[The characters and specific features of new human embryonic stem cells lines].

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

[Molecular genetic analysis of Y-chromosome micro deletions in men with severe spermatogenic defects]. / Molekuliarno-geneticheskii analiz mikrodeletsii Y-khromosomy u muzhchin s tiazhelymi narusheniiami spermatogeneza.

Microdeletions of the Y-chromosomal AZF loci were revealed in 10 (12%) of 82 patients with severe idiopathic spermatogenetic defects. Deletions involved AZFc in six patients, AZFa in one patient, AZFb + c in two patients, and AZFa + b + c in one patient. Microdeletion analysis employed multiplex PCR with 22 pairs of primers directed to Y-specific STS of deletion intervals 5, 6, and 7 (Yq11). Spermatogenesis in men with AZF microdeletions was assessed with semen analysis, microscopic examination of testicular aspirate, and quantitative karyotypic analysis of immature germline cells in ejaculate or aspirate. The character of spermatogenetic defects was correlated with the size and location of microdeletions in order to study the genotype-phenotype relationship.