Abstract
Prostaglandins (PGs) are critical regulators of a number of reproductive processes. To date, the presence and regulation of PGS in the rat endometrium have not yet been described. The objective of the present study was to investigate the expression of PGD synthase (PGDS) and prostacyclin synthase (PGIS) in the endometrium. Endometrial proteins and tissues were collected from cyclic non-pregnant, pregnant, and steroid-induced pseudopregnant rats. PGIS and PGDS were detected in the endometrium of cyclic, pregnant, and pseudopregnant rats but were not influenced by the estrous cycle. During early pregnancy, PGIS was significantly higher at day 5 and was gradually decreased from day 5.5 to 6.5. Later during pregnancy, PGIS was maximal on day 12 and gradually decreased to the end of pregnancy. PGDS expression was high during early and was maximal at the end of pregnancy. During pseudopregnancy, PGDS and PGIS were increased in a time-dependent manner and were maximal at day 5. Immunohistochemical analysis revealed that PGDS and PGIS were found in luminal as well as glandular epithelial cells and in stroma during late pregnancy. We also found a significant increase of PGD2 serum metabolite at days 21 and 22 of pregnancy. During steroid-induced pseudopregnancy, PGI2 serum metabolite was increased in a time-dependent manner and was maximal at day 7. These results suggest that PGDS and PGIS are present and could be regulated by steroids in the rat uterus during pregnancy, and that the endometrium could be a significant source of PGD2 and PGI2 at specific times during pregnancy.
Introduction
Prostanoids, which consist of prostaglandins (PGs) and thromboxanes (TXs), exert a variety of effects in diverse tissues and cell types. These prostanoid products are derived from C-20 unsaturated fatty acids and biosynthesized by PGH synthases (Narumiya et al. 1999). PGH synthases catalyze the formation of PGH2 from arachidonic acid. Newly formed PGH2 is subsequently converted to what are considered to be the biologically active prostanoids, PGD2, PGE2, PGF2α, TXA2, or PGI2. There are two PGH synthase isoforms called PGHS-1 (cyclooxygenase(COX)-1) and PGHS-2 (COX-2). These two isoforms share important similarities at the protein level; they are approximately the same size (70–72 kDa), and the important structural and functional domains are highly conserved (Xie et al. 1991, Williams & DuBois 1996). However, PGHS-1 and PGHS-2 transcripts are encoded by distinct genes located on different chromosomes and differ in size (Xie et al. 1991, Williams & DuBois 1996). PGHS-1 is a constitutive enzyme present in many mammalian cells (Kraemer et al. 1992, Kujubu & Herschman 1992, Kennedy 1994) and its expression appears to be regulated developmentally (Kirihara et al. 2005). PGHS-2 is undetectable in most mammalian tissues (Kennedy 1994), but its expression can be induced by growth factors, cytokines, and tumor promoters (Simmons et al. 1991, Evett et al. 1993, O’Neill & Ford-Hutchinson 1993).
PG receptors were identified and cloned in different species. They are G-protein-coupled receptors with seven transmembrane domains. There are eight types and subtypes of prostanoid receptors that are encoded by different genes but as a whole constitute a subfamily in the superfamily of the rhodopsin-type receptors. Receptors specific for TX, PGI, PGE, PGF, and PGD are named thromboxane A2 receptor (TP), prostacyclin receptor (IP), (prostaglandin E2 receptor EP; four different types identified EP1, EP2, EP3, and EP4), prostaglandin F2a receptor (FP), and D prostanoid receptor (DP) receptors respectively (DeWitt & Meade 1993, Coleman et al. 1994, Narumiya et al. 1999). It is believed, therefore, that PGs work locally (or closely to their site of production, i.e. originate from the uterus and act on the ovary), acting only at the site of their production (Narumiya et al. 1999). The presence and regulation of EPs and FP receptors were found to be regulated by sex steroids in the mouse (Yang et al. 1997) and rat uterus (Martel et al. 1989, Papay & Kennedy 2000, Shi et al. 2005).
In the rat uterus, attachment and invasion of embryonic trophoblast is accompanied by decidualization of the adjacent endometrial stroma. Decidualization involves the proliferation and differentiation of endometrial stromal cells into decidual cells, which ultimately form the maternal component of the placenta and is necessary for the successful establishment of pregnancy (Abrahamsohn & Zorn 1993). Decidualization can be initiated only when the endometrium is receptive, and this only occurs during a short period in pregnancy. Several decades of investigations have showed that PGs are needed to trigger the events of blastocyst implantation. Implantation processes include blastocyst–uterine attachment and stromal decidualization with vascular permeability changes (Rankin et al. 1979, Kennedy 1983). Indeed, Kennedy’s group and others have showed that PGE2 plays a major role in the implantation and decidualization in the rodent uterus (Zhang et al. 1996). COX-2 and PGE2 have also been shown to be important in the baboon endometrium at the time of implantation (for a review, see Fazleabas et al. (1999)). Later, S K Dey’s group established PGI2 as a key mediator in the process of implantation and decidualization in the mouse via the peroxisome proliferator-activated receptor (PPAR)δ and retinoid-x receptor (RXR)α signaling pathway (Lim et al. 1999). They also showed that PGI2 is the most abundant PG at the implantation sites (Lim et al. 1999). Another group showed that insulin-like growth factor-binding protein related protein-1 renders the uterine environment suitable for successful implantation by enhancing PGI2 production in a paracrine and/or autocrine fashion in the rat uterus (Tamura et al. 2004), indicating that might be an important factor for successful establishment of pregnancy.
PGD2 is synthesized by the PGD synthase (PGDS) enzyme and induces sleep, allergic responses, inhibition of platelet aggregation, and relaxation of vascular and non-vascular smooth muscle, and has some roles in reproduction. Two types of PGD2 synthase are known: lipocalin-type PGDS (L-PGDS) and hematopoietic PGDS (H-PGDS; Coleman et al. 1990, 1994). L-PGDS is an N-glycosylated dual functional monomeric protein which acts as a PGD2-producing enzyme, as well as a lipophilic ligand-binding protein. L-PGDS is present in cerebrospinal fluid, seminal plasma and may play an important role in male reproduction. H-PGDS is a cytosolic enzyme that isomerizes PGH2 to PGD2 in a glutathione-dependent manner (Coleman et al. 1990, Urade & Eguchi 2002). H-PGDS is present in the spleen, fallopian tube, endometrial gland cells, extravillous trophoblasts, and villous trophoblasts, and perhaps plays an important role in female reproduction (Urade & Eguchi 2002, Kanaoka & Urade 2003). PGI2 or prostacyclin is synthesized by the PGI synthase (PGIS) enzyme and has roles over and above relaxation in the lower uterine segment. PGIS is a membrane-bound hemoprotein that has been localized by immunohistochemical (IHC) techniques in endothelial cells and both vascular and in non-vascular smooth muscle cells (Giannoulias et al. 2002, Saito et al. 2002). PGIS is expressed in human myometrial smooth muscle (Tanabe & Ullrich 1995). In the mouse, COX-2 and PGIS coexist at the implantation site, suggesting the availability of PGI2 directly to uterine cells (for a review, see Lim & Dey (2002)).
The expression and regulation of PGDS and PGIS has never been described in the non-pregnant, pseudopregnant, and pregnant rat endometrium. Since these PG synthases might play a significant role in the production of PGI2 and PGD2, we investigated the expression of these enzymes and the possibility that they might be regulated by sex steroids during the estrous cycle, pregnancy, and in a model of pseudopregnancy.
Materials and Methods
Reagents
PGDS and PGIS antibodies were purchased from Oxford Biomedical Research (Oxford, MI, USA). PGD2 metabolite 11-β-PGF1α and PGI2 metabolite 6-keto-PGF1α were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Vectastain ABC Kit for rabbit IgG was purchased from Vector Laboratories Inc. (Burlingame, CA, USA). Protease Inhibitor Cocktail Tablets, peroxidase (POD), and 3,31-diaminobenzidene (DAB) substrate were purchased from Roche. 17β-Estradiol (E2) and progesterone were purchased from Laboratoire Mat (Québec, QC, Canada).
Animals
Sprague–Dawley female rats (200–225 g) were obtained from Charles River Laboratories Canada. Animals were maintained on standard chow and water, which were available ad libitum, in animal facilities illuminated between 0600 and 2000 h. All procedures were performed in accordance with guidelines of the Canadian Council on Animal Care for the handling and training of laboratory animals and the Good Health and Animal Care Committee of the Université du Québec à Trois-Rivières. Male and female rats were mated overnight and confirmation of mating was determined by vaginal smears and/or the presence of a vaginal plug (day 1). Rats were killed on days 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 21, and 22 of pregnancy at 1000 h in the morning and at 1800 h for days 5.5 and 6.5. Six to eight different rats were used for each stage of pregnancy. For the estrous cycle experiments, stages of the estrous cycle were confirmed by vaginal smears. Rats with three regular cycles of 4 days were used in these experiments and killed at various stages of the estrous cycle (diestrus, proestrus, estrus, and metestrus). Uteri were collected and fixed for IHC staining or endometrial protein extracts were collected for western blot analysis.
Model of pseudopregnancy
A total of three rats per day of pseudopregnancy were ovariectomized and then allowed to recover from surgery for a minimum of 10 days. They were pre-treated with physiological doses of E2 and progesterone to induce decidualization as described previously (Kennedy & Ross 1993): 1) 0.2 μg E2 per day for 3 days (in the morning, days −2, −1, and 0); 2) on the third day (day 0 of pseudopregnancy), another injection of E2 (0.2 μg) and progesterone (1 mg) was performed in the afternoon; 3) no treatment for 2 days (days 1 and 2 of pseudopregnancy); 4) injections of E2 (0.1 μg) and progesterone (4 mg) for 3 days (days 3, 4, and 5 of pseudopregnancy); 5) another injection of E2 (0.1 μg) in the afternoon on day 7 (day 4 of pseudopregnancy); and 6) rats were killed on days 1, 3, 5, 7, and 9 of pseudopregnancy. Uteri were collected and fixed for IHC staining or endometrial protein extracts were collected for western blot analysis.
Immunohistochemistry
The uterus was fixed in 4% paraformaldehyde solution and embedded in paraffin. Tissue sections of 7 μm thick were mounted on polylysine-coated slides, deparaffinized, rehydrated, and then heated in 10 mM citrate buffer (pH 6) containing 0.1% (v/v) Triton X-100 (Sigma–Aldrich). After two washes with PBS, slides were incubated with 0.3% hydrogen peroxide in methanol for 30 min to quench endogenous peroxidase activity. After washing with PBS, tissues were incubated with blocking serum (Vectastain ABC Kit) at room temperature for 1 h. Then, a primary antibody diluted in blocking serum (PGIS, 1:250; PGDS, 1:500 dilutions) was added to the slides and incubated at 4 °C overnight in a humidified chamber. After washing for 5 min in PBS, tissue sections were incubated for 30 min with 3 μg/ml biotinylated antibody (anti-rabbit or anti-mouse). Subsequently, slides were washed with PBS and incubated with avidin–biotin complex reagent containing horseradish peroxidase for 30 min. Slides were washed with PBS for 5 min and color development was achieved using DAB substrate. The tissue sections were counterstained with hematoxylin. Negative controls (day 20 tissue sections) were performed using the same protocol but substituting the primary antibody with normal rabbit IgG (Vector Laboratories Inc).
Protein extraction and western blot analysis
Protein homogenates from pregnant endometrium were isolated according to a protocol previously described (Dai & Ogle 1999). Briefly, uteri from days 2 to 20 pregnant rats were rapidly excised and placed in ice-cold saline until dissected. Uteri were carefully laid on a glass plate and placed on the stage of a dissecting microscope. In early pregnancy (days 2–5.5), total endometrium was scraped using a microscope glass and collected. Endometrium from days 6 to 10, because the placenta and decidua were at an early stage of differentiation, could not be separated. For this reason, decidua basalis (DB) dissected from animals between these days of pregnancy contained some chorioallantoic cells, but antimesometrial decidua, choriovitelline tissues, fetus, and myometrium were removed. Even though we carefully dissected DB from these tissues, there was a possibility that contamination with some antimesometrial decidual cells, which regress to form the deciduas caspularis (DC), would occur. In uteri collected from days 12 to 20, DB was isolated by gently separating the placenta and myometrial regions with 23-gauge needles. Additionally, the DB began to regress on day 14 and became too thin to reliably dissect after day 17. The protocol for DB isolation was described previously by Ogle & George (1995).
Endometrial cells from pregnant animals were homogenized using a pipette in RIPA lysis buffer (PBS 1X, pH 7.4; 1% nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS; Protease Inhibitor Cocktail Tablets). Homogenates were centrifuged (12 000 g for 20 min at 4 °C) to remove insoluble material. The supernatant was recovered and stored at −20 °C pending analysis. Protein content was determined with the Bio-Rad DC Protein Assay. Protein extracts (50 μg) were heated at 94 °C for 3 min, resolved by 10% SDS-PAGE, and electrotransferred to nitrocellulose membranes using a semi-dry transfer apparatus (Bio-Rad). The membranes were then blocked 2 h at room temperature with PBS containing 5% milk powder, then incubated with anti-PGDS 1:1000; and anti-PGIS 1:8000, and subsequently with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody respectively (1:3000; room temperature for 45 min). All membranes were stripped with restore western blot stripping buffer (Pierce, Arlington Heights, IL, USA), reprobed with an antibody specific to β-actin which was used as an internal standard. Peroxidase activity was visualized with the Super signal West Femto maximum sensitivity substrate (Pierce), according to manufacturer’s instructions. Signal was visualized using the Biochemi Imaging System (UVP, CA, USA). Densitometrical analyses were performed (protein of interest and β-actin) using Quantity One software (Bio-Rad). Results are expressed as a ratio (protein of interest/β-actin) to correct for loading for each endometrial sample.
PG metabolites enzyme immunoassay (EIA)
The animals were killed using a guillotine and blood was collected at the same time (n = 4 samples per group). After collection from the animals, blood was allowed to clot for 30–60 min at 37 °C. The serum was then separated from the clot and any remaining insoluble material removed by centrifugation at 2799 g for 10 min at 4 °C. After preparation, sera were kept at −80 °C until further use. EIAs for PG metabolites were used according to manufacturer’s instructions (Cayman Chemicals). Briefly, 50 μl serum obtained during experimentation were used for each of the PGD2 and PGI2 metabolites in 96-well plates coated with goat anti-rabbit IgG antibodies. The metabolite tracer (50 μl) and the metabolite-specific antibody (50 μl) were added to each well. The plates were incubated overnight at 4 °C or room temperature depending on the metabolite. Then, wells were washed five times with 10 mM phosphate buffer (pH 7.4) containing Tween 20 (0.05%) at pH 7.4, and 200 μl Ellman’s reagent (69 mM acetylthiocholine and 54 mM 5,5′-dithio-bis (2-nitrobenzoic acid) in 10 mM phosphate buffer, pH 7.4) were added to each well; plates were incubated in the dark at room temperature for 60–90 min. This allowed the bound enzyme tracer to react with Ellman’s reagent to yield a yellow solution that could be measured photometrically with a microplate reader at 420 nm. A standard curve was developed simultaneously with standards of the PG metabolites, and determination of metabolite concentrations relative to those standards was calculated. Samples were assayed in triplicates.
Statistical analysis
Western blot analyses of cycling, pregnant, and pseudopregnant animals were repeated four times (four different rats). Endometrial extracts from each rat were assessed individually. Results subjected to statistical analysis were expressed as mean ± s.e.m. Data were subjected to one-way ANOVA (PRISM software version 4.0; GraphPad, San Diego, CA, USA). Differences between experimental groups were determined by Tukey’s test. Statistical significance was accepted when P < 0.05.
Results
PGDS and PGIS are expressed in the female rat endometrium during the estrous cycle
PGD2 and PGI2 expression in the endometrium during the estrous cycle, pregnancy or pseudopregnancy remains unknown. We have found that PGDS and PGIS proteins were indeed present in the rat endometrium, at all stages of the estrous cycle (Fig. 1). However, the levels of PGDS and PGIS were not influenced by the estrous cycle (metestrus, diestrus, estrus, and proestrus), indicating that both enzymes might play a role in the maintenance of uterine function but are not regulated during this period in the absence of embryo.
Expression of PGIS and PGDS is regulated in the pregnant rat endometrium
To document the regulation of PGIS and PGDS enzymes in the uterus during pregnancy, western blot and IHC analyses were performed on uterine lysates and sections of pregnant rats. Immunodetection of PGIS by western blot resulted in a band with a molecular mass of 51 kDa (Fig. 2), while immunodetection of H-PGDS resulted in a single band with a molecular mass of 25 kDa (Fig. 3). We found that PGIS as well as PGDS was present in the rat uterus at all days of pregnancy. Noteworthy, protein levels of both enzymes were significantly maximal at day 5 of early pregnancy, suggesting a possible role for PGIS and PGDS in trophoblast implantation. The levels of PGIS also increased at days 12, 14, and 16 of pregnancy, which coincides with the initiation of DB regression, while the expression levels of PGDS gradually increased from day 12 to peak at day 20, suggesting another role for this enzyme in the preparation for parturition. L-PGDS was not detected in the endometrial protein extracts (data not shown). These results suggest that PGIS and PGDS are similarly regulated in the rat uterus throughout pregnancy, except in the days preceding parturition where PGDS would play a prominent role.
Expression of PGDS and PGIS in the model of pseudopregnancy
Since we have showed that PGDS and PGIS are regulated during pregnancy but not during the estrous cycle, we hypothesized that their expression could be influenced by sex steroids in a longer period of exposure as what is observed in true pregnancy. Indeed, in pseudopregnant uterus of rats stimulated with E2 and progesterone, PGDS and PGIS protein expressions were stimulated in a similar fashion (Fig. 4). The expression levels of both enzymes were increased significantly in a time-dependent manner from days 1 to 5 of steroid-induced pseudopregnancy and were significantly reduced at days 7 and 9. These results suggest that sex steroids influence the expression levels of PGDS and PGIS in the rat uterus in a similar situation as true pregnancy.
Localization of PGDS and PGIS proteins in rat endometrium by IHC
To localize PGDS and PGIS proteins, uterine tissues from pregnant rats were sectioned and IHC analysis was performed (Fig. 5). The results showed that both enzymes were expressed in luminal and glandular endometrial epithelial cells during pregnancy and expression was observed weakly in the stroma during late pregnancy.
Measurement of PG metabolites by EIA
To confirm the possible activity of PGDS and PGIS on the production of PGD2 and PGI2 in the rat endometrium, their metabolites (11β-PGF2α and 6-keto-PGF1α respectively) were measured in the rat sera during the estrous cycle (Fig. 6A and B), pregnancy (Fig. 6C and D), and pseudopregnancy (Fig. 7). PGE2 (PGEM) and PGF2α (13,14-dihydro-15-keto-PGF2α) metabolites were also measured at the same time. We found an inverse relationship between the levels of PGE2 and PGI2 metabolites, but no significant change in PGD2 metabolite levels, during the estrous cycle. In addition, there was a significant increase of PGE2 and PGF2α metabolites at proestrus and a decrease of PGI2 metabolite at proestrus (P < 0.05). During early pregnancy (Fig. 6), the results showed a significant increase of PGE2 metabolite on day 5.5 and an increase of PGI2 but not PGD2 metabolite on day 6. During late pregnancy, the results showed a significant increase of PGD2, but not PGI2, metabolite on days 21 and 22. An increase of PGE2 and of PGF2α metabolites was observed during DB regression on days 10, 12, and 14 (P < 0.05). PGF2α metabolites were also increased at days 21 and 22 of pregnancy. During pseudopregnancy (Fig. 7), PGE2 and PGI2 metabolites were increased in a similar fashion: the metabolites were increased in a time-dependent manner and were maximal on day 7 of pseudopregnancy. PGF2α metabolite was significantly (P < 0.05) increased at day 7 of pseudopregnancy when compared with all other days measured, while systemic levels of PGD2 metabolite were not influenced by the pseudopregnancy.
Discussion
Successful implantation is the result of reciprocal interactions between the implantation-competent blastocyst and receptive uterus, which is a multistep process including apposition of the blastocyst to the uterine luminal epithelium, adhesion to the epithelium, penetration through the epithelium and basal lamina, and invasion into the stromal vasculature (Enders et al. 1986). Due to the impracticalities of studying implantation in humans, implantation has been studied in mice and rats (Dey et al. 2004), guinea pigs (Enders & Schlafke 1969, Enders 2000), rabbits (Enders & Schlafke 1971, Hoffman et al. 1998), sheep (reviewed in Gray et al. 2001), and pigs (Bazer 1975, Geisert & Yelich 1997, Burghardt et al. 2002). Because PGs have been implicated in the implantation process (Dey et al. 2004), ovulation, fertilization, and parturition in different species, we hypothesized that PGDS and PGIS might also be important regulators of uterine function and contribute to the production of other key PGs such as PGD2 and PGI2. The present data are the first to show the presence, activity, and regulation of PGDS and PGIS in the pregnant, pseudopregnant, and the non-pregnant rat uterus.
During the estrous cycle, death of uterine cells occurs. It has been shown that apoptotic death was low at proestrus and increased at estrus (Sandow et al. 1979, Dery et al. 2003, Leblanc et al. 2003). This phenomenon is closely associated with the concentrations of ovarian steroids, which increase from diestrus to proestrus and decrease at estrus (Smith et al. 1975). We have demonstrated that PGDS and PGIS enzymes were present at elevated levels that remained unchanged in rat uterus throughout the estrous cycle, suggesting that their respective products PGD2 and PGI2 exert possibly important roles in uterine function and/or homeostasis. In accordance, we have measured constant levels of PGD2 metabolite in rat sera during the estrous cycle. Systemic levels of PGI2 metabolite however, which was the most abundant of all PG metabolites in the rat sera, was not constant during the estrous cycle as it was markedly decreased at proestrus, indicating that the source of increased PGI2 might not originate from the uterus. Our results have also highlighted an inverse relationship between systemic levels of PGI2 and PGE2 metabolites during the estrous cycle (data not shown). In this regard, recent studies have showed that PGE2 induces proliferation of human endometrial epithelial cells (Jabbour & Boddy 2003); the significant increase of PGE2 metabolite at proestrus may suggest its implication in cellular proliferation as seen in the endometrium during this period preceding embryo implantation, and argues for an anti-proliferative function for PGI2, as previously described (Kothapalli et al. 2003, 2004).
Our results also demonstrate that PGDS and PGIS are expressed in the endometrium of pregnant female rats throughout gestation, suggesting that local production of PGD2 and PGI2 might be important in the regulation of endometrial function. The increase of PGIS and PGDS levels at the time of establishment of pregnancy and implantation (days 5–7) and during regression of the DB (days 14–20) suggests a possible implication of these two PGs in these key stages of gestation. PGDS protein, which is high during early pregnancy, could alternatively be implicated in the recruitment of Th2 cells to the site of implantation in order to protect the embryo and the placenta from an attack by the immune system, similar to what was shown in human pregnancy (Michimata et al. 2002). We have also showed a significant increase of PGE2 metabolite on days 10, 12, and 14 of pregnancy (data not shown). Kennedy & Ross (1993) studies have clearly showed that PGE2 is involved in decidualization and PGE2 could also be involved in DB regression since it is significantly increased during this specific period of pregnancy. Recently, we have showed that transforming growth factor (TGF)-β isoforms are increased in the DB and induce apoptosis in decidual cells in vitro, suggesting that TGF-β might be involved in DB regression (Shooner et al. 2005). TGF-β isoforms have been shown to increase PGE2 secretion in several systems (Fong et al. 2000, Bradbury et al. 2002, Ishida et al. 2002, Chen et al. 2003). Whether TGF-β induces decidual cells apoptosis through the stimulation of PGE2 remains to be elucidated.
Since both PGIS and PGDS are upregulated in the endometrium on different days of rat pregnancy and are not regulated during the estrous cycle, we wondered whether sex steroids might be involved in this process and whether embryonic factors are involved. To answer these questions, we used ovariectomized rats treated with sex steroids to induce pseudopregnancy. The results clearly showed that PGIS, as well as its metabolite, is upregulated in response to sex steroids and in a dose-dependent manner. PGDS protein was also increased albeit to a lesser level as compared with PGIS, but its metabolite was not influenced by pseudopregnancy as opposed to pregnancy. These results suggest that regulation of PGDS expression is dependent on the presence of an embryo, while regulation of PGIS expression is not dependent on embryonic factors and is directly regulated by sex steroids. The fact that both PGDS and PGIS were not influenced by sex steroids in the estrous cycle but their expression was affected during pregnancy and in the model of steroid-induced pseudopregnancy indicates that exposure time and steroid concentration is important to induce PGDS and PGIS expression. The concentration and/or exposure time to steroids during the estrous cycle is probably not sufficient to act on gene expression, indicating that these enzymes might play a negligible role in the non-pregnant endometrium.
In conclusion, this study is the first to characterize the expression of PGIS and PGDS in the non-pregnant, pregnant, and steroid-induced pseudopregnant uterus and shows that these two enzymes might be key factors involved in embryo implantation and DB regression. PGDS might also be an important enzyme to induce PGD2 synthesis at the time of parturition. Whether these enzymes are directly or indirectly involved in the activation of luminal epithelial cell apoptosis during implantation and apoptosis activation during DB regression remains to be elucidated.
PGDS and PGIS expression in the female rat endometrium during the estrous cycle. Total endometrial proteins were collected from polyestrus cycling rats at each stage of the estrous cycle (diestrus, proestrus, estrus, and metestrus). Western blot analysis of (A) PGIS and (B) PGDS (one representative blot). Histograms show densitometric analysis of PGIS and PGDS proteins normalized to β-actin (42 kDa), which was used as an internal control for protein loading. Data represent the mean ± s.e.m. of four independent experiments (four different rats).
Citation: Journal of Endocrinology 195, 2; 10.1677/JOE-07-0353
PGIS expression in the female rat endometrium during pregnancy. Total endometrial proteins were collected at indicated days of pregnancy. Western blot analysis of PGIS during early (A) and late (B) pregnancy was performed (one representative blot presented). Histograms show densitometric analysis of PGIS protein normalized to β-actin (42 kDa), which was used as an internal control for protein loading. Data represent the mean ± s.e.m. of four independent experiments (four different rats). *P < 0.05 compared with day 6.5 (early pregnancy) and *P < 0.05 compared with days 8, 10, 18, and 20 (late pregnancy).
Citation: Journal of Endocrinology 195, 2; 10.1677/JOE-07-0353
PGDS expression in the female rat endometrium during pregnancy. Total endometrial proteins were collected at indicated days of pregnancy. Western blot analysis of PGDS during early (A) and late (B) pregnancy was performed (one representative blot presented). Histograms show densitometric analysis of PGDS protein normalized to β-actin (42 kDa), which was used as an internal control for protein loading. Data represent the mean ± s.e.m. of four independent experiments (four different rats). *P < 0.05 compared with day 6.5 (early pregnancy) and *P < 0.05 compared with all other days (late pregnancy). #P < 0.05 compared with days 8 and 10.
Citation: Journal of Endocrinology 195, 2; 10.1677/JOE-07-0353
PGDS and PGIS expression in the female rat endometrium during the pseudopregnancy. Total endometrial proteins were collected at days 1, 3, 5, 7, and 9 of the induced pseudopregnancy. Western blot analysis of (A) PGDS and (B) PGIS (one representative blot presented). Histograms show densitometric analysis of PGDS and PGIS protein normalized to β-actin (42 kDa), which was used as internal controls for protein loading. Data represent the mean ± s.e.m. of four independent experiments (four different rats). (A) *P < 0.05 compared with days 1, 7, and 9. (B) *P < 0.05 compared with days 1, 7, and 9.
Citation: Journal of Endocrinology 195, 2; 10.1677/JOE-07-0353
Immunohistochemical staining was performed to localize PGDS and PGIS proteins in the rat endometrium. PGDS and PGIS immunoreactivity was detected in the pregnant endometrium. They were both localized in the epithelial cells of endometrium and their expressions were absent in the stoma. A high level of PGDS immunostaining was observed in the luminal and glandular epithelium at day 14 (DB regression) and day 20 (parturition) of pregnancy. A strong staining of PGIS immunostaining in the luminal epithelium at day 14 (DB regression) of pregnancy was observed. LE, luminal epithelium; GE, glandular epithelium; and S, stroma.
Citation: Journal of Endocrinology 195, 2; 10.1677/JOE-07-0353
PGD2 and PGI2 metabolite concentrations in serum were determined during the estrous cycle (A and B) and pregnancy (C and D) by enzyme immunoassay (EIA). Serum samples were collected from polyestrus cycling rats at each stage of the estrous cycle (diestrus, proestrus, estrus, and metestrus) or pregnant rats at indicated days of pregnancy and were measured using a high-sensitivity enzyme immunoassay (EIA) kit obtained from Cayman Chemicals. Statistical significance was reached when P < 0.05; (A) and (B) *indicates values different from all the others. (C) *P < 0.05 compared with all other days. #P < 0.05 compared with day 5. (D) *P < 0.05 compared with days 5 and 5.5.
Citation: Journal of Endocrinology 195, 2; 10.1677/JOE-07-0353
(A) PGD2 and (B) PGI2 metabolite concentrations in serum were determined during pseudopregnancy by enzyme immunoassay (EIA). Serum samples were collected from pseudopregnant rats at indicated days of pregnancy and were measured using a high-sensitivity enzyme immunoassay (EIA) kit obtained from Cayman Chemicals. *P < 0.05 compared with days 1 and 9.
Citation: Journal of Endocrinology 195, 2; 10.1677/JOE-07-0353
This work has been supported by a grant from NSERC (238501-01). Eric Asselin is holder of the Canada research chair in Molecular Gyneco-oncology. We are grateful to Mrs Rollande Caron for the contribution of her precious time and expertise to our projects. We also thank Mrs Daphne Efford and Dr Céline Van Themsche for reviewing the manuscript. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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