Supplementary Materials and Methods
Generation of LPA3-deficient Mice. Isolation and characterization of LPA3 clones from
a 129/SvJ mouse genomic DNA library were described previously 1. To generate a
replacement vector, a 4.9 kb XhoI/NotI subclone of the LPA3 gene containing exon 2 in
pBluescript was used as the starting material. A 1286 bp fragment between BamHI and
HindIII sites was replaced with a ~1600 bp neomycin resistance cassette (excised with
BamHI and HindIII from the pNEOr vector). Thereby the intron 1 acceptor site, start
codon, and coding region for the first 26 amino acids of the LPA3 protein were deleted.
Twenty g of the linearized replacement vector was electroporated into R1 ES cells, and
colonies were screened by PCR. One homologous recombinant clone was identified by
screening 956 G418-resistant colonies. The correct integration was confirmed by
Southern blot analysis. This clone was injected into C57BL/6 blastocysts, producing
three chimeric males of 10-15% agouti color. These chimeric males were bred with
C57BL/6 females, resulting in germline transmission from one chimeric mouse. All
methods used in this study were approved by the Animal Subjects Program of the
University of California, San Diego, the Scripps Research Institute, and the University of
Tokyo, and conform to National Institutes of Health guidelines and public law.
Genotyping of LPA3-deficient Mice. Genotyping for LPA3 alleles was done by
Southern blot analysis or PCR. For Southern blot analysis, EcoRI/DraI-digested
genomic DNA was probed with two radiolabeled probes, 501T/5KO1 and 501O/501N.
Southern blots were performed as previously described 2. Probe 501T/5KO1 (382 bp)
was obtained by PCR amplification from genomic DNA using primers 501T (5´AGCTTGTGATTGCCAAACGGATGTGA-3´) and 5KO1 (5´AACGTCTCCTCCAACTGCACT-3´). Probe 501O/501N (1222 bp) was located
entirely within the deleted region and was obtained from a genomic clone by PCR
amplification using primers 501O (5´-CCAAAGGCAGACAGCTCAA-3´) and 501N (5´1
GAAAAAGTCCATGCGCTTGT-3´), and then 180 bp was removed by digestion of the
PCR product with BamHI. For PCR genotyping, genomic DNA was used as a template
in PCR reactions (36 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 60 s) using
three primers: A3e1b (5´-TGACAAGCGCATGGACTTTTTC-3´), A3e1c (5´GAAGAAATCCGCAGCAGCTAA-3´) and Neo1b (5´AGCGCCTCCCCTACCCGGTAGAAT-3´). Expected product sizes for WT and
targeted alleles were 216 bp and 170 bp, respectively.
Monoclonal Antibodies against Rat LPA3 and Western Blotting. The carboxylterminal 59 amino acids of rat LPA3 (296-354) was fused with glutathione S-transferase
and expressed in E.coli. The recombinant protein was injected into the hind foot-pads of
WKY/Izm rats using Freund's complete adjuvant. The enlarged medial iliac lymph nodes
from the rats were used for cell fusion with mouse myeloma cells, PAI. The antibodysecreting hybridoma cells were selected by screening with ELISA, immunofluorescence
and Western blotting. Several clones were established. In this study, monoclonal antibody
from clone 7 was used. The antibody was 1:500 diluted in skim milk for Western blot.
Proteins were extracted from WT and LPA3-deficient testis membrane fraction, and
separated by electrophoresis using gradient gel (15-25% acrylamide). Bands were
detected by enhanced chemiluminescence (ECL) for 10 min.
Plasmids and Intracellular Calcium Mobilization Assay. Mouse cDNA for full-length
LPA3, type 1 (presumably amino acid 87-354) and type 2 truncated LPA3 (presumably
amino acid 288-354) were amplified by RT-PCR using a mouse oviduct cDNA library
and introduced into the expression vector pCAGGS (kindly provided by Dr. Junichi
Miyazaki at Osaka University). Transient transfection into HeLa cells was performed
using LipofectAMINETM 2000 (Invitrogen) according to the manufacture’s protocol.
Twenty-four hours after the transfection, the cells were assayed for intracellular calcium
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mobilization as follows. After incubation in calcium Ringer solution (150 mM NaCl, 4
mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 5 mM HEPES) containing 5
µM of Fura-2 AM (Molecular Probes) and 0.1% BSA at 37 °C for 30 min, cells were
detached by trypsin-EDTA treatment, washed and resuspended in calcium Ringer
solution. After stimulation with various concentrations of LPA, cytosolic calcium was
measured by monitoring fluorescence intensity at an emission wavelength of 500 nm, and
excitation wavelengths of 340 and 380 nm using a CAF-110 (JASCO, Tokyo, Japan).
Superovulation and Fertilization Rate. Since there was no difference between Het and
WT females in phenotypes (Table S2, Supplementary Fig. S8b, S8c, and data not shown),
Het females were used as controls in these studies. Female littermates (6-7 weeks old,
mixed background) were ip injected with 5 IU (0.1 ml) pregnant mares’ serum
gonadotropin (PMSG, Sigma, G-4877) at 2 pm on day 1 and 5 IU (0.1 ml) human
chorionic gonadotropin (hCG, Sigma, C-1063) at 1 pm on day 3. After hCG injection,
each female was placed with a stud male overnight to determine fertilization rate. In the
morning of day 4, the females were anesthetized with Halothane inhalation followed by
cervical dislocation. Both oviducts were dissected and put in FHM (Specialty Media).
Eggs/zygotes were released from the enlarged ampulla and transferred to a Petri dish
containing hyaluronidase solution (Specialty Media) for a few minutes. After the
cumulus cells were shed, individual eggs/zygotes were counted. For the fertilization
study, zygotes were transferred to KSOM (Specialty Media) microdrop culture at 37oC,
5% CO2. Cell division was monitored the following day.
Ovum Transport and Blastocyst Development. E3.5 uteri from WT/Het controls and
LPA3-deficient female littermates mated with WT stud males were flushed with saline.
By counting the number and examining the morphology of blastocysts flushed, we could
retrospectively find out whether or not there was any defect with ovulation, ovum
transportation and blastocyst development.
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Decidualization. Het and LPA3-deficient female littermates (2-3 months old) were
mated with vasectomized males to get pseudopregnancy. The day a plug found was
defined as E0.5. Artificial decidualization was induced by intraluminal infusion of
sesame oil (~15 l) in one uterine horn at E3.5 of the pseudopregnant females. Uterine
horns were dissected at E7.5 and weights of the infused and non-infused horns were
recorded. The fold increases in uterine weights were used as an index of decidualization
as described 3.
Blastocyst Transfer. WT or LPA3-deficient fertilized eggs were cultured and injected
into the uteri of pseudopregnant WT or LPA3-deficient females at E2.5. Subsequently,
uteri at E4.5, E5.5, E10.5, and E18.5 were isolated. The numbers of implantation sites or
embryos were counted. Embryo weight was measured. Evans blue dye (Sigma, 200 l,
1% in 1xPBS) was i.v. injected and after 5 minutes uteri were observed to discern the
implantation sites at E4.5 and E5.5.
Measurement of cPLA2 enzyme activity. WT or LPA3-deficient uteri were
homogenized in buffer A (10 mM Tris-HCl , 1 mM EDTA, 250 mM sucrose, 1 mM
PMSF, 2.5 g/ml leupeptin, 5mM dithiothreitol). Phosphatidylethanolamine (PE from
bovine brain, mainly 18:0-20:4, 20 M, Avanti Polar Lipid) was used as a substrate. The
homogenates (50 g each) were incubated with PE in buffer A containing 4 mM CaCl2,
both in the presence or absence of EDTA (10 mM), for 2 hours at 37oC. Arachidonic
acids (20:4) liberated by phospholipase A2 reaction were extracted using the Bligh &
Dyer method and quantified using electro spray ionization mass spectrometry (ESI-MS).
The cPLA2 activity was calculated by subtracting the activity in the presence of EDTA
from that in the absence of EDTA.
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1.
Contos, J. J. & Chun, J. The mouse lp(A3)/Edg7 lysophosphatidic acid receptor
gene: genomic structure, chromosomal localization, and expression pattern. Gene 267,
243-53 (2001).
2.
Contos, J. J. & Chun, J. Complete cDNA sequence, genomic structure, and
chromosomal localization of the LPA receptor gene, lpA1/vzg-1/Gpcr26. Genomics 51,
364-78 (1998).
3.
Song, H. et al. Cytosolic phospholipase A2alpha is crucial [correction of A2alpha
deficiency is crucial] for 'on-time' embryo implantation that directs subsequent
development. Development 129, 2879-89 (2002).
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