This document contains abstracts from two studies: 1. The first study identifies a hypoxia/HIF/Kdm3a pathway that controls trophoblast stem cell differentiation and organization of the hemochorial placenta. This pathway regulates genes involved in trophoblast invasion and vascular remodeling during pregnancy. 2. The second study examines endometrial gene expression in lactating cows, dry cows, and heifers on day 19 of pregnancy. The study finds 135 differentially expressed genes between lactating cows and heifers, but only 17 between dry cows and heifers, suggesting lactation impacts the endometrial response to the developing embryo. Functional analysis found effects on vesicle transport and cytokine signaling

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2015 Abstracts – Page 44
115. A Hypoxia/HIF/Kdm3a Pathway Controls Trophoblast Stem Cell Lineage Decisions and Organization of the Hemochorial
Placenta.
Damayanti Chakraborty1
, Wei Cui2
, Regan Scott1
, Pramod Dhakal1
, Stephen Renaud1
, Gracy Rosario3
, Jay Vivian4
, Makoto Tachibana5
,
Mohammad K. Rumi4
, Michael Soares6
.
1
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA; 2
University of
Massachusetts, Amherst, Boston, MA, USA; 3
NA, Mumbai, Maharashtra, India; 4
Pathology and Laboratory Medicine, University of
Kansas Medical Center, Kansas City, KS, USA; 5
Institute for Enzyme Research,The University of Tokushima, Tokushima, Tokushima,
Japan; 6
IRHRM, University of Kansas Medical Center, Kansas City, KS, USA
The hemochorial placenta develops from the coordinated multi-lineage differentiation of trophoblast cells. Specific trophoblast
cell lineages are organized within the placentation site to perform specialized functions. The invasive trophoblast lineage remodels uterine
spiral arteries to convert them to flaccid low resistance vessels, facilitating the flow of nutrients to the placenta and fetus. Failure of
trophoblast invasion and vascular remodeling is associated with pathological conditions such as preeclampsia, intrauterine growth
restriction, and preterm birth. Oxygen delivery represents an environmental stimulus with an instructive role influencing trophoblast cell
differentiation and organization of the hemochorial placentation. In this study, we perform a series of in vitro and in vivo experiments
delineating a hypoxia- activated regulatory pathway controlling hemochorial placentation. Key downstream events are delineated using rat
trophoblast stem (TS) cells and tested in vivo using trophoblast-specific lentiviral gene delivery and genome editing. Initially, DNA
microarray analyses were performed on rat TS cells exposed to ambient or low oxygen (0.5%). Upregulation of genes characteristic of an
invasive/vascular remodeling/inflammatory phenotype and a marked downregulation of stem state-associated genes were
observed. Among the upregulated genes were a histone H3K9 demethylase (Kdm3a) and a matrix metalloelastase (Mmp12). Upregulation
of these transcripts was dependent upon the transcription factor, hypoxia inducible factor (HIF). We hypothesized that Kdm3a was a
mediator of hypoxia-directed trophoblast cell lineage differentiation and that Mmp12 was a key downstream target responsible for the
trophoblast cell invasive and vascular remodeling phenotype. Consistent with the hypothesis, knockdown of Kdm3a in rat TS cells
inhibited the expression of a subset of the hypoxia/HIF-dependent transcripts, including Mmp12, and altered locus specific as well as
global H3K9 methylation status. Conversely ectopic expression of Kdm3a upregulated a subset of hypoxia/HIF-dependent transcripts,
including Mmp12, and this upregulation was dependent on Kdm3a enzymatic activity. Furthermore, Kdm3a knockdown decreased
hypoxia-induced trophoblast cell invasion in both in vitro and in vivo experiments. Mmp12 possesses the capacity to degrade elastin and
modify the structure of arterial blood vessels. To further explore the functional importance of Mmp12 in trophoblast cell-directed uterine
spiral artery remodeling, we generated an Mmp12 mutant rat model using TALEN-mediated genome editing. A rat model was established
with a 609 bp deletion targeting exon 2 of Mmp12. Homozygous mutant rats showed reduced hypoxia-dependent endovascular trophoblast
invasion and impaired trophoblast-directed uterine spiral artery remodeling. In summary, we have discovered a hypoxia/HIF/Kdm3a
pathway modulating trophoblast cell lineage development, leading to acquisition of the invasive trophoblast cell phenotype, including
upregulation of the extracellular matrix-modifying enzyme Mmp12 and subsequent uterine spiral artery remodeling.
116. Endometrial gene expression in lactating and dry Holstein cows and Holstein heifers on Day 19 of pregnancy.
Stefan Bauersachs1
, Niamh Forde2
, Jochen Bick1
, Stefan Krebs3
, Helmut Blum3
, Eckhard Wolf3
, Patrick Lonergan2
.
1
ETH Zurich, Animal Physiology, Institute of Agricultural Sciences, Zurich, Zurich, Switzerland; 2
School of Agriculture and Food Science,
University College Dublin, Dublin, Leinster, Ireland; 3
Laboratory for Functional Genome Analysis, Gene Center, LMU Munich, Munich,
Bavaria, Germany
The compromised fertility of high producing dairy cows may be caused by negative energy balance. However, the point at which
this metabolic state impacts on impaired fertility is not known. We have previously shown that different types of embryos elicit different
transcriptomic responses from the endometrium. The aim of this study was to test the hypothesis that lactation alters the ability of the
endometrium to respond appropriately to the developing conceptus. Endometrial gene expression on Day 19 of pregnancy in a model of
metabolic stress was analyzed using RNA sequencing. Immediately after calving, primiparous Holstein cows with similar production and
fertility estimated breeding values (EBVs) were randomly divided into two groups, standard lactation (“Lact”) or dried off immediately
(“Dry”, i.e., never milked). Pregnancy was established by transferring grade 1 embryos recovered from superovulated Holstein heifers
(“Heif”) with similar EBVs for production and fertility into lactating and nonlactating recipients (n=1 per recipient). A control group of
Holstein heifers (“Heif”), with similar EBVs for production and fertility as groups 1 and 2, was artificially inseminated. Endometrial tissue
samples were collected after slaughter and recovery of a conceptus on Day 19 of pregnancy. Total RNA was isolated from intercaruncular
endometrium. Strand-specific RNA-Seq libraries (Heif n=4, Lact n=5, Dry n=8) were produced using the Encore Complete RNA-Seq DR
Multiplex System (NuGEN, San Carlos, CA) and run on an Illumina HiSeq 1500. Obtained 100 bp single-end reads (28-65 Mio reads per
sample) were processed using Trimmomatic (version 0.32.1) and mapped to the bovine genome assembly Btau_4.6.1 (bosTau7) with
TopHat2. Mapped reads were counted per gene by the use of QuasR qCount. Statistical analysis with DESeq2 revealed 17 differentially
expressed genes (DEG) between Dry and Heif (FDR 5%) and 135 DEG (FDR 5%) between Lact and Heif. The comparison of Lact and
Dry did not reveal differences at a significance threshold of 5%. Overall, gene expression differences were rather small and lower than two-
fold. Multi-dimensional scaling plots and principal component analysis confirmed the more pronounced differences in gene expression
between lactating cows and heifers and showed that gene expression in the endometrium of dry cows is intermediate compared to the Lact
and Heif groups. Furthermore, higher variation of gene expression in the Dry group was observed for the genes differential between Heif
and Lact. Functional annotation of the DEG between Lact and Heif revealed, e.g., endosome, cytoplasmic vesicle, endocytosis, regulation
of exocytosis, and cytokine receptor activity as overrepresented. The functional categories related to vesicle-mediated transport were
specifically enriched for genes with higher expression in lactating cows and defense response for genes with higher expression in heifers.
In conclusion, our data suggest that parity (cow vs. heifer) and metabolic condition (negative vs. normal energy balance) modulate the
ability of the endometrium to respond to a high-quality embryo on the level of the transcriptome. This research was supported by the
European Union (7th
Framework Program, KBBE.2012.1.3-04: “Optimised terrestrial farm animal reproduction systems and/or
technologies”, project FECUND).