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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 maintenance 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 described 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 primarily 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, EP2, EP3, and FP were detectable, but EP4 was undetectable, by RT-PCR Canada); EcoRI, EcoRV, BamHI, HindIII, and prestained protein markers in all tissue studied (data not shown). (New England Biolabs Inc., Mississauga, Ontario, Canada); enzyme cutting buffers and trypsin (Boehringer Mannheim Corp., Montreal, Quantitative RT-PCR (LightCycler) Quebec, Canada); T7 sequencing kit (USB Corp. Inc., Cleveland, OH); Bright Star-plus nylon membrane and UltraHyb (Ambion Inc., Austin Standard RT-PCR was used to confirm the specificity of the sets of TX); Trans-Blot nitrocellulose membrane (Bio-Rad Laboratories, Her- primers and amplification of single PCR product at the expected size as 32 32 cules, CA); [␥ P]ATP and [␣ P]deoxy-CTP (Perkin-Elmer life Sciences, described above. Then, the same sets of primers (Fig. 1) were used for Markham, Ontario, Canada); COX-1 and COX-2 antibodies (Merk-Frost, LightCycler using SYBR Green I. The reaction was performed in a total vol Montreal, Canada); goat antirabbit biotinylated Ig (Dako Diagnostics of of 20 ␮l in microcapillary tubes according to the manufacturer’s instruction. Canada Inc., Mississauga, Ontario, Canada); goat antirabbit or mouse Standard curve derivation was as follows. Recombinant plasmid containing IgG conjugated with horseradish peroxidase (Jackson Immunoresearch specific inserts of EP2, EP3, and FP, and the purified PCR product for Laboratories, West Grove, PA); monoclonal antimouse ␤-actin antibody GAPDH were used as templates. The plasmid DNA or PCR product was and antihuman rabbit EP2 polyclonal antibody (Cayman Chemicals, quantified and serially diluted from 100 pg to 0.01 pg/2 ␮l. Each reaction Ann Arbor, MI); Vectastain Elite ABC kit (Vector Laboratories Inc., mixture contained 2 ␮l of cDNAs, 2 ␮l FasterStart DNA Master SYBR Green Burlingame, CA); rabbit preimmune serum (Solution Recherche´ Inc., I mix, 2 ␮l of sense and antisense primers each (0.5 ␮m), 1.6 ␮lof25mm Quebec, Canada); Renaissance (Life Science Products Inc., Boston, MA); ␮ MgCl2 and 10.4 l of PCR-grade H2O. The LightCycler programs for each BioMax film (Eastman Kodak Corp., New York, NY); plasmid and gene were as follows: denaturation (95 C/10 min); PCR amplification and mRNA purification kits (QIAGEN Inc., Mississauga, Ontario, Canada); quantification (95 C/10 sec, 60 C/5 sec, 72 C/20 sec) with single fluores- and Mayer’s hematoxylin solution (Sigma-Aldrich Canada Ltd., cence measurement at specific temperature (acquisition) for 5 sec, repeated Oakville, Ontario); LightCycler FasterStart DNA Master SYBR Green I for 30–50 cycles respective to the gene studied; a melting program (70–95 mix and MgCl2 (Roche Diagnostics, Laval, Quebec, Canada). All oligo- C at the rate of 0.1 C/sec with continuous fluorescence measurement); and nucleotide primers were chemically synthesized using ABT 394 synthase finally, a cooling step to 40 C (Fig. 2). At all steps, the transition temperature (Perkin-Elmer, Foster City, CA). The other chemicals used were molec- was 20 C/sec. Quantification was performed using LightCycler analysis ular biological grade available from Laboratoire Mat or Fisher Biotech software. Second-derivative maximum analysis, arithmetic base line ad- (Quebec, Canada). justment, and polynomial calculation methods were used. Base line curve, melting curve, melting point, crossing point, slope, error (0.1–0.5), and Tissue collection correlation (r-1) were monitored (Fig. 2). For each gene, standard curves were repeated three to four times to obtain reproducible results. Earlier, Bovine pregnant uteri were collected at local abattoir immediately different concentrations of cDNA, MgCl2, and various steps in LightCycler after slaughter. Care was taken to eliminate pathological conditions (such as denaturation, PCR, melting and cooling programs) were tested and during processing as described previously (27). Uteri were opened lon- standardized. The amplified products were analyzed in 1.2% agarose gel. gitudinally along the greater curvature. Day of pregnancy was determined by measurement of the crown-rump length of fetuses present in Quantification of unknown. Based on analysis, a suitable PCR program the uterus (28). CAR, ICAR, and FM tissues were collected. CAR was was selected for each gene, and one appropriate standard curve was composed of endometrial CARs and fetal cotyledons (chorion and cho- exported and used as an external standard for quantification of the rioallantois); ICAR tissues consisted of endometrium and myometrium unknown. PCR, quantification and validation methods were as de- Arosh et al. • EP, FP, and COX in Pregnant Bovine Uterus Endocrinology, January 2004, 145(1):407–417 409

FIG. 1. A, PCR strategies. The gene structure analysis of PG receptors revealed no intron between ATG and the VIth transmembrane domain. Transmem- brane domains I–VI are encoded by exon 1, and transmembrane domain VII and the remaining part of the coding sequence by exon 2. B, Based on that,

the forward primer was selected within exon 1, and Downloaded from https://academic.oup.com/endo/article/145/1/407/2878588 by guest on 28 September 2021 the reverse primer was selected within exon 2, to eliminate nonspecific amplification of genomic DNA. C, Details of the primers used and cDNA size and position.

scribed above. For each reaction, 2 ␮l cDNA was used. The level of sections were blocked in 10% goat serum for 1 h at room temperature. mRNA was expressed as a ratio between EP2 or EP3 or FP and GAPDH. Incubation with the primary antibody EP2 (1:500) or COX-2 antiserum (1:4,000) was done overnight at 4 C. The sections were further incubated Northern blot analysis with the second antibody (goat antirabbit IgG biotinilated, 1:200) for 30 min at room temperature. For the negative control, preimmune rabbit Northern blotting and hybridization were performed as described serum was used instead of EP2 (1:20,000) antibody or COX-2 (1:4,000) previously (17, 27). Briefly, total RNA (ϳ20 ␮g) was loaded in each lane antiserum. Between each step, tissues were washed in PBS. Finally, and electrophoresed on 1.2% formaldehyde-agarose gel. RNA was trans- tissues were stained with Mayer’s hematoxylin. Photos were captured ferred overnight onto a nylon membrane in 10ϫ saline sodium citrate. using Spot program (Carsen Group Inc. Corp., Markham, Ontario, Blots were prepared separately for COX-1 and COX-2 and stored at Ϫ20 Canada). C until used. The cDNA probes for COX-1 and -2 were labeled with [␣32P]deoxy-CTP (3000 Ci/mmol) using the Ready-To-Go DNA labeling kit. Prehybridization was carried out for1hat45C,andhybridization Statistical analysis was carried out overnight at 45 C using UltraHyb. The blots were All numerical data were presented as the mean Ϯ sem. Data were stripped of COX-1 and COX-2 probes by boiling in 1% SDS for 30 min analyzed using two-way ANOVA followed by Fischer’s protected LSD and rehybridized with ␥-32P[ATP]-labeled oligoprobe specific to 18S and Duncan new multiple-range comparison and Scheffe´’s tests (SUPER ribosomal RNA to normalize each level of COX-1 and COX-2 mRNA. ANOVA, ABACUS Concepts, Inc., Berkeley, CA). The data were also The blots were exposed to BioMax film, and densitometry of autora- analyzed to study the effect of stage of gestation on tissue and gestation- diograms was performed using an Alpha Imager (Alpha Innotech Corp., tissue interaction. Differences were considered as statistically significant Montreal, Canada). Bovine COX-1 (777 bp) and COX-2 (449 bp) cDNA at 95% confidence level (P Ͻ 0.05). were obtained and used as probes as described previously (27, 30).

Western blot analysis Results Western blot analysis was performed as described previously (19, 27). Expression of EP2 receptor Briefly, total proteins (ϳ20 ␮g) were loaded in each lane and electrophoresed on 10% SDS-PAGE followed by electrotransfer onto nitrocel- EP2 mRNA and protein (Fig. 3) were expressed and mod- lulose membrane. Rabbit antihuman polyclonal EP2 antibody (1:500) ulated in CAR, ICAR, and FM tissues throughout pregnancy. and rabbit antisheep COX-1 and COX-2 (1:3,000) were used as the In CAR, the level of expression of EP2 mRNA and protein primary antibodies. Goat antirabbit IgG conjugated with horseradish was higher (P Ͻ 0.05) during the first trimester than at other peroxidase was used as the secondary antibody (1:20,000). Chemilumi- stages of pregnancy. However, expression of EP2 in ICAR nescent substrate was applied according to the manufacturer’s instruc- tions. The blots were exposed to BioMax film, and densitometry was was highest during midgestation but not affected by stage of done using an ␣ Imager. As an internal standard, ␤-actin (1:5,000) was gestation in FM. The level of expression of EP2 mRNA and measured. protein was higher (P Ͻ 0.05) (2- to 3-fold) in CAR and ICAR than in FM. Immunohistochemistry (see Fig. 5) showed that Immunohistochemistry in CAR, on the maternal side, EP2 protein was expressed in CAR and ICAR cross-sections (1 cm3) were taken. Tissues were fixed epithelial cells of caruncular crypts and stromal cells of ca- in 4% paraformaldehyde-buffered saline for4hat4Candprocessed runcular septa. On the fetal side, EP2 protein was expressed ␮ using standard procedures. Paraffin sections (3 m) were made. Im- in secondary branches of chorioallantoic villus; by contrast, munohistolocalization of EP2 and COX-2 protein was performed using Vectastain Elite ABC kit according to the manufacturer’s protocols and it was absent in its primary branches. EP2 protein was se- as described previously (19). Endogenous peroxidase activity was re- lectively expressed in mononuclear, binuclear, and giant moved by fixing sections in 0.3% hydrogen peroxide in methanol. Tissue cells of trophectoderm. In ICAR, EP2 protein expression was 410 Endocrinology, January 2004, 145(1):407–417 Arosh et al. • EP, FP, and COX in Pregnant Bovine Uterus Downloaded from https://academic.oup.com/endo/article/145/1/407/2878588 by guest on 28 September 2021 Arosh et al. • EP, FP, and COX in Pregnant Bovine Uterus Endocrinology, January 2004, 145(1):407–417 411 high in endometrial luminal epithelium and myometrial (days Ͼ 201), moderate at early (days Ͻ 50–100), and low at smooth muscle, moderate in glandular epithelium, and low mid (days 101–200) stages of pregnancy. The level of expres- in endometrial stroma. sion of COX-2 mRNA and protein were higher (P Ͻ 0.05) in CAR than in ICAR. Immunohistochemistry (Fig. 5) revealed Expression of EP3 receptor that COX-2 protein was expressed more intensely on the fetal Changes in EP3 mRNA (Fig. 3) expression in CAR, ICAR, than on the maternal compartment. In CAR, COX-2 protein and FM were tissue specific and vary with stage of gestation. was moderately and diffusely expressed in epithelial cells of In CAR, EP3 mRNA expression increased (P Ͻ 0.05) with caruncular crypts and stromal cells of caruncular septum. On advancing stage of gestation. In ICAR and FM, EP3 mRNA the fetal side, COX-2 protein was selectively expressed in levels were affected by stage of gestation, but changes in EP3 mononuclear, binuclear, and giant cells of trophectoderm. mRNA in CAR and FM were inversely related during preg- Moreover, COX-2 protein was expressed in secondary Ͻ branches of chorioallantoic villus; by contrast, it was absent nancy. Expression of EP3 mRNA was higher (P 0.05) in Downloaded from https://academic.oup.com/endo/article/145/1/407/2878588 by guest on 28 September 2021 CAR than in ICAR and FM at gestation days greater than 201. in its primary branches. In ICAR, COX-2 expression was high in endometrial luminal epithelial cells, moderate in myome- Expression of FP receptor trium and glandular epithelium, and low in endometrial stroma. Expression of FP mRNA differed (P Ͻ 0.05) among tissues (Fig. 3); high in CAR, moderate in ICAR, and low in FM. Discussion Expression pattern of FP mRNA did not change in any of the Ϯ tissues studied during pregnancy. In cattle, the duration of the pregnancy is 270 5d.The placenta is epitheliochorial, cotyledonary, and noninvasive. Temporal and tissue-specific expression of EP2, EP3, and In bovine species, 75–120 endometrial CARs take part in the FP receptors formation of the placentomes with fetal cotyledons for ex- change of nutrients, wastage, and gas. Intercaruncular re- In CAR, the expression levels of PG receptor mRNAs were Ͼ Ͼ gions are associated with maintenance of uterine quiescence FP EP2 EP3 (Fig. 3). The level of FP expression was and physiological hypertrophy of the uterus to accommodate higher (P Ͻ 0.05) than EP2 from days Ͼ 100 and EP3 from Ͻ Ͼ the growing fetus. The uterine glands present in intercarun- days 50–200 of pregnancy. Near-term (days 250), the cular regions secrete histotroph that is absorbed by placental expression level of EP3 was similar to that of FP. The ex- areolae by fluid phase pinocytosis and release these secre- pression patterns of EP2 and EP3 mRNAs were inversely tions into the fetal circulation (25, 26, 31–33). Thus, carun- related during pregnancy and differed (P Ͻ 0.05) between Ͻ Ͼ cular and intercaruncular tissues of the pregnant uterus play early (days 50–100) and late (days 200) stages of ges- selective and distinctive roles in the maintenance of success- tation. In ICAR, the expression levels indicated EP2 Ͼ EP3 Ն ful pregnancy (31–33). Uteroplacental PGE and PGF ␣ are FP throughout pregnancy. The level of expression of EP2 2 2 Ͻ considered important mediators involved in recognition and mRNA was higher (P 0.05) than FP and EP3, but no maintenance of pregnancy (4, 31–33). In the present study, differences were detected between EP2 and EP3 at any stage we have characterized the temporal and tissue-specific ex- of gestation. In FM, the levels of expression indicated EP3 Ն pression of PGE and PGF ␣ receptors and COX 1 and 2 in EP2 ϾϾ FP throughout pregnancy. Expression of EP2 and EP3 2 2 Ͻ uteroplacental tissues during bovine pregnancy. mRNAs were higher (P 0.05) than for FP throughout preg- In CAR, EP2 mRNA and protein are more highly ex- nancy, but no differences between EP2 and EP3 were pressed during early than at mid and late stages of preg- detected. nancy. EP2 protein is localized in maternal caruncular epithelial and stromal cells and in fetal trophoblast cells. EP2 Expression of COX-1 and COX-2 expression is absent in primary chorioallantoic villi (super- COX-2 mRNA and protein were highly regulated in CAR, ficial part of cotyledon) but is highly expressed in its sec- ICAR, and FM throughout pregnancy, whereas COX-1 ondary branches (deeper part of cotyledon interdigited with mRNA and protein were constantly expressed at low levels caruncular tissue). Activation of EP2 results in generation of (Fig. 4). In CAR, the levels of expression of COX-2 mRNA and cAMP, which, in turn, activates several signaling cascades Ͻ Ͻ protein were significantly (P 0.05) higher in early (days (6–8). The effects of PGE2 mediated by cAMP-dependent 50) and late (days Ͼ 251) than in midpregnancy (days 51– mechanisms are mitogenic, angiogenic, antiapoptotic, and 250). In ICAR, COX-2 mRNA and protein were expressed at immunomodulatory in different cell types (34, 35). More- low levels without change during gestation. In FM, the levels over, PGE2 has long been proposed as a temporary luteostatic of expression of COX-2 mRNA and protein were high at late and/or luteoprotective factor in ruminants (10–11). PGE2

FIG. 2. Real-time RT-PCR (LightCycler) quantification using SYBR Green I. Amplification quality was validated by analysis of (A) amplification curves, (B) melting curves, and agarose gels for, respectively, EP2, EP3, FP, and GAPDH transcripts. Recombinant plasmid DNA, containing specific inserts of EP2 or EP3 or FP receptors and purified PCR products for GAPDH, were serially diluted from 100 to 0.01 pg and used for derivation of the standard curve. PCR-grade water was used as negative control. Placentome CAR and/or intercaruncular tissues were used as unknown samples. Both melting curves and gel analysis show a single peak and band at the expected size, respectively. The acquisition temperatures for EP2, EP3, FP, and GAPDH are 87, 88, 80, and 88 C, respectively (indicated by vertical arrows). The numbers of PCR cycles were 40 for EP2, 50 for EP3, 36 for FP, and 30 for GAPDH. More details are given in Materials and Methods. 412 Endocrinology, January 2004, 145(1):407–417 Arosh et al. • EP, FP, and COX in Pregnant Bovine Uterus Downloaded from https://academic.oup.com/endo/article/145/1/407/2878588 by guest on 28 September 2021

FIG. 3. Expression and regulation of EP2, EP3, and FP receptors in placentome CAR, ICAR, and FM tissues throughout bovine pregnancy. A, Level of expression of EP2, EP3, and FP mRNAs based on real time RT-PCR (LightCycler) quantification. As an internal standard, GAPDH mRNA was measured. Values are expressed as mean Ϯ SEM of ratios between EP2 or EP3 or FP and GAPDH mRNAs. B, Analysis of PCR-amplified products of EP2, EP3, FP, and GAPDH, after the stipulated number of cycles using LightCycler in 1.2% agarose gel. C, Western analysis of EP2 protein. As an internal standard, ␤-actin was measured. D, Densitometry of EP2 protein is expressed as the mean Ϯ SEM of the ratio between EP2 and ␤-actin protein. Each group consisted of three to six samples; representative samples are shown. a and e, EP2—days Ͻ 50–100 vs. others; c and f, EP2—days 51–250 vs. others; b, EP3—days 201–Ͼ250 vs. others; d, EP3—days Ͻ 50 vs. others; P Ͻ 0.05. More details are given in Materials and Methods. Note: The scales used for relative expression are not the same for placentome CAR, ICAR, and FM. regulates its own production in uteroplacental tissues, de- maternal-fetal cross-talk between the different cell types to pending on stage of bovine gestation (36). PGE2 is involved maintain successful pregnancy. in placental functions and maintenance of pregnancy in ewes In ICAR, EP2 is expressed highly during midpregnancy,

(37, 38). Expression of EP2 in CAR supports a role for PGE2 and its level of expression is higher than that of EP3 and FP as paracrine and an autocrine factor involved in the regu- at all stages. EP2 protein is localized in luminal epithelium, lation of growth, differentiation, and function of the placen- stroma, and glandular epithelium of endometrium and myo- tome CAR. Furthermore, the presence of EP2 receptors in metrial smooth muscle cells. Other studies have demon- maternal caruncular epithelial, stromal, and fetal trophoblast strated the expression of EP2 in ovine endometrium at near- cells indicates that PGE2 could act through cAMP to effect the term pregnancy (22). Recently, we have reported increased Arosh et al. • EP, FP, and COX in Pregnant Bovine Uterus Endocrinology, January 2004, 145(1):407–417 413 Downloaded from https://academic.oup.com/endo/article/145/1/407/2878588 by guest on 28 September 2021

FIG. 4. Expression of COX-1 and COX-2 in placentome CAR, ICAR, and FM tissues throughout bovine pregnancy.A, Northern analysis of COX-1 and COX-2 mRNA expression. As an internal standard, 18S RNA was measured. B, Western analysis of COX-1 and COX-2 proteins. As an internal standard, ␤-actin was measured. C, Densitometric values for COX-1 and COX-2 mRNAs are expressed as the mean Ϯ SEM of ratios between COX-1 or COX-2 mRNA and 18S RNA. D, Densitometric values for COX-1 and COX-2 proteins are expressed as the mean Ϯ SEM of ratios between COX-1 or COX-2 and ␤-actin proteins. Each group consisted of three to six samples, representative samples are shown. a, b, and c, COX2 mRNA; d, e, and f, COX-2 protein; a and d, days 51–250 vs. others; b and e, days 101–200 vs. others; c and f, days Ͼ 201 vs. others; P Ͻ 0.05. More details are given in Materials and Methods. expression of EP2 in bovine endometrium and myometrium agonist of EP2, increases cAMP production and reduces during early pregnancy (19). The role of endometrial PGs myometrial contraction (41) and abolishes oxytocin-induced during pregnancy is not completely understood and may myometrial activity (15). The action of butaprost is signifi- include regulation of endometrial receptivity (39). The EP2 cantly greater in pregnant myometrium than in nonpregnant has been considered as a relaxant receptor in the myome- tissues, suggesting that there are more EP2 receptors in the trium of different species (4, 15, 22, 23, 40–42). Butaprost, an pregnant myometrium (15). Our results, showing increased 414 Endocrinology, January 2004, 145(1):407–417 Arosh et al. • EP, FP, and COX in Pregnant Bovine Uterus Downloaded from https://academic.oup.com/endo/article/145/1/407/2878588 by guest on 28 September 2021

FIG. 5. Cellular localization of EP2 and COX-2 proteins in placentome CAR and intercaruncular tissues during bovine pregnancy (50–150 d). A, Normal structure of placentome CAR. B, Immunohistochemistry. EP2 and COX-2 proteins are selectively expressed in epithelial cells of caruncular crypts, stromal cells of caruncular septum, and uninucleated, binucleated, and giant cells of trophoblast luminal epithelial, glandular epithelial, and stromal cells of endometrium and myometrial smooth muscle cells. EP2 and COX-2 proteins are colocalized in most cell types. BV, Blood vessels; CEP, caruncular epithelial cells; CC, caruncular crypts; CS, caruncular stalk; CSE, caruncular septum; CST, caruncular stromal cells; GLE, glandular epithelial cells; LE, luminal epithelium; LU, lumen; PCAV, primary chorioallantoic villus; SCAV, secondary chorioallantoic villus; SMC, smooth muscle cells; ST, stromal cells; TRV, trophoblastic-villous; UW, uterine wall. 1, Mononuclear; 2, binuclear; and 3, giant cells of trophectoderm. Immunohistochemistry was performed using Vectastain Elite ABC kit as described in Materials and Methods. Arosh et al. • EP, FP, and COX in Pregnant Bovine Uterus Endocrinology, January 2004, 145(1):407–417 415 expression of EP2 receptor in myometrium, support the con- (49, 50). Normally, FP expression increases just before labor cept that PGE2 of endometrial and/or myometrial origin is in most mammalian species (4, 20, 22, 42). In this study, high involved in maintenance of uterine quiescence during expression of FP in CAR is interesting but physiological pregnancy. intriguing. The physiological significance of this selective The EP3 receptor has different isoforms encoded by a tissue-specific expression during the course of pregnancy is single gene and generated by alternative C-terminal splicing not known. Whether the FPB isoform, the negative regulator (6, 7). Species variation does exist in the number of EP3 of FPA, is expressed in uteroplacental tissues remains to be subtypes: three in mouse, four in rat, four in bovine, five in elucidated. rabbit, and seven in human (43). Pharmacological and mo- In the present study, EP4 is undetectable in any of the lecular characterization of bovine EP3 isoforms indicated uteroplacental tissue during pregnancy. In rat, mouse, and that EP3B and EP3C increased, whereas EP3A inhibited human uterus, expression of EP4 along with EP2 was ob- cAMP production, and EP3D decreased cAMP and increased served during the implantation window and establishment Downloaded from https://academic.oup.com/endo/article/145/1/407/2878588 by guest on 28 September 2021 IP3 (6, 7, 43). Although some EP3 subtypes were pharmaco- of pregnancy (20, 21, 51). Recently, we found that EP2 was logically associated with cAMP formation by activation of highly expressed in endometrium and myometrium at the adenylate cyclase, there was no report of EP3-induced cAMP time of establishment of pregnancy, whereas expression of production in uterine tissues (44). In this study, we designed EP4 was very low or undetectable (19). Thus, changes in EP4 specific primers that did not distinguish the four isoforms of expression are species specific. Although EP4 is expressed in the bovine EP3. In CAR, EP3 mRNA is more highly expressed human uterus, it has either no or minimal effect on myo- during late than at mid and early stages of pregnancy. The metrial relaxation (43). Moreover, EP4 null mice reproduces expression pattern of EP3 mRNA is inversely related to that normally (8, 52), whereas EP2-deficient mice suffer from of EP2 receptor. In ICAR, EP3 mRNA expression is low multiple reproductive failures (8, 53). The present findings compared with EP2, and it is not modulated. In FM, EP3 is show that EP2 is the primary cAMP generating PGE2 recep- expressed at a level comparable with ICAR, and changes tor expressed and modulated in uterine and intrauterine during gestation are those of EP2. Abundant expression of tissues during pregnancy in cattle. EP3 in human uterus is associated with increased myome- During bovine pregnancy, COX-1 mRNA and protein are trial contraction at late stages of gestation (16). During hu- constitutively expressed, whereas COX-2 is highly modu- man pregnancy, EP3 receptor mRNA expression in the myo- lated in uteroplacental tissues. In CAR, COX-2 is highly metrium is reduced, suggesting that loss of EP3 may be an expressed at the beginning and at the end of the pregnancy. important regulatory mechanism to maintain quiescence of COX-2 protein is localized in maternal caruncular epithelial myometrium (45). In this study, there are contrasting ex- and stromal cells and fetal trophoblast. COX-2 is not expression patterns for EP3 and EP2. Distinct functions of EP2 pressed in primary chorioallantoic villi but is highly ex- (relaxation and vasodilation effects on smooth muscle cells) pressed in its secondary branches. Our findings support vs. effect of EP3 (contraction and vasoconstriction of smooth those reported for bovine (54), sheep (55, 56), and humans muscle) suggest that high levels of expression of EP3 mRNA (57). In ICAR tissues, COX-2 is more highly expressed in near the end of pregnancy contribute to mechanisms asso- luminal epithelium than in stroma, glandular epithelium, ciated with the initiation of parturition. However, the exis- and myometrial smooth muscle cells. Increased expression of tence of different isoforms with distinct functions does not COX-2 was reported in endometrium during the implanta- permit one to define the exact role of EP3. Indeed, it remains tion window and early pregnancy in bovine and ovine (25, to be determined whether different EP3 isoforms are ex- 58–60). Other results indicated that COX-2 expression was pressed in a cell-specific manner under different physiolog- decreased in myometrium during early and mid, compared ical conditions in bovine uteroplacental tissues during preg- with late and near term, pregnancy in several species (4, 54, nancy. Taken together, these results suggest a significant role 61–64). In FM, COX-2 expression is moderate during early, for PGE2 in uterine receptivity and quiescence, two prereq- low at mid, and high at late stages of pregnancy. Placental uisite mechanisms for successful maintenance of pregnancy. expression of COX-2 is gradually increased during the sec- FP is generally considered as a contractile receptor with ond half of pregnancy and at the time of labor in a variety two isoforms (FPA and FPB), and generated by alternative of species, including ruminants (4, 56, 62, 65). In a given splicing of C terminal of a single gene identified in ovine and tissue, the eventual production of PGE2 and PGF2␣ is deter- bovine (46, 47). Bovine FPB acts as a negative regulator to mined not only by COX isoenzymes but also by other en- attenuate the normal FPA-mediated protein kinase C func- zymes, such as cPLA2, PG synthases, and PGDH. The ex- tion (47). Information on FPB is at a preliminary stage. There- pression level of cPLA2 mRNA is not modulated, but PGFS fore, in this study, we evaluated only the bovine FPA mRNA. mRNA is highly regulated throughout pregnancy in mice In CAR, expression of FP mRNA is high and not affected by (66). PGES mRNA is expressed in ovine intrauterine tissues stage of gestation. In ICAR and FM, FP is expressed at very during pregnancy (67). PGDH is more highly expressed in low levels. Other studies have documented the expression of uteroplacental tissues during early and mid, than during late FP mRNA in endometrium and myometrium near-term in pregnancy, in ewe (68) and mice (66). Our recent studies ovine (22) and in myometrium in human (24) and rat (24, 42). demonstrate the expression of COX-1, COX-2, PGES, PGFS, In human, FP protein is expressed at a low level in smooth and PGDH in bovine endometrium during the implantation muscle cells of myometrium at all stages of pregnancy but window (17, 25, 69, 70). However, such coexpression of PG increases toward term (48). Increased placental apoptosis biosynthetic and catabolic enzymes in bovine uteroplacental and parturition failure were identified in FP-deficient mice tissues during pregnancy remains to be determined. Fur- 416 Endocrinology, January 2004, 145(1):407–417 Arosh et al. • EP, FP, and COX in Pregnant Bovine Uterus thermore, among the three tissues studied, COX-2 first in- 9. McCracken JA, Custer EE, Lamsa JC 1999 Luteolysis: a neuroendocrine mediated event. Physiol Rev 79:263–323 creases in fetal and then in maternal tissues at the late stage 10. Pratt BR, Butcher RL, Inskeep EK 1977 Antiluteolytic effect of the conceptus of pregnancy, supporting the notion that labor is initiated on and of PGE2 in ewes. J Anim Sci 45:784–791 the fetal side and then maternal tissues become involved (4, 11. Magness RR, Huie JM, Hoyer GL, Huecksteadt TP, Reynolds LP, Seperich 64). 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