Dose-dependent Effects of

Glucocorticoid on Prolactin
Production

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 A W A   Yo s h i h i r o ;
W ATA 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 ;
(2008)

Nada Almutairi
 The Objectives:

To Study the biphasic effects of corticosterone

on GH and PRL production in a rat pituitary
somatomammotropic cell line (MtT/SM) In
vitro.

To find out if glucocorticoids receptor (GR)
mediate the effects of corticosterone on
prolactin production.

To Study the effects of both E₂ estrogen and
corticosterone on PRL synthesis and PRL cell
proliferation.
Nada Almutairi
 Introduction
Anterior pituitary


cells:
§ Corticotropes,
§ Thyrotropes,
§ Gonadotropes,
§ Somatotropes
and
§ Mammotrophs
(Lactotroph).

Nada Almutairi
 Radioimmunoassay (RIA)
T h e Te ch n iq u e
•A m ixtu re is p re p a re d o f
•ra d io a ctive a n tig e n
•B e ca u se o f th e e a se w ith
w h ich io d in e a to m s ca n b e
in tro d u ce d in to tyrosine
 re sid u e s in a p ro te in , th e
ra d io a ctive  isotopes  125 I
or 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).
Nada Almutairi
 Materials and Methods

Cell Culture:


They used pituitary

clonal cell line
MtT/SM (known to
produce GH and
PRL), derived from
an estrogen-
induced
transplantable rat
prolactinoma tumor
line.
Nada Almutairi
 The Objectives:

To Study the biphasic effects of corticosterone

on GH and PRL production in a rat pituitary
somatomammotropic cell line (MtT/SM) In
vitro.

To find out if glucocorticoids receptor (GR)
mediate the effects of corticosterone on
prolactin production.

To Study the effects of both E₂ estrogen and
corticosterone on PRL synthesis and PRL cell
proliferation.
Nada Almutairi
 Quantitative analysis of GH and PRL mRNA
expression and PRL secretion on MtT/SM
Cells!
Materials and


Methods: Radioimmunoassay for



 medium conc. Of PRL:

Immunofluorescent
staining for GH
and PRL

Fluorescent - labeled 2 nd Ab :
→Goat anti monkey
→Donkey anti rabbit

Nada Almutairi
 Quantitative analysis of GH and PRL mRNA
expression and PRL secretion on MtT/SM
Cells!
Results:


º Quantitative real time PCR º medium conc. Of PRL:

was performed for GH and
PRL.


Nada Almutairi
 Results:
 Double Immunofluorescent staining for GH (red) and PRL
(green) on MtT/SM cells treated with several doses of
corticosterone.

Nada Almutairi
 The Objectives:

To Study the biphasic effects of corticosterone

on GH and PRL production in a rat pituitary
somatomammotropic cell line (MtT/SM) In
vitro.

To find out if glucocorticoids receptor (GR)
mediate the effects of corticosterone on
prolactin production.

To Study the effects of both E₂ estrogen and
corticosterone on PRL synthesis and PRL cell
proliferation.
Nada Almutairi
 The Effects of Corticosterone on GR and MR
mRNA expression

Materials and Methods



Cells +
corticosterone
(10⁻¹² ~ 10⁻⁷)

For 3 days



Then do real time
PCR.
Nada Almutairi
 The Effects of Corticosterone on GR and MR
mRNA expression
Results:


Nada Almutairi
 Effects of GR antagonist on
Corticosterone-induced PRL production!
 Materials and Methods:  Results:

ä Mifepristone inhibited the
change in the number of
PRL immunopositive cells
caused by Corticosterone.

The immunopositive cell

number was determined

Nada Almutairi
 The Objectives:

To Study the biphasic effects of corticosterone

on GH and PRL production in a rat pituitary
somatomammotropic cell line (MtT/SM) In
vitro.

To find out if glucocorticoids receptor (GR)
mediate the effects of corticosterone on
prolactin production.

To Study the effects of both E₂ estrogen and
corticosterone on PRL synthesis and PRL cell
proliferation.
Nada Almutairi
 Effects of Corticosterone on estrogen
receptor (ERα) mRNA expression.
Materials and Methods:

Results:


 


ä (~10⁻⁷) corticosterone
suppressed ERα mRNA
expression; while
(~10⁻¹²) Increased it.

Nada Almutairi
 Effects of Corticosterone and E₂ on PRL
production and PRL cell appearance!
Materials and Methods:

Results:


 

Nada Almutairi
 Effects of Corticosterone AND E₂ on PRL
cell proliferation.
Materials and Methods:


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

Nada Almutairi
 Effects of Corticosterone AND E₂ on PRL
cell proliferation.
Typical of double


Results:

immunofluorescent staining
for PRL(green) and BrdU(red).


Nada Almutairi
 Conclusions

Nada Almutairi
 Thank You!

Nada
Almutairi
Nada Almutairi