Crossover analysis using immunofluorescent detection of MLH1 foci in frozen–thawed testicular tissue

To date, the effects of freezing on spermatogenesis have not yet been fully investigated at a molecular level. Antibody localization studies have identified the MutL homolog 1 (MLH1) protein, a mis-match repair protein, at the prophase I stage of meiosis, which allows the detection of recombination foci during pachytene. This study investigated the effect of long-term testicular tissue cryopreservation on meiotic prophase I, identified by recombination foci frequency and synaptonemal complex (SC) integrity. Frozen–thawed testicular tissues from 12 males who had each fathered a child were used. Because vasectomy or reverse vasectomy procedures are rare in the locale of the investigation, it was not possible to obtain fresh testicular tissue and use the males as their own controls. Immunocytogenetic analysis of 612 spermatocytes at the pachytene stage was performed. The results indicated a mean number of MLH1 foci of 49.2 (SD ± 5.9), and no correlation was found between the freezing period, the MLH1 frequency and the SC integrity. The results suggest that freezing of testicular tissue taken post-puberty does not appear to be detrimental to the crossover process as identified by occurrence of MLH1 loci.