Deletions in the azoospermia factor region AZFa on the human Y chromosome and, more specifically, in the region that encompasses the ubiquitin-specific peptidase 9, Y-linked gene USP9Y have been implicated in infertility associated with oligospermia and azoospermia. We have characterized in detail a deletion in AZFa that results in an absence of USP9Y in a normospermic man and his brother and father. The association of this large deletion with normal fertility shows that USP9Y, hitherto considered a candidate gene for infertility and azoospermia, does not have a key role in male reproduction. These results suggest that it may not be necessary to consider USP9Y when screening the Y chromosome of infertile or subfertile men for microdeletions.
USP9Y spans 170 kb of DNA, consists of at least 46 exons, and occupies a small part of the AZFa interval. It encodes a protein reported to function as ubiquitin C-terminal hydrolase and is ubiquitously expressed.6,7 Deletions affecting USP9Y have been associated with azoospermia or severe oligospermia.6,8 Two partial deletions were recently found in men with a milder phenotype, oligoasthenoteratozoospermia, suggesting a minor role of this gene in spermatogenesis.5,9
Methods
The Patient
The patient, a 42-year-old man, underwent spermatologic and genetic analysis during an infertility evaluation solicited by him and his partner after miscarriage. He and other male members of his family provided written informed consent for participation in this study, as required by the institutional review board of the Siena Hospital.
Analysis of Semen
Three spermiograms were obtained at 3-month intervals for the patient. Semen samples were collected and volume, pH, and sperm concentration and motility were evaluated according to World Health Organization (WHO) guidelines.10 The brother and father did not provide semen samples.
Ultrastructural examination of ejaculated sperm was carried out by means of transmission electron microscopy. Semen specimens were fixed in cold Karnovsky's fixative and maintained at 4°C for 2 hours. Fixed semen samples were washed in 100 mM cacodylate buffer (pH 7.2) for 12 hours, postfixed in 1% buffered osmium tetroxide for 1 hour at 4°C, and dehydrated and embedded in Epon–Araldite. Ultrathin sections were cut with an ultramicrotome (Supernova, Reickert Jung), mounted on copper grids, stained with uranyl acetate and lead citrate, and observed and photographed with a transmission electron microscope (CM10, Philips). We analyzed ultrathin sections of 300 sperm specimens.
To evaluate the frequency of aneuploidy, fluorescence in situ hybridization (FISH) was carried out on sperm nuclei, according to Baccetti et al.11 A total of 2880 sperm were analyzed using a mix of satellite DNA probes (CEP, Vysis) for chromosomes 18, X, and Y, which were each directly labeled with different fluorochromes.
Molecular Analyses
Genomic DNA was isolated from peripheral-blood lymphocytes or spermatozoa with the use of a commercial extraction kit, according to the manufacturer's protocol. Screening for deletions was initially performed for AZFa, AZFb, and AZFc, according to the European Academy of Andrology–European Molecular Genetics Quality Network (EAA-EMQN) guidelines.12 In order to define the extent of deletion, we used several sequence-tagged sites (sY82, sY88, sY83, G64723 [GenBank] , AZFa-prox2, G66179 [GenBank] , G66183 [GenBank] , SHGC-3904, G65852 [GenBank] , G49201 [GenBank] , G65840 [GenBank] , G66201 [GenBank] , G49206 [GenBank] , G66189 [GenBank] , sY87, GY6, and G38346 [GenBank] ) and gene-specific primers to localize the breakpoints to an interval spanning less than 1 kb (see the Supplementary Appendix, available with the full text of this article at NEJM.org).
Polymerase-chain-reaction (PCR) assays were performed using 1 U of Taq polymerase with the supplier's buffer, 200 µM of each deoxyribonucleotide triphosphate and 0.3 mM of each primer, in a final volume of 20 µl. Thermocycling conditions were as follows: 30 seconds at 95°C, 30 seconds at 57 to 59°C, and 45 seconds at 72°C, for a total of 35 cycles. All PCR assays were performed on DNA derived from a woman as a negative control and DNA from a fertile male as a positive control. The results were considered negative only after three consecutive failures of amplification; occasionally the experiments were repeated on DNA extracted from the second blood samples obtained from the patient and his brother and father. Specific primers were also used to amplify USP9Y, exon 1 (GenBank accession number, G64987 [GenBank] ) and exon 46 (GenBank accession number, G34983 [GenBank] ), and DDX3Y, exon 1 (GenBank accession number, G38346 [GenBank] ) and exon 17 (GenBank accession number, G65240 [GenBank] ). For Y-haplotype analysis of the proband, the deep-rooting markers SRY-1532, M9, YAP, 12f2, M231, and 92R7 were typed by means of PCR amplification and DNA sequence analysis.13,14
DDX3Y Gene-Expression Analysis
We assayed the expression of DDX3Y in the patient's lymphocytes using a commercially available kit, according to the manufacturer's protocol. A blood specimen from a man with normal spermatogenesis was used as a positive control, and one from a woman was used as a negative control. For each sample, first-strand complementary DNA was synthesized from total RNA (previously treated with RQ1 RNase-Free DNase [Promega] to remove contaminant DNA), with the use of the reverse primer 5'-CTCGCTGTACTTGCTCCTCC-3' (targeting exon 2). DDX3Y transcripts were PCR-amplified with the same reverse primer and the 5'-AGTTCCGCTATTCGGTCTCA-3' primer (targeting exon 1). Thermocycling conditions were as follows: 40 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 45 seconds at 72°C.
Results
The analysis of a number of sperm specimens from the patient showed a normal sperm count, 54 to 66 million sperm per milliliter, with a mean of 330 million spermatozoa per ejaculate. The total progressive motility (the sum of rapid and slow) was slightly reduced, ranging from 28 to 34% of sperm, and the percentage of morphologically normal forms was approximately 30%. Apart from the reduction in sperm motility (mild asthenozoospermia), all other sperm characteristics were within the normal range, according to WHO guidelines.10 Normal sperm phenotype was confirmed by means of electron microscopy (Figure 1), which revealed well-shaped nuclei, condensed chromatin, tails with normal structure, and regular axonemes. Sperm with abnormal morphologic features showed structural anomalies typical of immaturity: altered acrosomal or nuclear molding, uncondensed chromatin, and cytoplasmic residues. A small percentage of sperm had necrotic features such as broken plasma membranes, reacted or missing acrosomes, and disrupted chromatin (Figure 1).
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Genetic screening for Y microdeletions was carried out by means of multiplex PCR analysis, according to EAA-EMQN guidelines.12 Analysis of DNA derived from peripheral-blood lymphocytes of the proband, his father, and his brother showed a deletion in the AZFa region (Figure 2).
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We also established that the deletion did not include any known coding or regulatory regions of DDX3Y, which lies downstream of the USP9Y (data not shown). To determine whether the deletion affects DDX3Y expression,15 we performed reverse-transcriptase–PCR analysis on RNA isolated from lymphocytes from the patient and found that gene expression was not affected (Figure 3).
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Discussion
Since deletions in USP9Y have been reported to cause mild-to-severe oligospermia or azoospermia,5,8,9 a phenotype not observed in our patient, we considered mosaicism as an explanation for the fertility of this subject. However, our analysis showed that DNA extracted from all ejaculated spermatozoa carried the same deletion. These results are in line with the previously postulated marginal role of the USP9Y gene in spermatogenesis9 and are also consistent with the presence of the same deletion in the father and the brother. The relatively normal spermatogenic phenotype of our patient, and the proven fertility of his father, show that previously described azoospermia and oligoasthenospermia cannot be due to deletion of USP9Y alone: additional genetic or nongenetic factors must influence the phenotype. Local testicular factors or the environmental or genetic background could be responsible for the phenotypic variability highlighted in previously reported cases. In particular, investigations of the Y haplotype in patients carrying a USP9Y deletion would be useful to determine whether there is a correlation between genetic background and phenotypic variation.
On the basis of the normal rate of USP9Y transcription in patients with spermatogenic failure and the absence of its correlation with the degree of sperm retrieval,16 we also infer that USP9Y has a marginal role or no role in spermatogenesis. Consistent with this hypothesis is the inactivation of the orthologous gene in chimpanzees and bonobos.17
In conclusion, we found that complete deletion of the USP9Y gene does not cause spermatogenic defects, nor does it preclude the natural conception of children. This gene was recently reported to be a "fine-tuner" of human spermatogenesis, improving its efficiency.9 Our findings indicate that USP9Y is not essential for normal sperm production and fertility in humans and that a revision of the diagnostic approach of screening for Y-chromosome microdeletions, according to EAA-EMQN guidelines,11 may be warranted. This approach does not detect deletions affecting DDX3Y alone.
Supported in part by a grant (0405) from Monte dei Paschi di Siena Bank.
No potential conflict of interest relevant to this article was reported.
We thank Csilla Krausz for helpful discussions, Elvira Costantino-Ceccarini for her extensive assistance in the preparation of a previous draft of the manuscript, and Carlo Alessandrini for participation in the ultrastructural studies.
Source Information
From the Department of Pediatrics, Obstetrics, and Reproductive Medicine (A.L., M.M., L.G., F.S., M.C., V.D.L., P.B.) and the Department of Biomedical Sciences, Applied Biology Section (P.P.), University of Siena; and the Center for Diagnosis and Treatment of Couple Sterility, Siena Hospital (L.G., F.S., V.D.L., P.P.) — all in Siena, Italy.
Address reprint requests to Dr. Piomboni at the Department of Biomedical Sciences, Applied Biology Section, University of Siena, Center for Diagnosis and Treatment of Couple Sterility–Siena Hospital, Policlinico S. Maria alle Scotte, Viale Bracci, 14, 53100 Siena, Italy, or at piomboni{at}unisi.it.
References
lu C, King R, Kivisild T, et al. Excavating Y-chromosome haplotype strata in Anatolia. Hum Genet 2004;114:127-148. [CrossRef][Web of Science][Medline]
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