Dose-dependent effects of a glucocorticoid on prolactin production.ppt

8/14/2019 Dose-dependent effects of a glucocorticoid on prolactin production.ppt

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Nada Almutairi

Y O K O YA M A K o t a r o ; H AYA S H I M a h o k o ;

M O G I C h i h i r o ; S A S A K AWA Yo s h i h i r o ;WATA N A B E G e n ; TAYA K a z u y o s h i ;D E V N AT H S u k u m a r ; I N O U E K i n j i ;

( 2 0 0 8 )

Dose-dependent Effects of Glucocorticoid on Prolactin

Production


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Nada Almutairi

The Objectives:

To Study the biphasic effects of corticosteroneon GH and PRL production in a rat pituitarysomatomammotropic cell line (MtT/SM) Invitro.

To find out if glucocorticoids receptor (GR)mediate the effects of corticosterone on

prolactin production.

To Study the effects of both E estrogen andcorticosterone on PRL synthesis and PRL cellproliferation.


3/20

Nada Almutairi

Introduction

Anterior pituitarycells: Corticotropes, Thyrotropes, Gonadotropes, Somatotropes

and Mammotrophs

(Lactotroph).


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Nada Almutairi

Radioimmunoassay (RIA)Th e Tech n iq u e A m ixtu re is p re p a re d o f

rad io a ctive a n tig e n B eca u se o f the ea se w ith w hich io d in e a to m s can b e

in tro d u ce d in to tyrosine,re sid u es in a p ro te in th e

ra d io a ctive isotopes 125 Ior 131 .I are often used

.antibodies against that antigen

(" ")Known amounts of unlabeled cold antigen are

.added to samples of the mixture These compete for the binding sites .of the antibodies

At increasing concentrations of unlabeled,antigen an increasing amount of radioactive antigen is displaced from the antibody

.molecules -The antibody bound antigen is separated from

,the free antigen in the supernatant fluid and

.the radioactivity of each is measured

, ,From these data a standard binding curve like, .this one shown in red can be drawn

( )The samples to be assayed the unknowns are.run in parallel

After determining the ratio of bound to free,antigen in each unknown the antigen

concentrations can be read directly from the( ).standard curve as shown above
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Tyr_phe.gifhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Elements.html#isotopeshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Ag_Ab.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/Ag_Ab.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Elements.html#isotopeshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Tyr_phe.gif


5/20

Nada Almutairi

Cell Culture:

They used pituitary

clonal cell lineMtT/SM (known toproduce GH andPRL), derived from

an estrogen-inducedtransplantable ratprolactinoma tumor

line.

Materials and Methods


6/20

Nada Almutairi

The Objectives:

To Study the biphasic effects of corticosterone To Study

the biphasic effects of corticosteroneon GH and PRL production in a rat pituitaryon GH and PRL p

roduction in a rat pituitarysomatomammotropic cell line (MtT/SM) Insomatomammotrop

ic cell line (MtT/SM) Invitro.vitro.





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Nada Almutairi

Immunofluorescent

staining

for

GH

and

PRL

Materials andMethods: Radioimmunoassay for

medium conc. Of PRL:

uan a ve ana ys s o an mexpression and PRL secretion on MtT/SM

Cells!

-l uor es ce nt l abe le d 2 nd :b oat ant i monkey onkey ant i rabbi t


8/20

Nada Almutairi

Results: medium conc. Of PRL: Quantitative real time PCRQuantitative real time PCRwas performed for GH andwas performed for GH andPRL.PRL.

uan a ve ana ys s o an mexpression and PRL secretion on MtT/SM

Cells!


9/20

Nada Almutairi

Results: Double Immunofluorescent staining for GH ( )red and PRL

( )green /on MtT SM cells treated with several doses of.corticosterone


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Nada Almutairi

The Objectives:


To find out if glucocorticoids receptor (GR) To find out if g lucocorticoids receptor (GR)mediate the effects of corticosterone onmediate the effects of corticosterone on

prolactin production.prolactin production.



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Nada Almutairi

Materials and Methods

Cells +

corticosterone(10 ~ 10)

Then do real time

PCR.

The Effects of Corticosterone on GR and MRmRNA e xpression

For 3 days


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Nada Almutairi

Results:

The Effects of Corticosterone on GR and MRmRNA e xpression


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Nada Almutairi

Mifepristone inhibited thechange in the number of PRL immunopositive cellscaused by Corticosterone.

Materials and Methods: Results:

Effects of GR antagonist onCorticosterone -induced PRL production !

h e i mm un op osi ti ve c el l umber was determined


14/20

Nada Almutairi

The Objectives:




To Study the effects of both E estrogen and To Study the effects of both E estrogen andcorticosterone on PRL synthesis and PRL cellcorticosterone on PRL sy nthesis and PRL cell

proliferation.proliferation.


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Nada Almutairi


(~10) corticosteronesuppressed ER mRNAexpression; while(~10) Increased it.

Effects of Corticosterone on estrogen(receptor ER ) mRNA expression .


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Nada Almutairi

Effects of Corticosterone and E on PRL!production and PR L cell appearance



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Nada Almutairi

Effects of Corticosterone AND E on PRL.cell pro liferation

Materials and Methods:

!o u b le im m u n o flu re sce n t s ta in in g fo r P R L a n d B rd U


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19/20

Nada Almutairi

Conclusions


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Nada Almutairi

T h a n k Y o u !

NadaadaAlmutair ilmutair i

Dose-dependent effects of a glucocorticoid on prolactin production.ppt

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