Lysophosphatic acid modulates prostaglandin secretion in the bovine uterus

in Reproduction

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. Expression of the receptor for LPA (LPAR1) was positively correlated with the expression of PGE2 synthase (PGES) and negatively correlated with the expression of PGF synthase (aldose reductase with 20 α-hydroxysteroid dehydrogenase activity – PGFS) during early pregnancy. In vivo LPA induced 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 gene expression in the endometrium during the estrous cycle and early pregnancy indicates that LPA may play autocrine and/or paracrine roles in the bovine uterus. LPAR1 gene expression 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/PGF2α ratio. It suggests that LPA may serve as an important factor in the maintenance of early pregnancy in cow.

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. Expression of the receptor for LPA (LPAR1) was positively correlated with the expression of PGE2 synthase (PGES) and negatively correlated with the expression of PGF synthase (aldose reductase with 20 α-hydroxysteroid dehydrogenase activity – PGFS) during early pregnancy. In vivo LPA induced 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 gene expression in the endometrium during the estrous cycle and early pregnancy indicates that LPA may play autocrine and/or paracrine roles in the bovine uterus. LPAR1 gene expression 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/PGF2α ratio. It suggests that LPA may serve as an important factor in the maintenance of early pregnancy in cow.

Introduction

Lysophosphatidic acid (LPA) is a simple phospholipid with a vast variety of physiological and pathological actions on many cell types, such as cell proliferation and differentiation (Goetzl et al. 1999, Pustilnik et al. 1999), cytoskeletal rearrangement (Moolenaar 1995), cell-to-cell interactions (Fukushima et al. 2002), and tumorigenesis (Kim et al. 2006). In the cells, LPA production is triggered by various agonists (Phorbol 12-myristate 13-acetate (Shen et al. 1998), intracellular calcium ions (Ca2+), ionophore, bombesin (Xie et al. 2002), LPA itself (Eder et al. 2000, etc.) and is accompanied by activation of phospholipase D (PLD2). Phospholipase D catalyzes the conversion of diacyl phospholipids to phosphatidic acid (PA; Exton 2002). PA is then converted to LPA by PLA1 and PLA2G1B (Eder et al. 2000, Aoki 2004). However, Snitko et al. (1997) demonstrated a preference of PLA2G1B for LPA. In mammals, LPA exerts its action via four high-affinity G-protein-coupled receptor types: LPAR1, LPAR2, LPAR3 and the recently identified LPAR4 (Bandoh et al. 1999, Im et al. 2000, Noguchi et al. 2003). However, the precise roles of each receptor in various LPA functions and various kinds of tissues are not known. In humans, LPA concentration gradually increases between weeks 5 and 40 of pregnancy (Tokumura et al. 2002). LPA stimulates oocyte maturation (Hinokio et al. 2002), preimplantation development of embryos to the blastocyst stage (Liu & Armant 2004), and embryo transport in the oviduct (Dey et al. 2004) in women. LPA3-deficient mice showed delayed implantations and a reduced number of implantation sites, despite comparable numbers of blastocysts available for implantation (Ye et al. 2005). The implantation sites in the LPA3-deficient uteri were clustered in the uterine segments proximal to the cervix. Moreover, embryos isolated from LPA3-deficient uteri were always smaller than those from the wild type. This study also proved that the administration of prostaglandin (PG) E2 and PGI2 could partially correct implantation defects in these animals.

In ruminants, PGs are crucial components in the regulation of estrous cycle and early pregnancy. Prostaglandin F is the major luteolytic agent, whereas PGE2 has luteoprotective and anti-luteolytic properties (Asselin et al. 1996, McCracken et al. 1999). Therefore, achieving an optimal PGF 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 before that in cattle, unlike in mice and pigs (Ye et al. 2005, Kaminska et al. 2008), LPA acts via LPAR1 receptor in the endometrium (Woclawek-Potocka et al. 2007). In the present study, we checked whether LPA may be locally produced and released from the bovine endometrium and examined if there exists gene expression for the enzymes involved in LPA synthesis (PLA2G1B and PLD2) in the bovine endometrium. However, the main goal of this study was to determine whether LPA regulates PG synthesis and secretion in the bovine endometrium in vivo. We also checked whether gene expression of LPA receptor is correlated with gene expression of the terminal enzymes responsible for PG production (PGES and aldose reductase with 20 α-hydroxysteroid dehydrogenase activity – PGFS) in the estrous cycle and early pregnancy in the bovine endometrium (Madore et al. 2003).

Results

Experiment 1. LPA concentration in the uterine and jugular vein

LPA concentration in the blood plasma from uterine and jugular vein

Figure 1 shows LPA concentration in the blood plasma taken from cows on days 17–19 of the estrous cycle, (at the hour 0, 6, 12, 18, and 24) from jugular and uterine vein. The total amount of LPA in the blood plasma taken from the jugular vein was significantly lower (79.54±18.0; arbitrary units, area under the curve) than that in the blood plasma from the uterine vein (541.2±90.0; P<0.05).

Figure 1

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Figure 1

Concentrations of LPA in the blood plasma taken from cows (n=3) on days 17 and 18 of the estrous cycle, five times for 24 h (at the hour 0, 6, 12, 18, and 24) from jugular (open circle) and uterine (black circle) veins. Small superscript letters: a and b indicate statistical differences in the respective LPA concentration between jugular and uterine veins respectively (P<0.05), as determined by repeated measures ANOVA with Bonferroni's Multiple Comparison Test.

Citation: REPRODUCTION 137, 1; 10.1530/REP-08-0209

Experiment 2. LPA production in the bovine endometrium during the estrous cycle and early pregnancy

LPA concentration in the bovine 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 cycle and pregnancy. LPA concentration in the bovine endometrium did not differ during either the estrous cycle or pregnancy (P>0.05). However, LPA concentration on 17–19 days of pregnancy was significantly higher on days 17–19 of the estrous cycle (P<0.05).

Figure 2

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Figure 2

(a) Concentrations of LPA or patterns of expression of (b) PLD2 and (c) PLA2G1B mRNA in the endometrial tissue on the selected days of the estrous cycle (white bars) and pregnancy (black bars). Data are expressed as ng/g tissue for LPA concentrations or arbitrary units of respective mRNA/mRNA GAPDH for mRNA quantitive expression. Small superscript letters: a and b indicate statistical differences in the respective LPA concentration or mRNA quantitative expression between groups of cyclic or pregnant animals respectively (P<0.05), as determined by one-way ANOVA followed by Bonferroni's multiple comparison test. Capital superscript letters: A and B indicate statistical differences in the respective mRNA quantitative expression between groups of animals on the same days of the estrous cycle and pregnancy respectively (P<0.05), as determined by one-way ANOVA followed by Bonferroni's multiple comparison test.

Citation: REPRODUCTION 137, 1; 10.1530/REP-08-0209

Expression of mRNA for PLD2 and PLA2G1B in the bovine endometrial tissue during the estrous cycle and early pregnancy

Figure 2b shows that the expression patterns of PLD2 mRNA on the days: 2–4, 8–10, and 17–19 of the estrous cycle and pregnancy. With specific primers, enabling amplification of PLD2, one strong band migrating at 106 bps (bp) was observed in the endometrial tissue at all examined stages (data not shown). PLD2 transcript abundance normalized to GAPDH mRNA expression did not differ either during the estrous cycle or pregnancy (P>0.05).

Figure 2c shows the expression patterns of PLA2G1B mRNA on the days: 2–4, 8–10, and 17–19 of the estrous cycle and pregnancy. With specific primers, enabling amplification of PLA2G1B, one strong band migrating at 107 bp was observed in the endometrial tissue at all examined stages (data not shown). PLA2G1B transcript abundance normalized to GAPDH mRNA expression did not differ during the estrous cycle (P>0.05), but increased on day 17–19 of pregnancy (P<0.05). Additionally, PLA2G1B transcript abundance on days 17–19 of pregnancy was significantly higher than on days 17–19 of the estrous cycle (P<0.05).

Experiment 3 Correlations between LPAR1, PGES and PGFS mRNA expression in the bovine endometrium during estrous cycle and early pregnancy

Expression of mRNA for LPAR1 and terminal PG synthesizing enzymes (PGES and PGFS) in the bovine endometrial tissue during the estrous cycle and early pregnancy

Figure 3a shows the expression patterns of LPAR1 mRNA on the days: 2–4, 8–10, and 17–19 of the estrous cycle and pregnancy. With specific primers, enabling amplification of LPAR1, one strong band migrating at 150 bps (bp) was observed in the endometrial tissue at all examined stages (data not shown). LPAR1 transcript abundance normalized to GAPDH mRNA expression revealed significant upregulation during the estrous cycle and was at the highest level on the days 17–19 (P<0.05). However, significant difference was shown only between days 2–4 and days 17–19 of the cycle (P<0.05), and between days 8–10 and days 17–19 of the cycle (P<0.05). LPAR1 transcript abundance on days 17–19 of pregnancy was higher on days 8–10 of pregnancy (P<0.05). Additionally, LPAR1 transcript abundance on days 8–10 of pregnancy was significantly higher on days 8–10 of the estrous cycle (P<0.05).

Figure 3

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Figure 3

Pattern of expression of (a) LPAR1, (b) PGES, and (c) PGFS mRNA on the selected days of the estrous cycle (white bars) and pregnancy (black bars). Data are expressed as arbitrary units of respective mRNA/mRNA GAPDH. Small superscript letters: a and b indicate statistical differences in the respective mRNA quantitative expression between groups of cyclic or pregnant animals respectively (P<0.05), as determined by one-way ANOVA followed by Bonferroni's multiple comparison test. Capital superscript letters: A and B indicate statistical differences in the respective mRNA quantitative expression between groups of animals on the same days of the estrous cycle and pregnancy respectively (P<0.05), as determined by one-way ANOVA followed by Bonferroni's multiple comparison test.

Citation: REPRODUCTION 137, 1; 10.1530/REP-08-0209

Figure 3b shows the expression patterns of PGES mRNA on the days: 2–4, 8–10, and 17–19 of the estrous cycle and pregnancy. With specific primers, enabling amplification of PGES, one strong band migrating at 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 (P>0.05) 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 (r=0.548; P<0.05). Additionally, there were negative correlations between LPAR1 and PGFS transcript abundance during the estrous cycle (r=−0.5923; P<0.05) and during pregnancy for PGES and PGFS (r=−0.517; P<0.05).

Experiment 4. The effect of LPA and LPAR1 antagonist (Ki 16425) on the secretion of progesterone and prostaglandins

Preliminary study

Figure 4a shows P4 concentrations in the blood plasma of control and experimental cows. Administration of all selected doses of LPA agonist (1, 10, 50, 100, and 250 μg) elevated the concentrations of P4 in peripheral blood during the 24 h after LPA infusion in comparison to the saline infused cows (P<0.05). However, all selected doses of LPA agonist also elevated the total amount of secreted P4 in the blood plasma, the highest, but not different from other doses of administered LPA, increase was noted after the infusion of 1 μg LPA agonist in comparison to the control cows (Table 1; P<0.01; area under the curve).

Figure 4

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Figure 4

Concentrations of (a) progesterone, (b) prostaglandin E2, and (c) 13,14-dihydro,15-keto-PGF2α in peripheral blood plasma of cows infused with saline (grey bars) and various doses of LPA agonist (lines). All reagents were infused into the aorta abdominalis. Different subscript letters indicate significant differences (P<0.05) between treated groups, as assessed by repeated-measures ANOVA test followed by Bonferroni's multiple comparison test.

Citation: REPRODUCTION 137, 1; 10.1530/REP-08-0209

Table 1

Effects of different doses of lysophosphatidic acid (LPA) administered into the aorta abdominalis on the mean (±s.e.m.) concentrations of released P4, PGE2, and PGFM in cows on day 17 of the estrous cycle*.

TreatmentP4PGE2PGFM
Control17.4±2.8a70.6±43.8a975±112a
LPA (1 μg) 62.6±5.8b8129±1126b1710±241b
LPA (10 μg) 40.5±7.5b3595±1085b1282±161b
LPA (50 μg) 49.4±6.1b5317±780b1370±640a
LPA (100 μg) 41.1±5.4b2191±533a1392±266b
LPA (250 μg)54.5±7.4b570.3±123a1410±431a

a,bDifferent subscript letters within a column indicate significant differences (P<0.05) between treated groups.

Values indicate the area under the curve (relative units, means±s.e.m). The baseline was defined on the basis of data from the first 2 h of the experiment. The area under the curve was measured using data from 24 h of the experimental period.

Figure 4b shows PGE2 concentrations in the blood plasma of control and experimental cows. Administration of the doses of 1, 10, and 50 μg LPA agonist strongly elevated the concentrations of PGE2 in peripheral blood during 24 h after LPA infusion in comparison to the saline infused cows (P<0.05). The highest, but not different from doses of 10 and 50 μg administered LPA, increase in the total amount of secreted PGE2 was noted after the infusion of 1 μg LPA agonist in comparison to the control cows (Table 1; P<0.01).

Figure 4c shows PGFM concentrations in the blood plasma of control and experimental cows. Administration of the doses of 1, 10, and 100 μg LPA agonist elevated the concentrations of PGFM in peripheral blood during 24 h after LPA infusion in comparison to the saline infused cows (P<0.05). The highest increase in the total amount of secreted PGFM was noted after the infusion of 1 μg LPA agonist in comparison to the control cows (Table 1; P<0.05).

On the basis of this preliminary study, the most effective dose of LPA agonist – 1 μg was chosen for the further studies.

Effects of LPA agonist and LPAR1 antagonist (Ki16425) on the progesterone and prostaglandin secretion in the bovine reproductive tract

Figure 5a shows P4 concentrations in the blood plasma of saline, LPA agonist (1 μg) and LPA antagonist (Ki16425; 10 μg) treated cows. Administration of LPA agonist strongly elevated the concentrations of P4 in peripheral blood during 24 h after LPA infusion in comparison to the saline infused cows (P<0.01). Administration of Ki16425 significantly decreased the concentrations of P4 in peripheral blood during 24 h in comparison to the saline and LPA agonist infused cows (P<0.05; P<0.01 respectively). Moreover, LPA agonist elevated the total amount of secreted P4 in the blood plasma in comparison to the control cows (Table 3; P<0.01), whereas Ki16425 significantly decreased the total amount of secreted P4 in the blood plasma in comparison to the saline and LPA agonist treated cows (Table 2; P<0.05; P<0.01 respectively).

Figure 5

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Figure 5

Concentrations of (a) progesterone, (b) prostaglandin E2, and (c) 13,14-dihydro,15-keto-PGF2α in peripheral blood plasma of cows infused with saline (grey bars), 1 μg LPA agonist (line) or 1 μg LPA antagonist (dotted line). All reagents were infused into the aorta abdominalis. Different subscript letters indicate significant differences (P<0.05) between treated groups, as assessed by repeated-measures ANOVA test followed by Bonferroni's multiple comparison test.

Citation: REPRODUCTION 137, 1; 10.1530/REP-08-0209

Table 2

Effects of lysophosphatidic acid agonist (LPA) and LPAR1 antagonist (Ki16425) administered into the aorta abdominalis on the mean (± s.e.m.) concentrations of released P4, PGE2, and PGFM in cows on day 17 of the estrous cycle*.

TreatmentP4PGE2PGFM
Control17.4±2.8a706.2±438a975±92a
LPA (1 μg)62.6±5.8b8129±1126b1710±141b
Ki16425 (1 μg)7.8±1.5c595±285c1082±111a

a,bDifferent subscript letters within a column indicate significant differences (P<0.05) between treated groups.

Values indicate the area under the curve (relative units, means±s.e.m). The baseline was defined on the basis of data from first 2 h of the experiment. The area under the curve was measured using data from 24 h of the experimental period.

Table 3

Primers used for real-time PCR.

GenePrimer sequencesGenBank (accession number)PositionPCR products (bp)
PLD2 (Bos taurus)5′-GCTTCAGCGTGATTCTAGGG-3′BC1235472537–2642106
5′-GCATTGCTCTCAGCTGTGTC-3′
PLA2G1B (Bos taurus)5′-AGGTGCACAACTTCATGCTG-3′BC1346101544–1650107
5′-GGCATCCAATTCGTCTTCAT-3′
LPAR1 (Bos taurus)5′-GGTGGGGTGTGAGAAAGAGA-3′U482361191–1336146
5′-AAAAGGAATGGGAGCAGGAT-3′
PGES (Bos taurus)5′-AGGACGCTCAGAGACATGGA-3′NM174443154–295142
5′-TTCGGTCCGAGGAAAGAGTA-3′
PGFS (Bos taurus)5′-GATCAAAGCGATTGCAGACA-3′S54973672–784113
5′-CAATGCGTTCAGGTGTCACT-3′
GAPDH (Bos taurus)5′-CACCCTCAAGATTGTCAGCA-3′BC102589492–594103
5′-GGTCATAAGTCCCTCCACGA-3′

Figure 5b shows PGE2 concentrations in the blood plasma of saline, LPA agonist (1 μg) and LPA antagonist (Ki16425; 10 μg) treated cows. Administration of LPA agonist strongly elevated the concentrations of PGE2 in peripheral blood during 24 h after LPA infusion in comparison to the saline infused cows (P<0.01). Administration of Ki16425 significantly decreased the concentrations of PGE2 in peripheral blood during 24 h in comparison to the saline and LPA agonist infused cows (P<0.05; P<0.01 respectively). Moreover, LPA agonist elevated the total amount of secreted PGE2 in the blood plasma in comparison to the control cows (Table 3; P<0.01), whereas Ki16425 significantly decreased the 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 μg) and LPA antagonist (Ki16425; 10 μg) 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). Administration of Ki16425 did not influence the concentrations of PGFM in peripheral blood during 24 h in comparison to the saline infused cows (P>0.05) 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).

Discussion

In the present study, we found that LPA is locally produced and released from the bovine endometrium. We found that the total concentration of LPA in the blood plasma taken from the uterine vein on days 17–19 of the estrous cycle for 24 h was significantly higher in the blood plasma from the jugular vein. We measured total LPA concentration, which is probably much higher than the concentration of LPA that is free and thus exerts some biological roles in the body. However, there is no possible way to measure free-unconjugated LPA in the body fluids and tissue. Moreover, LPA in the blood plasma is mainly bound to albumin or some other lipid binding protein (Aoki 2004) and thus we might suppose that the whole amount of LPA measured in our study after enzymatic hydrolysis is not active. We also found high concentrations of LPA in the endometrial tissue. Although, LPA concentration in the bovine endometrium did not significantly differ either during the estrous cycle or early pregnancy, it was significantly higher on days 17–19 of pregnancy than on days 17–19 of the estrous cycle. Moreover, PLD2 and PLA2G1B gene expression indicates that there is the possibility for LPA to be synthesized in the bovine endometrium. PLD2 was expressed at the constant level during estrous cycle and early pregnancy, however, PLA2G1B expression revealed the highest level on days 17–19 of pregnancy. Thus, LPA might be synthesized at all stages of the estrous cycle and pregnancy with the preference to days 17–19 of pregnancy. High LPA concentration, mRNA expression for its receptor (LPAR1), PLD2 and PLA2G1B during the estrous cycle and pregnancy might suggest that LPA plays an autocrine and/or paracrine role in the bovine endometrium during the estrous cycle and early pregnancy.

The pleiotropic roles of LPA in reproductive physiology and pathology are demonstrated by both the regulated expression of its receptors (Contos & Chun 2001, Moller et al. 2001, Contos et al. 2002, Ye et al. 2005) and the increased amount of LPA in body fluids (Tokumura et al. 2002, 2007). LPA exerts its biological effects via four G-protein coupled membrane receptors (LPAR1, LPAR2, LPAR3, and LPAR4; Bandoh et al. 1999, Im et al. 2000, Contos et al. 2002, Noguchi et al. 2003, Gardell et al. 2006). Although LPAR3 expression has been recently shown to be connected with PG synthesis and to play a crucial role in implantation in mice (Ye et al. 2005), we found only LPAR1 mRNA expression in the bovine endometrial tissue (Woclawek-Potocka et al. 2007). During the estrous cycle, LPAR1 mRNA expression increased from early to late luteal stage and reached the highest level at late luteal stage, suggesting that LPA might be involved in the first events of luteolysis. This LPA contribution in the luteolysis may be explained by its indirect stimulation of luteolytic PGF synthesis through the influence on prostaglandin like 2 synthase (PGFSL2) activity – the enzyme with 9-keto reductase activity, responsible for the conversion of PGE2 to PGF (Madore et al. 2003, Weems et al. 2006). However, this hypothesis needs further examination. The present study shows that LPA is mainly involved in the luteotropic mechanism during early pregnancy in cattle. Total LPAR1 gene expression on days 8–10 of pregnancy was lower than that on days 17–19 of pregnancy. Moreover, LPAR1 gene expression was higher on days 8–10 of pregnancy than on days 8–10 of the estrous cycle. These results seem to be consistent with the results of the first two experiments, where we showed higher LPA concentration in uterine vein than in jugular vein and the highest LPA concentrations in the endometrial tissue on the days 17–19 of pregnancy. The role of LPA in early pregnancy in cow may be similar to its role in humans in that LPA at the time of implantation induces blastocyst differentiation via a mechanism connected with Ca2+ transport (Liu & Armant 2004). Moreover, higher LPAR1 mRNA expression at each stage of early pregnancy than at each stage of the estrous cycle agrees with the data obtained by Gaits et al. (1997) who also proved that plasma LPA concentrations were much higher in pregnant women than in cyclic women.

In the present study, we demonstrated how PG synthesizing enzymes (PGES and PGFS) mRNA are expressed in the bovine endometrium and whether they are correlated with LPAR1 expression throughout the estrous cycle and early pregnancy. Our findings that PGES mRNA expression was much higher on days 2–4 of the cycle than on days 8–10 and days 17–19 (Fig. 3b) agree with the data of Arosh et al. (2002), who found high PGES expression at the beginning of the estrus cycle. In our study, we found that the high PGES expression at the beginning of the cycle may be responsible for its luteotropic action on the early development of the CL. Moreover, PGES mRNA expression was much higher at each stage of early pregnancy than at the each stage of the estrous cycle. These findings suggest that much more PGE2 is synthesized during early pregnancy than at any stage of the estrous cycle. We also showed that on days 8–10 of the cycle when PGES mRNA expression is low, there is an increase in mRNA expression for luteolytic PGF synthase (PGFS). These data suggest that on days 8–10 of the cycle the enzymatic mechanisms that change the ratio of the two main PGs toward luteolytic PGF are being turned on. A definite change of this ratio takes place on the 12th day of the estrous cycle, when the P4 level is the highest but the cyclic CL is already programmed for luteolysis (McCracken et al. 1999). The high PGFS mRNA expression on days 8–10 of the estrous cycle (Fig. 3c) agrees with the data of Madore et al. (2003). Interestingly, we found higher PGFS mRNA expression on days 8–10 than on days 17–19 of pregnancy. This finding might explain PGF-dependent motor activity of the uterus at this time, when the blastocyst moves around to find the most appropriate place for implantation.

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 that PGES expression was negatively correlated with the expression of PGFS at early pregnancy. These correlations may explain why PGE2 and LPA act similarly, and PGES with PGFS may act differently during early pregnancy in cows. The interactions also describe, on the enzymatic level, the mechanisms responsible for changing the ratio of luteotropic PGE2 to luteolytic PGF during early pregnancy in cows as well as suggest the influence of LPA on this process.

In the last experiment, we wanted to confirm the possible role of LPA and LPAR1 antagonist on the secretion of progesterone and prostaglandins. The dose of 1 μg LPA stimulated P4 and PGE2 concentration in the blood, and had weaker effect on PGFM level (Tables 1 and 2 area under the curve). The LPA-induced increase of PGF secretion was much less than the LPA-induced increase of PGE2 synthesis (1.75 vs 11.5 times). Moreover, we showed that the inhibition of endogenous LPA action via the infusion of LPAR1 receptor antagonist (Ki16425) caused the decrease of P4 and PGE2 concentrations. Summarizing this part of the study, LPA had strong effect on P4 and PGE2 concentrations in the blood, and much weaker effect on PGFM level. This suggests LPA influence on both endometrium and the CL. LPA-induced P4 stimulation suggests its influence not only on the endometrium – via LPAR1-dependent PGE2 stimulation (Woclawek-Potocka et al. 2007), but also a direct effect on the secretory functions of steroidogenic CL cells. However, this supposition needs further, thorough in vitro examination. The above data suggest that in ruminants, the possible LPA dependent development and maintenance of the CL during the estrous cycle and establishment of pregnancy might depend on the LPA influence on the CL and on the balance of luteolytic PGF and luteotropic PGE2 rather than absolute amounts of each prostaglandin.

In conclusion, the present study proved that LPA may be locally produced and released in the reproductive tract of cows including endometrium. Moreover, LPAR1 gene expression in the endometrium during the estrous cycle and early pregnancy indicates that LPA may play autocrine and/or paracrine roles in the bovine uterus. In the cow, LPA stimulates P4 and PGE2 secretion and its receptor gene expression (LPAR1) are positively correlated with the expression of the enzyme responsible for luteotropic PGE2 production (PGES) in endometrium. Finally, LPA in the bovine reproductive tract may indirectly (via endometrium) or directly support CL action via the stimulation of P4 synthesis and the increase of PGE2/PGF ratio. It suggests that LPA may serve as an important factor in the maintenance of early pregnancy in cow. However, this final supposition is now under in vivo and in vitro examinations.

Materials and Methods

Animals

All animal procedures were approved by the Local Animal Care and Use Committee in Olsztyn, Olsztyn, Poland (Agreement No. 34/2005/N).

For all experiments, normally cycling Holstein/Polish Black and White (75/25% respectively) cows (4–6 lactations; n=89) were chosen. The animals were eliminated by the owner from two dairy cow herds (Years 2004–2006) because of their low milk production. The estrus of the cows was synchronized using implants of a progesterone analog (Crestar, Intervet, Holland) with additional injection of an analog of PGF (dinoprost, Dinolytic; Upjohn- Pharmacia N V S A, Belgium), as recommended by Bah et al. (2006) for the estrus synchronization of multiparous cows. The onset of the estrus was determined by observing the signs of estrus (i.e. vaginal mucus, standing behavior), and was confirmed by a veterinarian via ultrasonography (USG) examination using Draminski Animal profi Scanner (Draminski Electronics in Agriculture, Olsztyn, Poland; ) and per rectum examination. Only the cows with behavioral signs of estrus were chosen for the study after positive USG and per rectum examination (n=77). The estrus was taken as day 0 of the estrous cycle.

Experimental procedure

Experiment 1: LPA concentration in uterine and jugular vein

The aim of this experiment was to confirm whether LPA is locally produced and released from the bovine endometrium. LPA concentrations were measured in the extracts from blood plasma taken from the jugular and uterine vein. The catheters were inserted into respective vessels according to the procedures described by Skarzynski et al. (2003) for jugular vein and by Acosta et al. (2000) for ovarian vein on the day 16 of the estrous cycle. In the procedure for uterine vein, a lateral laparotomy was performed. At surgery a catheter was inserted into the uterine vein ipsilateral to the functional CL and sutured (Acosta et al. 2000). The blood collections started 24 h after the surgery, at noon on the day 17 of the cycle and experiment was finished after 24 h (at noon on the day 18 of the cycle). Blood samples were collected into tubes with 5 μl EDTA, 1% aspirin solution (pH 7.3) for 24 h (5 times: at hour 0, after 6, 12, 18, and 24 h). The blood plasma was immediately separated by centrifugation (2000 g, 10 min at 4 °C) and stored at −20 °C until the time of lipid extraction. 1-oleoyl-LPA contained in plasma was extracted and measured according to the procedures described by Saulnier-Blache et al. (2000).

Experiment 2: LPA production in the bovine endometrium during the estrous cycle and early pregnancy

The aim of this experiment was to compare whether there are changes in LPA production in the bovine endometrium during estrous cycle and early pregnancy. LPA concentrations were measured in the extracts from endometrial tissues. Bovine uteri were obtained at a local slaughterhouse (Ubojnia Zwierzat Rzeznych, Gucin, Lukta, Poland) within 20 min of exsanguination and were transported on ice to the laboratory within 40 min. Before slaughter, the cows were divided into two groups, i.e. pregnant and cyclic animals. The animals chosen for the pregnant group underwent artificial insemination with the semen of the same bull. The tissues for this experiment were assigned for the following days of the estrous cycle and pregnancy: days 2–4 of the cycle (n=5), days 8–10 of the cycle (n=7) and pregnancy (n=12), days 17–19 of the cycle (n=4) and pregnancy (n=5). Gene expression for the enzymes involved in LPA synthesis (PLD2 and PLA2G1B) was quantitatively measured by real-time PCR in tissues. For real-time PCR, the small pieces of endometrial tissue were snap frozen in liquid nitrogen, before storage in −80°C. Estimation of the stages of the estrous cycle was confirmed by macroscopic observation of the ovaries and uterus (Miyamoto et al. 2000). The pregnancy was confirmed by flushing the uterus for the collection of the viable embryo as described previously (Leung et al. 2000). Moreover, the flushed uterine fluid was collected and the antiviral activity of interferon-τ was measured, at least at the later phase of early pregnancy (days 17–19), as recently described (Gierek et al. 2006). Moreover, we tested the sensitivity of cultured pure endometrial epithelial cells (days 17–19) to oxytocin (OT) treatment. Only non-pregnant bovine endometrium, at the late luteal and follicular phase of the cycle, is sensitive to OT treatment and PGF release (Miyamoto et al. 2000). Therefore, only the uteri that responded properly for the above tests were chosen for the respective pregnancy groups. 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 17/19) because of the lack of pregnancy.

1-oleoyl-LPA contained in tissues was extracted and measured according to the procedures described by Saulnier-Blache et al. (2000).

Experiment 3: Correlations between LPAR1, PGES, and PGFS mRNA expression in the bovine endometrium during estrous cycle and early pregnancy

The aim of the study was to examine whether there are any correlations between LPA receptor, PGES, and PGFS mRNA expression in the bovine endometrium during estrous cycle and early pregnancy. Gene expression for LPAR1, and terminal PG synthesizing enzymes (PGES and PGFS) were quantitatively measured by real-time PCR in the tissues used previously for the measurement of LPA concentration and PLD2 and PLA2G1B mRNA expression (experiment 2).

Experiment 4:The effect of LPA and LPAR1 antagonist (Ki 16425) on the secretion of progesterone and prostaglandins

Preliminary study

Twenty four cows were used to choose the effective dose of an LPA agonist. For infusion of either saline or different doses of LPA agonist, a catheter was inserted into the posterior aorta abdominalis through the coccygeal artery on day 16 of the estrous cycle according to the procedure described previously (Skarzynski et al. 2003). The animals were premedicated with xylazine at a dose of 25–30 mg/animal i.m. (Sedazin, Biowet Pulawy, Poland) and local epidural anesthesia was induced by injecting 4 ml of 2% procaine hydrochloride (Polocainum Hydrochloricum, Biowet Drwalew, Poland) between the 1st and 2nd coccygeal vertebrae. The tip of the cannula was positioned in the aorta 60–65 cm ahead of the point of insertion, just cranial to the origin of the ovarian artery and caudal to the renal artery (Skarzynski et al. 2003). This placement allowed infused reagents to be transported by the blood stream directly into the reproductive tract. A second catheter was inserted into the jugular vein for frequent collection of blood samples (Skarzynski et al. 2003).

The cows one day after the surgery were infused into the a. abdominalis with saline (n=4; control group) or five different doses of LPA agonist (1, 10, 50, 100, 250 μg; n=4 for each dose). Peripheral blood samples were collected in a tube with 5 μl EDTA, 1% aspirin solution (pH 7.3) from a jugular vein frequently for 24 h (18 times, beginning 2 h before the infusions). The blood plasma was immediately separated by centrifugation (2000 g, 10 min at 4 °C) and stored at −20 °C. Concentrations of P4, PGE2, and 13,14-dihydro,15-keto-prostaglandin F (PGFM) in the plasma samples were measured.

To confirm the possible role of LPA and LPAR1 antagonist on the secretion of progesterone and prostaglandins, 12 cows on day 17 of the estrous cycle were infused into the a. abdominalis with saline (n=4; control group), LPA agonist (1 μg; this group was taken from the preliminary study; n=4) or LPAR1 antagonist (Ki16425; 10 μg; n=4). Peripheral blood samples were collected from a jugular vein frequently for 24 h (18 times, beginning 2 h before the infusions), as described in the preliminary study. The blood plasma was immediately separated by centrifugation (2000 g, 10 min at 4 °C) and stored at −20 °C. Concentrations of P4, PGE2 and 13,14-dihydro,15-keto-prostaglandin F (PGFM) in the plasma samples were measured.

Lipid extraction from endometrial tissue and blood plasma and in vitro acylation of LPA

1-oleoyl-LPA or lipids contained in 100 mg of tissue and 100 μl of plasma were extracted with one volume of 1-butanol according to the procedures thoroughly described by Saulnier-Blache et al. (2000). After evaporation, lipids were incubated in the presence of semi-purified recombinant rat LPA acyltransferase (LPAAT) and [14C]oleoyl-CoA as described by Saulnier-Blache et al. (2000). The products of the reaction were separated by two-dimensional TLC and autoradiographed.

Hormone determination

The concentrations of P4, PGFM, and PGE2 in the plasma samples were determined with direct EIAs, as described 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 donated Dr W J Silvia, University of Kentucky, Lexington, KY, USA. The anti PGE2 serum was purchased from Sigma; #P5164. The P4 standard curve ranged from 0.39 pg/ml to 25 ng/ml and the effective dose for 50% inhibition (ID50) of the assay was 2.85 ng/ml. The intra- and inter-assay coefficients of variation averaged 6.6 and 8.4% respectively. The PGFM standard curve ranged from 32.5 to 8000 pg/ml and the ID50 of the assay was 315 pg/ml. The intra- and inter-assay coefficients of variation were on average 7.6 and 10.4% respectively. The PGE2 standard curve ranged from 0.07 to 20 ng/ml and the ID50 of the assay was 1.25 ng/ml. The intra- and inter-assay coefficients of variation were on average 6.9 and 9.7% respectively.

Total RNA extraction, RT and real-time PCR

Total RNA was extracted from endometrial tissues using TRIZOL according to the manufacturer's instructions. RNA samples were stored at −80 °C. Before use, RNA was verified by spectrophotometric measurement and agarose gel electrophoresis. Two micrograms of each sample of total RNA were reverse transcribed using a ThermoScript RT-PCR System (Invitrogen, Alab; #11146-016). The RT reaction was performed in total reaction volume of 20 μl, following manufacturer's instructions. RT products were stored at −20 °C until real-time PCR amplification.

The expression of mRNA for all examined genes was conducted by real-time PCR using specific primers for PLD2, PLA2G1B, LPAR1, PGES, and PGFS. Briefly, GAPDH expression was used as an internal control. The primers were chosen using an online software package (). The primers of all target genes are given in Table 3.

Real-time PCR was performed with an ABI Prism 7300 sequence detection system using Power SYBR Green PCR master mix (Applied Biosystems, Applera, Warsaw, Poland; #4367659). The PCRs were performed in 96-well plates. Each PCR well (25 μl) contained 2.5 μl of diluted RT product, 200 pM forward and reverse primers each, and 12.5 μl SYBR Green PCR master mix. As standard curves, serial dilutions of appropriate cDNA were used for gene quantification. For quantification of the mRNA expression levels, the primer length (20 bp) and GC-contents of each primer (50–60%) were selected. Real time PCR was performed under the following conditions: 95 °C for 10 min, followed by 40 cycles of 94 °C for 15 s, 55.6 °C (for GAPDH) or 56.3 °C (for LPAR1), or 56 °C (for PGES and PLD2) or 55.9 °C (for PLA2G1B) or 55.7 °C (for PGFS) for 28 s and 72 °C for 15 s. Each PCR was followed by obtaining melting curves by stepwise increase in the temperature from 60 °C to 95 °C to ensure single product amplification. In order to exclude the possibility of genomic DNA contamination in the RNA samples, the reactions were also run either on blank-only buffer samples or in the absence of the reverse transcriptase enzyme. The specificity of the PCR products for all examined genes was confirmed by gel electrophoresis and by sequencing. The obtained data were normalized on the basis of GAPDH mRNA content.

Statistical analysis

In Experiment 1, total concentration of LPA in the jugular and uterine veins during the 24 h of the experiment was expressed as the area under the curve (Y=0 was taken as a baseline) and analyzed by the Student's t test. The data are shown as means±s.e.m. (n=3). In experiments 2 and 3, all analyses were done using one way ANOVA tests followed by Bonferroni's Multiple Comparison Test (GraphPad PRISM; P<0.05 was considered significant). Additionally in experiment 3, different gene expression among the days was modeled by linear regression (GraphPad PRISM). In experiment 4, P4, PGE2, and PGFM concentrations in the jugular plasma were analyzed using a repeated measure design approach described previously (repeated measures ANOVA with Bonferroni's Multiple Comparison Test; GraphPad PRISM; Skarzynski et al. 2003). The total amounts of released P4, PGE2, and PGFM were shown by the area under the curve (relative units; Table 1) and were analyzed using one-way ANOVA followed by Bonferroni's Multiple Comparison Test (GraphPad PRISM).

Declaration of interest

I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the Grants-in-Aid for Scientific Research from the Polish Ministry of Scientific Research and Information Technology (KBN 2P06K 003 30) and the Japanese-Polish Joint Research Project under the agreement between JSPS and PAS.

Acknowledgements

The authors are indebted to Dr James Raymond for critical review of this manuscript and English correction. We thank Dr W J Silvia, University of Kentucky, Lexington, USA, for PGFM antisera; Dr Stanislaw Okrasa of University of Warmia and Mazury for progesterone antisera; and Dainippon Pharmaceutical Co., Ltd, Osaka, Japan for recombinant human TNF (HF-13). The authors are grateful to Draminski Electronics in Agriculture (Olsztyn, Poland, ) for their excellent cooperation and possibility to test and use the USG scanner. The authors also thank GENNUAS France, the owner of Animal Farm ‘Wroblik’ (Spółka Rolna ‘Wroblik’ Sp. z o.o, Lidzbark Warminski, Poland) for their excellent cooperation and agreement to let us use the animals for the present experiment. Two colleagues from the Reproductive Immunology Department – Anna Korzekwa and Marta Siemieniuch must be also mentioned in this section for their excellent assistance at the in vivo part of the study. Lastly, the authors are indebted to Estelle Wanecq who performed LPA quantification.

References

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  • GoetzlEJDolezalovaHKongYHuJLJaffeRBKalliKRConoverCA1999Distinctive expression and functions of the type 4 endothelial differentiation gene-encoded G protein-coupled receptor for lysophosphatidic acid in ovarian cancer. Cancer Research5953705375.

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  • The 11 th Annual Conference of European Society for Domestic Animal Reproduction

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Figures

  • View in gallery

    Concentrations of LPA in the blood plasma taken from cows (n=3) on days 17 and 18 of the estrous cycle, five times for 24 h (at the hour 0, 6, 12, 18, and 24) from jugular (open circle) and uterine (black circle) veins. Small superscript letters: a and b indicate statistical differences in the respective LPA concentration between jugular and uterine veins respectively (P<0.05), as determined by repeated measures ANOVA with Bonferroni's Multiple Comparison Test.

  • View in gallery

    (a) Concentrations of LPA or patterns of expression of (b) PLD2 and (c) PLA2G1B mRNA in the endometrial tissue on the selected days of the estrous cycle (white bars) and pregnancy (black bars). Data are expressed as ng/g tissue for LPA concentrations or arbitrary units of respective mRNA/mRNA GAPDH for mRNA quantitive expression. Small superscript letters: a and b indicate statistical differences in the respective LPA concentration or mRNA quantitative expression between groups of cyclic or pregnant animals respectively (P<0.05), as determined by one-way ANOVA followed by Bonferroni's multiple comparison test. Capital superscript letters: A and B indicate statistical differences in the respective mRNA quantitative expression between groups of animals on the same days of the estrous cycle and pregnancy respectively (P<0.05), as determined by one-way ANOVA followed by Bonferroni's multiple comparison test.

  • View in gallery

    Pattern of expression of (a) LPAR1, (b) PGES, and (c) PGFS mRNA on the selected days of the estrous cycle (white bars) and pregnancy (black bars). Data are expressed as arbitrary units of respective mRNA/mRNA GAPDH. Small superscript letters: a and b indicate statistical differences in the respective mRNA quantitative expression between groups of cyclic or pregnant animals respectively (P<0.05), as determined by one-way ANOVA followed by Bonferroni's multiple comparison test. Capital superscript letters: A and B indicate statistical differences in the respective mRNA quantitative expression between groups of animals on the same days of the estrous cycle and pregnancy respectively (P<0.05), as determined by one-way ANOVA followed by Bonferroni's multiple comparison test.

  • View in gallery

    Concentrations of (a) progesterone, (b) prostaglandin E2, and (c) 13,14-dihydro,15-keto-PGF2α in peripheral blood plasma of cows infused with saline (grey bars) and various doses of LPA agonist (lines). All reagents were infused into the aorta abdominalis. Different subscript letters indicate significant differences (P<0.05) between treated groups, as assessed by repeated-measures ANOVA test followed by Bonferroni's multiple comparison test.

  • View in gallery

    Concentrations of (a) progesterone, (b) prostaglandin E2, and (c) 13,14-dihydro,15-keto-PGF2α in peripheral blood plasma of cows infused with saline (grey bars), 1 μg LPA agonist (line) or 1 μg LPA antagonist (dotted line). All reagents were infused into the aorta abdominalis. Different subscript letters indicate significant differences (P<0.05) between treated groups, as assessed by repeated-measures ANOVA test followed by Bonferroni's multiple comparison test.

References

AcostaTJOzawaTKobayashiSHayashiKOhtaniMKraetzlWDSatoKSchamsDMiyamotoA2000Periovulatory changes in the local release of vasoactive peptides, prostaglandin f(2alpha), and steroid hormones from bovine mature follicles in vivo. Biology of Reproduction6312531261.

AokiJ2004Mechanisms of lysophosphatidic acid production. Seminars in Cell and Developmental Biology15477489.

AroshJAParentJChapdelainePSiroisJFortierMA2002Expression of cyclooxygenases 1 and 2 and prostaglandin E synthase in bovine endometrial tissue during the estrous cycle. Biology of Reproduction67161169.

AsselinEGoffAKBergeronHFortierMA1996Influence of sex steroids on the production of prostaglandins F and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Biology of Reproduction54371379.

BahMMAcostaTJPilawskiWDeptulaKMOkudaKSkarzynskiDJ2006Role of intraluteal prostaglandin F, progesterone and oxytocin in basal and pulsatile progesterone release from developing bovine corpus luteum. Prostaglandins & Other Lipid Mediators80218229.

BandohMAokiJHosonoHKobayashiiSMurakami-MurofushiiKAraiHInoueK1999Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid. Journal of Biological Chemistry2742777627785.

ContosJChunJ2001The mouse lpA3/Edg7 lysophosphatidic acid receptor gene: genomic structure, chromosomal localization, and expression pattern. Gene267243253.

ContosJIshiiIChunJYeX2002Characterization of LPA2 (edg4) and LPA1/LPA2 (edg2 and edg4) lysophosphatidic acid receptor knockout mice: signaling deficits without obvious phenotypic abnormality attributable to LPA2. Molecular and Cellular Biology2269216929.

DeySKLimHDasSKReeseJPariaBCDaikokuTWangH2004Molecular cues to implantation. Endocrine Reviews25341373.

EderAMSasagawaTMaoMAokiJMillsGB2000Constitutive and lysophosphatidic acid (LPA)-induced LPA production: role of phospholipase D and phospholipase A2. Clinical Cancer Research624822491.

ExtonJH2002Regulation of phospholipase D. FEBS Letters5315861.

FukushimaNWeinerJAContosJAKimKChunJ2002Lysophosphatidic acid influences the morphology and motility of young, postmitotic cortical neurons. Molecular and Cellular Neurosciences20271282.

GaitsFFourcadeOLe BelleFGuegruenGGaigeBGassama-DiagneAFauvelJSallesJPMaucoGSimonMF1997Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis. FEBS Letters4105458.

GardellSDubinAChunJ2006Emerging medical roles for lysophospolipid signaling. Trends in Molecular Medicine126575.

GierekDBaczynskaDUgorskiMBazerFKurpiszMBednarskiTGorczykowskiMChelmonska-SoytaA2006Differential effect of IFN-tau on proliferation and distribution of lymphocyte subsets in one-way mixed lymphocyte reaction in cows and heifers. Journal of Reproductive Immunology71126131.

GoetzlEJDolezalovaHKongYHuJLJaffeRBKalliKRConoverCA1999Distinctive expression and functions of the type 4 endothelial differentiation gene-encoded G protein-coupled receptor for lysophosphatidic acid in ovarian cancer. Cancer Research5953705375.

HinokioKYamanoSNakagawaKIraharaaMKamadaMTokumuraAAonoT2002Lysophosphatidic acid stimulates nuclear and cytoplasmic maturation of golden hamster immature oocytes in vitro via cumulus cells. Life Sciences70759767.

ImDSHeiseCHardingMGeorgeSTheodorescuDLynchK2000Molecular cloning and characterization of a lysophosphatidic acid receptor, Edg-7, expressed in prostate. Molecular Pharmacology57753759.

KaminskaKWasielakMBogackaIBlitekMBogackiM2008Quantitative expression of lysophosphatidic acid receptor 3 gene in porcine endometrium during the periimplantation period and estrous cycle. Prostaglandins and other Lipid Mediators852632.

KimKSSenguptaSBerkMKwakYGEscobarPFBelinsonJMokSCXuY2006Hypoxia enhances lysophosphatidic acid responsiveness in ovarian cancer cells and lysophosphatidic acid induces ovarian tumor metastasis in vivo. Cancer Research6679837990.

KotwicaJSkarzynskiDMlynarczukJRekawieckiR2003Role of prostaglandin E2 in basal and noradrenaline-induced progesterone secretion by the bovine corpus luteum. Prostaglandins & Other Lipid Mediators70351359.

LeungSTDereckaKMannGEFlintAPFWathesDC2000Uterine lymphocyte distribution and interleukin expression during early pregnancy in cows. Journal of Reproduction and Fertility1192533.

LiuZArmantDR2004Lysophosphatidic acid regulates murine blastocyst development by transactivation of receptors for heparin-binding EGF-like growth factor. Experimental Cell Research296317326.

MadoreEHarveyNParentJChapdelainePAroshJAFortierMA2003An aldose reductase with 20 α-hydroxysteroid dehydrogenase activity is most likely the enzyme responsible for the production of prostaglandin F in the bovine endometrium. Journal of Biological Chemistry2781120511212.

MagnessRRHuieJMHoyerGLHuecksteadtTPReynoldsLPSeperichGJWhysongGWeemsCW1981Effect of chronic ipsilateral or contralateral intrauterine infusion of prostaglandin E2 (PGE2) on luteal function of unilaterally ovariectomized ewes. Prostaglandins and Medicine6389401.

McCrackenJACusterEELamsaJC1999Luteolysis: a neuroendocrine-mediated event. Physiological Reviews79263323.

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The 11 th Annual Conference of European Society for Domestic Animal Reproduction

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