng Cheng1,2, Pao Lin Kuo3, Yen Ni Teng4, Ting Yi Kuo1, Chia Ling Chung3, Ying Hung Lin5, Rui Wen Liao3, Johnny Shinn Nan Lin1 and Yung Ming Lin1,6

1 Department of Urology 2 Institute of Clinical Medicine 3 Department of Obstetrics & Gynecology, National Cheng Kung University, College of Medicine 4 Department of Early Childhood Education and Nursery, Chia Nan University of Pharmacy and Science and 5 Institute of Basic Medical Science, National Cheng Kung University, College of Medicine, Tainan, Taiwan

6 To whom correspondence should be addressed at: Department of Urology, National Cheng Kung University, College of Medicine, 138, Sheng Li Road, Tainan, Taiwan. E-mail: linym{at}mail.ncku.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: DAZ gene family is crucial for human spermatogenesis that requires the precise co-ordination of cell cycle events. CDC25A is recognized as the downstream substrate of DAZ gene family and is thought to function on the M-phase regulation of cell cycles. We investigated the expression profiles of CDC25A in the testes of infertile men and evaluated the relationship between CDC25A levels and testicular phenotype, clinical hormonal parameters and sperm retrieval results. METHODS: The protein and mRNA transcript levels of CDC25A in the testes of 40 azoospermic men were determined by immunohistochemistry and quantitative real-time-PCR. CDC25A in human spermatozoa was investigated by western blotting and immunofluorescence staining. RESULTS: The CDC25A protein was expressed mainly in spermatocyte, spermatid and spermatozoa. CDC25A transcript levels were significantly decreased (P = 0.0009) in patients with spermatogenic failure, especially in men with meiotic arrest and Sertoli cell-only syndrome. Significantly higher CDC25A transcript levels were detected in patients with successful sperm retrieval than in patients with failed sperm retrieval (P = 0.005). CONCLUSIONS: Decreased CDC25A is associated with spermatogenic failure and failed sperm retrieval in infertile men. Further studies are necessary to explore the functional roles of CDC25A in human spermatozoa.

Key words: CDC25A/male infertility/testis


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In humans, there are increasing evidences that the DAZ gene family is required for spermatogenesis and is considered as sterility-related genes (Silber et al., 1998Go; Ferlin et al., 1999Go; Lin et al., 2002Go; Teng et al., 2002Go; Kuo et al., 2004Go; Luetjens et al., 2004Go). The human DAZ gene family contains at least three members represented by DAZ, DAZ-like (DAZL) and BOULE. All members of the DAZ gene family are expressed exclusively in germ cells (Xu et al., 2001Go). The proteins encoded by DAZ gene family contain an RNA-binding domain at their amino terminus, suggesting a functional role in mRNA stability or the translation regulation of their target RNA (Maines and Wasserman, 1999Go; Venables et al., 2001Go; Jiao et al., 2002Go). Recently, many putative substrates of Dazl or Boule have been proposed (Venables et al., 2001Go; Jiao et al., 2002Go; Fox et al., 2005Go; Reynolds et al., 2005Go), some of which are genes with known biological function. Among these known genes, the members of the CDC25 gene family are recognized as potential candidates.

In Drosophila, the expression of Twine protein, which is a Cdc25-type phosphatase, correlates with the intracellular accumulation of Boule and is significantly reduced by boule mutants, indicating that Boule could influence Twine expression through direct binding to the twine mRNA (Maines and Wasserman, 1999Go). By SELEX and tri-hybrid screening, the 5' untranslated region (UTR) of Cdc25C mRNA specifically bound Dazl protein in mice, indicating that Cdc25C mRNA acted downstream of Dazl (Venables et al., 2001Go). Using the approach of isolating specific nucleic acids associated with protein, it has been shown that Dazl bound the 3' UTR of Cdc25A mRNA, suggesting that Cdc25A is a putative downstream substrate of Dazl (Jiao et al., 2002Go). Additionally, in a subgroup of infertile men with meiotic arrest, BOULE was almost absent and CDC25A was concomitantly lacking, suggesting that CDC25A acting downstream of BOULE may be required for meiotic entry (Luetjens et al., 2004Go).

CDC25 gene family has been well recognized as cell cycle regulators. Spermatogenesis is a complex process that is highly ordered and requires the precise co-ordination of cell cycle events. A key player in cell cycle progression is M-phase promoting factor (MPF), which is composed of cyclin B and a catalytic subunit, Cdc2 (cell division cycle 2) (Masui and Markert, 1971Go). The Cdc25 proteins are threonine/tyrosine phosphatases that positively regulate the cell division cycle by activating Cdc2 (Galaktionov and Beach, 1991Go; Jinno et al., 1994Go). In the S and G2 phases, MPF activity is inhibited by the phosphorylation of Thr14 and Tyr15 residues (Lee et al., 1991Go; Lundgren et al., 1991Go); at the onset of M phase, this inhibition is removed by Cdc25 activity. Of the Cdc25 family, Cdc25A is abundantly expressed in the testis and functions at the G1–S transition and M-phase exit, suggesting that Cdc25A might play a role in mitotic or meiotic regulation of mammalian spermatogenesis (Wickramasinghe et al., 1995Go; Wu and Wolgemuth, 1995Go).

To date, little is known about the role of CDC25A in human spermatogenesis. Given that CDC25A is an important cell cycle regulator for both mitosis and meiosis and is a putative downstream substrate of the DAZ gene family, it is tempting to speculate that the CDC25A expression may correlate with testicular phenotypes. In this study, we investigated the CDC25A mRNA expression profiles in the testes of azoospermic patients. In addition, we investigated the presence of CDC25A protein in human sperm. We evaluated the relationships between CDC25A expression and the patients’ testicular phenotypes, clinical hormonal parameters and sperm retrieval results.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Patients
The experimental design was in accordance with the Helsinki Declaration of 1975 on human experimentation and was approved by The National Scientific Council of Taiwan and the Institutional Review Board of National Cheng Kung University Medical Centre. Informed consent was obtained from all patients enrolled in the study. Forty infertile men presenting with azoospermia and one fertile man who donated fresh semen were enrolled in this study. The diagnosis of azoospermia was based on the analysis of at least two semen samples collected at different times and two replicates of each sample which were centrifuged at 3000 x g for 15 min. All patients underwent 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 in the testicular biopsy, elevated serum FSH levels, or a total testicular volume <30 ml, and the absence of other applicable diagnoses.

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. The testicular histopathology was categorized according to the most advanced pattern of spermatogenesis present. Two specialists reviewed all testicular slices. The remaining two-thirds of the tissue volume was cryopreserved for RNA extraction.

Immunohistochemical staining of testicular tissue
Bouin’s fixed human testicular specimens were dehydrated, embedded in paraffin and sectioned at 5 µm. The tissue sections were immunostained using polyclonal antibodies against human CDC25A (sc-97; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were deparaffinized with 100% xylene and sequentially rehydrated with 96, 90, 80 and 70% ethanol. The slides were then blocked with 3% hydrogen peroxide in absolute methanol for 5 min, washed with water for 5 min and heated at 90°C for 5 min in pre-heated citrate buffer (pH 6.0). After cooling, the slides were washed twice with phosphate-buffered saline (PBS, pH 7.4) for 5 min each time. The slides were incubated with the primary antibody (1:1000) for 60 min at room temperature. Following the washing steps with PBS, sections were incubated with biotinylated mouse anti-rabbit immunoglobulin G (IgG) antibody (Dako, Carpinteria, CA, USA) for 30 min at room temperature, washed with PBS, then incubated with the avidin–biotin complex for 30 min at room temperature, followed by reaction with 0.035% diaminobenzidine tetrachloride/0.03% hydrogen peroxide. Sections were subsequently counterstained with haematoxylin, dehydrated, cleaned and mounted.

Western blotting, immunofluorescence staining and confocal microscopy analysis of spermatozoa
For western blotting analysis, semen sample was washed twice with PBS, followed by centrifugation. The cell pellets were mixed with lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). Approximately 20 µg of the total protein was then fractionated on a 12% sodium dodecyl sulphate–polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were washed with Tris-buffered saline (TBS, pH 7.4) and then incubated in a blocking solution for 1 h. The membranes were then incubated in a 1:1000 dilution of a primary antibody overnight and washed with PBS, followed by incubation with a 1:2000 dilution of secondary antibody (goat anti-rabbit IgG, conjugated with peroxidase). The filters were then washed three times, and the peroxidase activities were visualized using the SuperSignal substrate (Pierce, Rockford, IL, USA). For testing of specificity, testis tissue from an obstructive azoospermic man, antiserum pre-incubated with blocking peptide and a polyclonal antibody to beta-actin (A5060; Sigma, St Louis, MO, USA) were used as controls. For immunofluorescence staining, motile spermatozoa obtained after Percoll gradient centrifugation were spread on a slide and air-dried. The slides were treated with 0.1% Triton X-100, washed twice with TBS, followed by incubation with the primary antibody (1:500) for 60 min at room temperature. Following the washing steps with TBS, sections were exposed to swine anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) (Dako) for 60 min at room temperature and washed with TBS. The slides were subsequently counterstained with propidium iodide (PI) and mounted. Another slide stained with CDC25A antibody and its corresponding blocking peptide was used as a control. Labelled spermatozoa were examined and images recorded using a Leica TCS SP2 confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany). The images were visualized through two single-bandpass filters (488 nm for FITC and 543 nm for PI).

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. One microgram of RNA was treated with RNase-free DNase (Qiagen, Valencia, CA, USA) to remove contaminating genomic DNA. For complementary DNA (cDNA) synthesis, 12 µl aliquots of master mixture containing 2 µl (100 ng) of RNA, 1 µl of 500 ng/µl oligo(dT)12–18 primer (Gibco/BRL, Grand Island, NY, USA) and 9 µl of diethylpyrocarbonate-treated water were heated to 70°C for 10 min and put on ice. Reverse transcription reactions were performed in 20 µl volumes containing master mixture, 4 µl of 5x first-strand synthesis buffer, 0.1 M dithiothreitol, 10 mM 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 CDC25A 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. Because the CDC25A gene has been reported to have two transcript variants, the primer was designed to amplify a fragment in the common region of CDC25A (NM_001789 [GenBank] , 931–1133). The primer sequences were as follows: CDC25A, forward 5'-AGAT A GCAGTGAACCAGG-3' and reverse 5'-TGCATCGGTTGTCAAGG-3'; HMBS, forward 5'-AACGGCGGAAGAAAACAG-3' and reverse 5'-TCCAATCTTAGAGAGTGCA-3'. 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 reaction was 10 µl and contained 2 µl of cDNA template, 2 µl of primer mixture (final concentration: 2 µM), 4 mM MgCl2 and 1 µl of SYBR Green I. The RT–PCR conditions began with denaturation at 95°C for 7 min, followed by 40 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 performed 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 quantification 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 CDC25A in each testicular sample were normalized to the amount of HMBS mRNA.

Data analysis
The mRNA transcript levels of HMBS and transcript ratios of CDC25A 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 Dunn test. Pearson product moment correlation coefficients were calculated to determine the correlation between the CDC25A transcript ratios and the hormonal parameters. Receiver operating characteristic (ROC) curve analysis of the CDC25A transcript was used for distinguishing between patients with successful sperm retrieval and those without. Accuracy was measured as the area under the ROC curve. The threshold value was determined by Youden’s index, which equals per cent sensitivity plus per cent specificity – 100% (Skendzel and Youden, 1970Go). A P value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Patients
Of the 40 patients enrolled in this study, 10 were diagnosed with obstructive azoospermia and normal spermatogenesis, and 30 were diagnosed with non-obstructive azoospermia. The clinical characteristics of the study populations are summarized in Table I. Sperms were successfully retrieved from all 10 patients with obstructive azoospermia, either by microsurgical epididymal sperm aspiration (MESA) or by testicular sperm extraction (TESE). Testicular tissues were obtained by testicular biopsy (coinciding with MESA) in seven patients and by TESE in the other three patients. For 30 patients with non-obstructive azoospermia, sperms were successfully retrieved from all 15 patients with hypospermatogenesis, all three patients with maturation arrest at the spermatid stage and three of the 10 patients with Sertoli cell-only syndrome (SCOS).


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  		Table I. Clinical characteristics, testicular histologies, CDC25A expression and the results of sperm retrieval of the investigated patients

 
CDC25A protein in human testes and spermatozoa
In the human testis of normal spermatogenesis, high levels of CDC25A protein were found in the cytoplasm of meiotic spermatocyte and extended to elongated spermatid (Figure 1A and B). Weak staining of CDC25A can also be detected in Sertoli cells and spermatogonia (Figure 1C). These findings were consistent in testicular specimens from patients with hypospermatogenesis and maturation arrest at spermatid stage (Figure 1D and E). Of the two patients with maturation arrest at spermatocyte stage, one patient showed the presence of CDC25A protein (Figure 1F), whereas CDC25A was completely absent in the other patient (data not shown). Moderate to weak immunopositive reaction was found in 7 of 10 SCOS patients (Figure 1G). No signals were observed when the testicular specimens were stained with CDC25A antibody and its corresponding blocking peptide (Figure 1H).


Figure 1
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  		Figure 1. Immunohistochemistry of CDC25A in human testes. CDC25A was seen in testicular sections from normal spermatogenesis (A–C), hypospermatogenesis (D), maturation arrest at spermatid stage (E), maturation arrest at spermatocyte stage (F) and Sertoli cell-only syndrome (G). No staining was observed in tissue sections pre-incubated with CDC25A blocking peptide (H). Note that patient with strong CDC25A expression was detected from primary spermatocyte, round spermatid to elongated spermatid, a weaker but clear detectable signal was present in spermatogonia (C, arrow) and Sertoli cell (C, arrowhead). Scale bar = 200 µm (A, D–H), 100 µm (B) and 40 µm (C).

 
Western blot analysis revealed a band representing a protein of ~55 kDa, compatible with the predicted size of CDC25A protein in tissue extract (Figure 2A). Using antiserum pre-incubated with blocking peptide, we were unable to detect the protein band. Under a confocal microscope, positive FITC staining was observed in the tail and head of sperm, with faint staining in the nucleus region (Figure 2B). Control experiments by using the antibody pre-incubated with the blocking peptide revealed only a very weak background of fluorescent dye.


Figure 2
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  		Figure 2. (A) Western blot analysis of CDC25A. Proteins were extracted from human sperm and testis. A protein of ~55 kDa was detected in both testis and sperm extracts (upper zone). A polyclonal antibody to beta-actin was used as a positive control (lower zone). Lane 1: sperm protein pre-incubated with blocking peptide; lane 2: testis protein from a man with normal spermatogenesis; lane 3: sperm protein from a fertile man. (B) The localization of CDC25A protein on live human sperm. No signal was observed in sperm section pre-incubated with blocking peptide (a). Matched bright and dual-colour dark field confocal images (b–d) demonstrate that CDC25A protein is detected in the tail and head of sperm, with faint fluorescein isothiocyanate (FITC) signal in the nucleus region. Propidium iodide staining of chromatin is shown in red, FITC staining of CDC25A is shown in green, and (d) is the merged image. Scale bar = 5 µm.

 
mRNA transcript levels of CDC25A and HMBS in patients with different testicular histopathologies
Figure 3 shows the mRNA transcript levels of HMBS and CDC25A among the patients with different histopathologies. There was no significant difference in HMBS mRNA transcript levels between these groups (P = 0.627). Subdividing our patients into five groups according to the severity of testicular histopathology produced a progressive decrease in the CDC25A mRNA transcript ratios (CDC25A/HMBS) (P = 0.0009, Kruskal–Wallis test). Pairwise comparisons of the CDC25A mRNA transcript ratios among the five groups revealed that there were significant differences between the normal spermatogenesis group and the SCOS group, and the hypospermatogenesis group and the SCOS group (P < 0.01, Dunn test).


Figure 3
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  		Figure 3. Transcripts of hydroxymethylbilane synthase (HMBS) mRNA (A) and mRNA transcript ratios of CDC25A/HMBS (B) in different testicular histological groups. For HMBS, no significant difference was found among the groups (P = 0.627). For CDC25A, significant differences were found between the five groups (P = 0.0009, Kruskal–Wallis test). Histology abbreviations: HS, hypospermatogenesis; MA(spc), maturation arrest at spermatocyte stage; MA(spd), maturation arrest at spermatid stage; NR, normal spermatogenesis; SCOS, Sertoli cell-only syndrome.

 
Correlations between the CDC25A mRNA transcript ratios and hormonal parameters
Figure 4 shows the correlation between CDC25A mRNA transcript ratios and hormonal parameters, CDC25A mRNA transcript ratios and FSH levels (r = –0.332), LH levels (r = –0.334), prolactin levels (r = 0.089), and testosterone levels (r = 0.314) were low and categorized as not significant (P = 0.091, 0.089, 0.668 and 0.111, respectively).


Figure 4
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  		Figure 4. Correlation between the serum levels of FSH (A), LH (B), testosterone (C), prolactin (D) and the CDC25A/HMBS mRNA transcript ratio. The r value and significance value are shown on each graph.

 
mRNA transcript ratios of CDC25A and the results of sperm retrieval
The 40 patients were divided into two groups based on the presence or absence of mature sperm retrieved from MESA or TESE. The CDC25A mRNA transcript ratios ranged from 0.15 to 3.03 (0.97 ± 0.1) for 31 patients with successful sperm retrieval, and from 0.06 to 0.53 (0.28 ± 0.06) for 9 patients with failed sperm retrieval. A significant difference was noted between these two groups (P = 0.005, unpaired Student’s t-test). We hypothesize that there may be a threshold level of CDC25A mRNA transcripts required for the production of intratesticular spermatozoa. The ROC curve analysis of CDC25A mRNA transcript ratios indicates that the threshold that gave the maximum true-positive fraction (sensitivity) and false-positive fraction (1 – specificity) was 0.55 (Figure 5A and B). At this threshold value, the sensitivity and the specificity for predicting the presence of sperm in patients with spermatogenic failure were 84.4 and 100%, respectively. The calculated area under the curve was 0.944.


Figure 5
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  		Figure 5. Receiver operating characteristic curve of CDC25A mRNA transcripts (A). The fractions of true-positive results and false-positive results are shown. The calculated area under the curve was 0.944, and the 95% confidence interval was 0.664–1. This analysis showed that successful sperm retrieval by testicular sperm extraction (TESE) could be predicted with a sensitivity of 84.4% and specificity of 100% using a CDC25A mRNA transcript ratio threshold of 0.55 (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
In this study, we demonstrated the mRNA and protein levels for CDC25A in the testes of infertile men and, for the first time, showed that CDC25A protein is present in mature spermatozoa. The CDC25A protein is expressed mainly in spermatocyte, spermatid and spermatozoa; in addition, a lower intensity staining signal in Sertoli cells and spermatogonia was observed. The CDC25A protein is expressed in non-dividing, well-differentiated spermatozoa, suggesting that CDC25A may function not only in cell cycle events but also in the physiology of mature spermatozoa. The sperm tail exhibits a more intense signal than the head region. The sperm tail consists of helically arranged mitochondria, dense outer fibres and axonemes. A positive immunostaining signal in the tail of the spermatozoon might suggest an association of CDC25A protein with sperm motility. Moreover, the presence of the immunostaining signal in the head region that contains specialized enzymes necessary for the penetration of the oocyte suggests a role of CDC25A in sperm–oocyte fusion. It is also possible that CDC25A protein is located in the cytosol of spermatozoa and not associated with specific organelles. The roles of CDC25A protein in sperm motility and in the sperm–oocyte interaction await further investigation.

In this study, we demonstrated that CDC25A expressions were decreased in patients with spermatogenic failure at both RNA and protein levels (Table I). Significantly reduced CDC25A mRNA transcript levels were found in SCOS patients and in patients with maturation arrest at spermatocyte stage. Of the five patients who displayed maturation arrest, patients with maturation arrest at spermatid stage displayed higher CDC25A mRNA transcript levels than patients with maturation arrest at spermatocyte stage. Collectively, these results in human testes are in agreement with the biological role of CDC25A as an essential regulator of cell cycle progression or cell proliferation. Low CDC25A expression in infertile men is a result of reduced spermatogenic cycles within the seminiferous epithelium. Given that CDC25 phosphatase contributes to the hyperphosphorylation of MPF (Galaktionov and Beach, 1991Go; Jinno et al., 1994Go) and CDC25A is essential for the completion of meiosis (Luetjens et al., 2004Go; Wistuba et al., 2006Go), we can assume that low CDC25A phosphatase results in inhibitory activity of MPF, blocks the germ cell entering meiosis and/or mitosis and leads to germ cell arrest and degeneration.

Our results showed that the CDC25A transcripts did not have a significant correlation with hormonal parameters, indicating that CDC25A is not directly involved in the hormonal regulation pathway of spermatogenesis. The low expression of CDC25A transcript is usually commensurate with low protein levels, suggesting that the CDC25A content in human testis is likely regulated at the mRNA level. Factors upstream of CDC25A may be crucial for the CDC25A mRNA expression. As mentioned above, Boule could influence Twine levels through direct binding to the twine mRNA (Maines and Wasserman, 1999Go), and mouse Dazl has been shown to bind directly to the 3' UTR of the Cdc25A mRNA (Jiao et al., 2002Go), suggesting that DAZ gene family could regulate the CDC25A mRNA. In addition, Cdc25A might accumulate as a result of E2F-1- and c-myc-mediated transcriptional activation, and this increase in RNA and protein levels stimulates the cells to enter the cell cycle (Galaktionov et al., 1996Go; Blomberg and Hoffmann, 1999Go; Vigo et al., 1999Go). Therefore, the low expression of CDC25A in infertile men may be caused by mutation/polymorphism affecting DAZ gene family, E2F-1 and c-myc in the metabolic cascade or by other factors interfering with the mRNA stability or production. Further studies will be necessary to determine whether upstream defects are involved in our patients’ conditions.

Given that the expression of CDC25A is highly associated with the spermatogenic failure, CDC25A protein generally parallels its mRNA level and CDC25A protein is detectable in mature spermatozoa, we have speculated that CDC25A mRNA transcripts may be correlated with the presence of testicular sperm. In this study, higher levels of CDC25A mRNA transcripts have been shown to be correlated with the success of TESE in azoospermic men. Using the mRNA transcript threshold value of 0.55 to predict the success of TESE, the specificity and the sensitivity were 100 and 84%, respectively, and the calculated area under the ROC curve was >0.9. In our previous study, we have showed that CDC25A transcript levels were significantly decreased in men with spermatogenic failure, but the transcript levels did not differ between azoospermic men with and without successful sperm retrieval (Lin et al., 2006Go). The difference in the predictive role of CDC25A transcript levels between the two studies could be the result of different primer design and patient population. Two transcript variants (variant 1 and variant 2) encoding different isoforms have been found for CDC25A gene, and variant 2 lacks an alternate in-frame exon (6) when compared with variant 1. In our previous study, the CDC25A primer pair used could only detect variant 1, whereas in this study, a common primer pair was designed to amplify the two variants.

From the literature, many molecular markers, including cyclin A1, protamine and BOULE, have been shown to correlate with the sperm production or testicular histopathology (Schrader et al., 2002Go; Steger et al., 2003Go; Wolgemuth et al., 2004Go; Lin et al., 2005Go). Although the clinical significance of these markers in predicting the success of TESE has yet to be determined, it will be of great value to characterize those markers that could serve as targets for the development of a novel molecular classification system to supplement histological diagnosis or help predict the result of sperm retrieval prior to TESE-ICSI.

In summary, we have demonstrated the expression profiling of CDC25A in the testes of infertile men, and for the first time, we have also ensured the presence of CDC25A protein in human spermatozoa. Decreased CDC25A expressions are associated with spermatogenic failure and failed sperm retrieval in infertile men. Further studies have to be performed to explore the biological functions of CDC25A in human spermatozoa.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
This study is supported by grants from the National Cheng Kung University Hospital (NCKUH-95–56) and The Tainan Phoenix Urological Research and Education Foundation (TPUREF9501).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Kuo PL, Wang ST, Lin YM, Lin YH, Teng YN, Hsu CC. (2004) Expression profiles of the DAZ gene family in human testis with and without spermatogenic failure. Fertil Steril 81:1034–1040.[CrossRef][Web of Science][Medline]

Lee TH, Solomon MJ, Mumby MC, Kirschner MW. (1991) INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell 64:415–423.[CrossRef][Web of Science][Medline]

Lin YM, Lin YH, Teng YN, Hsu CC, Lin JSN, Kuo PL. (2002) Gene-based screening for Y chromosome deletions in Taiwanese men presenting with spermatogenic failure. Fertil Steril 77:897–903.[CrossRef][Web of Science][Medline]

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Submitted on January 24, 2006; resubmitted on April 8, 2006; accepted on April 19, 2006.


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