1 Department of Obstetrics and Gynecology, 3 Vascular Biology Center, Institute of Molecular Medicine and Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center-Houston and 2 Obstetrical and Gynecological Associates, Houston, TX, USA
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of Texas Health Science Center-Houston, 6431 Fannin Street, 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: embryo survival/embryotrophic factor/IVF/non-steroidal anti-inflammatory drugs/preimplantation embryo development
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Introduction |
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Blastocysts from farm animals such as cattle and sheep produce PGs; blastocysts from rabbits also produce PGs (reviewed by Lewis et al., 1989). Although PG production by mouse embryos was suggested by some studies, the direct evidence is lacking. Mouse embryos express COX mRNA (Takami et al., 1994
) and display immunoreactive COX (van der Weiden et al., 1996
). In addition, PGE2 was detected immunohistochemically in mouse embryos (Niimura and Ishida, 1987
). The exact eicosanoid profile, especially PGI2 production by mouse embryos, has not been reported. Furthermore, the role of embryo-derived eicosanoids in embryo hatching is unclear.
In this study, we determined eicosanoid production by mouse embryos and evaluated the role of PGI2 in embryo hatching. Our results confirmed earlier reports that 10 µmol/l of indomethacin inhibited mouse embryo hatching. Furthermore, using selective COX-1 and COX-2 inhibitors, we discovered that embryo hatching was suppressed selectively by COX-2 inhibitor and that adding back a PGI2 analogue abolished the effects of the COX-2 inhibitor. Analysis of eicosanoids by reverse phase high performance liquid chromatography (HPLC) revealed that mouse embryos synthesized predominantly PGI2 from exogenous arachidonic acid (AA). Western blot analysis showed that COX-1, COX-2 and PGI2 synthase (PGIS) proteins were expressed by mouse blastocysts. We concluded that COX-2-derived PGI2 enhanced embryo hatching.
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Materials and methods |
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Harvest and culture of mouse embryos
The care and the manipulation of mice were consistent with the Guide for the Care and Use of Laboratory Animals published by the US National Institute for Health (NIH publication No. 85-23, revised 1996). The research protocol was approved by the Animal Welfare Committee of the University of Texas Health Science Center-Houston.
Mouse embryos were harvested and cultured as described previously (Huang et al., 2003) with some modifications. Three-week-old C57Bl/6 female mice were purchased from Harlan (Indianapolis, IN) and 8-week-old C3H male mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Superovulation in the female mice was achieved by i.p. injection of pregnant mare's serum gonadotrophin (5 IU), followed by HCG (5 IU) 46 h later. After receiving HCG, each female mouse was paired with one fertile male mouse. Forty-eight hours later, 2-cell embryos were harvested from the oviducts into
-minimum essential medium (MEM) supplemented with 25 mmol/l HEPES and 1% bovine serum albumin (BSA) (Irvine Scientific, Santa Ana, CA).
Embryos (1720 per group) were cultured at 37°C under 5% CO2 in 3 cm plates containing 30 µl droplets of protein-free medium under oil (see next paragraph). Human tubal fluid medium (Sage Biopharma, Bedminster, NJ) was used during the first 48 h and -MEM, with Earle's salts and 2 mmol/l glutamine, was used during the second 48 h. After 96 h culture, each embryo was examined for the presence of the zona pellucida. Embryos completely free of the zona pellucida were counted as having completely hatched. The rate of complete hatching was determined by dividing the number of completely hatched embryos by the total number of embryos. Complete embryo hatching was chosen as the end-point because it correlates with the establishment of a viable pregnancy (
Huang et al., 2004).
Using 30 µl of culture medium per 1720 embryos was designed to reveal the autocrine/paracrine effects of embryo-derived factors. A similar approach was used previously to show the autocrine effects of embryo-derived transforming growth factor- (Brison and Schultz, 1997
) and platelet-activating factor (Lu et al., 2004
). We confirmed in our preliminary studies that embryos cultured in 30 µl of protein-free medium were optimal for our experiments as they had a significantly higher rate of complete hatching (48%) compared with those cultured in 600 µl of medium with or without protein supplement (30 and 27%, respectively).
Western blot analysis
Monoclonal antibody for COX-1, affinity-purified polyclonal peptide antibody for COX-2 (both from Cayman Chemical, Ann Arbor, MI) and affinity-purified polyclonal peptide antibody for PGIS (a gift from Dr Ke-He Ruan, The University of Texas Health Science Center) were used to detect the respective protein in the total cell lysate of mouse blastocysts. Details of the western blot analysis are described in the accompanying manuscript.
The total cell lysate of blastocysts was prepared as follows. Mouse blastocysts in 2 µl of medium were transferred to 1.5 ml Eppendorf tubes containing 30 µl of lysis buffer (150 mmol/l NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mmol/l sodium orthovanadate, 1 mmol/l EGTA and 1 mmol/l sodium fluoride) and protease inhibitors [1 mmol/l 4-(2-aminoethyl) benzene sulphonyl fluoride 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, Calbiochem-Novabiochem Corp., San Diego, CA]. The mixture was vortexed for 5 s, centrifuged for 10 s and stirred on ice for 30 min. After two bursts of sonication (Sonifier 250, Branson Co. Danbury, CT), of 1 s each, the protein loading buffer (4x) was added. Afterwards, the supernatant was used for western blot analysis. Lysates from 26, 75 and 67 mouse blastocysts were used for COX-1, COX-2 and PGIS detection, respectively.
Eicosanoid profile
The metabolites of [3H]AA generated by mouse embryos were analysed by a reverse phase HPLC based on a procedure described previously (Sanduja et al., 1991). Briefly, 200 mouse blastocysts were incubated in 100 µl of
-MEM containing 20 µmol/l [3H]AA (150240 Ci/mmol, ICN Biochemicals, Inc., Irvine, CA) in a 37°C water bath for 30 min. After extraction, the eicosanoids were separated and identified using HPLC as described in the accompanying manuscript. To confirm the identity of a peak, an equal amount of the standard was co-injected with the sample and a doubling in the height of the peak was observed.
Immunohistochemistry and fluorescence microscopy
Affinity-purified COX-1 and COX-2 polyclonal antibodies conjugated with rhodamine (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used to localize COX-1 and COX-2 in the mouse embryos. The affinity-purified polyclonal PGIS antibody (used in the western blot analysis) and a goat anti-rabbit immunoglobulin G (IgG) antibody coupled with Alexa 488 (Molecular Probes, Eugene, OR) were used to detect PGIS in the mouse embryos. The embryos were first fixed in 4% paraformaldehyde (pH7.4) at 4°C for 30 min. After three washes in phosphate-buffered saline (PBS), the embryos were blocked in Tris-buffered saline (pH7.4) containing 0.05% Tween-20, 5% powder milk and 0.1% Triton X-100 for 20 min at room temperature. The embryos were incubated at 37°C with the respective antibody in blocking buffer for 2 h. In the case of PGIS, there was an additional incubation with goat anti-rabbit IgG coupled with Alexa 488 (0.625 µg/ml) for 30 min at 37°C. Finally, the embryos were mounted in 0.1 mol/l TrisHCl (pH8.5) containing 16.6% Elvanol 50-42 (DuPont Wilmington, DE) and 2.5% 1,4-diazabicyclo-(2.2.2)-octane. An equal concentration of non-immune rabbit IgG conjugated with fluorescein isothiocyanate (Santa Cruz Biotechnology) in place of conjugated COX-1 and COX-2 antibody was used as negative controls for COX-1 and COX-2, respectively; an equal concentration of pre-absorbed PGIS antibody was used as negative control for PGIS.
Fluorescence microscopy was performed using a Zeiss AxioPlan 2 microscope (Carl Zeiss, Baden-Wuerttemberg, Germany) equipped with appropriate filters. Images were captured using a CCD camera and processed by the AxioVision program (Version 3.0.6).
Statistical analysis
Student's t-test or one-way analysis of variance followed by the indicated post hoc tests was used. P<0.05 was considered statistically significant. The analysis was performed using the InStat® software (GraphPad Prism Software Inc., San Diego, CA).
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Results |
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Discussion |
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Our results confirmed earlier reports by Biggers et al. (1978) and Hurst and MacFarlane (1981)
that indomethacin at a therapeutic concentration (
10 µmol/l) inhibited embryo hatching. The discrepancy between the aforementioned reports and the more recent reports by van der Weiden et al. (1993)
and Chida et al. (1986)
, which indicated that
100 µmol/l indomethacin was required to inhibit embryo hatching, may be attributable to the protein in the medium. Rat serum (30 mg/ml) and BSA (5 mg/ml) were used in the studies by van der Weiden and Chida, respectively, whereas protein-free medium was used in earlier studies.
Our data are in agreement with those of previous reports that indomethacin at 10 µmol/l inhibited embryo hatching by 4050% (Biggers et al., 1978
; Hurst and MacFarlane, 1981
). Prior to this study, it was unclear how indomethacin inhibits embryo hatching. Results from this study indicate that the inhibitory action of indomethacin was attributable to its suppression of COX-2 activity, as the selective COX-2 inhibitor but not the COX-1 inhibitor reduced embryo hatching to an extent similar to indomethacin. Our results further show that COX-2-derived PGI2 may be the key AA metabolite required for embryo hatching. This notion is supported further by the analysis of AA metabolites: PGI2 was the predominant metabolite generated from radiolabelled AA by the embryos. Thus, indomethacin blocks PGI2 synthesis depriving the embryo of one of its key mediators required for its growth and hatching.
It is well known that PGI2 plays an important role in maintaining vascular homeostasis and controlling blood cell activity. That PGI2 has physiological functions outside the cardiovascular system was first suggested by observations made in COX-2-deficient mice (Lim et al., 1997), which underscores the indispensable role of endometrial COX-2-derived PGI2 in normal decidualization and receptivity. Subsequently, it has been reported that human (Huang et al., 2002
) and mouse (Melham et al., 2004
) oviducts produce PGI2 and that embryos exposed to PGI2 analogue between 8-cell and morula stages undergo enhanced hatching (Huang et al., 2003
) and possess augmented potential for implantation and live birth (Huang et al., 2004
). Collectively, PGI2 produced by the embryo and the oviduct acts as an autocrine/paracrine factor to enhance mouse embryo hatching, implantation and live birth.
It may be speculated that mice deficient in phopholipase A2, COX-2, PGIS or PGI2 receptor may produce smaller litters than their wild-type counterparts because their embryos, lacking the autocrine/paracrine effects of PGI2, may have decreased hatching and consequently lower implantation potential. Phospholipase A2-deficient mice have smaller litters than the wild-type (Bonventre et al., 1997). COX-2-deficient mice are sterile because they do not ovulate (Lim et al., 1997
). The litter size of PGIS- (Yokoyama et al., 2002
) and PGI2 receptor- (Murata et al., 1997
) deficient mice has not been compared with that of the wild-types. However, it should be pointed out that breeding COX-2 heterozygous knockout mice (Morham et al., 1995
) or PGI2 receptor heterozygous knockout mice (Murata et al., 1997
) produced less homozygous offspring than expected based on Mendel's law. These observations suggest that PGI2 deficiency may compromise embryo hatching and decrease implantation potential.
Phenotypes of gene deficiency vary with the genetic backgrounds of the mice. For example, COX-2 deficiency causes sterility in C57BL/6J/129 mice (Lim et al., 1997) but subfertility in CD1 mice (Wang et al., 2004
). The diverse genetic pool of CD1 mice does not completely reverse the adverse impacts of COX-2 deficiency. As a result, COX-2-deficient CD1 mice still display significant problems in ovulation and decidualization: they have significantly smaller litters and display significantly less receptivity to transferred wild-type embryos. These observations underscore the importance of COX-2 in ovulation and decidualization. However, the potentials of hatching and implantation of COX-2-deficient embryos were not reported.
Embryo-derived PGs may have other physiological functions such as regulating the transport of embryos. Results from HPLC analysis indicate that, in addition to PGI2, mouse embryos also produce PGE2 and PGF2. Since oviducts express receptors for PGI2 (Arbab et al., 2002
) and PGE2 (Segi et al., 2003
), embryo-derived PGI2 and PGE2 may bind to these receptors, relax the muscle and regulate embryo transport.
The mechanisms by which PGI2 enhanced embryo hatching have not been elucidated. One possible mechanism is augmented ion/water transport across the tight junction between the inner cell mass and the trophectoderm (Biggers et al., 1988). Enhanced ion/water transport into blastocysts increases the blastocoel cavity and facilitates embryo hatching (Biggers et al., 1988
). Alternatively, endogenous PGI2 may increase embryonic cell number by decreasing cell death or increasing cell proliferation. This is supported by a recent report which showed that compared with embryo hatching in vivo, embryos hatching in vitro require more embryonic cells (Montag et al., 2000
). PGI2 has been shown to possess anti-apoptotic properties in renal glomerular mesangial cells (Ishaque et al., 2003
) and colonic epithelial cells (Cutler et al., 2003
). It is, therefore, possible that PGI2 enhances embryo hatching by promoting cell survival.
Although there is no direct evidence for the production of PG by human embryos, it is likely that human embryos, similar to their counterparts from sheep, cattle, rabbit and mouse, also produce PGs. Transcripts of phospholipase A2, COX-1 and COX-2 (Wang et al., 2002) and immunoreactive COX-2 protein (Wang et al., 2002
) and PGE2 (Holmes et al., 1990
) have been observed in human embryos. Embryo-derived PGs, together with oviduct-derived PGs (Huang et al., 2002
), may augment the hatching of human embryos in a paracrine/autocrine fashion analogous to their augmentation of the hatching of mouse embryos. Therefore, the safety of non-steroidal anti-inflammatory drugs, especially the selective COX-2 inhibitors, in women desiring pregnancy may need to be re-evaluated.
Our immunohistochemistry study shows that the trophectoderm and the inner cell mass of mouse blastocysts express COX-1, COX-2 and PGIS. This is different from a previous report based on observations of triploid human embryos which show that trophectoderm, but not inner cell mass, expresses COX-2 (Wang et al., 2002). The discrepancy could be due to species differences or abnormal embryo development as a result of triploidy of chromosomes. Alternatively, it may be due to different methodologies used in the immunohistochemistry. We used Triton X-100 to permeate the tight junction between the trophectoderm and the inner cell mass prior to immunostaining, whereas no detergent was used in the above-mentioned study.
In summary, our results show that mouse embryos express COX-1, COX-2 and PGIS which catalyse the formation of PGI2, and that COX-2-derived PGI2 plays a critical role in embryo hatching.
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Acknowledgements |
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References |
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Submitted on May 26, 2004; accepted on August 19, 2004.