No abstract

S U M M A R YWe performed basic research into quantifying in situ hybridization (ISH) signals in rat testis, a suitable organ for the quantification because germ cells undergo synchronized development and show stage-specific gene expression. In this model experiment, rRNA was selected as the hybridizable RNA in paraffin sections. Specimens fixed with Bouin's fixative and hybridized with digoxygenin-labeled probes could easily be analyzed quantitatively through "posterization" of the images. The amount of rRNA hybridized with the probe was greatest in early primary spermatocytes, followed by pachytene primary spermatocytes, then diplotene spermatocytes, and finally by secondary spermatocytes and spermatids. The amounts reached low levels in metaphase, anaphase, and telophase of meiotic division and early step 1 spermatids, and then slightly increased during spermiogenesis. ISH rRNA staining was a useful parameter for evaluation of the quantitative analysis of mRNA and the levels of hybridizable RNA in tissue sections.

We previously conducted basic research to quantify in situ hybridization (ISH) signals in rat testes. In this experimental model, we selected ribosomal RNA (rRNA) as the hybridizable RNA in paraffin sections, since it allowed us to easily analyze ISH signals expressed with digoxygenin (DIG)-labeled probes quantitatively through “posterization” of the images. We applied this method to analyze the quantification of transcript, PERF 15 mRNA. PERF 15 is expressed specifically in the testes and localized in the rigid cytoskeletal structure of the sperm head, and has been considered to be involved in the apoptotic process of spermatogenic cells. Quantification of the signals may help to clarify the detailed function of PERF 15. We further analyzed the signals concomitant with a confocal laser scanning microscope. The peak of PERF 15 mRNA expression was found in diplotene spermatocytes, and the amount of PERF 15 mRNA was greatest in late pachytene and diplotene spermatocytes and early spermatids, followed by early pachytene spermatocytes, and then late spermatids. PERF 15 may be involved in the events leading to meiotic division, in which apoptosis is also involved. The present study may help to determine the concentration of mRNA in tissue sections.