Accepted Manuscript Title: The contribution of leukemia inhibitory factor (LIF) for embryo implantation differs among strains of mice Author: Ryosuke Kobayashi Jumpei Terakawa Yasumasa Kato Shafiqullah Azimi Naoko Inoue Yasushige Ohmori Eiichi Hondo PII: DOI: Reference:

S0171-2985(14)00059-X http://dx.doi.org/doi:10.1016/j.imbio.2014.03.011 IMBIO 51133

To appear in: Received date: Revised date: Accepted date:

15-2-2014 12-3-2014 12-3-2014

Please cite this article as: Kobayashi, R., Terakawa, J., KATO, Y., Azimi, S., Inoue, N., Ohmori, Y., Hondo, E.,The contribution of leukemia inhibitory factor (LIF) for embryo implantation differs among strains of mice, Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.03.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The contribution of leukemia inhibitory factor (LIF) for embryo

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implantation differs among strains of mice

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Ryosuke KOBAYASHI1, Jumpei TERAKAWA1, Yasumasa KATO2, Shafiqullah AZIMI1, Naoko

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INOUE1, Yasushige OHMORI1, Eiichi HONDO1*

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Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

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Laboratory of Animal Morphology, Division of Biofunctional Development, Graduate School of

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Koriyama, Japan

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*corresponding author

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Department of Oral Function and Molecular Biology, Ohu University School of Dentistry, 963-8611,

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Correspondence to Eiichi HONDO

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Laboratory of Animal Morphology and Function, Division of Biofunctional

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Development, Graduate School of Bioagricultural Sciences, Nagoya University,

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Nagoya 464-8601, Japan Phone: +81-52-789-4185 Fax: +81-52-789-4185 Email: [email protected]

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Keywards: CT-1; Implantation; LIF; Strain differences; Uterus

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Abbreviations: CT-1, cardiotrophin 1; IL6, interleukin 6; LE, the luminal epithelium; LIF, leukemia

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inhibitory factor; STAT3, the signal transducer and activator of transcription 3.

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Abstract Despite of the claim that maternal leukemia inhibitory factor (LIF)—a member of

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interleukin 6 (IL6) family of cytokines—plays indispensable roles for murine embryo implantation,

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these roles remain undefined in humans because the potency of LIF on implantation appears to vary

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among individuals. Here, we showed that the contribution of LIF for murine implantation was

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dependent on the strains of mice (ICR, C57BL/6J (B6), ddY, BALB/c, DBA/2Cr and MF1 strains).

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Inhibition of LIF during the implantation period caused severe disruption of embryo implantation in

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B6 and MF1 strains. Implantation was partly disrupted in other strains, but some embryos were

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implanted successfully. We speculated that other IL6 family members compensate for LIF actions on

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implantation in ICR, ddY, BALB/c, and DBA/2Cr strains. Indeed, the expression level of Ctf1 was

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upregulated by blockage of LIF function. CT-1 (encoded by Ctf1) treatment induced successful

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implantation without LIF in delayed implantation mice (ICR and B6) via phosphorylation of the

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signal transducer and activator of transcription 3 (STAT3) in the uterine luminal epithelium.

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Simultaneous inhibition of LIF and CT-1 did not block implantation completely in ICR mice,

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indicating that embryo implantation in this strain was robustly protected by LIF, CT-1 and other

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potential STAT3 activators. The present study might provide an explanation for the individual

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variation in the potency of LIF for embryo implantation in humans.

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Introduction Embryo implantation is an essential event in mammalian early pregnancy. The precise

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feto-maternal responses to the interaction between active blastocysts and the receptive uterus are

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indispensable for successful implantation (Dey et al., 2004). Implantation occurs during a limited time

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known as the “implantation window”, which is regulated by the ovarian steroid hormones estrogen

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(E2) and progesterone (P4) (Wang et al., 2006; Cha et al., 2012).

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During early pregnancy, leukemia inhibitory factor (LIF) —a member of the interleukin 6

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(IL6) family of cytokines—has important functions in the endometrium, which is controlled by E2

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(Kimber et al., 2005). E2 triggers the secretion of LIF from the glandular epithelium on the fourth day

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of pregnancy (D4; the first day of pregnancy, D1, is defined as the day when a vaginal plug is first

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observed) (Yang et al., 1995, Song et al., 2000). Secreted LIF activates the JAK/STAT3 pathway in

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the luminal epithelium (LE), concurrent with the phosphorylation of STAT3 (Cheng et al., 2001).

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LIF-deficient females of C57BL/6J (B6) and MF1 mouse strains are infertile owing to implantation

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failure, although Lif-null embryos can develop normally up to the blastocyst stage (Stewart et al.,

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1992). Injection with LIF into Lif-deficient females on D4 rescues their infertile phenotype (Chen et

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al., 2000).

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IL6 family cytokines, including LIF, IL6, IL11, cardiotrophin-1 (CT-1), ciliary neurotropic

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factor (CNTF), and oncostatin M (OSM), share a common receptor, gp130, and are involved in

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several biological functions through JAK/STAT, MAPK, and/or PI3K pathways (Heinrich et al., 2003;

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Garbers et al., 2012). IL6 and IL11 induce homodimerization of gp130. LIF, CT-1, and CNTF

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promote heterodimerization of gp130 and the LIF receptor (LIFR) (Heinrich et al., 2003; Garbers et

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al., 2012). Murine OSM binds only to a heterodimer of gp130 and the OSM receptor (OSMR),

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whereas human OSM can interact with gp130/OSMR or gp130/LIFR heterodimers (Lindberg et al.,

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1998). In addition to LIF, CT-1 and/or CNTF can maintain pluripotency of embryonic stem (ES) cells

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(Niwa et al., 1998; Pennica et al., 1995b; Conover et al., 1993). LIF, CT-1, OSM, and IL11 cause

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cardiac myocyte hypertrophy in vitro via a receptor complex containing gp130 (Pennica et al., 1995b).

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In particular, LIF appears functionally similar to CT-1 (Pennica et al., 1995b). In white adipocytes,

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LIF and CT-1, but not CNTF, attenuate subsequent signaling of each cytokine, which correlates with

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the degradation of LIFR (White et al., 2011). Taken together, these data indicate that LIF and CT-1

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likely act on a common signaling pathway and crosstalk with each other. In humans, LIF mRNA is expressed in the endometrial glands during the luteal phase of

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the menstrual cycle (Charnock-Jones et al., 1994; Arici et al., 1995). The concentration of LIF in

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uterine flushing fluid obtained during the luteal phase has been found to be lower in infertile patients

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than in fertile patients (Laird et al., 1997). Heterozygous mutations in the LIF gene might be related to

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implantation failure in humans (Giess et al., 1999). However, one report showed no statistical

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difference in the LIF levels of uterine flushing fluids obtained from patients who had experienced

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recurrent miscarriages compared with multiparous women (Inagaki et al., 2003). These data indicate

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that the degree of dependence of human implantation on LIF varies between individuals. Our previous

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study suggested that the influence of LIF on embryo implantation differed between ICR and B6

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strains of mice (Terakawa et al., 2011). For the present study, we investigated strain differences in the

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response of murine embryo implantation to LIF inhibition and potential compensation by CT-1 for the

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disruption of implantation when LIF was blocked.

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Materials and Methods

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Animals

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B6 mice (Kyudo, Saga, Japan), ICR, ddY, BALB/c and DBA/2Cr female mice (Japan SLC,

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Shizuoka, Japan), and MF1 mice (maintained at Kanagawa Dental University, Japan) aged over

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6weeks were used in the experiments. These MF1 mice lacked Sparc genes. They had been

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maintained by the mating of homozygous males and females. The litter size of Sparc deficient mice

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was almost the same as B6 and BALB/c wildtype mice. The first day of pregnancy (D1) was

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determined as the morning when a vaginal plug was observed in the female that had been mated with

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males on the previous evening. For preparation of antibodies against LIF or CT-1, Japanese white

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rabbits (Japan SLC) were immunized. All animals were housed at 22 ± 3°C with controlled light-dark

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cycles (14 h light followed by 10 h dark) and were fed ad libitum. All experiments were performed

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according to the guidelines of the Committee for Animal Welfare at Nagoya University (approval

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number: 2012031402).

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Purification of recombinant proteins Recombinant LIF, CT-1, and CNTF proteins (rLIF, rCT-1, and rCNTF, respectively) were

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prepared as described in a previous report (Terakawa et al., 2011). The expression vectors were

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constructed using pET-46 (Merck, Darmstadt, Germany) with the corresponding proteins (LIF, 179

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amino

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PLPITPVNATCAIRHPCHGNLMNQIKNQLAQLNGSANALFISYYTAQGEPFPNNVEKLCAPNM

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TDFPSFHGNGTEKTKLVELYRMVAYLSASLTNITRDQKVLNPTAVSLQVKLNATIDVMRGLLS

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NVLCRLCNKYRVGHVDVPPVPDHSDKEAFQRKKLGCQLLGTYKQVISVLAQAF; CT-1, 203

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amino

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MSQREGSLEDHQTDSSISFLPHLEAKIRQTHNLARLLTKYAEQLLEEYVQQQGEPFGLPGFSP

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PRLPLAGLSGPAPSHAGLPVSERLRQDAAALSVLPALLDAVRRRQAELNPRAPRLLRSLEDA

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ARQVRALGAAVETVLAALGAAARGPGPEPVTVATLFTANSTAGIFSAKVLGFHVCGLYGEW

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VSRTEGDLGQLVPGGVA;

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MAFAEQSPLTLHRRDLCSRSIWLARKIRSDLTALMESYVKHQGLNKNISLDSVDGVPVASTD

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RWSEMTEAERLQENLQAYRTFQGMLTKLLEDQRVHFTPTEGDFHQAIHTLTLQVSAFAYQL

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EELMALLEQKVPEKEADGMPVTIGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRVISSHHM

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GISAHESHYGAKQM). Expression vectors were transformed into E. coli BL21 (DE3) cells, and the

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his-tagged recombinant proteins were induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside

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(Wako, Osaka, Japan) for 6 h at room temperature (RT). The proteins were purified using HisTrap FF

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(GE Healthcare, Little Chalfont, UK), followed by reversed-phase chromatography (trifluoroacctic

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acid/acetonitrile) with the Proteonavi apparatus (Shiseido, Tokyo, Japan).

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residues:

residues:

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amino

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residues:

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CNTF,

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Preparation of antibodies Normal rabbit immunoglobulin G (IgG) purified with HiTrap rProtein A FF (GE

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Healthcare) from rabbit serum collected before immunization was used as the control. Antibodies

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against LIF or CT-1 were prepared as described in a previous report (Terakawa et al., 2011). The reactivity of purified anti-CT-1 antibody against rCT-1 was determined by

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enzyme-linked immunosorbent assay (ELISA). Interaction between anti-CT-1 antibody and rCT-1 was

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detectable above 1.0 × 10−6 µg/mL. ELISA was performed using the following materials: TaKaRa

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Peptide Coating Kit (Takara, Kyoto, Japan), polyclonal goat anti-rabbit antibody conjugated with

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horseradish peroxidase (HRP; dilution 1:75,000; Bethyl, Montgomery, TX, USA), and

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3,3',5,5'-tetramethylbenzidine (TMB) Substrate (Bethyl). The specificity of purified anti-CT-1

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antibody against CT-1 protein in mice was confirmed by Western blot analysis. Crude protein (60 µg)

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from the uterine tissue at D4 was separated by sodium dodecyl sulfate polyacrylamide gel

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electrophoresis (SDS-PAGE) on a 12% gel. Proteins were transferred onto a polyvinylidene difluoride

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membrane (25 min) using Trans-Blot SD (Bio-Rad, Hercules, CA, USA). Blocking was performed in

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5% skim milk/Tris-buffered saline with Tween 20 (TBS-T) for 1 h at RT. Membranes were probed

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overnight at 4°C with anti-CT-1 antibody (0.40 µg/mL, diluted 1:10,000 in 5% skim milk/TBS-T).

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Subsequently, the membrane was treated with polyclonal swine anti-rabbit antibody conjugated with

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HRP (Dako, Glostrup, Denmark) diluted 1:2,000 in TBS-T. ECL Plus reagents (GE Healthcare) and

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LumiCube (Liponics, Tokyo, Japan) were used for signal detection.

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Blockage of embryo implantation

Pregnant mice of each strain were intraperitoneally injected with anti-LIF antibody (7.5

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µg/g body weight (BW) in 50 µL of 10 mM phosphate-buffered saline (PBS)), twice the amount of

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anti-LIF antibody (15 µg/g BW in 100 µL of 10 mM PBS), anti-CT-1 antibody (15 µg/g BW in 100

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µL of 10 mM PBS), or anti-LIF and anti-CT-1 antibodies (7.5 µg/g BW of each antibody in 100 µL of

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10 mM PBS) at 1200 h and 2200 h on D3 and 1000 h on D4. The same amount of normal rabbit IgG

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was injected as the control. Mice were sacrificed on D7, and the number of implantation sites was

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counted. The uterus was flushed with 10 mM PBS to collect blastocysts when no implantation site

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was recognized.

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Quantitative real-time polymerase chain reaction (PCR) Mice treated with anti-LIF antibody (or normal rabbit IgG) of each strain were sacrificed

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on the evening at D4, and the uteri were collected, incised, and scratched with a spoon to collect the

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endometrium. Samples were stored at −80°C until use. To analyze the expression of Ctf1 during early

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pregnancy, the endometrium was collected with same method described above from ICR mice at 1200

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h on D3, 1000 h on D4, 1800 h on D4, and 1200 h on D5. Total RNA was extracted using the RNeasy

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Mini Kit (Qiagen, Hilden, Germany) and purified with TURBO DNA-free Kit (Life Technologies,

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Carlsbad, CA, USA). cDNA was synthesized from 500 ng total RNA with the ReverTra Ace qPCR RT

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Kit (Toyobo, Osaka, Japan). Real-time PCR was performed using a StepOnePlus system (Life

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Technologies) with SYBR Green (Life Technologies) or LightCycler Nano system (Roche Applied

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Science, Penzberg, Germany) with FastStart Essential DNA Green Master (Roche Applied Science).

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Primers used in this study were as follows: Lif, 5′-GCCACGGCAACCTCATG-3′ and

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5′-ATTGGCGCTGCCATTGA-3′;

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5′-CGGGCAAGGTTGTGTGTCT-3′;

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5′-AGAGCAGTCAGGTCTGAACGAAT-3′; Osm, 5′-CCAGAGTACCAGGACCCAGTATG-3′ and

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5′-TGAGGGTCAAACTGAGCAGTGT-3′;

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5′-TTGGGAGTGGTATCCTCTGTGA-3′,

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5′-CGGCGCAGCCATTGTAC-3′,

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5′-CGACATCACAGAGCAGGC-3′ and 5′-CACCGAGGCAACAGTTGG-3′. Expression levels were

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normalized using the internal control, Rplp0. The average expression level of each gene in mice

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injected with normal rabbit IgG was given the value of 1.

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Ctf1,

5′-CAATCTCATTCCTACCCCATTTG-3′

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5′-GGGACCTCTGTAGCCGCTCTA-3′

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5′-CCACGGCCTTCCCTACTTC-3′

Il6,

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5′-TGCTGACAAGGCTTCGAGTAGA-3′

Il11,

(as

Rplp0

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internal

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control),

Induction of embryo implantation

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Pregnant mice of B6 or ICR strains were ovariectomized at 1800h on D3 under avertin

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anesthesia, and medroxyprogesterone acetate (1.0 mg/head; Pfizer Inc, New York, NY, USA) was

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injected subcutaneously and simultaneously to maintain delayed implantation (DI). Injection with

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recombinant LIF (25 µg/head), or CT-1 (25, 100, or 200 µg/head), or CNTF (200 µg/head) was

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performed at 1200 h on D6. The number of implantation sites was counted at 1200 h on D9.

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Immunohistochemistry For immunohistochemistry, DI mice of ICR strains treated with LIF, CT-1, or CNTF were

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perfused with paraformaldehyde (4% in 0.1 M phosphate buffer) at 1800h on D6. The specimens were

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embedded in paraffin, and 4-µm sections were prepared. Uterine sections were deparaffinized,

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autoclaved (121°C, 5 min), and treated with 0.3% H2O2/methanol for 20 min at RT. After blocking

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with blocking buffer (1% bovine serum albumin, 1.5% normal goat serum/PBS with Tween 20),

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monoclonal anti-pSTAT3 (1:50, diluted by blocking buffer; Cell Signaling Technology, Danvers, MA,

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USA) was applied (overnight at 4°C). The sections were incubated with biotinylated goat anti-rabbit

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antibody (1:800; Dako) for 1 h at RT, and avidin-biotin complex (Vector Labs, Burlingame, CA,

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USA) for 30 min at RT. The signal was visualized with diaminobenzidine and counterstained with

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hematoxylin.

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Reverse transcription-polymerase chain reaction (RT-PCR)

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Total RNA was extracted from the murine brain and pregnant uteri (on D3, D4, and D5) of

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ICR mice by RNeasy Mini Kit (Qiagen). cDNA was synthesized from total RNA (1 µg of each

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sample) using oligo-dT primer and ReverTra Ace reverse transcriptase (Toyobo). PCR was performed

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using the Expand High FidelityPLUS PCR System (Roche Applied Science). The thermal profile was as

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follows: an initial denaturation step (94°C, 5 min); 35 cycles of denaturation (94°C, 30 s), annealing

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(63°C, 30 s), extension (72°C, 1 min); and a final elongation at 72°C for 10 min. The following

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primers

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5′-GCTGCCAAGCTCCCCAGGGT-3′; Rplp0, 5′-GTGGGAGCAGACAACGTGGGCTCC-3′ and

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5′-GCAAATGCAGATGGATCAGCCAGGAAGGCCTTGACC-3′.

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electrophoresed on a 1.5% agarose gel.

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PCR:

Cntfr,

5′-CAGAAACACAGTCCACAGGA-3′

PCR

products

and

were

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Statistical analysis

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Two-tailed Student’s t-test was performed for statistical analysis. A value of p < 0.05 was considered statistically significant.

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The number of implantation sites was decreased by injection with anti-LIF antibody (7.5

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µg/g BW in 50 µL of 10 mM PBS) in ddY, BALB/c, DBA/2Cr, and MF1 mice (Fig. 1, 2 and Table 1).

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The reduction of implantation sites was observed in all strains and ranged from severe (no

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implantation) to moderate. In ddY, BALB/c and MF1 mice, the number of implantation sites was

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significantly reduced by injection with anti-LIF antibody compared to the control (Fig. 2A, B, D; p =

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1.59 × 10-6, p = 0.039, p = 6.53 × 10-5, respectively). Although DBA/2Cr mice tended towards a

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reduction in the number of implantation sites by anti-LIF treatment, no statistical difference from the

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control was recognized (Fig. 2C, p = 0.083). When no implantation site was detected macroscopically,

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the blastocysts were recovered by flushing the uterus and found to be alive in all mice except for the

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MF1 strain. In MF1 mice, we could not recover living blastocysts from most of uteri in which there

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was no implantation site; blastocysts were recovered from only one of seven mice without

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implantation sites. The implantation rate, which was defined as the ratio of the number of

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implantation sites injected with anti-LIF antibody to the number of implantation sites injected with

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normal rabbit IgG, was varied among mouse strains (ddY; 15.0 %, BALB/c; 44.7 %, DBA/2Cr;

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57.1 %, MF1; 11.3 %). Normal rabbit IgG did not block implantation in any strains (Fig. 1I, J, K, L

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and Fig. 2).

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In order to evaluate the compensation of IL6 family cytokines for LIF action, the gene

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expression of IL6 family cytokines (Lif, Ctf1 (coding CT-1), Cntf, Il6, Il11 and Osm) in the

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endometrium were examined in B6, ICR, ddY, BALB/c, DBA/2Cr, and MF1 mice that were injected

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with normal rabbit IgG (control group) or anti-LIF antibody (experimental group) (Fig. 3). Osm

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expression was decreased in B6 mice injected with anti-LIF antibody compared to control (p = 0.027).

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The expression levels of Ctf1 mRNA were increased in the experimental groups of B6, ICR, and MF1

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mice compared to controls (p values were 0.016, 0.024, and 0.015, respectively), and Cntf mRNA was

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increased in the experimental group of ICR mice compared to control (p = 0.0055). In ddY mice, a

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decreased level of Il6 expression was observed with anti-LIF antibody compared to control (p =

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0.029). Intraperitoneal administration of recombinant CT-1 (rCT-1) in DI mice induced embryo

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implantation (Table 2). Implantation was certainly induced by 200 µg of rCT-1 in ICR and B6 mice

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(Fig. 4A, B). Implantation sites were not found occasionally in DI mice injected with 25 or 100 µg of

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rCT-1, and the average numbers of implantation sites were less than those of DI mice injected with

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200 µg (Fig. 4C, Table 2). Administration of rCNTF in DI mice did not promote implantation (200

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µg/head, ICR mice (n = 3), Fig. 4D). In DI mice injected with LIF, phosphorylated STAT3 (pSTAT3)

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was uniformly observed in the LE (Fig. 5A). The localization of pSTAT3 in the nuclei of the LE was

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very sparse in the mice treated with the vehicle (Fig. 5B). CT-1 treatment also induced the

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phosphorylation of STAT3 in DI mice. The signal strength of pSTAT3 was the same in CT-1-treated

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mice and LIF-treated mice (Fig. 5C). CNTF treatment did not induce pSTAT3 in the nucleus of the LE

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(Fig. 5D). The expression of specific receptor for CNTF, Cntfr, was not observed in the endometrium

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of ICR strains (Fig. 6).

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The specificity of anti-CT-1 antibody produced in the present study was confirmed by

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Western blot analysis. The specific band of CT-1 protein was observed at 70 kDa (Fig. 7A).

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Simultaneous injection with anti-LIF and anti-CT-1 antibodies (7.5 µg/g BW of each antibody) or

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injection with the anti-LIF antibody only (15 µg/g BW) in ICR mice significantly decreased the

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number of embryo implantation sites (Fig. 7B). Implantation was not disrupted in mice injected with

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anti-CT-1 antibody alone or normal rabbit IgG.

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Discussion

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The importance of LIF for pregnancy has been reported in various animals, including

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humans, rhesus monkeys, mice, cattle, pigs, and sheep (Lass et al., 2001; Sengupta et al., 2006;

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Oshima et al., 2003; Modric et al., 2000; Vogiagis et al., 1997). The concentration of LIF in uterine

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fluid during the implantation period (between 7 and 9 days after ovulation) was significantly reduced

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in human patients, who had experienced repeated spontaneous and/or recurrent miscarriage during

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early pregnancy, but one report showed no statistical difference in the concentration of LIF between

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infertile and fertile women (Laird et al., 1997; Giess et al., 1999; Inagaki et al., 2003). These

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controversial data suggest that the contribution of LIF for promoting embryo implantation varies

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among human individuals.

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In our previous study, the effect of LIF on embryo implantation in ICR mice differed from

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that in B6 mice (Terakawa et al., 2011). Injection with anti-LIF antibody completely blocked embryo

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implantation in B6 mice, whereas in ICR mice, anti-LIF antibody caused a partial reduction in

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number of implantation sites (Terakawa et al., 2011). The present study showed that the number of

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implantation sites was dramatically decreased in ddY, BALB/c, and MF1 mice after injection with

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anti-LIF antibody (Fig. 2A, B and D). Although implantation was also reduced in DBA/2Cr mice,

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there was no statistical difference in the number of implantation sites compared to controls (Fig. 2C).

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These data suggest that the contribution of LIF to embryo implantation differs greatly among strains

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of mice. MF1 mice showed a distinct response from the other strains. Almost all uteri of this strain

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(seven mice) injected with anti-LIF antibody showed no implantation site at D7. Implantation sites

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were observed in the uterus from only one mouse that received antibody treatment, and the sites were

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very small compared to those in the control mice. A previous paper reported that no implantation sites

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were observed in most MF1 LIF knockout mice, and only one of eight mice showed increased

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vascular permeability at putative implantation sites by blue dye injection (Fouladi-Nashta et al., 2005).

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This observation corresponds to our findings in MF1 mice. Gene mutations can result in various

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phenotypes depending on the strain of mice (e.g., Tgfb1, Sox18, and Trp53) (Bonyadi et al., 1997;

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Hosking et al., 2009; Hu et al., 2007). Trp53-/- female mice are partially infertile, but the phenotype

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differs among strains (Hu et al., 2007). LIF-deficient mice were established exclusively in B6 and

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MF1 mice. Our data, showing that implantation was inhibited by anti-LIF antibody in B6 and MF1

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mice, were very similar to those of the corresponding knockouts. On the other hand, other strains used

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in this study were partly and/or completely fertile. This might be a cutting edge to explain individual

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differences in the relationship between LIF concentrations and infertility in humans.

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We could not recover blastocysts from the uteri of six of eight MF1 mice at D7 by

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intra-uterine flushing after treatment with anti-LIF antibody. Most normal pregnant MF1 females had

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blastocysts at 4 days after checking the vaginal plug (only one of nine females with a vaginal plug

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experienced abortion) in our study. In addition, live blastocysts could be collected from four MF1

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females at D4, following injection with anti-LIF antibody or normal rabbit IgG. We conclude that

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treatment with anti-LIF antibody caused a fatal development disorder in MF1 embryos, as previously

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reported (Cai et al., 2000).

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We hypothesized that other IL6 family cytokines could compensate for the action of LIF in

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embryo implantation when LIF was inhibited. IL6 family cytokines share a common signal

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transduction pathway through gp130 (Heinrich et al., 2003; Garbers et al., 2012), and have been

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proposed to functionally complement one another (White et al., 2011; Gritman et al., 2006). For

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example, Ctf1-deficient mice express increased levels of Il6 mRNA in cardiomyocytes compared to

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the wild type mice, and the expression of Lif mRNA is affected (Gritman et al., 2006). In this study,

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the expression levels of some Il6 family genes were affected by anti-LIF antibody injection, and

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expression patterns were different among strains. Notably, Ctf1 expression was significantly higher in

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ICR, B6 and MF1 mice, and Cntf mRNA was higher in ICR mice. We supposed that these genes

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might compensate for LIF function; therefore, DI mice were treated with rCT-1 or rCNTF to confirm

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whether these cytokines would induce embryo implantation. CT-1 treatment, but not CNTF treatment,

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resulted in successful implantation in ICR and B6 mice (Fig. 3). Although 25 µg of LIF was sufficient

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for implantation, 200 µg of CT-1 was required to achieve the same phenomenon in ICR mice,

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referring to our previous study (Table 2) (Terakawa et al., 2012). The ability of CT-1 to initiate

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implantation decreased dose-dependently in both ICR and B6 mice. Activation of the JAK/STAT3

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pathway is necessary to initiate implantation by LIF in the LE (Catalano et al., 2005). LIF and CT-1

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activated STAT3 after their administration to DI mice, which resulted in successful implantation;

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however, CNTF treatment did not cause implantation and STAT3 was not activated (Fig. 4). This

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result is supported by the report that phosphorylation of STAT3 was not induced in isolated LE by

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CNTF treatment (Cheng et al., 2000). LIF, CT-1, and CNTF have affinity for the gp130/LIFR

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heterodimeric receptor complex (Heinrich et al., 2003; Garbers et al., 2012). Sharing a common

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receptor system makes it possible for these cytokines to share the same function including the

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maintenance of ES cell pluripotency and the induction of neuronal differentiation (Pennica et al.,

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1995b). CNTF uses a unique non-signaling receptor, the CNTF receptor (CNTFR), in addition to the

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gp130/LIFR complex (Davis et al., 1993). LIF does not require cofactors to bind the gp130/LIFR

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complex (Heinrich et al., 2003; Garbers et al., 2012). One report has shown the existence of an

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additional receptor for CT-1 (Robledo et al., 1997), but further studies are necessary to substantiate

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these data. LIF and CT-1 have been shown to inhibit growth of the M1 mouse myeloid leukemia cell

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line, whereas CNTF did not show this activity in the absence of CNTFR (Pennica et al., 1995b; Davis

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et al., 1993). Cntfr mRNA was not detected in endometrium of ICR mice during D3-D5 (Fig. 6).

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CNTF does not have sufficient activity to phosphorylate STAT3 in the LE leading to implantation,

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because the unique CNTFR is not expressed in the LE during the normal implantation process.

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CT-1 was first identified as a cytokine that supports cardiomyocyte survival and

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hypertrophy (Pennica et al., 1995b). Murine Ctf1 mRNA is expressed in the heart, liver, and skeletal

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muscle (Pennica et al., 1995a). We also confirmed Ctf1 expression in murine endometrium during

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early pregnancy. To determine whether endogenous CT-1 compensated for the function of LIF, after

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inhibition of LIF, anti-LIF and anti-CT-1 antibodies were injected into ICR mice. Firstly, specificity of

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the anti-CT-1 antibody was analyzed by Western blot analysis. The specific band was observed at

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about 70kDa (Fig. 7A). Previous experiments in humans and rats showed that the plasma contains

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immunoreactive CT-1 sized 47-66 kDa, despite the fact that the monomeric molecular weight of CT-1

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is 21.5 kDa (Pemberton et al., 2005). Our Western blot data is consistent with this previous

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observation. There was no further reduction in implantation sites after anti-CT-1 antibody treatment in

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addition to that achieved with anti-LIF antibody (Fig. 7B) (Terakawa et al., 2011). Genetically

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modified Ctf1 null mice are fertile (Holtmann et al., 2005). Consistent with this phenotype, a single

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injection with anti-CT-1 antibody did not block embryo implantation (Fig. 7B). LIF and CNTF are

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primarily involved in maintaining the survival of motor neurons, whereas CT-1 also contributes to

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their development (Holtmann et al., 2005). Although deletion of gp130 or LIFR results in significant

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motor neuron loss and embryonic lethality, no obvious phenotype was identified in IL6 family

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ligand-knockout mice (Holtmann et al., 2005; Fasnacht and Müler, 2008). In Lif/Ctf1/Cntf triple null

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mice, the function and survival of motor neurons were not completely abolished (Holtmann et al.,

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2005). Therefore, it is hypothesized that additional unknown ligands bound to gp130/LIFR complex

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are involved in the function of motor neurons. A similar molecular response might be involved in our

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controversial result that administration of CT-1 leads to successful implantation, but suppression of

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both LIF and CT-1 did not show a synergistic effect. Increased expression of Ctf1 was also detected in B6 and MF1 mice; these two strains

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showed complete or almost complete lack of embryo implantation after treatment with anti-LIF

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antibody. It is still unclear why B6 and MF1 mice experienced more severe reductions in the number

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of implantation sites than other strains. As mentioned above, the function of LIF during implantation

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is to phosphorylate STAT3 in LE, which initiates the important events of pregnancy (Pawar et al.,

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2013). In the present study, CT-1 could also phosphorylate STAT3 and promote embryo implantation.

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This phosphorylation is the most important molecular response involved in embryo implantation, not

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only by IL6 family members, including LIF and CT-1, but also other potential molecules. These

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compensatory mechanisms for embryo implantation might be conserved in mammalian species, and

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they might account for individual differences in miscarriage rates among women.

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Acknowledgements

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This study was partly supported by Grants-in-Aid for Scientific Research (No. 23380172) from Japan

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Society for the Promotion of Science (JSPS). Other parts of funding are from internal budget of

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Nagoya University.

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Figure legends

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Figure 1

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Uteri from pregnant mice at D7 after anti-LIF antibody treatment. In all mouse strains, anti-LIF

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antibody reduced the number of implantation sites. Reduction ranged from moderate (A, B, C, D) to

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severe (E, F, G, H). Normal rabbit IgG was used as the control (I, J, K, and L). Arrowheads indicate

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the implantation site. Scale bar, 5 mm.

489 Figure 2

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Anti-LIF antibody blocks embryo implantation in mice. Significant reduction of embryo implantation

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sites was observed in ddY, BALB/c, and MF1 mice injected with anti-LIF antibody (A, B, D).

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Implantation tended to be reduced in DBA/2Cr mice injected with anti-LIF antibody but there was no

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statistical difference compared with control (C). Normal rabbit IgG was injected as a control. *p

The contribution of leukemia inhibitory factor (LIF) for embryo implantation differs among strains of mice.

Despite of the claim that maternal leukemia inhibitory factor (LIF) - a member of interleukin 6 (IL6) family of cytokines - plays indispensable roles ...
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