SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 283A

Scientific Abstracts

S-093
Leptin-Attenuated Luteinizing Hormone Release Requires Neuronal
Nitric Oxide Synthase (nNOS) Phosphorylation. Damian Guerra†,
Rachael Bok†, Cari Nicholas†, Joshua Johnson, K Joseph Hurt*. U of
Colorado SOM, Aurora, CO, United States.
INTRODUCTION: The pre-ovulatory release of luteinizing hormone
(LH) is a hallmark of normal ovulation. The LH surge may be abnormal
in subfertile populations. Acute leptin exposure stimulates nNOS
phosphorylation at serine 1412 to enhance NO production. However,
prior pharmacological studies have not determined conclusively if nNOS
activation increases or decreases LH release.
METHODS: We generated mice with a nNOS S1412A substitution
(nNOSS1412A) to abolish phosphorylation. We confirmed phosphorylation
blockade by kinase assays and phospho-immunoblotting. Female mouse
organs (including ovaries) were histomorphometrically assessed. We
quantified primordial and antral follicles, as well as corpora lutea, by
light microscopy from whole ovary serial sections. Estrous cycles were
assessed over a period of two weeks by vaginal swab. To determine if
acute leptin exposure and nNOS activation affects LH levels, we injected
mice in diestrous with intraperitoneal leptin (3mg/kg) or saline vehicle
and collected blood after 15 minutes. Serum LH, follicle-stimulating
hormone (FSH), estrogen (E2), and progesterone (P4) were quantified
by radioimmunoassay.
RESULTS: Kinase assays confirmed that the S1412A mutation blocks
nNOS phosphorylation. nNOSS1412A homozygotes had heavier brains (95%
CI: 460-500mg; n=5 mice) than wildtype (WT) mice (404-426mg; n=13).
Female nNOSS1412A mice were fertile, and their ovaries produced similar
numbers of primordial, antral follicles and corpora lutea as WT animals.
Estrous cycles were not altered by the nNOS S1412A mutation, and serum
levels of FSH, E2, or P4 did not differ between genotypes or with leptin
injection. Leptin decreased serum LH levels in WT (0.55-0.75ng/ml; n=14)
and nNOSS1412A heterozygotes (0.35-0.65ng/ml; n=8) compared with WT
vehicle control (1.0-1.4ng/ml; n=8). In contrast, nNOSS1412A homozygotes
showed no change in LH after leptin treatment (0.8-1.2ng/ml; n=9).
CONCLUSION: Our findings with the nNOSS1412A mouse suggest
leptin acutely suppresses pre-ovulatory LH levels. Leptin-mediated
LH suppression is likely NO-dependent. As such, therapies that target
nNOS phosphorylation could boost fertility in women who exhibit leptin
resistance and abnormal LH release.

283A

S-094
Vertical Transfer of Zika Virus (ZIKV) at Mid-Gestation Disrupts
Normal Development in the Cerebral Cortex in the Olive Baboon
(Papio anubis). Sunam Gurung†, Darlene N Reuter, Alisha Preno, James
Papin, Dean A Myers*. University of Oklahoma HSC, Oklahoma City,
OK, United States.
INTRODUCTION: ZIKV is vertically transmitted in humans and
associated with microcephaly and other fetal malformations (congenital
zika syndrome, CZS). The rate of microcephaly is estimated at ~10-15% of
infected pregnancies raising the question as to the potential range of fetal
CNS pathology arising from ZIKV. We have developed the olive baboons
as a non-human primate (NHP) translational model for ZIKV infection.
Baboons are similar to humans in terms of size, genetics, placentation,
immunology and critically, brain development. We hypothesize that ZIKV
infection of baboons at mid-gestation (the fist half of pregnancy in humans
is the highest risk for CZS) would result in neuroinflammation and/or
disrupted development of the fetal cerebral cortex (Ctx).
METHODS: Timed pregnant baboons were infected subcutaneously with
ZIKV (104 ffu; French Polynesian isolate:H/PF/2013) at mid-gestation
(97-107dG; n=3; term ~181dG). Blood was collected on 0, 3 and 7d postinfection (dpi). Animals were euthanized at 7, 14 and 21dpi (n=1/point).
A control (C) fetal brain was obtained at 116 dG. IHC was performed
on fetal Ctx for inflammatory markers (reactive microglia [Iba-1], IL6),
neural progenitor cells (NPCs; Nestin) and glia/radial glia (GFAP). vRNA
was extracted from samples and quantified by qRT-PCR.
RESULTS: Maternal viremia was observed in all dams through 7 dpi
(peak: 8.5x103-2.2x106 copies/ml blood). At 21 (but not 7 or 14) dpi,
ZIKV RNA was found in placenta (8.4x103 copies/mg) and fetal Ctx
(2.9x104copies/mg). In C fetal Ctx, GFAP staining showed a normal
pattern of glial/radial glia processes extending through the cortical plate/
subplate (CP/SP). A complete loss of GFAP+ glial processes with an
emergence of astrocytes in CP/SP was observed in the 21dpi Ctx. IL6+
cells and Iba1+ microglia were abundant in the 21 dpi Ctx vs. C. Nestin+
migrating NPCs were observed in C Ctx throughout the CP/SP, organized
along glial fibers. In the 21 dpi Ctx, Nestin+ NPCs were unorganized,
with regions devoid of Nestin+ NPCs. The 7 dpi Ctx was similar to C for
all staining; the 14 dpi Ctx exhibited normal GFAP and Nestin staining,
but increased IL6 and Iba1 microglia vs C.
CONCLUSION: Vertical transmission of ZIKV to the midgestation fetal
baboon Ctx resulted in disruption of migrating NPCs to upper cortical
layers possibly due to disruption of glial fibers and neuroinflammation.
Loss of glial fibers would likely result in reduction of cortical layers and
decreased sulci/gyri formation. Increased Ctx neuroinflammation at 14
dpi, despite the absence of ZIKV RNA, raises the possibility of fetal CNS
pathologies in the absence of vertical ZIKV transfer. NIH-NS103772,
OD010988

S-095
The TLR2/MYD88 and TLR4/MYD88 Signaling Pathways Activate
Notch Signaling in the Fetal Brain in Response to Intrauterine
Inflammation. Natalia Tulina, Amy Brown, Guillermo Barila, Michal
A Elovitz*. Maternal and Child Health Research Center, University of
Pennsylvania, Philadelphia, PA, United States.
INTRODUCTION: Exposure to intrauterine inflammation is associated
with fetal brain injury as well as long term adverse neurobehavioral
outcomes in exposed offspring. While innate immune responses have
been implicated in the production of inflammatory mediators in both
maternal and fetal tissues, how these pathways modify brain development
is not fully understood. One of the key signaling pathways involved in
the formation of the central nervous system is Notch. Disruptions in
Notch signaling lead to multiple cellular abnormalities during brain
development. The objective of this study was to determine if activation
of specific mediators of the innate immune response (TLR2, TLR4 and
MYD88) were responsible for subsequent alterations in Notch signaling
in the fetal brain.
METHODS: Using wild type C57B6 (WT), Tlr2-/-Tlr4-/- (Tlr DKO)
and Myd88-/- (Myd88 KO) strains of time-pregnant mice, we applied an
established model of intrauterine inflammation where LPS from E.coli

Saturday Posters

against vascular smooth muscle cells (VSMCs, α-SMA), endothelial cells
(ECs, CD31), AMPK, and P-AMPK. Placentas were grouped per animal
and homogenized for protein analysis by western blot, with antibodies
against AMPK, P-AMPK, and β-actin for normalization. Data are
expressed as mean ± SEM, and groups are compared by t-test.
RESULTS: Fetal weight was reduced at HA vs. SL (0.95 ± 0.02 vs. 1.17
± 0.01 g respectively, p<0.001) but placental weight was similar. Total
AMPK and P-AMPK were robustly expressed in murine UtA ECs and
VSMCs. Total AMPK expression was similar in HA- and SL-exposed
UtA and P-AMPK was abundant in SL-exposed UtA. However, P-AMPK
in both ECs and VSMCs was not detectable in UtA from HA-exposed
mice. The converse was seen in placenta with there being more P-AMPK
present in HA- than SL-exposed placenta (10.1 ± 1.0 vs. 5.03 ± 0.49 AU
respectively, p<0.05).
CONCLUSION: AMPK and P-AMPK are expressed in placenta and
UtA ECs and VSMCs during murine pregnancy. Activation of AMPK
appears to be diminished with HA exposure in pregnant murine UtA, but
increased in placenta. These findings are consistent with the possibility
that downregulation of AMPK activation in UtA may serve to reduce
UtA blood flow, and upregulation of AMPK activation in placenta may
contribute to mTOR inhibition, both contributing to a reduction in fetal
growth. Further studies will determine the effect of in vivo systemic
AMPK activation on UtA and placental AMPK activation and fetal growth.

Reproductive Sciences Vol. 25, Supplement 1, March 2018



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

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SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - Cover3
SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - Cover4
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_december2020
https://www.nxtbook.com/nxtbooks/sage/psychologicalscience_demo
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_october2020
https://www.nxtbook.com/nxtbooks/sage/fai_202009
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_august2020
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_june2020
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_april2020
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_february2020
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_december2019
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_october2019
https://www.nxtbook.com/nxtbooks/sage/fai_201909
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_july2019
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_june2019
https://www.nxtbook.com/nxtbooks/sage/canadianpharmacistsjournal_05062019
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_april2019
https://www.nxtbook.com/nxtbooks/sage/sri_supplement_201903
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_february2019
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_december2018
https://www.nxtbook.com/nxtbooks/sage/tec_20180810
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_october2018
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_julyaugust2018
https://www.nxtbook.com/nxtbooks/sage/fai_201807
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_june2018
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_april2018
https://www.nxtbook.com/nxtbooks/sage/sri_supplement_201803
https://www.nxtbook.com/nxtbooks/sage/slas_discovery_201712
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_february2018
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_december2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_november2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_october2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_september2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_julyaugust2017
https://www.nxtbook.com/nxtbooks/sage/fai_supplement_201709
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_june2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_may2017
https://www.nxtbook.com/nxtbooks/sage/fai_201706
https://www.nxtbook.com/nxtbooks/sage/fai_201607
https://www.nxtbookmedia.com