Cytogenetic Analysis Of Rhesus Macaque Oocytes And Embryos

Aneuploidy analysis can be done in two ways. First, standard karyotyping analysis can be performed on metaphase II oocytes exhibiting a first polar body. Karyotyping was previously used to examine age-related defects in rhesus monkey oocytes (Schramm et al., 2002). In that study, no statistically significant difference was found between young vs. old rhesus monkeys in aneuploidy frequency of oocytes. However, the number of oocytes examined was very small, totaling only 30 oocytes for both groups. We need to reexamine the question of whether aneuploidy increases with age in rhesus oocytes, using much larger numbers of oocytes obtained from ovaries excised at necropsy. The rhesus chromosome number is 42, and the homologies to the human karyotype are simple. There are two homologues of human chromosome 2 (HSA2), HSA14 and 15 as well as HSA 7 and 21, and HSA 20 and 22 are fused. Rhesus macaque BAC clones have now been generated in the laboratory of Dr. Leslie Lyons at the School of Veterinary Medicine, University of California-Davis. In collaboration with Dr. Lyons, our laboratory is trying to develop a macaque whole chromosome paint set. Meanwhile, we are developing three or four chromosome probes simultaneously, either by chromosome painting with human probes or by FISH with human BAC clones. Dr. Lyons' laboratory is making progress in developing a series of rhesus macaque FISH probes that we plan to compare against simple karyotyping methodologies using rhesus blood cells.

Interestingly, we have found that the AneuVysion® Assay (CEP 18, X, Y-alpha satellite, LSI 13 and 21) Multi-color Probe Panel from Vysis does not work on rhesus blood cells. Since these human centromeric probes from Vysis do not hybridize with highly repeated human satellite DNA sequences usually located at the centromeric region of the chromosome, Dr. Lyons' laboratory is now comparing repeat sequences in the monkey genome to the human genome. We found that Vysis LSI probes that consist of DNA probe sequences homologous to specific human genes or unique sequence FISH probes do hybridize quite well with human, chimpanzee and baboon as well as rhesus blood cell chromosomes (Figure 39.3).

In summary, because the FISH probes for detecting rhesus chromosomes are not fully developed yet, we recommend using standard karyotyping to ensure results. In a well-designed experiment, half of the oocytes, selected at random, from each cohort should be examined by karyotyping, while the other half is fertilized to study chromosome error rates in the embryos derived from the oocytes. After adequate FISH probe analysis methods become available for rhesus oocytes, then some oocytes should be examined in this way, to find if this is more suitable for future, larger-scale studies. Alternatively, for karyotype analysis, oocytes or embryo blas-tomeres can be processed and chromosomes prepared using methods previously described (Schramm and Bavister, 1999). Metaphase spreads are performed on mature (metaphase II) oocytes as described by Kamiguchi et al. (1993).

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