| Background Spermatogenesis is a complex and very efficient process which include spermatogonium proliferation, spermatocyte meiosis and spermeiogenesis. During meiotic prophase I, homologous chromosomes pair, synapse and recombine. These three events are essential for a successful meiotic process. Any alteration of the meiotic process results in a reduction of the quality and quantity of gamete, consequently cause infertility. To more accurately examine the events of pairing and recombination in meiosis, techniques that use ?uorescently labeled antibodies to specific meiotic structures in situ have been developed. Antibodies to synaptonemal complex proteins such as SCP3, MLH1 protein (a mismatch repair protein localized to events of recombination), and CREST (localized to the centromeric regions on all chromosomes), when applied to spreads of testicular cells, can be used to identify the events of pairing and recombination in cells at different stages of meiosis. With this new developed immunofluorescence technique, a new avenue was opened up to examined meiotic recombination directly in human spermatocyte, especially for infertile patients. Although intracytoplasmic sperm injection (ICSI) was invented to help thousands of male infertility father their biological offspring, this technique bypass nature fertilization process which might bring genetic defects to the new generation. Thus, it is critical necessary to prove up the cytobiology mechanism of male infertile patients.Objectives In this study, spermatocytes were immunolabelled in order to analyze the defective homologous chromosomes recombination of azoospermic patients.Material and Methods The testicular samples of seven control fertile men (control group) and seven azoospermic patients which included two obstructive azoospermic (OA) patients and five non-obstructive azoospermic (NOA) patients, were immunolabelled in the present study. Immunofluorescence technique was performed to categorize the early stage cells in meiosis prophase and analyze the chromosome pair and recombination of pachytene spermatocyte. Newly developed meiotic proteins antibodies (anti-SCP3, anti- synaptonemal complex proteins 3;anti-MLH1, anti-Mut-L Homolog 1;anti-CREST, chromosome centromere antibody) were used to identify synaptonemal complex (anti-SCP3), recombination sites (anti-MLH1) and centromere (anti-CREST). The stage of spermatocyte was identified according to the SCP3 formation progression. The MLH1 foci demonstrated recombination frequency. Qualitative data were compared by the chi-square test, while ANOVA was used to analyze quantitative data. p<0.05 was considered statistically different.Results1.For the control group, 2 346 spermatocytes were categorized and the mean proportions of leptotene, zygotene and pachytene cells were 14.0%, 4.8% and 81.4%, respectively. While, 2 932 spermatocytes were categorized in the seven azoospermic patients and the proportions of zygotene cells in two OA patients and four NOA patients were significantly higher than that in control group.2.1 867 spermatocytes from control group were carefully analyzed. The mean MLH1 foci per pachytene cell was 49.54. Another 354 pachytene cells were analyzed in the seven azoospermic patients and the mean MLH1 foci per pachytene cell were 44.1±4.9, 48.7±6.6, 48.4±6.4, 49.7±5.0, 13.2±13.5, 39.2±14.8 and 45.1±7.1, respectively. Statistical difference was found when compare the recombination frequency (the MLH1 foci per cell) of one OA patients with control group (44.1±4.9 versus 49.54±4.2, p<0.05), moreover, the mean MLH1 foci per cell in two NOA patients were significant lower than that in control group (13.2±13.5,39.2±14.8 vs 49.54±4.2,p<0.01). 3.The mean number of the autosomes which contained one, two, three, four MLH1 foci per pachytene cell in control person was 0.1, 3.47, 11.49, 5.54 and 1.29, respectively. The mean number of autosome without MLH1 foci per pachytene cell in two OA patients and four NOA patients were significantly higher than that in control group. Moreover, the same trend was observed when considered the proportion of cells contained at least one autosomal bivalent without MLH1 foci between control group and azoospermic patients. The proportion of cells with MLH1 foci in sex chromosome of two OA patients and three NOA patients were significantly lower than that in control group.4.The mean proportion of cells with gap (discontinuous chromosomal region) and split (unpaired chromosomal region) in control group were 13.9% (5%-21%), 2.3% (0-9%), respectively. No difference was found when compared the mean proportion of cells with gap and/or split between control group and OA patients, while statistical differences were found between control group and some of the NOA patients.Conclusions Immunofluorescence technique was a powerful method to categorize the early stages of spermatocytes in human. Azoospermic patients suffered from a delayed meiosis prophase compared with that in control fertile men. The mean MLH1 foci per pachytene cell decreased and pachytene cells with abnormal recombination increased in azoospermic patients. The delayed meiosis prophase and abnormal recombination events might be the possible reasons for azoospermia. |
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