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In situ aneuploidy assessment in human sperm: the use of primed in situ and peptide nucleic acid_fluorescence in situ hybridization techniques
Franck Pellestor
CNRS UPR 1142, Institute of Human Genetics, Montpellier Cedex 5, France
DOI: 10.1111/j.1745-7262.2006.00137.x
Abstract
Both the primed in situ (PRINS) and the peptide nucleic acid_fluorescence
in situ hybridization (PNA_FISH) techniques constitute alternatives to the conventional (fluorescence
in situ hybridization, FISH) procedure for
chromosomal investigations. The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an
in situ primer extension reaction. Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide
backbones. The two procedures present several advantages (specificity, rapidity and discriminating ability) that make
them very attractive for cytogenetic purposes. Their adaptation to human spermatozoa has allowed the development
of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements
to FISH for in situ assessment of aneuploidy in male
gametes. (Asian J Androl 2006 Jul; 8: 387_392)
Keywords: aneuploidy; chromosomal screening; peptide nucleic acid_fluorescence in situ hybridization; primed in situ; spermatozoa
Correspondence to: Dr Franck Pellestor, CNRS UPR 1142, Institute of Human Genetics, 141 rue de la Cardonille, F-34396 Montpellier
Cedex 5, France.
Tel: +33-4-9961-9912,
Fax: +33-4-9961-9901
E-mail: Franck.Pellestor@igh.cnrs.frReceived 2005-03-08
Accepted 2005-12-30
1 Introduction
The assessment of aneuploidy rate in human gametes is of critical importance because non-disjunction makes a
major contribution to the chromosomal abnormalities found in humans. In particular, the chromosomal analysis of
spermatozoa constitutes an essential approach for the investigation of the occurrence and etiology of chromosomal
abnormalities under a wide variety of clinical conditions. To date, a lot of data were gathered thanks to the human
sperm_hamster egg fusion technique [1, 2], but the procedure was so labor-intensive and time-consuming that its use
was limited to a few laboratories. The advent of molecular genetic techniques has brought forth new procedures for
in situ chromosomal analysis, and then has opened the way for comprehensive studies on aneuploidy occurrence.
Because of its relative simplicity and the availability of numerous probes, the fluorescence
in situ hybridization (FISH) technique has become the method of choice in molecular cytogenetic investigations. Numerous chromosomal
analyses on human sperm have been performed using FISH [3]. These reports have demonstrated the efficiency of
the in situ labeling procedure on male gametes, but also pointed out the limitations of FISH on this biological material,
which are essentially linked to the size of the probes and the reliability of the associated sperm decondensation
treatments [4, 5]. During the last decade, alternative methods to FISH have been introduced and have shown to be
valuable in detecting chromosomes and quantifying aneuploidies. These alternative procedures are the primed
in situ (PRINS) labeling and the peptide nucleic acid_FISH (PNA_FISH) probes. The two procedures present several
advantages for the in situ detection of nucleic acid sequences that make them very attractive for a number of
cytogenetic purposes. Several recent studies have demonstrated that PRINS and PNA_FISH were as efficient as FISH on
human gametes and exhibited higher specificity [6_8]. The present paper provides a brief overview of both the
PRINS and PNA_FISH methods, and highlights recent applications of these techniques on human spermatozoa.
2 Primed in
situ technique
2.1 Principles and methodology
Based on the use of chromosome-specific primers, the PRINS reaction combines the high sensitivity of polymerase
chain reaction (PCR) with the cytological localization of DNA sequences [9]. The chromosomal identification is
performed by in situ annealing of specific and unlabeled oligonucleotide primers to complementary sites on denatured
chromosome spreads, nuclei or tissue sections. Cells or tissue samples are fixed and denatured before PRINS
reaction, both to preserve morphology and to permit access of the reagents to the sequence target. The annealed
primers provide initiation sites for chain elongation catalyzed by a Taq DNA polymerase in the presence of free
nucleotides, of which at least one is labeled. The
in situ visualization of generated fragments results from the
incorporation of the labeled nucleotide (Figure 1) [10].
PRINS labeling of human chromosomes is obtained using primers for repeated DNA sequences. An advantage of
primers is their ability to differentiate between closely related sequences. This feature has been utilized for generating
chromosome-specific primers from the alpha-satellite DNA motif. The lengths of the PRINS primers range from 18
to 30 nucleotides. Compared to the size of DNA repetitive probes (250_600 bp), this small size greatly facilitates their
in situ accessibility to their genomic target sequences. This is particularly significant in cells with highly condensed
nuclei, such as spermatozoa. Because they are unlabeled, high amounts of primers can also be used in PRINS reaction
without inducing background signals. The complementation process between the primer and its centromeric target will
be so specific that a simple mismatch between the
3กฏ-end of the primer and the genomic sequence will prevent initiation
of the in situ elongation by the Taq DNA polymerase. Thus, it has been possible to define specific alpha-satellite
primers for some chromosomes undistinguishable by FISH with centromeric probes, such as chromosomes 13 and
21, which share 99.7% homology in their alpha-satellite DNA sequences [11].
Initially, PRINS reactions were performed either on a hotplate or a waterbath, but these procedures did not allow
precise and durable temperature control. The PRINS protocol has been considerably improved and simplified by the
introduction of programmable temperature cyclers equipped with a flat plate block. The use of automatic thermocyclers
allows an optimization of both annealing and extension conditions. Thus, semi automatic PRINS protocols have been
developed offering a high reproducibility in labeling reaction. An additional improvement was the direct use of
fluorochromes in sequential PRINS reactions. Recently, a new multicolor PRINS protocol has been reported, allowing
performance of ultra-rapid detection on several chromosomes, by mixing the different fluorochromes during the
chain elongation reaction [12]. Each PRINS reaction consists of a unique 4-min step for annealing and elongation of
each chromosome-specific primer. This new sequential procedure simplifies the PRINS technique and provides an
easy way to carry out multicolor labeling. The PRINS reaction can also be combined with conventional FISH labeling
protocols. No further denaturation is required before the FISH reaction. This combined use may be an efficient
approach to improve chromosomal detection.
2.2 Application to human sperm
In humans the PRINS method has been successfully tested for the assessment of aneuploidy in lymphocytes,
amniocytes and preimplantation embryos [13_15]. The use of PRINS has also been reported for analysis of structural
aberrations such as translocations and marker chromosomes as well as for the detection of fetal cells in peripheral venous
blood of pregnant women [16, 17]. Further applications of PRINS have also been found in tumoral cytogenetics. The
adaptation of PRINS to human spermatozoa has constituted a new step in the development of PRINS methodology and
an interesting challenge because of the particularities of the human sperm nucleus in terms of genomic compaction and
accessibility of DNA sequences.
The PRINS technique was combined with an efficient NaOH treatment of spermatozoa allowing the simultaneous
decondensation and denaturation of sperm nuclei. In the PRINS reaction, the decondensation of the sperm head is a
less limiting factor than in FISH because of the small size of the oligonucleotide primers. This greatly facilitates their
penetration into sperm nuclei, resulting in a more homogeneous and more rapid labeling of sperm nuclei [18]. This
might explain the extreme rapidity of PRINS labeling of human sperm. Indeed, a single-target PRINS reaction can be
performed in 10 min. The time of optimal NaOH treatment depends on the age of the sperm preparation slides and the
concentration of NaOH solution. Initially, we used 3 mol/L NaOH, but numerous experiments and a comparison of
the results in terms of quality of the preparation obtained led us to adopt a 0.5 mol/L NaOH solution. Best results in
labeling efficiency were obtained with 2-day-old slides and 5-min 0.5 mol/L NaOH pretreatment [18,19]. The NaOH
treatment induces uniform swelling of the sperm nucleus to 1.5_2-fold its normal size, and maintains the
characteristics and shape of the sperm nucleus, including the tail. This allows the differentiation between spermatozoa and other
cells such as leukocytes or immature germ cells present in the ejaculate.
Double and triple PRINS reactions have been performed using various combinations of chromosome-specific
primers (Figure 2) [10]. Both the efficiency and the reliability of the PRINS technique on sperm were tested by using
different primers to label individual chromosomes. Using this PRINS protocol, we have directly estimated diploidy and
disomy frequencies for 15 autosomes and sex chromosomes in sperm samples from several normal fertile donors [20].
The labeling efficiency usually ranges from 97% to 100% according to the primer tested. A minimum of 10 000 nuclei
per chromosome was analyzed. For the autosomes, the mean disomy rate ranged from 0.18% to 0.36%. Among all
donors, chromosome 21 exhibited the highest disomy rate (0.30_0.39%), suggesting that in male meiosis, chromosome
21 may be more susceptible to non-disjunction than the other autosomes. The frequency of disomy for gonosomes
ranged from 0.08% to 0.13%.
Interindividual variations in disomy rates were observed but these were statistically non-significant. Interindividual
heterogeneity was more evident in diploidy, where frequencies ranged from 0.08% to 0.45% among the normal
subjects tested.
The efficiency of the new multicolor PRINS protocol, based on the direct mixing of the color of fluorochromes, was
also tested and validated on human sperm [21]. Comparative estimates of disomy were performed for several
chromosomes on sperm samples from two donors, either by the new three-color protocol or by the conventional dual-color
PRINS procedure previously described. There was no statistical difference between the disomy rates obtained with the
conventional dual-color PRINS technique and the fast three-color procedure. This new protocol provides significant
simplifications in the multicolor PRINS protocol without modifying the efficiency and the specificity of the labeling
reaction. The use of this multicolor protocol has advantages of rapidity and low cost as compared to FISH.
The PRINS procedure has also been used to directly investigate in sperm the meiotic segregation patterns of
reciprocal translocations [22] and to investigate the occurrence of interchromosomal effect in sperm of chromosomal
rearrangement carriers [23].
3 Peptide nucleic
acid_fluorescence in situ hybridization technique
3.1 Definition and properties
Peptide nucleic acids (PNAs) constitute a new class of DNA probes, which provide an interesting complement to
FISH and PRINS. PNAs are synthetic mimics of DNA in which the deoxyribose phosphate backbone supporting the
nucleic acid bases is replaced by a non-charged peptide backbone (Figure 3) [24]. The unique chemical makeup of
these probes confers a number of beneficial properties, including enhanced hybridization rates, resistance to nucleases
and proteases and the abi-lity to penetrate condensed biological structures [25]. The neutral backbone of PNA
provides strong binding between PNA_DNA or PNA_RNA strands and greater specificity of interaction than their
DNA counterparts. While they hybridize according to normal Watson_Crick base pairing rules, PNA have been shown
to bind to DNA or RNA targets with higher affinity than the corresponding oligonucleotides. Unlike DNA probes,
which require high salt concentration to bind, PNA probes can bind to DNA or RNA under low ionic strength conditions that
do not favor reannealing of complementary strands [26]. Experiments with homopyrimidine strands have shown that
the melting temperature (Tm) of a 6-mer PNA deoxythymidine (PNAdT)_DNA deoxyadenosine (DNAdA) was 31ºC in
comparison to a DNAdT_DNAdA 6-mer duplex that denatures at a temperature less than 10ºC. Experiments done with
PNA probes containing all four bases have demonstrated that there is an increase of the Tm of approximately 1ºC per base
pair in PNA hybrids compared to DNA_DNA or RNA_RNA duplex. In addition, a PNA_DNA mismatch is more
destabilizing than a mismatch in a DNA_DNA duplex. A single mismatch in mixed PNA_DNA 15-mer decreases the Tm by 15ºC. In
the corresponding DNA_DNA duplex, a single mismatch decreases the Tm by only 1ºC. Moreover, the use of even shorter
PNA probes can further increase PNA specificity [27]. This advantage is particularly important for hybridization with short
probes targeting repetitive sequences, because both the length and the repetitive nature of these genomic targets will favor
renaturation over hybridization with probes.
This high level of discrimination at single-base level has indicated that short PNA probes could offer high
specificity and has thus allowed the further development of several PNA-based strategies for molecular investigations and
diagnosis. Short PNA oligomers, from17 to 22 base units, constitute efficient tools for detecting specific DNA
sequences with fast hybridization kinetics over a wide pH range. In addition, PNA probes are not limited to any
detection procedure. PNAs can be labeled with a large variety of reporter molecules (enzymes, haptens and fluorophores)
[28].
3.2 Application to human sperm
The unique properties of PNA probes as a DNA mimic have led to the development of numerous applications. Most
notably, PNAs find current uses in molecular biological techniques as specific and sensitive probes for complementary
nucleic acids [27]. The introduction of PNA technology in cytogenetics is recent. First, it has been demonstrated that PNA
probes were useful for detecting telomere repeat sequences [29]. Then, the availability of chromosome-specific centomeric
PNA probes, directly fluorochrome-labeled, has led to the development of rapid and easy multicolor PNA-FISH protocols
for the in situ detection and enumeration of human chromosomes in metaphases and interphase nuclei [30]. The procedure
has been recently adapted to human blastomeres, and gametes [7, 8].
On human spermatozoa, decondensation pretreatment is also indispensable with PNA probes. No PNA labeling of
sperm nuclei can be obtained without decondensation pretreatment. We have tested the two decondensation procedures
that give efficient and reproducible results with FISH and PRINS reactions, namely, the dithiothreitol (DTT) and the
NaOH treatments. The two procedures yielded satisfactory results with PNA probes and gave similar kinetics for the
labeling reaction [31]. To estimate and validate the efficiency of
PNA_FISH labeling on human sperm, comparative estimates of disomy X, Y and 1 were performed on sperm preparations from healthy subjects using multicolor FISH,
PRINS and PNA_FISH procedures in parallel. An equivalent quality of
in situ nuclear labeling (Figure 4) and similar
disomy rates were obtained with the three methods. However, the hybridization timing of PNA probes (i.e. 40 min) was
considerably shortened in comparison with the FISH reaction, which requires an overnight hybridization in order to be
efficiently completed for sperm preparations. The fast hybridization kinetics of
PNA_FISH labeling on sperm was comparable to the kinetics of the PRINS reaction. The similarity between
PNA_FISH and PRINS might essentially be due to the small size of both PNA oligoprobes (18_20 bp) and PRINS primers, which do not exceed 30 bases in length.
Similar performance of PNA_FISH and PRINS methods has already been reported for the
in situ detection and sizing of telomeric repeat sequences [32]. Both techniques presented comparable features in terms of specificity, staining
intensity and efficiency, but PRINS always displayed a faster turnaround reaction time. This could reflect the fact that
PRINS is an "active" reaction involving an ultra-fast biochemical reaction of
primer extension catalyzed by a Taq polymerase. In the case of
PNA_FISH reaction, the rapidity of the labeling is due to the neutral backbone of the PNA
molecules, which allows PNA_DNA binding to occur more rapidly and more tightly than DNA_DNA binding. The high
affinity of PNA probes to DNA constitutes an important feature for chromosomal analysis. Previous studies have
demonstrated that PNA probes could discriminate between two centromeric DNA repeats that differ by only a single base
pair [33]. Similar results were obtained with PRINS primers and oligonucleotide probes whereas standard FISH probes
are unable to discriminate between sequences with a single base resolution. The study of chromosomal polymorphism
could benefit from the discriminating power of PNA probes. For sperm, this could be useful for the
in situ distinction of autosomal non-disjunction occurring at meiosis I and meiosis II, when satellite polymorphisms exist between two
homologous chromosomes.
4 Conclusion
Since their introduction, both PRINS and PNA_FISH techniques have quickly evolved from basic research to
diagnosis procedures. The fast hybridization, penetration and discrimination of both PRINS primers and PNA probes
make them valuable tools for in situ chromosomal screening. Their successful use on human sperm has proven that
these reagents could be used on difficult biological material, and consequently they have a great potential for clinical
application. With the development of rapid and simplified protocols producing reliable and reproducible results, it has
been proven that both PRINS and PNA_FISH techniques could become powerful tools for cytogenetic investigations
and diagnosis. They can be used advantageously to complement FISH and PCR for the physical mapping of the human
genome, and the possibility of combining PNA_FISH or PRINS with conventional FISH on the same preparation opens
up interesting possibilities for multiplex assays.
Acknowledgment
This study was supported by a European INTAS project (No 03-51-4060) and a French research project (PHRC
No 7732) from the CHU of Montpellier.
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