icacgm18th International Colloquim of Animal Cytogenetics and Gene Mapping 2008

Session VII - Cytogenetics of reproduction


Sex chromosomes and male and female embryo development

W. Allan King

Department of Biomedical Sciences, University of Gueph, Guelph, On N1G 2W1, Canada

The fundamental differences between males and female are coded in the genes located on the sex chromosomes and can be traced to their initial expression. The Y-chromosome is variable in size and DNA content, largely heterochromatic and contains less than 100 coding sequences mostly related to sex determination and fertility. The X-chromosome consists of approximately 160 Mb (3 to 5% of the genome), codes for over 1000 genes with a diverse range of functions. In females, one of the two X-chromosomes is subject to epigenetic alteration in transcriptional potential (X-inactivation) to compensate for sex-specific differences in gene copy number.
Over the past 20 years, it has been recognized that sex related differences occur prior to implantation and the development of the fetal gonads. It has been shown that males develop faster, reach developmental milestones such as blastulation earlier and with more cells. This differential growth rate is more pronounced in vitro than in vivo. With the advent of gene expression technologies, it has been shown that bovine embryos produced by in vitro fertilization and cloning have altered levels of mRNA for a number of X-linked genes compared to in vivo produced ones. The differences between in vivo and in vitro produced embryos may allow us to identify the external triggers for this chromosome silencing (funded by NSERC, CIHR and Canada Research Chairs program).


Cytogenetics of Reproduction

Dino Di Berardino

Department of Soil, Plant, Environment and Animal Production Sciences, University of Naples "Federico II" , Via Università 100, 80055 Portici, Naples, Italy.

Chromosome analysis of embryos of domestic animals started around the 70’s, followed - ten years later- by analogous studies on sperm, synaptonemal complex, and oocytes.
In those years, conventional cytogenetic analysis was the only way to look at the chromosomes worldwide, but the relatively low efficiency in chromosome visualization, plus the chromosomal loss due to technical artefacts, required new technical approaches which came along around the end of 90’s with the fluorescent in situ hybridization techniques (FISH,PRINS) applied on interphase nuclei which, as known, do not require visualization of individual chromosomes. The laboratory production of chromosome-specific molecular probes by chromosome microdissection and DOP-PCR has further expanded the usefulness of cytogenetic investigation to other aspects, such as genome organization and meiotic segregation patterns. Recently, the advent -in humans- of a new immuofluorescent technique capable to visualize synaptonemal complex and recombination foci would make possible to investigate the recombination frequency in pachytene spermatocytes in various domestic animal species, breeds or genetic types. Other new interesting areas of research are now related to epigenetic changes, telomere integrity, X-inactivation, gene reprogramming.
Finally, Comparative Genomic Hybridization (CGH) and multicolor FISH or SKY-FISH for domestic animals are expected to come, like in humans, thus increasing the usefulness of Reproductive Cytogenetics for the implementation of genetic improvement programs in animal production.


Familial meiotic segregation analysis of a t(13 ;17) Robertsonian translocation in pigs

A. Pinton (1), N. Bonnet (1), A. Calgaro (1), S. Ferchaud (2) B. Billoux (1), V. Bacquie (1), N. Mary (1), A. Bonnet-Garnier (1), M. Yerle (1) and A. Ducos (1)

1. UMR 444 Genetique Cellulaire 23 chemin des Capelles, BP 87614, 31076 Toulouse Cedex 3 (France);
2. INRA / UEICP,Venours 86480 Rouille (France)

Robertsonian and reciprocal translocations are the most common structural rearrangements in Man. On the opposite, very few cases of Robertsonian translocations have been described in pigs compared to the reciprocal ones. Carriers of this kind of abnormality have a normal phenotype but can present reproduction troubles (gametogenesis disorders and/or unbalanced gametes production).
We report the analysis of the meiotic segregation of a t(13 ;17) Robertsonian translocation in related individuals : 3 males with normal spermatogenesis and 10 females. The "spermFISH" technique (fluorescent in situ hybridization on decondensed sperm heads) was used to determine the male segregation profiles and female meiosis analysis was carried out by chromosome painting on metaphases II of in vitro matured oocytes.

To our knowledge, this is the first study of the meiotic segregation analysis of a Robertsonian translocation in pigs. The results showed that the segregation profiles obtained for the three males were not statistically different and that the proportion of unbalanced gametes produced was low (3.29% on average) suggesting a limited impact of the translocation on the males’ reproduction. On the contrary, the proportion of unbalanced gametes was higher in females (19.18%). These results are coherent with the literature and suggest a higher effect of the translocation on the reproduction in females than in males.


Study of inter- and intra-individual variation of meiotic segregation patterns in t(3;15)(q27;q13) boars

K. Massip, H. Berland, N. Bonnet, A. Calgaro, S. Billoux, V. Baquie, N. Mary, A. Bonnet-Garnier, A. Ducos, M. Yerle and A. Pinton

UMR 444 INRA-ENVT Genetique Cellulaire, Ecole Nationale Veterinaire de Toulouse, 23 chemin des Capelles, BP 87614, 31076 Toulouse Cedex 3 (France)

Constitutional chromosomal rearrangements are relatively frequent genetic anomalies in both Man and pigs. These chromosomal rearrangements can be responsible for various developmental defects or for a degradation of the reproductive performance due to spermatogenesis impairments and/or production of genetically unbalanced gametes. Among them, reciprocal translocations are the most frequent in pigs, and present specific meiotic segregation patterns. The potential variation of segregation profiles between individuals carrying the same t(3;15)(q27,q13) translocation ("individual" effect) was studied using SpermFISH by comparing the meiotic segregation patterns of three boars. Otherwise, three samples were taken at different times from one of these boars to analyze a potential "time" effect.
Neither "time" nor "individual" effect was found in this study. This work brings new insights into reciprocal translocation meiotic segregation for related carriers, and then should allow a more efficient management of certain reciprocal translocations in pig population. However, these results are specific to the t(3;15), and need to be completed by the study of other kinds of chromosomal rearrangements.


MLH1 foci as an evidence of recombination events between B chromosomes in Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and red fox (Vulpes vulpes)*

J. Sosnowski (1), L. Migalska (1), A. Lukaszewicz (1), M. Wojnowska (1), A. Pienkowska-Schelling (2), Z. Polanski (3)

1. Department of Genetics and Animal Breeding, Agricultural University of Poznan (Poland)
2. Department of Animal Sciences, Federal Institute of Technology Zurich (Switzerland)
3. Department of Genetics and Evolution, Jagiellonian University, Cracow, (Poland)

In the karyotype of around 50 animal species, additional chromosomes were identified and called B chromosomes. They segregate randomly, vary in number in cells and form different configuration during meiotic prophase. Until our research there was no evidence for recombination events between B chromosomes.
We visualised synaptonemal complexes in pachytene spermatocytes, using antibody against axial element protein SCP3 and recombination foci using antibody against MLH1 (mismatch repair protein known to associate with late recombination nodules at the site of chiasmata). The FISH method, with specific painting probes for identification of raccoon dog’s B chromosomes, was used.
In 45 spermatocytes obtained from 3 male of Chinese raccoon dogs, 2 to 5 B chromosomes were observed. Among trivalents, the number of MLH1 foci varies from 1 to 2 and in quadrivalents from 0 to 3. Completely paired B chromosomes, distinguishable after FISH application, contained one MLH1 foci.
In 70 spermatocytes from testis of 3 male red foxes, from 1 to 4 B chromosomes were noticed. MLH1 foci were observed on bivalents (0-1 foci) and trivalents (0-2 foci).


Reproductive consequences of a new reciprocal translocation rcp(3;14) in a hypoprolific boar

A. Rodríguez (1), E. Sanz (1), E. De Mercado (1), E. Gómez (1), V. Domenech (2), M. Martín (2), C. Carrascosa (2), E. Gómez (2), R. Sánchez (2)

1. Instituto Tecnológico Agrario de Castilla y León, Valladolid (Spain).
2. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid (Spain).

Cytogenetic analysis of boars from an artificial insemination centre, revealed the presence of a reciprocal chromosomal translocation in a finish Duroc boar aged of 23 months. Peripheral blood lymphocytes culture and GTL banding techniques were used to identify the rearrangement as rcp(3;14)(p14;q23). The same abnormality was also detected in a littermate’s boar, located in the same artificial insemination centre, suggesting the parental origin of the abnormality. Both boars were phenotypically normal. The influence of the translocation on reproductive performance of boars was evaluated in 244 double mating records of sows in one herd, inseminated with one of two seminal doses of the translocated boars. Results were compared with records of contemporaries normal karyotype boars in the same herd. The reduction in litter size between both translocated boars and the normal karyotype boars was 11.82% (9.88 vs. 11.21 total piglets born, P<0.05), and fertility values decreased 16.01% (P=0.0001). Additionally, results in 17 mating events inseminating only with translocated boars were recorded, and the reduction of fertility and prolificacy observed was 41.84% and 15.10% respectively. This is the first time the translocation rcp(3;14)(p14;q23) is identified in pigs, and these results demonstrate the loss on reproductive performance even using boar’s seminal doses in doubles inseminations with normal karyotype boars.


A mosaicism XX/XY in a case of intersexuality in pigs

A. Rodríguez (1), E. Sanz (1), E. De Mercado (1), E. Gómez (1), V. Domenech (2), M. Martín (2), C. Carrascosa (2), E. Gómez (2), R. Sánchez (2)

1. Instituto Tecnológico Agrario de Castilla y León (ITACyL), Valladolid (Spain).
2. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid (Spain).

The present study describes a case of intersex pig include in a nutrition experience in the Pig Research Centre (ITACyL). Zootechnical parameters were measured from 20 to 100 kg, and cytogenetic analysis was carried out. Chromosome preparations from peripheral blood cultures were identified by GTL banding technique. This animal presented normal body conformation except for external genitalia, with the presence of completely developed testes and vulva with enlarged clitoris. Initially the animal presented a female phenotype, but until approximately 25 kg the testes had not descended into the scrotum. Lymphocyte’s karyotype revealed a mosaicism in sex chromosomes 38,XX/38,XY, and the result of analysis of one hundred metaphases showed a percentage of 30% of the cells with 38,XY karyotype and 70% of 38,XX cells. Compared with males and females, the intersex pig showed a lower weight gain (15.23%) until 55 kg, but to 100 kg it was higher (15.63%), corresponding with the appearance of a strongly boar taint and an increased aggressive behaviour of the animal. Cases of intersexuality usually consists of XX sex reversed animals with various degrees of masculinisation, and only a small proportion of detected intersex pigs are mosaics. Consequences of intersex condition in fattening pigs may result from an aggressive behaviour in groups and boar taint causing losses on meat quality.


A new reciprocal translocation rcp(3;18) in a boar and its effects on prolificacy and fertility

A. Rodríguez (1), E. Sanz (1), E. De Mercado (1), E . Gómez (1), V. Domenech (2), M. Martín (2), C. Carrascosa (2), E. Gómez (2), R. Sánchez (2)

1. Instituto Tecnológico Agrario de Castilla y León, Valladolid (Spain).
2. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid (Spain).

A new reciprocal chromosomal translocation was found in a Duroc boar of an AI centre, where all animals were cytogenetically analyzed. The translocation was located between chromosomes 3 and 18, and it was described as rcp(3;18)(q14;q21). The mitotic chromosomes were obtained from cultures of peripheral blood lymphocytes, and the rearrangements were highlighted using GTL banding techniques. Reproductive performance of this boar was studied in one herd, in 35 double mating records using one of two seminal doses of the translocated boar, and results were compared with normal karyotype boars contemporaries in the same herd. Fertility was not affected by the presence of seminal doses of the translocated boar, but an average reduction of prolificacy of 20.25% was observed in 27 litters of the boar. Statistical analyses indicated significantly differences between litter size (total piglets born) of translocated boars and normal karyotype boars (P=0.0095), and the mean difference was 2.22 piglets (10.96 vs. 8.74 total piglets born respectively). Carrier boar exhibited a perfectly normal body conformation. Only 4 reciprocal translocations involving acrocentric chromosome 18, the smallest in the pig karyotype, have been identified in pigs, but this is only the second study that demonstrate the effect on reproductive performance.


Meiotic segregation analysis of three different chromosome 2 inversions in pigs

K. Massip, M. Yerle, A. Ducos and A. Pinton

UMR 444 INRA-ENVT Genetique Cellulaire, Ecole Nationale Veterinaire de Toulouse, 23 chemin des Capelles, BP 87614, 31076 Toulouse Cedex 3 (France)

Inversions are structural chromosomal rearrangments originating from two breaks in the same chromosome, a subsequent 180° rotation of the chromosomal fragment, and a final joining of the different segments. This kind of rearrangement is considered to be harmless for the carrier, but can lead to spermatogenesis troubles and production of genetically unbalanced gametes.
In order to analyze the effects of different types of inversions (peri- and paracentric) and of the size of the inverted segment on the production of unbalanced gametes, the meiotic segregation patterns of pericentric inversions 2 (p1.1; q1.1) and (p1.1; q2.1) and paracentric (q1.3; q2.5) were studied. To the best of our knowledge, this is the first study of meiotic segregation of inversions in pigs using SpermFISH.
The percentage of unbalanced gametes were 0.33 %, 1.18 % and 3.48 % for inv 2(p1.1; q1.1), inv 2(p1.1; q2.1) and inv2(q1.3; q2.5), respectively. According to these results, it seems that the production of abnormal gametes is correlated with the size of the inverted segment. Despite the few studies reporting the analysis of meiotic segregation in men carriers of inversion, these results seem to be consistent with the data available.
Analysis of spermatocytes using immunocytological techniques will be carried out for the inv2 (q1.3; q2.5) to analyze meiotic pairing.


Comparative Study of Spontaneous and Folate SensitiveChromosomal Instability in Pregnant and Barren Ewes

M. E . Babar, Ahmad Ali, M. Abdullah

University of Veterinary and Animal Sciences, Lahore Pakistan

Correlation between infertility and fragile sites was tested by inducing folate sensitive chromosomal fragile sites in 8 pregnant and 10 barren ewes in Lohi sheep (Ovis aries). Control cultures were grown at 0 µg/ml FUdR and experimental cultures for all animals were synchronized with 5gm/ml 5-Fluorodeoxyuridine to induce chromosomal fragile sites. Aberrant cell count (AC) and number of aberrations (NoA) means per animal in pregnant group were 1.375  0.625 and 1.375  0.625 whereas the corresponding means of the same group were 10.38  1.93 and 16.25  3.12 in control and FUdR cultures respectively (P < 0.01). AC and NoA means in the barren group were 3.90  1.12 and 4.70  1.47 and the corresponding values in FUdR cultures were 9.70  1.11and 16.00  2.14 per animal respectively. The comparison of the AC and NoA means between the two groups inferred a significant variation (P < 0.05) between control and none in FUdR cultures. Fragile site distribution comparison revealed 33 and 49 FUdR unstable chromosomal bands in pregnant and barren groups respectively. Similarly the number of significant fragile sites was 11 and 15 in two groups respectively. X chromosome in the pregnant group expressed only three unstable and none significantly fragile bands. The barren group however, expressed lesions in five X-chromosome bands with Xq17 significantly fragile site.


The karyotype of round goby showed ancestral gobiid character with morphologically undifferentiated sex chromosomes

M., Sapota (1), K., Ocalewicz (2) , K., Dabrowski (3)

1. Department of Marine Biology and Ecology, University of Gdańsk, (Poland);
2. Department of Ichthyology, University of Warmia and Mazury in Olsztyn, 10-957 Olsztyn, ul. Oczapowskiego 5 (Poland);
3. School of Environment and Natural Resources, Ohio State University, Columbus, (USA).

Round goby (Neogobius melanostomus), the Ponto-Caspian invader which arrived to Europe in ballast water, can damage native aquatic comunities by competing for food and habitat and also eating eggs and fry of other species. Presumabely, sex ratio among round gobies influences the number of next generation fish and, consequently, the dynamics of invasion and the ability to colonize new territories. Chromosome set manipulation, namely gynogenesis has been utilized to study sex determination system in the round goby. Traditional and molecular cytogenetic analysis has been employed to search any differences between round goby female and male karyotypes. The diploid chromosome number was invariably 2n= 46 and karyotype composed of telocentric chromosomes gradually decreasing in size. The Nucleolus Organizer Regions (NORs) were observed in the pericentromeric regions of the two medium-sized chromosomes. Sequential staining (DAPI, CMA3 and Ag NO3) showed that the NORs are DAPI negative and CMA3 positive. AT-rich clusters of chromatin were observed in the terminal location of the short arms of NOR bearing chromosomes. PNA FISH with telomeric probe did not reveal any interstitial telomeric sites. Although female and male karyotypes were compared no differences were found.


Investigation of chromosome aneuploidies in early pig embryos by comparative genomic hybridization

M. Horňák, J.Rubeš

Veterinary Research Institute, Brno (Czech Republic)

Although numerical chromosome errors are known to be prevalent in early human embryos and are likely to be a considerable factor influencing the mortality of early embryos and implantation failure, in domestic animals, data about the frequency and nature of errors is rather limited. Several works concerning this topic hitherto, utilized FISH analysis, which has provided information about only a few specific chromosomes. We investigate the whole chromosome set in in-vivo early pig embryos applying methods of whole genome amplification and comparative genomic hybridization (CGH) and contribute to the comprehensive understanding of the issue. CGH allows neither the enumeration of all chromosomes nor determination of chromosomal breakages and partial aneuploidies in an unbalanced manner. Early pig embryos were lysed and underwent whole genome amplification by multiple displacement amplification (MDA). In a subsequent CGH, amplified DNA was compared to the control DNA using different fluorescent labeling and hybridization on male pig mitoses. So far we have examined over fifty pig embryos and detected various chromosomal errors encompassing the whole chromosome aneuploidies and arm-specific aberrations.


Origin of Diploidy in a Bull with Macrocephal Spermatozoa

T. Revay (1), C. Kopp (2), A. Flyckt (2), A. Hidas (1), W. Rens (3), D. Rath (4) and M. Andersson (2)

1. Research Institute for Animal Breeding and Nutrition, Godollo, (Hungary);
2. Department of Production Animal Medicine, University of Helsinki, Saarentaus, (Finland);
3. Department of Veterinary Medicine, University of Cambridge, Cambridge, (UK);
4. Institute of Animal Breeding, Neustadt, (Germany)

An AI bull with 20-25% macrocephal spermatozoa and 16% nuclear crest was investigated. Diploidy was assumed from the measurement of head area and proved by flow cytometry which was also used for sorting the haploid and diploid fractions. Fluorescence in situ hybridization was used to detect the sex chromosomes by an X-Y painting set. These diploid cells most likely originate from a defective meiosis II (M2 diploids) as only 0.7% XY-bearing spermatozoa were detected. The painting probes gave a single X or Y spot not only in the unsorted semen but during the investigation of the diploid fraction too. This indicates the very close vicinity of the non-partitioned sister chomatids even in the very differentiated cell type of spermatozoa. The BC1.2 probe - labeling BTAYp13-12 - was used to clarify the presence of the two chromatids in the singular signal of simultaneously hybridized Y-painting probe.


Application of fluorescence in situ hybridization (FISH) for analyses of the sex chromosomes in the mouse embryos

M. Bugno (1), Z. Jabłońska (2), E. Słota (1)

1. Department of Immuno- and Cytogenetics, National Research Institute of Animal Production, Balice/Kraków (Poland)
2. Faculty of Biology and Earth Sciences, Jagiellonian University of Krakow (Poland)

Mouse embryos were flushed from oviducts and cultured in vitro. The cultures were terminated in 2-, 4- and 8 blastomeres stages by addition of nokodazol. The FISH technique was applied on fixed preparations using commercial probes (Cambio) for X and Y chromosomes. Probes were labeled with FITC and Cy3 for the X and the Y chromosome, respectively. The FISH protocol included small modifications: the temperature of denaturation was 80°C for 5 minutes and hybridization lasted three days. We found that this method was useful for embryo sex identification and determination of the correct number of heterosomes in embryo’s cells.


The chromosomal instability in river buffalo females with hormonal stimulation of oestrus

I.Nicolae (1), M.Paraschivescu (1), A.Bota (2), F.Grigore (2), L.Harceaga (3), M.Roman (3)

1. Research and Development Institute for Bovine, Balotesti (Romania).
2. Research and Development Station for Buffaloes, Sercaia (Romania)
3. Semtest BVN-Tg.Mures (Romania).

In order to obtain a model of hormonal syncronization of oestrus 13 buffaloe females were selected and treated by Prosolvin, a hormonal product. With the aim to avoid the etiological role of chromosomal abnormalities in pathology of reproductive disturbances in the group of 13 buffaloe females the karoytype analysis had been performed. Peripheral blood lymphocytes were cultured for about 72 hours at 38,5˚ C in Minimal Essential Medium (Sigma) supplemented with 15 per cent fetal bovine serum (Sigma) and Con A as mitogen.

Metaphase chromosomes were prepared by conventional air-drying technique. The mitotic preparations were studied with a Nikon-Optiphot-2 microscope. The best images were taken with a CCD camera, processed and stored in specific files on the computer in order to study by using an Advanced American Biotechnology programme (AAB) with a specialized software in karyotype analysis.

The present study revealed that eight females had normal karyotype (2n=50,XX) and five females was found to carry frequent mono- and bi-chromatidic breakages on autosomes and heterosomes. The most unstable chromosome in almost metaphases was X chromosome. According to these results, the chromosomal instability identified in five of the investigated females could be the effect of the hormonal product treatment or the effect of the toxic agents of the environment, i.e.soil, water and forages.

(Acknowledgements: This study was supported by the MADR Sectorial Project 351/2006 )