Molecular Human Reproduction, Vol. 7, No. 1, 57-63,
January 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Vascular endothelial growth factor (VEGF) mRNA splice variants are differentially expressed in human blastocysts*
1 Department of Obstetrics and Gynecology, Heinrich-Heine-University Medical Center, Moorenstraße 5, D-40225, Düsseldorf, Germany, and 2 Department of GYN/OB, Reproductive Immunology Laboratory, Stanford University Medical Center, Stanford, CA, USA
Abstract
The aim of our study was to detect and characterize mRNA expression of VEGF isoforms VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206 in human blastocysts. We recently demonstrated VEGF mRNA expression during human preimplantation embryo development, and further information regarding the alternatively spliced mRNAs resulting in freely secreted proteins or proteins bound to cell surface heparan–sulphate proteoglycans is needed to better understand the process of angiogenesis during implantation. Human blastocysts unsuitable for transfer obtained from the IVF programme at Stanford University were examined by reverse transcription/hemi-nested polymerase chain reaction for their expression of VEGF mRNA splice variants. VEGF mRNA was expressed in 17 out of 19 (89%) blastocysts. Of the 17 blastocysts, VEGF121 mRNA was detected in 88%, VEGF145 mRNA in 100%, VEGF165 mRNA in 71%, and VEGF189 mRNA in 24% of blastocysts. There was co-expression of mRNA for VEGF121 and VEGF145 only in 29% blastocysts, of mRNA for VEGF165 and VEGF145 only in 12%, and of mRNA for VEGF121, VEGF145 and VEGF165 in 59% blastocysts. VEGF206 mRNA could not be detected. In conclusion, we demonstrated that blastocysts express the mRNAs encoding for the free VEGF proteins, enabling the implanting embryo to immediately induce angiogenesis at the implantation site.
angiogenesis/cytokines/gene expression/growth factors/implantation
Introduction
Angiogenesis, the growth of blood vessels by sprouting from a pre-existing endothelium (Klagsbrun and D'Amore, 1991
; Folkman and Shing, 1992), and vasculogenesis, the in-situ formation of primordial vessels from haemangioblasts (Coffin and Poole, 1988; Schwartz et al., 1990) are both essential mechanisms during early embryonic development following implantation into the maternal endometrium. The molecular mechanisms that lead to embryonic attachment to the endometrial epithelium, invasion into the endometrial stroma and angiogenesis to maintain nutrition and oxygenation of the implanting embryo are not yet fully understood. Growth factors and cytokines, however, appear to play an important role during these steps of embryonic implantation (Chard, 1995; Simón et al., 1995; Tazuke and Giudice, 1996; Krüssel et al., 1998a, 2000a).
Vascular endothelial growth factor (VEGF) is a highly specific mitogen for endothelial cells (Ferrara and Henzel, 1989). It induces angiogenesis and increases the permeabilization of blood vessels (Keck et al., 1989). The vascular endothelial growth factor A (VEGF-A) ligand system is composed of five isoforms created by alternative splicing of the VEGF mRNA. The human VEGF proteins have been characterized to consist of 121, 145, 165, 189 and 206 amino acids (VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206). As illustrated in Figure 1a, all isoforms contain exons 1–5 and 8. They differ only by various combinations of either no additional exon (VEGF121), or addition of exon 6 (VEGF145), exon 7 (VEGF165), exon 6 and exon 7 (VEGF189) or exon 6, exon 6' and exon 7 (VEGF206) (Houck et al., 1991; Charnock-Jones et al., 1993; Shibuya, 1995; Neufeld et al., 1996; Poltorak et al., 1997). Biologically, VEGF121, VEGF145 and VEGF165 are secreted forms, whereas VEGF189 and VEGF206 appear to be bound to the cell surface (for review, see Neufeld et al., 1999).
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There are three transmembraneous tyrosine kinase receptors: VEGFR-1 (flt-1), VEGFR-2 (KDR or flk-1) and VEGFR-3 (flt-4) which bind to all secreted VEGF isoforms (Neufeld et al., 1999
). Recently, the transmembraneous neuropilin-1 receptor, a receptor for the collapsin/semaphorin family that mediates neuronal cell guidance, was reportedly expressed on endothelial cells and specifically bound to VEGF isoform 165 (Soker et al., 1998). There is also one soluble receptor (sflt), created by alternative splicing of VEGFR-1 mRNA, that acts as a competitive antagonist to VEGF (for reviews, see Ferrara and Davis-Smyth, 1997; Neufeld et al., 1999). These receptors are expressed and, in the case of sflt, differentially regulated in human endometrium throughout the menstrual cycle (Krüssel et al., 1999).
We have previously described the expression of VEGF mRNA in human preimplantation embryos at various stages of development (Krüssel et al., 2000b). The question of whether the VEGF mRNA detected in human preimplantation embryos encodes for free or surface-bound isoforms is of great importance for understanding the biological process of implantation. Only the free VEGF protein should be able to induce angiogenesis at the implantation site and therefore might be involved in the process of embryonic nidation. Since the commercially available antibodies to human VEGF are not sensitive enough to detect secreted VEGF from preimplantation embryos, we have designed primers to differentiate between the alternatively spliced mRNAs for secreted or surface-bound VEGFs. The aim of this study, therefore, was to detect differential expression of VEGF isoforms in single human preimplantation embryos at the blastocyst stage.
Materials and methods
Patients
Patients from the Stanford University Medical Center (Stanford, CA, USA), undergoing IVF were asked to participate in this study by donating embryos that were unsuitable for freezing or transfer due to delayed development. All patients who chose to participate signed an informed consent form, which had been approved by the Institutional Review Board (Human Subjects in Medical Research Committee at Stanford University).
In-vitro culture
All embryos described in this study were fertilized in vitro by intracytoplasmic sperm injection (ICSI) following stimulation using standard gonadotrophin-releasing hormone (GnRH) agonist/FSH long protocols. Ovulation was triggered when at least two follicles were 17 mm in diameter. Oocytes were retrieved transvaginally under ultrasonographic guidance 35 h after administration of human chorionic gonadotrophin (HCG). All procedures were performed by the same physician. Oocytes were fertilized by ICSI 3–4 h after retrieval and were cultured in groups, under mineral oil in 150 µl droplets of P1 (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% synthetic serum substitute (Irvine Scientific) as described before (Milki et al., 1999) until day 3. The embryos were then transferred to blastocyst medium (Irvine Scientific), supplemented with 10% synthetic serum substitute, and were cultured for an additional 48–72 h.
When the embryos had reached blastocyst stage, embryos that appeared morphologically normal were either transferred into the recipient's uterus or frozen for subsequent transfers. Embryos that appeared delayed in development, i.e. not fully expanded, were unsuitable for transfer. If the patients decided to donate these embryos for research, they were cultured for another 24 h and assessed for their development. Expanded and hatching blastocysts were examined using a modification of previously described methods (Kumazaki et al., 1994; Tsai and Wiltbank, 1996; Huang et al., 1997; Krüssel et al., 1997, 1998b, ,c, 2000b) for their expression of ß-actin- and VEGF isoform mRNAs.
Primers for reverse transcription–polymerase chain reaction (RT–PCR)
Sequences of cDNA clones for the mRNAs that should be detected in single blastomeres (ß-actin; Ponte et al., 1984; and VEGF; Leung et al., 1989) were obtained from the GenBank Database of the National Center for Biotechnology Information (NCBI) of the National Institutes of Health (Internet address: http://www2.ncbi.nlm.nih.gov/cgi-bin/genbank). One set of corresponding outer primer sequences as well as one set of corresponding inner primer sequences for VEGF were constructed with the help of the program OLIGO 5.0 Primer Analysis Software (National Bioscience, Plymouth, MN, USA) and synthesized in the Beckman Center (Stanford University Medical Center, Stanford, CA, USA). The ß-actin primers were obtained from Clontech Laboratories Inc (Palo Alto, CA, USA). To ensure that the product detected resulted from amplification of cDNA rather than contaminating genomic DNA, primers were designed to cross intron/exon boundaries (Krüssel et al., 1998c). The primer-cDNA-sequences, GenBank accession numbers and locations of primers on the cDNAs are listed in Table I. As stated in the introduction, the VEGF isoforms all have common sequences (exons 1–5 and 8) and differ by additions of various exons between exon 5 and exon 8. We have designed a common 3' primer located on exon 8 and two nested 5' primers located on exons 1–5 (Figure 1a
) for the hemi-nested PCR. This enabled us to theoretically detect all five isoforms by simultaneous amplification. Primers were tested with cDNA obtained from luteal phase human endometrium, known to be a source of VEGF mRNA (Figure 1b) and the sequences were confirmed. The expected sizes of the amplified fragments for the various VEGF splice variants are listed in Table II.
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Downstream (3'-end) primers of the outer primer pairs were mixed and diluted in DEPC-treated H2O to a final concentration of 5 µmol/l for each primer. This primer-mix was used for the reverse transcription reaction instead of random primers to enrich for specific cDNA-products. For the first PCR, downstream (3'-end) and upstream (5'-end) primers of the outer primer pair and for the second PCR, downstream (3'-end) and upstream (5'-end) primers of the inner primer pair were mixed and diluted in DEPC-treated H2O to a final concentration of 5 µmol/l for each primer.
Reverse transcription
For each oocyte or embryo, 17.5 µl reverse transcription mastermix was prepared: 4 µl 25 mmol/l MgCl2 solution, 2 µl 10x PCR buffer, 2 µl DEPC-treated distilled H2O, 2 µl dATP, 2 µl dCTP, 2 µl dGTP, 2 µl dTTP (all Perkin-Elmer, Foster City, CA, USA), 1.5 µl outer 3' primer mix. The mix was placed into a 0.5 ml thin-walled PCR tube (Applied Scientific, South San Francisco, CA, USA), covered with 50 µl of light white mineral oil (Sigma, St Louis, MO, USA) and kept on ice until the RNA preparation. The single oocyte or embryo, was added to the reverse transcription mix allowing a carry-over of culture-medium of 1 µl. Samples were immediately heated up to 99°C for 1 min in a DNA Thermal Cycler 480 (Perkin-Elmer) to release the total RNA and denature the proteins. Samples were cooled down to 4°C and 0.5 µl RNase-Inhibitor (Perkin-Elmer) was added followed by 1.0 µl MuLV Reverse Transcriptase (Gibco BRL, Grand Island, NY, USA). The reverse transcription reaction was carried out in the DNA Thermal Cycler 480 (Perkin-Elmer) by using a program with the following parameters: 42°C, 60 min; 99°C, 5 min. After the reaction was complete, samples were cooled down to 4°C and immediately processed for PCR.
Hemi-nested PCR
RT-products were split for the first round of PCR. 2 µl of reverse transcription product were added to 48 µl of PCR-1-mix: 3.4 µl 25 mmol/l MgCl2 Solution, 4.7 µl 10x PCR buffer, 33.25 µl DEPC-treated distilled H2O, 1 µl dATP, 1 µl dCTP, 1 µl dGTP, 1 µl dTTP, 0.25 µl Polymerase GoldTM (all Perkin Elmer), and 2.4 µl outer 3' + 5' primer-mix (5 µmol/l each). After mixing all components in a 0.5 ml thin-walled PCR cup, the reaction-mix was covered with 50 µl light white mineral oil, heated in the DNA Thermal Cycler 480 to 95°C for 9 min to activate the Polymerase GoldTM and the first PCR was performed according to the parameters specified in Table III. After completion of the first PCR, the reaction was terminated at 72°C for 5 min and cooled down to 4°C. First-round PCR-products were stored at –20°C until agarose-gel electrophoresis (ß-actin) or immediately processed for the second PCR (VEGF).
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For the second PCR for VEGF, 5 µl of first round PCR-products were added to 95 µl PCR2-reaction-mix: 7.2 µl 25 mmol/l MgCl2 solution, 9.5 µl 10x PCR buffer, 65.0 µl DEPC-treated distilled H2O, 2.2 µl dATP, 2.2 µl dCTP, 2.2 µl dGTP, 2.2 µl dTTP, 0.5 µl Polymerase GoldTM, and 4 µl inner 3' + 5' primer-mix (5 µmol/l each) in a thin-walled PCR-tube and covered with 50 µl mineral oil. Activation of the Polymerase GoldTM was identical with the first round of PCR. After completing the second round of PCR according to the parameters specified in Table III
, samples were stored at –20°C until agarose-gel electrophoresis was carried out.
Agarose gel electrophoresis
Horizontal 2% agarose-gel electrophoresis was carried out in the presence of ethidium bromide solution (Sigma). After completion of electrophoresis, the agarose-gel was analysed (Figure 2a) on the GelDoc 1000 system (Bio-Rad Laboratories, Hercules, CA, USA). The cDNA-size calculation and densitometry (Figure 2b) was carried out by using the Molecular Analyst Software (Bio-Rad).
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Results
A total of 19 human blastocysts were examined using RT–PCR. All blastocysts expressed ß-actin mRNA and VEGF mRNA could be detected in 17 out of 19 blastocysts (89%). Of the blastocysts that expressed VEGF mRNA, VEGF121 mRNA was detected in 15 out of 17 (88%), VEGF145 mRNA was detected in 17 out of 17 (100%), VEGF165 mRNA was detected in 12 out of 17 (71%), and VEGF189 mRNA was detected in four out of 17 (24%) (Figure 3).
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There appeared to be three different patterns of co-expression for VEGF mRNA splice variants in the blastocysts. In five out of 17 (29%) blastocysts, there was co-expression of mRNA for VEGF121 and VEGF145 only and in two out of 17 (12%) co-expression of mRNA for VEGF165 and VEGF145 only. Ten out of 17 blastocysts (59%) expressed all three mRNA isoforms for the secreted VEGF proteins (VEGF121, VEGF145 and VEGF165) and four of these additionally expressed the mRNA for VEGF189. None of the embryos expressed mRNA for the surface-bound isoform VEGF206.
Since all VEGF isoforms were detected with the same primers, it can be assumed that the PCR characteristics for all splice variants were identical. This means that the different splice variants will compete for the primers and the polymerase during the PCR. Therefore, performing hemi-nested PCR enabled us to determine the relative amount of mRNA for each expressed isoform by densitometry analysis of the PCR products. The results are shown in Figure 4: in all observed patterns of mRNA expression, a lower amount of mRNA for VEGF145 was expressed, compared with mRNA for either VEGF121 and/or VEGF165. In the embryos co-expressing all four isoforms (VEGF121, VEGF145, VEGF165 and VEGF189), both VEGF145 and VEGF189 were expressed at relatively low levels compared with VEGF121 and VEGF165. In general, the isoforms with the highest relative expression levels were VEGF121 and VEGF165.
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Discussion
The present study is the first to demonstrate the expression of alternatively spliced VEGF mRNA isoforms in human preimplantation embryos. Alternative splicing is an important mechanism resulting in the creation of biological diversity in many growth factors and their receptors. All alternatively spliced isoforms generated from the VEGF gene encode proteins that induce angiogenesis and act as active mitogens for endothelial cells. The isoforms, however, differ in their molecular mass and in their ability to bind to cell-surface heparan–sulphate proteoglycans as well as in their binding to different receptors. It was shown (Keyt et al., 1996) that the protein sequence encoded by exons 1–5, which is expressed in all VEGF isoforms, contains information that is necessary for the recognition of VEGF receptors KDR and flt-1. It appears however, that VEGF121 and VEGF165 bind to both receptors, flt-1 and KDR, whereas VEGF145 predominantly binds to the KDR receptor (Neufeld et al., 1999).
The biological activity of VEGF is not only determined by the binding to the various VEGF receptors, but also by the ability to bind to heparan–sulphate proteoglycans on cell surfaces or extracellular matrix (Park et al., 1993). These proteoglycans are important regulators of several heparin binding growth factors (Schlessinger et al., 1995) and binding of VEGF to heparan–sulphate proteoglycans releases other angiogenic factors from the extracellular matrix (ECM), e.g. basic fibroblast growth factor (bFGF) (Jonca et al., 1997).
Since exon 8 is also expressed in all isoforms, the presence or absence of exons 6, 6' and 7 of the VEGF gene must explain the different biological properties of the splice variants. The ability of VEGF splice variants to bind to heparin is determined by exons 6 and 7 of the VEGF gene; both exons independently contain a heparin-binding domain (Park et al., 1993). Therefore, VEGF121 (which does not contain exons 6 or 7) does not bind to heparin or the ECM (Cohen et al., 1995). Insertion of the 24 amino acid peptide encoded by exon 6 or the 44 amino acid peptide encoded by exon 7 distinguishes VEGF145 and VEGF165 from VEGF121. Both VEGF145 and VEGF165 bind to ECM and to heparin and heparan–sulphate proteoglycans, but with a lower affinity than VEGF189 (Park et al., 1993; Cohen et al., 1995; Poltorak et al., 1997) which contains both the peptides encoded by exons 6 and 7. VEGF121, VEGF145 and VEGF165, therefore, are mostly secreted whereas VEGF189 remains bound to the cell surface and ECM, and this may be the explanation for its relatively low bioactivity in vivo (Cheng et al., 1997). Like VEGF189, VEGF206 (whose mRNA could not be detected in the present study) is bound to the cell surface and ECM (Houck et al., 1991).
There are several indications for a possible role of the VEGF system in embryonic implantation and development. VEGF and its receptors have been identified in several reproductive tissues, including corpus luteum, ovarian follicles, endometrial vessels and embryonic implantation sites in mice (Shweiki et al., 1993). They have also been detected in giant trophoblast cells and early yolk sac (Jakeman et al., 1993) and in the human endometrium (Charnock-Jones et al., 1993), placenta (Houck et al., 1991), Fallopian tube and ovary (Gordon et al., 1996). Recently, VEGF mRNA and protein was detected in the human endometrium throughout the menstrual cycle with maximal expression in secretory endometrium during the luteal phase and protein was localized in glandular epithelial cells (Shifren et al., 1996). Our previous studies showed that VEGF transmembraneous receptors KDR and Flt-1 are not regulated during the menstrual cycle in human endometrium, but interestingly, the soluble receptor, sflt, which acts as a competitive antagonist to VEGF was down-regulated in luteal phase human endometrium (Krüssel et al., 1999).
Mice with functional inactivation of one VEGF allele (VEGF+/–) have been generated and post-implantation mouse embryos of this species show several malformations in the vascular system resulting in lethality on days 11 and 12 of pregnancy. This strongly suggests a dose-dependent regulation of fetal vascular development by VEGF (Carmeliet et al., 1996; Ferrara et al., 1996).
VEGF145 was first described (Charnock-Jones et al., 1993) as a splice variant derived from the VEGF gene. In contrast to VEGF121, VEGF165 and VEGF189, which could be detected in most tissues and cells (Neufeld et al., 1999), VEGF145 expression seems to be restricted to reproductive tissue. It was shown, that this specific isoform is predominantly expressed in human endometrium (Charnock-Jones et al., 1993) and in tumour cell lines derived from female reproductive organs (Poltorak et al., 1997), as well as in ovine placenta and fetal membranes (Cheung et al., 1995). The fact that we were able to detect the mRNA for VEGF145 in human preimplantation embryos at the blastocyst stage again supports a possible role of this particular isoform in reproductive events.
In summary, we have demonstrated that the simultaneous detection of VEGF isoform mRNAs in human blastocysts is technically possible. Furthermore we showed that the VEGF mRNA isoforms that encode for the mostly secreted proteins are expressed in 100% of the human blastocysts examined in this study. In the context of our previous experiments (Krüssel et al., 1999, 2000b), and assuming that these mRNAs are at least partly translated into protein, these findings again support the hypothesis that VEGF plays an important role in human embryonic implantation.
Acknowledgments
The authors wish to thank and to acknowledge the contributions of Monika Branch, Janice Gebhardt and Douglas Moore from the Stanford University IVF Laboratory. We also wish to thank Linda C.Giudice, head of the REI-section at Stanford OB/GYN, for her friendly and supportive co-operation.
Notes
3 To whom correspondence should be addressed. E-mail: kruessel{at}uni-duesseldorf.de 
* Presented in part at the 16th annual meeting of ESHRE, Bologna, Italy, June 25–28, 2000.
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Submitted on July 20, 2000; accepted on October 11, 2000.
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 M. Stimpfl, D. Tong, B. Fasching, E. Schuster, A. Obermair, S. Leodolter, and R. Zeillinger Vascular Endothelial Growth Factor Splice Variants and Their Prognostic Value in Breast and Ovarian Cancer Clin. Cancer Res., July 1, 2002; 8(7): 2253 - 2259. [Abstract] [Full Text] [PDF]
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H. Wang, Y. Wen, S. Mooney, B. Behr, and M. L. Polan Phospholipase A2 and Cyclooxygenase Gene Expression in Human Preimplantation Embryos J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2629 - 2634. [Abstract] [Full Text] [PDF]
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 R.M. Sibug, F.M. Helmerhorst, A.M.I. Tijssen, E.R. de Kloet, and J. de Koning Gonadotrophin stimulation reduces VEGF120 expression in the mouse uterus during the peri-implantation period Hum. Reprod., June 1, 2002; 17(6): 1643 - 1648. [Abstract] [Full Text] [PDF]
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 D.J. Bloor, A.D. Metcalfe, A. Rutherford, D.R. Brison, and S.J. Kimber Expression of cell adhesion molecules during human preimplantation embryo development Mol. Hum. Reprod., March 1, 2002; 8(3): 237 - 245. [Abstract] [Full Text] [PDF]
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