REPRODUCTIONRESEARCH

Lysophosphatic acid modulates prostaglandin secretion in the bovine uterus

Izabela Woclawek-Potocka, Junichi Komiyama1, Jean Sebastian Saulnier-Blache2, Edyta Brzezicka, Mamadou Moussa Bah, Kiyoshi Okuda1 and Dariusz J Skarzynski Department of Reproductive Immunology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyn, Poland, 1Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Tsushima Naka 1-1-1, Okayama 700-8530, Japan and 2INSERM U317, Institut Louis Bugnard, Universite´ Paul Sabatier, CHU Rangueil, 31403 Toulouse, Cedex 04, France Correspondence should be addressed to I Woclawek-Potocka; Email: [email protected]

Abstract

Lysophosphatidic acid (LPA) modulates prostaglandin (PG) synthesis via LPA receptor 3 (LPAR3) in the murine endometrium. The lack of functional LPAR3 in mice may lead to embryo mortality. In the present study, we examined the role of LPA in the bovine uterus. We confirmed that LPA is locally produced and released from the bovine endometrium. Moreover, there are enzymes involved in LPA synthesis

(phospholipase (PL) D2 and PLA2G1B) in the bovine endometrium during estrous cycle and early pregnancy.Expressionof the receptor for LPA

(LPAR1) was positively correlated with the expression of PGE2 synthase (PGES) and negatively correlated with the expression of PGF2a

synthase (aldose reductasewith 20 a-hydroxysteroid dehydrogenase activity – PGFS) during early pregnancy. In vivo LPAinduced P4 and PGE2 secretion was inhibited by LPAR1 antagonist (Ki16425). The overall results indicate that LPA is locally produced and released from the bovine endometrium. Moreover, LPAR1 expression in the endometrium during the estrous cycle and early pregnancy indicates that LPAmay play autocrine and/or paracrine roles in the bovine uterus. LPAR1 is positively correlated with the expression of the enzyme

responsible for luteotropic PGE2 production (PGES) in endometrium. In cow, LPA stimulates P4 and PGE2 secretion. Thus, LPA in the bovine reproductive tract may indirectly (via endometrium) or directly support corpus luteum action via the increase of P4 synthesis and the increase

of PGE2/PGF2a ratio. It suggests that LPA may serve as an important factor in the maintenance of early pregnancy in cow. Reproduction (2009) 137 95–105

Introduction receptor in various LPA functions and various kinds of tissues are not known. In humans, LPA concentration (LPA) is a simple phospholipid with gradually increases between weeks 5 and 40 of pregnancy a vast variety of physiological and pathological actions on (Tokumura et al. 2002). LPA stimulates oocyte maturation many cell types, such as cell proliferation and differen- (Hinokio et al. 2002), preimplantation development of tiation (Goetzl et al. 1999, Pustilnik et al.1999), embryos to the blastocyst stage (Liu & Armant 2004), and cytoskeletal rearrangement (Moolenaar 1995), cell-to-cell embryo transport in the oviduct (Dey et al. 2004)in interactions (Fukushima et al. 2002), and tumorigenesis women. LPA3-deficient mice showed delayed implan- (Kim etal. 2006). In the cells, LPA production is triggered by tations and a reduced number of implantation sites, despite various agonists (Phorbol 12-myristate 13-acetate (Shen 2C comparable numbers of blastocysts available for implan- et al. 1998), intracellular calcium ions (Ca ), ionophore, tation (Ye et al. 2005). The implantation sites in the LPA3- bombesin (Xie et al. 2002), LPA itself (Eder et al. 2000,etc.) deficient uteri were clustered in the uterine segments and is accompanied by activation of phospholipase D proximal to the cervix. Moreover, embryos isolated from (PLD2). Phospholipase D catalyzes the conversion of diacyl LPA3-deficient uteri were always smaller than those phospholipids to phosphatidic acid (PA; Exton 2002). PA is from the wild type. This study also proved that the then converted to LPA by PLA1 and PLA2G1B (Eder et al. administration of prostaglandin (PG) E2 and PGI2 could 2000, Aoki 2004). However, Snitko et al. (1997) demon- partially correct implantation defects in these animals. strated a preference of PLA2G1B for LPA. In mammals, LPA In ruminants, PGs are crucial components in the exerts its action via four high-affinity G--coupled regulation of estrous cycle and early pregnancy. Prosta- receptor types: LPAR1, LPAR2, LPAR3 and the recently glandin F2a is the major luteolytic agent, whereas PGE2 has identified LPAR4 (Bandoh et al. 1999, Im et al. 2000, luteoprotective and anti-luteolytic properties (Asselin et al. Noguchi et al. 2003). However, the precise roles of each 1996, McCracken et al.1999). Therefore, achieving an

q 2009 Society for Reproduction and Fertility DOI: 10.1530/REP-08-0209 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access 96 I Woclawek-Potocka and others optimal PGF2a to PGE2 ratio is essential for endometrial receptivity, maintenance of corpus luteum (CL), and P4 secretion as well as accurate pregnancy establishment (Pratt et al.1977, Magness et al.1981, Milvae et al. 1996, Kotwica et al. 2003, Weems et al.2006). Taking into consideration, the interactions between LPA and PG action and the mechanisms of LPA synthesis (Shah & Catt 2005), we assume that LPA could have a direct role in endometrial secretory functions and signal transduction between embryos and endometrium, not only in mice (Ye et al. 2005) and human (Hinokio et al.2002, Dey et al. 2004, Liu & Armant 2004), but also in cattle. However, we proved Figure 1 Concentrations of LPA in the blood plasma taken from cows (nZ3) on days 17 and 18 of the estrous cycle, five times for 24 h (at the before that in cattle, unlike in mice and pigs (Ye et al. 2005, hour 0, 6, 12, 18, and 24) from jugular (open circle) and uterine (black Kaminska et al.2008), LPA acts via LPAR1 receptor in the circle) veins. Small superscript letters: a and b indicate statistical endometrium (Woclawek-Potocka et al.2007). In the differences in the respective LPA concentration between jugular and present study, we checked whether LPA may be locally uterine veins respectively (P!0.05), as determined by repeated produced and released from the bovine endometrium and measures ANOVA with Bonferroni’s Multiple Comparison Test. examined if there exists gene expression for the enzymes involved in LPA synthesis (PLA2G1B and PLD2)inthe Expression of mRNA for PLD2 and PLA2G1B in the bovine endometrium. However, the main goal of this study bovine endometrial tissue during the estrous cycle and was to determine whether LPA regulates PG synthesis and early pregnancy secretion in the bovine endometrium in vivo.Wealso Figure 2b shows thatthe expression patterns of PLD2 mRNA checked whether gene expression of LPA receptor is on the days: 2–4, 8–10, and 17–19 of the estrous cycle and correlated with gene expression of the terminal enzymes pregnancy. With specific primers, enabling amplification responsible for PG production (PGES and aldose reductase of PLD , one strong band migrating at 106 bps (bp) was a 2 with 20 -hydroxysteroid dehydrogenase activity – PGFS) observed in the endometrial tissue at all examined stages in the estrous cycle and early pregnancy in the bovine (data not shown). PLD2 transcript abundance normalized endometrium (Madore et al.2003). to GAPDH mRNA expression did not differ either during the estrous cycle or pregnancy (PO0.05). Results Figure 2c shows the expression patterns of PLA2G1B mRNA on the days: 2–4, 8–10, and 17–19 of the estrous Experiment 1. LPA concentration in the uterine and cycle and pregnancy. With specific primers, enabling jugular vein amplification of PLA2G1B, one strong band migrating at LPA concentration in the blood plasma from uterine and 107 bp was observed in the endometrial tissue at all jugular vein examined stages (data not shown). PLA2G1B transcript abundance normalized to GAPDH mRNA expression Figure 1 shows LPA concentration in the blood plasma did not differ during the estrous cycle (PO0.05), but taken from cows on days 17–19 of the estrous cycle, (at the increased on day 17–19 of pregnancy (P!0.05). hour 0, 6, 12, 18, and 24) from jugular and uterine vein. Additionally, PLA2G1B transcript abundance on days The total amount of LPA in the blood plasma taken from the 17–19 of pregnancy was significantly higher than on jugular vein was significantly lower (79.54G18.0; arbi- days 17–19 of the estrous cycle (P!0.05). trary units, area under the curve) than that in the blood plasma from the uterine vein (541.2G90.0; P!0.05). Experiment 3 Correlations between LPAR1, PGES and PGFS mRNA expression in the bovine endometrium Experiment 2. LPA production in the bovine endo- during estrous cycle and early pregnancy metrium during the estrous cycle and early pregnancy Expression of mRNA for LPAR1 and terminal PG LPA concentration in the bovine endometrial tissue synthesizing enzymes (PGES and PGFS) in the bovine during the estrous cycle and early pregnancy endometrial tissue during the estrous cycle and early pregnancy Figure 2a shows LPA concentration in the endometrial tissue on the days: 2–4, 8–10, and 17–19 of the estrous Figure 3a shows the expression patterns of LPAR1 mRNA cycle and pregnancy. LPA concentration in the bovine on the days: 2–4, 8–10, and 17–19 of the estrous cycle endometrium did not differ during either the estrous and pregnancy. With specific primers, enabling amplifi- cycle or pregnancy (PO0.05). However, LPA concen- cation of LPAR1, one strong band migrating at 150 bps tration on 17–19 days of pregnancy was significantly (bp) was observed in the endometrial tissue at all higher on days 17–19 of the estrous cycle (P!0.05). examined stages (data not shown). LPAR1 transcript

Reproduction (2009) 137 95–105 www.reproduction-online.org

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access LPA influence on the bovine reproduction 97

Figure 2 (a) Concentrations of LPA or patterns of expression of (b) PLD2 Figure 3 Pattern of expression of (a) LPAR1, (b) PGES, and (c) PGFS and (c) PLA2G1B mRNA in the endometrial tissue on the selected days mRNA on the selected days of the estrous cycle (white bars) and of the estrous cycle (white bars) and pregnancy (black bars). Data are pregnancy (black bars). Data are expressed as arbitrary units of expressed as ng/g tissue for LPA concentrations or arbitrary units of respective mRNA/mRNA GAPDH. Small superscript letters: a and b respective mRNA/mRNA GAPDH for mRNA quantitive expression. indicate statistical differences in the respective mRNA quantitative Small superscript letters: a and b indicate statistical differences in the expression between groups of cyclic or pregnant animals respectively respective LPA concentration or mRNA quantitative expression (P!0.05), as determined by one-way ANOVA followed by Bonferroni’s ! between groups of cyclic or pregnant animals respectively (P 0.05), as multiple comparison test. Capital superscript letters: A and B indicate determined by one-way ANOVA followed by Bonferroni’s multiple statistical differences in the respective mRNA quantitative expression comparison test. Capital superscript letters: A and B indicate statistical between groups of animals on the same days of the estrous cycle and differences in the respective mRNA quantitative expression between pregnancy respectively (P!0.05), as determined by one-way ANOVA groups of animals on the same days of the estrous cycle and pregnancy followed by Bonferroni’s multiple comparison test. respectively (P!0.05), as determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. 17–19 of pregnancy was higher on days 8–10 of abundance normalized to GAPDH mRNA expression pregnancy (P!0.05). Additionally, LPAR1 transcript revealed significant upregulation during the estrous abundance on days 8–10 of pregnancy was significantly cycle and was at the highest level on the days 17–19 higher on days 8–10 of the estrous cycle (P!0.05). (P!0.05). However, significant difference was shown Figure 3b shows the expression patterns of PGES only between days 2–4 and days 17–19 of the cycle mRNA on the days: 2–4, 8–10, and 17–19 of the estrous (P!0.05), and between days 8–10 and days 17–19 of the cycle and pregnancy. With specific primers, enabling cycle (P!0.05). LPAR1 transcript abundance on days amplification of PGES, one strong band migrating at www.reproduction-online.org Reproduction (2009) 137 95–105

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access 98 I Woclawek-Potocka and others

142 bps (bp) was observed in the endometrial tissue at all examined stages (data not shown). PGES transcript abundance normalized to GAPDH mRNA expression revealed significant downregulation during the estrous cycle, and was lower on days 8–10 and days 17–19 than on days 2–4 (P!0.05). The differences were observed between days 2–4 and days 8–10 of the cycle (P!0.05), and between days 2–4 and days 17–19 of the cycle (P!0.05). PGES transcript abundance on days 8–10 of pregnancy was significantly higher on days 8–10 of the estrous cycle (P!0.05) and on days 17–19 of pregnancy than on days 17–19 of the estrous cycle (P!0.05). Figure 3c shows the expression patterns of PGFS mRNA on the days: 2–4, 8–10 and 17–19 of the estrous cycle and pregnancy. With specific primers, enabling amplification of PGFS, one strong band migrating at 149 bps (bp) was observed in the endometrial tissue at all examined stages (data not shown). PGFS transcript abundance normalized to GAPDH mRNA expression revealed the highest level on days 8–10 of the estrous cycle and pregnancy (P!0.05). Additionally, PGFS transcript abundance on days 8–10 of pregnancy did not differ from the expression on days 8–10 of the estrous cycle (PO0.05) but was significantly higher on days 17–19 of the estrous cycle than on days 17–19 of pregnancy (P!0.05). In this experiment, there were positive correlations between PGES and LPAR1 transcript abundance during pregnancy (rZ0.548; P!0.05). Additionally, there were Figure 4 Concentrations of (a) progesterone, (b) prostaglandin E2, and negative correlations between LPAR1 and PGFS tran- (c) 13,14-dihydro,15-keto-PGF2a in peripheral blood plasma of cows script abundance during the estrous cycle (rZK0.5923; infused with saline (grey bars) and various doses of LPA agonist (lines). P!0.05) and during pregnancy for PGES and PGFS All reagents were infused into the aorta abdominalis. Different subscript ! (rZK0.517; P!0.05). letters indicate significant differences (P 0.05) between treated groups, as assessed by repeated-measures ANOVA test followed by Bonferroni’s multiple comparison test. Experiment 4. The effect of LPA and LPAR1 antagonist

(Ki 16425) on the secretion of progesterone and increase in the total amount of secreted PGE2 was noted prostaglandins after the infusion of 1 mg LPA agonist in comparison to ! Preliminary study the control cows (Table 1; P 0.01). Figure 4c shows PGFM concentrations in the blood Figure 4a shows P4 concentrations in the blood plasma of plasma of control and experimental cows. Admini- control and experimental cows. Administration of all stration of the doses of 1, 10, and 100 mg LPA agonist m selected doses of LPA agonist (1, 10, 50, 100, and 250 g) elevated the concentrations of PGFM in peripheral blood elevated the concentrations of P4 in peripheral blood during 24 h after LPA infusion in comparison to the during the 24 h after LPA infusion in comparison to the ! ! saline infused cows (P 0.05). The highest increase in saline infused cows (P 0.05). However, all selected doses the total amount of secreted PGFM was noted after the of LPA agonist also elevated the total amount of secreted P4 infusion of 1 mg LPA agonist in comparison to the control in the blood plasma, the highest, but not different from cows (Table 1; P!0.05). other doses of administered LPA, increase was noted after On the basis of this preliminary study, the most effective the infusion of 1 mg LPA agonist in comparison to the dose of LPA agonist – 1 mg was chosen for the further studies. control cows (Table 1; P!0.01; area under the curve). Figure 4b shows PGE concentrations in the blood 2 Effects of LPA agonist and LPAR1 antagonist (Ki16425) plasma of control and experimental cows. Admini- on the progesterone and prostaglandin secretion in the stration of the doses of 1, 10, and 50 mg LPA agonist bovine reproductive tract strongly elevated the concentrations of PGE2 in periph- eral blood during 24 h after LPA infusion in comparison Figure 5a shows P4 concentrations in the blood plasma to the saline infused cows (P!0.05). The highest, but not of saline, LPA agonist (1 mg) and LPA antagonist different from doses of 10 and 50 mg administered LPA, (Ki16425; 10 mg) treated cows. Administration of LPA

Reproduction (2009) 137 95–105 www.reproduction-online.org

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access LPA influence on the bovine reproduction 99

Table 1 Effects of different doses of lysophosphatidic acid (LPA) comparison to the saline and LPA agonist infused cows administered into the aorta abdominalis on the mean (GS.E.M.) (P!0.05; P!0.01 respectively). Moreover, LPA agonist concentrations of released P4, PGE2, and PGFM in cows on day 17 of * elevated the total amount of secreted P4 in the blood the estrous cycle . plasma in comparison to the control cows (Table 3; ! Treatment P4 PGE2 PGFM P 0.01), whereas Ki16425 significantly decreased the total amount of secreted P4 in the blood plasma in Control 17.4G2.8a 70.6G43.8a 975G112a LPA (1 mg) 62.6G5.8b 8129G1126b 1710G241b comparison to the saline and LPA agonist treated cows LPA (10 mg) 40.5G7.5b 3595G1085b 1282G161b (Table 2; P!0.05; P!0.01 respectively). b b a LPA (50 mg) 49.4G6.1 5317G780 1370G640 Figure 5b shows PGE concentrations in the blood LPA (100 mg) 41.1G5.4b 2191G533a 1392G266b 2 LPA (250 mg) 54.5G7.4b 570.3G123a 1410G431a plasma of saline, LPA agonist (1 mg) and LPA antagonist (Ki16425; 10 mg) treated cows. Administration of LPA a,bDifferent subscript letters within a column indicate significant agonist strongly elevated the concentrations of PGE2 in differences (P!0.05) between treated groups. * Values indicate the area under the curve (relative units, meansGS.E.M). peripheral blood during 24 h after LPA infusion in The baseline was defined on the basis of data from the first 2 h of the comparison to the saline infused cows (P!0.01). Admini- experiment. The area under the curve was measured using data from stration of Ki16425 significantly decreased the concen- 24 h of the experimental period. trations of PGE2 in peripheral blood during 24 h in comparison to the saline and LPA agonist infused cows agonist strongly elevated the concentrations of P4 in (P!0.05; P!0.01 respectively). Moreover, LPA agonist peripheral blood during 24 h after LPA infusion in ! elevated the total amount of secreted PGE2 in the blood comparison to the saline infused cows (P 0.01). plasma in comparison to the control cows (Table 3; Administration of Ki16425 significantly decreased the P!0.01), whereas Ki16425 significantly decreased the concentrations of P4 in peripheral blood during 24 h in total amount of secreted PGE2 in the blood plasma in comparison to the saline and LPA agonist treated cows (Table 2; P!0.05; P!0.01 respectively). Figure 5c shows PGFM concentrations in the blood plasma of saline, LPA agonist (1 mg) and LPA antagonist (Ki16425; 10 mg) treated cows. Administration of LPA agonist significantly elevated the concentrations of PGFM in peripheral blood during 24 h after LPA infusion in comparison to the saline infused cows (P!0.05). Admini- stration of Ki16425 did not influence the concentrations of PGFM in peripheral blood during 24 h in comparison to the saline infused cows (PO0.05) and decreased the concentrations of PGFM in peripheral blood during 24 h after Ki16425 infusion in comparison to LPA agonist infused cows (P!0.05). Moreover, LPA agonist elevated the total amount of secreted PGFM in the blood plasma in comparison to the control cows (Table 2; P!0.05). Ki16425 significantly decreased the total amount of secreted PGFM in the blood plasma in comparison to LPA agonist treated cows (Table 2; P!0.05).

Table 2 Effects of lysophosphatidic acid agonist (LPA) and LPAR1 antagonist (Ki16425) administered into the aorta abdominalis on the G mean ( S.E.M.) concentrations of released P4, PGE2, and PGFM in cows on day 17 of the estrous cycle*.

Treatment P4 PGE2 PGFM Control 17.4G2.8a 706.2G438a 975G92a LPA (1 mg) 62.6G5.8b 8129G1126b 1710G141b Ki16425 (1 mg) 7.8G1.5c 595G285c 1082G111a Figure 5 Concentrations of (a) progesterone, (b) prostaglandin E2, and (c) 13,14-dihydro,15-keto-PGF in peripheral blood plasma of cows 2a a,bDifferent subscript letters within a column indicate significant m m infused with saline (grey bars), 1 g LPA agonist (line) or 1 gLPA differences (P!0.05) between treated groups. * antagonist (dotted line). All reagents were infused into the aorta Values indicate the area under the curve (relative units, meansGS.E.M). abdominalis. Different subscript letters indicate significant differences The baseline was defined on the basis of data from first 2 h of the (P!0.05) between treated groups, as assessed by repeated-measures experiment. The area under the curve was measured using data from ANOVA test followed by Bonferroni’s multiple comparison test. 24 h of the experimental period. www.reproduction-online.org Reproduction (2009) 137 95–105

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access 100 I Woclawek-Potocka and others

Table 3 Primers used for real-time PCR.

GenBank Gene Primer sequences (accession number) Position PCR products (bp) 0 0 PLD2 (Bos taurus) 5 -GCTTCAGCGTGATTCTAGGG-3 BC123547 2537–2642 106 50-GCATTGCTCTCAGCTGTGTC-30 PLA2G1B (Bos taurus) 50-AGGTGCACAACTTCATGCTG-30 BC134610 1544–1650 107 50-GGCATCCAATTCGTCTTCAT-30 LPAR1 (Bos taurus) 50-GGTGGGGTGTGAGAAAGAGA-30 U48236 1191–1336 146 50-AAAAGGAATGGGAGCAGGAT-30 PGES (Bos taurus) 50-AGGACGCTCAGAGACATGGA-30 NM174443 154–295 142 50-TTCGGTCCGAGGAAAGAGTA-30 PGFS (Bos taurus) 50-GATCAAAGCGATTGCAGACA-30 S54973 672–784 113 50-CAATGCGTTCAGGTGTCACT-30 GAPDH (Bos taurus) 50-CACCCTCAAGATTGTCAGCA-30 BC102589 492–594 103 50-GGTCATAAGTCCCTCCACGA-30

Discussion Im et al. 2000, Contos et al. 2002, Noguchi et al. 2003, Gardell et al. 2006). Although LPAR3 expression has In the present study, we found that LPA is locally produced been recently shown to be connected with PG synthesis and released from the bovine endometrium. We found that and to play a crucial role in implantation in mice (Ye the total concentration of LPA in the blood plasma taken et al. 2005), we found only LPAR1 mRNA expression in from the uterine vein on days 17–19 of the estrous cycle for the bovine endometrial tissue (Woclawek-Potocka et al. 24 h was significantly higher in the blood plasma from the 2007). During the estrous cycle, LPAR1 mRNA jugular vein. We measured total LPA concentration, which expression increased from early to late luteal stage and is probably much higher than the concentration of LPA that reached the highest level at late luteal stage, suggesting is free and thus exerts some biological roles in the body. that LPA might be involved in the first events of However, there is no possible way to measure free- luteolysis. This LPA contribution in the luteolysis may unconjugated LPA in the body fluids and tissue. Moreover, be explained by its indirect stimulation of luteolytic LPA in the blood plasma is mainly bound to albumin or PGF2a synthesis through the influence on prostaglandin some other lipid binding protein (Aoki 2004)andthuswe like 2 synthase (PGFSL2) activity – the enzyme with might suppose that the whole amount of LPA measured in 9-keto reductase activity, responsible for the conversion our study after enzymatic hydrolysis is not active. We also of PGE2 to PGF2a (Madore et al. 2003, Weems et al. found high concentrations of LPA in the endometrial tissue. 2006). However, this hypothesis needs further exami- Although, LPA concentration in the bovine endometrium nation. The present study shows that LPA is mainly did not significantly differ either during the estrous cycle or involved in the luteotropic mechanism during early early pregnancy, it was significantly higher on days 17–19 pregnancy in cattle. Total LPAR1 gene expression on of pregnancy than on days 17–19 of the estrous cycle. days 8–10 of pregnancy was lower than that on days 17– Moreover, PLD2 and PLA2G1B gene expression indicates 19 of pregnancy. Moreover, LPAR1 gene expression was that there is the possibility for LPA to be synthesized in the higher on days 8–10 of pregnancy than on days 8–10 of bovine endometrium. PLD2 was expressed at the constant the estrous cycle. These results seem to be consistent level during estrous cycle and early pregnancy, however, with the results of the first two experiments, where we PLA2G1B expression revealed the highest level on days showed higher LPA concentration in uterine vein than in 17–19 of pregnancy. Thus, LPA might be synthesized at all jugular vein and the highest LPA concentrations in the stages of the estrous cycle and pregnancy with the endometrial tissue on the days 17–19 of pregnancy. The preference to days 17–19 of pregnancy. High LPA role of LPA in early pregnancy in cow may be similar to concentration, mRNA expression for its receptor its role in humans in that LPA at the time of implantation (LPAR1), PLD2 and PLA2G1B during the estrous cycle induces blastocyst differentiation via a mechanism C and pregnancy might suggest that LPA plays an autocrine connected with Ca2 transport (Liu & Armant 2004). and/or paracrine role in the bovine endometrium during Moreover, higher LPAR1 mRNA expression at each stage the estrous cycle and early pregnancy. of early pregnancy than at each stage of the estrous cycle The pleiotropic roles of LPA in reproductive physio- agrees with the data obtained by Gaits et al. (1997) who logy and pathology are demonstrated by both the also proved that plasma LPA concentrations were much regulated expression of its receptors (Contos & Chun higher in pregnant women than in cyclic women. 2001, Moller et al. 2001, Contos et al. 2002, Ye et al. In the present study, we demonstrated how PG 2005) and the increased amount of LPA in body fluids synthesizing enzymes (PGES and PGFS) mRNA are (Tokumura et al. 2002, 2007). LPA exerts its biological expressed in the bovine endometrium and whether effects via four G-protein coupled membrane receptors they are correlated with LPAR1 expression throughout (LPAR1, LPAR2, LPAR3, and LPAR4; Bandoh et al. 1999, the estrous cycle and early pregnancy. Our findings that

Reproduction (2009) 137 95–105 www.reproduction-online.org

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access LPA influence on the bovine reproduction 101

PGES mRNA expression was much higher on days 2–4 of This suggests LPA influence on both endometrium and the cycle than on days 8–10 and days 17–19 (Fig. 3b) the CL. LPA-induced P4 stimulation suggests its influ- agree with the data of Arosh et al. (2002), who found ence not only on the endometrium – via LPAR1- high PGES expression at the beginning of the estrus dependent PGE2 stimulation (Woclawek-Potocka et al. cycle. In our study, we found that the high PGES 2007), but also a direct effect on the secretory functions expression at the beginning of the cycle may be of steroidogenic CL cells. However, this supposition responsible for its luteotropic action on the early needs further, thorough in vitro examination. The above developmentoftheCL.Moreover,PGES mRNA data suggest that in ruminants, the possible LPA expression was much higher at each stage of early dependent development and maintenance of the CL pregnancy than at the each stage of the estrous cycle. during the estrous cycle and establishment of pregnancy These findings suggest that much more PGE2 is might depend on the LPA influence on the CL and on the synthesized during early pregnancy than at any stage of balance of luteolytic PGF2a and luteotropic PGE2 rather the estrous cycle. We also showed that on days 8–10 of than absolute amounts of each prostaglandin. the cycle when PGES mRNA expression is low, there is In conclusion, the present study proved that LPA may an increase in mRNA expression for luteolytic PGF2a be locally produced and released in the reproductive synthase (PGFS). These data suggest that on days 8–10 of tract of cows including endometrium. Moreover, LPAR1 the cycle the enzymatic mechanisms that change the gene expression in the endometrium during the estrous ratio of the two main PGs toward luteolytic PGF2a are cycle and early pregnancy indicates that LPA may play being turned on. A definite change of this ratio takes autocrine and/or paracrine roles in the bovine uterus. In place on the 12th day of the estrous cycle, when the P4 the cow, LPA stimulates P4 and PGE2 secretion and its level is the highest but the cyclic CL is already receptor gene expression (LPAR1) are positively corre- programmed for luteolysis (McCracken et al. 1999). lated with the expression of the enzyme responsible for The high PGFS mRNA expression on days 8–10 of the luteotropic PGE2 production (PGES) in endometrium. estrous cycle (Fig. 3c) agrees with the data of Madore Finally, LPA in the bovine reproductive tract may et al. (2003). Interestingly, we found higher PGFS mRNA indirectly (via endometrium) or directly support CL expression on days 8–10 than on days 17–19 of action via the stimulation of P4 synthesis and the pregnancy. This finding might explain PGF2a-dependent increase of PGE2/PGF2a ratio. It suggests that LPA may motor activity of the uterus at this time, when the serve as an important factor in the maintenance of early blastocyst moves around to find the most appropriate pregnancy in cow. However, this final supposition is now place for implantation. under in vivo and in vitro examinations. We showed a positive correlation between LPAR1 and PGES mRNA expression at early pregnancy. Moreover, we found that LPAR1 mRNA expression was negatively correlated with the expression of mRNA for PGFS and Materials and Methods that PGES expression was negatively correlated with the Animals expression of PGFS at early pregnancy. These corre- All animal procedures were approved by the Local Animal lations may explain why PGE2 and LPA act similarly, and PGES with PGFS may act differently during early Care and Use Committee in Olsztyn, Olsztyn, Poland pregnancy in cows. The interactions also describe, on (Agreement No. 34/2005/N). For all experiments, normally cycling Holstein/Polish Black the enzymatic level, the mechanisms responsible for Z changing the ratio of luteotropic PGE to luteolytic and White (75/25% respectively) cows (4–6 lactations; n 89) 2 were chosen. The animals were eliminated by the owner from PGF a during early pregnancy in cows as well as suggest 2 two dairy cow herds (Years 2004–2006) because of their low the influence of LPA on this process. milk production. The estrus of the cows was synchronized In the last experiment, we wanted to confirm the using implants of a progesterone analog (Crestar, Intervet, possible role of LPA and LPAR1 antagonist on the Holland) with additional injection of an analog of PGF2a secretion of progesterone and prostaglandins. The dose (dinoprost, Dinolytic; Upjohn- Pharmacia N V S A, Belgium), m of 1 g LPA stimulated P4 and PGE2 concentration in the as recommended by Bah et al. (2006) for the estrus blood, and had weaker effect on PGFM level (Tables 1 synchronization of multiparous cows. The onset of the and 2 area under the curve). The LPA-induced increase estrus was determined by observing the signs of estrus (i.e. of PGF2a secretion was much less than the LPA-induced vaginal mucus, standing behavior), and was confirmed by a increase of PGE2 synthesis (1.75 vs 11.5 times). More- veterinarian via ultrasonography (USG) examination using over, we showed that the inhibition of endogenous LPA Draminski Animal profi Scanner (Draminski Electronics in action via the infusion of LPAR1 receptor antagonist Agriculture, Olsztyn, Poland; www.draminski.com) and per (Ki16425) caused the decrease of P4 and PGE2 rectum examination. Only the cows with behavioral signs of concentrations. Summarizing this part of the study, LPA estrus were chosen for the study after positive USG and per had strong effect on P4 and PGE2 concentrations in rectum examination (nZ77). The estrus was taken as day 0 the blood, and much weaker effect on PGFM level. of the estrous cycle. www.reproduction-online.org Reproduction (2009) 137 95–105

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access 102 I Woclawek-Potocka and others

Experimental procedure Based on the above tests, in the early pregnancy groups, we excluded 4 uteri out of 12 (days 8/10) and further 2 out of 5 (days Experiment 1: LPA concentration in uterine and jugular vein 17/19) because of the lack of pregnancy. The aim of this experiment was to confirm whether LPA is 1-oleoyl-LPA contained in tissues was extracted and locally produced and released from the bovine endometrium. measured according to the procedures described by Saulnier- LPA concentrations were measured in the extracts from blood Blache et al. (2000). plasma taken from the jugular and uterine vein. The catheters were inserted into respective vessels according to the Experiment 3: Correlations between LPAR1, PGES, and PGFS procedures described by Skarzynski et al. (2003) for jugular mRNA expression in the bovine endometrium during estrous vein and by Acosta et al. (2000) for ovarian vein on the day 16 cycle and early pregnancy of the estrous cycle. In the procedure for uterine vein, a lateral The aim of the study was to examine whether there are any laparotomy was performed. At surgery a catheter was inserted correlations between LPA receptor, PGES, and PGFS mRNA into the uterine vein ipsilateral to the functional CL and sutured expression in the bovine endometrium during estrous cycle and (Acosta et al. 2000). The blood collections started 24 h after the early pregnancy. Gene expression for LPAR1, and terminal PG surgery, at noon on the day 17 of the cycle and experiment was synthesizing enzymes (PGES and PGFS) were quantitatively finished after 24 h (at noon on the day 18 of the cycle). Blood measured by real-time PCR in the tissues used previously for samples were collected into tubes with 5 ml EDTA, 1% aspirin the measurement of LPA concentration and PLD and solution (pH 7.3) for 24 h (5 times: at hour 0, after 6, 12, 18, 2 PLA2G1B mRNA expression (experiment 2). and 24 h). The blood plasma was immediately separated by centrifugation (2000 g, 10 min at 4 8C) and stored at K20 8C Experiment 4:The effect of LPA and LPAR1 antagonist (Ki until the time of lipid extraction. 1-oleoyl-LPA contained in 16425) on the secretion of progesterone and prostaglandins plasma was extracted and measured according to the procedures described by Saulnier-Blache et al. (2000). Preliminary study. Twenty four cows were used to choose the effective dose of an LPA agonist. For infusion of either saline or Experiment 2: LPA production in the bovine endometrium different doses of LPA agonist, a catheter was inserted into the during the estrous cycle and early pregnancy posterior aorta abdominalis through the coccygeal artery on The aim of this experiment was to compare whether there are day 16 of the estrous cycle according to the procedure changes in LPA production in the bovine endometrium during described previously (Skarzynski et al. 2003). The animals estrous cycle and early pregnancy. LPA concentrations were were premedicated with xylazine at a dose of 25–30 mg/ measured in the extracts from endometrial tissues. Bovine uteri animal i.m. (Sedazin, Biowet Pulawy, Poland) and local were obtained at a local slaughterhouse (Ubojnia Zwierzat epidural anesthesia was induced by injecting 4 ml of 2% Rzeznych, Gucin, Lukta, Poland) within 20 min of exsanguina- procaine hydrochloride (Polocainum Hydrochloricum, Biowet tion and were transported on ice to the laboratory within 40 min. Drwalew, Poland) between the 1st and 2nd coccygeal Before slaughter, the cows were divided into two groups, i.e. vertebrae. The tip of the cannula was positioned in the aorta pregnant and cyclic animals. The animals chosen for the 60–65 cm ahead of the point of insertion, just cranial to the pregnant group underwent artificial insemination with the origin of the ovarian artery and caudal to the renal artery semen of the same bull. The tissues for this experiment were (Skarzynski et al. 2003). This placement allowed infused assigned for the following days of the estrous cycle and reagents to be transported by the blood stream directly into pregnancy: days 2–4 of the cycle (nZ5), days 8–10 of the the reproductive tract. A second catheter was inserted into the cycle (nZ7) and pregnancy (nZ12), days 17–19 of the cycle jugular vein for frequent collection of blood samples (nZ4) and pregnancy (nZ5). Gene expression for the enzymes (Skarzynski et al. 2003). involved in LPA synthesis (PLD2 and PLA2G1B) was quan- The cows one day after the surgery were infused into the titatively measured by real-time PCR in tissues. For real-time a. abdominalis with saline (nZ4; control group) or five different PCR, the small pieces of endometrial tissue were snap frozen in doses of LPA agonist (1, 10, 50, 100, 250 mg; nZ4 for each liquid nitrogen, before storage in K808C. Estimation of the dose). Peripheral blood samples were collected in a tube with stages of the estrous cycle was confirmed by macroscopic 5 ml EDTA, 1% aspirin solution (pH 7.3) from a jugular vein observation of the ovaries and uterus (Miyamoto et al. 2000). frequently for 24 h (18 times, beginning 2 h before the The pregnancy was confirmed by flushing the uterus for the infusions). The blood plasma was immediately separated by collection of the viable embryo as described previously (Leung centrifugation (2000 g, 10 min at 4 8C) and stored at K20 8C. et al. 2000). Moreover, the flushed uterine fluid was collected Concentrations of P4, PGE2, and 13,14-dihydro,15-keto- and the antiviral activity of interferon-t was measured, at least at prostaglandin F2a (PGFM) in the plasma samples were the later phase of early pregnancy (days 17–19), as recently measured. described (Gierek et al.2006). Moreover, we tested the To confirm the possible role of LPA and LPAR1 antagonist on sensitivity of cultured pure endometrial epithelial cells (days the secretion of progesterone and prostaglandins, 12 cows on 17–19) to oxytocin (OT) treatment. Only non-pregnant bovine day 17 of the estrous cycle were infused into the a. abdominalis endometrium, at the late luteal and follicular phase of the cycle, with saline (nZ4; control group), LPA agonist (1 mg; this group Z is sensitive to OT treatment and PGF2a release (Miyamoto et al. was taken from the preliminary study; n 4) or LPAR1 2000). Therefore, only the uteri that responded properly for the antagonist (Ki16425; 10 mg; nZ4). Peripheral blood samples above tests were chosen for the respective pregnancy groups. were collected from a jugular vein frequently for 24 h

Reproduction (2009) 137 95–105 www.reproduction-online.org

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access LPA influence on the bovine reproduction 103

(18 times, beginning 2 h before the infusions), as described in Real-time PCR was performed with an ABI Prism 7300 the preliminary study. The blood plasma was immediately sequence detection system using Power SYBR Green PCR separated by centrifugation (2000 g, 10 min at 4 8C) and stored master mix (Applied Biosystems, Applera, Warsaw, Poland; K at 20 8C. Concentrations of P4, PGE2 and 13,14-dihydro,15- #4367659). The PCRs were performed in 96-well plates. Each keto-prostaglandin F2a (PGFM) in the plasma samples were PCR well (25 ml) contained 2.5 ml of diluted RT product, measured. 200 pM forward and reverse primers each, and 12.5 ml SYBR Green PCR master mix. As standard curves, serial dilutions of appropriate cDNA were used for gene quantification. For Lipid extraction from endometrial tissue and blood quantification of the mRNA expression levels, the primer plasma and in vitro acylation of LPA length (20 bp) and GC-contents of each primer (50–60%) were 1-oleoyl-LPA or lipids contained in 100 mg of tissue and 100 ml selected. Real time PCR was performed under the following conditions: 95 8C for 10 min, followed by 40 cycles of 94 8C for of plasma were extracted with one volume of 1-butanol 15 s, 55.6 8C (for GAPDH) or 56.3 8C (for LPAR1), or 56 8C (for according to the procedures thoroughly described by Saulnier- PGES and PLD ) or 55.9 8C (for PLA2G1B) or 55.7 8C (for PGFS) Blache et al. (2000). After evaporation, lipids were incubated in 2 for 28 s and 72 8C for 15 s. Each PCR was followed by obtaining the presence of semi-purified recombinant rat LPA acyl- melting curves by stepwise increase in the temperature from transferase (LPAAT) and [14C]oleoyl-CoA as described by 60 8Cto958C to ensure single product amplification. In order Saulnier-Blache et al. (2000). The products of the reaction to exclude the possibility of genomic DNA contamination in were separated by two-dimensional TLC and autoradiographed. the RNA samples, the reactions were also run either on blank- only buffer samples or in the absence of the reverse Hormone determination transcriptase enzyme. The specificity of the PCR products for all examined was confirmed by gel electrophoresis and The concentrations of P4, PGFM, and PGE2 in the plasma by sequencing. The obtained data were normalized on the samples were determined with direct EIAs, as described basis of GAPDH mRNA content. previously (Wocławek-Potocka et al. 2005). The anti-P4 serum was donated by Prof S Okrasa, University of Warmia and Mazury in Olsztyn. The anti-PGFM serum (WS4468-5) was Statistical analysis donated Dr W J Silvia, University of Kentucky, Lexington, KY, In Experiment 1, total concentration of LPA in the jugular and USA. The anti PGE2 serum was purchased from Sigma; #P5164. uterine veins during the 24 h of the experiment was expressed The P4 standard curve ranged from 0.39 pg/ml to 25 ng/ml and as the area under the curve (YZ0 was taken as a baseline) and the effective dose for 50% inhibition (ID50) of the assay was analyzed by the Student’s t test. The data are shown as meansG 2.85 ng/ml. The intra- and inter-assay coefficients of variation S.E.M.(nZ3). In experiments 2 and 3, all analyses were done averaged 6.6 and 8.4% respectively. The PGFM standard curve using one way ANOVA tests followed by Bonferroni’s Multiple ranged from 32.5 to 8000 pg/ml and the ID50 of the assay was Comparison Test (GraphPad PRISM; P!0.05 was considered 315 pg/ml. The intra- and inter-assay coefficients of variation significant). Additionally in experiment 3, different gene were on average 7.6 and 10.4% respectively. The PGE2 expression among the days was modeled by linear regression standard curve ranged from 0.07 to 20 ng/ml and the ID50 of (GraphPad PRISM). In experiment 4, P4, PGE2, and PGFM the assay was 1.25 ng/ml. The intra- and inter-assay coefficients concentrations in the jugular plasma were analyzed using a of variation were on average 6.9 and 9.7% respectively. repeated measure design approach described previously (repeated measures ANOVA with Bonferroni’s Multiple Comparison Test; GraphPad PRISM; Skarzynski et al. 2003). Total RNA extraction, RT and real-time PCR The total amounts of released P4, PGE2, and PGFM were shown Total RNA was extracted from endometrial tissues using by the area under the curve (relative units; Table 1) and were TRIZOL according to the manufacturer’s instructions. RNA analyzed using one-way ANOVA followed by Bonferroni’s samples were stored at K80 8C. Before use, RNA was verified Multiple Comparison Test (GraphPad PRISM). by spectrophotometric measurement and agarose gel electro- phoresis. Two micrograms of each sample of total RNA were Declaration of interest reverse transcribed using a ThermoScript RT-PCR System (Invitrogen, Alab; #11146-016). The RT reaction was per- I declare that there is no conflict of interest that could be formed in total reaction volume of 20 ml, following manufac- perceived as prejudicing the impartiality of the research turer’s instructions. RT products were stored at K20 8C until reported. real-time PCR amplification. The expression of mRNA for all examined genes was Funding conducted by real-time PCR using specific primers for PLD2, PLA2G1B, LPAR1, PGES,andPGFS.Briefly,GAPDH This work was supported by the Grants-in-Aid for Scientific expression was used as an internal control. The primers were Research from the Polish Ministry of Scientific Research and chosen using an online software package (http://www-genome. Information Technology (KBN 2P06K 003 30) and the wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The primers of Japanese-Polish Joint Research Project under the agreement all target genes are given in Table 3. between JSPS and PAS. www.reproduction-online.org Reproduction (2009) 137 95–105

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access 104 I Woclawek-Potocka and others

Acknowledgements Gardell S, Dubin A & Chun J 2006 Emerging medical roles for lysophospolipid signaling. Trends in Molecular Medicine 12 65–75. The authors are indebted to Dr James Raymond for critical Gierek D, Baczynska D, Ugorski M, Bazer F, Kurpisz M, Bednarski T, review of this manuscript and English correction. We thank Dr Gorczykowski M & Chelmonska-Soyta A 2006 Differential effect of IFN- W J Silvia, University of Kentucky, Lexington, USA, for PGFM tau on proliferation and distribution of lymphocyte subsets in one-way mixed lymphocyte reaction in cows and heifers. Journal of Reproductive antisera; Dr Stanislaw Okrasa of University of Warmia and Immunology 71 126–131. Mazury for progesterone antisera; and Dainippon Pharma- Goetzl EJ, Dolezalova H, Kong Y, Hu JL, Jaffe RB, Kalli KR & Conover CA ceutical Co., Ltd, Osaka, Japan for recombinant human TNF 1999 Distinctive expression and functions of the type 4 endothelial (HF-13). The authors are grateful to Draminski Electronics in differentiation gene-encoded G protein-coupled receptor for lysopho- Agriculture (Olsztyn, Poland, www.draminski.com) for their sphatidic acid in ovarian cancer. Cancer Research 59 5370–5375. Hinokio K, Yamano S, Nakagawa K, Iraharaa M, Kamada M, Tokumura A & excellent cooperation and possibility to test and use the USG Aono T 2002 Lysophosphatidic acid stimulates nuclear and cytoplasmic scanner. The authors also thank GENNUAS France, the owner maturation of golden hamster immature oocytes in vitro via cumulus of Animal Farm ‘Wroblik’ (Spo´łka Rolna ‘Wroblik’ Sp. z o.o, cells. Life Sciences 70 759–767. Lidzbark Warminski, Poland) for their excellent cooperation Im DS, Heise C, Harding M, George S, Theodorescu D & Lynch K 2000 and agreement to let us use the animals for the present Molecular cloning and characterization of a lysophosphatidic acid receptor, Edg-7, expressed in prostate. Molecular Pharmacology 57 experiment. Two colleagues from the Reproductive Immuno- 753–759. logy Department – Anna Korzekwa and Marta Siemieniuch Kaminska K, Wasielak M, Bogacka I, Blitek M & Bogacki M 2008 must be also mentioned in this section for their excellent Quantitative expression of lysophosphatidic acid receptor 3 gene in assistance at the in vivo part of the study. Lastly, the authors are porcine endometrium during the periimplantation period and estrous cycle. Prostaglandins and other Lipid Mediators 85 26–32. indebted to Estelle Wanecq who performed LPA quantification. Kim KS, Sengupta S, Berk M, Kwak YG, Escobar PF, Belinson J, Mok SC & Xu Y 2006 Hypoxia enhances lysophosphatidic acid responsiveness in ovarian cancer cells and lysophosphatidic acid induces ovarian tumor metastasis in vivo. Cancer Research 66 7983–7990. References Kotwica J, Skarzynski D, Mlynarczuk J & Rekawiecki R 2003 Role of prostaglandin E2 in basal and noradrenaline-induced progesterone Acosta TJ, Ozawa T, Kobayashi S, Hayashi K, Ohtani M, Kraetzl WD, secretion by the bovine corpus luteum. Prostaglandins & Other Lipid Sato K, Schams D & Miyamoto A 2000 Periovulatory changes in the local Mediators 70 351–359. release of vasoactive peptides, prostaglandin f(2alpha), and steroid Leung ST, Derecka K, Mann GE, Flint APF & Wathes DC 2000 Uterine hormones from bovine mature follicles in vivo. Biology of Reproduction lymphocyte distribution and interleukin expression during early 63 1253–1261. pregnancy in cows. Journal of Reproduction and Fertility 119 25–33. Aoki J 2004 Mechanisms of lysophosphatidic acid production. Seminars in Liu Z & Armant DR 2004 Lysophosphatidic acid regulates murine blastocyst Cell and Developmental Biology 15 477–489. development by transactivation of receptors for heparin-binding EGF-like Arosh JA, Parent J, Chapdelaine P, Sirois J & Fortier MA 2002 Expression of growth factor. Experimental Cell Research 296 317–326. cyclooxygenases 1 and 2 and prostaglandin E synthase in bovine Madore E, Harvey N, Parent J, Chapdelaine P,Arosh JA & Fortier MA 2003 An endometrial tissue during the estrous cycle. Biology of Reproduction 67 aldose reductase with 20 a-hydroxysteroid dehydrogenase activity is most 161–169. likely the enzyme responsible for the production of prostaglandin F in the Asselin E, Goff AK, Bergeron H & Fortier MA 1996 Influence of sex steroids 2a bovine endometrium. Journal of Biological Chemistry 278 11205–11212. on the production of prostaglandins F2a and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Magness RR, Huie JM, Hoyer GL, Huecksteadt TP, Reynolds LP, Seperich GJ, Biology of Reproduction 54 371–379. Whysong G & Weems CW 1981 Effect of chronic ipsilateral or contralateral Bah MM, Acosta TJ, Pilawski W, Deptula KM, Okuda K & Skarzynski DJ intrauterine infusion of prostaglandin E2 (PGE2) on luteal function of unilaterally ovariectomized ewes. Prostaglandins and Medicine 6 2006 Role of intraluteal prostaglandin F2a, progesterone and oxytocin in basal and pulsatile progesterone release from developing bovine corpus 389–401. luteum. Prostaglandins & Other Lipid Mediators 80 218–229. McCracken JA, Custer EE & Lamsa JC 1999 Luteolysis: a neuroendocrine- Bandoh M, Aoki J, Hosono H, Kobayashii S, Murakami-Murofushii K, mediated event. Physiological Reviews 79 263–323. Arai H & Inoue K 1999 Molecular cloning and characterization of a Milvae RA, Hinckley ST & Carlson JC 1996 Luteotropic and luteolytic novel human G-protein-coupled receptor, EDG7, for lysophosphatidic mechanisms in the bovine corpus luteum. Theriogenology 45 1327–1349. acid. Journal of Biological Chemistry 274 27776–27785. Miyamoto Y, Skarzynski DJ & Okuda K 2000 Is tumor necrosis factor-a a Contos J & Chun J 2001 The mouse lpA3/Edg7 lysophosphatidic acid trigger for the initiation of prostaglandin F2a release at luteolysis in cattle? receptor gene: genomic structure, chromosomal localization, and Biology of Reproduction 62 1109–1115. expression pattern. Gene 267 243–253. Moller T, Contos J, Musante DB, Chun J & Ransom BR 2001 Expression and Contos J, Ishii I, Chun J & Ye X 2002 Characterization of LPA2 (edg4) and function of lysophosphatidic acid receptors in cultured rodent microglial LPA1/LPA2 (edg2 and edg4) lysophosphatidic acid receptor knockout cells. Journal of Biological Chemistry 276 25946–25952. mice: signaling deficits without obvious phenotypic abnormality Moolenaar WH 1995 Lysophosphatidic acid, a multifunctional phospholid attributable to LPA2. Molecular and Cellular Biology 22 6921–6929. messenger. Journal of Biological Chemistry 270 12949–13001. Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T & Wang H 2004 Noguchi K, Ishii S & Shimizu T 2003 Identification of p2y9/GPR23 as a Molecular cues to implantation. Endocrine Reviews 25 341–373. novel G protein-coupled receptor for lysophosphatidic acid, structurally Eder AM, Sasagawa T, Mao M, Aoki J & Mills GB 2000 Constitutive and distant from the Edg family. Journal of Biological Chemistry 278 lysophosphatidic acid (LPA)-induced LPA production: role of phospho- 25600–25606. lipase D and phospholipase A2. Clinical Cancer Research 6 2482–2491. Pratt BR, Butcher RL & Inskeep EK 1977 Antiluteolytic effect of the Exton JH 2002 Regulation of phospholipase D. FEBS Letters 531 58–61. conceptus and of PGE2 in ewes. Journal of Animal Science 45 784–791. Fukushima N, Weiner JA, Contos JA, Kim K & Chun J 2002 Lysopho- Pustilnik TB, Estrella V, Wiener JR, Mao M, Eder A, Watt MA, Bast RC & sphatidic acid influences the morphology and motility of young, Mills GB 1999 Lysophosphatidic acid induces urokinase secretion by postmitotic cortical neurons. Molecular and Cellular Neurosciences 20 ovarian cancer cells. Clinical Cancer Research 5 3704–3710. 271–282. Saulnier-Blache JS, Girard A, Simon MF, Lafontan M & Valet P 2000 A Gaits F, Fourcade O, Le Belle F, Guegruen G, Gaige B, Gassama-Diagne A, simple and highly sensitive radioenzymatic assay for lysophosphatidic Fauvel J, Salles JP, Mauco G, Simon MF et al. 1997 Lysophosphatidic acid acid quantification. Journal of Lipid Research 41 1947–1951. as a phospholipid mediator: pathways of synthesis. FEBS Letters 410 Shah BH & Catt KJ 2005 Roles of LPA3 and COX-2 in implantation. Trends 54–58. in Endocrinology and Metabolism 9 397–399.

Reproduction (2009) 137 95–105 www.reproduction-online.org

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access LPA influence on the bovine reproduction 105

Shen Z, Belinson J, Morton RE, Xu Y & Xu Y 1998 Phorbol 12-myristate than phytoestrogens themselves in increasing prostaglandin F2a synthesis 13-acetate stimulates lysophosphatidic acid secretion from ovarian and via prostaglandin F2a synthase-like 2 stimulation in bovine endometrium. cervical cancer cells but not from breast or leukemia cells. Gynecologic Prostaglandins & Other Lipid Mediators 78 202–217. Oncology 71 364–368. Woclawek-Potocka I, Brzezicka E, Siemieniuch M, Komiyama J, Okuda K & Skarzynski DJ, Bah MM, Deptula KM, Woclawek-Potocka I, Korzekwa A, Skarz˙yn´ski DJ 2007 Prostaglandin synthases and lysophosphatidic acid Shibaya M, Pilawski W & Okuda K 2003 Roles of tumor necrosis factor-a receptor type 1 expression in the bovine endometrium during the estrous of the estrous cycle in cattle: an in vivo Study. Biology of Reproduction 69 cycle and early pregnancy. The 11 th Annual Conference of European 1907–1913. Society for Domestic Animal Reproduction. Celle-Germany, 21st to Snitko Y, Yoon ET & Cho W 1997 High specificity of human secretory class II 22nd September 2007. phospholipase A2for phosphatidic acid. Biochemical Journal321 737–741. Xie Y, Gibbs TC, Mukhin YV & Meier KE 2002 Role for 18:1 lysopho- Tokumura A, Kanaya Y, Miyake M, Yamano S & Fukuzawa K 2002 Increased sphatidic acid as an autocrine mediator in prostate cancer cells. production of bioactive lysophosphatidic acid by serum lysopho- Journal of Biological Chemistry 277 32516–32526. spholipase D in Human pregnancy. Biology of Reproduction 67 Ye X, Hama K, Contos JA, Suzuki H, Amano T, Arai H, Aoki J & Chun J 2005 1386–1392. LPA3-mediated lysophosphatidic acid signalling in embryo implantation Tokumura A, Kume T, Fukuzawa K, Tahara M, Tasaka K, Aoki J, Arai H, and spacing. Nature 435 104–108. Yasuda K & Kanzaki H 2007 Peritoneal fluids from patients with certain gynecologic tumor contain elevated levels of bioactive lysophospholi- pase D activity. Life Sciences 80 1641–1649. Received 16 May 2008 Weems CW, Weems YS & Randel RD 2006 Prostaglandins and First decision 17 July 2008 reproduction in female farm animals. Veterinary Journal 171 206–228. Wocławek-Potocka I, Okuda K, Acosta TJ, Korzekwa A, Pilawski W & Revised manuscript received 16 September 2008 Skarz˙yn´ski DJ 2005 Phytoestrogen metabolites are much more active Accepted 1 October 2008

www.reproduction-online.org Reproduction (2009) 137 95–105

Downloaded from Bioscientifica.com at 10/01/2021 01:11:30AM via free access