Induction of chromosomal fragile sites in goats: a preliminary study

Induction de sites chromosomiques fragiles chez les chevres: etude preliminaire. Cette etude decrit les resultats de differentes methodes de mise en evidence de sites chromosomiques fragiles sensibles au folate chez la chevre. Deux types differents d'expression de la fragilite chromosomique ont ete observes: des espaces telomeriques ne prenant pas la coloration, sur 20 des 21 animaux etudies, et des cassures chromatidiques sur 10 animaux. Les absences de coloration ont ete localisees principalement sur le chromosome 5 et leur frequence d'apparition allait de 30 a 66 % des cellules. Chez les porteurs de cassures, la frequence de ces dernieres allait de 2 a 5 % des cellules. D'apres les methodes utilisees et les frequences observees dans les cultures, les zones chromosomiques non colorees peuvent etre considerees comme appartenant a la categorie commune des sites fragiles sensibles au folate. Des differences significatives entre les methodes d'induction ont egalement ete observees.

Summary -The current study describes the results obtained from different methods of detection of folate-sensitive fragile sites in goat chromosomes. Two different types of expression of chromosomal fragility have been observed: telomeric non-staining gaps, in 20 out of 21 animals studied, and chromatidic breaks in ten animals. The non-staining gaps have been identified mainly in chromosome 5, and their frequency of occurrence ranged from 30 to 66% of the cells. The chromatidic break occurrence ranged from 2 to 5% of the cells among the break carriers. From the methods used and the observed frequency of expression in cultures, the gaps were classified as common folate-sensitive fragile sites. Significant differences between the induction methods used were obtained. ' goat / fragile site / folate deficiency Résumé -Induction de sites chromosomiques fragiles chez les chèvres : étude préliminaire. Cette étude décrit les résultats de différentes méthodes de mise en évidence de sites chromosomiques fragiles sensibles au folate chez la chèvre. Deux types différents d'expression de la fragilité chromosomique ont été observés : des espaces télomériques ne prenant pas la coloration, sur 20 des 21 animaux étudiés, et des cassures chromatidiques sur 10 animaux. Les absences de coloration ont été localisées principalement sur le chromosome 5 et leur fréquence d'apparition allait de 30 à 66 % des cellules. Chez les porteurs de cassures, la fréquence de ces dernières allait de 2 à 5 % des cellules. D'après les méthodes utilisées et les fréquences observées dans les cultures, les zones chromosomiques non colorées peuvent être considérées comme appartenant à la catégorie commune des sites fragiles sensibles au folate. Des différences significatives entre les méthodes d'induction ont également été observées. chèvre / site fragile / déficience en folate INTRODUCTION About one hundred chromosomal fragile sites have been detected in humans since the first description was made by Dekaban in 1965 (Sutherland, 1991). Human fragile sites have been successively related to different pathologies and one of the most well known is the association between the mental retardation syndrome and the fra.Xq27.3 (Sutherland and Baker, 1990;Vogel et al, 1990;Oberl6 et al, 1991;Craig, 1991). Furthermore, implications of chromosomal fragility in different processes like Bloom syndrome (Fundia et al, 1992), chromosomal viral integration points (Caporossi et al, 1991), chromosomal evolution (Mir6 et al, 1987;Popescu et al, 1990) and the relationship between fragile sites, oncogenesis and tumoral events (Yunis, 1983;Yunis and Soreng, 1984;DeBraeckeler, 1987;Dal Cin et al, 1991;Austin et al, 1991) have been well documented in human cytogenetics. In animals only a few chromosomal fragile sites have been reported, mainly in domestic species.
In pigs, Chrisman (1989, 1991) have described aphidicolin and folatesensitive fragile sites like the ones detected by Yan and Long (1993). More recently, folate, 5-BrdU and aphidicoline fragiles sites have been found in equine, rabbit, bovine, mole rat, dog and sheep karyotypes by Ronne (1992), Poulsen and Ronne (1991), Uchida et al (1986), Gripemberg (1991), Stone et al (1991a) and Matejka et al (1990). Although no close relationship with any pathology has yet been observed, Tewari et al (1987) have indicated a possible effect on the fertility of female rats and Stone et al (1991b) have suggested the implication of some fragile sites in tumoral chromosomal rearrangements in the mammary glands of dogs. These features and some of the published results, indicate that the fragile sites may be distributed in the majority of domestic species in a similar way as for humans. This highlights the importance of knowing the distribution and morphological characteristics of animal fragile sites, as a first step to finding the possible relationships between any defined pathology or syndrome and the presence of chromosomal fragility.
There is no knowledge of the induction methodologies or chromosomal fragility expression forms in goats and the aim of this work has been the adaptation of induction methodologies to begin studies of the detection and identification of folate-sensitive fragile sites in this karyotype.

MATERIAL AND METHODS
Twenty-one adult goats were used, including Saanen, Toggenburg and cross-bred animals.
An adaptation of published protocols (Sutherland et al (1985); Howard-Peebles (1991); Fisch et al (1991); Jacky et al (1991)) was used to induce the expression of fragile sites in lymphocyte cultures.
The cultures were harvested and fixed according to a standard technique (Moorhead et al, 1960). Two cultures for each treatment were made and 50 cells from each one were observed. Control cultures were used for each protocol according to the standard methodology: 1 mL of whole blood in 10 mL of RPMI 1640 (Gibco), supplemented with 20% SFB (bovine calf serum) (Gibco) and 1% penicillinestreptomycin (Gibco) and 1% L -glutamine (Sigma). The cultures were incubated at 37 °C in the absence of C0 2 and harvested after 72 h of growth.
The identification of chromosome pairs was accomplished by an adaptation of the original Seabright's G banding method (Seabright, 1971). An ANOVA test was used to establish the differences between treatments (Stat View, Macintosh).

RESULTS
Two different types of chromosomal alterations were observed. The numbers given below refer to protocol 3.

Non-staining gaps at the telomeric region
The results are shown in figure la and lb. The minimum expression value considered was 4% of the total observed cells, and only one animal presented an expression percentage below this. Among the remaining 20, the gaps were present at a frequency of 30-66% of the cells, with a mean value of 48.3 t 2.1%. Cells with more than one gap occurred at a frequency ranging from 0-38% of the total, with a mean value of 10.5 f 2.3% (table I)

Chromatidic breaks
The occurrence of chromatidic breaks ranged from 0% (breaks were detected only in ten animals) to 4%, with a mean value of 1.5 f 0.4% (table I). Unlike the gaps, the break locations were detected in different regions and chromosome pairs, and these ruptures were observed in only one chromatid in all the analyzed cases (fig 3a  and 3b).

Methodologies used
The three variants described were useful for detecting gaps and break induction. Considering the ANOVA test performed, treatment 3 showed significant differences (p < 0.05) from treatments 1 and 2, taking into account only gap expression (fig 4).
The proliferation cell rate was lower when protocol 2 was employed. For this reason, another comparison test between protocols 1 and 3 was carried out using 18 animals ( fig 5). The ANOVA test did not show significant differences, but a slight increase in gap expression in treatment 3 could be detected in both comparison tests (fig 4 and 5).

DISCUSSION
From a morphological point of view, the two types of lesions found can be considered as different chromosomal fragility expression forms in the goat karyotype. The high frequency of gaps and the constancy of their location are two features in agreement with the descriptions of other species whose gaps have been the most  (Voiculescou et al, 1991;Tommerup et al, 1981;Gollin et al, 1985). These high expression values are in agreement with the mean expression observed by us, and reinforce the fact that our results may represent a common folate-sensitive fragile site, which shows a chromosome gap as its form of expression in cell culture.
Furthermore, the three protocol modifications used were useful for gap induction. Even though some of the drugs used might have enhanced the expression, it can be pointed out that the absence of folic acid in the culture medium was the main factor in provoking the fragile expression. A slight positive effect on gap expression was detected only when methotrexate was added 24 h before harvesting (a difference was observed mainly when a comparison was made between treatments 1 and 3). If the effect produced by amethopterin on the condensation and chromosome elongation is considered (Laird et al, 1987), an increase of the degree of stretching at the fragile region, and consequently a clearer observation on optical microscopy, could be a logical explanation for the observed high level of expression when this drug is added.
Even though in some animals (three), different chromosome pairs were detected to be telomeric gap carriers, their expression levels were <4% of the cells and no chromosome repetitive location was confirmed; only in the pair 5 were the gap location and frequency expression values good enough to be considered a possible expression of chromosomal fragility. One important fact is that the gaps overlap the telomeric nucleolar organization region (NOR) in this pair. Consequently it is necessary to assume that any stretching of the NOR region could provoke a variable percentage of false-positive fragile detection. In this work no triple staining (Giemsa-G-Banding-NORs) was employed, and it is difficult to estimate what percentage of gaps might be false-positive due to active NOR regions being detected as fragile sites, or indeed if all the fragile sites described here are only NOR stretchings. There is some evidence that the latter is not the case. First, telomeric gaps were detected only in one (pair 5) of the five chromosome carriers of NORs in goat (according to Di Meo et al, 1991) pairs 2, 3, 4, 5 and 28 are NORs carriers in goats. Secondly, the gaps were not limited to the telomere only, the gap region involving all the telomeric positive R or negative G bands of the carrier chromosome. The gaps are bigger than the NOR region detected with normal AG-NOR (silver staining NOR) staining. These features indicate that it is not valid to assume that all the gaps are extensions of the NORs, though a percentage of false-positives due to this cannot be excluded.
Telomeric Structural Changes (TSC) have been indicated as another source of error when human telomeric fragile sites are analyzed (Butler et al, 1990). These TSC are chromosomal lesions which can be detected as fragile sites and the authors mentioned that about 10% of positive detection in human fra.X is due to this kind of alteration. According to this it would be necessary to take into account another variable percentage of error in the results observed. The expression rate and the different positions of the breaks are subjects for discussion. The breaks were not found in the control cultures and they presented morphological features similar to some of the fragile sites described in humans (isochromatidic breaks as reported by Sutherland et al, 1985, andSutherland, 1991).
On the other hand, in no case did the break frequency raise the minimum level of expression estimated in this work (4%) and only eleven animals expressed these breaks with no repetition established in the position (a variation was noted in the location within each chromosome and between chromosome pairs).
Taking into account that the main source of variation for the fragile expression is related to the induction methodology (as described by Fisch et al, 1991 for the fra.X syndrome in humans), the possibility that the methods used here caused a higher rate of break or, in some cases gap, inductions cannot be excluded.

CONCLUSIONS
Although this is a preliminary study, some conclusions may be drawn. The expression of chromosomal fragility and its induction in goat karyotype are similar to observations in related and unrelated species. Furthermore, gaps seem to be the main form of expression. Even though a combination of FdU and a low folate medium seems to be the only condition necessary for induction, some drugs like amethopterin and thymidine are important for improving fragility expression in this karyotype.
A telomeric gap could be detected successfully only in pair 5; according to its expression frequency and culture conditions it could be classified as a virtual folatesensitive fragile site. Further study is needed of the sources of error which can lead to a significant percentage of false-positive results (a combination of Telomeric Structural Changes and NORs). A study of its genetic inheritance is also required to reach a definitive conclusion.
It is not possible to draw a conclusion about the chromatidic breaks detected. The low frequency of expression and the variation in location on different chromosome pairs indicate that different induction methods are necessary for a better understanding of the nature of their expression.