Proceedings: Fluorescence microscopy of damage to the germinal epithelium of the rat testis.

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Acute heat damage to the rat testis causes cytoplasmic eosinophilia of late pachytene primary spermatocytes and the activation of tubular lysosomal enzymes within 1 to 2 hr. These cells desappear within 30 hr and spermatogenesis is disrupted. A number of fluorescent probes react with cytoplasmic and nuclear components of cells and these may help identify structural and chemical changes in the heat damaged testis. In aqueous solution, 2-p- toluidinyl-naphthalene-6-sulphonate (TNS) is non-fluorescent but it may bind hydrophobically to proteins when it fluoresces strongly. TNS bound strongly to the membranes of damaged primary spermatocytes and on excitation (365nm) there was a pale green fluorescence. The organic, mercurial, mercurochrome binds to SH groups and stains the normal testis with little differentiation. After heating, damaged cells showed fluorescent and absorption patterns indicative of increased numbers of available SH groups. Acridine orange (AO) shows inoin binding to phosphate groups of DNA and RNA and emits a green fluorescence 530NM) when excited at 365 NM. Non-ionic association of AO with RNA causes a red fluorescence (590nm). In the normal testis the latter was rapidly lost in heat-damaged spermatocytes and the green nuclear fluorescence became prominent. The trypanocidal dye, ethidium bromide, also binds to RNA and DNA probably by intercalation. Early spermatocyte nuclei fluoresced strongly at 590 nm in the normal testis but after heating the damaged pachytene primary spermatocytes showed a strong orange fluorescence of the nuclei. The results suggest that heating the testis causes early membrane damage (TNS) to the late pachytene primary spermatocytes with the reduction of S-S bonds to SH (mercurochrome). Changes in nucleic acids also occurred (AO and ethidium bromide) and these prececed actual neclear breakdown by several hours.