1 Service d'Histologie Embryologie Cytogénétique, Hôpital Jean Verdier, 93140 Bondy, 2 Service d'Histologie Embryologie Cytogénétique, Hôpital Robert Debré, 75019 Paris, 3 Service d'Histologie Embryologie, Faculté de Médecine Léonard de Vinci, 93017 Bobigny and 4 Service de Médecine de la Reproduction, Hôpital Jean Verdier, 93140 Bondy, France
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Abstract |
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Key words: chromosome abnormality/FISH/human spermatozoa/male infertility/spermatogenesis
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Introduction |
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In man, different authors have reported that morphological abnormalities in sperm heads are associated with defects of their chromosome content (Hassold et al., 1996; Lee et al., 1996
; Martin et al., 1996
). In't Veld and colleagues have described a case exhibiting unusual spermatozoal morphology with large sperm heads and multiple tails. Subsequent molecular cytogenetic analysis revealed a high level of polyploidy (In't Veld et al., 1997).
Here we describe a family in which two infertile brothers present with similar sperm abnormalities.
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Case report |
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Materials and methods |
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Ultrastructural study of spermatozoa by transmission electron microscopy (TEM)
After semen liquefaction spermatozoa were fixed for 1 h in 3% glutaraldehyde (550 mOsm) at room temperature. After centrifugation (10 min, 250 g), the pellet was embedded in agar. Post fixation was performed for 1 h in 4% osmic acid at 4°C. After dehydration in a graded series of ethanol, small pieces of agar were embedded in Epon, and sections 100 nm thick were cut on an ultramicrotome. They were stained with uranyl acetate and lead citrate, and analysed in a Philips EM 300 transmission electron microscope, at increasing magnifications from x500040 000.
Cytogenetic evaluation
Karyotype
Chromosome analysis was performed on peripheral blood lymphocytes; The slides were stained by Giemsa after G and R banding.
Semen preparation for FISH
Two ejaculates were washed three times in phosphate-buffered saline (PBS: 150 mmol/l NaCl, 10 mmol/l sodium phosphate, pH 7.2) and centrifuged at 280 g for 10 min (Martini et al., 1998).
The pellet was resuspended carefully in 1ml of fresh cold fixative solution (methanol:acetic acid, 3:1) and stored at -20°C for 1 h. The samples were then dropped onto clean microscope slides and air-dried (Chevret et al., 1995).
The slides were washed twice in standard saline citrate (SSC) for 30 min and then dehydrated in ethanol and air-dried. Sperm heads were decondensed for fluorescence in-situ hybridization (FISH) in 3 mol/l NaOH for 8 min. After washing in cold water, the slides were air-dried.
DNA probes for FISH
DNA probes coding for chromosomes X, Y, and 18, directly-labelled with fluorescent haptens (Vysis, Naperville, IL, USA), were used. The probes for chromosomes Y (DYZ1) and X (DXZ1) were labelled with red (CEP Spectrum orange; Vysis) and green (CEP Spectrum green; Vysis) fluorescent haptens respectively. The chromosome 18 probe was labelled with blue (CEP Aqua; Vysis). The use of an autosomal probe, in addition to X and Y probes, allowed the distinction between disomy and diploidy.
FISH studies
10 µl of the probes solutions were placed onto a microscope glass slide at 37°C. The slides then were covered with a coverslip, and incubated overnight at 37°C for hybridization. After post-hybridization washes in 0.4xSSC at 72°C for 2 min, slides were transferred to 1xSSC containing 0.1% Tween 20 and then counterstained with 4(,6-diamidino-2-phenylindole) (DAPI).
Slides were analysed using a Zeiss Axiophot equipped with a camera and connected to an Imaging System package (Applied Imaging, Newcastle-upon-Tyne, UK). A triple band pass filter for FITC, Texas red and DAPI was used for the simultaneous visualization of orange, green and DAPI. The aqua filter was used to visualize the blue spots. Hybridization signals were scored in the two separate ejaculates. Only spermatozoa with well defined boundaries were scored and signals in a specific colour were considered to be multiple when separated by at least one signal diameter, (Spriggs et al., 1995; Egozcue et al., 2000
; Soares et al., 2001
). DAPI-stained spermatozoa with no FISH signals were eliminated.
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Results |
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Ultrastructural sperm characteristics
Abnormalities observed by TEM were related to those observed by light microscopy. Examination of sperm heads showed the presence of 2 or 3 vacuolated nuclei, each nucleus being delimited by a nuclear membrane. The acrosome was sometimes partial. Nuclear chromatin was well condensed (75% of mature cells) and appeared rough. In some spermatozoa, a large amount of cytoplasm containing vacuoles was observed around the nucleus and the acrosome (Figure 2A,B,C,D). At the junction between head and midpiece up to 3 implantation fossae for one nucleus could be observed. The midpiece of each flagella showed all the expected structures with an axoneme associated with a set of 9 outer dense fibres.
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Cytogenetic Results
Karyotype
From an average of 20 analysed metaphase spreads, the patient's karyotype was considered to be normal.
FISH
A total of 390 spermatozoa was analysed from the first ejaculate, and 758 from the second. All the cells appeared polyploid, as a result of the nuclear abnormalities observed in light and electron microscopes. The green signal on the X chromosome and the red signal on the Y chromosome could be distinguished in the spermatozoa without any background (Figure 3ad). The aqua signal on chromosome 18 was stronger than the other spots and sometimes divided into fragments, but it was always interpretable. Among the 1148 spermatozoa observed, 241 were diploid (21.6%), 717 were triploid (62.4%), 160 were quadriploid (13.3%) and 30 (2.7%) were hyperploid (>4n). The specific signal corresponding to chromosome 18 confirmed the presence of diploid and hyperploid sperm cells.
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Discussion |
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Macrocephalic spermatozoa can be seen in several situations such as division failure during meiosis I or II or in cytoplasmic events like a disturbance of the sperm centriole or a cytokinesis defect (Escalier et al., 1993).
Because several nuclei were localized in the same cytoplasm, a disturbance of the first or the second or both meiotic divisions could explain the abnormal size of the heads and the excess of cellular components. Indeed, despite meiotic failure, spermatid differentiation can occur and results in dysmorphic spermatozoa with macrocephaly and multiple flagella. It has been reported in mouse and in drosophila that spermatid development can be achieved without completion of meiosis (Mori et al., 1999).
In man, Escalier showed that human tetraploid spermatozoa (named large heads) can differentiate into spermatids in spite of meiotic division alterations (Escalier, 1999a,b
). In the present study, the high rate of triploid spermatozoa (62.4%) is not consistent with a meiotic chromosomal segregation impairment which should lead preferentially to diploid or tetraploid sperm cells. Although extrusion of one nucleus cannot be excluded, triploid sperm cells are unlikely to arise from a defect during the meiotic chromosomal segregation process.
As suggested by the ultrastructural data from the spermatozoa with multiple nuclei, failure in cytokinesis could be responsible for the abnormal sperm cell phenotype in our patient. Genetic studies in animals have highlighted specific genes which might play a role in the function and regulation of both mitotic and meiotic division spindles. The beta tubulin isoforms are required for good meiotic spindle architecture (Maines and Wasserman, 1998). Mutations in the beta 2-tubulin gene disturbed both meiosis and spermatid differentiation (Hoyle and Raff, 1990
). In these mutants, chromosomes condensed normally but did not align at metaphase and failed to migrate to the opposite poles. Spindles were absent, cytokinesis did not occur and the nuclear envelope thickened. In humans, patients have been reported with macrocephalic spermatozoa apparently related to spermatogenic arrest at the meiosis stage (Escalier, 1983
). In our study, a cytokinesis defect cannot be excluded. A testicular biopsy would have been valuable to confirm this, but the two brothers declined this.
Other gene mutations have also been shown to lead to similar defects. In Drosophila melanogaster mutations diaphanous (dia) and peanut (pnut) and mutations of the gene coding for the kinesin-like protein (KLP3A) are associated with spermatids containing two or four nuclei of normal size (Maines and Wasserman, 1998). Since the proteins encoded by these genes localize to the contractile ring and intercellular bridge of dividing cells, they could be good candidates for explaining morphological abnormalities observed in the proband's spermatozoa. However, no mutations of these genes have been described in man.
In mice, spermatids containing >2 nuclei have been described in the recessive insertional mutation symplastic spermatids (sys). Homozygous males form spermatozoa exhibiting a multinucleated syncytia which fail to complete their differentiation leading to azoospermia (MacGregor et al., 1990). Although chromatin condensation and flagellar growth do not occur in the sys/sys phenotype, a defect in the formation of intercellular bridges that connect spermatids could also be responsible for the proband's sperm abnormalities. Unfortunately, DNA was no longer available to search for them in our patient.
Pedigree in our case is actually consistent with a familial genetic defect, the mode of inheritance of which remains to be elucidated. Despite the lack of consanguinity between the proband's parents, the association of several perinatal deaths and two identical established cases of male infertility could be due to a mutation present either in a homozygous or a heterozygous state in the offspring. Indeed, the former could be lethal while the later could be restricted to spermatogenesis, maybe by haploinsufficiency. Therefore, such a mutation might be considered as dominant with variable expressivity and incomplete penetrance as suggested by two other cases of male infertility in the proband's maternal uncles.
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Acknowledgements |
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Notes |
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References |
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Submitted on June 7, 2001; accepted on August 23, 2001.