In-vitro investigation of calcitonin associated effects on the trophoblastic cells

1. Manuela Ruiz Fernandez
Third semester 2020-1
Medicine faculty
Universidad Pontificia Bolivariana

2. Introduction
Calcitonin
Polypeptide hormone
Secretion by the
parafollicular cells of the
thyroid gland
Regulates blood calcium
levels (increases ca++ levels
inside the cell)
Decreases ca++
reabsorption in kidneys
Decreases ca++ uptake in
intestines
Increases ca++ deposition
in bones

3. Introduction
Outer layer of cells in the
blastocyst
Secretion of
immunosuppressive protein
(early pregnancy factor
appears within 24-48 hrs)
It originates the embryonic
side of the placenta
Trophoblastic cells

4. Introduction
Up regulation of calcitonin
expression in the epithelial cells
of the endometrium done by
progesterone secreted during the
implantation period
Expression of cadherin-11
modulates division of
trophoblastic cells into the
syncytiotrophoblast (BhCG) and
cytotrophoblast (stimulates
process of implantation)
Induced down regulation
of E-cadherin expression
(facilitates trophoblastic
invasion at the
endometrium)
β-catenin
dissociation from
adherent junctions.
Accumulation of β-catenin in the
cytoplasm associated with its
translocation to the nucleus (acts as
a transcriptional co-activator of the
transcription factor inducing growth
and invasion related genes)

5. Objective
“It was aimed to determine the effects of calcitonin on
the expressions of β-catenin and phospho-β-catenin in
a dose depended manner under the influence of
progesterone and estrogen hormones by using JAR cell
line as an in vitro model for trophoblasts”

6. Materials and methods
Cell culture:
-Analysis of JAR cells maintained in specific
conditions:
*High glucose without phenol red (pH indicator)
*10% FBS (Supplement for in vitro cells with
necessary macromolecules for growth)
*100 u/mL penicillin and 100 ug/mL streptomycin
*Humidified atmosphere 5% CO2 at 37° C
*Change to 2% FBS to limit exposure to
exogenous estrogen
-Microbiological culture method (detection
of mycoplasma contamination)
1)Control
2)Progesterone (P) + Estradiol (E2)
3)10 nM calcitonin
4)10nM calcitonin + P + E2
5)250 nM calcitonin
6)250 nM calcitonin + P + E2
Immunocytochemistry:
1)Identification of an antigen or protein in cells, through the specific interaction
between the antigen and an antibody (with prior labeling of the antibody, in
order to visualize the antigen under a fluorescence microscope).
2) The cell is stained to showcase the presence and cellular localization of the
molecule being assessed.
3)Primary antibodies: rabbit monoclonal anti-β-catenin, rabbit monoclonal
anti-phospho-β-catenin.
Secondary antibodies: biotinylated secondary antibody and horseradish
peroxidase (HRP) conjugated streptavidin.
Substrate: aminoethyl carbazole converts into a product that gives color for
future cell analysis

7. Materials and methods
Western blot analysis: Used to
identify specific proteins from a set
sample.
1)Proteins previously extracted from DNA are
separated with gel electrophoresis depending
on their size and charge.
2) Proteins (β-catenin and phospho-β-catenin)
are transferred to a filter (nitrocellulose
membrane previously incubated with rabbit
monoclonal anti-β-catenin and anti-phospho-
β-catenin antibodies), which will react with
the different proteins.

8. Materials and methods
Transmission electron microscopy: Used for ultra structuring of cells
and tissues. To determine the molecular structure, interactions and processes at a
cellular level.
It uses high energy electrons unlike regular microscopes. An electromagnetic lens
focuses the scattered electrons that passed through the specimen into a detailed
image.
In the study they used it to assess the involvement of cadherins-catenins complex
in the process of implantation, while keeping in mind the expression of calcitonin.

9. Results
250 nM
calcitonin
10 nM
calcitonin*
10 nM
calcitonin +
P + E
Control
group

10. Results
*

12. Results
*The amount of adherens junctions could indicate that
there was no dissociation of B-catenin from the complex
and thus no translocation to the nucleus

13. Discussion
Author What was said? Yes/No
Li et al
“Calcitonin is secreted by the endometrial
epithelial cells during the implantation window
under the effect of progesterone, and down
regulates the expression of E-cadherin which in
turn results in disruption of calcium dependent
adherens junctions”.
No
Getsios and
MacCalman
“Trophoblasts are epithelial-like cells and express
E-cadherin protein which is high in
cytotrophoblasts and decreases as the cells
undergo fusion to form syncytium, while
cadherin-11 expression is on increase”.
No
Klaus and Birchmeier
“Activation of canonical Wnt signaling pathway
induces the accumulation of β-catenin which in
turn translocate to nucleus and regulates the
expression of growth and invasion related genes”
No

14. Conclusion
1. The usual effect
that calcitonin has on
the epithelial cells of
the endometrium
changes with the
increased production
of progesterone and
estrogens during the
implantation.
2. The increased
expression of β-catenin
in the membrane and
the decreased
expression of phospho-
β-catenin in the
cytoplasm, accounts for
the presence of
adherens junctions and
thus less trophoblastic
cell invasion.