The mean fluorescence intensity of spermatogenic cells was measured with the ZEN-2010 software in conjunction with an LSM-700 microscope. Fluorescence brightness was classified into 255 levels.
To separate the sex chromosome signal from noise, we defined the signal as a fluorescence intensity that was greater than 50 at one or more point-like signals in the nucleus; a fluorescence intensity below 50 was characterized as “without signal”. In preleptotene and pachytene spermatocytes in stage VIII seminiferous tubules, if only X or Y was not detected, the spermatocytes were classified as “without signal.” to accurately calculate the hybridization efficiency. If step 8 spermatids had two or more signals, the cells were considered to be aneuploid. It was difficult to count the signals of elongated spermatids because many of them overlapped. In this study, therefore, we counted the signals of preleptotene spermatocytes, pachytene spermatocytes, and step 8 spermatids using the FISH method without proteinase K. We chose 12 stage VIII seminiferous tubules in one cross section for analysis. We evaluated aneuploidy as described by Komaki et al. [22 (link)]. A chi-squared test was used to investigate the deviation from the expected ratio of 50:50 (X:Y). Differences were considered significant at a level of P < 0.05.
To separate the sex chromosome signal from noise, we defined the signal as a fluorescence intensity that was greater than 50 at one or more point-like signals in the nucleus; a fluorescence intensity below 50 was characterized as “without signal”. In preleptotene and pachytene spermatocytes in stage VIII seminiferous tubules, if only X or Y was not detected, the spermatocytes were classified as “without signal.” to accurately calculate the hybridization efficiency. If step 8 spermatids had two or more signals, the cells were considered to be aneuploid. It was difficult to count the signals of elongated spermatids because many of them overlapped. In this study, therefore, we counted the signals of preleptotene spermatocytes, pachytene spermatocytes, and step 8 spermatids using the FISH method without proteinase K. We chose 12 stage VIII seminiferous tubules in one cross section for analysis. We evaluated aneuploidy as described by Komaki et al. [22 (link)]. A chi-squared test was used to investigate the deviation from the expected ratio of 50:50 (X:Y). Differences were considered significant at a level of P < 0.05.