The effect of cold storage on recombination frequencies in human male testicular cells

Abstract

Meiotic recombination is essential for the segregation of homologous chromosomes and formation of normal haploid gametes. Decreased recombination is associated with the production of aneuploid sperm in humans. MLH1, a DNA mismatch repair protein, was recently found to mark the sites of recombination in humans. Newly developed immunofluorescence techniques to identify MLH1 foci on synaptonemal complexes (SCs) in pachytene cells from testicular tissue have opened up a new avenue of research on meiotic recombination. Future studies on normal and abnormal recombination in early meiosis will further research in human reproduction and genetics. However, the availability of testicular material will always be a major limiting factor in this kind of study. In order to obtain an adequate number of samples and samples of particular research interest, it is often of benefit to obtain samples from distant regions. Therefore, it is necessary to determine whether the quality of samples and accuracy of MLH1 frequencies change after transporting testicular samples from a distance. In the present study, we examined the recombination frequencies (numbers of MLH1 foci using immunofluorescence techniques) in 6 normal testicular samples. Each sample was split and analyzed in the fresh state and after storage on ice for two days, mimicking overnight courier air transport. The results showed no significant difference in the quality of the SC preparations or in the number of MLH1 foci between these two groups. These results demonstrate that testicular specimens may be shipped on ice without compromising data on chromosome pairing and recombination in early meiosis.

Abnormal progression through meiosis in men with nonobstructive azoospermia

“…Studies in normal men have been reported (5,6,9,10), and a few studies in infertile men have been published as case reports (8,15) or small series (7,11,12,16).…”

“…We have previously demonstrated that cold storage of testicular tissue for 2 days does not affect recombination frequencies (6). Patients gave informed consent, and this study received ethical approval from the institutional review boards at the University of Calgary and the University of California San Francisco.…”

“…Recently developed immunocytogenetic techniques, which allow simultaneous analysis of synapsis and meiotic recombination by visualizing MLH1 recombination proteins along the synaptonemal complex, have opened up a new avenue for studying the early stages of meiosis during spermatogenesis (5,6). Antibodies to synaptonemal complex proteins SCP1 and SCP3 allow visualization of the axial (lateral) elements and transverse filaments, from the beginning of their assembly in early leptotene until their disassembly in diplotene.…”

Immunofluorescent synaptonemal complex analysis in azoospermic men

“…Testicular material from San Francisco was kept in PBS and transferred on ice by air courier to the lab in Calgary, Canada for fluorescence immunocytogenetic analysis. We have previously demonstrated that cold storage of testicular tissue does not affect chromosome synapsis and recombination (Sun et al, 2004c). This study received ethical approval from the institutional review boards of the University of California San Francisco and the University of Calgary.…”

Cryopreservation has no effect on meiotic recombination and synapsis in testicular tissues

“…Few attempts have been made to examine the effects of the freezing and thawing procedure itself on spermatogenesis, particularly on homologous synapsis and recombination during prophase I of meiosis. Sun et al (9) demonstrated that cold storage of testicular tissue on ice at 4 C for 2 days had no effect on the quality of preparations or on chromosome pairing or recombination. Lyrakou et al (10) reported that long-term cryopreservation had no effect on meiotic recombination and synapsis based on comparisons with the published data from fresh samples from different patients.…”