Research Article Lysophosphatidic Acid Signaling in Late ...downloads.hindawi.com/journals/mi/2014/678968.pdf · Research Article Lysophosphatidic Acid Signaling in Late Cleavage

Research ArticleLysophosphatidic Acid Signaling in Late Cleavage and BlastocystStage Bovine Embryos

Ana Catarina Torres1 Dorota Boruszewska2 Mariana Batista1 Ilona Kowalczyk-Zieba2

Patricia Diniz1 Emilia Sinderewicz2 Jean Sebastian Saulnier-Blache3

Izabela Woclawek-Potocka2 and Luis Lopes-da-Costa1

1 Faculty of Veterinary Medicine CIISAUniversity of Lisbon 1300-477 Lisbon Portugal2 Department of Reproductive Immunology and Pathology Institute of Animal Reproduction and Food ResearchPolish Academy of Sciences 10-747 Olsztyn Poland

3 Institute of Metabolic and Cardiovascular Diseases Inserm U1048 Paul Sabatier University 31432 Toulouse France

Correspondence should be addressed to Luis Lopes-da-Costa lcostafmvutlpt

Received 25 November 2013 Revised 4 March 2014 Accepted 4 March 2014 Published 15 April 2014

Academic Editor Antonio Miguel Galvao

Copyright copy 2014 Ana Catarina Torres et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Lysophosphatidic acid (LPA) is a known cell signaling lipid mediator in reproductive tissues In the cow LPA is involved in lutealand early pregnancy maintenance Here we evaluated the presence and role of LPA in bovine early embryonic developmentIn relevant aspects bovine embryos reflect more closely the scenario occurring in human embryos than the mouse modelTranscription of mRNA and protein expression of enzymes involved in LPA synthesis (ATX and cPLA

2) and of LPA receptors

(LPAR1ndash4) were detected in Days 5 and 8 in vitro produced embryos Embryonic LPA production into culture medium was alsodetected at both stages of development Supplementation of culture medium with LPA (10minus5M) between Days 2 and 8 had noeffect on embryo yield and quality and on blastocyst relative mRNA abundance of genes involved in prostaglandin synthesis(PTGS2 PGES and PGFS) and steroidogenesis (3120573HSD) However LPA treatment affected transcription levels of embryo qualitymarkers decreasing BAX (apoptotic) and increasing BCL2 (antiapoptotic) and IGF2R (growth marker) gene transcription levelsBlastocyst transcription ofOCT4 (pluripotency marker) was not affected by LPA stimulation In conclusion LPA is an early bovineembryonic autocrineparacrine signaling mediator and LPA action may be relevant in early embryo-maternal interactions leadingto embryonic survival

1 Introduction

Lysophosphatidic acid (LPA) is an extracellular lipid involvedin the cellular mediation of a plethora of physiological andpathological events in several tissues of vertebrates LPAsignaling was associated with a broad range of cellular eventsincluding survival differentiation proliferation migrationinvasion and adhesion [1ndash3]

Two major pathways of LPA production were pro-posed the intracellular LPA generation from phosphatidicacid by phospholipase A

1or PLA

2and the extracellular

LPA generation from lysophosphatidylcholine by autotaxin(ATX) which converts lysophosphatidylcholine to LPA [4 5]

The diversity of LPA effects on cells is explained by the activa-tion of different signaling pathways associated with differentG-protein coupled receptors (LPARs) and their interactionwith several types of G proteins (Gq Gi Gs and G1213) [6]Initially three subtypes of endothelial differentiation gene(Edg) family G protein-coupled receptors (LPAR1 LPAR2and LPAR3) were described Until the present date severalother LPARs have been identified structurally distinct fromEdg family G receptors including LPAR4 [7]

Mice null for one or more LPA receptors and for ATXwere generated in order to evaluate the role of LPA signaling[8] Mutant null mice presented disturbances in uterineembryo spacing and implantation and in spermatogenesis

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 678968 11 pageshttpdxdoiorg1011552014678968

2 Mediators of Inflammation

An increasing number of roles for LPA signaling in repro-ductive function have been described in several speciesincluding the mouse swine ovine bovine and humans [3]

In our previous studies we evaluated LPA productionand action in the bovine endometrium [9ndash11] and ovary[12 13] These studies demonstrated that LPA is a signalingmolecule involved in luteal and endometrial functions thatare relevant for pregnancy establishment in the cowHoweverit is unknown whether LPA can be synthesized by earlybovine embryos and whether LPA plays a role in bovineembryonic development quality and survival

Although embryo development is evolutionarily highlyconserved some critical steps of early development arespecies-specific Human andmouse preimplantation embryodevelopment shows significant differences in gene expressionpatterns programs of epigenetic modification susceptibilityto genetic instability and timing of embryo genome activa-tion [14] In relevant aspects such as the timing of epigeneticreprogramming and embryonic genome activation bovineembryos reflect more closely the scenario occurring inhuman embryos [15] Additionally bovine in vitro producedembryos can be generated from oocytes recovered fromcow ovaries collected postmortem at a local abattoir thuseliminating ethical concerns regarding mice manipulationand euthanasia

This study was designed to evaluate the main hypothesisthat early bovine embryos are a source and a target of LPAsignaling To test this hypothesis we evaluated embryonictranscription and expression of genes coding for enzymes ofLPA synthesis pathways and LPA receptors and measuredLPA concentrations in embryo culturemediumAdditionallywe evaluated the effect of in vitro LPA stimulation ontranscription of embryo quality marker genes and rate ofblastocyst development

2 Materials and Methods

21 In Vitro Embryo Production Bovine embryos were pro-duced in vitro as previously described [16] Briefly ovariesfrom Frisian crossbred heifers were collected postmortem atthe local abattoir and transported to the laboratory at 37∘Cwithin one hour Cumulus-oocyte complexes (COCs) wereobtained by aspiration of follicles with 2ndash8mm diameterCOCs with at least three layers of compact cumulus cells andeven cytoplasm were selected washed and placed in 400 120583Lof maturation medium in 4-well dishes (25 COCswell)overlaid with 400120583L mineral oil and incubated at 39∘Cin a 5 CO

2in humidified air atmosphere for 24 hours

Following maturation COCs were washed and placed in4-well dishes containing 400 120583L of fertilization mediumoverlaid with 400 120583Lmineral oil and coincubated with spermat 39∘C in a 5 CO

2in humidified air atmosphere for

48 hours For in vitro insemination frozen-thawed semenfrom one bull with previously proven in vitro and in vivofertility was used throughout the experiment After thawingsemen was recovered using the swim-up procedure Thesperm concentration per fertilization well was adjusted to1times106 spermmL and the day of in vitro insemination was

considered as Day 0 On Day 2 cleavage stage embryos weredenuded from remaining cumulus cells by vortexing andembryos with 4 or more blastomeres were selected for invitro culture These embryos were washed placed in 4-welldishes (25 per well) containing 400120583L of culture mediumoverlaid with 400 120583L mineral oil and incubated in a 5CO2plus 5 O

2in humidified air atmosphere until Day 5

or Day 8 Embryos were evaluated for stage of developmentand morphological quality according to IETS guidelines [17]Only embryos classified as quality grade 1 (excellent) or grade2 (good) were selected for immunohistochemistry (IHC) andReal-Time PCR (qRT-PCR)

22 LPA Measurement in Embryo Culture Medium Culturemedium from Day 5 and Day 8 embryo cultures was recov-ered and stored at minus80∘C until LPA measurement accordingto procedures described by Saulnier-Blache et al [18] Briefly1-Oleoyl-LPA contained in 400 120583L of culture medium wasextracted with one volume of 1-butanol and the reactionproducts were separated by two-dimensional thin-layer chro-matography (2D-TLC) and autoradiographed To convertradioactivity to picomoles lipids visualized under iodinevapors were scraped and counted with 3mL of scintillationcocktail LPA concentrations of control culture medium(blankmean concentrations = 95 pmolmL ie 38 pmolwellof 400 120583L) were subtracted from those of embryo culturemedium to calculate final LPA concentrations (pmolmL)This value was divided by the number of viable embryospresent inwells at the end of culture LPA concentrationswereexpressed as pmolviable embryo

23 LPA Stimulation of In Vitro Embryo Culture At Day 2embryos were randomly allocated to one of two experimentalgroups (i) control exposed to vehicle alone (PBS) and(ii) LPA exposed to a LPA agonist (1 oleoyl-snglycero-3-lysophosphatidic acid sodium salt Alexis) at a concentrationof 10minus5M in PBS Embryos were cultured until Day 8and evaluated for stage of development and morphologicalquality Only embryos classified as quality grade 1 (excellent)or grade 2 (good) were selected for RNA extraction

24 RNA Extraction and Real-Time PCR Analysis Embry-onic RNA was extracted from five pools of 3 embryos usingthe Arcturus PicoPure RNA Isolation Kit (Applied Biosys-tems Life Technologies) according to the manufacturerrsquosinstructions DNA digestion was performed with the RNase-free DNase Set (Promega Wood Hollow road MadisonUSA) Concentration and purity of RNA were determinedspectrophotometrically at 260 nm and 280 nm (NanoDrop1000 Thermo Scientific USA) and samples were storedat minus80∘C until processing Complementary DNA (cDNA)synthesis was obtained using the SuperScript III First-StrandSynthesis SuperMix (Invitrogen) the reverse transcriptase(RT) reaction being performed in a total reaction volume of20120583L The obtained RT products were stored at minus20∘C untilqRT-PCR amplification

Mediators of Inflammation 3

Table 1 Target genes and primer pair sequences used in qRT-PCR

Gene Primer sequence (51015840-31015840) Fragment size bp GenBank accession number

LPAR1 ACGGAATCGGGATACCATGACCAGTCCAGGAGTCCAGCAG 86 NM 1740472

LPAR2 TTCTATGTGAGGCGGCGAGTAGACCATCCAGGAGCAGCAC 161 NM 0011922351

LPAR3 TCCAACCTCATGGCCTTCTTGACCCACTCGTATGCGGAGA 101 NM 0011927412

LPAR4 CCACAGTACCTCCAGAAAGTTCATTGGAATTGGAAGTCAATGAATC 192 NM 0010981051

ATX ACCCCCTGATTGTCGATGTGTCTCCGCATCTGTCCTTGGT 120 NM 0010802931

PLA2 CTGCGTGCCACAAAAGTGACTCGGGGGTTGAAGAGATGAA 92 NM 0010758641

BCL2 GAGTTCGGAGGGGTCATGTGGCCTTCAGAGACAGCCAGGA 203 NM 0011664861

BAX GTGCCCGAGTTGATCAGGACCCATGTGGGTGTCCCAAAGT 126 NM 1738941

3120573HSD TCCCGGATGAGCCTTCCTATACTAGGTGGCGGTTGAAGCA 116 NM 1743432

COX-2 TGGGTGTGAAAGGGAGGAAAAAGTGCTGGGCAAAGAATGC 127 AF0049441

PTGES CCGAGGACGCTCAGAGACATAAAGCCCAGGAACAGGAAGG 122 NM 1744432

PGFS GGAGGACCCCAGGATCAAAGCTCAGCAATGCGTTCAGGTG 130 S549731

IGF2R ACCTCCGATCCTCAATCCCATGTAGTTGAAGTGCCGGTCC 89 NM 1743522

OCT4 GAGAAAGACGTGGTCCGAGTGGACCCAGCAGCCTCAAAATC 101 NM 1745802

GAPDH CACCCTCAAGATTGTCAGCAGGTCATAAGTCCCTCCACGA 103 NM 0010340342

Target genes included genes coding for enzymes of theprostaglandins (PGs PTGS2 PGES and PGFS) and proges-terone (P

4 3120573HSD) synthesis pathways genes associatedwith

apoptosis (BAX and BCL2) and quality markers (IGFR2 andOCT4) using GAPDH transcription as an internal controlThis housekeeping gene was chosen using the NormFindersoftware comparing three candidate genesGAPDH 120573-actinandH2A1 [19] Primers were chosen using an online softwarepackage (httpbioinfouteeprimer3) (Table 1) and werevalidated following the use of different concentrations and theobservation that each pair generated a single amplicon withthe predicted molecular size The qRT-PCR was performedwith an ABI Prism 7900 (Applied Biosystems Life 12 Tech-nologies USA) sequence detection system using MaximaSYBR GreenROX qPCR 13 Master Mix (number K0222FermentasThermo Scientific USA)The PCR reactions wereperformed in 96-well plates Each PCR reaction well (20120583L)contained 2 120583L of RT product 5 pmol120583L of forward andreverse primers and 10 120583L SYBR Green PCR master mixTheqRT-PCRwas performedunder the following conditions95∘C for 10min followed by 40 cycles at 94∘C for 15 secand 60∘C for 60 sec Melting curves were acquired (15 s at95∘C 30 s at 60∘C and 15 s at 95∘C) to ensure that a singleproduct was amplified in the reaction In order to exclude

the possibility of genomic DNA contamination in RNA sam-ples the reactions were also run either on blank-only buffersamples or in absence of the reverse transcriptase enzymeThe specificity of the PCR products for all examined geneswas confirmed by gel electrophoresis and by sequencingDataregarding relative mRNA quantification was analyzed withthe Real-Time PCR Miner algorithm [20]

25 Immunofluorescence Selected embryos were fixed in4 paraformaldehyde for 1 h at 4∘C washed in PBS andpermeabilized with 05 Triton X-100 for 10min at roomtemperature After washing in PBS embryos were incubatedfor 1 h in blocking solution (Novocastra Protein Blockrefa RE7102 Novocastra Laboratories Ltd Newcastle uponTyne UK) This blocking step was followed by overnightincubation at 4∘C with the following antibodies (Santa CruzBiotechnology) diluted 1 100 in blocking solution rabbitpolyclonal anti-ATX (sc-66813) PLA2 (sc438) LPAR1 (sc-22207) LPAR2 (sc-25490) LPAR3 (sc25492) and LPAR4(sc-46021) Antibodiesrsquo specificity in bovine tissue was pre-viously confirmed through WB and IHC [12 21] Negativecontrol sections were incubated with normal rabbit irrelevantIgG (concentration 1 100 sc-2027) After washing in PBS


Day 5 Day 8

0000

0001

0002

003

004

005

A

BATX

mRN

A (a

u)

(a)

Day 5 Day 80000

0001

0002

0003

A

B

cPLA

2 m

RNA

(au

)

0045

0040

0035

0030

0025

(b)

Figure 1 Transcription levels of genes coding for LPA synthesis enzymes ((a) ATX (b) cPLA2) in late cleavage (Day 5) and blastocyst (Day8) stage bovine in vitro produced embryos Columns with different superscripts differ significantly AB 119875 lt 005

embryos were incubated for 1 h at room temperature withAlexa Fluor 488 donkey anti-rabbit IgG antibody (con-centration 1 300 in blocking solution A-21206 InvitrogenLife Technologies Foster City CA USA) Following a finalwashing in PBS embryos were mounted in Vectashieldwith DAPI (ref H-1200 Vector Laboratories BrunschwigChemie Amsterdam Netherlands) and examined under aLeica DMRA fluorescence microscope

26 Statistical Analysis Data were analyzed through the sta-tistical software GraphPad PRISM 50 (GraphPad SoftwareInc La Jolla CA USA) Data are presented as mean plusmnSEM Quantitative data were analyzed by Studentrsquos t-test forindependent pairs whereas categorical data were analyzed byFisherrsquos exact test The differences were considered statisti-cally significant at the 95 confidence level (119875 lt 005)

3 Results

31 Late Cleavage and Blastocyst Stage Bovine EmbryosTranscribed and Expressed Genes Coding for Enzymes ofthe LPA Synthesis Pathway and Produced LPA into CultureMedium As shown in Figure 1 transcription of genes codingfor enzymes of the LPA synthesis pathway ((a) ATX and(b) cPLA2) was detected in Day 5 and Day 8 embryosTranscription levels of both genes were significantly higherin late cleavage than in blastocyst stage embryos Expressionof ATX and cPLA

2proteins was observed in cells of late

cleavage and blastocyst stage embryos (Figure 2) LPA wasdetected in culture medium of Day 5 and Day 8 embryosHowever LPA concentrations were significantly higher inmedium recovered from blastocyst culture than in mediumrecovered from late cleavage embryosrsquo culture (Figure 3)

32 Late Cleavage and Blastocyst Stage Embryos TranscribedandExpressedGenes Coding for LPAReceptors (LPAR1ndash4) Asshown in Figure 4 transcription of genes coding for LPAR1(a) LPAR2 (b) LPAR3 (c) and LPAR4 (d) was detectedin Day 5 and Day 8 bovine embryos Late cleavage stage

Table 2 Effect of LPA supplementation on bovine in vitro embryodevelopment and quality

Group 119899Day 8 embryos119899 ()

Qualities 1 and 2119899 ()

Control 304 101 (33) 92 (91)LPA 329 110 (33) 103 (93)

embryos showed significantly highermRNA abundance of allLPARs than blastocyst stage embryos Expression of LPAR1ndash4proteinswas observed inDay 5 andDay 8 embryos (Figure 5)

33 LPA Supplementation of CultureMediumHadNoEffect onEmbryo Development and Morphological Quality As shownin Table 2 the blastocyst rates on Day 8 were similar in LPA-stimulated and control embryos The proportions of qualitygrade 1 and grade 2 Day 8 blastocysts were also similar in theLPA and control groups

34 LPA Supplementation of CultureMediumHadNoEffect onBlastocyst Transcription Levels of Genes Coding for Enzymesof Prostaglandin and Steroidogenic Synthesis Pathways Asshown in Figure 6 transcription levels of PTGS2 PGESPGFS and 3120573HSD were similar in Day 8 LPA-stimulated andcontrol embryos

35 LPA Supplementation of Culture Medium Affected Blasto-cyst Transcription Levels of Quality Marker Genes As shownin Figure 7 LPA-stimulated blastocysts showed a significantlylower transcription level of BAX and significantly highertranscription levels of BCL2 and IGF2R than control blas-tocysts Transcription levels of OCT4 were similar in LPA-stimulated and control blastocysts

4 Discussion

This study demonstrates for the first time that late cleavageand blastocyst stage bovine embryos are able to transcribe


(a) (b) (c)

(d) (e) (f)

Figure 2 Expression of LPA synthesis enzymes ATX ((a) and (d)) and cPLA2((b) and (e)) in late cleavage (Day 5 upper row) and blastocyst

(Day 8 bottom row) stage bovine in vitro produced embryos (c) and (f) negative controls 200x magnification

Day 5 Day 8

0

5

10

15

LPA

(pm

olv

iabl

e em

bryo

)

A

B

Figure 3 LPA production by late cleavage (Day 5) and blastocyst(Day 8) stage bovine in vitro produced embryos LPA concentrationsrepresent the accumulated production sinceDay 2 and are expressedas pmolviable embryo Columns with different superscripts differsignificantly AB 119875 lt 005

and express genes coding for enzymes of LPA synthesispathways (ATX and cPLA2) and produce LPA into culturemedium This evidences that the early embryo is able to useboth pathways of LPA production Interestingly the PLA

2

pathway is mainly observed during inflammatory events [45] which may lead to the speculation that early embryo-maternal signaling includes an inflammatory componentTranscript abundance was higher in late cleavage (Day 5)than in blastocyst (Day 8) stage embryos In vivo Day 5cleaving uncompacted morulae travel through the oviduct

whereas Day 8 expandingecloding blastocysts are shed inthe endometrium In mice LPA stimulated oviduct smoothmuscle contraction and ovum transport through the oviduct[22] In bovine active LPA production by embryos of laterstages of cleavage may also indicate a paracrine role in theoviduct to enhance embryo transport into the uterus

Embryonic LPA production was higher in blastocyst thanin late cleavage stage embryos This may reflect the highercell content of blastocysts compared to late cleavage stagesandor the accumulated production from Day 5 to Day 8Active LPA production by bovine blastocysts is suggestive ofan early paracrine interaction between the embryo and theuterus We demonstrated that the bovine uterus is a sourceand a target of LPA through LPAR1-mediated actions [9 10]In vitro LPA stimulation of endometrial cells recovered fromcows either on Days 8ndash10 of the estrous cycle or on Days 8ndash10of pregnancy showed that LPA inhibited endometrial PGF2120572production only in early pregnancy [23] Taken togetherthese results lead us to the proposal that blastocyst LPAparacrine signaling in the uterus attenuates endometrialPGF2120572 production contributing to luteal maintenance andmaternal recognition of pregnancy

Late cleavage and blastocyst stage bovine embryos tran-scribed and expressed genes coding for LPARs (1ndash4) and genetranscription was higher in late cleavage than in blastocyststage embryos This indicates that preeclosion embryos arealso a target for LPA autocrine (within the embryo) andparacrine (from the oviductuterus) actions In mice embry-onic transcription of LPAR1-2 was detected in differentiatingblastocysts [24] and LPAR1ndash4 transcripts were detected in65 to 105 dpc embryos [25] In sheep transcription and


006

004

002

000

Day 5 Day 8

A

B

LPAR

1m

RNA

(au

)

(a)

010

008

006

004

002

000

Day 5 Day 8

A

B

LPAR

2m

RNA

(au

)

(b)

0003

0002

0001

0000

Day 5 Day 8

A

B

LPAR

3m

RNA

(au

)

(c)

025

020

015

010

005

000

Day 5 Day 8

BLPAR

4m

RNA

(au

)

A

(d)

Figure 4 Transcription levels of genes coding for LPA receptors ((a) LPAR1 (b) LPAR2 (c) LPAR3 (d) LPAR4) in late cleavage (Day 5) andblastocyst (Day 8) stage bovine in vitro produced embryos Columns with different superscripts differ significantly AB 119875 lt 005

expression of LPARs were evaluated in Day 12 to Day 18conceptuses [26] LPAR1 and LPAR3 transcripts peaked onDay 14 whereas expression of LPAR proteins peaked on Day17

Uterine concentrations of LPA were estimated to be 08ndash1 120583M in the ovine [26] and porcine [27] In vitro LPAsynthesis is decreased due to the lower availability of substrate(lysophosphatidylcholine) and synthesized LPA is degradedalong culture time [26] This also holds true to exogenouslyadded LPA therefore we chose a high LPA treatment con-centration to counterbalance degradation along culture timeIn this study LPA stimulation had no effect on embryonicdevelopment and morphological quality In mice LPA stim-ulation at the pronuclear stage improved blastocyst rate butstimulation at later stages had no effect on development[28] This is in line with the present results Therefore theembryonic rate of development is probably not regulated bythe paracrine action of oviductuterus secreted LPA

Blastocyst stage bovine embryos are able to transcribegenes coding for PGs synthesis pathway enzymes and forP4synthesis enzymes and produce PGs (PGF

2120572and PGE

2)

and P4into culture medium [16] In vitro we found a

stimulatory effect of LPA on P4synthesis and interferon

(IFN)120591 action in bovine luteal cells [12] and on luteotropicPGE2secretion in the bovine endometrium [9 10] In vivo

we also found that LPA infusion prevented spontaneousluteolysis prolonged the functional lifespan of the corpusluteum and stimulated luteotropic PGE

2synthesis in heifers

[11] Additionally administration of a LPARs antagonistdecreased pregnancy rate in cows [11] These results indicatethat LPA is relevant in early pregnancy maintenance in thebovine mainly through its luteotropic action In the ovineLPA stimulation of trophectoderm cells in culture induced a2-fold increase in PGF

2120572and PGE

2synthesis not associated

with an increase in cPLA2120572 and PTGS2 transcription [26]Authors [26] suggested that LPA-mediated signaling occursthrough the release of PGs probably due to a stimulationof arachidonic acid mobilization Here LPA stimulation hadno effect on transcription of genes coding for enzymes ofPG synthesis (PTGS2 PGES and PGFS) and steroidogenic(3120573HSD) pathways However we cannot exclude the possibil-ity of an LPA-induced embryonic release of PGs independentof PTGS2 PGES and PGFS mRNA abundance as we didnot measure PGs in culture medium Also the functional


(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j)

Figure 5 Expression of LPARs (LPAR1 LPAR2 LPAR3 and LPAR4) in late cleavage (Day 5 (a) (b) (c) and (d) resp) and blastocyst (Day8 (f) (g) (h) and (i) resp) stage bovine in vitro produced embryos (e) and (j) negative controls 200x magnification

role of embryonic P4production remains unclear Here LPA

stimulation had no effect on 3120573HSD transcription contraryto what was observed in luteal cells [12]

LPA stimulation induced a decrease in blastocyst tran-scription of BAX and an increase in transcription of BCL2Balanced cellular expression of BAX and BCL2 pro- andantiapoptotic factors respectively is determinant for thecontrol of cell survival [29] The BAXBCL2 protein ratio

was decreased in fragmented bovine blastocysts leading tothe suggestion that this ratio might be used to evaluate thetendency of oocytes and embryos towards either survival orapoptosis [30] Also LPA stimulation of cell culture regulatedexpression of BCL2 and BAX enhancing cell survival [31ndash33] In bovine cultured luteal cells LPA stimulation inhibitedTNF120572 and IFN120574 (proapoptoticmediators) induced apoptosisthis effect was obtained through the inhibition of BAX


Control LPA00

05

10

15PT

GS2

mRN

A (a

u)

(a)

Control LPA

006

004

002

000

PGES

mRN

A (a

u)

(b)

Control LPA

03

02

01

00

PGFS

mRN

A (a

u)

(c)

Control LPA

008

006

004

002

000

3120573HSD

mRN

A (a

u)

(d)

Figure 6 Effect of LPA stimulation on transcription levels of genes coding for PG ((a) PTGS2 (b) PGES (c) PGFS) and P4((d) 3120573-HSD)

synthesis enzymes in bovine in vitro produced blastocysts

TNFR1 Fas FasL and caspase 3 activity [34] Here blastocystgene transcription was shifted towards high BCL2 and lowBAX levels by LPA stimulation Although LPA treatmentdid not affect embryo development and quality until Day 8inhibition of apoptosis in blastocyst cells may be beneficialfor subsequent in vivo survival

We evaluated LPA stimulation effect on blastocyst tran-scription of two embryo quality marker genes IGF2R andOCT4 Several studies reported expression of IGF systemgenes (IGF1 IGF2 IGF1R and IGF2R) in bovine embryos[35ndash39] IGF-system genes were proposed as bovine embryoquality marker genes as transcription of these genes wasassociatedwithmorphological assessment and growth poten-tial of embryos [35] In the present study LPA stimulatedblastocyst IGF2R transcription Again although LPA stimu-lation did not affect embryo development and quality untilDay 8 stimulation of blastocyst growth may be beneficial forsubsequent in vivo survival

In mice OCT4-deficient embryos develop to blastocystbut ICM cells lose pluripotency [40] Although transcriptionfactor OCT4 is a cornerstone of pluripotency a recent study[41] showed that OCT4 major activity in mouse blastocysts

is to support primitive endoderm differentiation In bovineIVF and SCNT blastocysts showed significantly differentexpression ofOCT4 [42] Here LPA stimulation had no effecton OCT4 blastocyst transcription which indicates that LPAdoes not interfere with OCT4 pathway mediated blastocystdifferentiation

In conclusion late cleavage and blastocyst stage bovineembryos transcribed and expressed genes coding for LPAsynthesis enzymes and produced LPA into culture mediumAdditionally late cleavage and blastocyst stage bovineembryos transcribed and expressed genes coding for LPARsThis turns early bovine embryos into a potential source andtarget of autocrineparacrine LPA mediated cell signalingLPA stimulation had no effect on in vitro embryo develop-ment and quality until Day 8 but affected blastocyst transcrip-tion of apoptosis (BAX BCL2) and growth (IGF2R) relatedgenes Inhibition of apoptosis and promotion of growth inblastocyst cellsmay be relevant for subsequent in vivo embryosurvival Altogether the present results lead us to proposethat LPA mediated cell signaling may operate in an auto-paracrine way during bovine early embryonic developmentbeing involved in early embryo-maternal interactions leading


015

010

005

000

Control LPA

A

B

BAX

mRN

A (a

u)

(a)

0010

0008

0006

0004

0002

0000

Control LPA

A

B

IGF2

Rm

RNA

(au

)

(b)

06

04

02

00

Control LPA

A

B

BCL2

mRN

A (a

u)

times10minus4

(c)

015

010

005

000

Control LPA

OCT

4m

RNA

(au

)

(d)

Figure 7 Effect of LPA stimulation on transcription levels of genes coding for embryo quality markers ((a) BAX (b) IGF2R (c) BCL2 and(d) OCT4) in bovine in vitro produced blastocysts Columns with different superscripts differ significantly AB 119875 lt 005

to embryo survival Due to similarities between bovine andhuman early embryonic development [15] results describedhere may also reflect events occurring in the human embryo

Disclosure

Data presented in this paper are part of PhD thesis of DorotaBoruszewska

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ana Catarina Torres and Dorota Boruszewska both con-tributed equally to this work

Acknowledgments

This research was funded by Grant PTDCCVT656902006from Foundation for Science and Technology (FCT) This

research was supported by Grants-in-Aid for ScientificResearch from the Polish National Science Centre(201205ENZ903480) Dorota Boruszewska and IlonaKowalczyk-Zieba were supported by the European Unionwithin the European Social Fund (DrINNO3)

References

[1] X Ye ldquoLysophospholipid signaling in the function and pathol-ogy of the reproductive systemrdquo Human Reproduction Updatevol 14 no 5 pp 519ndash536 2008

[2] A Skoura and T Hla ldquoLysophospholipid receptors in verte-brate development physiology and pathologyrdquo Journal of LipidResearch vol 50 pp S293ndash298 2009

[3] X Ye and J Chun ldquoLysophosphatidic acid (LPA) signal-ing in vertebrate reproductionrdquo Trends in Endocrinology andMetabolism vol 21 no 1 pp 17ndash24 2010

[4] J Aoki A Inoue and S Okudaira ldquoTwo pathways for lysophos-phatidic acid productionrdquo Biochimica et Biophysica ActamdashMolecular and Cell Biology of Lipids vol 1781 no 9 pp 513ndash5182008

[5] N Samadi R Bekele D Capatos G Venkatraman M Sariah-metoglu and D N Brindley ldquoRegulation of lysophosphatidatesignaling by autotaxin and lipid phosphate phosphatases with


respect to tumor progression angiogenesis metastasis andchemo-resistancerdquo Biochimie vol 93 no 1 pp 61ndash70 2011

[6] E J Van Corven A Groenink K Jalink T Eichholtz and WH Moolenaar ldquoLysophosphatidate-induced cell proliferationidentification and dissection of signaling pathways mediated byG proteinsrdquo Cell vol 59 no 1 pp 45ndash54 1989

[7] G Tigyi ldquoAiming drug discovery at lysophosphatidic acidtargetsrdquo British Journal of Pharmacology vol 161 no 2 pp 241ndash270 2010

[8] JW Choi C-W Lee and J Chun ldquoBiological roles of lysophos-pholipid receptors revealed by genetic null mice an updaterdquoBiochimica et Biophysica ActamdashMolecular and Cell Biology ofLipids vol 1781 no 9 pp 531ndash539 2008

[9] I Woclawek-Potocka J Komiyama J S Saulnier-Blache et alldquoLysophosphatic acid modulates prostaglandin secretion in thebovine uterusrdquo Reproduction vol 137 no 1 pp 95ndash105 2009

[10] I Woclawek-Potocka K Kondraciuk and D J SkarzynskildquoLysophosphatidic acid stimulates prostaglandin E2 productionin cultured stromal endometrial cells through LPA1 receptorrdquoExperimental Biology andMedicine vol 234 no 8 pp 986ndash9932009

[11] I Woclawek-Potocka I Kowalczyk-Zieba and D J SkarzynskildquoLysophosphatidic acid action during early pregnancy in thecow in vivo and in vitro studiesrdquo Journal of Reproduction andDevelopment vol 56 no 4 pp 411ndash420 2010

[12] Kowalczyk-Zieba D Boruszewska J S Saulnier-Blache et alldquoLysophosphatidic acid action in the bovine corpus luteum -anin vitro studyrdquo Journal of Reproduction and Development vol58 no 6 pp 661ndash671 2012

[13] D Boruszewska E Sinderewicz I Kowalczyk-Zieba D JSkarzynski and I Woclawek-Potocka ldquoInfluence of lysophos-phatidic acid on estradiol production and follicle stimulatinghormone action in bovine granulosa cellsrdquo Reproductive Biol-ogy vol 13 no 4 pp 344ndash347 2013

[14] K K Niakan J Han R A Pedersen C Simon and RA R Pera ldquoHuman pre-implantation embryo developmentrdquoDevelopment vol 139 no 5 pp 829ndash841 2012

[15] S Leidenfrost M BoelhauveM Reichenbach et al ldquoCell arrestand cell death in mammalian preimplantation developmentlessons from the bovine modelrdquo PLoS ONE vol 6 no 7 ArticleID e22121 2011

[16] A Torres M Batista P Diniz L Mateus and L Lopes-da-Costa ldquoEmbryo-luteal cells co-culture an in vitro model toevaluate steroidogenic and prostanoid bovine early embryo-maternal interactionsrdquo In Vitro Cellular amp DevelopmentalBiologymdashAnimal vol 49 no 2 pp 134ndash146 2013

[17] D A Stringfellow and S M Seidel Eds Manual of theInternational Embryo Transfer Society International EmbryoTransfer Society Savoy 1998

[18] J S Saulnier-Blache A Girard M-F Simon M Lafontan andP Valet ldquoA simple and highly sensitive radioenzymatic assay forlysophosphatidic acid quantificationrdquo Journal of Lipid Researchvol 41 no 12 pp 1947ndash1951 2000

[19] C L Andersen J L Jensen and T F Oslashrntoft ldquoNormalization ofreal-time quantitative reverse transcription-PCR data a model-based variance estimation approach to identify genes suited fornormalization applied to bladder and colon cancer data setsrdquoCancer Research vol 64 no 15 pp 5245ndash5250 2004

[20] S Zhao and R D Fernald ldquoComprehensive algorithm forquantitative real-time polymerase chain reactionrdquo Journal ofComputational Biology vol 12 no 8 pp 1047ndash1064 2005

[21] D Boruszewska I Kowalczyk-Zieba K Piotrowska-Tomala etal ldquoWhich bovine endometrial cells are the source of and targetfor lysophosphatidic acidrdquo Reproductive Biology vol 13 no 1pp 100ndash103 2013

[22] K Kunikata S Yamano A Tokumura and T Aono ldquoEffectof lysophosphatidic acid on the ovum transport in mouseoviductsrdquo Life Sciences vol 65 no 8 pp 833ndash840 1999

[23] I Woclawek-Potocka E Brzezicka and D J SkarzynskildquoLysophosphatic acid modulates prostaglandin secretion inthe bovine endometrial cells differently on days 8-10 of theestrous cycle and early pregnancyrdquo Journal of Reproduction andDevelopment vol 55 no 4 pp 393ndash399 2009

[24] Z Liu and D R Armant ldquoLysophosphatidic acid regulatesmurine blastocyst development by transactivation of receptorsfor heparin-binding EGF-like growth factorrdquo Experimental CellResearch vol 296 no 2 pp 317ndash326 2004

[25] L A Van Meeteren P Ruurs C Stortelers et al ldquoAutotaxina secreted lysophospholipase D is essential for blood vesselformation during developmentrdquoMolecular andCellular Biologyvol 26 no 13 pp 5015ndash5022 2006

[26] E Liszewska P Reinaud E Billon-Denis O Dubois P Robinand G Charpigny ldquoLysophosphatidic acid signaling duringembryo development in sheep involvement in prostaglandinsynthesisrdquo Endocrinology vol 150 no 1 pp 422ndash434 2009

[27] H Seo M Kim Y Choi C-K Lee and H Ka ldquoAnalysisof lysophosphatidic acid (LPA) receptor and LPA-inducedendometrial prostaglandin-endoperoxide synthase 2 expres-sion in the porcine uterusrdquo Endocrinology vol 149 no 12 pp6166ndash6175 2008

[28] TKobayashi ldquoEffect of lysophosphatidic acid on the preimplan-tation development of mouse embryosrdquo FEBS Letters vol 351no 1 pp 38ndash40 1994

[29] I Boumela S Assou A Aouacheria et al ldquoInvolvement ofBCL2 family members in the regulation of human oocyte andearly embryo survival and death gene expression and beyondrdquoReproduction vol 141 no 5 pp 549ndash561 2011

[30] MY Yang andR Rajamahendran ldquoExpression of Bcl-2 andBaxproteins in relation to quality of bovine oocytes and embryosproduced in vitrordquo Animal Reproduction Science vol 70 no 3-4 pp 159ndash169 2002

[31] E J Goetzl Y Kong and B Mei ldquoLysophosphatidic acid andsphingosine 1-phosphate protection of T cells from apoptosisin association with suppression of Baxrdquo Journal of Immunologyvol 162 no 4 pp 2049ndash2056 1999

[32] J Hurst-Kennedy B D Boyan and Z Schwartz ldquoLysophospha-tidic acid signaling promotes proliferation differentiation andcell survival in rat growth plate chondrocytesrdquo Biochimica etBiophysica ActamdashMolecular Cell Research vol 1793 no 5 pp836ndash846 2009

[33] M Gotoh K Sano-Maeda H Murofushi and K Murakami-Murofushi ldquoProtection of neuroblastoma Neuro2A cells fromhypoxia-induced apoptosis by cyclic phosphatidic acid (cPA)rdquoPLoS ONE vol 7 no 12 Article ID e51093 2012

[34] I Woclawek-Potocka I Kowalczyk-Zieba M Tylingo DBoruszewska E Sinderewicz and D J Skarzynski ldquoEffects oflysophopatidic acid on tumor necrosis factor 120572 and interferon120574 action in the bovine corpus luteumrdquo Molecular and CellularEndocrinology vol 377 no 1-2 pp 103ndash111 2013

[35] H-C Liu Z-Y He C A Mele L L Veeck O K Davisand Z Rosenwaks ldquoExpression of IGFs and their receptors isa potential marker for embryo qualityrdquo American Journal ofReproductive Immunology vol 38 no 4 pp 237ndash245 1997


[36] M A Yaseen C Wrenzycki D Herrmann J W Carnwathand H Niemann ldquoChanges in the relative abundance of mRNAtranscripts for insulin-like growth factor (IGF-I and IGF-II)ligands and their receptors (IGF-IRIGF-IIR) in preimplanta-tion bovine embryos derived from different in vitro systemsrdquoReproduction vol 122 no 4 pp 601ndash610 2001

[37] AVMakarevich andMMarkkula ldquoApoptosis and cell prolifer-ation potential of bovine embryos stimulated with insulin-likegrowth factor I during in vitro maturation and culturerdquo Biologyof Reproduction vol 66 no 2 pp 386ndash392 2002

[38] A T Byrne J Southgate D R Brison and H J LeeseldquoRegulation of apoptosis in the bovine blastocyst by insulinand the insulin-like growth factor (IGF) superfamilyrdquoMolecularReproduction and Development vol 62 no 4 pp 489ndash4952002

[39] J A Neira D Tainturier M A Pena and J Martal ldquoEffect ofthe association of IGF-I IGF-II bFGF TGF-1205731 GM-CSF andLIF on the development of bovine embryos produced in vitrordquoTheriogenology vol 73 no 5 pp 595ndash604 2010

[40] J Nichols B Zevnik K Anastassiadis et al ldquoFormation ofpluripotent stem cells in the mammalian embryo depends onthe POU transcription factor Oct4rdquo Cell vol 95 no 3 pp 379ndash391 1998

[41] T Frum M A Halbisen C Wang H Amiri P Robson andA Ralston ldquoOct4 cell-autonomously promotes primitive endo-derm development in the mouse blastocystrdquo DevelopmentalCell vol 25 no 6 pp 610ndash622 2013

[42] K I Aston G-P Li B A Hicks et al ldquoAbnormal levels oftranscript abundance of developmentally important genes invarious stages of preimplantation bovine somatic cell nucleartransfer embryosrdquoCellular Reprogramming vol 12 no 1 pp 23ndash32 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


An increasing number of roles for LPA signaling in repro-ductive function have been described in several speciesincluding the mouse swine ovine bovine and humans [3]

In our previous studies we evaluated LPA productionand action in the bovine endometrium [9ndash11] and ovary[12 13] These studies demonstrated that LPA is a signalingmolecule involved in luteal and endometrial functions thatare relevant for pregnancy establishment in the cowHoweverit is unknown whether LPA can be synthesized by earlybovine embryos and whether LPA plays a role in bovineembryonic development quality and survival

Although embryo development is evolutionarily highlyconserved some critical steps of early development arespecies-specific Human andmouse preimplantation embryodevelopment shows significant differences in gene expressionpatterns programs of epigenetic modification susceptibilityto genetic instability and timing of embryo genome activa-tion [14] In relevant aspects such as the timing of epigeneticreprogramming and embryonic genome activation bovineembryos reflect more closely the scenario occurring inhuman embryos [15] Additionally bovine in vitro producedembryos can be generated from oocytes recovered fromcow ovaries collected postmortem at a local abattoir thuseliminating ethical concerns regarding mice manipulationand euthanasia

This study was designed to evaluate the main hypothesisthat early bovine embryos are a source and a target of LPAsignaling To test this hypothesis we evaluated embryonictranscription and expression of genes coding for enzymes ofLPA synthesis pathways and LPA receptors and measuredLPA concentrations in embryo culturemediumAdditionallywe evaluated the effect of in vitro LPA stimulation ontranscription of embryo quality marker genes and rate ofblastocyst development

2 Materials and Methods

21 In Vitro Embryo Production Bovine embryos were pro-duced in vitro as previously described [16] Briefly ovariesfrom Frisian crossbred heifers were collected postmortem atthe local abattoir and transported to the laboratory at 37∘Cwithin one hour Cumulus-oocyte complexes (COCs) wereobtained by aspiration of follicles with 2ndash8mm diameterCOCs with at least three layers of compact cumulus cells andeven cytoplasm were selected washed and placed in 400 120583Lof maturation medium in 4-well dishes (25 COCswell)overlaid with 400120583L mineral oil and incubated at 39∘Cin a 5 CO

2in humidified air atmosphere for 24 hours

Following maturation COCs were washed and placed in4-well dishes containing 400 120583L of fertilization mediumoverlaid with 400 120583Lmineral oil and coincubated with spermat 39∘C in a 5 CO

2in humidified air atmosphere for

48 hours For in vitro insemination frozen-thawed semenfrom one bull with previously proven in vitro and in vivofertility was used throughout the experiment After thawingsemen was recovered using the swim-up procedure Thesperm concentration per fertilization well was adjusted to1times106 spermmL and the day of in vitro insemination was

considered as Day 0 On Day 2 cleavage stage embryos weredenuded from remaining cumulus cells by vortexing andembryos with 4 or more blastomeres were selected for invitro culture These embryos were washed placed in 4-welldishes (25 per well) containing 400120583L of culture mediumoverlaid with 400 120583L mineral oil and incubated in a 5CO2plus 5 O

2in humidified air atmosphere until Day 5

or Day 8 Embryos were evaluated for stage of developmentand morphological quality according to IETS guidelines [17]Only embryos classified as quality grade 1 (excellent) or grade2 (good) were selected for immunohistochemistry (IHC) andReal-Time PCR (qRT-PCR)

22 LPA Measurement in Embryo Culture Medium Culturemedium from Day 5 and Day 8 embryo cultures was recov-ered and stored at minus80∘C until LPA measurement accordingto procedures described by Saulnier-Blache et al [18] Briefly1-Oleoyl-LPA contained in 400 120583L of culture medium wasextracted with one volume of 1-butanol and the reactionproducts were separated by two-dimensional thin-layer chro-matography (2D-TLC) and autoradiographed To convertradioactivity to picomoles lipids visualized under iodinevapors were scraped and counted with 3mL of scintillationcocktail LPA concentrations of control culture medium(blankmean concentrations = 95 pmolmL ie 38 pmolwellof 400 120583L) were subtracted from those of embryo culturemedium to calculate final LPA concentrations (pmolmL)This value was divided by the number of viable embryospresent inwells at the end of culture LPA concentrationswereexpressed as pmolviable embryo

23 LPA Stimulation of In Vitro Embryo Culture At Day 2embryos were randomly allocated to one of two experimentalgroups (i) control exposed to vehicle alone (PBS) and(ii) LPA exposed to a LPA agonist (1 oleoyl-snglycero-3-lysophosphatidic acid sodium salt Alexis) at a concentrationof 10minus5M in PBS Embryos were cultured until Day 8and evaluated for stage of development and morphologicalquality Only embryos classified as quality grade 1 (excellent)or grade 2 (good) were selected for RNA extraction

24 RNA Extraction and Real-Time PCR Analysis Embry-onic RNA was extracted from five pools of 3 embryos usingthe Arcturus PicoPure RNA Isolation Kit (Applied Biosys-tems Life Technologies) according to the manufacturerrsquosinstructions DNA digestion was performed with the RNase-free DNase Set (Promega Wood Hollow road MadisonUSA) Concentration and purity of RNA were determinedspectrophotometrically at 260 nm and 280 nm (NanoDrop1000 Thermo Scientific USA) and samples were storedat minus80∘C until processing Complementary DNA (cDNA)synthesis was obtained using the SuperScript III First-StrandSynthesis SuperMix (Invitrogen) the reverse transcriptase(RT) reaction being performed in a total reaction volume of20120583L The obtained RT products were stored at minus20∘C untilqRT-PCR amplification

























Day 5 Day 8

0000

0001

0002

003

004

005

A

BATX

mRN

A (a

u)

(a)

Day 5 Day 80000

0001

0002

0003

A

B

cPLA

2 m

RNA

(au

)

0045

0040

0035

0030

0025

(b)




3 Results








Control 304 101 (33) 92 (91)LPA 329 110 (33) 103 (93)





4 Discussion



(a) (b) (c)

(d) (e) (f)



Day 5 Day 8

0

5

10

15

LPA

(pm

olv

iabl

e em

bryo

)

A

B



2






006

004

002

000

Day 5 Day 8

A

B

LPAR

1m

RNA

(au

)

(a)

010

008

006

004

002

000

Day 5 Day 8

A

B

LPAR

2m

RNA

(au

)

(b)

0003

0002

0001

0000

Day 5 Day 8

A

B

LPAR

3m

RNA

(au

)

(c)

025

020

015

010

005

000

Day 5 Day 8

BLPAR

4m

RNA

(au

)

A

(d)





2120572and PGE

2)







2120572and PGE




(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j)







Control LPA00

05

10

15PT

GS2

mRN

A (a

u)

(a)

Control LPA

006

004

002

000

PGES

mRN

A (a

u)

(b)

Control LPA

03

02

01

00

PGFS

mRN

A (a

u)

(c)

Control LPA

008

006

004

002

000

3120573HSD

mRN

A (a

u)

(d)









015

010

005

000

Control LPA

A

B

BAX

mRN

A (a

u)

(a)

0010

0008

0006

0004

0002

0000

Control LPA

A

B

IGF2

Rm

RNA

(au

)

(b)

06

04

02

00

Control LPA

A

B

BCL2

mRN

A (a

u)

times10minus4

(c)

015

010

005

000

Control LPA

OCT

4m

RNA

(au

)

(d)



Disclosure






Acknowledgments



References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































Day 5 Day 8

0000

0001

0002

003

004

005

A

BATX

mRN

A (a

u)

(a)

Day 5 Day 80000

0001

0002

0003

A

B

cPLA

2 m

RNA

(au

)

0045

0040

0035

0030

0025

(b)




3 Results








Control 304 101 (33) 92 (91)LPA 329 110 (33) 103 (93)





4 Discussion



(a) (b) (c)

(d) (e) (f)



Day 5 Day 8

0

5

10

15

LPA

(pm

olv

iabl

e em

bryo

)

A

B



2






006

004

002

000

Day 5 Day 8

A

B

LPAR

1m

RNA

(au

)

(a)

010

008

006

004

002

000

Day 5 Day 8

A

B

LPAR

2m

RNA

(au

)

(b)

0003

0002

0001

0000

Day 5 Day 8

A

B

LPAR

3m

RNA

(au

)

(c)

025

020

015

010

005

000

Day 5 Day 8

BLPAR

4m

RNA

(au

)

A

(d)





2120572and PGE

2)







2120572and PGE




(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j)







Control LPA00

05

10

15PT

GS2

mRN

A (a

u)

(a)

Control LPA

006

004

002

000

PGES

mRN

A (a

u)

(b)

Control LPA

03

02

01

00

PGFS

mRN

A (a

u)

(c)

Control LPA

008

006

004

002

000

3120573HSD

mRN

A (a

u)

(d)









015

010

005

000

Control LPA

A

B

BAX

mRN

A (a

u)

(a)

0010

0008

0006

0004

0002

0000

Control LPA

A

B

IGF2

Rm

RNA

(au

)

(b)

06

04

02

00

Control LPA

A

B

BCL2

mRN

A (a

u)

times10minus4

(c)

015

010

005

000

Control LPA

OCT

4m

RNA

(au

)

(d)



Disclosure






Acknowledgments



References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Day 5 Day 8

0000

0001

0002

003

004

005

A

BATX

mRN

A (a

u)

(a)

Day 5 Day 80000

0001

0002

0003

A

B

cPLA

2 m

RNA

(au

)

0045

0040

0035

0030

0025

(b)




3 Results








Control 304 101 (33) 92 (91)LPA 329 110 (33) 103 (93)





4 Discussion



(a) (b) (c)

(d) (e) (f)



Day 5 Day 8

0

5

10

15

LPA

(pm

olv

iabl

e em

bryo

)

A

B



2






006

004

002

000

Day 5 Day 8

A

B

LPAR

1m

RNA

(au

)

(a)

010

008

006

004

002

000

Day 5 Day 8

A

B

LPAR

2m

RNA

(au

)

(b)

0003

0002

0001

0000

Day 5 Day 8

A

B

LPAR

3m

RNA

(au

)

(c)

025

020

015

010

005

000

Day 5 Day 8

BLPAR

4m

RNA

(au

)

A

(d)





2120572and PGE

2)







2120572and PGE




(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j)







Control LPA00

05

10

15PT

GS2

mRN

A (a

u)

(a)

Control LPA

006

004

002

000

PGES

mRN

A (a

u)

(b)

Control LPA

03

02

01

00

PGFS

mRN

A (a

u)

(c)

Control LPA

008

006

004

002

000

3120573HSD

mRN

A (a

u)

(d)









015

010

005

000

Control LPA

A

B

BAX

mRN

A (a

u)

(a)

0010

0008

0006

0004

0002

0000

Control LPA

A

B

IGF2

Rm

RNA

(au

)

(b)

06

04

02

00

Control LPA

A

B

BCL2

mRN

A (a

u)

times10minus4

(c)

015

010

005

000

Control LPA

OCT

4m

RNA

(au

)

(d)



Disclosure






Acknowledgments



References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e) (f)



Day 5 Day 8

0

5

10

15

LPA

(pm

olv

iabl

e em

bryo

)

A

B



2






006

004

002

000

Day 5 Day 8

A

B

LPAR

1m

RNA

(au

)

(a)

010

008

006

004

002

000

Day 5 Day 8

A

B

LPAR

2m

RNA

(au

)

(b)

0003

0002

0001

0000

Day 5 Day 8

A

B

LPAR

3m

RNA

(au

)

(c)

025

020

015

010

005

000

Day 5 Day 8

BLPAR

4m

RNA

(au

)

A

(d)





2120572and PGE

2)







2120572and PGE




(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j)







Control LPA00

05

10

15PT

GS2

mRN

A (a

u)

(a)

Control LPA

006

004

002

000

PGES

mRN

A (a

u)

(b)

Control LPA

03

02

01

00

PGFS

mRN

A (a

u)

(c)

Control LPA

008

006

004

002

000

3120573HSD

mRN

A (a

u)

(d)









015

010

005

000

Control LPA

A

B

BAX

mRN

A (a

u)

(a)

0010

0008

0006

0004

0002

0000

Control LPA

A

B

IGF2

Rm

RNA

(au

)

(b)

06

04

02

00

Control LPA

A

B

BCL2

mRN

A (a

u)

times10minus4

(c)

015

010

005

000

Control LPA

OCT

4m

RNA

(au

)

(d)



Disclosure






Acknowledgments



References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















006

004

002

000

Day 5 Day 8

A

B

LPAR

1m

RNA

(au

)

(a)

010

008

006

004

002

000

Day 5 Day 8

A

B

LPAR

2m

RNA

(au

)

(b)

0003

0002

0001

0000

Day 5 Day 8

A

B

LPAR

3m

RNA

(au

)

(c)

025

020

015

010

005

000

Day 5 Day 8

BLPAR

4m

RNA

(au

)

A

(d)





2120572and PGE

2)







2120572and PGE




(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j)







Control LPA00

05

10

15PT

GS2

mRN

A (a

u)

(a)

Control LPA

006

004

002

000

PGES

mRN

A (a

u)

(b)

Control LPA

03

02

01

00

PGFS

mRN

A (a

u)

(c)

Control LPA

008

006

004

002

000

3120573HSD

mRN

A (a

u)

(d)









015

010

005

000

Control LPA

A

B

BAX

mRN

A (a

u)

(a)

0010

0008

0006

0004

0002

0000

Control LPA

A

B

IGF2

Rm

RNA

(au

)

(b)

06

04

02

00

Control LPA

A

B

BCL2

mRN

A (a

u)

times10minus4

(c)

015

010

005

000

Control LPA

OCT

4m

RNA

(au

)

(d)



Disclosure






Acknowledgments



References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j)







Control LPA00

05

10

15PT

GS2

mRN

A (a

u)

(a)

Control LPA

006

004

002

000

PGES

mRN

A (a

u)

(b)

Control LPA

03

02

01

00

PGFS

mRN

A (a

u)

(c)

Control LPA

008

006

004

002

000

3120573HSD

mRN

A (a

u)

(d)









015

010

005

000

Control LPA

A

B

BAX

mRN

A (a

u)

(a)

0010

0008

0006

0004

0002

0000

Control LPA

A

B

IGF2

Rm

RNA

(au

)

(b)

06

04

02

00

Control LPA

A

B

BCL2

mRN

A (a

u)

times10minus4

(c)

015

010

005

000

Control LPA

OCT

4m

RNA

(au

)

(d)



Disclosure






Acknowledgments



References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control LPA00

05

10

15PT

GS2

mRN

A (a

u)

(a)

Control LPA

006

004

002

000

PGES

mRN

A (a

u)

(b)

Control LPA

03

02

01

00

PGFS

mRN

A (a

u)

(c)

Control LPA

008

006

004

002

000

3120573HSD

mRN

A (a

u)

(d)









015

010

005

000

Control LPA

A

B

BAX

mRN

A (a

u)

(a)

0010

0008

0006

0004

0002

0000

Control LPA

A

B

IGF2

Rm

RNA

(au

)

(b)

06

04

02

00

Control LPA

A

B

BCL2

mRN

A (a

u)

times10minus4

(c)

015

010

005

000

Control LPA

OCT

4m

RNA

(au

)

(d)



Disclosure






Acknowledgments



References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















015

010

005

000

Control LPA

A

B

BAX

mRN

A (a

u)

(a)

0010

0008

0006

0004

0002

0000

Control LPA

A

B

IGF2

Rm

RNA

(au

)

(b)

06

04

02

00

Control LPA

A

B

BCL2

mRN

A (a

u)

times10minus4

(c)

015

010

005

000

Control LPA

OCT

4m

RNA

(au

)

(d)



Disclosure






Acknowledgments



References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Lysophosphatidic Acid Signaling in Late ...downloads.hindawi.com/journals/mi/2014/678968.pdf · Research Article Lysophosphatidic Acid Signaling in Late Cleavage