Mol. Hum. Reprod. Advance Access originally published online on May 3, 2007
Molecular Human Reproduction 2007 13(7):455-460; doi:10.1093/molehr/gam024
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Development of a non-human primate sperm aneuploidy assay tested in the rhesus macaque (Macaca mulatta)
1 School of Veterinary Medicine, Department of Population Health and Reproduction, University of California Davis, One Shields Avenue, Davis, CA, USA 2 California National Primate Research Center, University of California Davis, Davis, CA, USA
3 Correspondence address. Tel: +1 530 752-7127; Fax: +1 614 386 8611; E-mail: lfroenicke{at}ucdavis.edu
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Abstract |
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Numerical chromosome aberrations in germ cells are important factors contributing to abnormal reproductive outcomes. Fluorescence in situ hybridization onto spermatozoa (sperm-FISH) has allowed the study of the influence of a wide range of biological factors and chemical exposure on aneuploidy incidences in human sperm as well as in mouse and rat animal models. The assay presented here extends the applicability of the sperm-FISH method to non-human primates and was tested in the prevalent model species, the rhesus macaque. The assay provides probes for macaque chromosomes 17, 18, 19, 20, X and Y, the homologues of human chromosomes 13, 18, 19, 16, X and Y, respectively. The analysis of 11 000 spermatozoa each from five individuals revealed spontaneous sex chromosomal disomy frequencies (X: 0.08%; Y: 0.09%) and an average autosomal disomy frequency (0.03%) coinciding with some of the lowest incidences scored in human studies. The non-human primate sperm-FISH assay provides a fast and efficient tool complementing the available analysis methods in non-human primate exposure studies. Since the assay employs large locus-specific FISH probes representing evolutionary conserved DNA sequences, it can be expected that the assay is also applicable to other cercopithecoid and hominoid non-human primate species.
Key words: cross-species fluorescence in situ hybridization/sperm-FISH/chromosome abnormalities/infertility
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Introduction |
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Numerical and structural chromosomal aberrations in sperm are considered to be main factors contributing to infertility, early embryonic losses and aneuploidy syndromes. The average incidence of aneuploid sperm in healthy men is estimated to be about 6.5% (Egozcue et al., 1997) and it has been shown that the aneuploidy incidence is elevated in association with risk factors such as age (Rousseaux et al., 1998), medical drugs (Frias et al., 2003) or even lifestyle factors like caffeine or alcohol consumption and smoking (Robbins et al., 1997, 2005).
In the last 10 y, virtually all sperm aneuploidy investigations have been carried out by consecutively applying sperm decondensation and fluorescence in situ hybridization (FISH) techniques (Guttenbach and Schmid, 1990; Wyrobek et al., 1990). The introduction of these techniques has greatly improved the speed and the feasibility of sperm analysis for chromosomal aberrations as compared to earlier methods involving human-sperm/hamster-oocyte hybrids (for review see Wyrobek et al., 2005). In humans, sperm-FISH studies have revealed associations of numerical chromosomal abnormalities with paternal risk factors such as age, drugs and environmental and occupational exposures. Although the majority of sperm-FISH experiments have been conducted on human specimens, sperm-FISH assays have been developed for species as diverse as hamster (Ward et al., 1996), pig (Kawarasaki et al., 1998; Rubes et al., 1999), cattle (Rens et al., 2001), river buffalo, goat and sheep (Di Berardino et al., 2004) and mink (Christensen, 1998). In general, these studies have been very limited and have focused mostly on the determination of the sex chromosome content. The only extensive studies in animal models have been undertaken with sperm-FISH assays developed for mice (Adler et al., 1996) and rats (Lowe et al., 1998). In a single case it has been possible to compare the effects of chemicals on the chromosomal content of sperm in different species (Baumgartner et al., 2001). This study revealed that the sensitivity of spermatocytes to diazepam is 10–100-fold higher in humans as compared to mice. Although a singular finding, this comparative study indicates that non-human primate sperm-FISH assays would be of great value to allow for data of animal models physiologically much closer to humans. To our knowledge, a recent investigation by O'Brien and collaborators (2005) is the first report of sperm-FISH analyses in non-human primate models. In this study successful hybridizations were carried out with probes corresponding to three different human chromosomes (18, 21 and X) on gorilla sperm nuclei as well as experiments with two probes corresponding to the human Y chromosome and chromosome 21 to hamadryas baboon and common marmoset sperm nuclei (O'Brien et al., 2005). Similar to some other animal sperm-FISH studies, the main focus of this investigation was to determine X/Y chromosome ratio in flow-sorted sperm.
Thus, currently there is no sperm-FISH assay available that would be applicable to a greater variety of non-human primate species and that is able to interrogate both sex chromosomes and a larger set of autosomes. Such an assay would be a prerequisite for the assessment of paternal risk factors in non-human primate models since the limitations of the sperm-FISH method requires the use of internal controls. For example, a minimum of two probes are needed to confidently evaluate autosomal aneuploidies and three probes to analyse gonosomal aneuploidies (the two sex chromosomes, one autosomal control; Egozcue et al., 1997; Shi and Martin, 2000). To facilitate non-human primate sperm aneuploidy testing, we have developed a six chromosome sperm-FISH assay and have established a protocol for the analysis of rhesus macaque sperm samples. The rhesus macaque is the most important non-human primate animal model in biomedical research. However, by design the new sperm-FISH assay should also be applicable to other cercopithecoid and hominoid non-human primate species since the probe sets are assembled of human bacterial artificial chromosome (BAC) pools representing evolutionarily highly conserved DNA sequences. To assess the performance of the assay and to establish a baseline of chromosomal aneuploidy incidences in rhesus macaques we have scored aneuploidy incidences for all chromosomes represented in the assay in 11 000 spermatozoa of four or five individuals. In addition, differences between ejaculates from the same individuals have been investigated.
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Collection of sperm and preparation for in situ hybridization
The rhesus macaques (Macaca mulatta) were individually housed at the California National Primate Research Center (CNPRC). CNPRC is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC); all animal protocols and experiments were reviewed and approved in advance by the Institutional Animal Care and Use Committee. Semen was collected from five adult 5–16-y-old rhesus macaques by electroejaculation (Sarason et al., 1991). Semen was allowed to stand at room temperature for 30 min and the liquid portion was transferred to a fresh centrifuge tube and washed three times in 15 ml Tyrode's medium supplemented with bovine serum albumin (TL-BSA) medium at 400 x g as previously described (Schramm and Bavister, 1994). After an additional wash in phosphate-buffered saline (PBS)/0.5 mM EDTA the sperm cells were stored at –20°C. As a biological safety precaution, the thawed samples were fixed in 70% methanol for 2 h before transport. Experiments included decondensation and hybridization protocols according to the most often used technique developed by Robbins et al. (1993) and a protocol employing decondensation and denaturation through NaOH treatment (Malan et al., 2006). For the majority of experiments we followed the latter protocol with slight modifications as follows: The methanol was removed and the sperm cells were washed once in PBS and then resuspended in PBS/0.1% Tween20. Ten µl aliquots of these suspensions were spread onto ethanol pre-cleaned microscope slides and allowed to air-dry. The addition of the Tween detergent allowed for a more even spreading of the sperm. The spreads were fixed by a 10 min incubation in methanol/acetic acid (3:1) at room temperature and allowed to air-dry. The sperm heads were decondensed and denatured by a 3 min incubation in 1 M NaOH, and rinsed consecutively in distilled water, 0.2 x PBS, again in distilled water and then air dried.
DNA probes
Four to six human BAC DNAs were pooled for use as FISH probes for each of the human chromosomes 13, 16, 18, 19, X and Y. BACs were selected from the RPCI-11 and RPCI-13 libraries (Osoegawa et al., 2001) to represent highly conserved regions of the genome as delineated by genome conservation scores in the UCSC Genome Browser (Kent et al., 2002). At the time of the selection, the conservation data were based on human and mouse genome sequence data. In a browser, view displaying the complete length of a BAC even the selected Y chromosome BACs achieved a maximum conservation rating for about 20% of their length. The BACs were chosen to represent centromere near regions and are spanning genomic distances of up to 1.2 Mbp per pool.
BAC DNAs were extracted using the PhasePrep BAC DNA kit (Sigma, St. Louis, Missouri) and then PCR amplified using a simplified version of the tagged-PCR protocol (T-PCR; (Grothues et al., 1993). The PCR products of BAC DNA templates generated by this protocol display a homogenous smear, free of bands, after agarose gel electrophoresis, indicating a more uniform amplification of the target DNA than can be achieved by degenerate oligonucleotide primed PCR primer (DOP)-PCR. Briefly, the primary PCR was conducted from 20 ng BAC DNA in 50 µl of the following reaction mixture (final concentrations): 1 x TAPS2-PCR buffer (Fiegler et al., 2003), 200 µM dNTPs, 2.5 mM MgCl2, 0.1% BSA, 0.6 U TAQ (AbGene, Epsom, United Kingdom) and 0.2 µM degenerated R1 primer (TAG CTC TTG ATC AGA GGN NNN S). The first part of the primary PCR was conducted for 5 min at 94°C and six cycles of 40 s at 94°C, 3 min at 15°C, 30 s at 22 °C, 30 s at 35°C and 1.5 min at 72°C. After the addition of 1 µl 100 µM R2 primer the second part of the primary PCR was run with seven cycles of 40 s at 94°C, 1 min at 45°C and 1.5 min at 72°C and 20 cycles of 40 s at 94°C, 1 min at 58°C and 1.5 min at 72°C. Three microliter of these BAC probes were labeled during a secondary 50 µl PCR reaction with either biotin-16 dUTP (Biotium, Hayward, California), digoxigenin-11-dUTP (Roche) or Cy5-dUTP (Amersham, Piscataway, New Jersey) under the same conditions as the primary PCR except that only primer R2 was used, and the dNTP concentrations were modified as described previously (Froenicke et al., 2002). The secondary PCR was run at 30 cycles of 40 s at 94°C, 1 min58°C and 1.5 min at 68°C. The BAC probes were hybridized individually to rhesus macaque chromosome preparations prepared from lymphoblastoid cells (Rabin et al., 1977) to map their signals to macaque chromosomes and to check for non-ambiguous FISH signals. Labeling of the T-PCR probes with DNP-11-dUTP (Perkin Elmer, Waltham, Massachusetts) was carried out with a bioprime random priming kit (Invitrogen, Carlsbad, California) as described by the manufacturer with the exception that biotin-16-dUTP was replaced by the DNP-dUTP. FISH probes producing strong signals on rhesus macaque chromosomes were assembled by pooling primary PCR products of BACs representing the following human chromosome regions: 13q14 (930N23, 1069N3, 245D16, 78J21, 435C23); 16p11 (301D18, 114A14, 612G2, 1079F19, 2C24); 18q21 (360B14, 940O22, 430M13, 715O3, 976G14), 19q13 (880C20, 447A19, 662E5, 664M2, 787F3), Xq13 (26D14, 528B10, 291O7, 753F2, 485H23, 624G23) and Yq11 (125B2, 460B21, 113K10, 1093F5, 936M3). All BACs except 26D14 originated from the RPCL-11 BAC library. These BAC pool probes were labeled in secondary PCR reactions as described above and differentially labeled probes (0.5 µg each) were combined to two- or three-color probe sets by co-precipitation along with 20 µg of human Cot-1 DNA (Invitrogen) as described previously (Froenicke et al., 2002).
FISH and analysis
The hybridizations were conducted as described by Robbins et al. (1995) except that the hybridization solution consisted of 50% formamide/2 x SSC and a 30 min pre-annealing incubation of the denatured probes at 37 °C was employed before a 36 h-long hybridization. The post-hybridization washes in formamide-free buffer and the detection of the digoxigenin-, DNP- and biotin-labeled probes with rhodamine conjugated goat antibodies (Roche, Indianapolis, Indiana), FITC conjugated rabbit antibodies and Alexa488 conjugated avidin (both Invitrogen), respectively, were carried out as otherwise described by Scherthan et al. (1994). Slides were analysed using an Olympus BX60 microscope equipped with a monochrome CCD camera (Cohu 4912; COHU, San Diego, CA, USA) connected to Applied Imaging (San Jose, CA, USA) software. Appropriate fluorescence filter sets for FITC/Alexa 488, Rhodamine, Cy5 and DAPI and a triband filter (DAPI, FITC and Rhodamine) were used to visualize the signals. For each sperm, sample hybridization signals were scored for at least 2000 non-overlapping sperm heads with clearly defined borders. Spermatozoa without FISH signals were not included into the sex chromosome analysis as were spermatozoa displaying two signals of the chromosome 19 control probe (diploid cells). The autosomal chromosomes were scored in two-color FISH experiments combining differentially labeled probes for human chromosomes (HSA) 13 and 16 as well as HSA18 and 19, respectively.
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Sperm-FISH protocol
All six chromosome specific FISH probes assembled from BAC pools produce strong spot-like FISH signals, which can easily be distinguished from the background fluorescence in hybridizations onto spermatozoa. In three-color assays using human chromosomes probes X, Y and 19, the signal strength is sufficient to score the Alexa 488 and rhodamine labeled sex chromosome probes simultaneously using a triband filter set, speeding up the scoring considerably. The hybridization efficiency of the protocol was higher than 99% for each of the probes. Thus, the BAC pool probes representing HSA 13, 16, 18, 19, X and Y do provide an efficient assay to assess aneuploidies of the orthologous macaque chromosomes (MMUL) 17, 20, 18, 19, X and Y, respectively. The strongest FISH signals were achieved by a protocol employing two denaturation steps; the NaOH treatment and a subsequent standard FISH denaturation in 70% formamide/2 x SSC at 76°C. This treatment resulted in approximately a 1.5–2-fold enlargement of the sperm head. The sperm decondensation protocol most often used in human assays, involving dithiothreitol and diiodosalicilate treatments (Robbins et al., 1995), failed to produce an enlargement of the macaque sperm heads and resulted in unspecific FISH signals. These problems could be due to the additional methanol fixation step required for this study or they might indicate differences in the chromatin structure between rhesus macaques and human sperm. In hybridizations to metaphase chromosomes the probes produce strong unique signals on each chromatid in centromere near regions in both human and rhesus macaque karyotypes (Fig. 1). In interphase cells single or double spots are observed as expected for G1 or G2 phase cells, respectively (Fig. 1).
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Aneuploidy incidences
Eleven thousand spermatozoa from five rhesus macaques were analysed with a three-color FISH assay employing X chromosome (DNP-label; FITC detection) and Y chromosome (dig-label; rhodamine detection) probes while using the HSA19 probe (biotin-labeled; Cy5 detection) as internal ploidy control (Fig. 1a and b). Since the far-red Cy5 signals are only visible upon digital image capture, the HSA19 signals were only analysed for cells displaying abnormal sex chromosome signals. Nullisomies of the sex chromosomes were observed with an incidence of 0.19% (Table 1). Disomies of the X and Y chromosomes were detected in 0.06 and 0.08% of the sperm, respectively. Diploid sperm made up 0.04% of the analysed sperm samples whereas haploid sperm containing both X and Y chromosomes made up 0.05% (Table 1). In experiments with the autosomal FISH probes on 11 000 spermatozoa from four macaques (Fig. 1c and d; Table 2) an average disomy frequency of 0.034% per autosome was observed. For all probes no significant inter-individual differences in the aneuploidy incidences were noted.
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The analysis of three further semen collections from two individuals in consecutive weeks investigated the variability of between ejaculates detected only very minor differences in aneuploidy incidences (Table 3). Semen of these individuals had also been collected weekly or twice weekly for other projects in the months before the sperm-FISH samples were taken.
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Discussion |
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Applicability of the sperm-FISH assay
The sperm-FISH aneuploidy assay presented here provides an efficient way of screening for chromosomal aneuploidies by means of cross-species hybridization with human probes. Since the probes, assembled from pooled BAC DNAs, produce strong and discrete signals they fulfill the criteria for FISH probes preferred for aneuploidy assays as described in Egozcue et al. (1997). The probes generate spot like signals (Fig. 1) as they span only regions of up to 1.2 Mbp per chromosome. Such genomic distances cannot be resolved by conventional FISH to chromosomes or to sperm heads. Since the probes do map close to the centromeres in all cases, the assay can be expected to be minimally affected by clastogenic agents and should be well suited to delineate meiotic or mitotic segregation failures. The probe set is not only applicable to sperm studies but should be equally well suited for interphase FISH applications like aneuploidy studies in uncultured amniocytes (Lev et al., 2005) or embryonic cells (Rambags et al., 2005).
A previous report of FISH hybridizations to non-human primate sperm nuclei employed 
-satellite DNA probes or smaller commercially available locus-specific DNA probes (O'Brien et al., 2005). Whereas three out of five of the human probes were applied successfully in cross-species hybridizations to chimpanzee sperm, only the human chromosome 21 locus specific probe displayed FISH signals in the more distantly related baboon and marmoset (O'Brien et al., 2005). Satellite DNA FISH probes has traditionally been the preferred tool for interphase FISH studies and sperm-FISH assays because they usually produce very bright FISH signals. However, because of the high evolutionary plasticity of the satellite DNA sequences their performance in cross-species hybridizations is hard to predict. For example a macaque -satellite DNA probe generated by PCR with Y-chromosome specific primers [analogous to the successful experiments with baboon samples by O'Brien et al. (2005)] produced unspecific FISH signals on 10 macaque chromosomes even under highly stringent conditions (data not shown). Because of the potential advantages of a universal sperm-FISH assay with applicability to the majority of non-human primate model species our experiments focused on increasing the efficiency of locus-specific probes in sperm-FISH experiments. Our protocol improves on the previous experiments (O'Brien et al., 2005) in two ways: (i) the FISH probes do represent about five times larger genomic regions than the ones used previously and (ii) the probes have been selected for a high content of evolutionary conserved DNA sequences.
Since the BAC-pool probe sets do not rely on pericentromeric satellite DNA sequences and because of the BAC clone selection criteria, it can be expected that the assay is equally functional in all other cercopithecoid and hominoid non-human primate species as they display similar or shorter evolutionary distances to humans than the analysed rhesus macaque. So far the applicability to other primate species can only be inferred, but the high efficiency of human chromosome paint probes generated by a similar protocol (DOP-PCR) in FISH experiments to the evolutionary even more distant new world monkey chromosomes (de Oliveira et al., 2002) as well as the positive sperm-FISH results with a single smaller locus-specific probe in the marmoset (O'Brien et al., 2005) does corroborate the proposed applicability.
The most often, employed human sperm-FISH assay interrogates the ploidy status of chromosomes 13, 18, 21, X and Y. These chromosomes have been selected because they are most often involved in human aneuploidy syndromes. However it has been shown that aneuploidies of the respective autosomes are most often contributed by the maternal side (Egozcue et al., 1997). The non-human primate sperm-FISH assay presented here interrogates the same chromosomes as the standard human assay, except HSA21, and adds probes for HSA16 and 19. The orthologue of HSA21 is found translocated into the telomeric region of a big chromosome in the macaque (MMUL3; (Moore et al., 1999) and thus is very unlikely to display a similar meiotic behavior as in humans. The presented six chromosomes probe set provides twice as many probes as the ones currently used for aneuploidy studies in rats and mice (Adler et al., 2002; Wyrobek et al., 2005). In addition, it can be expected that, using the probe selection criteria described here, the assay can be quickly extended to cover additional monkey chromosomes.
Comparison of aneuploidy incidences
To gain first insights into the incidence of chromosomal aneuploidies in the rhesus macaque we analyse at least 11 000 spermatozoa originating from four or five macaques with each of the six sperm-FISH probes (Tables 1 and 2). The observed rates for spontaneous chromosomal aneuploidies in the macaque fall into the range of human incidences (Egozcue et al., 1997; Shi and Martin, 2000), however they do coincide with the lowest incidences reported. Sperm-FISH studies in humans have consistently shown that sex chromosomes are about 1.7 times more often involved in aneuploidies than autosomes (e.g. Egozcue et al., 1997; Shi and Martin, 2000; Wyrobek et al., 2005). Our first macaque data indicate that gonosomal aneuploidies occur about twice as often as in macaque chromosomes 17, 18, 19 and 20.
Non-human primate model for aneuploidy induction
The rhesus macaque is the most important non-human model in biomedical research and many medical exposure studies are currently undertaken in macaques and related species as African green monkeys. For these studies the presented sperm-FISH assay should provide a valuable tool supplementing the available analyses methods delineating effects of drug treatments and environmental exposures.
In addition to the similarities in spontaneous aneuploidy incidences between humans and macaques, the macaque exhibits two other important qualities: (i) much higher degree of physiological similarity to humans than mouse and rat animal models and (ii) a genome organization very similar to the human genome. For example the syntenic groups of human chromosomes are found conserved in the macaques and baboons with the exceptions of only two chromosome fusions and a single fission (e.g. Moore et al., 1999). Furthermore the rapidly evolving centromeric DNA sequences in humans and macaques do share the similar basic repeat structure common to all primates (alphoid DNA sequences e.g. Schueler et al., 2005). These alphoid DNA sequences do interact directly with the centromere binding proteins during meiosis and mitosis. Thus the physiological similarities between human and cercopithecoid monkeys do extend to the mechanisms involved in promoting or hindering the correct distribution of chromosomes during gametogenesis potentially making them ideal models for important aneuploidy related research.
The agreement of the first macaque baseline data with human aneuploidy incidences, together with the physiological and cytological similarities to humans, suggest the rhesus macaque to be an excellent model for human aneuploidy induction tests. The apparently very limited variance between the rhesus macaque ejaculates might facilitate the statistical analysis of such experiments. To further validate the model, the analysis of a higher number of individuals as well as comparative exposure studies between humans and macaques would be desirable. Aliquots of the primate aneuploidy assay probes are available free of charge to the research community.
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Acknowledgments |
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This research was funded by NIH-Grants NCRR RR17584, RR00169 and RR13439 and by Philip Morris USA Inc. and by Philip Morris International. The lymphoblastoid rhesus macaque cell line was a kind gift of Mike McChesney (California National Primate Research Center, University of California-Davis, Davis, CA, USA).
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Submitted on June 16, 2006; resubmitted on September 22, 2006; accepted on September 26, 2006.
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