Defective oogenesis in mice with pristane-induced model of systemic lupus.

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by generation of autoantibodies and severe damage of various organs. The hormonal changes associated with pregnancy and especially estrogen might lead to damage of reproductive function and ovarian quality. We employed a pristane-induced lupus model of Balb/c mice which resembles human lupus in an attempt to follow oogenesis disruption during the disease progression. The integrity of cytoskeletal and chromatin structures was estimated in oocytes derived by hormonally stimulated ovulation in lupus mice and the results were compared with those from healthy mice. Chromatin, tubulin and actin structures in oocytes were detected by Hoechst 33258, anti-alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. All available meiotic spindles were analyzed - in immature (metaphase I) and mature oocytes (metaphase II). The total number of mature oocytes obtained from lupus mice was lower compared to healthy controls. The maturation rate was 9.8 % for lupus mice, 12.7 % for 7-month old controls, and 14.3 % for the young control mice (4 weeks old). Another major difference between the studied groups was the higher percentage of defective metaphase I spindles registered in oocytes derived from lupus mice (60 % normal spindles), while for the young and older controls this proportion was 86 % and 81 %, respectively. No such difference was registered for metaphase II spindles. For both metaphase I and metaphase II oocytes, the proportions of normal actin cap and chromosomal condensation were similar between the experimental groups.

Analysis of human unfertilized oocytes and pronuclear zygotes--correlation between chromosome/chromatin status and patient-related factors.

OBJECTIVE: The objective was to evaluate the relationship between ploidy and chromatin status of human unfertilized oocytes/zygotes and infertility history, female age, and stimulation regimens. STUDY DESIGN: Two hundred and eighty-nine unfertilized oocytes and 63 zygotes were subjected to cytogenetic analysis: karyotyping for oocytes and fluorescent in situ hybridization (FISH) analysis for zygotes. Ploidy and chromosome/chromatin status were analyzed according to stimulation regimen, female age, and infertility history. The correlation coefficient was estimated and data were interpreted using a five-grade scale. RESULTS: Aneuploidy in karyotyped oocytes (19.7% hyperhaploidy, 18.8% hypohaploidy, and 6.3% haploid abnormal) was associated with chromosome fragmentation and lesions due to chromosome aging in culture. Premature chromosome condensation and cytoskeletal defects were significantly higher in unexplained infertility (34.7% and 52.9%, respectively; p<0.05). Chromatin quality was most important for successful ploidy analysis of zygotes. FISH analysis of abnormal zygotes elucidated genetic aspects of pronuclear number aberrations and raised questions about the current selection criteria. Abnormalities were found to correlate moderately with stimulation strategy and female age and significantly with infertility history. CONCLUSION: Genetic analysis of human oocytes and zygotes showed that poor chromatin quality and patient-related factors contribute to aneuploidy and pronuclear number aberrations.

Chromosomal disorders and nuclear and cell destruction in cleaving human embryos.

Three types of defects of preimplantation embryogenesis contribute to the developmental arrest of cleaving human embryos: blastomere fragmentation, abnormal nuclear status and chromosomal disorders. Data concerning the relation and succession of these abnormalities during first mitotic cycles of the human zygote are controversial and mainly empirical at present. In this study we have performed simultaneous evaluation of blastomere fragmentation, nuclear apoptotic changes and the ploidy of four chromosomes (1, 5, 19 and X or 18, 21, X and Y) in 193 human embryos. Another group of 28 embryos was subjected to TUNEL for confirmation of apoptosis in blastomere nuclei. Nuclei with apoptotic chromatin were seen in nearly 1/10 of blastomeres of embryos with good morphology and in more than 1/5 of blastomeres of embryos with more than 20% fragmentation. The correct number of investigated chromosomes was registered in 85.2% of successfully tested embryos. Chromatin apoptotic changes are the only limiting factor for the success of chromosomal FISH tests. Nearly 1/2 of embryos with at least one apoptotic nucleus were chromosomally abnormal. For the embryos that contain only normal nuclei, the rate of ploid normality was more than 89%. The rate of euploidy was higher (66%) in embryos with a significant degree of cell fragmentation. Moderate cell fragmentation was not related to significant increase of chromatin and chromosomal disorders. In a substantial portion of abnormal blastomeres, chromatin damage preceded cell fragmentation. Nuclear destruction in human blastomeres was illustrated by fluorograms of different stages of chromatin lesions.