SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 262A

262A

Reproductive Sciences Vol. 25, Supplement 1, March 2018

were examined by immunofluorescence, qPCR and western blot analysis.
Collagen contraction assays were performed to determine the functional
relevance of the alternatively spliced ERα splice isoforms.
RESULTS: Lentiviral driven shRNA mediated knockdown of hnRNPG
confirmed its role as an ERα splice factor responsible for the generation
of ERΔ7 by driving exon7 exclusion. Furthermore, human myometrial
tissues demonstrated elevated levels of hnRNPG and ERΔ7 in the preterm
non-laboring samples, which decline to barely detectable levels at term.
Collagen contraction assays demonstrate that overexpression of ERΔ7
decreases uterine myocyte contractility and is associated with the ablation
of ERα66-ERα46 mediated connexin43 and oxytocin receptor expression.
CONCLUSION: In this study, we demonstrate a novel tocolytic role
for the dominant negative myometrial ERΔ7 isoform. Across gestation
ERΔ7 isoform predominates due to increased splicing events mediated
by the RNA-binding splice factor hnRNPG and thereby preventing the
uterotonic action of ERα66 and ERα46. At term the decline in hnRNPG
eliminates ERΔ7 allowing for the uterotonic action of ERα66 and ERα46.
This novel discovery with respect to the functional significance of ERΔ7
in myometrial quiescence and its regulation through hnRNPG opens
up exciting new therapeutic direction for tocolysis and prevention of
preterm birth.

S-026
Adiponectin Receptor (AdipoR) Mediates Uterine Tocolysis via a
Non-Canonical Pathway. Vibhuti Vyas, Jenae Luellen, Rachael Bok,
Theresa Powell, Thomas Jansson, K Joseph Hurt. University of Colorado,
Aurora, CO, United States.
INTRODUCTION: Circulating adiponectin (AdipoQ) levels are
reportedly lower in patients with preterm labor. Using mouse myometrial
strips in physiologic organ bath, we have previously reported a potent
uterine tocolytic effect of AdipoQ (SRI 2017). In this study, we determined
the expression of AdipoR1 and AdipoR2 in mouse and human uterus
and investigated the signaling pathways mediating uterine relaxation
by AdipoQ.
METHODS: AdipoQ, AdipoR1 and R2 mRNA was determined by
quantitative real-time PCR in non-pregnant (NP) and pregnant C57Bl/6
mouse myometrium (gestational day [GD] 9, 12, 15-19, laboring, and
post-partum), and in human myometrium obtained at hysterectomy or
at scheduled cesarean delivery (IRB#03-1270). Protein expression of
myometrial AdipoRs was determined by Western blot. In organ bath,
we used selective inhibitors or vehicle to evaluate signaling pathways
mediating adiponectin tocolysis (Table). NP myometrial strips were
stimulated with 4nM oxytocin then AdipoR agonist AdipoRon (45µM)
was added. Contractility was quantified using area under the curve (AUC)
and analyzed with Kruskal-Wallis followed by Mann Whitney test.
RESULTS: In both murine (n=5) and human (n=6-7) myometrium,
Adiponectin mRNA was absent; AdipoR1 and R2 mRNA were present
and increased during pregnancy (p<0.05). In murine uterus, AdipoR2
protein increased during pregnancy; in human samples, there was no
difference between NP and pregnant AdipoR2. None of the inhibitors
tested (n=3-6; Table) affected the AdipoRon-mediated inhibition of
myometrial contractility.
CONCLUSION: Our data suggests that adiponectin exerts its tocolytic
effect mediated by AdipoR1 and/or R2 and a signaling pathway not
involving mediators well-established to regulate myometrial contractility
such as PPAR-α, NO and K+ channels. We speculate these findings could
identify novel therapeutic targets to prevent preterm birth.

Scientific Abstracts
Effect of various inhibitors on AdipoRon-mediated inhibition of myometrial
contractility.
AUC % of baseline
Inhibitor

Target

contractility
(Mean ± SD)

Vehicle

---

46% ± 18

Dorsomorphin
(50µM)

AMP-activated protein kinase
(AMPK)

11 ± 12

GW6471
(4µM)

Peroxisome proliferator-activated
receptor alpha (PPAR-α)

58 ± 8

L-NAME
(1mM)

Nitric oxide synthases

20 ± 7

Iberiotoxin
(100nM)

K+ channels

52 ± 14

BaCl2 (1mM)

K+ channels

35 ± 11

TEA (5mM)

K+ channels

21 ± 3

The effect of AdipoRon (45µM) ± Inhibitors on myometrial contractility
(baseline = 100%) was studied.

S-027
Selection of Optimal Myometrial Cells for High-Throughput
Screening for Novel Regulators of Uterine Contractility. Shajila
Siricilla†,1 Kelsi Knapp†,1 Jennifer Condon,2 Dehui Mi,1 Paige Vinson,1
Bibhash C Paria, 1 Jeff Reese, 1 Jennifer Herington*. 1 1Vanderbilt
University, Nashville, TN, United States; 2Wayne State University, Detroit,
MI, United States.
INTRODUCTION: The uterine myometrium (UT-myo) is a therapeutic
target for regulating uterine contractions. A final pathway controlling
myometrial contractions involves stimulation of intracellular calcium
[Ca2+]i-release. Therefore, we developed an assay for high-throughput
screening (HTS) of small-molecular compounds to regulate Ca2+mobilization in UT-myo cells. In order to select the optimal UT-myo
cells for our drug discovery assay, we compared the gene expression of
contraction-associated proteins (CAPs) as well as functional response
to oxytocin (OT) between: 1) term-pregnant human and mouse
myometrial tissue, 2) isolated primary human and mouse UT-myo cells,
3) commercially-available pregnant human myometrial (PHM1) cells and
4) human telomerase immortalized (hTERT) myometrial cells.
METHODS: Primary human and mouse UT-myo cells were isolated from
tissue collected at the time of c-section during term pregnancy (≥37 weeks
or 19 days, respectively). Quantitative RT-PCR was used to determine the
gene expression levels of CAPs: OT-receptor, prostaglandin F receptor
and connexin-43. Cell density and passage number (low and high) were
optimized, and cell viability was monitored for each automated step used
during the 384-well format HTS Ca2+-mobilization assay. Thirteen-point,
3-fold dilutions of OT were tested in the Ca2+-mobilization assays,
followed by calculations of the OT EC50 and %induction, to determine
the responsiveness and sensitivity of myometrial cells. Finally, data were
analyzed by 1-way ANOVA.
RESULTS: The relative mRNA expression levels of CAP genes were
significantly (p<0.05) less in all four myometrial cell types examined
compared to endogenous levels measured from human tissue. However,
the mRNA expression of CAP genes in PHM1 and hTERT cells were
similar to that measured from primary human myometrial cells. Moreover,
PHM1 and hTERT cells were more responsive to OT-induced [Ca2+]
i-release (EC50 = 60nM and 40nM, %induction = 200% and 70%,
respectively) compared to human and mouse primary myometrial cells.
CONCLUSION: While there is a benefit of using primary cells in
HTS due to their retention of many in vivo functions and endogenous
expression of mechanisms/targets of interest, primary cells must be
proven reproducible for reliable use in HTS and available at similar robust
numbers to that of cell lines. Among the 4 different myometrial cells
examined, both PHM1 and hTERT cells exhibit similar gene expression
levels of CAPs found in primary isolated cells from term pregnant human
myometrial tissue, and are the most responsive to OT-induced [Ca2+]
i-release.

Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018