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Pregnancy |
a College of Life Sciences, Northeast Agricultural University, Harbin 150030, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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decidua, female reproductive tract, implantation, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PGD2, PGI2, and TXA2 (thromboxane) [1]. PGs have been shown to be important for implantation and decidualization in laboratory rodents [2, 3]. PGE2 synthase (PGES) is a terminal prostanoid synthase and can enzymatically convert the COX product PGH2 to PGE2. Two isoforms of PGES exist; microsomal PGES (mPGES) and cytosolic PGES (cPGES). Cytosolic PGES is constitutively expressed in a wide variety of cells and tissues and is predominantly linked with COX-1 to promote the immediate response, during which a relatively high concentration of arachidonic acid is released in a short period [4]. Microsomal PGES, a membrane-associated and inducible perinuclear enzyme with glutathione-dependent activity, is expressed in a variety of tissues, including prostate, testes, and small intestine [5]. Microsomal PGES is preferentially coupled with the inducible COX-2 to promote delayed PGE2 generation, and if COX-2 already exists in cells, it also regulates immediate PGE2 generation [6]. Although COX-1-deficient mice have normal fertility and litter size [7], COX-1-derived PGs are important for uterine cellular differentiation and proliferation, and for uterine edema and luminal closure [8]. COX-2 compensation occurs in COX-1-deficient mice [8], which may explain the normal fertility and litter size in COX-1-deficient mice. Although the expression and regulation of the mPGES gene in mouse uterus was recently characterized [9], the expression and regulation of cPGES in mouse uterus during early pregnancy is still undefined. The aim of this study was to investigate the expression and regulation of the cPGES gene in mouse uterus during early pregnancy using in situ hybridization and immunohistochemistry.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature mice (Kongmin White outbred strain) were caged in a controlled environment with a 14L:10D cycle. All animal procedures were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. Adult females were mated with fertile or vasectomized males of the same strain to induce pregnancy or pseudopregnancy, respectively (Day 1 = the day of vaginal plug). Pregnancy on Days 1–4 was confirmed by recovering embryos from the reproductive tracts. The implantation sites on Days 5–6 were identified by i.v. injection of 0.1 ml of 1% Chicago blue (Sigma Chemical Company, St. Louis, MO) in 0.85% sodium chloride.
To induce delayed implantation, pregnant mice were ovariectomized under ether anesthesia at 08:30–09:00 h on Day 4 of pregnancy. Progesterone (1 mg/mouse) was injected to maintain delayed implantation from Days 5–7. 17ß-Estradiol (25 ng/mouse) was given to progesterone-primed delayed-implantation mice to terminate delayed implantation. The mice were killed in order to collect uteri 24 h after estrogen treatment. The implantation sites were identified by i.v. injection of Chicago blue solution. Delayed implantation was confirmed by flushing the blastocysts from the uterus.
Artificial decidualization was induced by intraluminally infusing 25 µl of sesame oil into one uterine horn on Day 4 of pseudopregnancy, whereas the contralateral, uninjected horn served as a control. The uteri were collected on Day 8 of pseudopregnancy. Decidualization was confirmed by weighing the uterine horn and performing a histological examination of uterine sections [10].
Immunohistochemistry
Mouse uteri were immediately cut into small pieces, fixed in Bouins solution, dehydrated, and embedded in paraffin. Sections (7 µm) were cut, deparaffined, and rehydrated. Nonspecific binding was blocked in 10% normal horse serum in PBS for 1 h. The sections were incubated with rabbit anti-human cPGES (1 µg/ml; Cayman Chemical, Ann Arbor, MI) in 10% horse serum overnight at 4°C. After washing in PBS three times for 5 min each, the sections were incubated with biotinylated goat anti-rabbit immunoglobulin G (IgG) followed by an avidin-alkaline phosphatase complex and Vector Red according to the manufacturer's protocol (Vectastain ABC-AP kit, Vector Laboratories, Burlingame, CA). Vector Red was visualized as a red color. Endogenous alkaline phosphatase activity was inhibited by supplementing 1 mM levamisole (Sigma) into the Vector Red substrate solution. In some sections, rabbit anti-human cPGES was replaced with normal rabbit IgG as a negative control. The sections were counterstained with hematoxylin and mounted. The degree of staining of the slides was subjectively assessed by two investigators who were blind to the experiment and expressed as basal (±), low (+), moderate (++), or strong (+++).
In Situ Hybridization
Total RNAs from mouse uterus on Day 7 of pregnancy were reverse transcribed and amplified with forward primer (5'-ATGCAGCCTGCTTCTGCA) and reverse primer (5'-TTACTCCAGATCTGGCAT) designed according to human p23 (233–715 base pairs [bp], GenBank accession number L24804) [4]. The amplification of cPGES cDNA was performed for 35 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 45 sec. The amplified fragment (483 bp) of cPGES was recovered from the agarose gel and cloned into pGEM-T plasmid (pGEM-T Vector System 1, Promega, Madison, WI). The orientation of cPGES fragment in the pGEM-T plasmid was determined by a combination of the primers for T7, SP6, and p23. The cloned cPGES fragment was further verified by sequencing and was compared with the corresponding region of human p23 sequence (233–715 bp, GenBank accession number L24804). There was 95% homology between mouse cPGES and human p23. These plasmids were linearized with appropriate enzymes for labeling. Digoxigenin (DIG)-labeled antisense or sense cRNA probes were transcribed in vitro using a DIG RNA labeling kit (T7 for sense and SP6 for antisense; Boehringer-Mannheim, Mannheim, Germany).
Uteri were cut into 4–6 mm pieces and flash-frozen in liquid nitrogen. Frozen sections (10 µm) were mounted on slides coated with 3-aminopropyltriethoxy-silane (Sigma) and fixed in 4% paraformaldehyde solution in PBS. The sections were washed in PBS twice, treated in 1% Triton X-100 for 20 min, and washed again in PBS three times. Following the prehybridization in the solution of 50% formamide and 5x SSC (1x SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) at room temperature for 15 min, the sections were hybridized in the hybridization buffer (5x SSC, 50% formamide, 0.02% BSA, 250 µg/ml yeast tRNA, 10% dextran sulfate, and 1 µg/ml of denatured DIG-labeled antisense or sense RNA probe for mouse cPGES) at 55°C for 16 h. After hybridization, the sections were washed in 50% formamide/5x SSC at 55°C for 15 min, 50% formamide/2x SSC at 55°C for 30 min, 50% formamide/0.2x SSC at 55°C twice for 30 min each, and in 0.2x SSC at room temperature for 5 min. After nonspecific binding was blocked in 1% block mix (Boehringer-Mannheim) for 1 h, the sections were incubated in sheep anti-DIG antibody conjugated with alkaline phosphatase (1:5000, Boehringer-Mannheim) in 1% block mix overnight at 4°C. The signal was visualized with 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate and 0.4 mM nitroblue tetrazolium in the buffer containing 100 mM Tris-HCl pH 9.5, 100 mM NaCl, and 50 mM MgCl2. Endogenous alkaline phosphatase activity was inhibited with 2 mM levamisole (Sigma). All sections were counterstained with 1% methyl green in 0.12 M glacial acetic acid and 0.08 M sodium acetate for 30 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A basal level of cPGES appeared in the luminal epithelium from Days 1 to 4 of pregnancy (Fig. 1, A and B). On Day 5 of pregnancy, a strong level of cPGES signal was observed in the stromal cells near the lumen at the implantation site (Fig. 1C). However, no signal was observed at the implantation site on Day 5 of pregnancy after DIG-labeled sense probe was used for hybridization (Fig. 1D). On Day 6, cPGES signal was detected in the decidual cells near the myometrium, and was stronger at the mesometrial pole (Fig. 1F). On Days 7 and 8, a strong level of cPGES was observed in the secondary decidual zone and in the implanted embryos(Fig. 1, G and H).
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Cytosolic PGES Immunostaining During Early Pregnancy
Cytosolic PGES immunostaining was strongly detected in the luminal epithelium on Day 1 and it dropped to a low level on Days 2 and 3 (Fig. 2, A and B). On Day 4, a low level of cPGES immunostaining was observed in both luminal and glandular epithelium (Fig. 2C). On Day 5, cPGES immunostaining was strongly detected in the luminal epithelium, but it was weak in the glandular epithelium and stroma (Fig. 2D). On Day 6, a low level of cPGES immunostaining was observed in the primary decidua (Fig. 2F). A strong level of cPGES immunostaining was observed in the whole decidua on Days 7 and 8 of pregnancy (Fig. 2G). However, no immunostaining was observed after rabbit anti-human cPGES was replaced with normal rabbit IgG (Fig. 2H).
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Cytosolic PGES mRNA and Immunostaining During Pseudopregnancy
A cPGES mRNA signal was not detectable in the uterus from Days 1 to 4 of pseudopregnancy. Only a basal level of cPGES mRNA signal was observed in the luminal epithelium from Days 4 to 8 of pseudopregnancy (Fig. 1E). A basal level of cPGES immunostaining was detected in the luminal epithelium on Days 1 and 2 of pseudopregnancy. However, a low level of cPGES immunostaining was detected in both luminal and glandular epithelium from Days 3 to 6 of psedopregnancy (Fig. 2E).
Expression of cPGES mRNA and Protein in the Delayed Implanting Uterus Before and After the Initiation of Implantation
No cPGES mRNA signal was observed in the uterus under delayed implantation (Fig. 3A). After delayed implantation was terminated by estrogen treatment and embryo implantation was initiated, cPGES mRNA signal was strongly detected in the stroma underlying the luminal epithelium at the implantation site (Fig. 3B). In the uterus with progesterone-primed, delayed implantation, a basal level of cPGES immunostaining was observed in the luminal epithelium (Fig. 3C). However, cPGES immunostaining was strongly detected in the luminal epithelium surrounding the implanting blastocyst after delayed implantation was terminated by estrogen treatment and embryo implantation was initiated (Fig. 3D).
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Cytosolic PGES mRNA and Protein Under Artificial Decidualization
A basal level of cPGES mRNA signal was detected in the luminal epithelium in the uninjected horn (Fig. 3E), whereas cPGES mRNA signal was strongly detected in the decidualized cells under artificial decidualization (Fig. 3F). A basal level of cPGES immunostaining was detected in the luminal epithelium of the uninjected horn (Fig. 3G). After decidualization was artificially induced, cPGES immunostaining was strongly observed in the decidualized cells (Fig. 3H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytosolic PGES mRNA and immunostaining were both highly expressed in decidualized cells on Days 6–8 of pregnancy. Similarly, the high level of cPGES mRNA and immunostaining could be induced in decidualized cells under artificial decidualization. Cytosolic PGES expression in decidual cells may suggest a role during decidualization. It has been shown that cPGES is predominantly linked with COX-1 to promote an immediate response in order to produce enough PGE2 in order to maintain tissue homeostasis [4]. Although COX-1 expression remained low on Day 6 of pregnancy, COX-1 expression increased dramatically in secondary decidual cells at both the mesometrial and antimesometrial poles on Day 8 [11]. In rats, prostacyclin (PGI2) and PGE2 are higher in implantation sites than in the surrounding uterus [12, 13]. PGE2 has a major role for hatching and inducing implantation of mouse blastocysts [14, 15]. Among various PGs, PGE2 and prostacyclin have been considered as primary candidates involved in implantation and decidualization in rodents [3]. PGE2 produced through the COX-1/cPGES system may act through the vasodilating receptor subtype EP2, resulting in a local increase in endometrial vascular permeability and preparing for angiogenesis and placentation. EP2 mRNA was coexpressed with cPGES protein in the luminal epithelium at the implantation site [16], suggesting that cPGES could mediate the synthesis of PGE2 functioning as an autocoid in these cells.
The linkage between the three constitutive enzymes of the biosynthetic cascade (i.e., cytosolic phospholipase A2 [PLA2], COX-1, and cPGES) implies that this pathway is crucial for the production of PGE2, which is required to maintain tissue homeostasis [6]. Pregnancy in cytosolic PLA2-deficient females mated by cytosolic PLA2-deficient males often fails near the time of implantation. Furthermore, the lower number of pups born to cytosolic PLA2-deficient mothers must be the result of maternal problems that affect ovulation, oocyte transport, or implantation because the litter size is the same for cytosolic PLA2 (+/+), (±), or (-/-) male matings with cytosolic PLA2 (-/-) females [17]. Cytosolic PLA2 is present in uterine tissue [17], and cytosolic PLA2 activity in the cytosol was significantly induced during artificial decidualization. In vivo administration of cytosolic PLA2 inhibitor caused a dose-dependent inhibition of decidualization [18]. Although COX-1-deficient females are fertile except for specific parturition defects [7], reduced PG concentrations were observed in COX-1-deficient mice [8]. In addition, COX-1-deficient mice had approximately 32% less uterine vascular permeability than wild-type females did [8]. Whether cPGES-deficient mice have any defects in reproduction still remains to be determined.
In this study, cPGES was strongly observed only in the luminal epithelium at the implantation site. However, we previously showed that mPGES immunostaining was highly detected in subluminal stromal cells [9]. This differential localization between mPGES and cPGES immunostaining may reflect a functional cooperation for PGE2 production in the endometrium at the implantation site. In addition, a compensation was reported between COX-1 and COX-2 in COX-1-deficient mice in that COX-2 was unexpectedly expressed in the luminal epithelium on the morning of Day 4 in COX-1-deficient mice. The presence of COX-2 during this time may partially compensate for the loss of COX-1 activity [8]. Even so, the expression of COX-1 in the uteri of COX-2-deficient mice on Day 4 was unchanged [10]. Furthermore, a compensatory up-regulation of respective COX isoforms was also reported in lung fibroblasts from COX-1-deficient or COX-2-deficient mice [19].
In summary, the strong cPGES expression at the implantation site and decidual cells in mouse uterus suggests that cPGES might be important for the greater vascular permeability at the first stage of attachment.
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FOOTNOTES |
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2 Correspondence. FAX: 86 451 5303336; e-mail: zmyang{at}mail.neau.edu.cn
Received: 10 May 2002.
First decision: 29 May 2002.
Accepted: 10 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Genes Dev 1999 13:1561-1574 is markedly elevated at the site of blastocyst implantation in the rat. Prostaglandins 1978 16:599-606[CrossRef][Medline]This article has been cited by other articles:
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 J. Kang, P. Chapdelaine, P.Y. Laberge, and M.A. Fortier Functional characterization of prostaglandin transporter and terminal prostaglandin synthases during decidualization of human endometrial stromal cells Hum. Reprod., March 1, 2006; 21(3): 592 - 599. [Abstract] [Full Text] [PDF]
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J. Cong, H.-L. Diao, Y.-C. Zhao, H. Ni, Y.-Q. Yan, and Z.-M. Yang Differential expression and regulation of cylooxygenases, prostaglandin E synthases and prostacyclin synthase in rat uterus during the peri-implantation period Reproduction, January 1, 2006; 131(1): 139 - 151. [Abstract] [Full Text] [PDF]
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 S. Chandrasekharan, N. A. Foley, L. Jania, P. Clark, L. P. Audoly, and B. H. Koller Coupling of COX-1 to mPGES1 for prostaglandin E2 biosynthesis in the murine mammary gland J. Lipid Res., December 1, 2005; 46(12): 2636 - 2648. [Abstract] [Full Text] [PDF]
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