Oviduct prostacyclin functions as a paracrine factor to augment the development of embryos

Jaou-Chen Huang1,4, Jennifer S. Goldsby1, Farinaz Arbab3, Ziad Melhem1, Nena Aleksic2 and Kenneth K. Wu2

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 Center–Houston, 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 Center–Houston, Department of Obstetrics and Gynecology–Division of Reproductive Endocrinology, 6431 Fannin, MSB 3.604, Houston, TX 77030, USA. Email: jaou-chen.huang{at}uth.tmc.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Recently we discovered that human oviducts produce a significant amount of prostacyclin (prostaglandin I2, PGI2) and that PGI2 enhances the potentials of live birth of mouse embryos. However, the eicosanoid profile of mouse oviducts remains unknown. METHODS: The metabolites of [14C]arachidonic acid by mouse oviducts were analysed by high-performance liquid chromatography. The expression of cyclooxygenase (COX)-1, COX-2 and PGI2 synthase (PGIS) was analysed by western blot analysis and immunohistochemistry. The PGI2 synthetic capacities and the COX transcripts during the preimplantation period were compared. The effects of COX-2 inhibitor on PGI2 production were ascertained. RESULTS: Mouse oviducts produced, in order of abundance, PGI2, PGD2 and PGE2. Western blot analysis confirmed the expression of COX-1, -2 and PGIS which were expressed by luminal epithelia and smooth muscle cells. Day 2–3 post-coitus (p.c.) oviducts produced PGI2 10-fold higher than day 4 p.c. oviducts (P=0.0087); day 1 p.c. oviducts expressed COX-2 transcript 5-fold higher than day 3 p.c. oviducts (P=0.0004). The PGI2 production was markedly reduced by a selective COX-2 inhibitor. CONCLUSIONS: Mouse oviducts synthesized maximal PGI2 during day 2–3 p.c., coinciding with the transformation of 2-cell embryos to morulae. The results suggest that oviduct-derived PGI2 may enhance embryo development in a paracrine fashion.

Key words: cyclooxygenase/embryotrophic factor/embryo co-culture/embryo transport/non-steroidal anti-inflammatory drugs


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Until recently, prostacyclin (prostaglandin I2, PGI2) was considered a molecule in the cardiovascular system responsible for maintaining blood homeostasis and vascular tone. Observations made in cyclooxygenase (COX)-2 knockout mice suggest that COX-2-derived, endometrial PGI2 is critical to endometrial decidualization and receptivity (Lim et al., 1999Go). We recently discovered that human oviducts synthesize abundant PGI2 (Huang et al., 2002Go) and that supplementing culture media with PGI2 enhances the complete hatching (Huang et al., 2003Go) and the potentials of implantation and live birth (Huang et al., 2004Go) of cultured mouse embryos. These findings suggest that oviduct-derived PGI2 may enhance the development of embryos and augment the likelihood of pregnancy. However, the eicosanoid profile of mouse oviducts has not been reported. Furthermore, the capacity of mouse oviducts to synthesize PGI2 during the preimplantation period has not been examined.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Source of reagents and institutional approval
Unless specified otherwise, all reagents were purchased from Sigma–Aldrich Co. (USA). The research protocol was approved by the Animal Welfare Committee of the University of Texas Health Science Center. 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).

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., 2002Go) with minor modifications. Briefly, 42 oviducts minced to 1 x 1 mm were suspended in 250 µl incubation buffer (50 mmol/l Tris–HCl 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 ({beta}-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., 2002Go) 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 Tris–HCl 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 Tris–HCl 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., 2003Go) 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 RT–PCR, the DNAse was inactivated by 10 min of incubation at 75°C.

The RT–PCR 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, 1303–1321; 1394–1414), COX-2 (gene bank NM_011198, 1464–1483; 1521–1541) and {beta}-actin (gene bank, 1108–1125; 1018–1035). 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 {beta}-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 {beta}-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., 2002Go). 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., 2001Go). Briefly, paraffin-embedded sections were de-paraffinized and rinsed well with double-distilled water. The sections were heated to 100°C in Tris–EDTA 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Mouse oviducts produce abundant PGI2
Eicosanoids produced by the mouse oviducts were determined by incubating 42 mouse oviducts with 20 µmol/l [14C]AA and analysing the extracted eicosanoids by HPLC. The results show that 6-keto PGF1{alpha}, the stable metabolite of PGI2, was most abundant. PGE2 and PGD2 were also produced, but in a lesser amount. Lipoxygenase products were also detected (Figure 1).



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Figure 1. Mouse oviducts synthesize abundant prostaglandin I2 (PGI2). Forty-two mouse oviducts were minced and incubated with [14C]arachidonic acid (AA) (20 mmol/l) for 30 min at 37°C. The eicosanoids were extracted from the supernatant and separated by high-performance liquid chromatography. The retention time of each eicosanoid was determined previously using individual standards. I = 6-keto PGF1{alpha}, the stable metabolite of PGI2; B = thromboxane B2, the stable metabolite of thromboxane A2; F = PGF2{alpha}; E = PGE2; D = PGD2; AA = arachidonic acid.

 
Mouse oviducts express COX-1, COX-2 and PGIS
The biosynthesis of PGI2 involves the conversion of AA to PGH2 by COX and the conversion of PGH2 to PGI2 by PGIS. In order to confirm that mouse oviducts possess the necessary enzymatic machinery to produce PGI2, we performed western blot analysis on microsomes of oviducts. Our results show that mouse oviducts express COX-1, COX-2 and PGIS (Figure 2). We also performed immunohistochemistry to determine the types of cells expressing these proteins. The results show that luminal epithelia, smooth muscle cells and vascular endothelial cells expressed COX-1, COX-2 and PGIS (Figure 3).



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Figure 2. Mouse oviducts express both cyclooxygenase (COX) isoforms and prostaglandin I2 synthase (PGIS). Microsomes (30 µg) of mouse oviducts were analysed by western blot analysis. Monoclonal antibody against COX-1, affinity-purified polyclonal antibodies against COX-2 and PGIS were used for the detection. Lanes 1, 3, 5 are ovine COX-1, recombinant human COX-2 and PGIS respectively; lanes 2, 4, 6 are the microsomes of mouse oviducts.

 


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Figure 3. The luminal epithelia, vascular endothelia and the smooth muscle cells of mouse oviducts express cyclooxygenase (COX)-1, COX-2 and prostaglandin I2 synthase (PGIS). COX-1, COX-2 and PGIS were localized by indirect immunohistochemistry using monoclonal antibody against COX-1 and affinity-purified polyclonal antibodies against COX-2 and PGIS. Red or brown colour indicates positive staining. All three enzymes were detected in luminal epithelia (arrow), smooth muscle cells (arrow head) and vascular endothelial cells (dashed arrow). Same concentration of non-immune IgG instead of the primary antibody was used as a negative control. One of the negative controls is shown. Bar {approx} 100 µm.

 
Day 2–3 p.c. oviducts produce the most PGI2
We reasoned that, for oviduct-derived PGI2 to act as a paracrine factor, oviduct PGI2 synthesis may increase on day 2–3 p.c., because during this period 2-cell embryos transform to morulae inside the oviducts. Therefore, we compared PGI2 synthetic capacities between day 2–3 p.c. oviducts, day 4 p.c. oviducts and non-pregnant oviducts. Our results show that day 2–3 p.c. oviducts synthesized ~10-fold more PGI2 than day 4 p.c. oviducts or non-pregnant oviducts (Figure 4).



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Figure 4. Day 2–3 post-coitus (p.c.) oviducts synthesize the maximum prostacyclin I2 (PGI2). The ability to synthesize PGI2 was compared among day 2–3 p.c., day 4 p.c. and non-pregnant oviducts. Microsomes were incubated with 20 mmol/l AA at 37°C for 30 min. The PGI2 in the supernatant was determined by an enzyme immunoassay and normalized by the amount of protein. Day 2–3 p.c. oviducts produced ~10-fold more PGI2 than day 4 p.c. oviducts or non-pregnant oviducts. The mean±SD of PGI2 synthetic capacities of days 2 and 3 p.c. oviducts (two mice each), day 4 p.c. oviducts (four mice) and non-pregnant oviducts (five mice) is shown. P=0.0087 based on analysis of variance; *P<0.05, post hoc Bonferroni test.

 
COX-2 induction precedes the enhanced PGI2 synthetic capacity
Since COX catalyses the rate-limiting step of PG synthesis, the augmented PGI2 synthetic capacity in day 2–3 p.c. oviducts was likely due to an increased COX activity. We speculated that levels of COX-2 transcript were augmented and that levels of COX-1 transcript remained unchanged, because the former is inducible and the latter is ‘house-keeping’. We compared the mRNA levels of COX-1 and COX-2 in day 1 p.c. and day 3 p.c. oviducts. As expected, levels of COX-1 mRNA remained unchanged whereas levels of COX-2 mRNA in day 1 p.c. oviducts were 5-fold higher than that of day 3 p.c. oviducts (Figure 5).



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Figure 5. Transcripts of COX isoforms during the preimplantation period. Levels of COX mRNA in day 1 p.c., day 3 p.c. and non-pregnant oviducts were determined by real-time quantitative RT–PCR and normalized to that of {beta}-actin. (a) The levels of COX-1 mRNA remained unchanged during the preimplantation period. (b) The level of COX-2 mRNA in day 1 p.c. oviduct was 5-fold higher than that of day 3 p.c. or non-pregnant oviducts. The mean±SD of COX mRNA from day 1 p.c. oviducts (two mice), day 3 p.c. oviducts (three mice) and non-pregnant oviducts (four mice) is shown. P> 0.05 for COX-1 and P=0.0004 for COX-2, based on analysis of variance; *P< 0.01, post hoc Bonferroni test.

 
Oviduct COX-2 contributed to PGI2 synthesis
In order to confirm the biological activity of COX-2, we compared the PGI2 synthetic capacities of paired microsome samples where one was incubated with a specific COX-2 inhibitor, NS-398. Our results confirm that oviduct COX-2 was biologically active and contributed greatly to PGI2 synthesis by the oviducts (Figure 6).



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Figure 6. Oviduct cyclooxygenase (COX)-2 contributes greatly to prostacyclin I2 (PGI2) synthesis. To determine the contribution of COX-2 to PGI2 synthesis, aliquots of microsomes from three randomly selected, non-pregnant oviducts were incubated with and without a specific COX-2 inhibitor, NS-398, before incubating with arachidonic acid (20 mmol/l). The levels of PGI2 in the supernatant were determined by an enzyme immunoassay and normalized by the amount of microsome protein. PGI2 synthesis was greatly reduced by the COX-2 inhibitor. The mean±SD of PGI2 synthesized is shown.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Our findings reveal, for the first time, that mouse oviducts express COX-1, COX-2 and PGIS and synthesize abundant PGI2. Our results also show that mouse oviducts produce the maximal amount of PGI2 on day 2–3 p.c., coinciding with the transformation of 2-cell embryos to morulae inside the oviduct.

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., 2000Go). 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, 2001Go), brain (Hoffmann, 2000Go), stomach (Halter et al., 2001Go) and human oviducts (Huang et al., 2002Go). 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., 1997Go). Receptors for LH are expressed in the luminal epithelia of human (Lei et al., 1993Go) and mouse (Zheng et al., 2001Go) oviducts; hCG, which shares the same receptor as LH, induces COX-2 expression in cultured epithelial cells from human oviducts (Han et al., 1996Go).

Augmented PGI2 production by oviducts during day 2–3 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., 2003Go), implantation and live birth (Huang et al., 2004Go). Since the transformation of 2-cell embryos to morulae takes place in the oviduct during day 2–3 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., 2002Go). 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., 1997Go; Arbab et al., 2002Go; Huang et al., 2003Go, 2002Go, 2004Go), 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 2–3 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank Dorene M.Rudman, HT (ASCP), Texas Children's Hospital, for technical assistance. The author also wishes to thank Dana Whittaker for secretarial assistance. J.-C.H. is a Women's Reproductive Health Research Scholar (HD01277).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on June 9, 2004; accepted on August 19, 2004.





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