1 Clinica Medica 3, Department of Medical and Surgical Sciences and 2 Institute of Histology and Embryology, University of Padova, Italy
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Abstract |
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Key words: DAZexpression/DAZ gene/RTPCR/testiculopathy/Y chromosome
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Introduction |
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Following our previous study on Yq PCR screening of infertile men (Ferlin et al., 1999), we have analysed the expression of DAZ and, for comparison, of RBM (RNA binding motif) and SRY (sex determining region) genes in testicular cells from infertile men carrying an apparently normal DAZ gene constitution as assessed by PCR on genomic DNA, in order to investigate these aspects and in general to better clarify the role of this gene in idiopathic testiculopathies.
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Materials and methods |
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RNA extraction from testicular cells
The presence of DAZ, RBM and SRY mRNA was analysed by means of reverse transcription (RT)PCR in testicular cells retrieved by FNA. Part of the material retrieved from both testes by FNA for diagnostic purpose was immediately frozen at 20°C and used for RNA extraction. Total RNA was extracted from testicular samples using the RNeasy Method (Qiagen, Germany). Briefly, the cells were recovered by means of centrifugation at 1000 g for 5 min, rapidly homogenized and lysed in the presence of a highly denaturing guanidinium isothiocyanate-containing buffer that immediately inactivates RNases to ensure isolation of intact RNA. Ethanol was added to provide appropriate binding conditions and the sample was then applied to an RNeasy mini spin column where the total RNA binds to the membrane and contaminants are efficiently washed away. Total RNA was then eluted in 30 µl of DEPC-treated water and concentrated to 5 µl by means of Centricon 3 (Amicon, Beverly, MA, USA).
cDNA synthesis
RT of total RNA was performed using 0.2 µg of oligo-dT primer for 1 h at 42°C utilizing 25 U of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim, Germany) in Tris 50 mmol/l (pH 8.3), KCl 75 mmol/l, MgCl2 3 mmol/l, dithiothreitol (DTT) 10 mmol/l, RNasin 1 U (Promega, USA) and 1 mmol/l of dATP, dGTP, dCTP and dTTP in a total volume of 20 µl.
Polymerase chain reaction
PCR was performed using the following primer pairs: sY254 for the DAZ gene (Reijo et al., 1995), which amplify the region 14091789 (from exon 2 to exon 3) of the genomic DNA of the DAZ gene (clone 63C9); sY14 for the SRY gene (Vollrath et al., 1992
), which amplify the region 479948 of the SRY gene; F19/E355 for the RBM gene (Ma et al., 1993
; Kobayashi et al., 1995
), which amplify the region 12921733 of the RBM-I coding sequence, corresponding to the distal part of exon 11 and the most part of exon 12. PCR was carried out for each gene in 20 µl of reaction volume containing: 6 µl of testicular cDNA diluted 1:20 from total cDNA obtained, Taq polymerase (0.8 U), dNTP (0.2 mM dTTP, dCTP, dGTP, dATP), oligonucleotide primers (10 pmol each) made up in a final concentration of 1x PCR reaction buffer (TrisHCl 10 mmol/l pH 8.3, MgCl2 1.5 mmol/l, KCl 50 mmol/l). All reagents were obtained from Pharmacia (Milan, Italy). Amplification was performed for 35 sequential cycles, each of them including 1 min of denaturation at 94°C, 1 min of primer annealing at 60°C and 1 min of extension at 72°C; before the first cycle, all samples were incubated for 10 min at 94°C. PCR reaction products were eventually stored at 4°C and then separated on 2% agarose gel by electrophoresis in TAE (Trisacetic acidEDTA) buffer at room temperature using a voltage gradient of 8 V/cm for 3060 min. Experiments were also performed on mRNA samples without AMVRT to amplify the full length (unspliced) products of SRY, RBM and DAZ. Co-amplification of genomic DNA was also observed due to a contamination that naturally occurs in these circumstances. Furthermore, RTPCR of glyceraldehyde-6-phosphate dehydrogenase (GAPDH) was used as internal control. In order to obtain a good quality image for Figure 1
, the RTPCR fragments from agarose gel were purified and run separately.
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Results |
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Patients were considered normal for the DAZ gene when PCR on genomic DNA normally amplified primer pairs sY277, sY254, sY279, sY283 and sY255; deletions of this gene were considered when all five primers repeatedly failed to amplify. Similarly, RBM and SRY were studied by PCR using primer pairs F19/E355 and sY14 respectively.
The SRY gene is expressed in different testicular cells (Sinclair et al., 1990; Clepet et al., 1993
; Tricoli et al., 1993
) and therefore its analysis by RTPCR has been chosen as an internal control for the presence of testicular mRNA. However, sY14 does not span an intron and therefore it cannot distinguish between amplification of DNA and RNA. On the contrary, the RBM gene, like DAZ, is expressed only in germ cells (above all spermatogonia and spermatocytes) (Elliott et al., 1997
, 1998
) and the primers used for its detection amplify across regions containing introns, therefore being an internal control for the presence of mRNA from spermatogenic cells. Primer pairs sY14 and F19/E355 utilized for RTPCR analysis of SRY and RBM genes produced amplification products of 472 bp and 441 bp, respectively, which correspond to the normal lengths based on cDNA sequences (Sinclair et al., 1990
; Ma et al., 1993
). As expected, the product size of sY14 in the absence of AMVRT was again 472 bp, while that of F19/E355 was ~800 bp, as previously reported. Primers sY254 amplify part of exons 2 and 3 of the DAZ gene and its RTPCR product was of 106 bp, confirming DAZ cDNA sequence (Reijo et al., 1995
), while the length of the unspliced (testicular DNA) product was 380 bp, corresponding to the genomic DAZ sequence. Table I
shows the primers used for RTPCR with their product size. In all samples a normal RTPCR amplification was obtained for a basic gene, such as GAPDH.
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Discussion |
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To better understand the functional activity of the DAZ gene and to overcome misleading results obtained by PCR analysis on genomic DNA, in this study we analysed and compared by RTPCR the expression of DAZ, and for comparison that of RBM and SRY genes, in testicular cells from infertile men affected by obstructive azoospermia, Sertoli cell-only syndrome and severe hypospermatogenesis. This latter testiculopathy was selected, and not maturation arrests, since DAZ deletions have been so far more clearly correlated with a quantitative reduction of germ cells instead of with impairment of the maturation progression. Sertoli cell-only syndrome was selected as control. All subjects presented a normal Yq, as shown by PCR analysis on genomic DNA extracted from peripheral leukocytes. Normal amplification of such genes was observed both when spermatogenesis was completely normal (obstructive forms) and when it was quantitatively reduced, but with presence of all germ cell subtypes (severe hypospermatogenesis), whereas only SRY was detected when only Sertoli cells were present. These findings confirmed previous studies, clearly demonstrating that RBM and DAZ are transcribed in a germ cell-specific manner (Elliott et al., 1997, 1998
; Menke et al., 1997
; Habermann et al., 1998
; Lee et al., 1998
), even if RTPCR did not enable us to distinguish in which spermatogenic cell subtype these genes are specifically expressed. The expression of SRY cannot be determined in our experiments, since this gene does not contain introns; and therefore RTPCR analysis could not distinguish between amplification of DNA or RNA; however, since no genomic DNA was amplified with RBM and DAZ primers in these reactions, we can assume RNA has been identified.
Nine out of 10 patients affected by idiopathic severe hypospermatogenesis showed the presence of normal testicular mRNA for RBM and DAZ, while in one patient no DAZ transcript was detected, suggesting that in this case the testiculopathy was likely to be related to the absence of DAZ expression. The lack of DAZ mRNA detection in testicular cells with an apparently normal DAZ gene constitution on DNA extracted from leukocytes may be explained by different hypotheses.
At the moment none of these hypotheses (deletion of the functional DAZ copies, mosaicism, abnormalities of transcription or post-transcription) can be verified, and confirmation of the association between absent DAZ expression and severe testiculopathy is difficult. Northern or Western blot experiments on testicular tissue were not possible since the material retrieved by FNA was not sufficient for these analyses. Furthermore, no experiments were possible on spermatozoa, as the patient was repeatedly found to be azoospermic.
The testicular picture of the reported patient is very similar to that reported in the presence of a specific genomic DAZ deletion (Reijo et al., 1995, 1996
; Najmabadi et al., 1996
; Vogt et al., 1996
; Foresta et al., 1997
, 1998
; Girardi et al., 1997
; Pryor et al., 1997
; McLachlan et al., 1998
; Silber et al., 1998
; Vogt, 1998
; Ferlin et al., 1999
). Several lines of evidence indicate that the absence of DAZ is more likely to produce a depopulation of germ cells, and both cytological (by FNA) and histological (by open biopsy) examination in the reported patient showed a picture of severe hypospermatogenesis, characterized by a quantitative reduction of spermatogenic cells with conserved relative proportions. These data further suggest that DAZ may induce, by unknown mechanisms, a depopulation of spermatogonia without affecting mitotic and meiotic phases.
Expression of RBM has been observed in all patients affected by idiopathic hypospermatogenesis. Given the small number of patients at risk for Yq microdeletions examined in this study and the low prevalence of RBM deletions in infertile men (Reijo et al., 1995, 1996
; Najmabadi et al., 1996
; Qureshi et al., 1996
; Vogt et al., 1996
; Foresta et al., 1997
, 1998
; Girardi et al., 1997
; Pryor et al., 1997
; Liow et al., 1998
; McLachlan et al., 1998
; Silber et al., 1998
; Vogt, 1998
; Ferlin, 1999), this finding is not surprising. However, this gene is in multiple copies both in the short and in the long arm of the Y chromosome, representing active genes as well as pseudogenes (Chai et al., 1997
, 1998
; Elliott et al., 1997
; Gläser et al., 1997
). Primer pairs used for RTPCR in this study amplify a region between exon 11 and 12 (outside the SRGY box) and we observed a single amplification product that may originate from a single gene or from different genes with sequence homology in this region. In fact, the RBM transcripts substantially differ only in the number of copies of the SRGY box (Prosser et al., 1996
; Chai et al., 1997
), and it is possible that the region examined in the present study may not harbour significant differences among the RBM copies detected by RTPCR.
Finally, the finding that patients with severe hypospermatogenesis may be infertile because of the lack of DAZ activity despite an apparent normal peripheral constitution of this gene has two other major consequences. It highlights the intrinsic interpretative difficulties of normal PCR analysis for DAZ, as well as for RBM, on leukocytes and it further suggests caution in the use of ejaculated spermatozoa and intratesticular cells for assisted reproductive techniques, since it has been demonstrated that spermatozoa carrying Yq deletions are able to fertilize and to give rise to pregnancies by means of these techniques (Kent-First et al., 1996a,b
; Mulhall et al., 1997
; Rossato et al., 1998
; Silber et al., 1998
).
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Acknowledgments |
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Notes |
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References |
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Submitted on February 1, 1999; accepted on May 24, 1999.