1 UMR INRA-ENVT Cytogénétique des Populations Animales, 31076 Toulouse, 2 Laboratoire de Génétique Cellulaire, INRA, 31326 Castanet Tolosan, 3 Unité de sélection porcine SESP, INRA, 86480 Rouillé and 4 CNRS, UPR 1142, 34396 Montpellier, France
5 To whom correspondence should be addressed at: UMR INRA-ENVT Cytogénétique des Populations Animales, Ecole Nationale Vétérinaire de Toulouse, BP 87614, 23 chemin des Capelles, 31076 Toulouse Cedex 3, France. Email: a.pinton{at}envt.fr
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Abstract |
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Key words: chromosome/meiosis/pig/reciprocal translocation/segregation
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Introduction |
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Epidemiological studies in humans have frequently underlined the effect of sex of the carrier parent on the risk of imbalance. Although the general picture of the differences between male and female meiotic processes has been known for a long time, the impact of these differences on chromosome segregation remains unclear. In the case of reciprocal translocations, most imbalances at birth result from a rearrangement carried by the mother (Boue and Gallano, 1984; Daniel et al., 1988
). However, this apparently higher predisposition of female meiosis than male meiosis to the production of imbalance has not been conclusively established and certain studies suggest that such predisposition would be limited to specific types of imbalance, notably the 3:1 (Mackie Ogilvie and Scriven, 2002
). According to Faraut et al. (2000)
, the observed sex-ratio deviation in the parental origin of the chromosomal imbalances would not be due to a predisposition of female meiosis but rather would result from the male infertility frequently associated with reciprocal translocations (Luciani and Guichaoua, 1990
). Thus, it seems important to be able to dissociate the different processes (synapsis, formation of chiasmata, segregation during the first and second meiotic divisions) when studying the meiotic behaviour of chromosomes. Analysis of the differences between the meiotic products of females and males for a given chromosomal rearrangement could provide clues as to the respective roles of the mechanisms in operation.
Numerous experimental meiotic segregation studies have been carried out in males using the spermFISH technique (fluorescent in situ hybridization on decondensed sperm nuclei) (Guttenbach et al., 1997). In contrast, the limited accessibility of human biological material has restricted the study of female meiosis. The precise determination of female segregation patterns in carriers of structural chromosomal rearrangements has been rarely reported. The development of preimplantation genetic diagnosis (PGD) has brought forth new procedures for chromosomal segregation studies, such as polar body chromosome painting analysis or comparative genomic hybridization assays on biopsied blastomeres (e.g. Munné et al., 1998
; Wells and Delhanty, 2000
). However, the cytogenetic analysis of female gametes and embryos remains extremely difficult in humans for ethical reasons. Consequently, recourse to an animal model for the study of female meiosis is of great interest. The main comparative study of segregation products in male and female carriers of identical reciprocal translocations has been performed in the mouse, using analyses of metaphases I and II of spermatocytes and oocytes (Tease, 1998
). The karyotypic structure of the domestic pig (Sus scrofa domestica L.) being more similar to human than that of the mouse, this species could provide another relevant animal model for studying the mechanisms of meiosis in the presence of chromosomal rearrangements. The females of this species are relatively prolific (12 progeny per litter on average). The number of oocytes (embryos) that can be analysed per female is therefore relatively high. In addition, the generation interval is relatively short (
2 years), and the experimental production of individuals with particular karyotypes is possible at reasonable expense. Finally, a programme for systematic control of the karyotypes of individuals destined for reproduction exists in this species, which permits the identification of new chromosomal rearrangements (515 per year; incidence in the order of 0.4%; Ducos et al., 2002b
).
Here, we investigate the segregation patterns determined from metaphase II oocyte samples obtained from sows heterozygotes for one of the two following reciprocal translocations: 38, XX, rcp(3;15)(q27;q13) and 38, XX, rcp(12;14)(q13;q21). These data were compared with the segregation patterns previously determined in boars carrying the same chromosomal rearrangements (Pinton et al., 2004). Hypotheses to interpret the observed differences between the two translocations, as well as between the male and female segregation patterns are formulated and discussed.
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Materials and methods |
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In vitro maturation of oocytes
The metaphase II oocytes were obtained after in vitro maturation of oocytes (Marchal et al., 2001). Briefly, the oocytes were removed from ovaries obtained from the abattoir. The cumulusoocyte complexes (COC) from follicles of 36 mm were initially washed four times in phosphate-buffered saline +0.5 mg/ml of bovine serum albumin (Sigma, St Louis, MO, USA) +50 µg/ml of Gentamycin (Sigma), then cultured in 500 µl of maturation medium composed of culture medium 199 (Sigma) supplemented with epidermal growth factor (10 ng/ml final; Sigma) and Cysteamine (570 µmol/l final; Sigma). The duration of maturation was fixed at 44 h at 39°C in a 5% CO2 oven.
Harvesting of metaphase II oocytes
After maturation, the oocytes were mechanically cleaned of cumulus cells (by sucking and blowing with a micropipette). Maturation was determined by the presence of polar bodies at the oocyte periphery. The mature oocytes were then spread on slides as described by Tarkowski (1966).
Chromosome painting on metaphase II oocytes and lymphocytes
Two types of painting probes were used. For chromosomes 3, 12 and 14, the probes were produced from flow-sorted chromosomes amplified using priming authorizing random mismatches (PARM)PCR (Schmitz et al., 1992; Yerle et al., 1993
). The probe for chromosome 15 was produced by chromosomal microdissection followed by degenerate oligonucleotide-primed (DOP)PCR amplification (Telenius et al., 1992
) of the microdissected material (Pinton et al., 2003
). Probe labelling was carried out either by PARMPCR or DOPPCR using 2 µl of amplified chromosomal products. For this, 50 µl of a reaction mixture consisting of: Taq 1xbuffer; 2 IU AmpliTaq (Applied Biosystems, Foster City, CA, USA); MgCl2 2 mmol/l; 0.2 mmol/l of each dAGC/TP; dTTP 20 µmol/l; biotin-16-dUTP or digoxigenin-11-dUTP (Roche Applied Science, Meylan, 38, France) 100 µmol/l; 50 pmol of primers: (GAG)7 for PARM PCR or 5'-CCGACTCGAGNNNNNNTGTGG-3' for DOP PCR; and H2O quantity sufficient for (q.s.f.) were added to 2 µl of amplified chromosomal products. Twenty PCR cycles were performed (56°C, 1 min; 72°C, 1 min; 94°C, 1 min) followed by a terminal elongation (72°C, 5 min). Once mixed (i.e. either chromosomes 3 and 15 or chromosomes 12 and 14) and purified by G50 column chromatography, the PCR products were precipitated in the presence of 15 µg of porcine competitor DNA (Hybloc Competitor DNA; Applied Genetics Laboratories, Melbourne, FL, USA). The probes were then re-suspended in 25 µl of conventional hybridization solution and 5 µl were placed (under a 22 mmx22 mm cover slip) on each preparation that had been previously denaturated (in 70% formamide at 70°C for 2 min) and treated with proteinase K for 3 min. The biotin-labelled probes were revealed using a red Alexa 594 fluorochrome (Molecular Probes, Eugene, OR, USA), and the digoxigenin-labelled probes using a green Alexa 488 fluorochrome (Molecular Probes) as in the protocol described by Pinton et al., (2003)
.
Only the metaphase II oocytes showing clear and homogeneous hybridization signals were further considered in this study. The presence of differentially labelled chromatids for the same chromosome allowed the detection of interstitial crossing-over.
The same sets of probes and hybridization protocols were used for chromosome painting on metaphases of the boars obtained from lymphocyte cultures (Ducos et al., 2002b; Pinton et al., 2003
) (Figures 1 and 2).
Sperm preparation and analysis
The classical methods used to determine the male segregation patterns (fluorescent in situ hybridization on decondensed sperm heads: spermFISH) have been presented in detail by Pinton et al. (2004).
Data analysis
A conventional 2x2 2-test with Yates' correction (Dagnélie, 1975
) was used to compare the proportions of the different segregation products (comparison between translocations, for both males and females, as well as comparison between males and females carrying the same translocation). P<0.05 was considered statistically significant.
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Results |
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Comparison of these results with those previously obtained in a male carrier of the same rearrangement reveals important differences in the segregation profiles between the two sexes (Table III). The overall percentage of balanced gametes is higher in females (67.6%) than in the male (52.2%, P<0.004). The modes of production of the chromosomally unbalanced gametes are also different. In the male, these unbalanced gametes are mainly produced by adjacent-I segregation (31.4%), whereas they are mainly derived from adjacent-II and 3:1 segregations in females: 14.3% for each, compared with 3.8%=1.1%+(0.5x5.5%) for the adjacent-I segregation. The percentages of the 3:1 segregation products did not differ significantly between the males (13.50%) and females (14.3%; P>0.5).
Finally, no diploid spermatozoid were detected in the boar semen, whereas 22.9% of the metaphase II oocytes were diploids.
12/14 translocation
A total of 206 metaphase II oocytes were analysed by FISH after in vitro maturation and spreading; of these, 11 (5.3%) were diploids. Some representative images of the main meiosis I products are presented in Figure 5. The relative proportions of the different female segregation products are indicated in Table II. As for the previous translocation, the results obtained in the boar using spermFISH (3006 sperm studied: Pinton et al., 2004) are given for comparison in Table III.
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As with the previous translocation, large differences were observed between the meiotic segregation profiles of the two sexes (Table III). The total proportion of balanced gametes was lower in the females than in the males (59.4%=40.5%+0.5x37.9%, compared with 75.9%; P<0.001). The adjacent-I segregation was the main mode of production of unbalanced gametes in both sexes, but was more frequent in the females (36.4%=17.4+0.5x37.9%) than in the males (14.9%; P<0.001). In contrast, the estimated percentage of adjacent-II segregation products was higher in the males (5.7%) than in females (2.1%; P<0.05). The percentages of the 3:1 segregation products did not differ significantly between the male (3.5%) and females (2.1%) (P>0.38).
A single diploid spermatozoid had been identified out of the 3006 analysed in the semen of a boar carrier of translocation 12/14. The estimated percentage was higher (5.3%) in the oocyte samples.
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Discussion |
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In the case of the 12/14 translocation, the frequency of interstitial crossing-over is higher on chromosome 14 than on chromosome 12. For the 3/15 translocation, this frequency is highest on chromosome 3. These results are consistent with the hypothesis of a positive correlation between the size of the chromosomal segments and the probability of crossing-over (Figure 3).
Comparison of the segregation patterns between sexes
The 11q;22q translocation seems to be the only recurrent reciprocal translocation in humans. Preimplantation genetic diagnosis was carried out for several couples carrying this particular chromosomal rearrangement. To our knowledge, this is the unique source of data allowing a direct comparison of segregation patterns between males and females carrying an identical reciprocal translocation in humans (Van Assche et al., 1999; Mackie Ogilvie and Scriven, 2002
). However, due to the very low number of embryos studied, the significance of these results is rather limited. Still in humans, it would be worthwhile to consider PGD data on female and male translocation carriers even though the comparison relates to different translocations. In a first review of 35 cases of PGD of translocations with several methods, Munné et al. (2000)
found no differences in the rates of chromosome abnormalities between male and female carriers. So far there have been close to 500 PGD cycles for translocations performed worldwide (Verlinsky et al., 2004
), and the initial conclusions seem to be confirmed. This would indicate that, on average, male and female meioses generate comparable proportions of unbalanced gametes in the case of reciprocal translocations. However, such data should be considered cautiously because patients undergoing PGD usually have a history of recurrent miscarriage which could bias the results obtained.
The most complete and important study aimed at comparing segregation patterns for males and females carrying identical reciprocal translocations has been carried out in the mouse (Tease, 1998). Very different segregation patterns between males and females were demonstrated in this study. Our results, obtained in another mammalian species, also reveal marked differences between the two sexes, for the two translocations studied. In the reciprocal translocation rcp(12;14)(q13;q21) for instance, the proportion of products derived from alternate segregation (i.e.chromosomally balanced) was significantly higher in the male than in females. For translocation, rcp(3;15)(q27;q13), the production of unbalanced gametes was very different between the two sexes, with a significantly higher proportion of products derived from adjacent-I segregation in males than in females. These results confirm those previously reported by Tease (1998)
in the mouse. They may partially be explained by differences in the frequency and localization of chiasmata (crossing-over) between the two sexes. Indeed, both parameters determine the meiotic configurations (ring, chain etc.) which form during prophase of meiosis I, as well as the orientation of multivalents, and consequently affect the types of segregation which occur, as already mentioned (Goldman and Hultén, 1993a
,b
; Tease, 1996
, 1998
). As postulated by McClintock (1945)
, the presence of an interstitial chiasma might have a direct effect on the percentage of products derived from adjacent-II segregation (Faraut et al., 2000
). Such an event favours the co-orientation of homologous centromeres to the opposite poles, thus limiting the probability of adjacent-II segregation. Globally, the frequency of adjacent-II segregations is inversely proportional to the frequency of crossing-over on interstitial segments (Rickards, 1983
). In our study, the translocation 12/14 exhibits the highest frequency of interstitial crossing-over (37.9%, compared with 5.5% for the 3/15 translocation; P<0.001; Tables I and II). Therefore it is logical that the lowest proportion of adjacent-II segregation is observed for this translocation (2.1%, compared with 14.3% for the 3/15 translocation; P<0.001). Genetic mapping data (e.g. Broman et al., 1998
) and localizations of chiasmata by immunohistochemistry in humans (Barlow and Hulten, 1998
) and mice (Anderson et al., 1999
; Froenicke et al., 2002
), have indicated that the frequency of crossing-over is higher and their distribution more interstitial in females than in males. A lower proportion of adjacent-II segregation products would therefore be expected in females than in males. This was observed in the case of the 12/14 translocation. In contrast, a reverse distribution was observed for the 3/15 translocation.
Finally it can be noted that the proportions of 3:1 segregation products in males and females for both translocations are not significantly different. These results are consistent with those obtained by Tease (1998) in mice but disagree with the hypothesis of a strong predisposition of the female meiosis to 3:1 segregation (Faraut et al., 2000
; Mackie Ogilvie and Scriven, 2002
). For the 12/14 translocation, as already mentioned, the symmetrical pachytene configuration (long pairing segments) as well as the relatively high proportion of interstitial chiasmatas (37.9%) clearly favours the occurrence of 2:2 segregations. This leads to a very low proportion of 3:1 segregation products, even in the females, and can explain the lack of difference between the two sexes. In constrast, in the 3/15 translocation, a higher proportion of III+I configuration in prophase I is expected. In such a situation, a non-negligible proportion of 3:1 segregation products should be observed. Moreover, this proportion should be higher in females than in males because of the apparent predisposition of female meiosis to produce this kind of imbalance (lower meiotic quality controls: Hunt and Hassold, 2002
). However, a high chiasma frequency in the translocated and interstitial segments may incline the prophase I meiotic configurations towards chain-IV and ring configurations instead of the expected III+I, and therefore may incline the segregation towards 2:2 segregations instead of 3:1. The chiasma frequency being more important in females than in males (especially in the interstitial segments), this could partially compensate the basic predisposition of the female meiosis to produce 3:1 segregation products, and partially explain the low difference between the two sexes. Another possible explanation could be the relatively young age of the sows used in our study. Indeed, the data showing that human female meiosis appears highly error-prone (post-natal and gametic studies) were obtained on adult women whose oocytes were arrested in prophase I for a very long period (generally >20 years). This is not the case of the females used in our study, the majority of which have been slaughtered just after puberty before 1 year of age.
The estimated frequency of diploid oocytes was relatively high in the case of the 3/15 translocation (22.9%). Although this value is much higher than that of the 12/14 translocation (5.3%; P<0.001) or than several results obtained in humans (e.g. 3.5%, Plachot, 2001; 5.4%, Pellestor et al., 2002
) it is nevertheless consistent with earlier results obtained by Vozdova et al. (2001)
and Sosnowski et al. (2003)
in sows with normal karyotypes (27.7 and 12.8% respectively). Otherwise, the higher diploidy rate observed in females compared to males for both translocations suggests a more stringent meiotic quality control affecting spermatocytes as compared with oocytes during the meiotic process (Hunt and Hassold, 2002
).
In conclusion, this study is a new scientific contribution to compare the segregation profiles of male and female carriers of identical chromosomal rearrangements, a topic that has been rarely investigated to date. This approach could be extended to an analysis of other translocations in pigs, even of other types of chromosomal rearrangements (such as inversions). An interesting complement to this work could be provided by new immunohistochemical techniques, based on the use of anti-MLH1 and anti-SCP3 antibodies, which allow an accurate determination of the distribution of crossing-over in oocytes and spermatocytes (Baker et al., 1996). This would enable the effects of the rearrangements on chiasma distribution in the two sexes to be analysed and compared. Also, complementary studies difficult to carry out in humans could be envisaged in pigs, such as the analysis of the effect of age on male and female segregation profiles in the presence of a chromosomal rearrangement, or the investigation of inter-individual variability of male segregation profiles.
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Acknowledgements |
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Submitted on February 7, 2005; resubmitted on April 1, 2005; accepted on April 11, 2005.
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