Abstract
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 35S-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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Cox KH, DeLeon DV, Angerer LM, Angerer RC (1984) Detection of mRNAs in sea urchin embryos by in situ hybridisation using asymmetric RNA probes. Dev Biol 101: 485–502
Erickson-Lawrence M, Zabludoff SD, Wright WW (1991) Cyclic protein 2, a secretory product of rat Sertoli cells, is the proenzyme form of cathepsin L. Mol Endo 5: 1789–1798
Fraggioni G, Borgioli G (1979) Polystyrene embedding: a new method for light and electron microscopy. Stain Tech 54: 167–172
Giaid A, Hamid Q, Adams C, Springall DR, Terenghi G, Polak JR (1989) Non isotopic RNA probes: comparison between different labels and detection systems. Histochemistry 93: 191–196
Gordon KC (1982) Tissue processing. In: Bancroft JD, Stevens A (eds) Theory and practice of histological techniques. Churchill Livingstone, New York, pp 41–60
Grosskopf R, Feldman H (1981) Analysis of a DNA segment from rat liver mitochondria containing the genes for the cytochrome oxidase subunits i, ii, iii, atpase subunit 6, and several tRNA genes. Curr Genetics 4: 191–196
Jamrich M, Mahon KA, Gavis ER, Gall JG (1984) Histone RNA in amphibian oocytes visualised by in-situ hybridisation to methacrylate embedded tissue sections. EMBO J 3: 1939–1943
Larsson L-I, Hougaard DM (1990) Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment, hybridization and probe labelling conditions. Histochemistry 93: 347–354
Leblond CP, Clermont Y (1952) Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci 55: 548–573
Luerssen H, Maier WM, Hoyer-Fender S, Engel E (1989) The nucleotide sequence of rat transition protein 2 (TP2) cDNA. Nucl Acids Res 17: 3585
Parvinen M (1986) Regulation of the seminiferous epithelium. Endocr Rev 3: 404–417
Parvinen M (1993) Stage-dependent changes in Sertoli cell function. In: Russell LD, Griswold MD (eds) The Sertoli Cell. Cache River Press, pp 331–347
Saiki PK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning. A laboratory manual. 2nd edn. New York, Cold Spring Harbor Press
Saunders PTK, Millar MR, Maguire SM, Sharpe RM (1992) Stage specific expression of rat transition protein 2 mRNA and possible localisation to the chromatoid body of step 7 spermatids by in situ hybridisation using a non radioactive riboprobe. Mol Reprod Dev 33: 385–391
Saunders PTK, Millar MR, West AP, Sharpe RM (1993) Mitochondrial cytochrome C oxidase is expressed at a high level in pachytene spermatocytes and in a stage dependant manner during spermatogenesis in the rat. Biol Reprod 48: 57–67
Sharpe RM (1990) Intratesticular control of steroidogenesis. Clin Endocrinol 33: 787–807
Sharpe RM (1993) Experimental evidence for Sertoli-germ cell and Sertoli-Leydig cell interactions. In: Russell LD, Griswold MD(eds) The Sertoli Cell. Cache River Press, pp 391–418
Stevens A (1982) Resin embedding media. In: Brancroft JD, Stevens A (eds) Theory and practice of histological techniques. Churchill Livingstone, New York, pp 458–466
Sue W, Griffin T (1987) Methods for hybridization and quantitation of mRNA in individual brain cells. In: Valentino KL, Eberwine JH, Barchas JD (eds) In situ hybridization. Applications to Neurobiology. Oxford University Press, pp 97–110
Wright WW (1988) Germ cell-Sertoli cell interactions: analysis of the biosynthesis and secretion of cyclic protein-2. Dev Biol 130: 45–56
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Millar, M.R., Sharpe, R.M., Maguire, S.M. et al. Cellular localisation of messenger RNAs in rat testis: application of digoxigenin-labelled ribonucleotide probes to embedded tissue. Cell Tissue Res 273, 269–277 (1993). https://doi.org/10.1007/BF00312828
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00312828