Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Biology, Siena University, Regional Referral Center for Male Infertility, Azienda Ospedaliera Universitaria Senese, Siena and 1 Institute of Clinical Physiology, Siena Branch, National Research Council, Siena, Italy
2 To whom correspondence should be addressed. Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Biology, University of Siena, Policlinico Le Scotte, Viale Bracci, 14, 53100 Siena, Italy. E-mail: piomboni{at}unisi.it.
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Abstract |
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Key words: Akap3/Akap4 deletions/DFS/FISH analysis/male infertility/TEM
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Introduction |
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Genetic male infertility may also be associated with deletions of specific regions of the long arm of the Y chromosome. Y microdeletion screening is especially worthwhile in patients with a sperm count below 5 x 106/ml or even below 10 x 106/ml. PCR analysis for Yq microdeletions in 392 normospermic men did not reveal any deletions (Krausz et al., 2003). A new model of deletions in which AZFb and AZFc regions overlap has emerged. Repping et al. (2002)
suggested at least three different deletion patterns as a consequence of AZFb and AZFc deletions.
Most studies using multicolour fluorescence in situ hybridization (FISH) demonstrate that infertile men may also have a higher frequency of sperm chromosomal abnormalities (Martin et al., 1996; Bernardini et al., 1997
; Rives et al., 1998
), in particular when DFS sperm defect is present (Baccetti et al., 2005
).
In this paper we report the results of different molecular analyses performed in an infertile man with DFS. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse specific Y microdeletions and partial sequences of the AKAP4 and AKAP3 genes. Meiotic segregation pattern was also investigated by three-colour FISH sperm analysis.
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Materials and methods |
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Semen analysis
Light and electron microscopy
A semen sample was collected by masturbation after 4 days of sexual abstinence and examined after liquefaction for 30 min at 37°C. Volume, pH, concentration and motility were evaluated according to WHO guidelines (World Health Organization, 1999).
For electron microscopy, semen was fixed in cold Karnovsky fixative and maintained at 4°C for 2 h. Fixed semen was washed in 0.1 mol/l cacodylate buffer (pH 7.2) for 12 h, postfixed in 1% buffered osmium tetroxide for 1 h at 4°C, dehydrated and embedded in Epon Araldite. Ultra-thin sections were cut with a Supernova ultramicrotome (Reickert Jung, Vienna, Austria), mounted on copper grids, stained with uranyl acetate and lead citrate, and observed and photographed with a Philips CM10 transmission electron microscope (TEM; Philips Scientifics, Eindhoven, The Netherlands). Three hundred sperm in ultra-thin sections were analysed.
An aliquot from the same sperm sample was also processed for scanning electron microscopy, fixing the spermatozoa as described above and smearing them on polylysine (1%)-coated coverslides. After dehydration specimens were dried by the critical point technique, coated in gold and observed with a Philips CM 515 scanning electron microscope (Philips Scientifics, Eindhoven, The Netherlands).
PCR analysis
DNA was extracted from peripheral blood lymphocytes using the QIAamp DNA Blood Kit (Qiagen).
PCR analysis for Y microdeletion screening was performed according to European Academy of Andrology (EAA) guidelines (Simoni et al., 2004).
Control DNA was extracted from blood of 10 male donors, age 3040 years, with a documented history of fertility. DNA extracted from blood of two fertile females was used as negative control.
PCR product corresponding to the region of pro-hAKAP82 that encodes the RII-binding domain was amplified according to Turner et al. (2001a). Oligonucleotide primers flanking the RII-binding site were used: sense primer, 5'-GCGATTATCACAGAGCATCC-3'; antisense primer. 5'-TGCTCTCTCTCTGGGACATC-3'.
PCR products corresponding to a region of hAKAP4 involved in binding to AKAP3 (site 1) and to a region of AKAP3 involved in binding to hAKAP4 (site 2) were amplified according to Turner et al. (2001b). Oligonucleotide primers flanking the respective binding sites, were used: site 1, sense primer, 5'-TCAGTGCCCTTATAGGTGAG-3'; antisense primer, 5'-GCAGAGCTTCATCACAGATTC-3'; site 2, sense primer, 5'-TTGAGGAATCTCCACAGCG-3'; antisense primer, 5'-CCAACGGTCTTTCACACAACTTC-3'.
Control DNA was extracted from blood of a fertile man and four infertile men with DFS sperm defect.
Immunofluorescence
Semen samples of the patient and fertile men, used as controls, were washed twice in phosphate buffered saline (PBS), smeared on glass slides, air-dried, rinsed in PBS and fixed for 15 min in methanol at 20°C. Slides were then treated with blocking solution (PBS, 1% BSA, 5% normal goat serum [NGS]) for 20 min at room temperature and incubated overnight at 4°C with mouse monoclonal anti--tubulin (Sigma Chemical, St Louis, MO, USA) and mouse monoclonal anti-AKAP 82 (BD Biosciences, Erembodegem, Belgium) diluted 1:100 and 1:50 respectively in PBS, 0.1% BSA, 1%NGS. After three washes in PBS, the samples were treated with goat anti-mouse IgG-Texas Red-conjugated antibody (Southern Biotechnology Associates, Birmingham, AL, USA). Finally, the samples were washed three times in PBS and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). Incubation in primary antibodies was omitted in procedure control samples. Observations and photographs were made with a Leitz Aristoplan light microscope equipped with fluorescence apparatus.
FISH sperm analysis
FISH sperm analysis was carried out according to Baccetti et al. (2003) to evaluate aneuploidy frequency. A mix of
-satellite DNA probes (CEP, Vysis, IL) for chromosomes 18, X and Y, directly labelled with different fluorochromes, was used.
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Results |
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The genotypic sperm defect DFS was demonstrated by TEM (Figure 1a) in the whole sperm population. Sperm tails were extremely short, as confirmed also by scanning electron microscopy (Figure 1b). A disorganized fibrous sheath and altered axonemal structure, sometimes lacking dynein arms and microtubular doublets including the central pair, were observed. Although spermatozoa with these defects usually have well formed acrosomes and nuclei (Baccetti et al., 1993), in this case we found severe nuclear alterations, particularly in chromatin texture, in almost the entire sperm population.
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Immunofluorescence staining using monoclonal anti--tubulin confirmed short, thick axonemal structure (Figure 2a, b). Immunofluorescence staining, using a monoclonal anti-AKAP82 antibody specific for human AKAP4 protein (previously named AKAP82), was completely negative (Figure 2c, d). In control samples (Figure 2e), the same antibody labelled the tail principal piece. Anti-tubulin antibody recognized the centriolar region and principal piece of the sperm tail in control samples (Figure 2f).
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FISH was performed on sperm nuclei to evaluate aneuploidy frequency (Table I). We examined 4233 sperm. Only XY disomy was more frequent than in controls (Baccetti et al., 2003); the frequency of other disomies and diploidies was normal.
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PCR screening of specific Y chromosome according to EAA guidelines (Simoni et al., 2004) did not evidence any deletions in the regions investigated.
When we performed PCR using two pairs of primers flanking sites 1 and 2, as described by Turner et al. (2001b), the expected PCR products corresponding to a region of hAKAP4 involved in binding to AKAP3 (site 1) and to a region of AKAP3 involved in binding to hAKAP4 (site 2) were both absent (Figure 3, lanes C2, 3). This was confirmed by three subsequent PCR attempts. The same sequences were present in a fertile man (Figure 3, lanes A2, 3) and four infertile men with DFS (Figure 3, lanes B2,3, D2,3, E2,3, F2,3), who were analysed as controls (Figure 3). In all DFS patients, including our studied case, and in the control fertile man, PCR products corresponding to pro-hAkap82, which encodes the RII-binding domain, were present (Figure 3, lane 1, AF)
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Discussion |
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TEM demonstrated the presence of a peculiar ultrastructural defect of the sperm tail, namely dysplasia of the fibrous sheath. This defect, like other genetic sperm defects, is more frequent in consanguineous patients and is related to the degree of consanguinity (Baccetti et al., 2001). DFS includes a heterogeneous array of structural defects of the sperm tail (Chemes and Rawe, 2003
) and causes almost complete immotility, but nuclear and acrosomal structure are generally unaffected. In our patient, however, we observed severe nuclear alterations, particularly in chromatin texture and condensation.
For this reason, screening for Y microdeletion was carried out even though the patient had a normal sperm count. No Y microdeletions were detected. This coincides with the results of Krausz et al. (2003), who failed to detect Y microdeletions in normospermic individuals. On the other hand, we found an absence of the potential AKAP4/AKAP3 binding regions in the patient under study while the presence of these PCR products was detected in fertile men and other infertile men with DFS, analysed as controls. Turner et al. (2001b)
did not find evidence to support the hypothesis that mutations in Akap4 or Akap3 are responsible for DFS in humans. However, in our case, deletion of the Akap4/Akap3 binding regions and failure to detect AKAP4 protein by immunofluorescence in the fibrous sheath of sperm tails suggests that, in this case, the lack of AKAP4 could be pathogenically responsible for the DFS phenotype.
Partial deletions of Akap3 and Akap4 gene sequences could therefore be related to defective assembly of fibrous sheath components and failure of compartmentalization of AKAP3 and AKAP4 proteins in the tail, causing sperm immotility. AKAP-3 is involved in organizing the basic structure of the fibrous sheath, while AKAP4 has a major role in completing fibrous sheath assembly. Their absence is linked to sperm immotility because the fibrous sheath lacks any signal transduction system (Miki et al., 2002; Brown et al., 2003
).
Our findings suggest that, in some cases, the DFS sperm defect could be associated with alterations of Akap3 and Akap4 gene sequences.
Our results would require future confirmation. It would be useful to perform an immunoblot with the anti-AKAP4 antibody, in order to show whether there is any AKAP4 protein being formed.
Finally, FISH analysis highlighted an increased frequency of sex chromosome (XY) disomy in our patient, indicating a segregation anomaly at the first meiotic division. This rare combination of genetic and sperm defects is responsible for fertilization failure in vivo.
Intracytoplasmic sperm injection could be the only tool able to bypass the reproductive problem in these kinds of patients. Nevertheless, the possibility of transmission of different genetic anomalies to the offspring should be considered in genetic counselling before undergoing micromanipulative assisted reproduction.
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Acknowledgements |
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References |
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Baccetti B, Capitani S, Collodel G, Di Cairano G, Gambera L, Moretti E and Piomboni P (2001) Genetic sperm defects and consanguinity. Hum Reprod 16,13651371.
Baccetti B, Bruni E, Collodel G, Gambera L, Moretti E, Marzella R and Piomboni P (2003) 10,15 reciprocal translocation in an infertile man: ultrastructural and fluorescence in-situ hybridization sperm study: case report. Hum Reprod 18,23022308.
Baccetti B, Collodel G, Gambera L, Moretti E, Serafini F and Piomboni P (2005) Fluorescence in situ hybridization and molecular studies in infertile men with dysplasia of the fibrous sheath. Fertil Steril, in press.
Bernardini L, Martini E, Geraedts JP, Hopman AH, Lanteri S, Conte N and Capitanio GL (1997) Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severe male factor detected by in situ hybridization. Mol Hum Reprod, 3,431438.[Abstract]
Bhasin S, de Kretser DM and Baker HW (1994) Clinical review 64: Pathophysiology and natural history of male infertility. J Clin Endocrinol Metab 79, 15251529.[CrossRef][ISI][Medline]
Brown PR, Miki K, Harper DB and Eddy EM (2003) A-kinase anchoring protein 4 binding proteins in the fibrous sheath of the sperm flagellum. Biol Reprod 68,22412248.
Carrera A, Moos J, Ning XP, Gerton GL, Tesarik J, Kopf GS and Moss SB. (1996) Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. Dev Biol 180,284296.[CrossRef][ISI][Medline]
Chemes, HE. Phenotypes of sperm pathology: genetic and acquired forms in infertile men (2000) J Androl 21,799808.
Chemes HE and Rawe VY (2003) Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men. Hum Reprod 9,405428.[CrossRef]
Chemes HE, Brugo S, Zanchetti C, Carrere C and Lavieri JC (1987) Dysplasia of the fibrous sheath: an ultrastructural defect of human spermatozoa associated with sperm immotility and primary sterility. Fertil Steril 48,664669.[ISI][Medline]
Chemes HE, Olmedo SB, Carrere C, Oses R, Carizza C, Leisner M and Blanquier J (1998) Ultrastructural pathology of the sperm flagellum: association between flagellar pathology and fertility prognosis in severely asthenozoospermic men. Hum Reprod 9,25212526.[CrossRef]
Eddy EM, Toshimori K and OBrien DA (2003) Fibrous sheath of mammalian spermatozoa. Microsc Res Tech 61,103115.[CrossRef][ISI][Medline]
Krausz C, Forti G and McElreavey K (2003) The Y chromosome and male fertility and infertility. Int J Androl 26,7075.[CrossRef][ISI][Medline]
Martin RH, Spriggs E and Rademaker AW (1996) Multicolor fluorescence in situ hybridization analysis of aneuploidy and diploidy frequencies in 225846 sperm from normal men. Biol Reprod 54,394398.[Abstract]
Miki K, Willis WD, Brown PR, Goulding EH, Fulcher KD and Eddy EM (2002) Targeted disruption of the AKAP4 gene causes defects in sperm flagellum and motility. Dev Biol 248,331342.[CrossRef][ISI][Medline]
Repping S, Skaletsky H, Lange J, Silber S, van der Veen F, Oates RD, Page DC and Rozen S (2002) Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet 71,906922.[CrossRef][ISI][Medline]
Rives N, Mazurier S, Sibert L, Simeon N, Joly G and Mace B (1998) Incidence of aneuploidy in sperm nuclei of infertile men. Hum Reprod 13,126127, O245.
Simoni M, Bakker E and Krausz C (2004) EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State of the art 2004. Int J Androl 27,240249.[CrossRef][ISI][Medline]
Turner RM, Johnson LR, Haig-Ladewig L, Gerton GL and Moss SB (1998) An X-linked gene encodes a major human sperm fibrous sheath protein, hAKAP82. Genomic organization, protein kinase A-RII binding, and distribution of the precursor in the sperm tail. J Biol Chem 273,3213532141.
Turner RM, Foster JA, Gerton GL, Moss SB and Patrizio P (2001a) Molecular evaluation of two major human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82, in stump tail sperm. Fertil Steril 76,267274.[CrossRef][ISI][Medline]
Turner RMO, Musse MP, Mandal A, Klotz K, Jayes FCL, Herr JC, Gerton GL, Moss SB and Chemes HE (2001b) Molecular genetic analysis of two human sperm fibrous sheath proteins, AKAP4 and AKAP3, in men with dysplasia of the fibrous sheath. J Androl 22,302315.
World Health Organization (1999) WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 4th ed. Cambridge: Cambridge University Press.
Submitted on December 10, 2004; resubmitted on May 5, 2005; accepted on May 12, 2005.
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