1 Department of Zoology, University of Toronto, 25 Harbord St., Toronto, Ontario, Canada M4S 3G5, 2 Rosewell Park Cancer Institute, Department of Molecular and Cellular Biology, Buffalo, NY 14263-0001, USA and 3 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM, Université Louis Pasteur, Illkirch-Strasbourg, France, 67404
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Abstract |
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Key words: aneuploidy/immunocytochemistry/spermatid/spermatogenesis/testis
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Introduction |
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Approximately half of couples who present themselves at infertility clinics have problems that can be attributed to the male partner (Aitken and Stewart, 1996; Cooke et al., 1998
; Huang et al., 1999
; Rives et al., 1999
). Causes of male factor infertility include prior infections, primary endocrine failure and immunological disorders (Diemer and Desjardins, 1999
). However, the largest single class of male factor infertility cannot be attributed to any of these disorders and is classified as idiopathic (Elliott and Cooke, 1997
; Diemer and Desjardins, 1999
). Idiopathic infertile men routinely show symptoms of severe oligozoospermia or azoospermia (Diemer and Desjardins, 1999
).
While the underlying aetiologies of idiopathic azoospermia are unknown, attempts are being made to shed light on this issue. A significant proportion of azoospermia cases have been associated with microdeletions of the Y chromosome (Oliva et al., 1998), and many numerical or constitutional chromosome abnormalities can lead to a block in meiosis, presumably through pairing dysfunction (Diemer and Desjardins, 1999
). However, a large proportion of idiopathic azoospermia remains unexplained, leading some reviewers to speculate that as yet unidentified X-linked or autosomal mutations in genes involved in spermatogenesis may be the underlying cause (Elliott and Cooke, 1997
; Tuerlings et al., 1997
; Chandley, 1998
; McLachlan et al., 1998
; Okabe et al., 1998
). A vertical genetic study (Liliford et al., 1994
) has provided statistical support for this view.
Despite its success, concern over the use of immature germ cells in ICSI and round spermatid injection (ROSI) procedures has been raised, and long-term implications of this technique are still unclear. Success rates with ROSI and elongated spermatid injection are significantly lower than that with ICSI (Prapas et al., 1999), possibly due to the immature nature of the gametes being used (Sousa et al., 1998
). Another risk factor associated with these procedures is the possibility that gametes with chromosome abnormalities may be used for fertilization, thus generating embryos with unbalanced chromosome complements (Bernardini et al., 1998
). Chromosomally unbalanced embryos can result in early spontaneous abortion, stillbirth, or birth with severe developmental and/or congenital problems (Giltay et al., 1999
). Many studies have reported elevated levels of aneuploidy in sperm from infertile men (Colombero et al., 1999
; Pfeffer et al., 1999
; Huang et al., 1999
; Rives et al., 1999
; Ushijima et al., 2000
). Further, paternally derived aneuploidy has been reported to be a leading cause of pregnancy loss, particularly in the case of (45,X) embryos (Hassold, 1998
). Studies have been published which attempt to assess the effects and success of these procedures specifically in the context of these risk factors in humans (Feichtinger et al., 1995
; In't Veld et al., 1995
; Kurinczuk and Bower, 1997
; Mansour et al., 1997
; Bonduelle et al., 1998
; Munné et al., 1998
).
We report here the results of an analysis performed on spermatids retrieved from testicular cell suspensions from three different types of infertile male mice whose sterility is genetically based. PP1c-/- males are completely sterile, with reduced production of condensing and elongating spermatids, very few mature testicular sperm, and no sperm in the epididymides (Varmuza et al., 1999
). CREM -/- males show complete arrest at the round spermatid stage (Blendy et al., 1996
; Nantel et al., 1996
). C57BL/6J.MAC17023 males are sterile with a histopathological profile similar to PP1c
-/- males (R.Elliott, unpublished data). The absence of sperm in all three types of males led us to devise an alternative methodology for assessing haploid chromosome numbers based on immunocytochemical detection of centromeres in round spermatids. We show that the levels of numerical chromosome abnormalities in the spermatids from two of the three different sterile males are elevated. Our observations are consistent with the hypothesis that a compromised testicular environment may play a role in the increased levels of chromosomal abnormalities (Mroz et al., 1998
), but that not all types of sterility increase the frequency of meiotic non-disjunction.
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Materials and methods |
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Immunocytochemistry
The CREST antiserum, obtained from patients with Calcinosis Raynaud's phenomenon Esophageal dysmotility Sclerodactyly Telangiectasia, a syndrome of scleroderma, recognizes an epitope in kinetochores and produces a distinctive, and highly reproducible, punctate signal in immunocytochemical preparations. The number of signals reflects indirectly the number of chromosomes (Figure 1). The reliability of the methodology makes it possible to obtain large amounts of data with very small amounts of material. Because round spermatids are the most abundant cell type in testicular cell suspensions, it is possible to perform extensive analyses on small amounts of testicular tissue.
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Data analysis
Slides were examined under fluorescent light at x1000 using oil immersion objectives on an Olympus BX60 microscope. Spermatids were identified as those cells that stained positive for Histone H1T and CREST, negative for COR-1, and positive for an autofluorescent acrosome signal (see Figure 1). Spermatids represent approximately half of all non-condensed cells in the testicular cell suspension, with the other half comprising diploid, tetraploid and octaploid germ cells in various stages of mitosis and meiosis, and somatic cells. Spermatids with indistinct or excessively clumped centromeres, or overlapping or very closely apposed cells were not recorded. Slides were systematically scanned until 100 images per subject had been captured. All scorable spermatids in a field of view were recorded to reduce observer bias in sampling. The images were captured and recorded using a CCD camera and stored on writeable compact discs as TIFF files. Some images contained several well-separated spermatids in the field of view. These spermatids were scored individually. Following transformation of images into greyscale, centromeres were counted manually and recorded using Quantity One image analysis software (BioRad Laboratories, Hercules, CA, USA). The number of mice assayed in each group was 9 (+/+), 9 (+/-), 14 (-/-), four (CREM-/-) and five (B6.MAC).
Scoring criteria were based on the high degree of uniformity of CREST signals from cell to cell, and experiment to experiment. Many cells contained centromeres that were close together. However, shape and intensity of the aggregate signals were used as cues for scoring clumped centromeres (Figure 2). Singletons in each field acted as internal controls for size and intensity. No correlation between clumping and chromosome scores was detected (P = 0.712).
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Results |
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Some of the observed variation may be skewed by the high numbers of centromere signals in some cells. Therefore it was informative to look at frequencies of cells with numerical chromosome abnormalities rather than variation in centromere numbers. However, the actual rate of aneuploidy cannot be determined by our approach because of the uncertainty in distinguishing experimental error from biological chromosome abnormality. Only comparative rates of numerical chromosome abnormalities can be estimated in relation to the wild-type group, if it is assumed that all groups are subject to the same degree of experimental error (debris on the slide, addition or loss of chromosomes from burst nuclei, overlapping centromeres, counting errors).
We adopted a conservative approach to establishing `normal' numbers of centromere signals by arbitrarily setting a range of the mean ± 2 SD of the wild-type data. This convention revealed rates of numerical chromosome abnormality of 4.16% (wild type), 3.78% (PP1c+/-, and 15.39% (PP1c
-/-).
2-Analysis revealed that only the mutant PP1c
-/- numerical chromosome abnormality rate was statistically significant, even after accounting for diploidy by removing cells with
35 centromere signals, or cells with
30 centromere signals from the data set (Table II
). Interestingly, the rate of chromosome abnormality declined markedly after removing potentially diploid cells from the data set. This observation is consistent with the presence of both aneuploid and diploid spermatids in the PP1c
-/- gamete pool.
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The frequency of numerical chromosome numbers in CREM-/- spermatids was 13.06%. This elevated frequency is statistically significantly different from wild type, even after removal of potentially diploid cells (Table II). The latter exercise had a much less dramatic effect on the proportion of chromosomally abnormal cells in CREM-/- spermatids than in PP1c
-/- spermatids, suggesting that there are far fewer diploid cells in these mice. This may be an artefact of image capture if diploid or polyploid spermatids do not display the same features (histone H1T positive, presence of autofluorescent acrosome) as haploid spermatids. Interestingly, spermatids from B6.MAC mice displayed a low frequency of numerical chromosome numbers (4.47%) that was not statistically different from wild type (Table II
).
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Discussion |
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The variation in chromosome numbers in PP1c-/- spermatids was significantly elevated when compared with wild-type controls. These results indicate that a large proportion of germ cells in PP1c
-/- males is either aneuploid or diploid. While our approach did not allow us unequivocally to measure absolute rates of aneuploidy, the frequency of spermatids in the PP1c
-/- sample with numerical chromosome abnormalities was 3.7-fold higher than the frequency in the wild-type sample (4.16 versus 15.39%). Even after removal of potentially diploid cells from the data set, a significantly elevated frequency of cells with abnormal chromosome numbers was observed. Heterozygous PP1c
+/- spermatids were indistinguishable from wild type when the frequency of abnormal cells was compared.
The increased level of aneuploidy/diploidy observed in PP1c mutants may have been a direct consequence of the mutation, since it is known that type 1 protein phosphatases are involved in both meiosis and mitosis in yeast and fruit flies (Ohkura et al., 1989
; Axton et al., 1990
; Baksa et al., 1993
; Tu et al., 1996
). Alternatively, altered testicular environment may have contributed to numerical chromosome abnormalities by adversely affecting some aspects of meiosis. We investigated this latter hypothesis by examining two other strains of sterile mice that appear to progress through meiosis, and arrest or become dysfunctional during spermiogenesis. Both CREM-/- and B6.MAC spermatids displayed increased variation in chromosome numbers when compared with wild type. However, when frequencies of abnormal cells were analysed in these two data sets, only CREM-/- spermatids continued to display statistically significant differences from wild type. These results argue that testis pathology plays an important role in maintaining normal meiotic divisions, but that different mutations may have different effects on the general pathology.
It was evident from both the quantitative data and the visual data that some proportion of PP1c spermatids was diploid. In contrast, very few spermatids from CREM-/- and B6.MAC mice appeared to be diploid. This observation argues further for different responses of testicular pathology to different genetic lesions. In the case of the PP1c
mutation, the presence of both diploid and aneuploid spermatids argues for two defective responsesone involving failure of one meiotic division in a small proportion of spermatocytes, and another involving non-disjunction in a somewhat larger proportion of spermatocytes.
A large number of studies have recently applied the technique of multicolour FISH using centromeric probes specific for a small subset of chromosomes to assess aneuploidy in sperm of infertile men. The majority of these studies suggest that germ cells derived from infertile males have a higher incidence of chromosomal abnormalities compared to normal (fertile) men (Colombero et al., 1999; Pfeffer et al., 1999
; Huang et al., 1999
; Rives et al., 1999
; Ushijima et al., 2000
). However, other studies have reported no statistically significant difference (Damri et al., 2000
; Martin et al., 2000
). These studies may reflect variation in the aetiology of infertility among the subjects, rather than inherent methodological differences in the analyses, although the impact of the latter cannot be ignored. Nevertheless, the potential risk of using aneuploid gametes for ICSI when the male partner displays abnormal spermatogenesis cannot be underestimated. One of the limiting factors in these studies is availability of suitable material for analysis (i.e. sperm). For men with azoospermia, FISH studies on sperm are not possible. It may prove useful therefore to adapt the procedure we have described here, since it is possible with a very small testicular biopsy to obtain sufficient round spermatids for an extensive analysis (S.Varmuza, unpublished data). Suitable markers that would identify early stage round spermatids would need to be used. The autofluorescent acrosome marker that we found so very useful in our studies of mice was a specific feature of the particular batch of CREST antiserum used throughout the study, and is not seen with other CREST antisera (S.Varmuza, unpublished data).
Some studies have shown that children born from ICSI may be at risk for increased levels of sex chromosome aneuploidy (In't Veld et al., 1995; Andrews and Elster, 1998
; Bonduelle et al., 1998
; Givens, 2000
), whereas others report no significant increase in risk of aneuploid embryos (Feichtinger et al., 1995
; Munné et al., 1998
). However, a prenatal chromosomal analysis performed on ICSI fetuses (Van Opstal et al., 1997
) determined that all of the sex chromosome aneuploidy (six out of 71 ICSI-derived fetuses) was of paternal origin. None of these studies, however, distinguished between male partners with normal spermatogenesis and those with abnormal spermatogenesis. The possibility therefore exists that germ cells acquired from males suffering from non-obstructive azoospermia are less capable of supporting development and producing normal children than ICSI procedures performed on obstructive azoospermic patients where spermatogenesis appears normal (Chandley and Hargreave, 1996
; Lange et al., 1997
; Zech et al., 2000
). Further, Hassold (1998) reports that paternally derived aneuploidy involving the X chromosome (45,X) is one of the leading known causes of spontaneous pregnancy loss.
It should be noted that the centromeres in CREM-/- round spermatids appeared slightly more ragged than those in spermatids from wild-type or PP1c mutant testes. This is a function of the early block in spermiogenesis; very early spermatids in wild-type testicular cell preparations with a similar appearance can be found at much lower frequency. However, these cells are passed over for image capture in favour of the more advanced spermatids with a strong autofluorescent acrosome and more distinct centromeres. In recording CREM mutant spermatids, attempts were made to choose those cells with the most distinct centromeres. Inter-observer variation in scoring was slightly higher for the CREM images than for the PP1c
images, but was lower than for the B6.MAC images, which had clearer centromere signals.
Quantitative data produced by visual scoring of cytological characteristics such as our centromere signals, or FISH signals, are inherently error-prone, especially where subjective evaluation of spots on a slide is required. Even with guidance by strict scoring criteria, it is inevitable that some error will creep in. This point is well illustrated by the high variability in aneuploidy rates reported by various laboratories (Shi and Martin, 2000). We would argue that cytological approaches such as ours, and FISH, are most useful in comparative studies, and perhaps less useful in establishing absolute rates of aneuploidy.
We have described a series of experiments in which chromosome numbers in round spermatids from infertile male mice were quantified using immunofluorescent localization of centromeres. An elevated level of numerical chromosome abnormalities was observed in two of three different sterile strains. Our results are consistent with the hypothesis that abnormal testicular environment can adversely affect meiosis, leading to elevated non-disjunction. This observation suggests that the use of ICSI for treatment of male factor infertility may carry an increased risk of producing a chromosomally unbalanced zygote. Adaptation of the methodology described here to analysis of human testicular biopsies may provide an additional level of understanding of the risk of aneuploidy.
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Acknowledgements |
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Notes |
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References |
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Submitted on June 6, 2001; accepted on November 2, 2001.