SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 226A

226A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

is significantly downregulated in IUGR placentas, which is believed
to contribute to reduced fetal amino acid delivery and restricted fetal
growth. Peroxisome proliferator-activated receptor gamma (PPARγ), a
ligand-inducible transcription factor belonging to the nuclear receptor
superfamily, is a key regulator of placental development and PPAR γ
mRNA levels are reduced in placentas of small-for-gestational age fetuses.
Rostaglitazone is a known activator of PPARγ, stimulates amino acid
transport in 3T3-L adipocytes. We hypothesized that PPARγ activation
in primary human trophoblast (PHT) cells isolated from IUGR placentas
restores normal placental PPARγ signaling and amino acid uptake capacity.
METHODS: PHT cells were isolated from term pregnancies placentas
delivered by Caesarean with appropriate gestational age (AGA,
Gestational Age; GA, 38.0 ± 0.45 wks; 3490 ± 170 g, n=4) or IUGR
infants (GA, 37.8 ± 0.5; 2298 ± 70 g, n=4). Both AGA and IUGR PHT
cells were cultured for 66 h before treatment with the PPAR γ activator
rostaglitazone (50 nM) or Dimethyl sulfoxide (DMSO, vehicle control)
with or without rapamycin (100 nM, mTOR inhibitor) for 24 hr. System
A amino acid transport was determined by measuring Na+-dependent
uptake of 14C-methyl-aminoisobutyric acid and System L transport activity
measured as 2-amino-2-norbornane-carboxylic acid (BCH)-inhibitable
uptake of 3H-leucine at 90 hours. PHT cells PPAR γ expression was
determined by Western Blot.
RESULTS: Basal System A (-73%, p<0.01; n=4) and L uptake (-70%,
p<0.01; n=4) was decreased in PHT cells isolated from IUGR as compared
to PHT from AGA pregnancies. Rostaglitazone treatment restored PPARγ
expression and System A uptake in IUGR PHT cells as compared to AGA
PHT cells treated with vehicle control (p<0.001; n=4). There was also a
trend for increased System L uptake following Rostaglitazone treatment
(p=0.07) in AGA and IUGR PHT cells. Rapamycin inhibited System
A (-45 %, p<0.001, n=4) and System L (-40 %, p<0.01; n=4) activity
in AGA PHT cells as we have previously shown. However, rapamycin
(mTOR inhibitor) did not inhibit rostaglitazone mediated System A uptake
in AGA PHT cells.
CONCLUSION: Restoration of PPARγ expression in PHT cells from
IUGR pregnancies restored basal System A but not System L amino acid
transport activity. PPARγ mediated activation of System A transport is
independent of mTOR signaling pathway. Intervention strategies aiming at
restoring normal placental PPARγ signaling may be effective to improve
placental nutrient transport and fetal growth in IUGR.

F-135
Impaired Trophoblast Nutrient Transport and Pancreatic β Cell
Defects in Intra Uterine Growth Restriction (IUGR) Are Alleviated
by Activation of Trophoblast mTOR Signaling. Fredrick J Rosario†,
Powell L Theresa*, Thomas Jansson*. University of Colorado Anschutz
Medical Campus, Aurora, CO, United States.
INTRODUCTION: IUGR due to placental insufficiency is associated
with down-regulation of placental transport of nutrients such as amino
acids and impaired fetal pancreatic β cell development and function.
Mechanistic target of rapamycin complex (mTORC) 1 and mTORC2
signaling are positive regulators of trophoblast amino acid transport and
placental mTOR signaling is inhibited in IUGR, strongly implicating
decreased placental mTOR activity in the development of IUGR. DEPTOR
is an endogenous inhibitor of mTORC1 and 2 signaling. We hypothesized
that mTOR activation in primary human trophoblast (PHT) cells isolated
from IUGR placentas restores normal placental mTORC1 and mTORC2
signaling and nutrient uptake capacity and that trophoblast secretome
(conditioned media, CM) from IUGR PHT cells inhibits β cell function.
METHODS: PHT cells were isolated from term pregnancies placentas
complicated by IUGR (GA, 38.0 ± 0.45 wks; 2298 ± 70 g, n=4) and
appropriate for gestational age (AGA) infants (GA, 37.8 ± 0.5; 3490 ±
170 g, n=4). PHT cells were cultured for 18 h and transfected with siRNA
targeting DEPTOR (activating mTORC1 and 2 signaling) or Scrambled
siRNA (Sc). System A amino acid transport was determined by measuring
Na+-dependent uptake of 14C-methyl-aminoisobutyric acid and System
L transport activity measured as 2-amino-2-norbornane-carboxylic acid
-inhibitable uptake of 3H-leucine at 90 hr. In subsequent experiments,

Scientific Abstracts

INS-1 cells (a rat β cell line) were incubated in AGA or IUGR PHT cell
CM for 24 hr. PDX1 and mitochondrial electron transport chain complex
(ETC) expression in INS-1 cells were measured by Western Blot.
RESULTS: Basal System A (-73%, p<0.01; n=4) and System L (-70%,
p<0.01; n=4) uptake were decreased in IUGR PHT cells as compared to
AGA PHT cells transfected with scramble siRNA (AGA-Sc). Silencing
DEPTOR restored basal mTORC1 and C2 signaling and System A
(p<0.001; n=4) and L (p<0.01; n=4) uptake in IUGR PHT cell as
compared to AGA-Sc. Incubation of INS-1 cells with CM from IUGR
PHT cells reduced PDX-1 and ETC-2 expression as compared to INS-1
cells incubated with CM from AGA-Sc. Importantly, PDX-1 and ETC-2
expression was not affected in INS-1 cells incubated in CM from IUGR
PHT cells in which basal mTORC1 and mTORC2 signaling had been
restored with DEPTOR siRNA.
CONCLUSION: DEPTOR silencing in IUGR PHT cells restored
mTORC1 and mTORC2 signaling and System A/L nutrient transport to
levels similar to AGA PHT cells. In addition, our data suggest that the
trophoblast secretome contributes to the pancreatic β cell defect in IUGR
and that activation of trophoblast mTORC1/C2 signaling could prevent
IUGR placenta mediated β cell defects. Intervention strategies aiming at
activating placental mTOR signaling may be effective to improve placental
nutrient transport, β cell function and fetal growth in IUGR.

F-136
Multiscale Imaging of Placental Villous Fibroblasts. Eleni Palaiologou,
Patricia Goggin, David S Chatelet, Christopher Torrens, Bram G Sengers,
Jane K Cleal, Anton Page, Rohan M Lewis*. University of Southampton,
Southampton, United Kingdom.
INTRODUCTION: The placenta is formed of multiple cell types,
but research has focused mainly on the trophoblast. The roles of the
non-trophoblastic cell types in the villi and their possible connection to
disease processes is poorly understood. Villous fibroblasts sit in the stroma
between the trophoblast and the fetal vascular system. These fibroblasts
are believed to be involved in the maintenance of the extracellular matrix.
The advent of new imaging techniques allows 3D imaging of cells across
a range of scales and can provide an insight into the relationship between
cellular structure and function. The objective of this study was to undertake
multiscale three-dimensional imaging of placental villous fibroblasts.
METHODS: Term human placental villi were collected following
delivery and processed for wholemount confocal immunohistochemistry,
serial block-face scanning electron microscopy (SBFSEM) or transmission
electron microscopy (TEM) and imaged. Segmentation of confocal and
SBFSEM image stacks was performed in Amira (Ver. 6) to construct
three-dimensional images.
RESULTS: Whole-mount confocal imaging demonstrated that the
fibroblasts formed networks of 2-12 connected cells. 3D reconstruction
of a fibroblast from a SBFSEM stack demonstrated the stellate processes,
sail-like structures and their relationship to surrounding structures
including the capillary, trophoblast, membrane-bound extracellular
stromal vesicles and cell-cell connections to neighbouring fibroblasts.
TEM images of the stellate processes showed rough endoplasmic
reticulum. TEM imaging of the connections between fibroblast processes
and stromal vesicles indicated a close association, but each had discrete
plasma membranes.
CONCLUSION: Multiscale 3D imaging demonstrates that fibroblasts
form networks of connected cells. Each cell has a complex structure,
with processes that are closely associated with other villous structures.
This study poses questions about the functions of these cells and suggests
novel roles such as the association with stromal vesicles. How the function
of these cells might be altered in disease states and how this may affect
placental function needs to be determined.
*Figure(s) will be available online.
Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018
