LOCALIZATION OF MESSENGER-RNAS BY IN-SITU HYBRIDIZATION TO THE RESIDUAL BODY AT STAGES IX-X OF THE CYCLE OF THE RAT SEMINIFEROUS EPITHELIUM- FACT OR ARTIFACT

Several recent articles have reported localization of specific mRNAs i n the rat testis to stage IX and X seminiferous tubules using in-situ hybridization. In all cases the expression was located basally in the tubules and appeared as discrete round clusters of grains close to the lamina propria. The localization was interpreted as being in Sertoli cells or leptotene spermatocytes. In this study we demonstrate that th is pattern is most probably due to artefactual binding of probes to th e residual body (RB). In the present study testicular tissue, perfusio n-fixed with Bouin's and embedded in paraffin, was used, as this resul ted in excellent morphological preservation such that RBs within tubul es at stages VIII-X were clearly distinguishable. RNA content of the R Bs was demonstrated at stages VIII-X using methyl green pyronin staini ng, and could be eliminated by pretreatment with RNAse or trichloroace tic acid. Localization of mRNAs for 11 seminiferous tubule proteins wa s assessed using S-35-labelled and digoxigenin-labelled riboprobes (ac tivin receptor-II, alpha-inhibin, transferrin, androgen-binding protei n (ABP), cyclic protein-2 (CP-2), CREM, sulphated glycoproteins 1 and 2 (SGP-1 and SGP-2), transition protein 2 (TP-2) and cystatin-C), and digoxigenin-labelled oligonucleotide probes (transition protein-1 (TP- 1), TP-2 and protamine-1). All of these probes showed localization to the correct cell type(s) within the seminiferous epithelium. In additi on, six antisense riboprobes (activin receptor-II, CREM, SGP-2, CP-2, cystatin C and alpha-inhibin) showed hybridization to basally located residual bodies in tubules at stages IX-X on one or more occasions, wh ereas residual bodies around the edge of the lumen (stage VIII) or in transit through the seminiferous epithelium showed no hybridization; s ense probes showed no localization to residual bodies. A common featur e of the probes which localized to the basal Rbs was that they had pre pared using cDNA cloned into Bluescript SK- vector such that the antis ense strand was generated from the T7 polymerase promotor. A cRNA prep ared using T7 polymerase and Bluescript vector alone and a GC-rich 27m er oligonucleotide corresponding to the region of the multiple cloning site of Bluescript adjacent to the T7 site both localized uniquely to basal RB. It is concluded that the hybridization seen within RBs is p robably a subtle artefact unique to RBs undergoing dissolution followi ng fusion with Sertoli cell lysosomes, and may reflect nonspecific hyb ridization to GC-rich fragments of RNA.