ANALYSIS OF MEIOTIC SEGREGATION PATTERNS AND INTERCHROMOSOMAL EFFECTS IN SPERM FROM 13 ROBERTSONIAN TRANSLOCATIONS
Wang B1,*, Nie B1,*, Tang D2,*, Li R3,*, Liu X1, Song J1, Wang W1, Liu Z1,** *These authors contributed equally to this study.
*Corresponding Author: Dr. Zhi Liu, Department of Clinical Laboratory, Hubei Maternal and Child Health Hospital, 745 WuLuo Road, Wuhan, Hubei, Peoples Republic of China. Tel: +86-27-8716-9190. Fax: +86-27-8716-9097. E-mail: wangbo1005@163.com.
page: 43

MATERIALS AND METHODS

Patients. Thirteen male carriers of ROB translocations (designated P1-13) consulting for infertility or repeated abortions were included in this study. Twelve were heterozygous ROB translocation and one was a homozygous ROB translocation. Except P1 and P2 (P2 was P1s father), all the patients were unrelated. Three males with normal 46,XY karyotype and normal sperm characteristics were recruited as controls. The local ethics committee approved this protocol and all patients and controls had previously given informed consent for the study. Sperm Preparation, Fluorescent in Situ Hybridization and Scoring. All semen samples were first analyzed to evaluate volume, concentration and motility according to the World Health Organization (WHO) criteria [16]. After semen analysis, sperm with progressive motility was isolated and washed twice in phosphate buffered saline (pH 7.4) by centrifugation at 1500 rpm for 11 min. Final pellets were fixed with 5 mL of acetic acid/methanol mixture (1:3) for at least 30 min. at 4 C. Aliquots (40-50 L) of the resulting suspension of nuclei were smeared on cold pre-cleaned slides. Nuclei decondensation was performed in 1 N NaOH for 2 min. After dehydration in ethanol series (70.0, 90.0, 100.0%), denaturation was performed in 0.25% formamide in 2 × saline sodium citrate (SSC) followed by overnight hybridization with a combination of commercially available probes. To assess the segregation behavior of each rearrangement, a dual-color FISH using locus-specific probes and/ or telomere probes and/or chromosome enumeration probes, was carried out on spermatozoa of the translocation carriers. Below we list the probes that we used to identify the segregation products for each karyotype. For a der(13;14) carrier, LSI13 (13q14, Spectrum Green) and Tel14 (14q32.33, Spectrum Red) were used. For a der(14;15) carrier, Tel14 (14q32.33, Spectrum Red) and Tel15 (15q26.3, Spectrum Green) were used. For a der(14;21) carrier, Tel14 (14q32.33, Spectrum Red) and LSI21 (21q22.13-q22.2, Spectrum Green) were used. For a der(13;22) carrier, LSI13 (13q14, Spectrum Green) and LSI22 (22q12.3, Spectrum Red) were used. For a der(21;22) carrier, LSI21 (21q22.13-q22.2, Spectrum Green) and LSI22 (22q12.3, Spectrum Red) were used. Regarding ICE evaluation, triple-color FISH was performed using the probe mixture including commercial satellite (DNA) probes for chromosomes 18, X and Y (CEP 18, Spectrum Blue/CEP X, Spectrum Green/CEP Y, Spectrum Red) from Vysis (Abbott Park, IL, USA). Post-hybridization washes included 2 min. in 0.4 × SSC/0.3% NP-40 (pH 7) at 72 C, followed by 1 min. in 2 × SSC/0.1% NP-40 (pH 7) at room temperature. Slides were covered with DAPI II (Vysis). Only intact spermatozoa bearing a similar degree of decondensation and clear hybridization signals were scored. Disrupted or overlapping spermatozoa were excluded from analysis. Only slides with hybridization efficiency of 99.0% and more were analyzed. One thousand sperm nuclei per patient were analyzed. Statistical analyses. The χ2 test was used to compare the frequencies of segregation products. A p value of less than 0.05 was regarded as statistically significant.



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