1 Department of Obstetrics, Gynecology and Reproductive Sciences, 2 Vascular Biology Center, Institute of Molecular Medicine and Division of Hematology, Department of Internal Medicine, University of Texas Health Science CenterHouston, Houston, TX 77030 and 3 Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
4 To whom correspondence should be addressed at: The University of Texas Health Science CenterHouston, Department of Obstetrics and GynecologyDivision of Reproductive Endocrinology, 6431 Fannin, MSB 3.604, Houston, TX 77030, USA. Email: jaou-chen.huang{at}uth.tmc.edu
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Abstract |
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Key words: cyclooxygenase/embryotrophic factor/embryo co-culture/embryo transport/non-steroidal anti-inflammatory drugs
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Introduction |
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In this report, we analysed eicosanoids synthesized by mouse oviducts and determined the expression of COX-1, COX-2 and PGI2 synthase (PGIS) by western blot analysis and immunohistochemistry. We also compared the PGI2 synthetic capacity and the transcripts of COX isoforms in the oviducts during the preimplantation period. We found that PGI2 was the most abundant PG synthesized and that mouse oviducts expressed COX-1, COX-2 and PGIS. All three proteins were detected in luminal epithelia, smooth muscle cells and vascular endothelial cells by immunohistochemistry. The capacity to synthesize PGI2 by days 2 and 3 post coitus (p.c.) oviducts was 10-fold higher than that by day 4 p.c. oviducts; the levels of COX-2 transcripts in day 1 p.c. oviducts were 5-fold higher than that of day 3 p.c. oviducts. The production of PGI2 by mouse oviducts was greatly reduced by a selective COX-2 inhibitor.
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Materials and methods |
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Oviduct preparation
Oviducts were obtained from randomly selected, non-pregnant, 8 week old C3B6F1 female mice (Harlan, USA) as described below. Additional oviducts were obtained from mice at days 1, 2, 3 and 4 p.c. (day 1 p.c. was defined as the day after the female mouse mated with a vasectomized male mouse, confirmed by the presence of a vaginal plug). Immediately after euthanasia, the oviduct and the uterus were removed en bloc. The uterus and other unwanted tissues such as fat and mesosalpinx were removed under a dissecting microscope; the uterus was removed with a small piece of the oviduct to ensure that only oviducts were used for the experiment. Both oviducts from the same mouse were examined together.
Eicosanoid profile
Eicosanoids were analysed by reverse-phase high-performance liquid chromatography (HPLC) as previously described (Huang et al., 2002) with minor modifications. Briefly, 42 oviducts minced to 1 x 1 mm were suspended in 250 µl incubation buffer (50 mmol/l TrisHCl pH 8.0, 2 mmol/l EDTA, 1 mmol/l glutathione, 1 mmol/l tryptophan) containing 20 µmol/l [14C]arachidonic acid (AA) (56 mCi/mmol, 50 µCi/ml; Amersham Pharmacia Biotech, Inc., USA) and incubated at 37°C water bath for 30 min. The eicosanoids were extracted from the supernatant using a C18 column (Sep-Pak cartridges C18; Waters Corp. USA), separated by reverse-phase HPLC (Waters Corp.) and detected by an in-line radio-detector (
-Ram; Inus Systems Inc., USA). The data were acquired and analysed using the Millenium 32TM software (Waters). The retention time of each eicosanoid was determined previously using individual standards.
Microsome preparation
The microsomes were prepared from mouse oviducts based on methods described previously (Huang et al., 2002) with some modifications. Each pair of oviducts was homogenized using a 2 ml tissue homogenizer (Kontes; Fisher Scientific, USA) in 200 µl homogenization buffer (50 mmol/l TrisHCl pH 8.0, 2 mmol/l EDTA, 0.25 mol/l sucrose) and protease inhibitors (1 mmol/l 4-(2-aminoethyl)benzenesulphonylfluoride hydrochloride, 0.8 µmol/l aprotinin, 50 µmol/l betastatin, 15 µmol/l E-64, 20 µmol/l leupeptin hemisulphate, 10 µmol/l pepstatin A). The homogenate was centrifuged (14 000 g) at 4°C for 2 min. The supernatant was ultracentrifuged (100 000 g) for 50 min at 8°C (TL-100 Ultracentrifuge; Beckman Coulter, USA). The pellet was suspended in a resuspension buffer (50 mmol/l TrisHCl pH 8.0, 2 mmol/l EDTA, 1 mmol/l diethyldithiocarbamate, 0.5 mg/ml tryptophan) using a hand-held homogenizer. An aliquot was removed for protein determination using bovine serum albumin (BSA) as standards (Micro BCA; Pierce Chemical Co., USA); the remainder was stored at 80°C using glycerol (15%) as a cryoprotectant.
Total RNA extraction, RT and real-time quantitative PCR
The RT and the real-time quantitative PCR were performed based on methods described previously (Helmer et al., 2003) with some modifications. Total RNA was extracted from mouse oviducts using a commercial kit (RNAeasy; Qiagen, USA). The RNA (50 µl) was incubated with 0.5 µl of DNAse (10 U/ml; Roche Applied Science, USA) and 6 µl of MgCl2 (25 mmol/l) at 37°C for 30 min. Prior to RTPCR, the DNAse was inactivated by 10 min of incubation at 75°C.
The RTPCR was performed in triplicates using a commercial kit (QuantiTect SYBR Green; Qiagen) in a Smart Cycler® (Cepheid, USA) according to the manufacturer's protocols. Briefly, 10 ml of DNAse-treated RNA sample was reverse-transcribed (50°C for 30 min), followed immediately by RNAse deactivation (85°C for 15 min) and complementary DNA (cDNA) amplification (95°C for 15 s, 60°C for 30 s and 72°C for 30 s). Fluorescence signals generated by SYBR Green bound to the amplicons were recorded real-time during the 72°C cycle.
The primer sequences were: COX-1 (gene bank NM_008969, 13031321; 13941414), COX-2 (gene bank NM_011198, 14641483; 15211541) and -actin (gene bank, 11081125; 10181035). The amount of mRNA was estimated based on the quantity of the respective cDNA, which was, in turn, calculated based on its threshold cycles (CT) against a calibration curve generated from the CT values of DNA standards. The DNA standards were synthetic DNA based on the sequences of respective PCR product. For each DNA standard, a five-log calibration curve was generated. For COX-1 and COX-2, the curve ranged from 20 ag/µl to 2 pg/µl; for
-actin, from 200 ag/µl to 20 pg/µl. The r2 of the calibration curves was >0.998. The mean of the triplicates was used for calculation. The values of COX-1 and COX-2 mRNA were normalized against that of the
-actin.
PGI2 synthetic capacity
The PGI2 synthetic capacity was defined as the maximum PGI2 converted from 20 µmol/l AA by a given amount of microsomes. Preliminary experiments showed PGI2 production reached its maximum after 30 min of incubation. Therefore, the PGI2 synthetic capacity was determined as follows. Aliquots of microsomes (10 µg) were incubated at 37°C for 30 min in an incubation buffer containing 20 µmol/l of AA. The reaction was terminated by rapidly freezing the reaction tube to 20°C. The levels of PGI2 in the supernatant were determined in duplicates using a commercial enzyme immunoassay kit (Cayman Chemical Co., USA). The amount of protein in the microsomes was determined separately by Micro BCA assay using BSA as standards (Pierce Biotechnology, USA). The PGI2 synthetic capacity was expressed as the amount of PGI2 (fmol) produced by 1 µg of microsomes during the 30 min period.
Western blot analysis
Western blot analysis was performed based on methods described previously (Huang et al., 2002). A monoclonal antibody against COX-1, an affinity-purified polyclonal antibody against COX-2 (both from Cayman Chemical Co.) and an affinity-purified peptide antibody against PGIS (a gift from Dr Ke-He Ruan, The University of Texas Health Science Center) were used to detect the respective protein. The COX-1 antibody was raised against purified ovine COX-1, the COX-2 antibody, a human COX-2 peptide sequence, and the PGIS antibody, a human PGIS peptide sequence. All antibodies show cross-reactivity with their murine counterparts. Briefly, microsome protein (30 mg) was electrophoresed in 10% acrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., USA). Each target protein was detected by the respective antibody using enhanced chemi-fluorescence (Amersham Biosciences, USA) and visualized by a STORM 860 laser scanner (Amersham Biosciences). Ovine COX-1 (a gift from Dr A.-L.Tsai, The University of Texas Health Science Center), recombinant human COX-2 (a gift from Dr Richard Kulmacz, The University of Texas Health Science Center) and recombinant human PGIS (a gift from Dr L.-H.Wang, The University of Texas Health Science Center) were used as positive controls.
Immunohistochemistry
The immunohistochemical localization of COX-1, COX-2 and PGIS in mouse oviducts was performed according to methods described previously (Parker et al., 2001). Briefly, paraffin-embedded sections were de-paraffinized and rinsed well with double-distilled water. The sections were heated to 100°C in TrisEDTA buffer (containing 1 mmol/l Tris and 0.1 mmol/l EDTA) for 45 min using a Black and DeckerTM steamer. After cooling down, the sections were rinsed in double-distilled water and blocked with 3% H2O2 for 15 min. The remaining steps were carried out manually at room temperature with phosphate-buffered saline rinse between steps. The sections were blocked for 5 min using Power BlockTM (Biogenex, USA) followed by 15 min each of avidin and biotin block (Vector Laboratories, USA). Following the 2 h incubation with primary antibody (1:200 dilution), the sections were incubated with secondary antibody (MultiLink®; Biogenex) and horseradish peroxidase conjugate, each for 20 min. After the final incubation with substrate (AEC, 3-amino-9-ethylcarbazole; Biogenex) for 15 min, the sections were counterstained with haematoxylin. Non-immune IgG was used for negative controls.
Estimation of COX-2 contribution to PGI2 synthesis
Since both COX isoforms undergo auto-inactivation during catalysis, the PGI2 synthetic capacity reflected the total COX activity to convert AA to PGH2, the rate-limiting step in PGI2 synthesis. Therefore, a reduction of PGI2 synthetic capacity after blocking COX-2 activity with a specific COX-2 inhibitor reflected the contribution of COX-2 to total COX activity. The PGI2 synthetic capacities of microsomes were determined in sets of two. One sample was incubated with 20 mmol/l AA; the other was incubated with COX-2 inhibitor NS-398 (5 mmol/l) for 30 min before incubation with AA and COX-2 inhibitor. The levels of PGI2 in the two samples were compared. The reduction in PGI2 in the latter sample reflected the contribution of COX-2 to the total COX activity.
Statistical analysis
Student's t-test or one-way analysis of variance followed by post hoc tests was performed. P<0.05 was considered statistically significant. The statistical analysis was performed using InStat® software (GraphPad Prism Software Inc., USA).
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Results |
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Discussion |
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Results from immunohistochemistry show that COX-1, COX-2 and PGIS are constitutively expressed in the luminal epithelia, smooth muscle cells and vascular endothelial cells. It has been suggested that co-localization of COX-1 or COX-2 with PGIS in the endoplasmic reticulum and nuclear envelope of vascular endothelial cells may enhance functional coupling of COX with PGIS and facilitate PGI2 production (Liou et al., 2000). We suspect that a similar functional coupling may also occur in mouse oviducts.
COX-2 is generally considered to be involved in pathological processes such as inflammation. Recent reports indicate that COX-2 is constitutively expressed in kidney (Harris and Breyer, 2001), brain (Hoffmann, 2000
), stomach (Halter et al., 2001
) and human oviducts (Huang et al., 2002
). This is in contrast to the conventional belief that COX-2 is associated with pathological conditions. The physiological functions of the constitutively expressed COX-2 and factors responsible for its expression remain to be explored.
Our results show that the level of COX-2 mRNA in day 1 p.c. oviducts was 5-fold higher than that of day 3 p.c. oviducts. We speculate that the increased COX-2 mRNA expression was induced by the pre-ovulatory surge of LH, which reportedly induces the expression of COX-2 in the pre-ovulatory follicles (Liu et al., 1997). Receptors for LH are expressed in the luminal epithelia of human (Lei et al., 1993
) and mouse (Zheng et al., 2001
) oviducts; hCG, which shares the same receptor as LH, induces COX-2 expression in cultured epithelial cells from human oviducts (Han et al., 1996
).
Augmented PGI2 production by oviducts during day 23 p.c. may provide an environment to ensure a successful pregnancy. Cultured mouse embryos exposed to PGI2 analogue between 4-cell and morula stages had higher rates of complete hatching (Huang et al., 2003), implantation and live birth (Huang et al., 2004
). Since the transformation of 2-cell embryos to morulae takes place in the oviduct during day 23 p.c., a maximal PGI2 output from the oviducts during the same period will likely enhance the potentials of hatching, implantation and live birth.
In addition to augmenting the development of embryos, oviduct-derived PGI2 may have other physiological functions such as regulating embryo transport. Results of immunohistochemistry show that smooth muscle cells of mouse oviducts also expressed the receptor for PGI2 (data not shown). We speculate that PGI2 may relax the smooth muscle contractions of mouse oviducts similar to that of human oviducts (Arbab et al., 2002). Furthermore, significant amounts of PGE2 and PGD2 were produced by mouse oviducts. They may also affect the development and the transport of embryos.
In light of this and other reports (Lim et al., 1997; Arbab et al., 2002
; Huang et al., 2003
, 2002
, 2004
), the safety of non-steroidal anti-inflammatory drugs, especially the COX-2 inhibitors, in women desiring pregnancy may need to be re-evaluated.
In summary, mouse oviducts expressed COX-1, COX-2 and PGIS and synthesized abundant PGI2. They synthesized the maximal PGI2 during day 23 p.c., coinciding with the transformation of 2-cell embryos to morulae. These results suggest that oviduct-derived PGI2 may affect preimplantation embryos in a paracrine fashion.
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Acknowledgements |
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References |
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Submitted on June 9, 2004; accepted on August 19, 2004.
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