CELLULAR-LOCALIZATION OF MESSENGER-RNAS IN RAT TESTIS - APPLICATION OF DIGOXIGENIN-LABELED RIBONUCLEOTIDE PROBES TO EMBEDDED TISSUE

In an attempt to identify key changes involved in normal spermatogenesis we have developed methods to enable the study of gene expression by the various subpopulations of testicular cells by use of in-situ hybridisation histochemistry. The use of digoxigenin-labelled ribonucleotide and oligonucleotide probes on testicular tissue perfusion-fixed with Bouin's fixative and embedded in paraffin, polystyrene or methacrylate, has been used to accurately localise three transcripts to three different cell types (Sertoli cells, pachytene spermatocytes, and step 7-12 spermatids) within the seminiferous tubule. The ability to produce semi-thin sections of polystyrene- or methacrylate-embedded tissue and successfully to apply digoxigenin-labelled ribonucleotide or oligonucleotide probes resulted in far greater resolution and unequivocal localisation of mRNA in testicular cells than was previously possible by use of thicker paraffin or frozen sections hybridised with S-35-labelled riboprobes. A comparison of the different embedding media versus digoxigenin-labelled oligonucleotide or ribonucleotide probes is made and we demonstrate the relative sensitivities and merits of each combination.