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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 6 2629-2634
Copyright © 2002 by The Endocrine Society

The Impact of the Human Genome on Endocrinology: Original Articles

Phospholipase A2 and Cyclooxygenase Gene Expression in Human Preimplantation Embryos

Hongbo Wang, Yan Wen, Stephen Mooney, Barry Behr and Mary Lake Polan

Department of Gynecology and Obstetrics (H.W., Y.W., S.M., B.B., M.L.P.), Stanford University School of Medicine, Stanford, California 94305; and Department of Gynecology and Obstetrics (H.W.), Huazhong University of Science and Technology, Tongji Medical School Union Hospital, Wuhan 430022, China

Address all correspondence and requests for reprints to: Hongbo Wang, M.D., Stanford University School of Medicine, Department of Gynecology and Obstetrics, Polan Laboratory, 300 Pasteur Drive, Room HH333, Stanford, California 94305-5317. E-mail: . hongbo99{at}leland.stanford.edu

Abstract

Phospholipase A2 (PLA2) and cyclooxygenase (COX) are two key enzymes in PG synthesis; the latter has two forms, COX-1 and COX-2. mRNA was extracted from single preimplantation embryos and examined for PLA2, COX-1, and COX-2 gene expression by RT-PCR to investigate whether PLA2 and COX genes are expressed in human preimplantation conceptuses from zygote to blastocyst stage and to compare COX-1 and COX-2 gene expression within the same stage of embryonic development. Expression of PLA2, COX-1, and COX-2 was detected in 48, 37, and 45%, respectively, of total embryos examined. COX-1 was expressed in approximately 66% of early human preimplantation embryos from zygote to two-cell stage, whereas COX-2 was expressed in about 58% of later stage embryos from eight-cell to blastocyst stage (P < 0.05). Furthermore, COX-2 mRNA and protein were localized to trophectoderm in blastocyst stage embryos. In conclusion, PLA2, COX-1, and COX-2 are expressed during early human embryonic development and may contribute to the production of PGs such as PGE2 in human embryogenesis. COX-1 and COX-2 are differentially expressed, with COX-2 being primarily expressed by trophectoderm in late-stage human preimplantation embryos, which may promote embryonic differentiation and implantation.

PROSTAGLANDIN SYNTHESIS REQUIRES three stages: a rate-limiting, stimulus-induced mobilization of arachidonic acid (AA) from membrane phosphoglycerides, conversion of AA to the PG endoperoxide PGH2, followed by isomerization of PGH2 to biologically active end-products (1). Phospholipase A2 (PLA2) and cyclooxygenase (COX) are two key enzymes in this PG pathway (2).

The synthesis of eicosanoids requires the activation of one or more PLA2 enzymes that catalyze the release of free AA from the sn-2 position of membrane phospholipids. Cytosolic PLA2 (cPLA2) is specific for arachidonyl-containing phospholipids and has been shown to be a key intracellular mediator of hormone-stimulated eicosanoid synthesis (3). Two distinct COX isoenzymes have been described and designated COX-1 and COX-2. COX-1 is produced constitutively by most cells and so is primarily responsible for the immediate synthesis of PGs in response to agonist stimulation (4). COX-2, in contrast, is induced only in response to agonists such as inflammatory cytokines, catalyzing PG synthesis several hours after the inflammatory insult (5). The results of numerous studies indicate that COX-1 and COX-2 have discrete functions (6, 7, 8, 9, 10).

As a first step in exploring the function of cPLA2 in developing embryos, Farber et al. (11) identified that embryonic cPLA2 activity remained constant in zebrafish from the one-cell stage until the onset of somitogenesis and suggested that cPLA2 is an important mediator of stimulus-induced AA release and subsequent eicosanoid synthesis. The functions of COX-1 and COX-2 gene in mouse reproduction have been demonstrated through gene knockout experiments (5). COX-1 knockouts are fertile; in contrast, COX-2 knockouts are sterile.

Although advances in assisted reproductive technology have occurred rapidly in recent years, the implantation rate of embryos in in vitro fertilization (IVF) programs remains unsatisfactory. For successful implantation to occur, several factors must be present, including an embryo that is able to develop to the blastocyst stage with subsequent hatching, adequate endometrial receptivity, and successful interaction between the embryo and endometrium. Studies in animal models have revealed that PGs are involved in multiple aspects of reproduction, including ovulation, fertilization, implantation, decidualization, and parturition (7, 8, 9). During the implantation of ovine embryos, trophoblast cells become adhesive, and invasion is directly related to the level of COX-2 expression (9). Thus, COX-2 may play an important role in embryo implantation. In humans, PLA2 and COX gene expression have been found in placenta and uterine endometrium (10), and PGE2 has been detected in the medium of cultured human embryos (12). No information on the expression of cPLA2 and COX in human embryos during the preimplantation period is available. The goal of this investigation was to examine the expression of cPLA2 and COX genes in human preimplantation conceptuses from zygote to blastocyst stage and to compare differential COX gene expression within the same embryo.

Materials and Methods

Embryo selection

Human preimplantation embryos were obtained from patients undergoing IVF-embryo transfer at Stanford University Department of Gynecology and Obstetrics. For both ethical and practical reasons, we have conducted our study mostly on triploid human embryos. All embryos were considered unsuitable for embryo transfer. Embryos donated by patients were cultured under normal IVF conditions and processed for research at different developmental stages.

Although, this investigation was performed on pathologically fertilized embryos, there are indications that such embryos may be capable of relatively normal gene expression, and there are enough of them to provide convincing information (13, 14, 15, 16). All patients who participated in this study signed an informed consent approved by the Human Subjects in Medical Research Committee at Stanford University.

Primers for RT-PCR

Single embryos [zygote (n = 39), two cells (n = 15), four cells (n = 12), eight cells (n = 15), morula (n = 31), and blastocyst (n = 49)] were examined for PLA2, COX-1, and COX-2 mRNAs by one round of RT followed by two rounds of PCR using a modification of methods described previously (14, 17). In addition, the ß-actin transcript was amplified and identified to ensure the presence of intact mRNA. Sequences of cDNA clones for the mRNA that should be detected in single embryos were obtained from the GenBank database of the National Center for Biotechnology Information of the NIH. One set of corresponding outer primer sequences were synthesized according to published human cPLA2, COX-1, COX-2, and ß-actin cDNA sequences (18, 19, 20). A second set of corresponding inner primer sequences were constructed with the help of the program OLIGO primer Analysis software (National Bioscience, Plymouth, MN) and synthesized at the Beckman Center (Stanford University Medical Center, Stanford, CA). Paired outer primers were used for the first round PCR, whereas the inner primers were used for the second round PCR. Primer sets used were designed to span at least two exons to exclude the possibility of amplifying genomic DNA from contamination during RNA extraction. The identify of all PCR products was confirmed by sequence analysis. The primer cDNA sequences and the size of the amplified fragments are listed in Table 1Go.


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  		Table 1. Primers used for RT-PCR

 
RT

RT was modified for single embryo analysis according to previous protocols (21). In brief, for each embryo, 15 µl RT master mix was prepared as follows: 4 µl 25 mmol/liter MgCl2 solution, 2 µl 10x PCR buffer, 2 µl each dATP, dCTP, dGTP, and dTTP (Perkin-Elmer Corp., Foster City, CA); 0.75 µM one of the three amplifying outer 3'-primers and 0.75 µM ß-actin outer 3'-primer were mixed in a 0.5-ml PCR tube (Applied Scientific, South San Francisco, CA). RT master mix in PCR tubes was covered with 50 µl light white mineral oil (Sigma, St. Louis, MO) and kept on ice until RNA extraction.

One embryo was added to the RT mix, allowing a culture medium carryover of 1 µl. Samples were immediately heated to 99 C for 1 min in a DNA Thermal cycle 480 (Perkin-Elmer Corp.) to release total RNA and denature protein. Samples were cooled to 4 C, and 20 U RNase inhibitor (Perkin-Elmer Corp.) was added, followed by 100 U Maloney murine leukemia virus (Life Technologies, Inc., Grand Island, NY). The RT was performed in the DNA Thermal cycle 480 as follows: 42 C for 15 min, 99 C for 5 min, and 5 C for 5 min. After the reaction was complete, samples were stored at -20 C until the PCR. In all cases, the negative control of culture medium without an embryo was used in RT.

Two rounds of PCR

For the first PCR, 5 µl of RT product from an individual embryo was added to the first PCR master mix to a total volume of 80 µl containing 2 mM MgCl2, 10x PCR Buffer II, 0.24 µM 3' and 5' primers mixture of each specific outer pair and 2.5 U AmpliTaq DNA polymerase (Applied Biosystem, Foster City, CA). Then the mixture was subjected to 40 cycles of amplification for PLA2, COX-1, and COX-2. For PLA2 amplification, the reaction involved denaturation at 94 C for 1 min, annealing at 55 C for 45 sec, and extension at 72 C for 2 min. For COX-1 and COX-2, the PCR with denaturation at 94 C for 1 min, 10 sec; annealing at 55 C for 1 min, 50 sec; and extension at 72 C for 1 min, 10 sec, were performed. First-round PCR products were stored at -20 C until the second round of PCR.

For the second round PCR, 5 µl of the initial PCR products were added to the second PCR master mix to a total volume of 80 µl containing 2 mM MgCl2, 10x PCR Buffer II, 0.2 mM of each dNTP (dATP, dCTP, dGTP, and dTTP), 0.24 µM 3' and 5' primers mixture of each corresponding inner pair, and 2.5 U AmpliTaq DNA polymerase. After the second round PCR using the same program was completed, samples were stored at -20 C until electrophoresis.

Agarose gel electrophoresis

PCR product (20 µl) was size-fractionated on a 2% agarose gel and visualized using ethidium bromide. Photocopies of the agarose gel were printed on the GelDoc 1000 system (Bio-Rad Laboratories, Inc., Hercules, CA).

Isolation of trophectoderm (TE) and inner cell mass (ICM)

Blastocysts were placed in a single drop of medium (50 µl) under a micromanipulator, and the zona pellucida was partially dissolved by the punctual application of acidified Tyrode’s solition adjusted to pH 2.0 (Sigma). ICM was separated from TE, using the process commonly described for blastomere biopsy (22).

Briefly, in microdrops under oil, the embryo was stabilized by a pipette, and the cells of ICM were successively removed by a biopsy pipette. The ICM and TE were then moved to different parts of the drop by the pipette and sucked individually into two tubes preloaded with RT mixture solution. The method of RT-PCR was described above.

Immunohistochemical staining

Single blastocysts were examined by an avidin-biotin alkaline phosphatase method (13) to localize the COX-2 at the protein level. Embryos were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature in microdrops under oil and then treated with acid Tyrode’s solution (pH 2.5) to induce permeabilization of the zona pellucida. Endogenous peroxidase was quenched in 3% hydrogen peroxide for 15 min at room temperature. To reduce the nonspecific binding, 2% normal goat serum in PBS was applied to the embryos for 30 min, then rinsed twice in PBS, and incubated for 60 min at 37 C with the primary antibody rabbit antihuman COX-2 (1:50 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After rinsing with PBS, the embryos were incubated for 60 min at 37 C with a secondary antibody, biotinylated antirabbit IgG (1:800 dilution, Sigma). Negative controls were incubated with PBS containing 2% goat serum without primary antibody. To amplify the signal, embryos were washed with PBS, and then with the avidin-biotin alkaline phosphatase-staining method (Vector Laboratories, Inc., Burlingame, CA) was used. Finally, embryos were incubated in alkaline phosphatase substrate solution until the red color had developed; then the reaction was stopped in all of the embryos simultaneously. A red precipitate indicated positive staining by the primary antibody. Embryos in microdrops under oil were visualized and photographed by a 35-mm camera.

Statistical analysis

The number of embryos in which each gene was detected was recorded. We observed the PG genes in early stage (one-cell to two-cell) and late stage (eight-cell to blastocyst) embryo during the preimplantation period. The data obtained in these studies were analyzed using {chi}2 test, and P value less than 0.05 was considered statistically significant.

Results

PLA2, COX-1, and COX-2 mRNA were detected in all stages of human preimplantation embryos (Fig. 1Go). Respective 236-bp, 207-bp, 120-bp, and 248-bp signals consistent with the expected sizes of PLA2, COX-1, COX-2, and ß-actin fragments were demonstrated. In two-cell stage embryos, COX-1 mRNA was expressed at higher levels than it was in blastocyst stage.



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  		Figure 1. RT-PCR analysis of cPLA2, COX-1, and COX-2 gene expressed in single human embryos. Agarose gels were stained with ethidium bromide showing electrophoretic bands corresponding to the cDNA amplification products derived from the second PCR amplification using cPLA2, COX-1, and COX-2 inner primers. The ß-actin transcript also was amplified to ensure the presence of intact mRNA. Respective 236-bp, 207-bp, 120-bp, and 248-bp signals are consistent with the expected sizes of cPLA2, COX-1, COX-2, and ß-actin fragments.

 
PLA2, COX-1, and COX-2 were detected in 48.7% (19 of 39), 37.7% (26 of 69), and 45.3% (24 of 53) of preimplantation embryos, respectively (Table 2Go). The percentage of transcripts detected in each stage (from zygote to blastocyst) varied. Because of the small numbers of embryos at each stage, definitive statistical analysis was not possible. However, PLA2 expression does not appear to vary as a function of embryo stage.


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  		Table 2. Number of embryos at each stage expressing detectable levels of PLA2, COX-1, and COX-2 mRNA

 
Two different patterns of COX gene expression are evident during the development of human preimplantation embryos (Fig. 2Go). In early stages from zygote to two-cell, COX-1 was expressed in 66.7% (16 of 24) of embryos examined. But it was only expressed in 22% (9 of 41) of later stages from eight-cell to blastocyst (P < 0.05). In contrast, COX-2 was expressed in 58% (18 of 31) of later stage embryos but in only 27% (5 of 18) of early stage embryos (P < 0.05). However, no statement can be made about the inducibility of COX expression in developing human embryos.



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  		Figure 2. COX-1 and COX-2 were expressed in different stages of preimplantation human embryos. COX-1 was mainly expressed in early embryos from one-cell to two-cell stages (66.7%), whereas it was expressed in 21.9% of later embryos from eight-cell to blastocyst stages (P < 0.05). COX-2 was expressed in 54.5% of embryos from eight-cell to blastocyst, but only 29.4% in early embryos from one-cell to two-cell (P < 0.05).

 
We separated TE and ICM in four embryos, submitting both samples to RT-PCR for the COX-2 gene, under the same conditions. COX-2 mRNA was detected in two embryonic TEs, but in none of the ICM samples (Fig. 3Go). Single embryo immunohistochemistry revealed that COX-2 protein localized on TE. No staining of COX-2 was observed in ICM (Fig. 4Go).



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  		Figure 3. ICM was separated from TE and submitted to RT-PCR for the COX-2 gene. COX-2 mRNA was only detected in TE (two of four embryos examined) but not ICM. The ß-actin transcript also was amplified to ensure the presence of intact mRNA.

 


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  		Figure 4. Immunohistochemical localization of COX-2 protein in a human blastocyst. A, An expanded blastocyst stained positive for COX-2 in TE but not ICM. B, Negative control, no positive staining in TE or ICM.

 
Discussion

The present study clearly demonstrates that human embryos express PLA2 and COX gene transcripts, two key enzymes of PG synthesis, throughout the preimplantation period. Thus, human preimplantation embryos are assumed to be able to synthesize PGs, which are involved in multiple aspects of reproduction including fertilization, implantation, and decidualization.

PGs have been implicated in a number of embryonic developmental processes, including the shedding of the zona pellucida (hatching), accumulation of blastocoelic fluid, and elongation of trophoblast (23). On the other hand, implantation is a complex process requiring the interaction of the blastocyst and the subsequently developing embryo with the endometrium (24). Psychoyos et al. (25) suggested that PGs produced by the blastocyst may serve as embryonic signals to the uterus. The role of PG synthesis in embryo implantation was first demonstrated by Lau et al. (26). There is clear evidence that PGs, primarily PGE2, are necessary for increased vascular permeability at the site of implantation and for increased local blood flow (27).

PGs produced by embryos may be involved in other functions during the preimplantation period such as modulation of the endometrial implantation site (28). Immunologically, the conceptus is a hemiallograft and, thus, should be rejected unless immunoregulatory mechanisms intervene to protect the allogenic pregnancy. Recently, it has become apparent that immune effector mechanisms of maternal but also embryonic origin are critical determinants of successful blastocyst implantation. PGE2 has been detected in the medium of cultured human embryos and has been demonstrated to down-regulate IL-2 receptors on T-lymphocytes, a function that would effectively inhibit the proliferation and cytotoxic activation of T-lymphocytes (29). Through this mechanism, the blastocyst could defend itself from attack by maternal immune cells, while possibly also providing an immunosuppressed implantation site.

PLA2 and COX are two key enzymes in PG synthesis. PLA2 has been shown to function in digestion of lipids, microbial degradation, inflammation, cell signaling, and membrane remodeling (30). The cPLA2 protein is ubiquitously expressed in all adult human tissues but is subject to complex regulation that enables the immediate generation of PGs in response to physiological stimuli. cPLA2 has been identified in equine conceptuses between days 12 and 15 (31). In humans, cPLA2 mRNA and protein have been detected in fetal membranes at term, where they may contribute to production of PG (32). In the present study, cPLA2 was expressed in preimplantation human embryos from one cell to blastocyst. This is of particular significance because AA released by PLA2 activity is the precursor for the synthesis of PGs.

Our study suggests that the COX gene is expressed in all the stages of preimplantation in human embryos, but with two different patterns. COX-1 was mainly expressed during early stages of embryogenesis, whereas COX-2 was predominately expressed later in eight-cell, morula, and blastocyst stages. Herschman (4) suggested that COX-1 is expressed constitutively and is responsible for the immediate synthesis of PGs in response to agonist stimulation. However, COX-2, the more recently identified isoform, can be specifically induced by a wide variety of factors. In humans, COX-1 and COX-2 are expressed in fetal membranes and placenta. However, Slater et al. (33) reported COX-2 alone was up-regulated during labor, primarily participating in PG synthesis.

COX-2 was found to be important for each stage of pregnancy, and its expression is regulated by platelet-derived growth factor, epidermal growth factor, and IL-1 (34, 35). Those factors also play an important role in embryo differentiation, development, and endometrial decidualization. Charpigny et al. (9) studied COX in ovine embryos during the implantation period and found COX-2 to be highly expressed in ovine embryo, while low levels of COX-1 were detected. In the present study, COX-2 was mainly expressed in eight-cell, morula, and blastocyst stages. Moreover, COX-2 mRNA and protein were observed only in the TE of the developing embryo, suggesting that COX-2 may be related to embryonic differentiation and implantation in humans.

The contribution of COX-1 in reproduction is less clear. Our study demonstrates that COX-1 is highly expressed in early human preimplantation embryos from zygote to two-cell stages, indicating that COX-1 mRNA might be of maternal origin. Evidence supporting this speculation originates from reports showing that embryonic gene expression first occurs between the four-cell and eight-cell stages of embryos in humans (36).

For both ethical and practical reasons, we have conducted our study on pathological human embryos. The most crucial question regarding the biological significance of these findings is whether or not they are influenced by triploidy of these embryos. However, indications are that such embryos may be capable of relatively normal gene expression, as seen in polyploid embryos that are capable of an extensive, morphologically normal development far beyond the eight-cell stage (37, 38) and reports of triploid birds (39). It therefore seems unlikely that the pattern of transcriptional activity in these embryos varies significantly from that of regularly fertilized, diploid embryos (40).

At present, we cannot explain the lack of expression of these genes in many embryos. It is conceivable that this subpopulation of poor-quality embryos is not representative of normal embryos. On the other hand, it may demonstrate some of the specific features of poor embryos. Therefore, our findings may not be generally applicable to all embryos. The expression of PLA2 and COX genes in human preimplantation embryos could provide more clues as to the possible role of PGs in multiple aspects of human reproduction including fertilization, implantation, and decidualization. We may be able to translate this knowledge into facilitating preimplantation embryonic development and enhancing embryonic implantation when we fully understand the function of these genes in normally developing embryos.

In conclusion, PLA2, COX-1, and COX-2 are expressed during early human embryonic development. These enzymes may contribute to the production of PGs. Differences in gene expression between COX-1 and COX-2 were observed in different stages of human early conceptus development with COX-2 preferentially expressed in late preimplantation human embryos. The localization of COX-2 mRNA and protein on TE in blastocysts suggests that it may promote the process of differentiation and implantation in human embryo.

Acknowledgments

We thank and acknowledge the contributions of Janice Gebhart, Jennifer Lyon, and Danny Dasig from the Stanford University IVF laboratory. We also thank Mary Peterson for her excellent help in preparing this manuscript.

Footnotes

This work was supported by a grant from the Children’s Health Initiative of the Lucille Salter Packard Children’s Hospital and the Packard Foundation.

Abbreviations: AA, Arachidonic acid; COX, cyclooxygenase; cPLA2, cytosolic PLA2; ICM, inner cell mass; IVF, in vitro fertilization; PLA2, phospholipase A2; TE, trophectoderm.

Received July 23, 2001.

Accepted December 26, 2001.

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D. M. J. Curfs, S. A. I. Ghesquiere, M. N. Vergouwe, I. van der Made, M. J. J. Gijbels, D. R. Greaves, J. S. Verbeek, M. H. Hofker, and M. P. J. de Winther
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A. El-Sayed, M. Hoelker, F. Rings, D. Salilew, D. Jennen, E. Tholen, M.-A. Sirard, K. Schellander, and D. Tesfaye
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P. K. JAIN
Epigenetics: The Role of Methylation in the Mechanism of Action of Tumor Suppressor Genes
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