Hum. Reprod. Advance Access originally published online on August 26, 2006
Human Reproduction 2007 22(1):151-158; doi:10.1093/humrep/del341
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Expression profile of AZF genes in testicular biopsies of azoospermic men
1 Institute for the Study of Fertility, Lis Maternity Hospital and 2 Institute of Pathology, Tel Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
3 To whom correspondence should be addressed at: Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman Street, Tel Aviv 64239, Israel. E-mail: ser{at}tasmc.health.gov.il
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Abstract |
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BACKGROUND: The Y-chromosome AZF regions include genes whose functions and specific roles in spermatogenesis have not been fully clarified. This study investigated the expression of several AZF (USP9Y, DDX3Y/DDX3Yt1, EIF1AY and PRY) and USP9X transcripts in testicular biopsies of 89 azoospermic men who had been classified by histology and cytology assessments. METHODS: Expression was analysed by RT–PCR, and some biopsies were evaluated by multiplex RT–PCR. Quantitative PCR was performed in some biopsies to determine the ratio of the testis-specific transcript DDX3Yt1 to the total DDX3Y transcription. RESULTS: The expression of USP9Y, USP9X and DDX3Y was found in all the specimens tested, whereas DDX3Yt1 expression was diminished or undetectable in several biopsies with impaired spermatogenesis. EIF1AY was detected in all except two of the specimens. Noteworthy, PRY expression was detected mainly in biopsies with germ cells, and this association was significant (P < 0.001). An identical expression profile was obtained by either single or multiplex RT–PCR. CONCLUSIONS: These findings suggest that PRY is usually expressed in germ cells, whereas the other transcripts are also expressed in testicular somatic cells. The absence of EIF1AY expression might sporadically contribute to azoospermia. The decreased ratio of DDX3Yt1/DDX3Y transcript in impaired spermatogenesis suggests that the DDX3Yt1 transcript is under-expressed in impaired spermatogenesis. The findings contribute to the search and selection of the most valuable gene markers potentially useful as additional tools for predicting complete spermatogenesis by multiplex expression analysis.
Key words: AZF gene expression/azoospermia/infertility/markers of spermatogenesis/testicular expression
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Introduction |
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Y-Chromosome de-novo microdeletions have been identified in 7–13% of men with azoospermia and severe oligozoospermia (Reijo et al., 1995
, 1996; Pryor et al., 1997; Kleiman et al., 1999). The microdeleted regions were historically named AZFa, AZFb and AZFc (Vogt et al., 1996). Now, following the complete characterization of the Y-chromosome structure, the AZFb-deleted region is more accurately called P5/proximal-P1 deletion (Repping et al., 2002). The AZF regions include genes that are expressed during spermatogenesis and encode proteins necessary for specific stages of spermatogenesis as well as for maintaining the general housekeeping functions of the cells involved (Lahn and Page, 1997). The Dead Box Y gene (DBY, recently renamed DDX3Y) encodes a putative RNA helicase. The ubiquitin-specific protease 9Y gene (USP9Y, previously known as DFFRY) encodes a protease with activity specific to ubiquitin that is involved in the regulation of protein metabolism (protein turnover) (Lee et al., 2003; Ginalski et al., 2004). Both genes are located at the AZFa region and have homologous genes on the X chromosome. Lahn and Page (1997) detected two DDX3Y transcripts differentially expressed: one ubiquitously and the other (DDX3Yt1) testis specific. Both the translation Initiation Factor 1A Y isoform gene (EIF1AY) and the RNA binding motif (RBM) are found on AZFb region. EIF1AY encodes an essential translation factor. The PTP-BL-related Y (PRY) family of genes is mapped to AZFb and AZFc regions and encodes proteins proposed to be involved in apoptosis (Stouffs et al., 2004). RBM and deleted-in-azoospermia (DAZ) genes encode RNA-binding proteins that are exclusively expressed in germ cells (Ma et al., 1993; Reijo et al., 1995). In addition to DAZ, chromodomain Y genes (CDY1) are found on the AZFc region and encode a protein involved in DNA remodelling that can acetylate histone H4 in vitro (Lahn et al., 2002). Alteration in gene expression patterns often accompanies various disease states. Thus, understanding the expression of genes involved in spermatogenesis in fertile and infertile men will assist in understanding spermatogenic failure. Multiplex RT–PCR can be applied to analyse the expression of a modest number of genes, making it potentially useful for testing gene expression for diagnostic purposes.
We had earlier analysed the testicular expression of CDY family, DAZ and RBM transcripts and found that CDY1 transcripts significantly correlate with complete spermatogenesis (Kleiman et al., 2001, 2003). In this study, to characterize the expression of additional AZF genes (USP9Y and its homologous gene on the X-chromosome USP9X, DDX3Y/DDX3Yt1, EIF1AY and PRY), testicular messenger RNA was analysed in men with normal (obstructive azoospermia) and impaired spermatogenesis by RT–PCR.
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Materials and methods |
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The study cohort
Eighty-nine azoospermic men who underwent testicular sperm extraction and consented to undergo genetic evaluation were included in this study, which was approved by the local Institutional Review Board committee in accordance with the Helsinki Declaration of 1975. No microdeletion was detected in 85 men who underwent Y-chromosome microdeletion test. Four men—two with normal spermatogenesis, one with hypospermatogenesis and one with Sertoli cell-only—did not consent to be tested for Y-chromosome deletions. The study cohort included three additional men with AZF microdeletions as controls for validation of the specificity of the PCR—one man with AZFc microdeletion, one with AZFb-c (P5/terminal) microdeletion and one with AZFa-c microdeletion. Men with an abnormal karyotype were excluded from the expression study.
Testicular tissue evaluation and analysis
Testicular tissue was histologically and cytologically evaluated as previously reported (Kleiman et al., 2001, 2003). The most advanced spermatogenic cell that was identified by the combined evaluation determined the specimen definition. Accordingly, biopsies were sorted into four groups: normal spermatogenesis (n = 22), hypospermatogenesis (n = 33), complete spermatocyte maturation arrest (n = 10) and Sertoli cell-only (n = 24).
Paraffin-embedded sections were stained with haematoxylin and eosin, and the total number of mature spermatids was counted in 20 seminiferous tubules. The mean value of mature spermatids per tubule was calculated.
Genetic evaluation
The expression of the transcripts USP9Y, USP9X, DDX3Y, DDX3Yt1, EIF1AY, RBM, DAZ, PRY, CDY2, CDY1 and 
-ACTIN was assessed in testicular tissue by reverse transcriptase with poly-dT oligonucleotides followed by single-gene PCR (single RT–PCR) and/or multiplex gene amplification (multiplex RT–PCR). The expression of RBM, DAZ, CDY2 and CDY1 in 84 of the 89 analysed specimens was reported earlier (Kleiman et al., 2003). RNA from men with detected Y-chromosome AZF microdeletions was assessed to confirm the primer-specific amplification of the tested Y-chromosome transcripts. We did not expect to find specific transcripts included in the deletions.
The expression of -ACTIN was evaluated to have an internal control for the RNA isolation and efficiency of the RT–PCR. DAZ, RBM and CDY2 expressions were indicative of the presence of germ cells (Kleiman et al., 2001), and CDY1 correlated with complete spermatogenesis.
Oligonucleotide primer sets were generally designed to amplify sequences up to 1000 bp from the polyA tail to diminish potential variations resulting from the efficiency of reverse transcriptase processing in the individual samples. The only exception was the set for USP9Y/X amplification. Their specificity was tested by in silico PCR (http://genome.ucsc.edu/cgi-bin/hgPcr?db=hg16) to rule out a possible similar or retroposed sequence that might be amplified. The oligonucleotide primer sets that were used for DDX3Yt1 (accession number G49470
[GenBank]
), CDY2, CDY1 (minor + short), DAZ, RBM and -ACTIN expression were described elsewhere (Foresta et al., 2000; Kleiman et al., 2001, 2003). The unpublished oligonucleotide primer sets designated for the expression studies are presented in Table I. Primer sets for USP9Y/X, DDX3Y (which amplify both the ubiquitous and the testis-specific transcripts), DDX3Yt1 (which amplify the testis-specific transcript), EIF1AY, PRY1 (which amplify both the short and the long transcripts), CDY1 (minor + short), DAZ and RBM transcripts amplify size-specific PCR products for complementary DNA (cDNA). No genomic DNA (gDNA) and cDNA of DDX3Y amplification could be observed with DDX3Yt1 primers because the reverse primer included the polyA tail and was homologous to the DDX3Y sequence in only the first 13 of the 20 base pairs. In addition, DDX3Yt1 annealing was performed at 65°C. Because the same PCR product size was obtained for cDNA and gDNA with -ACTIN and CDY2-1 primers, they were tested in each case with and without the RT step to detect any gDNA contamination. The PCR was repeated twice more whenever there were exceptional results.
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Direct sequencing of agarose gel-purified USP9Y/X, DDX3Y, DDX3Y-t1 and both PRY PCR products was performed with the Thermo Sequenase kit (Amersham, Cleveland, USA) to confirm the specificity of the amplified fragment. PCR products were purified with the High Pure PCR Product Purification kit (Roche, Mannhein, Germany).
Multiplex RT–PCR
Two PCR mixes were established: mix I included -ACTIN, CDY1 (minor + short) and EIF1AY and mix II included -ACTIN, DAZ, RBM and PRY1 (Figure 1). The reliability of the multiplex amplification was tested in 24 RT samples from the four histocytological groups previously analysed by single RT–PCR. Eight additional samples were analysed for the first time. Whenever gene expression was not detected by multiplex RT–PCR, the result was reconfirmed by single RT–PCR. Based on previous (Kleiman et al., 2001, 2003) and current expression analyses, markers that were expressed ubiquitously (-ACTIN and EIF1AY), in germ cells (RBM, DAZ and PRY) and only in spermatids (CDY1) were chosen to be included in the mixes. A reliable identical expression profile was obtained by both methods, and it was in concordance with the testicular histological and cytological evaluations. Because the long transcript of PRY1 was not reliably amplified in the multiplex mix, only the PRY1 short transcript was taken into account. Only in two specimens, identified as Sertoli cell-only, was a disparity in PRY expression detected by multiplex RT–PCR and single RT–PCR. PRY was negative by the multiplex methodology and positive by single RT–PCR.
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Quantitative PCR
An equal amount of total RNA (0.7 µg) was taken for the RT step. RT samples were diluted 1:3 before PCR preparation to prevent the inhibition of the reaction. Amplification was followed by the increase of fluorescence with SYBR Green I dye in a four-step PCR program (denaturation at 95°C, annealing at 65°C or 62°C, extension at 72°C and fluorescence assessment at 87 or 81°C for DDX3Yt1 or DDX3Y, respectively). SYBR Green I dye is a double-strain DNA dye that increases over 100-fold in fluorescence on binding. Each assay included a standard curve of four serial dilutions of a long RT–PCR product of known concentration as a PCR template for extrapolation and determination of either total DDX3Y or DDX3Yt1 transcript concentration. This template was a cleaned RT–PCR product obtained by using DDX3Y-forward primer and DDX3Yt1-reverse primer. None of the PCR transcript products had a secondary structure that could affect the PCR amplification efficiency (assessed by MFOLD: http://www.bioinfo.rpi.edu/applications/mfold/old/dna/form1.cgi). Each sample was assessed in duplicate, and the mean was calculated. The negative control was RNA from female placenta. The real-time PCR LightCycler System (Roche) monitored the cycle-by-cycle increase of fluorescence and calculated the concentration of PCR products by the second-derivative method. Similar slopes were observed in DDX3Y and DDX3Yt1 amplifications (slope values: –3.59 to –3.580 and –3.63 to –3.769, respectively). The ratio of DDX3Yt1/DDX3Y transcripts was computed.
Statistical analysis
Pearson’s chi-square test was performed to assess the significance of the relationship between the expression of the transcripts in the study groups. Fisher’s exact test was applied for assessment of the association between the expression of the transcripts and the presence of either germ cells or sperm cells. Kruskal–Wallis test assessed the significance of the difference in the ratio of DDX3Yt1/DDX3Y total gene expression among the groups. Mann–Whitney test assessed the significance of the difference in the ratio of DDX3Yt1/DDX3Y gene expression between the normal and the impaired groups. All statistics were performed at the Statistical Department of Tel Aviv University.
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Results |
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Testicular expression of AZF-related transcripts was assessed in biopsies from 89 idiopathic azoospermic men divided into four groups according to their testicular findings (see Materials and Methods).
Expression of AZFa genes
The sequence analysis of PCR products that were obtained with two different primer sets supposed to be specific for USP9Y also co-amplified USP9X. The final identification of each of the two homologous genes was feasible based on the presence of an NspI restriction site at base pair 6426 of the USP9Y transcript, which did not exist at USP9X (accession number AF000986
[GenBank]
and NM_004652, respectively) (Figure 2). Both genes were expressed in all the testicular specimens analysed by non-quantitative RT–PCR (Figures 3 and 4; Table II).
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Two bands were observed by PCR amplification with primers for DDX3Y (Figure 3). The sequence analysis of both PCR products revealed that the strong band corresponded to the reported DDX3Y sequence (AF000984 [GenBank] and AF000985 [GenBank] ) and that the faint one was probably an incompletely spliced or unreported DDX3Y transcript that included intron 9 but not introns 10 and 11. Similar to USP9Y, DDX3Y transcript was detected in all the testicular specimens (Figure 4; Table II). The testis-specific polyadenylated transcript DDX3Yt1, however, was not ubiquitously expressed, and it was detected at variable frequencies in the different groups: 100, 73, 75 and 62% in normal spermatogenesis, hypospermatogenesis, complete spermatocyte maturation arrest and Sertoli cell-only, respectively (Figure 4). The association between DDX3Yt1 expression and complete spermatogenesis (normal and hypo-spermatogenesis groups) and the presence of germ cells (normal, hypo-spermatogenesis and spermatocyte maturation arrest groups) were not significant (P = 0.098 and P = 0.125, respectively, Fisher’s exact test). DDX3Yt1 expression was not tested in 17 specimens in which no further RNA was available (Table II).
Among the AZF-deleted specimens, the AZFa-c was the one that did not express all the AZFa transcripts (USP9Y, DDX3Y and DDX3Yt1) (Table III). The DDX3Yt1 transcript was also not detected in the AZFb-c specimen with spermatocyte maturation arrest, as observed in 25% of the specimens with similar histological and cytological findings. To improve our understanding of the variable expression of DDX3Yt1, we measured the level of expression of DDX3Yt1 with reference to the total DDX3Y expression. The expression ratio of DDX3Yt1/DDX3Y was evaluated by quantitative PCR in 14 specimens (Table IV). The relative amount of DDX3Yt1 transcript was 16–23% of the total DDX3Y transcripts in men with normal spermatogenesis. In contrast, the DDX3Yt1 transcript comprised a maximum of 12% of the total DDX3Y transcript in specimens with impaired spermatogenesis. Differences in the number of mature spermatids per tubule were significant among the normal, hypospermatogenesis and Sertoli cell-only groups (P = 0.005, Kruskal–Wallis test). The only specimen with complete maturation arrest was not included in the statistical calculations. Significant differences in the DDX3Yt1/DDX3Y expression ratio were found between the group with normal spermatogenesis and the two groups with impaired spermatogenesis (hypospermatogenesis and Sertoli cell-only) (Table IV).
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Expression of AZFb and AZFc genes
EIF1AY transcript was detected ubiquitously in almost all 89 testicular specimens (Figures 3 and 4): the exceptions were two specimens—one with hypospermatogenesis and the other with Sertoli cell-only (Table II). The absence of expression was not due to a genomic deletion because none was observed in these two specimens by checking with the sequence tagged site markers, sY136, sY134 and sY143, that are mapped 255 kb, 246 kb proximal and 176 kb distal to the gene, and M274_M275 and SHGC-36325 mapped at the 5' and 3' end of EIF1AY gene, respectively.
The primer set of PRY amplified both reported alternative spliced transcripts (GI:22770597 and GI:22507416). Accordingly, the dual expression of both transcripts was observed in almost all cases: there were 14 (16%) in which only the short transcript was expressed (Table II). These 14 specimens were mainly spread among the three groups with impaired spermatogenesis. Neither of the PRY transcripts was expressed in 23 of the 67 specimens with impaired spermatogenesis that included the two without EIF1AY expression. For statistical calculations, the expression was considered positive if either of the PRY transcripts was detected. A highly significant correlation was observed among the expression of PRY transcripts and the four groups of biopsies (P < 0.001, Pearson’s chi–square). The association was highly significant between PRY expression and the presence of germ cells or sperm cells (P < 0.001, Fisher’s exact test). Nevertheless, PRY transcripts were found in 5 of the 24 specimens with Sertoli cell-only. The positive expression of three of them was observed by both multiplex and single RT–PCR. In the other two specimens, PRY expression was observed only in single RT–PCR but not in multiplex RT–PCR.
EIF1AY and PRY gene expression was not detected in AZFb-deleted specimens (Table III).
DAZ and RBM expressions were consistently detected in all the men with germ cells (normal spermatogenesis, hypospermatogenesis and spermatocyte maturation arrest), except in five whose histology showed Sertoli cell-only, although mature sperm cells were cytologically identified in other locations (Table II).
CDY1 expression was observed in almost all men with sperm cells (normal spermatogenesis and hypospermatogenesis), with the exception of six whose histology showed Sertoli cell-only and four whose histology showed most of the tubules with Sertoli cell-only and a few with only some spermatocyte, although mature sperm cells were cytologically identified in other locations (Table II).
Among the four men not screened for Y-chromosome microdeletions, the three with complete spermatogenesis expressed all the genes tested and the one with Sertoli cell-only did not express any of the RBM/DAZ markers.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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This study was conducted to better understand the role of the AZF genes and their transcripts in the spermatogenesis process. For this purpose, the expression of AZF-related transcripts (USP9, DDX3Y, DDX3t1, EIF1AY and PRY) in specimens with normal and impaired spermatogenesis was studied. This approach had already been successfully applied for the study of the multiple transcripts of the CDY family of genes and the DAZ and RBM genes (Kleiman et al., 2001
, 2003). Changes in gene expression at the tissue level can reflect changes in the concentration of its messenger RNA in a specific cell type, as well as changes in volume of specific cells and changes in the cell-type composition.
The USP9Y gene and its homologous gene USP9X were previously reported to be expressed ubiquitously in nine different human tissues (Lahn and Page, 1997). The regulation of expression of ubiquitously expressed genes is not necessarily accomplished in the same way for different tissues, particularly in testis, and these genes may not be expressed at all in some spermatogenetic impairments. A single de-novo mutation in USP9Y gene was detected in a healthy man exhibiting azoospermia (Sun et al., 1999), suggesting a putative testis-specific role for this gene. The presence of USP9Y transcript was previously reported in a group of 28 azoospermic patients, but their specific histology findings were not reported (Friel et al., 2002). In this study, USP9Y and USP9X transcripts were detected in all the 89 tested specimens, indicating that their impairment is at least infrequent in spermatogenesis failure. USP9Y expression was not impaired in the specimen with AZFb-c and AZFc deletions, thus refuting any hypothetical presence of testicular AZFb or AZFc factor(s) that might prevent AZFa gene expression. Indeed, these results imply that USP9Y can effectively serve as a ubiquitously expressed control for RT–PCR assessments in specimens without AZFa deletions.
Lahn and Page (1997) detected two transcripts encoded by the DDX3Y gene by northern blot. The long one was expressed ubiquitously, and the short one (DDX3Yt1) produced by the use of an alternative polyadenylation site was expressed exclusively in testicular tissue. Foresta et al. (2000) also observed testis-specific expression of DDX3Yt1 by RT–PCR and suggested that the testis-specific transcript DDX3Yt1 plays a crucial role in the spermatogenetic process. Additional DDX3Y transcripts were recently reported by Ditton et al. (2004). None of these articles, however, assessed DDX3Y and DDX3Yt1 expression in impaired spermatogenesis. In this study, RT–PCR expression of DDX3Y was found in all the specimens analysed, whereas DDX3Yt1 transcript was detected in only 78% of the testicular specimens that included 62% of the Sertoli cell-only specimens. The observed DDX3Yt1 expression in Sertoli cell-only specimens was not as a result of the cross amplification of the DDX3Y because the sequence of the reverse primer was homologous only in 10 of the 20 base pairs. Additionally, DDX3Yt1 was not detected ubiquitously in the specimens tested. It can be speculated that basal levels of expression have been detected in some Sertoli cell-only specimens. To assess this supposition, it would be interesting to check whether a basal level of DDX3Yt1 expression is detectable by RT–PCR methodology in somatic tissues.
Some specimens were further tested by quantitative PCR, and significant differences were observed in the expression levels of DDX3Yt1/DDX3Y when comparing a group of normal spermatogenesis biopsies with both groups with impaired spermatogenesis tested (P = 0.011 and P = 0.034 for hypospermatogenesis and Sertoli cell-only, respectively). The lower expression levels of DDX3Yt1/DDX3Y among the groups with impaired spermatogenesis (2-fold at most) imply a decreased use of the DDX3Yt1 alternative polyadenylation site. This lower expression obviously did not reflect the absence of germ cells because no significant differences in the expression ratio were found between the hypospermatogenesis and the Sertoli cell-only groups. Testing additional specimens will strengthen our preliminary results and allow us to draw conclusions about the importance of this transcript.
Alternative polyadenylation is a mechanism for controlling gene expression (Yu et al., 2006). Both DDX3Y transcripts differ only in the length of the 3'-untranslated region, which is a non-coding region involved in the regulation of translation in many different ways (Kuersten and Goodwin, 2003). The DDX3Y protein was recently reported to be detectable only in testicular tissue, predominantly in the cytoplasm of spermatogonia, although the long DDX3Y transcript is expressed in many different tissues (Ditton et al., 2004). Although DDX3Yt1 transcription was detected in half of the specimens with Sertoli cell-only, its translation is probably delayed by the absence of the required specific translation factors. Checking these peculiar Sertoli cell-only specimens with the Ditton’s antibodies that recognize DDX3Y protein could confirm this assumption. Our results suggest that the transcription level of DDX3Yt1 is down-regulated in impaired spermatogenesis. It is well known that the deletions of the AZFa region that includes the DDX3Y gene cause severe spermatogenic impairment (Foresta et al., 2000) and that the complete deletion of the AZFa region is usually associated with the total depletion of spermatogenetic cells (Kleiman et al., 2001). All these findings taken together suggest that the undetectable or abnormal expression of DDX3Yt1 is frequently encountered among men suffering from non-obstructive azoospermia.
Northern blot analysis with normal testicular tissue had shown that DDX3Yt1 is the major transcript in testis (Lahn and Page, 1997; Ditton et al., 2004). Our quantitative analysis, however, showed that DDX3Yt1 accounts for up to 23% of the total number of DDX3Y transcripts. This disparity can be partially explained by our having considered obstructive azoospermic specimens as ‘normal specimens’. Recent studies have shown a significant decrease in sperm yields/gr testis and even reduced recombination frequencies in obstructive azoospermia compared with normal fertile controls, suggesting that these specimens are not completely normal (McVicar et al., 2005; Sun et al., 2005). Another explanation for the discrepancy between the quantitative analysis and the northern blot data might be the presence of an undetected subset of DDX3Yt1 transcripts with additional polyadenylation sites close to the one analysed in the study. Indeed, additional DDX3Yt1 transcript (accession number AA437293
[GenBank]
), with a nearby polyadenylation site five nucleotides downstream, had been reported in the GeneBank Human EST database. One more EST (accession number DB514160
[GenBank]
) without a polyA track was also described. However, it may correspond to the DDX3Yt1 transcript as described by Foresta et al. (2000) or to a truncated transcript.
Similar to the AZFa transcripts (USP9Y and DDX3Y), EIF1AY, which had been previously found to be expressed in several tissues (Lahn and Page, 1997), was detected in all biopsies with two exceptions. Testing EIF1AY expression with additional sets of primers could have confirmed these results if additional RNA from these specimens had been available. The absence of EIF1AY may have contributed to the spermatogenic failure in these specimens. The presence of microdeletions in the EIF1AY gene region was excluded, but additional assessments, such as sequencing the gene and its upstream regions and testing its transcription in somatic tissue, would help clarify the absence of EIF1AY expression in these two specimens. In any event, different testicular histological and cytological findings observed in these two specimens implicate additional factors as being involved in this process. The expression in almost all specimens together with its absence in two support the idea that EIF1AY has a role as an essential transcription factor during spermatogenesis with housekeeping functions that are probably also required in the somatic cells of the testis (Lahn and Page, 1997; Ginalski et al., 2004).
The expression of the PRY protein was detected in testis and in ejaculated sperm by immunohistochemistry (Stouffs et al., 2004). In the testis, PRY protein was observed in only a small fraction of the elongating and mature spermatids. Stouffs et al. (2004) suggested that PRY is involved in the apoptosis of spermatids and spermatozoa, but we found the expression of PRY transcripts in most of the specimens in which germ cells were observed. These results imply that PRY is expressed before the spermatid stage. PRY is probably transcribed early during spermatogenesis and translated particularly when apoptotic steps are activated or during spermiogenesis. This temporal delay in translation of specific transcripts had been already observed in other genes expressed in testis (Steger et al., 1998).
The detection of PRY in several biopsies with Sertoli cell-only might indicate the presence of foci of spermatogenesis in these biopsies. The absence of the DAZ and RBM germ cell markers (Kleiman et al., 2001) in these particular biopsies, however, refutes this possibility. This seldom-occurring atypical expression of PRY in somatic cells of the testis suggests that it should not be used as the exclusive PCR-germ cell marker because its expression is occasionally altered. Similarly, altered expression of CDY2 in some Sertoli cell-only specimens was observed as well.
Variations in RT–PCR results due to the individual quality of RNA isolation cannot be completely excluded as a potential factor affecting the expression of DDX3Yt1 and PRY. This possibility, however, seems remote because additional transcripts that were similarly amplified had been detected. In addition, to prevent variation in RT–PCR results due to the individual quality of RNA isolation, we reported herein two multiplex RT–PCR mixes established and verified for reliability to be used in future analysis. The co-amplification of the transcripts with a control locus that amplifies to a similar extent is also a very reliable control of RNA quality after their expression has been well characterized. Still, interference is sometimes detected when multiplex PCR is performed.
The specificity of the PCR test may also introduce variations in the results. This possibility was ruled out by various tests: the absence of amplification by in silico genomic PCR, sequence analysis verification and the absence of expression in specimens with deletions that included the genes tested. In addition, the findings were similar by both single and multiplex RT–PCR in three specimens with atypical PRY expression.
Previous studies have highlighted the need for more sensitive methods for diagnosing spermatogenesis in the testes of men with azoospermia (Lee et al., 1998; Song et al., 2000; Kleiman et al., 2001, 2003; Schrader et al., 2002a,b). We previously found that DAZ and RBM are reliable markers for germ cell presence and that CDY1, particularly CDY1 minor and short transcripts, appeared to be a good marker of complete spermatogenesis (Kleiman, 2001, 2003). The previously analysed CDY2 and the PRY are less reliable markers for the detection of spermatogenesis because they are also identified in over 20% of the specimens with Sertoli cell-only (Kleiman, 2003). DDX3Yt1 cannot be applicable as a marker for spermatogenesis because it is detected in a large fraction of specimens with Sertoli cell-only. Lastly, CDYL, USP9Y/X, DDX3Y and EIF1AY can serve only as ubiquitously expressed control markers. The availability of a large battery of genes with a step-specific expression pattern will be of help in choosing the most promising markers for the detection of complete spermatogenesis. The findings in this study demonstrated that carefully selected stage-specific expression markers could be applicable to routine laboratory analysis by establishing sensitive and quick methods, such as multiplex RT–PCR, for diagnosing spermatogenesis. Men with germinal failure, i.e. Sertoli cell-only, might have minute foci of spermatogenesis sparsely distributed in the testis (Silber et al., 1997). Applying a complementary methodology, such as the approach described here, will improve the prediction of hidden foci of complete spermatogenesis.
Although the transcripts assessed herein are not good molecular markers of spermatogenesis for use in biomedical consultation, our findings contribute to the understanding of the complex gene expression process during spermatogenesis.
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The authors thank Tovi Morad for her excellent technical assistance, Esther Eshkol for editorial assistance and Ilana Galernter (Statistical Department, Tel Aviv University) for the expert statistical analysis. The Chief Scientific Office, Ministry of Health (Israel) grant number 4823, supported this study. The research was carried out under the auspices of the Alan and Ada Selwyn Chair in Clinical Infertility Research and Molecular Medicine (Melbourne, Australia) granted to one of the authors (G.Paz).
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References |
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Submitted on August 11, 2005; resubmitted on November 30, 2005; resubmitted on February 23, 2006; resubmitted on June 4, 2006; resubmitted on July 9, 2006; accepted on July 27, 2006.
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