Departments of 1 Urology, 2 Obstetrics & Gynecology and 3 Institute of Basic Medical Science, National Cheng Kung University, College of Medicine, Tainan, Taiwan and Department of 4 Early Childhood Education and Nursery, Chia Nan University of Pharmacy and Science, Tainan, Taiwan
5 To whom correspondence should be addressed at: Department of Urology, National Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan, Taiwan 704. Email: linym{at}mail.ncku.edu.tw
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Abstract |
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Key words: azoospermia/BOULE/testis
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Introduction |
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The human BOULE gene, a germ cell-specific cell cycle regulator, was recently identified and mapped to chromosome 2 (Xu et al., 2001). BOULE belongs to the DAZ (Deleted in AZoospermia) gene family, which consists of DAZ on the Y chromosome and DAZL (DAZ-Like) on chromosome 3. It is believed that the BOULE gene is highly conserved from flies to humans. Human BOULE and fly boule share 80% similarity in RNA binding domains and 42% similarity in protein sequence (Xu et al., 2001
). In the fly, boule is required for completion of meiosis in male germ cells (Castrillon et al., 1993
; Eberhart et al., 1996
). Mutation of boule resulted in germ cell meiotic arrest and infertility; in addition, human BOULE could rescue this meiotic defect in infertile flies (Xu et al., 2003
). Therefore, the human BOULE is considered to have a similar function to the fly boule, as a meiosis regulator.
In humans, BOULE is expressed exclusively in the male germline, and the BOULE protein can be detected mainly in the cytoplasm of the meiotic spermatocytes (Xu et al., 2001; Luetjens et al., 2004
). The unique BOULE protein expression pattern strongly suggests that human BOULE is involved in the progression of cell cycles during meiosis. As meiosis is the key process for haploid germ cell production, it is tempting to speculate that the amount of BOULE transcripts correlates with the amount of sperm production as well as testicular phenotypes. To gain a better understanding of the role of BOULE in human spermatogenesis, we determined the BOULE mRNA transcript levels in the testes of azoospermic patients, and evaluated the relationship between BOULE mRNA transcript levels and patients' testicular phenotypes, clinical parameters as well as the results of sperm retrieval.
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Materials and methods |
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Testicular samples
For paternity purposes, the patients underwent sperm retrieval, either by microsurgical epididymal sperm aspiration (MESA) or TESE, and ICSI. Before sperm retrieval, these patients were asked to provide a small piece of testicular tissue (diameter 5 mm) for further study. For men undergoing MESA, an additional testicular incision was performed for sampling on the same side as the MESA procedure. For men undergoing TESE, a tiny sample was obtained from the pooling of testicular tissues. One third of the testicular tissue volume obtained 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 diagnosis of the testicular histopathology was confirmed by two specialists, and was categorized according to the most advanced pattern of spermatogenesis present. The remaining two thirds of the tissue volume was cryopreserved for RNA extraction. Following sperm retrieval, positive sperm retrieval was defined as the presence of spermatozoa on wet preparation of the testicular tissues under an inverted microscopic examination (400x magnification).
Primer design
Primers specific for the human BOULE gene and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were designed. The nucleotide sequences spanning different exons were identified in GenBank using accession number BC023632 for GAPDH and accession number AF272858 for BOULE (National Center for Biotechnology Information). Another two sets of competitive reverse primers for BOULE and GAPDH were designed, based on the same nucleotide sequences and an additional short sequence upstream of the target cDNA incorporated into the 5' ends of the original reverse primers. The primer sequences, positions on the cDNAs, and expected sizes of the PCR products are listed in Table I.
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Quantitative competitive polymerase chain reaction (QCPCR)
For QCPCR, we synthesized wild and competitor RNA templates for internal standards. For the synthesis of internal standards, RNA extracted from the testicular tissue with normal spermatogenesis was subjected to RTPCR. The products were separated by polyacrylamide gel electrophoresis using 1x TBE buffer (90 mM TrisHCl, 90 mM boric acid, 1 mM EDTA, pH 8) at 110 V for 3050 min. After initial confirmation by gel electrophoresis, competitor and wild PCR products were purified using the ConcertTM Rapid PCR Purification System, (Gibco/BRL) and subcloned into a pT7Blue T-vector (Novagen, Madison, WI). The inserts were confirmed by DNA sequencing using an automatic sequencer (ABI 377, Applied Biosystems/PE, Foster City, CA). The cDNA products were quantified by measuring the absorbance at 260 nm. To determine the equivalence of testicular cDNA and competitor cDNA, serial dilutions of competitor cDNA were co-amplified with testicular cDNA from tissue exhibiting normal spermatogenesis. The point at which the two lines crossed indicated the amount of competitor cDNA that should be added for subsequent standard curve construction and QCPCR reaction. To produce the standard curve, a fixed amount of competitor cDNA was co-amplified with serial dilutions of wild cDNA, and the logarithm of the ratio of wild to competitor cDNA was plotted against the logarithm of the initial amounts of wild cDNA. The standard curves were highly reproducible and the R2 values for the GAPDH and BOULE standard curves were 0.9966 and 0.993, respectively (Figure 1).
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Data analysis
We used GraphPad Prism 4 statistical software (GraphPad Software, San Diego, CA) for data analysis. The copy numbers of GAPDH and transcript ratios of BOULE in five histological groups (normal spermatogenesis, hypospermatogenesis, maturation arrest at spermatid stage, maturation arrest at spermatocyte stage and Sertoli cell-only syndrome) were presented as the mean±SEM. Transcript differences in patients with normal spermatogenesis and patients with spermatogenic failure (i.e. hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome) were analyzed using an unpaired Student's t-test. Transcript differences or transcript ratios in patients with varying degrees of spermatogenic failure were analyzed using the KruskalWallis test, and multiple pairwise comparisons were performed using Dunnett's test. Pearson product moment correlation coefficients were calculated to determine the correlation between BOULE transcript ratios and clinical parameters, i.e. serum FSH, LH, prolactin and testosterone level. Receiver operating characteristic (ROC) curve analysis of BOULE transcript ratios was used to determine the cut-off value that maximized the sensitivity and specificity for distinguishing between patients with successful sperm retrieval from those without. A P-value <0.05 was considered significant. Where appropriate, Bonferroni's method was used to correct significance levels for multiple testing of data.
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Results |
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BOULE mRNA transcript ratios and hormonal parameters
Figure 3 shows the correlation between BOULE mRNA transcript ratios and hormonal parameters. The correlation between BOULE mRNA transcript ratios and FSH levels (r=0.267), BOULE mRNA transcript ratios and LH levels (r=0.014), BOULE mRNA transcript ratios and prolactin levels (r=0.203), and BOULE mRNA transcript ratios and testosterone levels (r=0.155) were low and categorized as not significant (P=0.104, 0.475, 0.341, and 0.234, respectively).
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Discussion |
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It is also possible that meiosis can be regulated by other factors acting upstream or downstream of BOULE. In Drosophia, Cdc25 is considered to be a general target for BOULE protein (Xu et al., 2003), and sa (spermatocyte arrest) and mia (meiosis I arrest) might act upstream of boule (Maines and Wasserman, 1999
). A mutation in Cdc25 completely blocks meiosis in the male fly (Courtot et al., 1992
), and mutations in sa and mia have been shown to result in the failure of BOULE protein to accumulate normally in the fly (Maines and Wasserman, 1999
). Although, to date, human homologs of sa and mia have not been identified, further studies will be performed to identify the molecules upstream of the human BOULECDC25CDC2/cyclin A (B) pathway, and to determine whether defects in components upstream or downstream of BOULE are associated with specific patient phenotypes.
In this study, BOULE mRNA transcripts were significantly decreased in patients with spermatogenic failure, especially in the Sertoli cell-only group. This result is not surprising given the germ-line specificity of BOULE. Higher BOULE mRNA transcript levels reflect increased numbers of germ cells entering the meiosis processes, and lower transcript levels may simply reflect a general loss of germ cells. However, it is interesting to note that BOULE mRNA transcripts were also detectable in all Sertoli cell-only specimens. Possible explanations for this include the expression of BOULE in the nucleus or cytoplasm of some type of non-germ lineage in the testis. It is possible that the BOULE protein level is too low to be detected by immunostaining or it is difficult to identify due to its nuclear location. A similar phenomenon occurred with other members of DAZ gene family. DAZ and DAZL mRNAs have been detected by RTPCR in patients with a diagnosis of Sertoli cell-only syndrome (Kuo et al., 2004). To resolve this question, laser-capture microdissection, in combination with RTPCR or western blotting analysis, may be useful. A second explanation for the detection of BOULE mRNA transcripts in Sertoli cell-only specimens is that these specimens may contain some germ cell foci, i.e. incomplete Sertoli cell-only (Devroey et al., 1995
; Silber et al., 1996
). There have been many reports of patients with a Sertoli cell-only diagnosis who have undergone successful TESE (Silber et al., 1995
; Tournaye et al., 1997
). We believe that our histological diagnosis of Sertoli cell-only is correct because there were generally >100 seminiferous tubule cross-sections available for examination in each patient, the advanced category system was applied, and the histological diagnoses were confirmed by two experienced specialists. Our results also indicate the limited predictability of histological diagnosis at TESE. A third explanation for the detectable BOULE expression in these patients might be due to sample contamination. However, we believe that the presence of BOULE mRNA transcripts is not due to tissue contamination because BOULE mRNA transcripts were invariably detected in all 14 Sertoli cell-only specimens and DNase was routinely added before each cDNA synthesis.
In the present study, BOULE mRNA transcripts have been shown to correlate with the results of TESE. Similar predictive value tests have been evaluated for the other members of the DAZ gene family (DAZ and DAZL), but less predictive value was noted. Using a certain mRNA transcript level as a cut-off value, the sensitivity and specificity were 82.8% and 100%, respectively, for DAZ (Kuo et al., 2004), and 78.1% and 94%, respectively, for DAZL (our unpublished data). These divergent predictive values for the DAZ family might be explained by their distinct expression patterns. DAZL and DAZ are expressed mainly from germ-line stem cells to meiotic spermatocytes, whereas BOULE protein exists in the cytoplasm of pachytene spermatocytes, and persists through meiosis to round spermatids (Xu et al., 2001
). The timeline for BOULE expression may be the reason for its higher predictive value for sperm production.
Although a number of studies have attempted to test the predictive powers of variable parameters, clinically, testicular samples for quantitative and/or qualitative evaluations are currently used to predict the presence of sperm at TESE. Qualitative measurement based on the most advanced pattern of spermatogenesis was 5258% accurate (Su et al., 1999; Seo and Ko, 2001
), and a positive result for quantitative measurement of mature spermatids predicted an 85% chance for subsequent success with TESE (Silber et al., 1997
). In this study, BOULE mRNA transcripts have been shown to yield a predictive power of 100%, therefore, we believe that the measurement of BOULE mRNA transcripts may potentially be useful for predicting the presence of sperm in testis. Conversely, it is also noted that the sample size is limited in the present study, and the possibility exists that more samples may reduce the 100% predictive value reported.
Given that diagnostic testicular biopsy is less invasive than TESE, we recommend diagnostic testicular biopsy before attempting TESE for patients with non-obstructive azoospermia. A tiny piece of testicular tissue is sufficient for both histopathological examination and QCPCR to detect BOULE mRNA transcripts. If we fail to identify spermatozoa, despite detecting high BOULE transcript levels from a tiny piece of biopsied sample, we hypothesize that it may be worthwhile to try TESE for sperm retrieval. Alternatively, if we fail to identify spermatozoa and the BOULE transcript levels are low in the biopsied sample, it may not be worthwhile to try more invasive TESE procedures. The BOULE transcript levels would also provide technicians in the ART laboratories with useful information to guide further attempts to search for sperm. This information will be highly valuable for counselling infertile couples before TESEICSI procedures. It would help avoid unnecessary, invasive TESE for men, and decrease the risks of potential complications from assisted reproductive techniques for women. Furthermore, we believe that a less invasive, more cost-effective, and outpatient-based method of diagnostic testicular sampling is necessary. A study is now ongoing to test the feasibility of applying this BOULE mRNA transcript assay to the testicular sample from a fine needle aspirate.
Because the testicular histology may be highly variable throughout the testis, it is possible that the biopsied sample may contain foci without germ cells. In this case, the BOULE transcript levels would be low. The same tissue may also contain other foci with many germ cells, which would give rise to high BOULE transcript levels. Although our QCPCR assay has been shown to be highly predictable for successful sperm retrieval, the reliability of BOULE transcript levels obtained from diagnostic testicular biopsy should be further evaluated in future.
In summary, we have determined that BOULE mRNA transcript levels are significantly decreased in patients with spermatogenic failure and these mRNA transcript levels correlate with the success of sperm retrieval. Because BOULE is an important regulator of the human meiotic cell cycle, we believe that the level of BOULE mRNA transcript in the testis of azoospermic patients is informative. Using a cut-off value of 0.5 for the BOULE mRNA transcript ratio, the predictability for the success of sperm retrieval is ideal. Therefore, we recommend a prior diagnostic testicular biopsy, evaluating both testicular histology and BOULE mRNA transcript levels, for all couples attempting TESEICSI.
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Acknowledgements |
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References |
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Submitted on July 28, 2004; accepted on November 11, 2004.