In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1, PGE2) prevent luteolysis in cows. II: mRNA for PGF2α, EP1, EP2, EP3 (A–D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue

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Prostaglandins and Other Lipid Mediators

In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1 , PGE2 ) prevent luteolysis in cows. II: mRNA for PGF2␣ , EP1, EP2, EP3 (A–D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue Yoshie S. Weems a , Phillip J. Bridges b,c , Myoungkun Jeoung b , J. Alejandro Arreguin-Arevalo d , Torrance M. Nett d , Rhonda C. Vann e , Stephen P. Ford f , Andrew W. Lewis h , Don A. Neuendorff h , Thomas H. Welsh Jr. g , Ronald D. Randel h , Charles W. Weems a,∗ a

Dept. of HNFAS, University of Hawaii, Honolulu, United States Division of Clinical and Reproductive Sciences, University of Kentucky, Lexington, United States c Dept. of Animal and Food Sciences, University of Kentucky, Lexington, United States d College of Veterinary and Biomedical Sciences, Colorado State University, Fort Collins, United States e Dept. of Animal Science, Mississippi State University, Brown Loam, United States f Dept. of Animal Science, University of Wyoming, Laramie, United States g Dept. of Animal Science, Texas A&M University, College Station, United States h Texas AgriLife Research, Texas A&M University System, Overton, United States b

a r t i c l e

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Article history: Received 21 August 2011 Received in revised form 10 November 2011 Accepted 14 November 2011 Available online 22 November 2011 Keywords: PGE1 PGE2 Cow Corpus luteum Brahman Angus mRNA prostanoid receptors

a b s t r a c t Previously, it was reported that chronic intra-uterine infusion of PGE1 or PGE2 every 4 h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17␤ or PGE2 given intra-uterine every 8 h did not inhibit luteolysis in cows, but infusion of estradiol + PGE2 inhibited luteolysis. In contrast, intra-luteal implants containing PGE1 or PGE2 in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE1 or PGE2 alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE1 , or PGE2 and corpora lutea were retrieved on day19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A–D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE1 and PGE2 up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE1 and PGE2 down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE1 or PGE2 up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE1 and PGE2 support their agonistic effects. We conclude that both PGE1 and PGE2 may prevent luteolysis by altering expression of mRNA for prostanoid receptors, which is correlated with changes in luteal mRNA for LH receptors reported previously in these same cows to prevent luteolysis. Published by Elsevier Inc.

1. Introduction Approximately one-third of ewe (female sheep) and cow (female cattle) embryos are lost during the first third of pregnancy [1-for review]. Additional losses of 6–8 percent occur after the first third of pregnancy in ewes [1-for review]. These losses may

∗ Corresponding author. Tel.: +1 808 388 1344; fax: +1 808 956 4883. E-mail address: [email protected] (C.W. Weems). 1098-8823/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.prostaglandins.2011.11.006

be due to deficiencies in luteal progesterone secretion, since progesterone is required throughout gestation to maintain pregnancy [1-for review]. The corpus luteum is the source of progesterone during the estrous cycle [2-for review]. Sources of progesterone during pregnancy differ in cows and ewes [2]. In cows, concentrations of circulating progesterone increase two-fold from day-12 to day-18 post-breeding and do not change from days 20 to 280 [3]. The placenta of cows does not secrete progesterone when the corpus luteum is functional [4]. Ovaries can be removed after day55 of pregnancy in ewes, since the placenta secretes sufficient

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progesterone to maintain pregnancy [2-for review]. In ewes, half of the progesterone circulating at day-90 is from the corpus luteum and half is from the placenta [1-for review]. In ewes, circulating progesterone increased after day-50 and until day-130 of pregnancy, and then decreased by day-135 when luteal steroidogenic enzymes decreased [2-for review]. Secretion of progesterone by bovine and ovine luteal tissue during the estrous cycle is regulated by LH [5]. Bovine luteal tissue regresses when cows are given antisera to LH in vivo [6]. Receptors for LH are on both small and large steroidogenic luteal cells (SLC, LLC) in the corpus luteum; however, LH increases SLC progesterone secretion via adenylate cyclase activity, cAMP, and protein kinase A (PKA), but not by LLC in ewes and cows [7–12]. Loss of progesterone secretion at the end of the estrous cycle in ewes is via uterine PGF2␣ secretion [1-for review]. However, PGF2␣ in endometrium, uterine or ovarian venous blood, luteal tissue, binding of PGF2␣ to luteal membranes, or transport of PGF2␣ from the uterine vein to the adjacent ovarian artery of the luteal-containing ovary are not decreased to explain prevention of luteolysis during early pregnancy in ewes [2,13]. In cows, PGF2␣ is decreased in uterine venous blood on day-18 of pregnancy, but not in ovarian arterial or venous blood or in luteal tissue [14]. In ewes, uterine secretion of PGF2␣ in vitro does not decrease until days 50–60 of pregnancy when the ovaries are not needed to maintain pregnancy [2-for review]. In ewes, the embryo–endometrial interaction provides resistance to PGF2␣ -induced luteolysis during early pregnancy [2-for review]. This resistance to PGF2␣ is due to the 2-fold increase in PGE1 and PGE2 in the endometrium on day-13 of pregnancy in ewes [2-for review]. Concentrations of PGE are increased in uterine venous blood and the PGE:PGF2␣ ratio increases from 0.1:1 on day-8 to 1:1 during early pregnancy in ewes [2-for review]. During the estrous cycle of ewes, luteal tissue does not secrete detectable PGE or PGF2␣ in vitro [15], but the corpus luteum secretes detectable levels of PGE in vitro by day-50 of pregnancy, luteal secretion of PGE increases further by day-90 in vitro, and by day-90 of pregnancy concentrations of PGE in ovarian venous blood draining the luteal-containing ovary 72 h after hysterectomy ranges from 4 to 6 ng/ml [2,14]. This is equivalent to concentrations of PGE in uterine venous blood at day-90 of pregnancy in ewes [1,2-for review]. In cows, luteal tissue on day-15 of the estrous cycle or day-200 of pregnancy secretes both PGE and PGF2␣ in vitro at a 1:1 ratio and both PGE and PGF2␣ increased with time in culture, but the ratio remains 1:1 [15,16]. In ewes, the luteotropin switches from LH to PGE1 and PGE2 by day-50 to day90 in ewes, and by day-200 of pregnancy in cows [2,16,17]. LH does not stimulate ovine or luteal progesterone secretion after day-50 of pregnancy in ewes or at day-200 of pregnancy in cows [2,16,17]. However, PGE1 or PGE2 stimulated luteal progesterone secretion in vitro after day-50 of pregnancy in ewes or at day-200 of pregnancy in cows [2,16,17]. PGE1 and PGE2 stimulate bovine luteal progesterone secretion in vitro via cAMP [18]. Indomethacin given in vivo to 90-day pregnant ewes lowers circulating progesterone and PGE in inferior vena cava blood draining the luteal-containing ovary, reduces day-90 ovine luteal tissue of pregnancy secretion of PGE and progesterone in vitro, and indomethacin + PGE2 restores luteal tissue progesterone secretion in vitro [2]. Ovine luteal progesterone secretion is regulated by LH until day-50 of pregnancy and PGE regulates luteal and placental progesterone secretion after day 50 [2-for review]. Pregnancy specific protein B (PSPB) regulates luteal and placental tissue PGE secretion after day-50 of pregnancy in ewes and after day-200 of pregnancy in the cow and PGE regulates ovine luteal and placental progesterone secretion after day-50 of pregnancy in ewes [2,16,17]. These data fit changes in LH observed during pregnancy in ruminants where pituitary LH content, concentrations of LH in blood, and LH pulse amplitude and pulse frequency in ewes or cows decreased as pregnancy

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progressed [19]. In cows, indomethacin in vivo decreased circulating progesterone [20]. Chronic intrauterine infusion of PGE1 or PGE2 adjacent to the corpus luteum-containing ovary prevents spontaneous or premature luteolysis induced by estradiol-17␤, IUD, or PGF2␣ [1,2-for review]. In addition, PGE1 given intramuscularly in cows increased circulating progesterone for the duration of the 72 h sampling period [21]. Acute treatment with PGE1 in the interstitial tissue of the ovarian vascular pedicle of the corpus luteum-containing ovary of ewes increased occupied and unoccupied luteal LH receptors, while PGE2 does not increase occupied or unoccupied LH receptors in luteal tissue [22,23]. Moreover, chronic intrauterine treatment with PGE1 from days 10 to 16 post-estrus increased luteal occupied and unoccupied LH receptors, mRNA for LH receptors, and circulating progesterone, while PGE2 only prevented a decrease in mRNA for LH receptors, occupied and unoccupied luteal LH receptors, and circulating progesterone in ewes [24]. These data indicate differences in PGE1 and PGE2 actions to prevent luteolysis in ewes. In addition, intra-luteal implants of PGE1 or PGE2 on days 13–19 prevented the decline in circulating progesterone, luteal mRNA for LH receptors, and unoccupied and occupied receptors for LH in Angus or Brahman cows [25]. Subtypes of PGE receptors (EP1, EP2, EP3 (A–D), EP3A, EP3B, EP3C, EP3D, and EP4) and an FP receptor have been identified [26–29]. Data on PGE receptor subtypes have been primarily from in vitro binding rather than in vivo functional studies [26–29]. However, a single treatment with an EP3 or FP specific receptor agonist, but not treatment with an EP1, EP2 [30] or an EP4 [31] specific receptor agonist, given into the interstitial tissue of the ovarian vascular pedicle adjacent to the corpus luteum-containing ovary during the estrous cycle of ewes decreased circulating progesterone below 1 ng/ml, decreased luteal mRNA for LH receptors, and luteal unoccupied and unoccupied receptors for LH within 48 h. The objective of this experiment was to determine whether intraluteal implants containing PGE1 or PGE2 affect luteal mRNA for FP, EP1, EP2, EP3 (A–D), EP3A, EP3B, EP3C, EP3D, or EP4 to explain the anti-luteolytic effect of PGE1 or PGE2 in Angus or Brahman cows. 2. Materials and methods 2.1. General procedures This experiment was approved by Texas A&M University Animal Care and Use Committee (TAMU AUC 2008-129). Cows were checked twice daily (05:00 and 17:00 h) for estrus. Twenty nonlactating multiparous Angus cows and twelve nonlactating multiparous Brahman cows from 5 to 8 years of age were maintained on pasture and withdrawn from feed and water 12 h prior to surgery. Prior to surgery, the flank of cows was clipped, washed with soap and water, scrubbed with Betadine (Purdue Frederick, Stamford, CT), and given Lidocaine (Hospira Inc., Lake Forest, I) was administered subcutaneously into the flank as a local anesthesia. Ten cm3 of penicillin were given around the incision and intramuscularly (Bimeda Inc., Le Sueur, MN) after surgery [25]. 2.2. Intra-luteal implants and collection of samples One-half ml of Dow 382 silastic elastomer (Dow Corning Chemical Co., Midland, MI) was mixed in a sterile tube with 1 mg of PGE1 or PGE2 , then mixed with 0.5 ml medical grade fluid #360 (Dow Corning Chemical Co.; via Factor II Inc., Lakeside, AZ), and then mixed with 20 drops of stannous octoate (Spectrum Chemical Co., Gardena, CA) as a catalyst. 0.5 ml of the mixture was loaded into a sterile 1-cm3 syringe with a 16-gauge needle for injection into the corpus luteum via the ovarian stroma. If size of the

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Table 1 Primer sequences for real time PCR. FP EP1 EP2 EP3 (A–D) EP3A EP3B EP3C EP3D EP4 GAPDH bRPL19

F 5 -TTGGTGTTCTGTGAGCTGAAGT-3 R 5 -CATGACCAGAGCGACATCAT-3 F 5 -GGCCGCTGTTTTTGGCCGTG-3 R 5 -CCTCCATGGCTGCCCTTGGC-3 F 5 -TTTCCAGGGAAGGGTGTATG-3 R 5 -GAGCATGAGTCAAGCCATGT-3 F 5 -ACTCCGACCCGGGCATCTGG-3 R 5 -TTCTTGCGCTTGCCCTCCCG-3 F 5 -CGGAGGGTGGGACAGAGAACAAAGA-3 R 5 -TGCTTCTGTGAAATGGCCTAAAAGGAT-3 F 5 -GTCGCCGTTGCTGATAATGA-3 R 5 -GTGAGGCCTGGCAAAACTT-3 F 5 -GTCGCCGTTGCTGATAATG-3 R 5 -CCACATGCTGGCAAAACTT-3 F 5 -CGTCAGTTGAGCACTGCAAG-3 R 5 -GTCGGCACTTTAGGTCCATC-3 F 5 -TCCCAGTGAAACGCTGAAC-3 R 5 -CTCGTCTGTCTGCAAAGTGC-3 F 5 -ACATCAAGTGGGGTGATGCT-3 R 5 -GGCATTGCTGACAATCTTGA-3 F 5 -CACAAGCTGAAGGCAGACAA-3 R 5 -CGGGCTTCCTTGGTCTTAGG-3

intra-luteal implant weighs more than 500 mg, circulating progesterone will decline to less than 1 ng/ml within 24 h [25]. Implants with PGE1 or PGE2 secreted an average of 5.1 and 4.7 ng/4 h, respectively, in vitro. On day-13 post-estrus, Angus cows received no intra-luteal implant and corpora lutea were retrieved. Angus and Brahman cows received intra-luteal implants of Dow Corning 382 silastic elastomer containing Vehicle, PGE1 , or PGE2 on day-13 postestrus via a flank laparotomy and corpora lutea were retrieved on day-19 [25]. Corpora lutea retrieved on day-13 or day-19 were weighed, quartered, and frozen in liquid nitrogen until analysis for mRNA for prostanoid receptors in luteal tissue. Day-13 Angus luteal weights served as pre-luteolytic controls. These are the same cows, treatments, and procedures reported previously for changes in circulating progesterone, mRNA for luteal LH receptors, and unoccupied and occupied luteal LH receptors [25]. 2.3. Real-time PCR analysis for prostanoid mRNA Total RNA was isolated from each sample with Trizol (Invitrogen, Carlsbad, CA) and then purified using RNeasy (Qiagen, Valencia, CA), following the manufacturer’s directions. The concentration and integrity of each sample of RNA was assessed by UV spectroscopy using an Eppendorf Biophotometer-plus (Eppendorf, Hauppauge, NY) as well as by visual distinction of 18S and 28S rRNA bands after ethidium bromide staining in an agarose gel. cDNA was reverse transcribed from each sample of RNA and real-time PCR performed, as described previously (111–114), to determine expression of mRNA for FP, EP1, EP2, EP3 (A–D), EP3A, EP3B, EP3C, EP3D, and EP4, and the housekeeping genes GAPDH, and bRPL19 using SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA) and gene specific primer pairs (Table 1) on a Bio-Rad IQ5 system. The specificity of each primer set was confirmed by both running the PCR products on a 2.0% agarose gel and analyzing the melting (dissociation) curve after each PCR reaction. Protocol conditions consisted of denaturation at 95 ◦ C for 30 s, followed by 40 cycles at 94 ◦ C for 30 s, 53 ◦ C for 30 s and 72 ◦ C for 45 s with a final dissociation analysis. The relative level of expression of each mRNA was standardized against GAPDH and bRPL19 as housekeeping genes [25,32–34]. 2.4. Statistical analyses All data were analyzed for normality and for homogeneity of variance by Bartlett’s Box F Test and data were transformed by log 10 when necessary [35]. Data for luteal weights were analyzed

Fig. 1. Effect of intra-luteal silastic implants of vehicle, PGE1 , or PGE2 in Angus or Brahman cows on luteal mRNA for FP receptors/normalized GAPDH/bRPL19 during the estrous cycle.

by a 2 × 3 Factorial design for ANOVA [35] and published previously [25]. mRNA data for prostanoid receptor subtypes were analyzed by REST-2008 software and expressed as relative expression ratios compared to control groups [36]. Changes in mRNA copy number were analyzed by a Factorial design for ANOVA (breed × PG receptor subtype; 35). When differences among treatment means were detected, differences were compared by a Least Significant Difference test [35]. 3. Results Day-13 Angus luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased (P ≤ 0.05) in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE1 and PGE2 up-regulated (P ≤ 0.05) FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed and gene expression was greater (P ≤ 0.05) in Angus than Brahman cows treated with PGE1 , but was greater (P ≤ 0.05) in Brahman than Angus cows on day-19 treated with PGE2 implants (Fig. 1). EP1 receptor mRNA was not altered (P ≥ 0.05) by any treatment compared to Vehicle controls on day-19 (data not shown). PGE1 or PGE2 down-regulated (P ≤ 0.05) mRNA for EP2 receptors when compared to Vehicle implants on days 13–19 regardless of breed (Fig. 2). PGE1 and PGE2 up-regulated (P ≤ 0.05) mRNA for EP3B receptors (Fig. 3), mRNA for compared to Vehicle controls on days 13–19 regardless of breed. PGE1 and PGE2 down-regulated (P ≤ 0.05) mRNA for EP4 receptors compared to Vehicle controls regardless of breed (Fig. 4). The similarities in relative gene expression profiles induced by PGE1 and PGE2 support their agonistic effects. 4. Discussion Decreased luteal weights, circulating progesterone, loss of luteal mRNA for LH receptors, and loss of luteal unoccupied and occupied receptors for LH in these same Angus and Brahman cows with control intra-luteal implants from days 13 to 19 post-estrus have been reported previously [25]. In addition, in these same Angus and Brahman cows, treated with intra-luteal implants containing PGE1 or PGE2 from days 13 to 19 post-estrus prevented the decrease in luteal weights, circulating progesterone, loss of luteal mRNA for LH receptors, and loss of luteal unoccupied and occupied receptors for LH was reported previously [25]. These data are supported further by similar data in ewes treated with chronic intra-uterine

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Fig. 2. Effect of intra-luteal silastic implants of vehicle, PGE1 , or PGE2 in Angus or Brahman cows on luteal mRNA for EP2 receptor expression ratio compared to Angus + Brahman implant/normalized GAPDH/bRPL19 during the estrous cycle.

Fig. 3. Effect of intra-luteal silastic implants of vehicle, PGE1 , or PGE2 in Angus or Brahman cows on luteal mRNA for EP3B receptor expression ratio compared to Angus + Brahman implant/normalized GAPDH/bRPL19 during the estrous cycle.

Fig. 4. Effect of intra-luteal silastic implants of vehicle, PGE1 , or PGE2 in Angus or Brahman cows on luteal mRNA for EP4 receptor expression ratio compared to Angus + Brahman implant/normalized GAPDH/bRPL19 during the estrous cycle.

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infusions of PGE1 or PGE2 where PGE1 or PGE2 also prevented the decrease in luteal weights, circulating progesterone, loss of luteal mRNA for LH receptors, and loss of luteal unoccupied and occupied receptors for LH [24]. The decrease in mRNA for FP receptors in luteal tissue on day-19 of Angus and Brahman cows treated with control intra-luteal implants reported herein is not due to the intraluteal implant and is consistent with decreased FP receptor mRNA in luteal tissue when luteolysis is induced with an exogenous luteolytic dose of PGF2␣ given intramuscularly in ewes [37]. Treatment with a luteolytic dose of PGF2␣ at mid-cycle in ewes decreased the mRNA for FP receptor expression by fifty percent [37]. Although intra-luteal implants containing PGE1 or PGE2 prevented luteolysis in Angus or Brahman cows reported herein, PGE1 or PGE2 prevented loss of mRNA for FP receptors leaving the bovine luteolytic mechanism intact. Receptors for EP3 may also play a role in luteolysis. PGF2␣ given at mid-cycle to ewes decreased mRNA for FP receptors, but did not alter expression of mRNA for EP3 receptors [37]. Support for both FP as well as EP3 receptors being involved in luteolysis has been reported in ewes [30]. Treatment with an EP3 receptor agonist was as effective as exogenous PGF2␣ in inducing luteolysis in ewes [30] and PGF2␣ does not affect expression of mRNA for EP3 receptors in ewes [37]. A single treatment with an EP3 receptor agonist given at mid-cycle into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary of ewes was as effective as a luteolytic dose of PGF2␣ given via the same route in decreasing luteal weight, circulating progesterone, luteal mRNA for LH receptors, and luteal unoccupied and occupied receptors for LH over the 48 h treatment period [30]. This effect of an EP3 receptor agonist is not due to interaction with the FP receptor, since there is little cross reactivity with the FP receptor [27]. Genes encoding both EP3 and FP receptors are on the same chromosome and are in close proximity [27]. EP3B receptor mRNA was decreased in day-19 control cows; however, PGE1 or PGE2 increased mRNA for EP3B receptor mRNA in bovine luteal tissue on day-19 to maintain the EP3B receptor-mediated luteolytic mechanism. PGE1 , but not necessarily PGE2 , increased other EP3 mRNA subtypes (data not shown), which may be related to the smaller luteal weights reported previously for Brahman cows than Angus cows during the estrous cycle [16,17]. However, at day-200 of pregnancy luteal weights are similar in Angus and Brahman cows [16,17]. This may indicate that lack of luteal PGE1 may explain the lighter Brahman cow luteal weights than in Angus cows during the estrous cycle [16,17]. The lack of an effect of PGE1 or PGE2 on mRNA for EP1 receptors in Angus or Brahman cows is consistent with the lack of an effect of an EP1 receptor agonist given in vivo on luteal weight, circulating progesterone, mRNA for luteal LH receptors, and luteal unoccupied and occupied receptors in ewes [30]. The decrease in mRNA for EP2 receptors in luteal tissue of both Angus and Brahman cows treated with intra-luteal implants containing PGE1 or PGE2 is interesting. While PGE1 , but not PGE2 , has some cross reactivity with IP receptors albeit at a magnitude of concentration 3-fold above that for a highly specific IP receptor agonist in the cow [29]. Thus, some of the effect of PGE1 , but not PGE2 , on mRNA for EP2 receptors could have been mediated via the IP receptor. This may indicate a unique interaction to prevent too many EP2 or EP4 receptors to limit growth of luteal tissue, since a plethora of papers have been associated with EP2 and EP4 receptors, second messengers, growth factors, or oncogene products with carcinoma in reproductive tissues [38–51]. PGE1 or PGE2 alter luteal angiopoietin-1 and fibroblast growth factor-2, but not vascular endothelial growth factor in these same cows with intra-luteal implants [52]. This could be due to the immuno-suppressive and vasodilatory effects of PGE1 and PGE2 to promote abnormal growth of tissues and decrease or inhibit cytokines involved in PGF2␣ -activated luteolysis [53,54]. Increases in the EP3 prostanoid receptor mRNA by PGE1 or PGE2

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reported herein could be related to the decrease in EP2 and EP4 receptor mRNA to control luteal growth. Knockout of the EP3 receptor gene in mice results in massive numbers of tumors in the colon when the knockout mice were challenged with a carcinogen, and it was concluded that the tumors were a result of a lack of the EP3 receptor gene to control the EP1 gene [55,56]. In addition, the decrease in mRNA for EP4 receptors in Angus and Brahman cows treated with intra-luteal implants containing PGE1 or PGE2 is also interesting. While PGE1 , but not PGE2 , has some cross reactivity with IP receptors albeit at a magnitude of concentration 3-fold above that for a highly specific IP receptor agonist. Thus, some of the effect of PGE1 , but not PGE2 , on mRNA for EP4 receptors could have also been mediated via the IP receptor [55,56]. Induction of luteolysis in cows treated in vivo with a PGF2␣ analogue rapidly increased mRNA for EP4 receptors, which was interpreted that EP4 receptors were associated with the luteolytic process (Berisha and Schams, personal communication). However, a single treatment with the EP4 receptor agonist CAY10580 into the interstitial tissue adjacent to the luteal-containing ovary of ewes at mid-cycle did not affect circulating progesterone over the 48 h experimental period and did not affect luteal weight or occupied luteal LH receptors at 48 h [31]. However, the EP4 receptor agonist increased luteal mRNA for LH receptors and luteal unoccupied receptors for LH at 48 h [31]. The EP4 receptor agonist CAY10580 also increased PGE secretion and tended to decrease luteal PGF2␣ secretion by ovine luteal tissue of the estrous cycle in vitro [31]. Cystic follicles and cystic corpora lutea decrease fertility in cows [57,58]. EP2 and EP4 receptors may be involved in the pathogenesis of cystic follicles or cystic corpora lutea in cows. The EP4 gene determined by RT-PCR from microarray analysis of gene expression in granolosa cells is upregulated in cystic dairy cows along with angiogenin and G-coupled receptor genes [59]. Moreover, cyclooxygenase-2 and EP2 and EP4 receptors have been reported to be involved with human uterine endometrial and stromal endometriosis and their invasiveness of these tissues. Selective inhibition of EP2 and EP4 receptors inhibit invasion in these tissues via suppression of metalloproteinases [60–63]. We conclude that both PGE1 and PGE2 may alter expression of mRNA for prostanoid receptors as well as luteal mRNA for LH receptors reported previously in these same cows and these effects may act in concert to prevent luteolysis.

Acknowledgements The authors would like to thank Mr. Wayne Toma (University of Hawaii) for assistance with statistical analysis and graphics. This paper is dedicated to the memory of Dr. Hal R. Behrman who has been a long time collaborator with Dr. Weems. This work was supported by USDA-CSREES Special Grants Program (TSTAR) administrated by the Pacific Basin Advisory Group (PBAG) grant to C.W. Weems (CSREES 2006-04752) and USDA Regional Research Project W-112 to C.W. Weems (HAW-259) at the College of Tropical Agriculture and Human Resources.

References [1] Weems CW, Weems YS, Vincent DL. Maternal recognition of pregnancy and maintenance of gestation in sheep. In: Enne G, Greppi GF, Lauria A, editors. Reproduction and animal breeding: advances and strategies. Proceedings XXX Intl Simposia Societa Italiana Zootechnica. Amsterdam, Holland: Elsevier Press; 1995. p. 277–93. [2] Weems CW, Weems YS, Randel RD. Prostaglandins and reproduction in female farm animals. Vet J 2006;171:206–28. [3] Randel RD, Erb RE. Reproductive steroids in the bovine. VI. Changes and interrelationships from 0 to 260 days of pregnancy. J Anim Sci 1971;33:115–23. [4] Conley AJ, Ford SP. Effect of prostaglandin F2␣ -induced luteolysis in vivo and in vitro progesterone production by individual placentomes in cows. J Anim Sci 1987;65:500–7.

[5] Niswender GD, Farin CE, Gamboni F, Sawyer HR, Nett TM. Role of luteinizing hormone in regulating luteal function in ruminants. J Anim Sci 1986;2(62 (Suppl.)):1–13. [6] Snook RB, Brunner MA, Saatman RB, Hansel W. The effect of antisera to bovine LH in hysterectomized and intact heifers. Biol Reprod 1969;1:49–58. [7] Harrison LM, Kenny N, Niswender GD. Progesterone production, LH receptors, and oxytocin secretion by ovine luteal cell types on days 6, 10 and 15 of the oestrous cycle and day 25 of pregnancy. J Reprod Fertil 1987;79:539–48. [8] Hansel W, Alila HW, Dowd JP, Milvae RA. Differential origin and control mechanisms in small and large bovine luteal cells. J Reprod Fertil 1991;43(Suppl.):77–89. [9] Niswender GD. Molecular control of luteal secretion of progesterone. Reproduction 2002;123:333–9. [10] Juengel JL, Niswender GD. Molecular regulation of luteal progesterone synthesis in domestic ruminants. J Reprod Fertil 1999;54(Suppl.):193–205. [11] Wiltbank M, Diskin M, Niswender GD. Differential actions of second messenger systems in the corpus luteum. J Reprod Fertil 1991;43(Suppl.): 65–75. [12] Niswender GD, Davis TL, Griffith RJ, et al. Judge, jury, and executioner: the auto-regulation of luteal function. In: Juengel JL, Murray JF, Smith MF, editors. Reproduction in domestic ruminants VI. Nottingham University Press; 2007. p. 191–206. [13] Weipz GJ, Wiltbank MC, Nett T, Niswender GD, Sawyer HR. Receptors for prostaglandin F2 ␣ and E2 in ovine corpora lutea during maternal recognition of pregnancy. Biol Reprod 1992;47:984–91. [14] Lukaszewska J, Hansel W. Corpus luteum maintenance during early pregnancy in the cow. J Reprod Fertil 1980;59:485–93. [15] Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine luteal tissue of the estrous cycle and pregnancy or placental secretion of prostaglandins E2 (PGE2 ) and F2␣ (PGF2␣ ), and progesterone in vitro. Prostaglandins Other Lipid Mediat 2007;84:163–73. [16] Weems YS, Lammoglia MA, Vera-Avilla H, Randel RD, Sasser RG, Weems CW. Effect of luteinizing hormone (LH), PGE2, 8-Epi-PGE1 , 8-Epi-PGE2 , trichosanthin, and pregnancy specific protein B (PSPB) on secretion of prostaglandin E (PGE) or PGF2 ␣ in vitro by corpora lutea (CL) from nonpregnant and pregnant cows. Prostaglandins 1998;55:359–76. [17] Weems YS, Lammoglia MA, Vera-Avila H, Randel RD, Sasser RG, Weems CW. Effect of luteinizing hormone (LH) PGE2 , 8-Epi-PGE1 , 8-Epi-PGE2 , trichosanthin and pregnancy specific protein B (PSPB) on secretion of progesterone in vitro by the corpus luteum (CL) from nonpregnant and pregnant cows. Prostaglandins 1998;55:28–43. [18] Marsh J. The effect of prostaglandins on the adenyl cyclase of the bovine corpus luteum. Ann NY Acad Sci 1971;180:416–25. [19] Nett TM. Function of the hypothalamic–hypophyseal axis during gestation in ewes and cows. J Reprod Fertil 1987;34(Suppl.):201–13. [20] Milvae RA, Hansel W. Inhibition of bovine luteal function by indomethacin. J Anim Sci 1985;60:528–31. [21] Kimball F, Lauderdale J. Prostaglandins E1 and F2 ␣ specific binding sites in. bovine corpora lutea: comparison with luteolytic effects. Prostaglandins 1975;10:313–9. [22] Reynolds LP, Stigler J, Hoyer GL, et al. Effects of PGE1 or PGE2 on PGF2 ␣-induced luteolysis in nonbred ewes. Prostaglandins 1981;21:957–72. [23] Weems CW, Reynolds LP, Huie JM, Hoyer GL, Behrman HR. Effects of prostaglandins E1 or E2 (PGE1 ; PGE2 ) on luteal function and binding of luteinizing hormone in nonpregnant ewes. Prostaglandins 1985;29:161–73. [24] Weems YS, Nett TM, Davis TL, et al. Prostaglandin E1 (PGE1 ), but not prostaglandin E2 (PGE2 ), alters luteal and endometrial luteinizing hormone (LH) occupied and unoccupied receptors and mRNA LH receptor:␤ actin ratio in ovine luteal tissue to prevent luteolysis. Prostaglandins Other Lipid Mediat 2010;92:67–72. [25] Weems YS, Arreguin-Arevalo A, Nett TM, et al. In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1 , PGE2 ) prevent luteolysis in cows. I. Luteal weight, circulating progesterone, mRNA for luteinizing hormone (LH) receptors, and occupied and unoccupied luteal receptors for LH. Prostaglandins Other Lipid Mediat 2011;95:35–44. [26] Sugimoto Y, Narumiya S. Prostaglandin E receptors. J Biol Chem 2007;282:11613–7. [27] Narumiya S, Fitzgerald GA. Genetic and pharmacological analysis of prostanoid receptor function. J Clin Invest 2001;108:25–30. [28] Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structure, properties, and functions. Physiol Rev 1999;79:1193–226. [29] Shariff NA, Xu SX, Williams GW, Crider JY, Griffin BW, Davis TL. Pharmacology of [3 H] prostaglandin E1 /[3 H] prostaglandin E2 and [3 H] prostaglandin F2␣ binding to EP3 and FP prostaglandin receptor binding sites in bovine corpus luteum: characterization and correlation with functional data. Pharmacol Exp Ther 1998;286:1094–102. [30] Weems YS, Nett TM, Davis TL, et al. Effects of prostaglandin E and F receptor agonists in vivo on luteal function in ewes. Prostaglandins Other Lipid Mediat 2010;92:67–72. [31] LaPorte ME. Effects of LPA2, LPA3, EP1, 2, 3 and 4, sirtuins, 1, 2 and 3, or estradiol17␤ receptor agonists or antagonists on luteal or endometrial function in ewes. M.S. Thesis. Manoa: University of Hawaii; 2010, 178 pp. [32] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real Time quantitative PCR and the 2-[Delta][Delta]CT method. Methods 2001;25:402–8.

Y.S. Weems et al. / Prostaglandins & other Lipid Mediators 97 (2012) 60–65 [33] Whelan JA, Russell NB, Whelan MA. A method for the absolute quantification of cDNA using real-time PCR. J Immunol Methods 2003;278:261–9. [34] Jeoung M, Bridges PJ. Cyclic regulation of apoptotic gene expression in the mouse oviduct. Reprod Fertil Dev 2011;23:638–44. [35] Steel RGD, Torrie JH. Principles and procedures of statistics. NY: McGraw-Hill Book Co., Inc.; 1960. [36] Pfaffl M. Relative expression software tool 2008 (REST 2008). Munich: Technical University; 2008, http://www.gene-quantification.de/pfaffl-nar-2001.pdf. [37] Tsai SJ, Anderson LE, Juengel J, Niswender GD, Wiltbank MC. Regulation of prostaglandin F2 ␣ and E receptor mRNA by prostaglandin F2 ␣ in ovine corpora lutea. J Reprod Fertil 1998;114:69–75. [38] Lau MT, Wong AS, Leung PC. Gonadotropins induce tumor cell migration and invasion by increasing cyclooxygenases expression and prostaglandin E2 production in human ovarian cancer cells. Endocrinology 2010;151:2593–985. [39] Rask K, Zhu Y, Wang W, Hedin L, Sundfeldt K. Ovarian epithelial cancer: a role for PGE2 -synthesis and signalling in malignant transformation and progression. Mol Cancer 2006;5:62–8. [40] Sales KJ, Katz AA, Howard B, Soeters RP, Millar RP, Jabbour HN. Cyclooxygenase1 is up-regulated in cervical carcinomas: autocrine/paracrine regulation of cyclooxygenase-2, prostaglandin E receptors, and angiogenic factors by cyclooxygenase-1. Cancer Res 2002;62:424–32. [41] Liu H, Yang Y, Xiao J, Liu Y, Yang H, Zhao L. COX-2-mediated regulation of VEGFC in association with lymphangiogenesis and lymph node metastasis in lung cancer. Anat Rec (Hoboken) 2010;293:1838–46. [42] Baratelli F, Lee JM, Hazra S, et al. PGE2 contributes to TGF-beta induced T regulatory cell function in human non-small cell lung cancer. Am J Transl Res 2010;2:356–67. [43] Pack SD, Alper OM, Stromberg K, Augustus M, et al. Simultaneous suppression of epidermal growth factor receptor and c-erbB-2 reverses aneuploidy and malignant phenotype of a human ovarian carcinoma cell line. Cancer Res 2004;64:789–94. [44] Gupta RA, Tejada LV, Tong BJ, et al. Cyclooxygenase-1 is overexpressed and promotes angiogenic growth factor production in ovarian cancer. Cancer Res 2003;63:906–11. [45] Fujino H, Toyomura K, Chen XB, Regan JW, Murayama T. Prostaglandin E2 regulates cellular migration via induction of vascular endothelial growth factor receptor-1 in HCA-7 human colon cancer cells. Biochem Pharmacol 2011;81:379–87. [46] Baratelli F, Lee JM, Hazra S, et al. PGE2 contributes to TGF-beta-induced T regulatory cell function in human non-small cell lung cancer. Am J Transl Res 2010;2:356–67. [47] Oshima H, Popivanova BK, Oguma K, Kong D, Ishikawa TO, Oshima M. Activation of epidermal growth factor receptor signaling by the prostaglandin E2 receptor EP4 pathway during gastric tumorigenesis. Cancer Sci 2011;102:713–9. [48] Kim JI, Lakshmikanthan V, Frilot N, Daaka Y. Prostaglandin E2 promotes lung cancer cell migration via EP4-betaArrestin1-c-Src signalsome. Mol Cancer Res 2010;8:569–77.

65

[49] Qian X, Zhang J, Liu J. Tumor-secreted PGE2 inhibits CCL5 production in activated macrophages through cAMP/PKA signaling pathway. J Biol Chem 2011;286:2111–20. [50] Chandramouli A, Mercado-Pimentel ME, Hutchinson A, et al. The induction of S100p expression by the Prostaglandin E2 (PGE2 )/EP4 receptor signaling pathway in colon cancer cells. Cancer Biol Ther 2010;10:1056–66. [51] Timoshenko AV, Xu G, Chakrabarti S, Lala PK, Chakraborty C. Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells. Exp Cell Res 2003;289:265–74. [52] Weems CW, Weems YS, Ma Y, et al. Effects of prostaglandin (PG) E1 or E2 (PGE1, PGE2) intra-luteal implants on vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), and angiopoietin 1 and 2 (ANG-1; ANG-2) receptor:B-actin ratio to prevent luteolysis in cows. Proc Soc Study Reprod 2011 [Abstr. 211]. [53] Penny L. Monocyte chemoattractant protein 1 in luteolysis. Rev Reprod 2000;5:63–79. [54] Ford SP. Maternal recognition of pregnancy in the ewe, cow, and sow: vascular and immunological aspects. Theriogenology 1984;23:145–61. [55] Narumiya S. Physiology and pathophysiology of disease. Proc Japan Acad 2007;83:296–319. [56] Matsuoka T, Sugimoto Y, Narumiya S. Prostaglandin E receptors. J Biol Chem 2007;170(282):11613–7. [57] Hatler TB, Hayes SH, Laranja da Fonseca LF, Silvia WJ. Relationship between endogenous progesterone and follicular dynamics in lactating dairy cows with ovarian follicular cysts. Biol Reprod 2003;69:218–23. [58] Vanholder T, Opsomer G, DeKruif A. Aetiology and pathogenesis of cystic ovarian follicles in dairy cattle: a review. Reprod Nutr Dev 2006;46:105–19. [59] Grado-Ahuir JA, Aad PY, Spicer LJ. New insights into the pathogenesis of cystic follicles in cattle: microarray analysis of gene expression in granulosa cell. J Anim Sci 2011;89:1769–86. [60] Banu SK, Lee J, Speights Jr VO, Starzinski-Powitz A, Arosh JA. Cyclooxygenase2 regulates survival, migration, and invasion of human endometriotic cells through multiple mechanisms. Endocrinology 2008;149:1180–9. [61] Banu SK, Lee J, Speights Jr VO, Starzinski-Powitz A, Arosh JA. Selective inhibition of prostaglandin E2 receptors EP2 and EP4 induces apoptosis of human endometriotic cells through suppression of erk1/2, akt, NFkappaB, and betacatenin pathways and activation of intrinsic apoptotic mechanisms. Mol Endocrinol 2009:1291–305. [62] Lee J, Banu SK, Rodriguez R, Starzinski-Powitz A, Arosh JA. Selective blockade of prostaglandin E2 receptors EP2 and EP4 signalling inhibits proliferation of human endometriotic epithelial cells and stromal cells through distinct cell cycle arrest. Fertil Steril 2010;93:2498–506. [63] Lee J, Banu SK, Subbarao T, Starzinski-Powitz A, Arosh JA. Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits invasion of human immortalized endometriotic epithelial and stromal cells through suppression of metalloproteinases. Mol Cell Endocrinol 2011;332: 306–13.