Unique uterine localization and regulation may differentiate LPA3 from other lysophospholipid receptors for its role in embryo implantation

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1 Unique uteine localization and egulation may diffeentiate LPA3 fom othe lysohosholiid ecetos fo its ole in embyo imlantation Xiaoqin Ye, M.D., Ph.D., a,b Deon R. He, Ph.D., c Honglu Diao, Ph.D., a Richad Rivea, Ph.D., c and Jeold Chun, M.D., Ph.D. c a Deatment of Physiology and Phamacology, College of Veteinay Medicine, and b Intediscilinay Toxicology Pogam, Univesity of Geogia, Athens, Geogia; and c Deatment of Molecula Biology, Helen L. Dois Child and Adolescent Neuosychiatic Disode Institute, The cis Reseach Institute, La Jolla, Califonia Objective: To detemine factos diffeentiating LPA3 fom othe lysohosholiid (LP) ecetos fo its ole in embyo imlantation. Design: Exeimental mouse models. etting: Institute/univesity eseach laboatoies. Animal(s): Wild-tye, La3 ( / ), La ( / ) La ( / ), and ( / ) 3 ( / ) mice. Intevention(s): Ovaiectomy. Main Outcome Measue(s): Blue dye injection fo detemining imlantation sites on gestation day 4.. Real-time olymease chain eaction fo measuing gene exession in whole uteus and seaated uteine layes. In situ hybidization fo detecting ogesteone (P) induced La3 exession in the uteine luminal eithelium (LE). Result(s): Nomal imlantation was obseved in La ( / ) La ( / ) and ( / ) 3 ( / ) females. Temoal exession showed eak exession of La3 in the eimlantation uteus and constitutive exession of the othe nine LP ecetos in the eiimlantation uteus. atial localization evealed main exession of La3 in the LE and boad exession of the emaining LP ecetos in all thee main uteine layes: LE, stomal, and myometial layes. Homonal egulation in ovaiectomized uteus indicated u-egulation of La3 but downegulation o no effect of the emaining nine LP ecetos by P, and down-egulation of most LP ecetos, including La3, by7b-estadiol. Conclusion(s): LE localization and u-egulation by P diffeentiate LPA3 fom the othe nine LP ecetos and may undelie its essential ole in embyo imlantation. (Fetil teil Ò ;9:7 3. Ó by Ameican ociety fo Reoductive Medicine.) Key Wods: Lysohosholiid ecetos, uteine luminal eithelium, ogesteone, embyo imlantation Lysohosholiids (LPs) ae quantitatively mino liid secies that ae well known as comonents in the biosynthesis of membane hosholiids and as metabolic intemediates (). A few LPs, mainly lysohoshatidic acid (LPA) and shingosine -hoshate (P), have been oven to function as extacellula signaling molecules though cell suface G otein couled ecetos (), including LPA and P (3 ). Othe otential LP ecetos have also been eoted (6 9). eveal LP eceto secific in vivo functions have been identified in eceto-secific knockout mice, e.g., LPA in oe caniofacial fomation, neual develoment, and neuoathic ain ( ), P in vascula develoment (3), P in auditoy and vestibula function (4, ), P4 in shaing the teminal diffeentiation of megakayocytes (6), LPA 3 in sematogenesis (7), and LPA3 in embyo imlantation (8 ). Received Decembe 3, ; evised Januay 7, ; acceted Febuay, ; ublished online Mach,. X.Y. has nothing to disclose. D.R.H. has nothing to disclose. H.D. has nothing to disclose. R.R. has nothing to disclose. J.C. has nothing to disclose. uoted by the Office of the Vice Pesident fo Reseach at Univesity of Geogia and National Institutes of Health gants RHD663 (to X.Y.) and RHD68 (to J.C.). Reint equests: Xiaoqin Ye, M.D., Ph.D., Deatment of Physiology and Phamacology, College of Veteinay Medicine and Intediscilinay Toxicology Pogam, Univesity of Geogia, DW Books D., Athens, GA 36 ( ye@uga.edu). Embyo imlantation is a cucial ste fo the successful establishment of egnancy in mammals. It equies effective eciocal signaling between a cometent blastocyst and a ecetive uteus duing a discete imlantation window. The imlantation window is a limited time duing which the uteine envionment favos blastocyst gowth, attachment, and imlantation into the uteine wall ( ). We have eviously shown that deletion of La3 in mice delays embyo imlantation and altes embyo sacing. These defects ae caused by uteine athe than embyonic loss of La3, indicating uteine defects (8). Defective embyo imlantation has not been eoted in othe available LP eceto deficient females (, 3, 6, 6 9), and in the esent study we confim nomal embyo imlantation in mice lacking LPA, LPA, P, o P3. ome LP ecetos, e.g., LPA, LPA, and LPA3 (3), shae high sequence homology and have simila exession levels in the eimlantation uteus, yet only deletion of La3 but not of La and La affects embyo imlantation. What factos contibute to LPA3 eceto-secific ole in embyo imlantation? Heein, we evaluate the ten bonafide LP ecetos fo thei temoal exession attens in the eiimlantation uteus, thei localization in the eimlantation day 3. uteus, and thei uteine egulation by ovaian homones to identify the uniqueness of La3 among the LP ecetos, which may undescoe LPA3 eceto secificity in embyo imlantation, esecially in the establishment of uteine ecetivity. The infomation fom this study can also ovide guidance -8/$36. Fetility and teility â Vol. 9, No. 6, May 7 doi:.6/j.fetnstet...4 Coyight ª Ameican ociety fo Reoductive Medicine, Published by Elsevie Inc.

2 fo identifying candidate genes that ae otentially involved in the establishment of uteine ecetivity. MATERIAL AND METHOD Animals Wild-tye (WT) and La, La, La3,, and 3 knockout mice (9/vJ and C7BL/6 mixed backgound) wee geneated and genotyed as descibed elsewhee (, 8, 6, 7, 3). The animal facility is on a -hou light-dak cycle (6: AM to 6: PM) at 3 C with 3% % elative humidity. All methods used in this study wee aoved by the Animal ubjects Pogams of The cis Reseach Institute and the Univesity of Geogia and confom with National Institutes of Health guidelines and ublic law. Localization of Imlantation ites and Uteine amle Collection Imlantation sites wee localized as eviously descibed (8). Uteine tissues fom gestation days., 3., and 4. WT females between : AM and : PM wee flash-fozen. Pegnancy status was detemined by the esence of eggs and sems in the oviduct (day.), o blastocysts in the uteus (day 3.) o imlantation sites (day 4.). Utei fom egnant WT females wee included in the study. Isolation of Thee Uteine Layes Peviously eoted ocedues wee modified to seaate luminal eithelium (LE), stomal laye, and myometium fom thee gestation day 3. WT utei (3, 33). Biefly, sliced-oen uteine hons wee submeged in.% disase in calcium- and magnesium-fee Hanks balanced salt solution (HB, Invitogen) and digested fo hous at oom temeatue. LE sheets wee gently scaed. The stomal laye (t) with glandula eithelium was isolated with a stonge scaing foce. LE and t wee collected fo RNA isolation. The emaining myometial laye (Myo) was ulveized in liquid nitogen fo RNA isolation. Homonal Teatment Homonal teatment was administeed as eviously descibed (34). Each gou included 4 6 ovaiectomized females. Uteine hons wee flash-fozen. Real-time Revese Tanscition Polymease Chain Reaction RNA isolation and eal-time olymease chain eaction (PCR) wee done as eviously descibed (8, 3). To quantify the elative exession levels of each LP eceto, cdna oduct fom each ime ai (ulemental Table, available online at was subcloned into GEM vecto (Pomega). A standad cuve was eaed fo each ime ai fom seial dilution of the GEM-cDNA lasmid ( to 6 fmol). The elative tanscit numbe of each gene was quantified and then nomalized to b-actin as a loading contol. In itu Hybidization In situ hybidization was done as eviously descibed (36, 37). tatistical Analyses Data wee exessed as mean D. tatistical analyses wee done using tudent unequal-vaiance t test. The significance level was set at P<.. FIGURE Exession of lysohosholiid (LP) ecetos in gestation days., 3., and 4. wild-tye mouse uteus by eal-time olymease chain eaction. P<., comaed with day. o day 4.. n ¼ 3 6. Eo bas eesent standad deviation. ( x L P R / - a c t i n a L a L 3 a L Day. Day 3. Day 4. 4 a L a L REULT Exession Levels of LP Recetos in the Day 3. Uteus La3 shows the highest exession level in the WT uteus at gestation day 3. (8). To comae the elative exession of othe LP ecetos in the uteus, day 3. WT uteus was chosen to quantify the exession levels of La and. Figue shows that La, La,,, and 4 mrna levels wee simila to La3 level in gestation day 3. WT uteus. The exession level of 3 was about 4% of La3. The othe thee LP ecetos, La4, La, and, had significantly lowe exession levels (Fig. ). Embyo Imlantation in Diffeent LP Receto Knockout Mice Because othe LP ecetos ae also highly exessed in the eimlantation WT uteus, to identify othe LP eceto subtyes (besides LPA3) that might secifically influence imlantation, we evaluated embyo imlantation on the available and elevant eceto knockout mice. Of the ten LP ecetos examined, seven (La 3 and 4) wee exessed at notably highe levels than the othe thee (La4 and ) in the day 3. eimlantation uteus (Fig. ). These seven highly exessed LPs have been genetically deleted and mice deficient of six of them (excet ) oduced viable offsing: La, La,, 3, 4, and the eviously analyzed La3. Embyo imlantation was evaluated in females deficient of these LP ecetos excet P4. Imlantation sites wee detected at day 4. by Evans blue dye in La ( / ),La ( / ), ( / ),and3 ( / ) females, as well as La ( / ) La ( / ) and ( / ) 3 ( / ) females. On-time imlantation was detected in the single-knockout mice (data not shown) as well as the double-knockout mice (Fig. A). The numbes of imlantation sites fom La ( / ) La ( / ) and ( / ) 3 ( / ) double-knockout mice detected at gestation day 4. wee comaable to that fom WT (Fig. B). In addition, nomal imlantation sacing was obseved in these mice, suggesting that these fou LP ecetos, La, La,,and3, ae not citical fo embyo sacing eithe (Fig. A). These obsevations contast with delayed imlantation and embyo cowding in La3 ( / ) females (Fig. A) (8). These esults suggest that LPA, LPA, P, and P3 ae not citical fo embyo imlantation timing and sacing, desite thei significant exession levels in the eimlantation day 3. uteus. 3 Ye. Uteine LP eceto exession and egulation. Fetil teil. 4 8 Ye et al. Uteine LP eceto exession and egulation Vol. 9, No. 6, May

3 FIGURE Detection of imlantation sites in wild-tye, La ( / ) La ( / ), La3 ( / ), and ( / ) 3 ( / ) females on gestation day 4. by Evans blue dye injection. (A) Uteine images. Red aows indicate imlantation sites. No imlantation sites wee detected in the La3 ( / ) uteus, but healthy-looking blastocysts wee flushed fom the uteine hons (data not shown). (B) Numbe of imlantation sites. n ¼ 9. Eo bas eesent standad deviation. Ye. Uteine LP eceto exession and egulation. Fetil teil. Receto mrna Localization in the Day 3. Uteus La3 mrna in the uteus is mainly exessed in the LE (8). The LE is the fist laye of cells with which a blastocyst communicates befoe imlanting in the uteine wall. This LE-secific exession atten undescoes the imotance of LPA3 in embyo imlantation. To detemine if any othe LP ecetos shae this exession atten, we quantified mrna levels of the ten LP ecetos in the thee uteine layes: LE, t, and Myo. In addition to La3, all of the emaining nine LP ecetos wee also detectable to some extent in the LE. Howeve, wheeas La3 was mainly detected in the LE, the emaining nine LP ecetos had simila levels of exession in all thee layes (Fig. 3). Tansthyetin (TTR) is mainly exessed in the glandula eithelium in the t (37). TTR was used as a make fo stomal laye. Postaglandin F a eceto (FP) is mainly exessed in the myometium of day 3. uteus, and it seved as a make fo myometium (Fig. 3) (38). These esults indicate that La3 has a unique LE localization that is diffeent fom the othe LP ecetos examined. Receto Regulation by Ovaian Homones Pogesteone and Estogen Ovaian homones ogesteone (P) and estogen ae the maste contols of the embyo imlantation ocess in mice (39). LP eceto gene exession was theefoe assessed fo ovaian homonal influences. It had been demonstated that La3 is egulated by both P and 7b-estodiol (E ) (4). To detemine the egulation of La and by ovaian homones, ealie-ublished homonal teatment aoaches (34) wee combined with eal-time PCR to quantify the exession levels of LP ecetos. La exession was not changed by P (4 hous) o P (4 hous) þ E. It was tansiently down-egulated by E only ( h and 6 h; Fig. 4A and ulemental Fig. A [available online at La exession was down-egulated by both P and E (6 h and 4 h; Fig. 4A and ulemental Fig. B). La3 exession was u-egulated by P and down-egulated by E (6 h and 4 h). The P-induced u-egulation of La3 could be evesed by E teatment fo 6 hous and 4 hous (Fig. 4A and ulemental Fig. C). La3 Fetility and teility â 9

4 FIGURE 3 Localization of lysohosholiid (LP) ecetos in isolated uteine luminal eithelium (LE), stomal laye (t) with glandula eithelium, and myometium (Myo) fom eimlantation day 3. wild-tye uteus by eal-time olymease chain eaction. LEsecific La3 (8), glandula eithelium-secific TTR (tansthyetin) (37), and myometium-secific FP (ostaglandin F a eceto) (38) seved as makes fo LE, t, and Myo, esectively. P<. comaed with t and Myo fo La3, LE and Myo fo TTR, LE and t fo FP. n¼ 3. Eo bas eesent standad deviation. ct i n ( x L P R / - a La La was also u-egulated by P afte 4 hous of teatment, and this u-egulation could be abolished by ogesteone eceto (PR) antagonist RU486 (data not shown), indicating the involvement of PR in egulating P-induced uteine La3 exession. P induced La3 exession mainly in the LE (Fig. 4B 4G). La4 exession was down-egulated by P þ E (6 h) and E (6 h and 4 h) but not P alone (Fig. 4A and ulemental Fig. D). La exession was tansiently down-egulated by P þ E and E teatments fo 6 hous only (Fig. 4A and ulemental Fig. E). exession was deceased only by P þ E and E teatments fo 4 hous and not affected by P teatment, indicating a main ole of E in this egulation (Fig. 4A and ulemental Fig. A). exession was suessed by E teatment at all of the time oints examined ( h, 6 h, and 4 h) but not changed by P o P þ E teatments, indicating that although P did not affect the exession of, it could block the effect of E on exession (Fig. 4A and ulemental Fig. B). 3 exession was down-egulated by P, P þ E,oE (6 h and 4 h) teatment, indicating that both P and E affected 3 exession (Fig. 4A and ulemental Fig. C). 4 had a simila esonse to P and E teatments as that of 3 and La (Fig. 4A and ulemental Figs. B, C and D). exession was not affected by eithe P o E (Fig. 4A and ulemental Fig. E). Leukemia inhibitoy facto (Lif) was u-egulated by E and seved as a contol to demonstate that the down-egulation of LP ecetos by E was secific (Fig. 4A and ulemental Fig. F) (4). Although LP eceto mrna levels can be diffeentially egulated by P and E, La3 was the only one u-egulated by P teatment (Fig. 4A andulemental Figs. and ). LE t Myo La3 La4 La 3 4 TTR FP Ye. Uteine LP eceto exession and egulation. Fetil teil. The u-egulation of uteine La3 by P is anothe unique featue among the ten LP ecetos. Temoal Exession of LP Recetos in the Peiimlantation Uteus La3 has its eak exession in the eimlantation day 3. WT uteus duing egnancy (8). The exession of La and at gestation days., 3. (eimlantation), and 4. (ostimlantation) in WT uteus was also assessed. Inteestingly, only La3 showed a dynamic exession atten, wheeas the othe nine LP ecetos maintained simila exession levels in the uteus duing eiimlantation (Fig. ). DICUION Lase-catue micodissection couled with eal-time PCR and in situ hybidization wee eviously used to localize La3 in uteus (8). Both methods gave ecise gene localization, but they wee inefficient fo sceening lage numbe of genes and wee not amenable to simultaneously detemining thei elative exession levels. The simle method used in the esent study fo seaating the thee main uteine layes has the advantages of oughly localizing uteine gene exession and quantifying elative exession levels of diffeent genes in the same uteine layes. This aoach can be validated using La3, TTR, and FP as makes fo LE, t, and Myo, esectively (Fig. 3) (8, 37, 38). In addition,.. mg total RNA fom gestation day 3. LE can be obtained, geatly exceeding that fom lase-catue micodissection, which is in the ng ange (4). The ecise mrna localization of candidate genes could be futhe analyzed by using the two eviously mentioned techniques (8). Evidence suggests that the LE lays imotant oles in embyo imlantation, esecially the establishment of uteine ecetivity: It is the fist laye of cells that a blastocyst communicates with duing the initial stage of imlantation; LE cells develo midsecetoy uteodomes o inoodes, an attibute of ecetive uteine tissue; and the LE acts as a tansduce of the embyo s esence to elicit undelying stomal esonses (33, 4 4). Theefoe, the localization of La3 in the LE undescoes its imotant ole in the establishment of uteine ecetivity. All ten LP ecetos ae detectable in the LE; howeve, only La3 shows nealy exclusive exession in the LE. Theefoe, although othe LP ecetos may have oles in the uteus, only LPA3 eceto has the identified ole in the establishment of uteine ecetivity (8). La3 is the only LP eceto u-egulated by P PR, secifically in the LE (Fig. 4 and data not shown), which may be anothe citical deteminant of its function in the establishment of uteine ecetivity. P is a maste contol of embyo imlantation, and PR-mediated P signaling is indisensable fo embyo imlantation (39, 46 48). Results fom Indian hedgehog (Ihh, P-PR taget gene) uteine eithelium conditional knockout mice einfoce the citical ole of P-PR signaling in LE fo embyo imlantation (49 ). Howeve, sustained PR exession in the uteine eithelium beyond the exected imlantation window is detimental to embyo imlantation (46, 47, ). We have eviously demonstated that PR emains exessed in the LE befoe decidualization becomes manifested and disaeas fom LE aftewad (6), suggesting an active ole of PR in the LE duing the initial imlantation ocess. The obsevations that La3 exession eaks in the eimlantation LE and etuns to basal level in the ostimlantation uteus (Fig. ) (8) and that La3 is u-egulated by P-PR signaling in the LE (Fig. 4 and data not Ye et al. Uteine LP eceto exession and egulation Vol. 9, No. 6, May

5 FIGURE 4 FIGURE 4 Continued Regulation of lysohosholiid (LP) ecetos by ogesteone (P) and 7b-estadiol (E ) in ovaiectomized wild-tye mouse uteus. (A) Effects of P and E on LP eceto exession detemined by eal-time olymease chain eaction. Wild-tye vigin females (6 weeks old) wee ovaiectomized and allowed to ecove fo weeks. Oil ¼ daily injection of vehicle (. ml sesame oil) fo 3 days, killed 6 hous afte the final injection (4 hous afte the fist injection). P ¼ daily injection of P ( mg/mouse) fo 3 days, killed 6 hous afte the final injection (4 hous afte the fist injection). P þ E ¼ daily injection of P fo 3 days, lus a single dose of E ( ng/ mouse) on day 3, killed hou, 6 hous, and 4 hous late. E ¼ daily injection of vehicle fo days, lus a single dose of E on day 3, killed hou, 6 hous, and 4 hous late. A comlete set of data is esented in ulemental Figs. and. Abitay scale. Leukemia inhibitoy facto (Lif) was used as a contol fo egulation of LP ecetos by E, b-actin as a loading contol. P<. (downegulation); #P<. (u-egulation); comaed with contol (Oil). n ¼ 4 6. Eo bas eesent standad deviation. 4h indicates the duation of P teatment (4 hous); 6h indicates 6 hous afte E injection. In situ hybidization on ovaiectomized utei ae shown in B G. (B) La3 antisense obe, oil-injected. (C) Enlaged view of the ectangle aea in B. (D) La3 sense obe, oil-injected. (E) La3 antisense obe, P-injected. (F) Enlaged view of the ectangle aea in E. (G) La3 sense obe, P-injected. The duation of teatment was 4 hous. The sections wee countestained with % methyl geen. ecific signal (dak bown) is detected in the uteine luminal eithelium (LE). No secific signals wee detected in the negative contol using a sense La3 obe (D, G). Positive contol (day 3. uteus, La3 antisense obe) is shown in ulemental Fig. 3.n¼ 3. cale ba: mm. Ye. Uteine LP eceto exession and egulation. Fetil teil. shown) suggest that these two ecetos, PR and LPA3, may communicate with each othe in the LE fo the establishment of uteine ecetivity. P and E diffeentially egulate LP ecetos (Fig. 4A and ulemental Figs. and ). uisingly, the down-egulation of LP ecetos by P and/o E seen in ovaiectomized uteus was not obseved in the eiimlantation uteus, esecially at day 3., when the P level is high in addition to an estogen suge (7). One ossible exlanation is that the uteine local levels and atios of P and E eceived in the ovaiectomized mice did not mimic the hysiological conditions in the eiimlantation uteus. Howeve, uteine La3 eaks at day 3. (Fig. ) (8), which agees with the u-egulation of La3 by P in the ovaiectomized uteus (Fig. 4). The infomation obtained fom this study, e.g., diffeential satiotemoal uteine exession and homonal egulation, can otentially be used as a guide to identify uteine genes citical fo embyo imlantation. LPA, LPA, P, and P3 wee demonstated to be not individually citical fo embyo imlantation (Fig. ). Othe eots indicate no imlantation defects due to loss of LPA4 and P (8, 9). Thee was no mention of imlantation defects in the 4 ( / ) mice, eithe (6). The in vivo ole of P in embyo imlantation could not be assessed, because of embyonic lethality of ( / ) mice and the lack of uteine-secific ( / ) mice (3). Although La ( / ) mice ae not yet available, the lack of distinct satiotemoal exession attens in the eiimlantation uteus and its low levels of exession in the eiimlantation uteus (Figs. and 3) suggest that LPA may not be citical fo embyo imlantation. imila imlantation defects between La ( / ) La ( / ) La3 ( / ) and La3 ( / ) females () einfoces the citical ole of LPA3 in the establishment of uteine ecetivity. Howeve, these obsevations do not exclude othe otential uteine functions of LP ecetos, e.g., P, P, and P3 may lay a ole in decidualization (8). Two inteesting studies suggest that human LPA3 may lay a ole in the establishment of uteine ecetivity (9, 6). Futhe analyses of human uteine micoaay data (identifie GE6364) (9) indicate that LPAR3 (EDG7) mrna eaks in the nomal ealy secetoyhase endometium (EE, eimlantation stage), aalleling data obtained fom mice (8). LPAR3 mrna levels ae significantly down-egulated in the EE of endometiosis atients, wheeas LPA3 otein levels ae maginally down-egulated in the EE and significantly down-egulated in the middle and late secetoyhase endometium (9, 6). The diffeential exession attens Fetility and teility â

6 of LPAR3 in nomal and endometiosis atients ae not obseved in othe LP ecetos that wee included in the database (9). The dysegulation of uteine LPA3 in endometiosis atients suggests that LPA3 may be involved in endometiosis-associated defective uteine ecetivity. Acknowledgments: The authos thank Ms. Gace Kennedy fo technical assistance and Ms. Danielle Letouneau at the cis Reseach Institute fo editoial assistance. The authos also thank D. James N. Mooe and D. Zhen Fu at the College of Veteinay Medicine, Univesity of Geogia, fo access to the ABI 79 Real-time PCR machine and the imaging system, esectively. REFERENCE. Pieinge RA, Bonne H J, Kunnes R. Biosynthesis of hoshatidic acid, lysohoshatidic acid, diglyceide, and tiglyceide by fatty acyltansfease athways in Escheichia coli. J Biol Chem 967;4: Ishii I, Fukushima N, Ye X, Chun J. Lysohosholiid ecetos: signaling and biology. Annu Rev Biochem 4;73: Hecht JH, Weine JA, Post R, Chun J. Venticula zone gene- (vzg-) encodes a lysohoshatidic acid eceto exessed in neuogenic egions of the develoing ceebal cotex. J Cell Biol 996;3: Mutoh T, Chun J. Lysohosholiid activation of G otein couled ecetos. ubcell Biochem 8;49: Choi JW, He DR, Noguchi K, Yung YC, Lee CW, Mutoh T, et al. LPA ecetos: subtyes and biological actions. Annu Rev Phamacol Toxicol ;: Pastenack M, von Kugelgen I, Aboud KA, Lee YA, Ruschendof F, Voss K, et al. G otein couled eceto PY and its ligand LPA ae involved in maintenance of human hai gowth. Nat Genet 8;4: Yanagida K, Masago K, Nakanishi H, Kihaa Y, Hamano F, Tajima Y, et al. Identification and chaacteization of a novel lysohoshatidic acid eceto, y/lpa6. J Biol Chem 9;84: Tabata K, Baba K, hiaishi A, Ito M, Fujita N. The ohan GPCR GPR87 was deohanized and shown to be a lysohoshatidic acid eceto. Biochem Biohys Res Commun 7;363: Muakami M, hiaishi A, Tabata K, Fujita N. Identification of the ohan GPCR, PY() eceto as the shingosine--hoshate and lysohoshatidic acid eceto. Biochem Biohys Res Commun 8;37:77.. Contos JJ, Fukushima N, Weine JA, Kaushal D, Chun J. Requiement fo the la lysohoshatidic acid eceto gene in nomal suckling behavio. Poc Natl Acad ci U A ;97: Kingsbuy MA, Rehen K, Contos JJ, Higgins CM, Chun J. Nonolifeative effects of lysohoshatidic acid enhance cotical gowth and folding. Nat Neuosci 3;6:9 9.. Inoue M, Rashid MH, Fujita R, Contos JJ, Chun J, Ueda H. Initiation of neuoathic ain equies lysohoshatidic acid eceto signaling. Nat Med 4;: Liu Y, Wada R, Yamashita T, Mi Y, Deng CX, Hobson JP, et al. Edg-, the G otein couled eceto fo shingosine--hoshate, is essential fo vascula matuation. J Clin Invest ;6: He DR, Gillet N, chwande M, Rivea R, Mulle U, Chun J. hingosine -hoshate (P) signaling is equied fo maintenance of hai cells mainly via activation of P. J Neuosci 7;7: MacLennan AJ, Benne J, Andinga A, Chaves AH, Rosing JL, Vesey R, et al. The P shingosine - hoshate eceto is essential fo auditoy and vestibula function. Hea Res 6;: Golfie, Kondo, chulze T, Takeuchi T, Vassileva G, Achtman AH, et al. haing of teminal megakayocyte diffeentiation and olatelet develoment by shingosine--hoshate eceto P4. FAEB J ;4: Ye X, kinne MK, Kennedy G, Chun J. Age-deendent loss of sem oduction in mice via imaied lysohoshatidic acid signaling. Biol Reod 8;79: Ye X, Hama K, Contos JJ, Anlike B, Inoue A, kinne MK, et al. LPA3-mediated lysohoshatidic acid signalling in embyo imlantation and sacing. Natue ;43: Hama K, Aoki J, Inoue A, Endo T, Amano T, Motoki R, et al. Embyo sacing and imlantation timing ae diffeentially egulated by LPA3- mediated lysohoshatidic acid signaling in mice. Biol Reod 7;77: Ye X. Lysohosholiid signaling in the function and athology of the eoductive system. Hum Reod Udate 8;4: Ye X, Chun J. Lysohoshatidic acid (LPA) signaling in vetebate eoduction. Tends Endocinol Metab ;:7 4.. Cason DD, Bagchi I, Dey K, Endes AC, Fazleabas AT, Lessey BA, et al. Embyo imlantation. Dev Biol ;3: Dey K, Lim H, Das K, Reese J, Paia BC, Daikoku T, et al. Molecula cues to imlantation. Endoc Rev 4;: hakey AM, mith K. The endometium as a cause of imlantation failue. Best Pact Res Clin Obstet Gynaecol 3;7: Paia BC, Reese J, Das K, Dey K. Deciheing the coss-talk of imlantation: advances and challenges. cience ;96: Contos JJ, Ishii I, Fukushima N, Kingsbuy MA, Ye X, Kawamua, et al. Chaacteization of la() (Edg4) and la()/la() (Edg/Edg4) lysohoshatidic acid eceto knockout mice: signaling deficits without obvious henotyic abnomality attibutable to la(). Mol Cell Biol ;: Ishii I, Ye X, Fiedman B, Kawamua, Contos JJ, Kingsbuy MA, et al. Maked einatal lethality and cellula signaling deficits in mice null fo the two shingosine -hoshate (P) ecetos, P()/LP (B)/EDG- and P(3)/LP(B3)/EDG-3. J Biol Chem ;77: Jaillad C, Haison, tankoff B, Aigot M, Calve AR, Duddy G, et al. Edg8/P: an oligodendoglial eceto with dual function on ocess etaction and cell suvival. J Neuosci ;: Lee Z, Cheng CT, Zhang H, uble MA, Wu J, Mukhejee A, et al. Role of LPA4/y9/GPR3 in negative egulation of cell motility. Mol Biol Cell 8;9: Contos JJ, Chun J. The mouse l(a3)/edg7 lysohoshatidic acid eceto gene: genomic stuctue, chomosomal localization, and exession atten. Gene ;67: Ishii I, Fiedman B, Ye X, Kawamua, McGiffet C, Contos JJ, et al. elective loss of shingosine -hoshate signaling with no obvious henotyic abnomality in mice lacking its G otein couled eceto, LP(B3)/EDG-3. J Biol Chem ;76: idhu, Kimbe J. Homonal contol of H-tye alha(-)fucosyltansfease messenge ibonucleic acid in the mouse uteus. Biol Reod 999;6: Cambell EA, O Haa L, Catalano RD, hakey AM, Feeman TC, Johnson MH. Temoal exession ofiling of the uteine luminal eithelium of the seudo-egnant mouse suggests ecetivity to the fetilized egg is associated with comlex tanscitional changes. Hum Reod 6;: Bown N, Deb K, Paia BC, Das K, Reese J. Embyo-uteine inteactions via the neuegulin family of gowth factos duing imlantation in the mouse. Biol Reod 4;7:3. 3. Diao H, Alin JD, Xiao, Chun J, Li Z, Chen, et al. Alteed satiotemoal exession of collagen tyes I, III, IV, and VI in La3-deficient ei-imlantation mouse uteus. Biol Reod ;84: Diao H, Xiao, Zhao F, Ye X. Uteine luminal eithelium secific oline-ich acidic otein (PRAP) as a make fo successful embyo imlantation. Fetil teil ;94:88. e. 37. Diao H, Xiao, Cui J, Chun J, Xu Y, Ye X. Pogesteone eceto mediated u-egulation of tansthyetin in eimlantation mouse uteus. Fetil teil ;93: Yang ZM, Das K, Wang J, ugimoto Y, Ichikawa A, Dey K. Potential sites of ostaglandin actions in the eiimlantation mouse uteus: diffeential exession and egulation of ostaglandin eceto genes. Biol Reod 997;6: Wang H, Dey K. Roadma to embyo imlantation: clues fom mouse models. Nat Rev Genet 6;7: Hama K, Aoki J, Bandoh K, Inoue A, Endo T, Amano T, et al. Lysohoshatidic eceto, LPA3, is ositively and negatively egulated by ogesteone and estogen in the mouse uteus. Life ci 6;79: Yang ZM, Chen DB, Le P, Hae MJ. Diffeential homonal egulation of leukemia inhibitoy facto (LIF) in abbit and mouse uteus. Mol Reod Dev 996;43: Niklaus AL, Pollad JW. Mining the mouse tanscitome of ecetive endometium eveals distinct molecula signatues fo the luminal and glandula eithelium. Endocinology 6;47: Alin JD. Embyo imlantation: the molecula mechanism emains elusive. Reod Biomed Online 6;3: Muhy CR. Undestanding the aical suface makes of uteine ecetivity: inoods o uteodomes? Hum Reod ;: Cave J, Matin K, yooulou I, Balow D, agent I, Madon H. An in-vito model fo stomal invasion duing imlantation of the human blastocyst. Hum Reod 3;8: Baze FW, layden OD. Pogesteone-induced gene exession in uteine eithelia: a myth eetuated by conventional wisdom. Biol Reod 8;79: Mulac-Jeicevic B, Conneely OM. Reoductive tissue selective actions of ogesteone ecetos. Reoduction 4;8: Ye et al. Uteine LP eceto exession and egulation Vol. 9, No. 6, May

7 48. Lydon JP, DeMayo FJ, Funk CR, Mani K, Hughes AR, Montgomey CA J, et al. Mice lacking ogesteone eceto exhibit leiotoic eoductive abnomalities. Genes Dev 99;9: Takamoto N, Zhao B, Tsai Y, DeMayo FJ. Identification of Indian hedgehog as a ogesteoneesonsive gene in the muine uteus. Mol Endocinol ;6: Jeong JW, Lee KY, Kwak I, White LD, Hilsenbeck G, Lydon JP, et al. Identification of muine uteine genes egulated in a ligand-deendent manne by the ogesteone eceto. Endocinology ;46:349.. Lee K, Jeong J, Kwak I, Yu CT, Lanske B, oegiato DW, et al. Indian hedgehog is a majo mediato of ogesteone signaling in the mouse uteus. Nat Genet 6;38:4 9.. Palomino WA, Fuentes A, Gonzalez RR, Gable F, Boic MA, Vega M, et al. Diffeential exession of endometial integins and ogesteone eceto duing the window of imlantation in nomo-ovulatoy women teated with clomihene citate. Fetil teil ;83: Lessey BA, Killam AP, Metzge DA, Haney AF, Geene GL, McCaty K J. Immunohistochemical analysis of human uteine estogen and ogesteone ecetos thoughout the menstual cycle. J Clin Endocinol Metab 988;67: Wakitani, Hondo E, Phichitasli T, tewat CL, Kiso Y. Uegulation of Indian hedgehog gene in the uteine eithelium by leukemia inhibitoy facto duing mouse imlantation. J Reod Dev 8;4:3 6.. Baze FW, Wu G, ence TE, Johnson GA, Bughadt RC, Bayless K. Novel athways fo imlantation and establishment and maintenance of egnancy in mammals. Mol Hum Reod ;6:3. 6. Diao H, Paia BC, Xiao, Ye X. Temoal exession atten of ogesteone eceto in the uteine luminal eithelium suggests its equiement duing ealy events of imlantation. Fetil teil ;9: McComack JT, Geenwald G. Evidence fo a eimlantation ise in oestadiol-7beta levels on day 4 of egnancy in the mouse. J Reod Fetil 974;4: kaznik-wikiel ME, Kaneko-Taui T, Kashiwagi A, Pu JK. hingosine--hoshate eceto exession and signaling coelate with uteine ostaglandin endoeoxide synthase exession and angiogenesis duing ealy egnancy. Biol Reod 6;74: Buney RO, Talbi, Hamilton AE, Vo KC, Nyegaad M, Nezhat CR, et al. Gene exession analysis of endometium eveals ogesteone esistance and candidate suscetibility genes in women with endometiosis. Endocinology 7;48: Wei Q, t Clai JB, Fu T, tatton P, Nieman LK. Reduced exession of biomakes associated with the imlantation window in women with endometiosis. Fetil teil 9;9: Fetility and teility â 3

8 UPPLEMENTAL FIGURE Regulation of LPA ecetos by ogesteone (P) and 7b-estadiol (E ) in ovaiectomized mouse uteus detemined by eal-time olymease chain eaction. (A) La; (B) La; (C) La3; (D) La4; (E) La. P<., down-egulation; P<., down-egulation; #P<., uegulation; ##P<., u-egulation; comaed with contol (Oil). n ¼ 4 6. Eo bas eesent standad deviation. h, 6h, and 4h indicate hous afte E injection. x L a / - acti n ( - actin ( x L a / x L a3 / - acti n ( ( x L a4 / -acti n.. 3 ## # - actin ( x 4 ). L a /. Oil P P+E h P+E 6 h + h P+E 4 E h E 6h E 4h Oil P P+E h P+E 6h P+E 4h E h E 6h E 4h Ye. Uteine LP eceto exession and egulation. Fetil teil. 3.e Ye et al. Uteine LP eceto exession and egulation Vol. 9, No. 6, May

9 UPPLEMENTAL FIGURE Regulation of P ecetos by ogesteone (P) and 7b-estodiol (E ) in ovaiectomized mouse uteus detemined by eal-time olymease chain eaction. (A) ; (B) ; (C) 3; (D) 4; (E) ; (F) Leukemia inhibitoy facto (Lif) was a contol fo egulation of LP ecetos by E ; abitay scale. P<., down-egulation; P<., down-egulation; #P<., u-egulation; ##P<., u-egulation; comaed with contol (Oil). n ¼ 4 6. Eo bas eesent standad deviation. h, 6h, and 4h indicate hous afte E injection. / -a c tin ( x / - acti n (x / n ( x - acti L if / -acti n / - ac ti n ( x ) Oil P P+E h P+E 6h P+E 4h E h E 6h E 4h 3 / - acti n (x ## # ## # ## Oil P4 P+E h P+E 6h E h E 6h E 4h P+E Ye. Uteine LP eceto exession and egulation. Fetil teil. Fetility and teility â 3.e

10 UPPLEMENTAL FIGURE 3 Positive contol fo in situ hybidization. (A) La3 antisense obe, gestation day 3. wild-tye uteus. (B) Enlaged view of the ectangle aea in A. The section was countestained with % methyl geen. ecific signal (dak bown) is detected in the uteine luminal eithelium (LE). cale ba: mm. Ye. Uteine LP eceto exession and egulation. Fetil teil. 3.e3 Ye et al. Uteine LP eceto exession and egulation Vol. 9, No. 6, May

11 UPPLEMENTAL TABLE Pimes used in the study. Gene Pime sequences ( -3 ) Accession no. ize (b) La La La3 La4 La 3 4 b-actin TTR Lif FP TCT TCT GGG CCA TTT TCA AC TGC CTG AAG GTG GCG CTC AT ACC ACA CTC AGC CTA GTC AAG AC CTG AGT AAC GGG CAG ACT TG ACA CCA GTG GCT CCA TCA G GTT CAT GAC GGA GTT GAG CAG AGG CAT GAG CAC ATT CTC TC CAA CCT GGG TCT GAG ACT TG AGG AAG AGC AAC CGA TCA TCA CAG ACC ACC ATA TGC AAA CGA TGT G TGT AGA CCC AGA GTC CTG C TAG AGG GCG AGG TTG AGT G ATA GAC CGA GCA CAG CCA AC GTG TTC CAG AAC CTT CTC AGG CCA GAT GCG CCT TGC AGA A CAT GGC TTC CTA GAG ACA GA ACC TTC AGT CTG CTC TTC ACG AAG AGC ACA TAG CCC TTG GAG AGA TTT CCA ATA GCC GCT CTC AGC TTG CCG GTG TAG TTG TAG TGG AAT CCT GTG GCA TCC ATG AAA C TAA AAC GCA GCT CAG TAA CAG TCC G GCT GGA CTG GTA TTT GTG TCT GAG TCG TTG GCT GTG AAA GGC AAC CTC ATG AAC CAG ATC AGT GGG GTT CAG GAC CTT CT CGG GAG TCA CAC TCT TAA GAG T ACG TTG CCA TGC GGA GAG CAA AA NM_ NM_ NM_983.3 NM_ ENMUT NM_ NM_ NM_.3 7 NM_. NM_39. 3 NM_ NM_ NM_8. 63 NM_ Ye. Uteine LP eceto exession and egulation. Fetil teil. Fetility and teility â 3.e4