0013-7227/04/$15.00/0 Endocrinology 145(1):407–417 Printed in U.S.A. Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2003-1007 Temporal and Tissue-Specific Expression of Prostaglandin Receptors EP2, EP3, EP4, FP, and Cyclooxygenases 1 and 2 in Uterus and Fetal Membranes during Bovine Pregnancy J. A. AROSH, S. K. BANU, P. CHAPDELAINE, AND M. A. FORTIER Downloaded from https://academic.oup.com/endo/article/145/1/407/2878588 by guest on 28 September 2021 Unite´ d’Ontoge´nie & Reproduction, Centre de Recherche du Centre Hospitalier de l’Universite´ Laval, Ste-Foy, Que´bec, Canada G1V 4G2; and Centre de Recherche en Biologie de la Reproduction and De´partement d’Obste´trique et Gyne´cologie, Universite´ Laval, Ste-Foy, Que´bec, Canada GIV 4G2 Uteroplacental prostaglandins (PGs) play pivotal roles in EP2 ϾϾ FP. EP2 and EP3 expressions were modulated in utero- maintenance and /or termination of pregnancy in mammals. placental tissues, depending on days of pregnancy, whereas Regulation of PG biosynthetic and signaling mechanisms in FP was uniformly expressed. COX-1 mRNA and protein were uteroplacental tissues during maintenance of pregnancy is constitutively expressed, whereas COX-2 was highly modu- largely unknown. In the present study, we have characterized lated in uteroplacental tissues throughout pregnancy. Immu- the expression of PGE2 receptors (EP2, EP3, EP4), PGF2␣ re- nohistochemistry showed that EP2 and COX-2 proteins were ceptor (FP), and cyclooxygenase (COX) types 1 and 2 in pla- colocalized in most cell types of placentome CAR, endome- centome caruncle (CAR), intercaruncle, and fetal membrane trium, and myometrium. Our study indicates that EP2 is the tissues during pregnancy in cattle. Pregnant bovine uteri primary cAMP-generating PGE2 receptor expressed in utero- were collected and classified into six groups covering the en- placental tissues during bovine pregnancy. Temporal and tis- tire gestational length. The levels of expression of EP2, EP3, sue-specific expression of PGE2 and PGF2␣ receptors and and FP mRNAs differ depending on tissues and days of ges- COX-1 and -2 at the maternal-fetal interface suggests a selec- tation (days < 50 to > 250). EP4 mRNA was undetectable in all tive and distinctive role for PGE2 and PGF2␣ in uterine activ- the tissues studied. The expression levels of PG receptor ities during pregnancy in bovine. (Endocrinology 145: 407–417, mRNAs were as follows: placentome CAR FP > EP2 >EP3, 2004) intercaruncle EP2 > EP3 > FP, and fetal membranes EP3 > ROSTAGLANDINS (PGs) ARE central mediators in- exist in four isoforms (A–D) having a wide range of action, P volved in several female reproductive functions such from inhibition of cAMP production to increases in intra- as ovulation, fertilization, establishment and maintenance of cellular calcium, and IP (3) and are termed “inhibitory” re- pregnancy, and parturition (1–4). Arachidonic acid (AA), an ceptors (6–8). essential fatty acid stored in membrane phospholipids, is the In most mammals, including ruminants, PGF2␣ is the lu- primary precursor of PGs. AA is converted into PGH2 by the teolytic hormone (9) and a myometrial stimulant (3–4). PGE2 rate-limiting enzymes cyclooxygenase (COX) 1 and 2. PGH2 has been proposed to have multiple roles as a temporary is then converted into different primary PGs, including PGE2, luteotrophic, luteostatic, or luteoprotective signal at the time PGF2␣, PGD2, PGI2, and TxA2, by cell-specific isomerases and of establishment of pregnancy (10, 11); as an immunomodu- synthases (5). PGs exert their effects primarily through G latory mediator at fetal-maternal interface (12); as a mito- protein-coupled receptors designated EP, FP, DP, IP, and TP, genic, antiapoptotic, and angiogenic factor (13, 14); and ei- respectively. EP receptor has four subtypes (EP1, EP2, EP3, ther as a myometrial relaxant (15) or stimulant (16). Bovine and EP4). EP2, EP4, IP, and DP receptors are coupled to EP3 and FP receptors have been cloned (17, 18) but not EP1. adenylate cyclase and generate cAMP that activates the PKA We recently cloned bovine EP2 and EP4 receptors and stud- signaling pathway, and have been termed “relaxant” recep- ied their regulation in the uterus during the estrous cycle and tors. TP, FP, and EP1 receptors are coupled to phospholipase early pregnancy (19). Several studies documented the selec- C, generating two second messengers, inositol triphosphate tive expression of COXs, and PG’s relaxant and contractile (IP3) involved in the liberation of intracellular calcium and receptors in uterine and intrauterine tissues at the time of diacyl glycerol, an activator of protein kinase C, and consti- establishment of pregnancy (20, 21) and at term pregnancy tute the “contractile” receptor group. Bovine EP3 receptors and parturition (3, 4, 22, 23) in a variety of species. However, no detailed information is available in relation to mainte- nance of pregnancy. It has been suggested that changes in the Abbreviations: AA, Arachidonic acid; CAR, caruncle; COX, cycloox- expression of PG relaxant or contractile receptors could be ygenase; FM, fetal membrane; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAR, intercaruncle; PG, prostaglandin. involved in the maintenance of uterine quiescence for the Endocrinology is published monthly by The Endocrine Society (http:// majority of gestation and activate the uterus to contract at the www.endo-society.org), the foremost professional society serving the time of parturition for expulsion of the fetus (24). Therefore, endocrine community. it is necessary to obtain information on tissue-specific and 407 408 Endocrinology, January 2004, 145(1):407–417 Arosh et al. • EP, FP, and COX in Pregnant Bovine Uterus temporal expression and regulation of PG receptors in utero- (full thickness of uterus); and FM was composed of intercotyledonary placental tissues to understand the mechanisms by which portions of amnion, chorion, and chorioallantois. Cross-sections of tis- PGs regulate uterine activity during pregnancy. sues were prepared and processed for immunohistochemistry as de- scribed below. Tissues were cut into small pieces and snap-frozen in In ruminants, endometrial caruncles (CARs) take part in liquid nitrogen and stored at Ϫ80 C until used. the formation of placentomes with fetal cotyledons and are involved in fetal-maternal communication and maintenance Experimental design of pregnancy. The intercaruncular regions have been pri- marily associated with maintenance of uterine quiescence Based on the days of pregnancy, the CAR, ICAR, and FM tissues were classified into six groups as days less than 50 (n ϭ 3), 51–100 (n ϭ 6), and also involved in other essential fetal-maternal interac- 101–150 (n ϭ 4), 151–200 (n ϭ 5), 201–250 (n ϭ 4), and more than 250 (n ϭ tions (25, 26). No information is available on selective ex- 3). Total RNA was isolated using TRIzol according to the manufacturer’s pression and regulation of PGE2 and PGF2␣ receptors in protocol. Total proteins were extracted and quantified (29). Expression placentome CAR, intercaruncle (ICAR), and fetal membrane of EP2, EP3, EP4, and FP mRNAs was studied using RT-PCR and real time quantitative RT-PCR (LightCycler). Expression of COX-1 and Downloaded from https://academic.oup.com/endo/article/145/1/407/2878588 by guest on 28 September 2021 (FM) tissues throughout the pregnancy in cattle. Further- COX-2 mRNA was studied using Northern blot. EP2, COX-1, and COX-2 more, expression of COX enzymes is poorly studied in these proteins were analyzed by Western blot. Cellular localization of EP2 and uteroplacental tissues during the same period. Therefore, the COX-2 proteins were performed by immunohistochemistry. objectives of the present investigation were: 1) to study the expression of PGE2 receptors (EP2, EP3, EP4) and PGF2␣ RT-PCR receptor (FP) in CAR, ICAR, and FM throughout the bovine Total RNA isolated from bovine endometrium and corpus luteum pregnancy; 2) to study the coexpression of COX-1 and COX-2 was used as templates for EP2, EP3, EP4, and FP, respectively. The with PGE2 and PGF2␣ receptors in these uteroplacental tis- PCR-cloning strategies were as described previously (19). Briefly, total sues at different stages of pregnancy. RNA (1 ␮g) was reverse transcribed using random primer and Super- script II RT. Sets of specific primers were deduced from the known sequences of bovine EP2, EP3, EP4, and FP (Fig. 1). Based on gene Materials and Methods structure analysis of PG receptors, the forward primer was selected Materials within exon 1, and the reverse primer was selected within exon 2 to eliminate nonspecific amplification of genomic DNA (Fig. 1) (6–8). The Reagents used for this study were purchased from the following RT-PCR products were cloned into pCR 2.1 Vector. First, the expression suppliers: Superscript II RT, DNA ladder, RNA ladder, dithiothreitol, T4 of EP2, EP3, EP4, and FP mRNAs in CAR, ICAR, and FM was studied kinase, 5ϫ forward reaction buffer, 5ϫ first-strand buffer, TRIzol and by standard RT-PCR. As an internal standard, bovine ␤-actin or glyc- Topo cloning kits (Invitrogen Life Technologies Inc., Burlington, On- eraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using tario, Canada); Random primer-pd(N)6, deoxynucleotide triphosphates, specific primers. The PCR conditions were: 94 C/1 min, 60 C/30 sec, and RNA guard, rTaq DNA polymerase, PCR 10ϫ buffer and Ready-To-Go 72 C/1 min for 35 cycles for each gene. The results demonstrated that DNA labeling kit (Amersham Pharmacia Biotech Montreal, Quebec,