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Titolo:
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
Autore:
MILLAR MR; SHARPE RM; MAGUIRE SM; GAUGHAN J; WEST AP; SAUNDERS PTK;
Indirizzi:
MRC,REPROD BIOL UNIT,37 CHALMERS ST EDINBURGH EH3 9EW SCOTLAND
Titolo Testata:
International journal of andrology
fascicolo: 3, volume: 17, anno: 1994,
pagine: 149 - 160
SICI:
0105-6263(1994)17:3<149:LOMBIH>2.0.ZU;2-1
Fonte:
ISI
Lingua:
ENG
Soggetto:
MESSENGER-RIBONUCLEIC-ACID; SERTOLI CELLS; INSITU HYBRIDIZATION; MOLECULAR-CLONING; HORMONAL-REGULATION; RNA EXPRESSION; SPERMATOGENESIS; SEQUENCE; BIOSYNTHESIS; SPERMATIDS;
Keywords:
IN-SITU HYBRIDIZATION; RESIDUAL BODIES; ACTIVIN RECEPTOR-II; ALPHA-INHIBIN; CYCLIC PROTEIN-2; CREM; SGP-2; TRANSITION PROTEINS 1 AND 2; BLUESCRIPT;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
30
Recensione:
Indirizzi per estratti:
Citazione:
M.R. Millar et al., "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", International journal of andrology, 17(3), 1994, pp. 149-160

Abstract

Several recent articles have reported localization of specific mRNAs in the rat testis to stage IX and X seminiferous tubules using in-situhybridization. In all cases the expression was located basally in thetubules and appeared as discrete round clusters of grains close to the lamina propria. The localization was interpreted as being in Sertolicells or leptotene spermatocytes. In this study we demonstrate that this pattern is most probably due to artefactual binding of probes to the residual body (RB). In the present study testicular tissue, perfusion-fixed with Bouin's and embedded in paraffin, was used, as this resulted in excellent morphological preservation such that RBs within tubules at stages VIII-X were clearly distinguishable. RNA content of the RBs was demonstrated at stages VIII-X using methyl green pyronin staining, and could be eliminated by pretreatment with RNAse or trichloroacetic acid. Localization of mRNAs for 11 seminiferous tubule proteins was assessed using S-35-labelled and digoxigenin-labelled riboprobes (activin receptor-II, alpha-inhibin, transferrin, androgen-binding protein (ABP), cyclic protein-2 (CP-2), CREM, sulphated glycoproteins 1 and2 (SGP-1 and SGP-2), transition protein 2 (TP-2) and cystatin-C), anddigoxigenin-labelled oligonucleotide probes (transition protein-1 (TP-1), TP-2 and protamine-1). All of these probes showed localization tothe correct cell type(s) within the seminiferous epithelium. In addition, six antisense riboprobes (activin receptor-II, CREM, SGP-2, CP-2,cystatin C and alpha-inhibin) showed hybridization to basally locatedresidual bodies in tubules at stages IX-X on one or more occasions, whereas residual bodies around the edge of the lumen (stage VIII) or intransit through the seminiferous epithelium showed no hybridization; sense probes showed no localization to residual bodies. A common feature of the probes which localized to the basal Rbs was that they had prepared using cDNA cloned into Bluescript SK- vector such that the antisense strand was generated from the T7 polymerase promotor. A cRNA prepared using T7 polymerase and Bluescript vector alone and a GC-rich 27mer 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 probably a subtle artefact unique to RBs undergoing dissolution following fusion with Sertoli cell lysosomes, and may reflect nonspecific hybridization to GC-rich fragments of RNA.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 11/07/20 alle ore 11:16:59