SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 139A

Scientific Abstracts

T-088
Late Gestation Hypoxia Stimulates Hepatic Expression of
Gluconeogenic Genes in Fetal Sheep. Amanda K Jones†,2 Nathan M
Kanner,2 Taylor H Reynolds,2 William W Hay Jr,2 Laura D Brown,2 Paul
J Rozance,2 Sean W Limesand,1 Stephanie R Wesolowski.2 1University
of Arizona, Tuscon, AZ, United States; 2University of Colorado School
of Medicine, Aurora, CO, United States.
INTRODUCTION: Fetuses with placental insufficiency and intrauterine
growth restriction are exposed to hypoxia, reduced nutrient supply, and
altered endocrine signals. These fetuses also have an early activation of
hepatic glucose production. We hypothesized that late gestation hypoxia
is critical for activating the mechanisms responsible for this pathway in
the fetus.
METHODS: Surgeries were performed in late gestation pregnancies
(~120 days) to place chronic indwelling catheters in the maternal and
fetal vasculature and maternal trachea. A variable rate maternal tracheal
nitrogen infusion was used to maintain a 25% reduction in fetal PO2 (HOX;
n=9) compared to fetuses from ewes receiving compressed air (CON;
n=5). Fetal arterial blood gasses, blood oximetry, plasma glucose, lactate,
and hormones were measured followed by necropsies and collection of
fetal liver after ~9 days of treatment.
RESULTS: Fetal PO2 was 18% lower in HOX versus CON fetuses
(14.2 +/- 0.6 vs 17.3 +/- 1.2 mmHg; P<0.05) with similar reductions
in O2 content, SO2, and CO2 content (P<0.05) and no difference in
hematocrit or pH. Plasma glucose and insulin concentrations were similar
between HOX and CON fetuses, but plasma lactate concentrations were
3-fold greater in HOX versus CON fetuses (P<0.05). Plasma cortisol
concentrations were not greater in HOX versus CON fetuses, yet were
inversely correlated with fetal PO2 (R2=0.41, P<0.05). Expression of
the gluconeogenic genes phosphoenolpyruvate carboxylase (PCK1) and
glucose 6 phosphatase (G6PC) were 4- and 13-fold greater, respectively,
in HOX versus CON livers (P<0.05). Expression of lactate dehydrogenase
(LDHA) and O2 sensitive prolyl hydroxylase (PHD3) was 2-fold greater
in HOX than in CON livers (P<0.05). Hepatic glycogen contents and
fetal weights were not different.
CONCLUSION: Late gestation fetal hypoxia increased expression of
key hepatic gluconeogenic genes and the concentrations of lactate, a
gluconeogenic precursor. This fetal metabolic adaptation occurred without
a change in glucose or insulin concentrations or hepatic glycogen content.
Given the relationship between fetal oxygen and cortisol, we speculate
that endocrine signals act synergistically with hypoxia to initiate an early
activation of fetal hepatic glucose production.

T-089
Maternal Nutrient Excess and Obesity (MO) in Ewes during
Pregnancy Leads to Alterations in Hepatic Mitochondrial Biology and
Redox Balance. Susana P Pereira†,1,2 Chiara H Cavallaro†,1 Luis Grilo†,1
Ines Cardoso†,1 Ines Baldeiras,1 Teresa Cunha-Oliveira,1 Ashley Smith†,3
Stephen P Ford*,3 Peter W Nathanielsz*,3 Paulo J Oliveira*.1 1University
of Coimbra, Coimbra, Portugal; 2University of Porto, Porto, Portugal;
3
University of Wyoming, Laramie, WY, United States.
INTRODUCTION: Pregnancy is a critical period of physiological change
in mother and fetus, exposing both to short and long-term health risks. MO
is increasing exponentially in pregnant women. A better understanding

139A

of the MO induced maternal physiological changes is needed to prevent
adverse outcomes. We characterized liver mitochondrial activity profile
and redox network in term pregnant MO ewes.
METHODS: Ewes consumed an obesogenic (MO: 150% of NRC
requirements; n=8), or control diet (C: 100% NRC; n=10) from 60 days
prior to conception through pregnancy. Maternal livers were removed
at 0.9 gestation at euthanasia under general anesthesia for right lobe
measurements. Mitochondrial and antioxidant defense system proteins
were determined by Western blot. Mitochondrial respiratory chain
complex activities were determined in isolated fractions, catalase,
superoxide dismutase, glutathione peroxidase and glutathione reductase
activities in whole liver tissue by spectrophotometry. Lipid peroxidation
was assessed by fluorometry for malondialdehyde (MDA) formation
and reduced and oxidized glutathione determined using a commercial
kit. Data were expressed as mean ± SE and comparison between groups
was performed using the t-test with P-value less than 0.05 considered
as significant.
RESULTS: MO increased MDA levels indicating greater lipid
peroxidation, mirroring an imbalance in the general endogenous
antioxidant defense system with decreased levels of reduced glutathione.
These alterations in the MO redox state were accompanied by altered
content of proteins implicated in mitochondrial metabolism, namely by
increased VDAC1, a key protein in mitochondria-mediated apoptosis,
augmented cyclophilin D, a modulator of mitochondrial ATP synthase
and enriched cytochrome c, a major regulator of mitochondrial function.
Succinate dehydrogenase complex subunit B (SDHB, complex II) protein
was decreased in MO and was noticed a possible post-translational
modification of NDUFB8 (complex I) induced by MO, despite unchanged
mitochondrial DNA copy number.
CONCLUSION: MO in pregnancy alters maternal hepatic mitochondrial
biology potentially predisposing the mother to metabolic diseases, including
non-alcoholic fatty liver disease. Funded by FEDER/COMPETE/FCTPortugal (PTDC/DTP-DES/1082/2014, POCI-01-0145-FEDER-007440,
OCI-01-0145-FEDER-016657, SFRH/BPD/116061/2016 and SFRH/
BPD/101169/2014), and NIH (R01HD070096-01A1).

T-090
Redox Ratio is Higher in White Adipose Tissue Compared to Brown
Adipose Tissue. Jack RT Darby†,3 Alexandra Sorvina,3 Christie Bader,3
Mitchell Lock†,3 Jia Yin Soo†,3 Mike Seed,2 Tim Kuchel,1 Doug Brooks,3
Sally Plush,3 Janna L Morrison*.3 1SAHMRI, Adelaide, Australia; 2The
Hospital for Sick Children, Toronto, ON, Canada; 3University of South
Australia, Adelaide, Australia.
INTRODUCTION: During the first few weeks after birth, perirenal
adipose tissue undergoes a transition from brown adipose tissue (BAT) to
white adipose tissue (WAT). Adipose tissue plays a key role in metabolic
homeostasis through lipid accumulation and the secretion of various
endocrine molecules. The morphology and function of these two types
of adipose tissue are distinct. Two-photon microscopy can be employed
to measure the fluorescence of the metabolic coenzymes: pyridine
nucleotides (NAD(P)H) and flavin adenine dinucleotide (FAD). In doing
so a redox ratio may be obtained to characterize the metabolic state of
a specific tissue. Herein, we aimed to use two-photon microscopy to
determine the difference in redox ratio between these distinct fat depots.
METHODS: At 119-120 days gestation perirenal adipose tissue was
collected from the ewe (WAT) and her fetus (BAT) and either frozen in
liquid nitrogen for gene expression analysis using qRT-PCR or placed
in ice-cold phosphate buffered saline for live tissue imaging using
two-photon microscopy. Using two-photon microscopy, the emission
of NAD(P)H was determined at 489 nm (474-504 nm interval) and
the emission of FAD was detected at 548 nm (533-562 nm interval) by
exposure to two-photon illumination. To avoid crosstalk between these
two fluorophores, different excitation wavelengths were used; 740 nm
for NAD(P)H and 900 nm for FAD.Data presented as mean±SEM and
P<0.05 was deemed significant (Student's t-test).
RESULTS: Compared to fetal perirenal fat (BAT), maternal perirenal fat
(WAT) had decreased mRNA expression of mitochondrial units COX5A,
COX5B and COX7A, demonstrating metabolic differences between the

Thursday Posters

(Illumina) to sequence 94 single nucleotide variants and 59 short tandem
repeats. FISH for chromosomes 13, 18, 21, X and Y, and a Bacs on Beads
(BOBS) assay that determines relative levels of all 24 chromosomes were
used to evaluate ploidy status of the isolated cells and DNA, respectively.
RESULTS: One thousand one hundred EVT cells were isolated. Purity
of the isolated EVT cells was 90.4%. ForenSeq demonstrated that the
fetal fraction of DNA obtained from the EVT cells was 97%. FISH assay
detected 25 XY/trisomy 21 cells, and BOBS assay confirmed XY +21.
CONCLUSION: This is the first report of aneuploidy identified using
TRIC. TRIC provides a means of isolating EVT cells as early as 5 wk
GA with a high degree of purity, and could provide an earlier alternative
for NIPS of aneuploidy.

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