Evolution du nucleole dans les ovocytes de rat au stade de follicule preovulatoire
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Colloque franco-belgede M.E.. mai 1988 AUTORADIOGRAPHIC AND IMMUNOCYTOCHEMICAL STUDIES OF PERICHROMATIN GRANULES ACCUMULATED IN THE NUCLEI OF HEAT-SHOCKED CELLS. Edmond PUVION (1), Annie VIRON ( 1 ) , Francis HARPER (]) and Jean-Paul FUCHS (2). (]) Groupe de BioJogie et G~n~tique Mol~culaires IRSC BP 8 94802 VillejuiF Cedex (France) and (2) Laboratoire de G6n~tique Mol~culaiee des Eucaryotes 1], rue Humann 67085 Strasbourg Cede×. During recovery at 37°C of HeLa cells previously submitted to a brief heat shock, large clusters of perichromatin 9ranules (PG) accumulate in the extranucleolar area of the nucleus. Electron microscope autoradiographs following labeling of cells with tritiated uridine in the presence or not of low doses actinomycin D, clearly demonstrate that the accumulated PG contain extranucleolar RNA synthesized during recovery at 37°C. This RNA remains stored For several hours at the level of PG in conditions of blockage of its transport to the cytoplasm, The nature of PG was further analyzed by indirect immunolabeling carried out on thin sections of cells embedded in Lowicryl K4M. Anti-Sm and anti-RNP antibodies against nucleoplasmic small nuclear ribonucleoproteins (SnRNP) as well as an antibody against proteins of hnRNP clearly associate with PG. These observations represent a direct confirmation that PG a c c u m u l a t e d in heat- shocked cell nuclei are hnRNP structures involved in the storage and processing of hnRNA. Our results are discussed in view of the known effects of heat shocks on cellular metabolism; they may lead to a better knowledge of the Functional significance of PG regularly observed in relative low quantities in normal cells. IMMUNOELECTRON MICROSCOPE DETECTION OF REPLICATED DNA BY MONOCLONAL ANTI-BROMODEOXYURIDINE ANTIBODY ON ULTRATHIN SECTIONS. Marc THIRY. Laboratoire de Biologic cellulaire et tissulaire, Institut A. Swaen, 20, rue de PiLteurs, ~-4020 Liege, Belgique. A new method for the identification of replicated DNA at the ultrastructural level has been develo- ped. In a first step, cells are exposed in vitro to 5-bromodeoxyuridine (BUdR) in conjunction with S-flu- orodeoxyuridine (FUdR) in order to privilege the incorporation of the exogenously supplied BUdR and to avoid exposure to high doses of BUdR known as a mutagenic substance. In a second step, cells are fixed, dehydrated and embedded either in Lowicryl K4M or in Epon 812. Finally, the BUdR incorpora- ted into DNA is detected on ultrathin sections by indirect immunogold technique using a monoclonal an- ti-BUdR antibody. Further, in order to increase the accessibility of BUdR binding sites to the antibo- dies, a prior HCI hydrolysis is recommended. Under these conditions, a very intense and specific labeling with high resolution is observed. All major cellular sites known to contain DNA (nucleus, chromosomes, mitochondria} can be revealed accor- ding to the BUdR exposure time. After a very short pulse of BUdR, this relatively simple method al- lows to visualize the replication sites within cells. Further, when most cells in the examined population are exposed to BUdR at least during one S phase, the labeling pattern reflects, to a large extent, a general distribution of DNA. This recent approach has been used to study the spatial repartition of DNA w~thin the nucleolus. Gold particles are essentially observed over dense perinucleolar and Intranucleolar chromatin. In addi- tion, some gold particles are also localized over the fibrillar centres preferentially at their periphery. EVOLUTION DU NUCLEOLE DANS LES OVOCYTES DE RAT AU STADE DE FOLLICULE PREOVULATOIRE. Nadine ANTOINE, Alain VIGNERON et Guy GOESSENS. Laboratoire de Biologie cellulaire et tissulaire, Instltut A. Swaen, 20, rue de Pitteurs, B-qO20 Li6ge, Belgique. Le nucl~ole subit dqmportantes transformations morphologiques au cours de I'ovogen6se. De struc- ture r6ticul~e, il devient, au stade de follicule antral, enti6rement compact. II est alors constitu6 d'une masse homog6ne et dense occupant tout le volume nucl6olaire au d6triment des constituants clas- siqueso La nature et les fonctions de cette masse nucl6olaire sont encore controvers6es. Des 6tudes cytochimiques nous ont permis d'y mettre en 6vidence des prot6ines non argyrophiles riches en groupements thiols et en ponts disulfures. A I'aide de la microanalyse en rayons X, une quantit6 abondante de S a 6t6 d6tect6e dans cette masse homog6ne confirmant ainsi les r6sultats cy- tochimiques. La technique RNase-gold a permis quant 6 elle de r6v61er une faible quantit6 de RNA au sein de la masse nucl6olaire. Ces r6sultats nous ont permis de postuler que la masse nucl6olaire correspondrait 6 un stockage de prot~ines et de RNA. Lorsque I'on provoque la maturation ovocytaire 6 I'aide d'hormones lut6inisantes jusqu16 un stade proche de I'ovulation, le nucl6ole se vacuolise et, simultan6ment, des corps extra-nucl6olaires appa- raissent au sein de la v6sicule germinale. Ceux-ci semblent ~tre d'origine nucl6olaire et pourraient correspondre 6 un largage de RNP dans la v6sicule germinale alors que I'activit6 transcriptionnelle y est stopp6e et ne reprendra qu'aux premiers stades de Imembryogen~se. Une 6tude biochimique pr61iminaire semble indiquer que la masse nucl6olaire serait essentiellement constitu6e d'une prot6ine d'environ 65,000 MW. 15a
Transcript of Evolution du nucleole dans les ovocytes de rat au stade de follicule preovulatoire
Colloque franco-belge de M.E.. mai 1988
AUTORADIOGRAPHIC AND IMMUNOCYTOCHEMICAL STUDIES OF PERICHROMATIN GRANULES ACCUMULATED IN THE NUCLEI OF HEAT-SHOCKED CELLS. Edmond PUVION (1 ) , Annie VIRON ( 1 ) , F ranc i s HARPER ( ] ) and Jean-Paul FUCHS ( 2 ) . ( ] ) Groupe de B ioJogie et G~n~tique M o l ~ c u l a i r e s IRSC BP 8 94802 V i l l e j u i F Cedex (France) and (2) Labora to i re de G6n~t ique Mol~cu la iee des Eucaryotes 1] , rue Humann 67085 St rasbourg Cede×.
Dur ing recovery at 37°C o f HeLa c e l l s p r e v i o u s l y submi t ted to a b r i e f heat shock, la rge c l u s t e r s o f p e r i c h r o m a t i n 9 ranu les (PG) accumulate in the e x t r a n u c l e o l a r area o f the nuc leus . E lec t ron microscope au to rad iographs f o l l o w i n g l a b e l i n g o f c e l l s w i th t r i t i a t e d u r i d i n e in the presence or not o f low doses ac t inomyc in D, c l e a r l y demonst ra te t h a t the accumulated PG con ta in e x t r a n u c l e o l a r RNA s y n t h e s i z e d du r i ng recovery at 37°C. This RNA remains s to red For severa l hours at the leve l o f PG in c o n d i t i o n s o f blockage of i t s t r a n s p o r t to the cytop lasm, The nature o f PG was f u r t h e r ana lyzed by i n d i r e c t immunolabel ing c a r r i e d out on t h i n sec t i ons o f c e l l s embedded in Lowicry l K4M. Anti-Sm and ant i -RNP a n t i b o d i e s aga ins t nuc leop lasmic small nuc lea r r i b o n u c l e o p r o t e i n s (SnRNP) as wel l as an an t ibody aga ins t p r o t e i n s o f hnRNP c l e a r l y assoc ia te w i th PG.
These o b s e r v a t i o n s rep resen t a d i r e c t c o n f i r m a t i o n t h a t PG accumulated in hea t - shocked c e l l nuc le i are hnRNP s t r u c t u r e s invo lved in the s torage and process ing of hnRNA. Our r e s u l t s are d iscussed in view of the known e f f e c t s o f heat shocks on c e l l u l a r metabol ism; they may lead to a b e t t e r knowledge of the Funct iona l s i g n i f i c a n c e o f PG r e g u l a r l y observed in r e l a t i v e low q u a n t i t i e s in normal c e l l s .
IMMUNOELECTRON MICROSCOPE DETECTION OF REPLICATED DNA BY MONOCLONAL ANTI-BROMODEOXYURIDINE ANTIBODY ON ULTRATHIN SECTIONS. Marc THIRY. Laboratoire de Biologic cellulaire et tissulaire, Insti tut A. Swaen, 20, rue de PiLteurs, ~-4020 Liege, Belgique.
A new method for the identification of replicated DNA at the ultrastructural level has been develo- ped.
In a f i rst step, cells are exposed in vitro to 5-bromodeoxyuridine (BUdR) in conjunction with S-flu- orodeoxyuridine (FUdR) in order to privilege the incorporation of the exogenously supplied BUdR and to avoid exposure to high doses of BUdR known as a mutagenic substance. In a second step, cells are fixed, dehydrated and embedded either in Lowicryl K4M or in Epon 812. Finally, the BUdR incorpora- ted into DNA is detected on ultrathin sections by indirect immunogold technique using a monoclonal an- ti-BUdR antibody. Further, in order to increase the accessibility of BUdR binding sites to the antibo- dies, a prior HCI hydrolysis is recommended.
Under these conditions, a very intense and specific labeling with high resolution is observed. All major cellular sites known to contain DNA (nucleus, chromosomes, mitochondria} can be revealed accor- ding to the BUdR exposure time. After a very short pulse of BUdR, this relatively simple method al- lows to visualize the replication sites within cells. Further, when most cells in the examined population are exposed to BUdR at least during one S phase, the labeling pattern reflects, to a large extent, a general distribution of DNA.
This recent approach has been used to study the spatial repartition of DNA w~thin the nucleolus. Gold particles are essentially observed over dense perinucleolar and Intranucleolar chromatin. In addi- tion, some gold particles are also localized over the fibri l lar centres preferentially at their periphery.
EVOLUTION DU NUCLEOLE DANS LES OVOCYTES DE RAT AU STADE DE FOLLICULE PREOVULATOIRE. Nadine ANTOINE, Alain VIGNERON et Guy GOESSENS. Laboratoire de Biologie cellulaire et tissulaire, Instltut A. Swaen, 20, rue de Pitteurs, B-qO20 Li6ge, Belgique.
Le nucl~ole subit dqmportantes transformations morphologiques au cours de I'ovogen6se. De struc- ture r6ticul~e, il devient, au stade de follicule antral, enti6rement compact. II est alors constitu6 d'une masse homog6ne et dense occupant tout le volume nucl6olaire au d6triment des constituants clas- siqueso La nature et les fonctions de cette masse nucl6olaire sont encore controvers6es.
Des 6tudes cytochimiques nous ont permis d'y mettre en 6vidence des prot6ines non argyrophiles riches en groupements thiols et en ponts disulfures. A I'aide de la microanalyse en rayons X, une quantit6 abondante de S a 6t6 d6tect6e dans cette masse homog6ne confirmant ainsi les r6sultats cy- tochimiques. La technique RNase-gold a permis quant 6 elle de r6v61er une faible quantit6 de RNA au sein de la masse nucl6olaire. Ces r6sultats nous ont permis de postuler que la masse nucl6olaire correspondrait 6 un stockage de prot~ines et de RNA.
Lorsque I'on provoque la maturation ovocytaire 6 I'aide d'hormones lut6inisantes jusqu16 un stade proche de I'ovulation, le nucl6ole se vacuolise et, simultan6ment, des corps extra-nucl6olaires appa- raissent au sein de la v6sicule germinale. Ceux-ci semblent ~tre d'origine nucl6olaire et pourraient correspondre 6 un largage de RNP dans la v6sicule germinale alors que I'activit6 transcriptionnelle y est stopp6e et ne reprendra qu'aux premiers stades de Imembryogen~se.
Une 6tude biochimique pr61iminaire semble indiquer que la masse nucl6olaire serait essentiellement constitu6e d'une prot6ine d'environ 65,000 MW.
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