Fetal brain tissue was obtained from 10 fetuses at the time of selective terminations of intrauterine pregnancies from healthy females. The permission of the Ethics Com­mittee of Mental Health Research Center of the Russian Academy of Medical Sciences (RAMS) was obtained. Written informed consent was obtained from the abortion-seeking women. The sterile suction abortion was per­formed at pressures 0.3-0.6 bar by a12 mm vacuum curette (Laboratoire Juneau, Paris, France). The criteria for inclu­sion were: age from 25 to 35 years; gestation age 9-11 weeks; negative test for the most common infectious dis­eases; absence of systemic and genetic diseases in both parents; absence of treatment by drugs with teratogenic effect. Gestational age was determined by a number of parameters (date of last menstrual period, uterine size, ultrasonography, and after abortion, by fetal foot length measurement).

The fetal tissue was transferred to a laminar flow hood. The brain was rinsed with EBSS (Cat #24010-035; Gibco Invitrogene Corporation, SARL, Cergy Pontoise Cedex, France). Medulla oblongata was isolated from the surrounding tissue by a transverse cut through the brain stem at the level of the pons and of the lower medulla using a dissecting microscope (Wild/Leitz M 32; Ernst Leitz Wetzlar GmbH, Wetzlar, Germany). Meninges and major blood vessels were removed. The isolated medulla was placed in a sterile plastic petri dish with 2 mL EBSS, and transversely cut into 300-400 mL slices by hand with razor blades. The slices were then divided into about 20 pieces, rinsed in EBSS and transferred to rubber silicon stoppered pyrex glass bottles containing 10 mL of me­dium. Samples were cultivated in high-speed mini-roller under rotation at 60 rpm for 4 weeks without changing the medium [7]. One hundred mL of medium consists of 97.3 mL MEM (Cat. #32260-018, Gibco Invitrogene Corporation); 5 mL of fetal bovine serum (Cat. #10106-169; Gibco Invitrogene Corporation); 5 mL human pla­centa serum; 2 mL of 40% glucose; 1 mL of 200 mM L-glutamine; 0.5 mL of insulin; 0.2 mL of antibiotic/ antimyotic (Cat. #15240-039; Gibco Invitrogene Corpora­tion). A culture series was defined as the total number of cultures from a single medulla oblongata.

Chromosome 1-, 18-, 13/21-, X-, and Y-specific DNA probes were cloned in the Laboratory of Cytogenetics of National Research Center of Mental Health, Moscow, Russia, and prepared as described previously [8-10]. Chro­mosome 1-specific classical satellite DNA pUC1.77 (re­gion 1q12) was kindly supplied by Dr. Howard Cooke (MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland). Mixtures of (i) biotinylated chro­mosome 1-specific probe, Cy3-labeled chromosome Y-specific probe, fluoresceine-labeled chromosome X-spe­cific probe, of (ii) biotinylated chromosome-specific probe, Cy3-labeled chromosome 13/21-specific probe, fluoresceine-labeled chromosome X-specific probe, and (iii) biotinylated chromosome 18-specific probes, Cy3-labeled chromosome Y-specific probe, fluoresceine-la­beled chromosome X-specific probe each taken at final concentration of 50 ng/10 mL of hybridizing solution (20% dextran sulfate and 2 x SSC in 55% formamide, pH 7) was used. The in situ hybridization and detection proto­cols were performed as described earlier [8,11]. Aliquots of DNA probe (10 mL per each slide) were placed onto the slides, covered by coverslips and denatured for 5 min. at 72°C. After hybridization at 37°C for 4 hours or over­night, the slides were washed for 15 min. in 50% forma­mide, 2 x SSC (pH 7) at 42°C, followed by two washes of 5 min. each in 2 x SSC, 0.1% Tween 20 at 37°C. The slides were then incubated with 5 mg/mL AMCA-avidin (Vector Laboratories, Burlingame, CA, USA) to detect the biotin-labelled DNA probes Finally, interphase nuclei were counterstained with diluted (1:10) DAPI to allow simultaneous observation of total DNA and hybridized probe. The DNA stain was carried in anti-fade solution to preserve the fluorescence during extended microscopy sessions. Hybridization signals were detected simulta­neously using a Zeiss-Axioscope fluorescence microscope (Carl Zeiss GMBH, Jena, Germany) with objectives of 63X or 100X. One thousand interphase nuclei were ana­lyzed for each specimen.