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Endocrinology Vol. 145, No. 5 2551-2560
Copyright © 2004 by The Endocrine Society

Prostaglandin Biosynthesis, Transport, and Signaling in Corpus Luteum: A Basis for Autoregulation of Luteal Function

J. A. Arosh, S. K. Banu, P. Chapdelaine, E. Madore, J. Sirois and M. A. Fortier

Unité d’Ontogénie et Reproduction (J.A.A., S.K.B., P.C., E.M., M.A.F.), Centre Hospitalier Universitaire de Québec, Centre Hospitalier de l’Université Laval, Centre de Recherche en Biologie de la Reproduction, and Département d’Obstétrique et Gynécologie, Université Laval (M.A.F.), Québec, G1K 704, Canada; and Centre de Recherche en Reproduction Animale (J.S.), Département de biomédecine vétérinaire, Université de Montréal, Québec J2S 7C6, Canada

Address all correspondence and requests for reprints to: Dr. Michel A. Fortier, Ph.D., Unité d’Ontogénie et Reproduction, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Centre Hospitalier de l’Université Laval, 2705 Boul Laurier, Ste-Foy, Québec GIV 4G2, Canada. E-mail: mafortier{at}crchul.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The corpus luteum (CL) is a transient ovarian endocrine gland formed from the ovulated follicle. Progesterone is the primary secretory product of CL and is essential for establishment of pregnancy in mammals. In the cyclic female, the life span of CL is characterized by luteal development, maintenance, and regression regulated by complex interactions between luteotrophic and luteolytic mediators. It is universally accepted that prostaglandin (PG) F2a is the luteolysin whereas PGE2 is considered as a luteotropin in most mammals. New emerging concepts emphasize the autocrine and paracrine actions of luteal PGs in CL function. However, there is no report on selective biosynthesis and cellular transport of luteal PGE2 and PGF2{alpha} in the CL of any species. We have studied the expression of enzymes involved in the metabolism of PGE2 and PGF2{alpha}, cyclooxygenase (COX)-1 and -2, PGE and F synthases, PG 15-dehydrogenase, and PG transporter as well as receptors (EP2, EP3, and FP) throughout the CL life span using a bovine model. COX-1, PGF synthase, and PG 15-dehydrogenase are expressed at constant levels whereas COX-2, PGE synthase, PG transporter, EP2, EP3, and FP are highly modulated during different phases of the CL life span. The PG components are preferentially expressed in large luteal cells. The results indicate that PGE2 biosynthesis, transport, and signaling cascades are selectively activated during luteal maintenance. By contrast the PGF2{alpha} system is activated during luteal regression. Collectively, our results suggest an integrated role for luteal PGE2 and PGF2{alpha} in autoregulation of CL function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CORPUS LUTEUM (CL) is a transient ovarian endocrine gland formed from the ovulated follicle. The mature CL is composed of heterogeneous cell population. There are at least two steroidogenic cell types: large luteal cells (LLCs) of granulosa cell origin and small luteal cells (SLCs) of theca cell origin. The primary secretory product of the CL is progesterone (P4), which is essential for establishment and maintenance of pregnancy as well as ovarian cyclicity in most mammals. Defects in luteal function have been associated with infertility, abortion, and ovarian cycle disorders. Mechanisms involved in the control of CL life span and function have been reviewed extensively (1, 2, 3, 4, 5). In the cyclic female, the life span of CL is characterized by luteal development, maintenance, and regression (luteolysis). The life span and function of the CL is regulated by complex interactions between stimulatory (luteotrophic) and inhibitory (luteolytic) mediators. It is universally accepted that prostaglandin (PG) F2{alpha} is the luteolysin in most mammals investigated. Surgical removal of the uterus (hysterectomy) prolongs the life span of the CL in cyclic sheep, cattle, pig, horse, and guinea pig and in the pseudopregnant hamster, rabbit, and rat providing evidence that luteolytic PGF2{alpha} is of uterine/endometrial origin in these species. The endometrial PGs reach the CL by local, systemic, or a combination of both mechanisms depending on the species. By contrast, in primates and humans, hysterectomy does not prolong either the length of the ovarian cycle or the life span of the CL suggesting that luteolytic PGF2{alpha} is not of uterine origin. In these species, it has been proposed that autocrine and paracrine action of luteal PGs may be involved in the control of CL life span and function. Intraluteal production of PGs has been reported in cattle, sheep, pigs, and rats, supporting a role for luteal PGs in CL function in these species as well. Luteal regression proceeds in two steps: the decrease in P4 is considered as functional luteolysis, and the luteal involution is described as structural luteolysis (1, 2, 3, 4). Recent studies have proposed that endometrial/extraluteal PGF2{alpha} initiates functional luteolysis whereas luteal PGF2{alpha} may contribute to structural luteolysis (4, 6).

PGE2 plays many roles in different mammalian systems: mitogenesis, angiogenesis, antiapoptosis, and vasodilation etc. In the female reproductive system, PGE2 has been considered as luteoprotective or luteotrophic in a variety of species since the late 1970s (7, 8). Intrauterine administration of PGE2 protects the CL from spontaneous and/or induced luteolysis in ruminants (7, 8, 9, 10). PGE2 stimulates luteal P4 secretion through a cAMP-mediated pathway in human, rabbit, and ruminant CL (11, 12, 13, 14, 15). When indomethacin was used to inhibit luteal PG production, it reduced P4 secretion in ewes (16). Moreover, a positive correlation between luteal PGE2 and P4 has been demonstrated during the menstrual cycle in humans (17) and the estrous cycle in cows (13). Furthermore, intraluteal metabolism of P4 is an important factor determining the circulating concentration of P4 (4). In rat and rabbit CLs, it has been demonstrated that 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}-HSD) converts P4 into the inactive progestin 20{alpha}-hydroxyprogesterone (18, 19, 20). This functional alteration of P4 secretion and CL function through 20{alpha} HSD activity has not been described in other species (4).

It is evident from the available information that both PGE2 and PGF2{alpha} play key roles in CL function. It has been suggested that luteal tissues or cells possess an inherent capacity to produce PGs in most mammalian species (21). The CL has a rich source of arachidonic acid (AA) (5), an essential fatty acid stored in membrane phospholipids and the primary precursor of all PGs (22). The net production of individual PGs is controlled by several enzymes such as cytosolic phospholipase A2 (cPLA2), cyclooxygenases (COX), PG synthases, and PG 15-dehydrogenase (PGDH). cPLA2 liberates AA from phospholipids. COX-1 and COX-2 convert AA into PGH2, the common intermediate metabolite for various forms of PGs including PGE2 and PGF2{alpha}. The downstream enzymes, PGE synthase (PGES) and PGF synthase (PGFS), catalyze the conversion of PGH2 to PGE2 and PGF2{alpha}, respectively (22). Current evidence suggests three forms of PGES; among them, microsomal PGES-1 (mPGES-1) is highly inducible along with COX-2 (23). We have found that an aldoketoreductase 1B5 (AKR1B5) possessing 20{alpha}-HSD activity is the PGFS involved in the production of PGF2{alpha} in bovine endometrium at the time of luteolysis (24). Catabolism of PGs is governed by PGDH converting PGE2 and PGF2{alpha} into inactive PGEM (PGE2 metabolite) and PGFM (PGF2{alpha} metabolite), respectively (25). COX-1, COX-2, and PGDH mRNAs are expressed in the sheep CL (26, 27). It has been demonstrated that PGF2{alpha} treatment increases COX-2 expression and luteal production of PGF2{alpha} in ruminants (26). However, no information is available on expression and regulation of PGES and PGFS in CL.

PGE2 and PGF2{alpha} exert their effects primarily through the G protein-coupled receptors designated EP and FP, respectively. There are four EP receptor subtypes EP1, EP2, EP3, and EP4, and two FP receptor subtypes FPA and FPB. EP2 and EP4 receptors are coupled to adenylate cyclase activating cAMP that in turn activates the protein kinase A (PKA) signaling pathway. EP1 and FP receptors are coupled to phospholipase C (PLC) generating two second messengers, inositol triphosphate (IP3) involved in the liberation of intracellular calcium (Ca2+) and diacyl glycerol, an activator of protein kinase C (PKC). There are four isoforms of EP3 receptors designated from A to D. These EP3 receptors exhibit a wide range of action from inhibition of cAMP production to increases in Ca2+ and IP3 (28). Expression and regulation of FP has been extensively studied in luteal steroidogenic cells, but information pertaining to EP is largely unknown (29, 30, 31).

It was initially thought that due to their lipid nature PGs would freely cross cell membranes like steroids. However, PGs predominate as charged anions and diffuse poorly through plasma membranes. Prostaglandin transporter (PGT) mediates both the efflux of newly synthesized PGs to effect their biological actions through their cell surface receptors, and influx of PGs from the extracellular milieu for their inactivation (32, 33). We have unraveled the role of PGT in reproductive function for the first time in mammals (34). Our findings have suggested that PGT is involved in cellular transport of PGE2 and PGF2{alpha} within the uterine tissues and in their transport from the uterus to the ovary through the uteroovarian plexus in bovine. Because of the strategic role played by PGs in the regulation of CL function, it appears important to evaluate the contribution of PGT in luteal tissue.

The bovine species is ideally suited to study the role of PGs in the regulation of CL function. In cyclic cow, the CL goes through distinct growing, mature, and regressing phases during its life span, and these have to be studied individually. The cow is a nonseasonal polyestrous animal relatively having a long cycle of 21 d similar to the 28-d human cycle. The bovine CL provides a large volume of luteal tissue easily accessible without ethical concerns. Recently, we have developed most of the molecular tools necessary to explore PGs functions in the bovine reproductive system. We have documented previously the expression and regulation of COX-1, COX-2, PGFS, PGES, PGDH, and PGT as well as PG receptors EP2, EP3, EP4, and FP in uterine tissues during the estrous cycle and pregnancy in the bovine (24, 34, 35, 36, 37, 38).

New concepts that have emerged in recent years regarding the autocrine and paracrine roles of PGs in CL might have important implications in luteal physiology (3, 4, 21). However, lack of evidence on cell-specific expression of various components of PGE2 and PGF2{alpha} biosynthetic, transporting, and signaling machineries remains a major limitation in our understanding of CL function. Therefore, the objectives of the present study were to evaluate concurrently the expression of 1) PG’s biosynthetic enzymes COX-1, COX-2, mPGES-1, and PGFS (AKR1B5); 2) PG’s catabolic enzyme PGDH; 3) PG’s transporter PGT, and 4) PG receptors EP2, EP3, and FP in CL during different phases of its life span.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The numerous reagents necessary for this study were purchased from the following suppliers: Superscript II RT, DNA and RNA ladders, dithiothreitol, T4 kinase, 5x forward reaction and first strand buffers, and TRIzol (Invitrogen Life Technologies Inc., Burlington, Ontario, Canada); Random primer-pd(N)6, deoxynucleotide triphosphates, RNA guard, rTaq DNA polymerase, PCR 10x buffer, and Ready-To-Go DNA Labeling Kit (Amersham Pharmacia Biotech, Montreal, Québec, Canada); prestained protein markers (New England Biolabs Inc., Mississauga, Ontario, Canada); Bright Star-Plus nylon membrane and UltraHyb (Ambion Inc., Austin, TX); Trans-Blot nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA); [{gamma}32P]ATP and [{alpha}32P]dCTP (PerkinElmer Life Sciences, Markham, Ontario, Canada); Renaissance (Life Science Products Inc., Boston, MA); BioMax film (Eastman Kodak Corp, New York, NY); plasmid and mRNA purification kits (QIAGEN Inc., Mississauga, Ontario, Canada); Mayer’s hematoxylin (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada); LightCycler FasterStart DNA Master SYBR Green I mix and MgCl2 (Roche Diagnostics, Laval, Québec, Canada); and Vectastain Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA). All oligonucleotide primers were chemically synthesized using ABT 394 synthase (PerkinElmer, Foster City, CA). The other chemicals used were molecular biological grade available from Laboratoire Mat or Fisher Biotech (Québec City, Québec, Canada). Goat antirabbit biotinylated Ig (Dako Diagnostics of Canada Inc., Mississauga, Ontario, Canada); goat antirabbit or mouse IgG conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA); monoclonal antimouse ß-actin antibody and antihuman rabbit EP2 polyclonal antibody (Cayman Chemicals, Ann Arbor, MI) were purchased. Antibodies against bovine PGES (39), PGFS (24), PGDH (38), and PGT (34) were produced in our laboratories as described previously. Antisheep COX-1 and COX-2 antibodies were donated by Dr. Stacia Kargman (Merck-Frost, Montreal, Canada).

Experimental design
Uteri were collected along with the ovaries from cyclic cows at a local abattoir. Days of the estrous cycle were determined by uteroovarian morphology as described previously (36) and classified into seven groups covering the entire cycle length: d 1–3 (n = 4); 4–6 (n = 3); 7–9 (n = 3); 10–12 (n = 3); 13–15 (n = 6); 16–17 (n = 7); and 18–21 (n = 5). CLs were separated by blunt dissection from the surrounding ovarian tissues. Cross-sections of tissues were prepared and processed for immunohistochemistry as described below. Tissues were cut into small pieces and snap-frozen in liquid nitrogen and stored at –80 C until used. Total RNA was isolated using TRIzol according to the manufacturer’s protocol. Total proteins were extracted and quantified as we described (36). Expression of COX-1, COX-2, and PGT mRNA was studied using Northern blot analysis. COX-1, COX-2, PGES (mPGES-1), PGFS (AKR1B5), PGDH, PGT, and EP2 proteins were studied by Western blot analysis. EP2, EP3, and FP mRNAs were studied using real time quantitative RT-PCR (LightCycler). Cellular localization of COX-2, PGES, PGFS, PGDH, PGT, and EP2 proteins were performed by immunohistochemistry.

Quantitative RT-PCR (LightCycler)
LightCycler reaction using SYBR Green I (Roche Applied Science) and quantifications were performed as we described (37). In brief, total RNA (1 µg) was reverse transcribed using random primer and Superscript II RT. Sets of specific primers were deduced from the known sequences of bovine EP2, EP3, EP4, and FP. LightCycler reactions were performed in a total volume of 20 µl in microcapillary tubes according to the manufacturer’s instructions. Recombinant plasmids containing specific inserts of EP2, EP3, and FP, and the purified PCR product for glyceraldehyde-3-phosphate dehydrogenase were used as templates. The plasmid DNAs or PCR products were quantified and serially diluted from 100–0.01 pg /2 µl. Each reaction mixture contained 2 µl of cDNAs, 2 µl FasterStart DNA Master SYBR Green I mix, 2 µl of sense and antisense primers each (0.5 µM), 1.6 µl of 25 mM MgCl2, and 10.4 µl of PCR-grade H2O. The LightCycler programs for each gene were as follows: denaturation (95 C/10 min); PCR amplification and quantification (95 C/10 sec, 60 C/5 sec, 72 C/20 sec) with single fluorescence measurement at specific temperature (acquisition) for 5 sec repeated for 30–50 cycles depending on the gene studied (see Fig. 2Go); a melting program (70–95 C at rate of 0.1 C/sec with continuous fluorescence measurement), and finally a cooling step to 40 C. At all steps the transition temperature was 20 C/sec.



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  		FIG. 2. Expression of PGES, PGFS, and PGDH in CL. A, Western blot analysis. Densitometry of PGES, PGFS, and PGDH (B); PGES:PGFS (C); PGES:PGDH (D); and PGFS:PGDH (E). All values are expressed as the mean ± SEM of ratios relative to ß-actin. Each group consisted of three to six samples. Data were analyzed using ANOVA followed by Scheffé’s post hoc analysis; P < 0.05. a, PGES, d 1–12 vs. others; b, PGES, d 18–21 vs. 1–12; c, PGES:PGFS, d 1–12 vs. 13–21; d, PGES:PGDH, d 1–12 vs. 13–21; e, PGFS:PGDH, d 4–21 vs. 1–3.

 
Northern blot analysis
Northern blotting and hybridization were performed as we described (36) Briefly, total RNA (~20 µg) was loaded in each lane and electrophoresed on 1.2% formaldehyde-agarose gel. RNA was transferred overnight onto a nylon membrane in 10x saline sodium citrate (SSC). The cDNA probes for COX-1, COX-2, and PGT were labeled with [{alpha}32P] dCTP (3000 Ci /mmol) using the Ready-To-Go DNA Labeling Kit. Prehybridization (2–3 h) and hybridization (overnight) were carried out at 45 C using UltraHyb. The blots were stripped off by boiling in 1% SDS for 30 min and rehybridized with {gamma}32P[ATP]-labeled oligoprobe specific to 18S ribosomal RNA. The blots were exposed to BioMax film and densitometry of autoradiograms was performed using an Alpha Imager (Alpha InnotechCorp., Montreal, Québec, Canada). Bovine COX-1 and COX-2 (36) and PGT cDNAs (34) were obtained and used as probes as described previously.

Western blot analysis
Western blot analysis was performed as we described (36). Briefly, total proteins (~20 µg) were loaded in each lane and electrophoresed on 10% SDS-PAGE followed by electrotransfer onto nitrocellulose membrane. The following primary antibodies (raised in rabbit) were used for the respective protein: antisheep COX-1 and COX-2 (1:3000), antibovine PGES (1:2000), PGFS (1:3000), PGDH (1:2000), PGT (1:1000), and antihuman EP2 (1:500). Goat antirabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody (1:20000). Chemiluminescent substrate was applied according to the manufacturer’s instructions (Renaissance, Life Science Products, Inc.). The blots were exposed to BioMax films and densitometry was done using an Alpha Imager. As an internal standard, ß-actin (1:5000) was measured.

Immunohistochemistry
Cross-sections were made in the middle portion of the CL. Tissues were fixed in 4% buffered paraformaldehyde for 4 h at 4 C and processed using standard procedures. Paraffin sections (3 µm) were made. Immunohistolocalization was performed using Vectastain Elite ABC Kit according to the manufacturer’s protocols, and as described (35, 36). Endogenous peroxidase activity was removed by fixing sections in 0.3% hydrogen peroxide in methanol. Tissue sections were blocked in 10% goat serum for 1 h at room temperature. The primary antibodies were the same as described above. The following concentrations were used: COX-2 (1:2000), PGES (1:500), PGFS (1:1000), PGDH (1:2000), PGT (1:500), and EP2 (1:500). Incubation with the primary antibodies was done overnight at 4 C. The sections were further incubated with the second antibody (goat antirabbit IgG biotinylated, 1:200) for 30 min at room temperature. For the negative control, preimmune or control rabbit serum was used at the respective dilution used for primary antibodies. Between each step, tissues were washed in PBS. Finally, tissues were stained with Mayer’s hematoxylin. Photos were captured using the Spot Program (Carsen Group Inc., Markham, Ontario, Canada). [Preimmune serum was used for the antibodies produced in our laboratory (PGFS, PGDH, PGT), and control serum was used for commercial and donated antibodies (COX-2, EP2, PGES) (control serum is the one collected without immunization from the same species in which the antibody was raised)].

Statistical analysis
All numerical data were presented as the mean ± SEM. Data were analyzed using two-way ANOVA followed by Scheffé’s tests (Super Anova, Abacus Concepts, Inc., Berkeley, CA). Differences were considered as statistically significant at the 95% confidence level (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of COX-1 and COX-2
Maximal (P < 0.05) expression of COX-2 mRNA and protein occurs on d 7–17 and 10–21 of the estrous cycle, respectively. By contrast, COX-1 mRNA and protein are expressed at constant low levels during the entire life span of CL (Fig. 1Go). Cellular localization shows that COX-2 protein is expressed in LLCs but not in SLCs and other cell types (see Fig. 5Go). COX-2 staining intensity does not differ among growing (d 1–12), mature (d 13–17), and regressing (d 18–21) CLs.



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  		FIG. 1. Expression of COX-1 and -2 in CL. A, Northern blot analysis. B, Western blot analysis. Densitometric values for COX-1 and-2 mRNAs (C), and COX-1 and -2 proteins (D) are expressed as the mean ± SEM of ratios with 18S RNA/ß-actin. Each group consisted of three to six samples. Data were analyzed using ANOVA followed by Scheffé’s post hoc analysis; P < 0.05. a: COX-2 mRNA; d 7–18 vs. others; b: COX-2 protein, d 10–21 vs. others.

 


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  		FIG. 5. Cellular localization of COX-2, PGES, PGFS, PGT, and EP2 proteins in CL. Only representative days during growing, mature, and regressing phases of CL life span are shown. All components were selectively localized in LLC. In growing CL (d 10–12), LLCs and SLCs, and capillaries are distinguishable. In mature CL (d 15–17), SLCs were progressively replaced by LLCs. In regressing CL (d 18–20), loss of cellular integrity is more evident. Staining for PGES and EP2 was higher in growing CL than at other stages.

 
Expression of PGES, PGFS, and PGDH
Relative expression shows that PGES protein is highly modulated whereas PGFS and PGDH proteins are expressed at steady-state levels during different phases of the CL life span (Fig. 2Go). The expression level of PGES is higher than that of PGFS in growing, lower in regressing, and equal in mature CLs. Immunohistochemistry shows that PGES and PGFS proteins are localized exclusively in LLCs (see Fig. 5Go), whereas PGDH is undetectable (data not shown). Comparatively, PGES staining intensity decreases in mature and regressing CL compared with growing CL whereas no difference is observed for PGFS. As an indication of the relative importance of PGE2 and PGF2{alpha} production, the PGES to PGFS ratio is presented. Similarly, the relative PG anabolism to catabolism is shown as PG synthases to PGDH ratio. The data indicate that the PGES to PGFS ratio is high (P < 0.05) on d 1–12 compared with d 13–21 of the estrous cycle. The PGES to PGDH ratio is elevated between d 1 and 12, whereas the PGFS to PGDH ratio is stable and high at all times except on d 1–3 (P < 0.05).

Expression of PGT
The level of expression of PGT mRNA and protein is higher (P < 0.05) on d 7–21 than d 1–6 of the estrous cycle (Fig. 3Go). The maximal (P < 0.05) expression is observed on d 13–15. PGT protein is highly expressed in LLCs (see Fig. 5Go). The staining intensity for PGT protein is similar in growing, mature, and regressing CLs.



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  		FIG. 3. Expression of PGT in CL. A, Northern blot analysis. B, Western blot analysis. Densitometric values for PGT mRNA (C) and protein (D) are expressed as the mean ± SEM of ratios relative to 18S RNA or ß-actin. Each group consisted of three to six samples. Data were analyzed using ANOVA followed by Scheffé’s post hoc analysis; P < 0.05. a and b, PGT mRNA; c and d, PGT protein. a and c, d 7–21 vs. 1–6; b and d, d 13–17 vs. others.

 
Expression of EP2, EP3 and FP
The level of expression of PG receptor mRNAs are FP >> EP3 > EP2 (P < 0.05) throughout the CL life span (Fig. 4Go). EP2 mRNA expression is high (P < 0.05) on d 4–15, moderate on d 16–21, and low on d 1–3 of the estrous cycle. EP2 protein expression follows that of mRNA except on d 13–15, on which it begins to decrease. Cellular localization shows that EP2 protein is highly expressed in LLC (Fig. 5Go). Comparatively, the staining intensity for EP2 protein is higher in growing than in mature and regressing CLs. EP3 mRNA expression is low on d 1–6, increases on d 7, and reaches its maximum between days 10 and 21 (P < 0.05). The concentration of FP mRNA progressively increases from d 1–9, reaches peak on d 10–12 (P < 0.05), and decreases from d 13 onward.



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  		FIG. 4. Expression of EP2, EP3, and FP in CL. A, LightCycler quantification of EP2, EP3, and FP mRNAs, expressed as the mean ± SEM of ratios relative to glyceraldehyde-3-phosphate dehydrogenase. B, Western blot analysis of EP2. Densitometric values are expressed as the mean ± SEM of ratios relative to ß-actin (C). Each group consisted of three to six samples. Data were analyzed using ANOVA followed by Scheffé’s post hoc analysis; P < 0.05. a, EP2 mRNA, d 4–15 vs. others; b, EP3 mRNA, d 7–21 vs. others; c, FP mRNA, d 10–12 vs. others; d and e, EP2 protein; d, 4–21 vs. others; e, d 7–12 vs. others.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We present here an integrated view of PGE2 and PGF2{alpha} biosynthesis, transport, and signaling in the CL. Our results show that the bovine CL possesses all of the PGE2 and PGF2{alpha} machineries necessary for autoregulation of its function. The net production and action of PGE2 and PGF2{alpha} in a given tissue is governed by several components such as anabolic and catabolic enzymes, transporters, and receptors. Among those, cPLA2, COX-1, COX-2, PGDH, and PGT are involved in nonselective production and transport of PGs whereas PG synthases E and F (PGES and PGFS) and receptors (EP and FP) control the selective production and the associated distinct biological actions. We have characterized the expression of both selective and nonselective components in the CL and found that PGE2 and PGF2{alpha} machineries are selectively and specifically expressed in CL. Our findings suggest a contribution of locally produced luteal PGs that may complement the classical paradigm of endometrial/extraluteal PG influence on CL function.

COX-2 and PGES are highly modulated whereas COX-1, PGFS, and PGDH are expressed at a constant level during the CL life span. Relative expression of these enzymes demonstrates that, relative to PGF2{alpha}, PGE2 biosynthesis is higher in growing, lower in regressing, and similar in mature CL. Cellular localization of COX-2, PGES, and PGFS proteins indicates that PGE2 and PGF2{alpha} biosynthesis preferentially occurs in LLCs compared with other cell types. Other studies reported that COX-1 required higher concentration of AA than does COX-2 to exert its function, and different pools of AA could be used by the two isoforms of COXs (22, 23, 40). This may explain the distinct role of COX-1 and -2 in the conversion of AA to PGH2. It has been estimated based on specificity constant (Kcat/Km) that conversion of PGH2 to PGE2 by mPGES-1 is 150-fold higher than conversion of PGH2 to PGF2{alpha} by PGFS (AKR1B5) (23, 24). It suggests that although PGES and PGFS are expressed at the same level in a given tissue, PG biosynthesis is preferentially directed toward PGE2 rather than PGF2{alpha} production. Together, expression patterns of PGs metabolic enzymes in the CL suggest that intraluteal PG biosynthesis is selectively directed toward PGE2 during growth, toward PGF2{alpha} during regression, and is nonselective during the mature phase of the CL life span.

Newly synthesized luteal PGE2 and PGF2{alpha} must flow out of luteal cells to bring forth their autocrine and paracrine actions. Recently, it has been shown that although PGs cross the membrane by simple diffusion, the estimated flow rate is too low to exert a biological function (41). Therefore, a carrier-mediated transport mechanism is needed for PGs to cross the biological membranes (32, 33). We have very recently demonstrated that bovine PGT facilitates efflux and influx of PGE2 and PGF2{alpha} with equal affinities in a competitive manner (34). Interestingly, in bovine endometrium PGT is highly expressed when PG production and action is higher. PGT expression also closely matches the pattern of expression of PG biosynthetic enzymes and receptors (24, 34, 35, 36). In the present study, luteal expression of PGT is lower in early growing (d 1–6) than in late growing, mature, and regressing phases. PGT is almost exclusively expressed in LLCs. The expression pattern of PGT along with PG metabolic enzymes suggests that PGT expression is not associated with a particular PG pathway. Rather, we believe that PGT facilitates both efflux and influx of available luteal PGE2 and/or PGF2{alpha} in a competitive manner to affect their autocrine and paracrine actions, as well as their catabolism during the different phases of CL life span. However, a detailed functional study in luteal cells would be needed to define PGT contribution to CL function.

Expression of EP2 and EP3 shows a contrasting pattern in growing, mature, and regressing CL. EP2 protein is highly expressed in LLCs and to a lesser extent in SLCs. The EP2 expression pattern closely matches that of PGES. Recently, we have identified EP2 and not EP4 as the major cAMP-generating PGE2 receptor expressed in bovine reproductive tissues including the CL (35). In this study, we have analyzed EP3 mRNA with no distinction of the four isoforms. The existence of different EP3 subtypes does not permit at the present time to relate our results to a specific signaling pathway in CL. Others have found that EP3 mRNA is not expressed in LLCs of ovine CL (31). Taken together, available evidence suggests that PGE2 probably uses both EP2 and EP3 receptors subtypes to accomplish distinct purposes at different phases of CL life span as it has been shown in other systems (42). In the present investigation, FP expression increases in growing and decreases in mature and regressing CLs as it was reported by others (30, 31). They have found that FP receptor is primarily localized in LLCs and to some extent in SLCs in ruminants where PGF2{alpha} treatment also decreases FP mRNA in LLCs.

Maintenance of CL and P4 secretion is the prerequisite for establishment of pregnancy in most mammals. PGE2 stimulates P4 secretion with an efficiency comparable to LH in bovine and ovine CL (12, 16, 43). Accumulating evidence indicates that PGE2 increases luteal P4 secretion by activating the cAMP-PKA pathway in human, rabbit, and ruminant CLs (11, 12, 13, 14, 15). Moreover, a positive correlation between luteal PGE2 and P4 has been demonstrated during the menstrual cycle in humans (17) and the estrous cycle in cows (13). Most mitogenic, angiogenic, vasodilatory, and antiapoptotic activities of PGE2 are mediated through cAMP in a variety of cells and tissues (44, 45). PGE2 increases COX-2 and EP2 expression, thereby autoregulating its own production and action in different cell types (44, 45, 46). On the other hand, regular ovarian cyclicity requires the regression of CL and decrease in P4. The signaling pathways leading to luteal regression are more complex and greatly differ among species. The pleiotropic action of PGF2{alpha} and FP signaling in luteal regression has been reviewed extensively (3, 29). Activation of FP increases intracellular PKC and Ca2+ to effect luteal regression (1, 2, 3, 4). Increase in PKC results in decreased secretion of P4 (functional luteolysis) and Ca2+ is involved in apoptosis and cell death (structural luteolysis) (3, 4, 29, 31). It has been reported that FP mRNA is temporally associated with the decrease in luteal mass and luteal P4 concentration (47, 48). A new FP receptor (FPB) has been identified recently. Although the physiological role of the conventional FPA is well defined, a possible role for FPB has not been explored in the CL (49). It has been demonstrated that FPB increases COX-2 expression through a Rho, but not PKC, signaling pathway (50). Recent studies have suggested that endometrial/extraluteal PGF2{alpha} triggers intraluteal production of PGF2{alpha} to complete the luteolytic process (6, 26). Others have proposed that PGF2{alpha} increases luteal PGE2, which augments the luteolytic effect of PGF2{alpha} through EP3 by an autocrine/paracrine action in the regressing CL (6, 51). The present findings showing selective activation of PGE2 biosynthesis and EP2 signaling during luteal maintenance and increased expression of PGFS and EP3 during luteal regression greatly support the new emerging concept that, after action of extraluteal/endometrial PGs, contribution from local (luteal) sources participate to the regulation of CL function and life span.

Intraluteal metabolism of P4 may be an important factor contributing to luteal regression. It has been suggested that, in the rat, CL 20{alpha}-HSD activity is down-regulated by P4 and up-regulated by PGF2{alpha} (19, 20). The PGFS (AKR1B5) we have evaluated in this study also possesses 20{alpha}-HSD activity. In the cow, although AKR1B5 expression is constant throughout the CL life span, high intraluteal concentration of P4 may block both the 20{alpha}-HSD and PGFS activities during the growing and mature phases. When endometrial/extraluteal PGF2{alpha} initiates functional luteolysis, the decrease in P4 would clear the inhibition and allow this enzyme to further decrease P4 and increase luteal PGF2{alpha} production, thus creating an amplification loop leading to luteal regression.

In cyclic cows, endometrial PGF2{alpha} initiates the luteolytic process between d 15 and 17; by contrast, endometrial PGE2 may rescue the CL in the presence of a viable embryo (1, 2). Intrauterine or intraluteal administration of PGE2 during this window counteracts the luteolytic effect of PGF2{alpha} and increases life span of the CL (1, 2, 7, 8, 9, 10). The underlying regulatory mechanisms may involve intraluteal factors. It is evident from the present study that PGE2 and PGF2{alpha} factories are selectively localized in LLCs. It is well known from other studies that, in ruminants, more than 80% of total P4 is produced from LLCs and independent of LH (4, 52). The mechanism responsible for the large amount of P4 produced by LLCs remains to be elucidated (52). With accumulating evidence on PGE2 and luteal P4 in other species (11, 12, 13, 14, 15), the presence of PGE2 biosynthetic, transporting, and EP2 signaling machineries in LLCs point to PGE2 as the candidate involved in the stimulation of P4 production from LLCs during luteal maintenance in cattle. On the other hand, down-regulation of PGE2 biosynthesis and EP2 signaling gives way to develop PGF2{alpha} biosynthesis and FP signaling during luteal regression. In ruminants, the mechanism responsible for refractoriness of early CL to luteolytic PGF2{alpha} is poorly understood. It has been proposed that higher PGDH activity could account for low intraluteal concentration of PGF2{alpha} (26, 27). But, in the present study, we have shown that PGDH protein is present throughout the estrous cycle at constant low levels. On the other hand, we show here that expression of PGT is low and that PGE2 biosynthesis and signaling pathways are high in the growing CL. Thus, limited availability of PGT and increased PGE2 production and action may explain refractoriness of early CL to luteolysis in response to extraluteal PGF2{alpha}.

Overall, the present study strongly supports the emerging concept that, in addition to extraluteal/endometrial sources, locally produced luteal PGs contribute to the regulation of CL function and life span. The information obtained from this study may be particularly relevant to the human, in which extraluteal PGs do not appear to contribute to luteolysis. Our results showing the expression of PGT, PGES, and PGFS open a new research avenue in the field of CL biology. Future studies are required in this arena to unravel the molecular mechanisms involved in the regulation of luteal production and action of PGs. Based on our findings and the available information, we propose a model integrating the role of luteal PGE2 and PGF2{alpha} in maintenance and regression of the CL (Fig. 6Go). Selectively produced luteal PGE2 and/or PGF2{alpha} are transported extracellularly by PGT. Preferential production of PGE2 and PGF2{alpha} during distinct stages of CL life leads to luteal maintenance or demise. After accomplishing biological functions, PGs are transported into cells for metabolism by cellular oxidation.



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  		FIG. 6. Proposed model to illustrate the integrated role of luteal PGE2 and PGF2{alpha} in CL life and function. After luteolytic process initiated by extraluteal or endometrial PGs (extensively reviewed in Refs. 1 2 3 4 ), in LLCs AA is metabolized into PGH2 by COX-1 and COX-2. PGH2 is then converted into either PGE2 or PGF2{alpha} by PGES or PGFS, respectively. Newly synthesized luteal PGs are transported through the PGT to bring forth their autocrine and paracrine effects on LLCs, SLCs, immune cells (IC), blood vessels (BV), endothelial cells (EC), and smooth muscle cells (SMC). Activation of EP2 and FP increases intracellular cAMP and PKA, and Ca2+, IP3, and PKC, respectively. EP3 can use either one or both of these signaling pathways depending on the isoform expressed. Autoamplification of luteal PGE2 or PGF2{alpha} production occurs by controlling any of the PG biosynthetic, transporting, and signaling steps. The PGE2-EP2 signaling pathway leads to increase in luteal production of P4, mitogenesis, angiogenesis, vasodilation, immunomodulation, and decrease in apoptosis and 20{alpha}-HSD activity, thus promoting luteal development, growth, and maintenance. The PGF2{alpha}-FP signaling pathway leads to effects opposite to PGE2, culminating in luteal regression. Finally, PGs are transported into cells for catabolism by cellular oxidation.

 
In conclusion, the present data demonstrate the existence of PGE2 and PGF2{alpha} metabolic, transporting, and signaling machineries in CL, suggesting that luteal PGs may contribute to the autoregulation of CL function.


   Footnotes
 
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to M.A.F.) and Canadian Institutes for Health Research (CIHR) (to M.A.F. and J.S.). J.S. is supported by a CIHR Investigator Award. J.A.A. and S.K.B. contributed equally to this work.

Abbreviations: AA, Arachidonic acid; AKR1B5, aldoketoreductase 1B5; Ca2+, intracellular calcium; CL, corpus luteum; COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; 20{alpha}-HSD, 20{alpha}-hydroxysteroid dehydrogenase; IP3, inositol triphosphate; LLC, large luteal cell; mPGES, microsomal PGES-1; P4, progesterone; PG, prostaglandin; PGDH, PG 15-dehydrogenase; PGES, PG E synthase; PGFS, PG F synthase; PGT, PG transporter; PLC, phospholipase C; SLC, small luteal cell.

Received November 26, 2003.

Accepted for publication January 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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