Masses Preimplantation Embryos and Inner Cell Spliced Transcripts Of

Differential Expression of Alternatively Spliced Transcripts of HLA-G in Human Preimplantation Embryos and Inner Cell Masses This information is current as of September 23, 2021. Yuan Q. Yao, David H. Barlow and Ian L. Sargent J Immunol 2005; 175:8379-8385; ; doi: 10.4049/jimmunol.175.12.8379 http://www.jimmunol.org/content/175/12/8379 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Expression of Alternatively Spliced Transcripts of HLA-G in Human Preimplantation Embryos and Inner Cell Masses

Yuan Q. Yao, David H. Barlow, and Ian L. Sargent1,2

It has been reported that preimplantation human embryos secrete HLA-G, and the levels may be predictive of their ability to implant. However, it is not known which of the membrane-bound (HLA-G 1–4) and soluble (HLA-G 5–6) alternatively spliced forms are present, nor the developmental stage at which they appear. Therefore, we have investigated HLA-G mRNA isoform expression on single embryos at the two-, four-, six-, and eight-cell, morula, and blastocyst stages. The percentage of embryos expressing each HLA-G isoform mRNA increased with developmental stage, but contrary to expectation, HLA-G5 mRNA was not detected in single two- to eight-cell embryos and was only expressed by 20% of morulae and blastocysts. Similarly, soluble Downloaded from HLA-G6 mRNA was not detected until the blastocyst stage and then in only one-third of embryos. In contrast, labeling with MEM G/9 Ab (speciﬁc for HLA-G1 and -G5) was observed in 15 of 20 two- to eight-cell embryos and 5 of 5 blastocysts. This disparity between mRNA and protein may be due to HLA-G protein remaining from maternal oocyte stores produced before embryonic genome activation and brings into question the measurement of soluble HLA-G for clinical evaluation of embryo quality. Although HLA-G is expressed in the preimplantation embryo, later it is primarily expressed in the invasive trophoblast of the placenta

rather than the fetus. Therefore, we have investigated whether down-regulation of HLA-G ﬁrst occurs in the inner cell mass http://www.jimmunol.org/ (precursor fetal cells) of the blastocyst and, in support of this concept, have shown the absence HLA-G1 and -G5 protein and mRNA. The Journal of Immunology, 2005, 175: 8379–8385.

uccessful implantation in the human is dependent on the the ␣1 domain; and HLA-G4 lacks exon 4 and hence the ␣3 do- early embryo avoiding immune recognition and destruc- main. HLA-G5 and -G6 retain intron 4, which contains a stop S tion by the maternal immune system. One of the key pro- codon that prevents the transcription of the transmembrane region, tective mechanisms is thought to be the selective expression of the resulting in the expression of the soluble proteins. A further splice nonclassical HLA class I gene HLA-G by the trophoblast in the variant of HLA-G (HLA-G7) has also been reported (2). This iso- absence of classical class I (HLA-A and HLA-B) Ags. In contrast form contains intron 2, which has a stop codon, so that the result- by guest on September 23, 2021 to the classical class I Ags, HLA-G is virtually nonpolymorphic ing G7 protein would be soluble HLA-G comprised of only the ␣1 with a small number of HLA-G alleles encoding only three dif- region. However, while the authors were able to demonstrate the ferent amino acid primary sequences (1). In addition, HLA-G presence of G7 proteins in extracts of transfected cells by Western mRNA can be alternatively spliced into at least six transcripts, blot analysis and immunoprecipitation, it could not be detected as which encode four membrane bound isoforms (G1, G2, G3, and a secreted protein and is therefore not considered further here. G4) and two soluble isoforms (G5 and G6, otherwise known as Although the full range of functions of HLA-G remain to be elu- soluble HLA-G1 and soluble HLA-G2) (1). HLA-G1 is the full- cidated, both full-length membrane-bound (HLA-G1) and soluble length isoform containing eight exons and seven introns. Exons 2, (HLA-G5) forms have been shown to have immunoregulatory 3, and 4 encode the ␣1, ␣2, and ␣3 domains, respectively. Exon 5 functions, including the inhibition of T cell activation and stimu- encodes the transmembrane region, and exon 6 encodes the intra- lating decidual NK cells and macrophages to produce cytokines cellular region. The other HLA-G isoforms are alternatively that are beneficial to implantation (3). spliced, shorter transcripts lacking regions complementary to one HLA-G mRNA and protein have previously been shown to be or more entire exons. Thus, HLA-G2 lacks exon 3, corresponding expressed by RT-PCR and immunofluorescence staining in a pro- to the ␣2 domain; HLA-G3 lacks exon 3 and 4 and thus only has portion of cleavage stage (two- to eight-cell, day 2/3) human embryos and blastocysts (day 5/6) created by in vitro fertilization (IVF)2 (4, 5). The primers used for the RT-PCR in these original Oxford Fertility Unit, Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom studies amplified all isoforms of HLA-G, so it was not known which specific isoforms (membrane bound or soluble) were Received for publication December 17, 2004. Accepted for publication September 30, 2005. present. Interestingly, it was noted that sibling embryos from pa- The costs of publication of this article were defrayed in part by the payment of page tients that became pregnant were more likely to express HLA-G charges. This article must therefore be hereby marked advertisement in accordance than embryos from patients that did not conceive as a result of their with 18 U.S.C. Section 1734 solely to indicate this fact. IVF cycles (5), suggesting that HLA-G expression enhances the 1 This work was supported by the Oxford Fertility Unit. Prof. Sargent is a member ability of embryos to implant. This is supported by several recent of Embryo Implantation Control, a European Network of Excellence (www. embic.org) within the Framework Programme of the European Union (LSKM-CT- reports showing that a proportion of cleavage stage IVF embryos 2004-512040). secrete the soluble form of HLA-G into the culture medium. It was 2 Address correspondence and reprint requests to Prof. Ian L. Sargent, Oxford Fer- tility Unit, Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, U.K. E-mail address: ian.sargent@obs- 3 Abbreviations used in this paper: IVF, in vitro fertilization; ICM, inner cell mass; ␤ ␤ gyn.ox.ac.uk 2m, 2-microglobulin; hCG, human chorionic gonadotrophin.

found that, in general, embryos that produced soluble HLA-G were grade A–C embryos and grade 4 blastocysts were used for mRNA isolation much more likely to produce pregnancies when transferred back or immunofluorescence studies. into the uterus than those that did not (6–10). This is an important JEG-3 cell culture new finding, which, if confirmed, has significant implications for the selection of the best embryos for transfer in IVF. Currently, The HLA-G expressing choriocarcinoma cell line, JEG-3 (American Type embryos are selected for transfer at the cleavage stage (day 2/3) Culture Collection), was used as a positive control for these studies. JEG-3 principally on the basis of morphology, which is not always reli- cells were grown in DMEM:Ham’s F-12 (Sigma-Aldrich) supplemented with 10% FCS, glutamine, and antibiotics. able. The availability of a noninvasive, quantitative assay for a marker of implantation potential could revolutionize IVF practice Messenger RNA isolation by increasing success rates and lowering the risk of multiple preg- The zona pellucida was removed by briefly exposing the embryo to Ty- nancy and by reducing the number of embryos transferred. How- rode’s acid solution (Sigma-Aldrich). Individual human embryos were ever, to be clinically useful, a better understanding of which lysed in 300 ␮l of lysis/binding buffer (Dynabeads mRNA DIRECT kit; HLA-G isoforms are expressed at each stage of development and Dynal Biotech) and stored at Ϫ70°C until mRNA isolation. Embryo in what proportion of embryos they are found is required, and this mRNA was isolated using the Dynabeads mRNA DIRECT kit following ␮ was one aim of the current study. the manufacturer’s instructions (13). The mRNA was taken up in 20 lof diethylpyrocarbonate-treated water. HLA-G appears to be expressed throughout the early embryo developmental stages (cleavage stages, morula, and blastocyst), Nested RT-PCR for HLA-G isoforms but later on in development, its expression is predominantly con- Eight sets of specific primers (Table I) for HLA-G mRNA isoform-nested Downloaded from fined to the invasive extravillous cytotrophoblast of the placenta. PCR were designed using software Primer3 (14) based on published se- Thus, at some point, HLA-G expression must be switched off in quences retrieved from GenBank (accession no. NM_002127). In the first the cells that are destined to form the fetus and the villous core of round of the nested PCR, the forward primer was located in exon 1 and the the placenta. The most likely time for this to happen is at the reverse primer was across exons 5 and 6 so that all HLA-G mRNA iso- blastocyst stage when differentiation into the trophectoderm forms could be amplified. In the second round of PCR, primers were designed to span the link between two neighboring exons to individually (which goes on to form the trophoblast) and the inner cell mass distinguish each HLA-G mRNA isoform, namely G1, G2, G3, G4, G5, and

(ICM) (which forms the fetus) first occurs. Therefore, we have G6. An additional set of primers was designed to amplify all isoforms of http://www.jimmunol.org/ investigated the expression of HLA-G isoforms in isolated ICM to HLA-G together (designated pan HLA-G). All primers were synthesized determine whether its down-regulation in fetal tissues begins at commercially by Invitrogen Life Technologies. Both cDNA synthesis and the first round of PCR were performed in a single tube using SuperScript this stage. One-Step RT-PCR with the Platinum Taq kit (Invitrogen Life Technolo- gies) and conducted using a PerkinElmer DNA thermal cycler 480. mRNA Materials and Methods was reverse transcribed and amplified in a 50-␮l volume containing 2ϫ Human embryo collection and culture reaction mix, 0.4 ␮M forward and reverse primer, and RT/Platinum Taq Mix. The conditions for first-strand cDNA synthesis and predenaturation The human embryos in this study were donated with informed consent by were 1 cycle of 45°C for 45 min and 94°C for 2 min. PCR amplification couples attending the Oxford Fertility Unit, John Radcliffe Hospital. Ethics was conducted for 40 cycles using the following conditions: denaturation approval for the study was obtained from the Central Oxford Research for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 2 min at by guest on September 23, 2021 Ethics Committee and the Human Fertilization and Embryology Authority. 68°C. Amplification was completed by a final extension at 68°C for 5 min. Ovarian stimulation was achieved by a combination of pituitary desensi- The second round of the nested PCR was performed using the Platinum tization and gonadotropin stimulation protocol. Oocyte retrieval was per- PCR SuperMix High Fidelity kit (Invitrogen Life Technologies). Two mi- formed 35 h following human chorionic gonadotropin (hCG). Embryos croliters of the first-round PCR products were added to the second PCR were initially cultured singly in 1 ml of universal IVF medium (MediCult) Mastermix to a total volume of 50 ␮l containing Platinum PCR SuperMix ␮ in a humidified atmosphere of 5% CO2 in air at 37°C. On day 2 following HF and 0.4 M forward and reverse primers. PCR was conducted for one insemination, embryos surplus to treatment and freezing, which had been cycle of 3 min at 94°C and then 40 cycles of 30 s at 94°C, 30 s at 58°C, donated for research, was transferred to culture medium 2 of the Blastassist and 1 min at 68°C. All PCR products were separated on 1.5% agarose gels System (MediCult) for an additional 4 days, with the medium being and visualized with ethidium bromide. As a control, ␤-actin was amplified changed on day 4 (11). Embryos were cultured singly throughout to retain in parallel in all embryos using nested RT-PCR. The corresponding primer their identity for morphological assessment and were harvested at the two-, pairs are shown in Table I. four-, six-, eight-cell, morula, and blastocyst stages. Day 6 blastocysts were The JEG-3 choriocarcinoma cell line was used to validate the HLA-G graded according to the criteria of Gardner and Schoolcraft (12). Only nested RT-PCR. Amplicons of the nested PCR from JEG-3 cells were

Table I. Nested RT-PCR primers for ampliﬁcation of HLA-G mRNA isoforms

Target Gene Primer Sequence (5Ј-3Ј) Position in Sequence Product Size (bp)

First PCR primer AAC CCT CTT CCT GCT GCT CT Exon 1 1004 CTC CTT TTC AAT CTG AGC TCT TCT Exon 5–6 HLA-G1 GAG CGA GGC CAG TTC TCA Exon 2–3 576 AGG GAA GAC TGC TTC CAT CTC Exon 4–5 HLA-G2 ACC AGA GCG AGG CCA ACC Exon 2–4 304 AGG GAA GAC TGC TTC CAT CTC Exon 4–5 HLA-G3 GCT CCC ACT CCA TGA GGT ATT Exon 2 276 ACT GCT TGG CCT CGC TCT Exon 2–5 HLA-G4 GGC CAG TTC TCA CAC CCT CCA Exon 2–3 290 AAG ACT GCT CCG CGC GCT Exon 3–5 HAL-G5 ATA CCT GGA GAA CGG GAA GG Exon 3 363 AGG CTC CTG CTT TCC CTA AC Intron 4 HLA-G6 ACC AGA GCG AGG CCA ACC Exon 2–4 339 GGC TCC TGC TTT CCC TAA CAG Intron 4 Pan HLA-G CTG ACC CTG ACC GAG ACC T Exon 1 291 CTC GCT CTG GTT GTA GTA GCC Exon 2 The Journal of Immunology 8381 sequenced for confirmation using the BigDye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems). Determination of RT-PCR sensitivity The sensitivity of the nested RT-PCR was examined in two ways. First, total RNA was isolated from JEG-3 cells using the RNeasy Mini kit (Qia- gen) according to the manufacturer’s instructions. Serial dilutions of 106, 105,104,103,102,101, and 1 pg of total RNA were subjected to HLA-G mRNA isoform nested RT-PCR, as above. Second, a suspension of 105 JEG cells was serially diluted to 104,103, and 102, and micromanipulation was used to obtain suspensions containing 10 cells and 1 cell. The cells were transferred to lysing/binding buffer (Dynabeads mRNA DIRECT kit; Dynal Biotech), and messenger RNA was isolated using Dynabeads mRNA DIRECT kit (Dynal Biotech) and subjected to HLA-G mRNA isoform-nested RT-PCR. Immunosurgery ICM were isolated from human blastocysts using a standard immunosurgery technique (15). The blastocysts were briefly exposed to 0.5% protease (Sigma-Aldrich) to remove the zona pellucida. The zona-free blastocyst was then incubated with 50% broad-specificity rabbit anti-human serum (Sigma-Aldrich) for 30 min, which binds to Ags on the trophectoderm. The Downloaded from addition of 10% guinea pig complement (Sigma-Aldrich) for an additional 30 min causes Ab-dependent complement-mediated lysis of the trophectoderm. Lysed trophectoderm cells were removed by sucking the embryo through a small-bore glass pipette. The intact isolated ICM were either stained by immunofluorescence or lysed in 300 ␮l of lysis/binding buffer and stored at Ϫ70°C until mRNA isolation as above. http://www.jimmunol.org/ Immunofluorescence staining of human preimplantation embryos FIGURE 2. Determination of RT-PCR sensitivity. A, HLA-G mRNA Human embryos with intact zona pellucidae or ICM were fixed in 3% isoforms from different concentrations of total RNA from JEG-3 cells. B, paraformaldehyde and permeabilized with 0.1% Triton X-100. They were HLA-G mRNA isoforms from different numbers of JEG-3 cells. then incubated with the 25 ␮g/ml primary anti-HLA-G mAb MEM-G/9 (Serotec) overnight at 4°C, followed by a 1/75 dilution of the secondary donkey anti-mouse IgG Ab conjugated to FITC (The Jackson Laboratory) for 45 min at room temperature. Control embryos were stained using mouse IgG (DakoCytomation) in place of the MEM-G/9 Ab. JEG-3 cells Expression of HLA-G mRNA isoforms in human preimplantation were used as positive controls. The embryos and ICM were mounted in embryos Ј Ј

Vectashield (Vector Laboratories) mounting medium containing 4 ,6 -dia- by guest on September 23, 2021 midino-2-phenylindole to counterstain the nuclei, and images were cap- A total of 20 individual cleavage stage human embryos was ex- tured using a Leitz DMRBE microscope (Leica Microsystems) and Open- amined for HLA-G mRNA isoform expression by nested RT-PCR lab imaging software (Improvision). (five two-cell, five four-cell, five six-cell, and five eight-cell). Fig. 3A shows a typical HLA-G mRNA isoform RT-PCR from an Results eight-cell embryo, and Fig. 3B shows representative results of em- Expression of HLA-G mRNA isoforms in JEG-3 cells bryos from each cleavage stage. Only 7 of 20 embryos expressed The JEG-3 cell line was shown to express all HLA-G alternatively HLA-G as determined by the pan-HLA-G primers (summarized in spliced mRNA variants (Fig. 1) with the PCR products corre- Fig. 5). However, not all isoforms were expressed by the one em- sponding to the predicted sizes shown in Table I. Complementary bryo. One two cell embryo expressed G3 and G4, one four-cell DNA sequencing confirmed that the nested RT-PCR amplicons embryo expressed G1, G3, and G4, and one six-cell expressed G3 were identical to the regions spanned by the appropriate forward and G4 while another expressed G1, G3, and G4. Three eight-cell and reverse primers (data not shown). embryos were positive, with two expressing G1, one expressing G2, three expressing G3, and two expressing G4. No soluble G5 Determination of RT-PCR sensitivity and G6 were detected in any of these embryos. Using JEG-3 cells, all HLA-G isoforms could be detected at RNA In addition, we made two pools of two- to four-cell human em- concentrations as low as 10 pg, and HLA-G3 and -G4 were still bryos (each containing mRNA from 10 embryos). G4 was found in detectable in 1 pg of total RNA (Fig. 2). All HLA-G mRNA iso- one pool and G5 in the other (data not shown). Two similar mRNA forms were amplified successfully from single JEG-3 cells. pools of 10 six- to eight-cell embryos were made and HLA-G1, -G3, and -G4 expression was found in both (data not shown). Five individual day 4 human morulas were next examined. All of them expressed HLA-G as detected by the pan-G primers. G1 was detected in three morulas, G2 in one, G3 in all five, and G4 in four. Soluble HLA-G5 was found in one morula. Fig. 3B shows two typical results. Finally, 25 individual day 6-expanded blastocysts (grade 4) were studied. All of them were found to express HLA-G mRNA (Fig. 4, A and B), but the isoforms were differentially expressed. G1 was detected in 20 of 25 (80.0%), G2 in 4 of FIGURE 1. HLA-G mRNA isoforms in the JEG-3 cell line. The lanes 25 (16.0%), G3 in 25 of 25 (100%), G4 in 24 of 25 (96.0%), G5 are marked as follows: M, 1-kb DNA marker; G1, HLA-G1; G2, HLA-G2; in 5 of 25 (20%), and G6 in 8 of 25 (32%). G3, HLA-G3; G4, HLA-G4; G5, HLA-G5; G6, HLA-G6; PG, Pan Overall, there was an increase in HLA-G mRNA expression HLA-G; and -ve, negative control. with developmental stage with the most abundant isoforms being 8382 HLA-G mRNA ISOFORM EXPRESSION IN HUMAN EMBRYOS Downloaded from

FIGURE 4. HLA-G mRNA isoforms in human blastocysts. A, HLA-G mRNA isoform expression in a typical day 6 human blastocyst. The lanes are marked as follows: M, 1-kb DNA marker; G1, HLA-G1; G2, HLA-G2; G3, HLA-G3; G4, HLA-G4; G5, HLA-G5; G6, HLA-G6; PG, Pan

FIGURE 3. http://www.jimmunol.org/ HLA-G mRNA isoforms in human embryos from 2-cell to HLA-G; and -ve, negative control. B, HLA-G mRNA isoforms from 10 A morula stage. , HLA-G mRNA isoform expression in a typical human representative blastocysts. 8-cell embryo. The lanes are marked as follows: M, 1-kb DNA marker; G1, HLA-G1; G2, HLA-G2; G3, HLA-G3; G4, HLA-G4; G5, HLA-G5; G6, HLA-G6; PG, Pan HLA-G; and -ve, negative control. B, HLA-G mRNA implantation embryos. Studying single embryos, these were found isoforms from representative 2-cell (2c), 4-cell (4c), 6-cell (6c), and 8-cell to be differentially expressed with the predominant forms being (8c) embryos and morulas (Mo). HLA-G3 and -G4 throughout. The full-length membrane bound (G1) and soluble forms (G5) and the truncated G2 and G6 were more varied in their expression, with G1 mRNA being present in the truncated G3 and G4 forms (Fig. 5). The full-length G1 iso- 80% of blastocysts, while the soluble G5 was only present in 20% by guest on September 23, 2021 form was expressed in the majority of embryos by the blastocyst and soluble G6 in 32%. stage, but the full-length soluble form G5 was not expressed until Jurisicova et al. (4, 5) reported the expression of HLA-G mRNA the morula stage and was poorly expressed even at the blastocyst in 43.3% of 148 of human blastocysts tested, which was much stage. lower than in our study. They also reported that HLA-G mRNA was found in all preblastocyst development stages, including 2- to Detection of HLA-G protein in human preimplantation embryos 4-, 5- to 8-, and 9- to 16-cell embryos and morulas. Surprisingly, Twenty two- to eight-cell human embryos were stained with no data on the proportions of embryos expressing HLA-G in each MEM-G/9, a mAb specific for HLA-G1 and G5 (Fig. 6). HLA-G group was given, so comparison with our study is not possible. In labeling was found in 15 of 20 two- to eight-cell human embryos. another report, Hiby et al. (16) found no HLA-G mRNA in 11 Two of two two-cell embryos, five of eight four-cell embryos, two preimplantation embryos ranging from the 2 cell to the blastocyst of three six-cell embryos, and six of seven eight-cell embryos were stage using nested primers for full-length HLA-G. Their method- HLA-G positive. HLA-G-positive labeling was observed in all ology differed from ours in that they isolated RNA from zona three morulas and five blastocysts examined (Fig. 6). No staining intact embryos rather than removing the zona first and used stan- was found when nonspecific mouse IgG was used as a control for dard phenol-chloroform extraction instead of the magnetic bead the primary Ab (Fig. 6). method, both of which may affect the yields of RNA. The primers used in both the studies of Jurisicova and Hiby would have am- Detection of HLA-G mRNA isoforms and protein in ICM plified all the different isoforms, giving the total HLA-G mRNA Three ICM were isolated from day 6 human blastocysts and sub- expression, but the hemi-nested RT-PCR system used by Jurisi- jected to mRNA extraction and nested RT-PCR for HLA-G cova et al. (4, 5) was probably less sensitive than our specific mRNA isoforms. The only isoforms found were HLA-G3 and -G4, nested RT-PCR. Hiby et al. (16) also used nested RT-PCR with the which were present in all three ICM. Fig. 7 shows a typical nested outside forward primer located at exon 3 and the reverse primer RT-PCR from one ICM. No immunofluorescence staining of the located at 3Ј untranslated region. The inside forward primer was ICM with the MEM-G/9 was seen (n ϭ 2) (Fig. 6). located at exon 5, and the reverse primer was located at 3Ј untranslated region. This primer set cannot amplify HLA-G2 and -G3 Discussion because G2 lacks exon 3 and G3 lacks exon 3 and 4. Another This study has confirmed that human preimplantation embryos ex- possible reason is the differences in the quality of the embryos used press HLA-G mRNA and has shown that the proportion of em- as only better grade embryos were included in our study. bryos expressing it increases with developmental stage. To our Using immunofluorescence staining with the HLA-G-specific knowledge, this is the first report investigating the expression pro- mAb MEM-G/9, we found that all blastocysts and 75% (15 of 20) files of alternatively spliced transcripts of HLA-G in human pre- of the preblastocyst human embryos examined expressed HLA-G The Journal of Immunology 8383

in the 2 cell stage (18). The store of proteins in the fertilized oocyte is sufficient to support embryo development to the 8 cell stage (21). Embryonic gene activation occurs at the 4-8 cell stage in human embryos. Consistent with this, our results show that the expression rate of HLA-G mRNA increased from the six- to eight-cell stage onward. Thus, before the four- to eight-cell stage, HLA-G protein may come from the oocyte stores while during and after the four- to eight-cell stage, new HLA-G transcription and translation occurs with embryonic genome activation. There is however conflicting evidence concerning HLA-G protein expression by oocytes. Dohr et al. (22) found no staining with the pan class I Ab W6/32 used immunofluorescence on three unfixed oocytes which FIGURE 5. Summary of HLA-G mRNA isoform expression in human preimplantation embryos. 1, HLA-G1; 2, HLA-G2; 3, HLA-G3; 4, HLA- had either failed to fertilize or had fertilized abnormally, while G4; 5, HLA-G5; 6, HLA-G6; and P, Pan HLA-G. Roberts et al. (20) using the same Ab on acetone fixed oocytes found 2 of 11 to be positive. Jurisicova et al. (4) used both W6/32 and the anti-HLA-G monoclonal 1B8 (raised against the ␣1 re- protein. This was much higher than HLA-G mRNA expression for gion) on paraformaldehyde-fixed oocytes and found positive stain- the preblastocyst embryos (7 of 20, 35.0%). The Ab MEM-G/9 ing on 6 of 13 and 15 of 20 oocytes, respectively. Their findings specifically recognizes full-length HLA-G1 and soluble HLA-G5 were supported by parallel studies that showed the presence of Downloaded from ␤ ␤ in association with 2-microglobulin ( 2m) and does not react HLA-G mRNA in 17 of 21 pools of 5-8 unfertilized oocytes (4). with HLA-G2, -G3, or -G4 (17). The relative absence of G1 and Unfortunately, it is not currently possible to measure HLA-G G5 mRNA, compared with protein in early embryos is unlikely to mRNA and membrane-bound protein in the same embryos, which be due to a lack of sensitivity of the amplification because our would help to clarify these issues, but further studies measuring dilution experiments showed that it is possible to amplify all mRNA and secreted soluble HLA-G5 are in progress in our HLA-G isoforms from a single JEG-3 cell and from as little as 10 laboratory. http://www.jimmunol.org/ pg of total RNA. Although there are no data for humans, the av- These findings have implications for studies of soluble HLA-G erage total RNA in mouse 2-, 4-, 8- to 16-, and 32-cell embryos is secretion as a marker of human embryo developmental potential. 350, 240, 690, and 1470 pg, respectively (18), and in addition, the One study (6) has reported that soluble HLA-G was detectable in volume of the human embryo is four times larger than that of the day 2–3 culture supernatants of preimplantation (6–10 cells) hu- mouse. HLA-G5 mRNA was found in two- to four-cell embryos man embryos. Supernatants from 285 embryo cultures from 101 when pools of 10 embryos each were used (thereby increasing the IVF treatment cycles were examined. As some embryos were cul- cell numbers to between 20 and 80), but only in one of the four tured in groups, it was not possible to study individual embryos, pools studied. Although, it is not possible to know how many

and therefore, the results were divided into patients who had em- by guest on September 23, 2021 embryos in each pool were producing HLA-G5 mRNA, this is bryos that secreted soluble HLA-G (n ϭ 75) and those that did not consistent with a low frequency of expression in these early de- (n ϭ 26). No pregnancies were obtained in the patients whose velopmental stages. embryos did not secrete HLA-G, but 18 of 75 of the patients whose There is conflicting evidence from other studies of class I MHC protein expression on human embryos. Desoye et al. (19) were embryos produced soluble HLA-G became pregnant, suggesting unable to detect staining with the pan class I Ab W6/32 (which that soluble HLA-G secretion is a prerequisite for successful im- recognizes both HLA-G1 and -G5) staining on three unfixed plantation. In more recent studies, only embryos that had been polyploid embryos at the two-, five-, and eight-cell stages. Using cultured singly were examined, making the data easier to interpret the same Ab, Roberts et al. (20) found no staining on three blas- (7, 8). In one study, 72 of 101 (71%) of women under 39 years old tocysts. In contrast, Jurisicova et al. (4) used both W6/32 and the who had at least one HLA-G secreting embryo transferred anti-HLA-G monoclonal 1B8 (raised against the ␣1 region) and achieved a pregnancy, compared with 13 of 58 (22%) in the sol- found W6/32 to stain all preblastocyst development stages, includ- uble HLA-G-negative group (7). In the other, no pregnancies were ing 2- to 4-, 5- to 8-, and 9- to 16-cell embryos, morulas, and two obtained in 26 of 66 women whose transferred embryos did not of five blastocysts. Similarly, the 1B8 Ab stained 2-, 4-, and 16- secrete soluble HLA-G, while nine pregnancies occurred in the 40 cell embryos and seven of nine blastocysts. Possible reasons for of 66 women whose embryos did secrete soluble HLA-G (8). It has these discrepancies are differences in methodology and the quality also been reported that soluble HLA-G secretion was independent of the embryos used. Previous studies have used unfixed (19), of embryo grade, but the cleavage rate of embryos secreting sol- acetone fixed (20), and paraformaldehyde fixed embryos (4) with uble HLA-G was significantly higher than that of those lacking it, primary Ab incubation times between 30 min and 1 h. In our study, as was the live birth rate (9). A prospective study, in which at least we used the HLA-G-specific mAb MEMG/9 on fixed and perme- one embryo known to be producing soluble HLA-G was trans- abilized embryos, which will reveal both cytoplasmic and surface ferred to the mother, has also shown significantly improved im- expression, together with an overnight incubation with the Ab to plantation and pregnancy rates (10). increase the sensitivity of the technique. Furthermore, all of the The majority of these studies used an ELISA based on the Ab embryos studied were good-quality diploid embryos (grade A–C MEM-G/9 and W6/32 (which recognizes all class I H chains in ␤ embryos and grade 4 blastocysts), which was not the case in all the association with 2m) and hence would only specifically measure previous studies (19). HLA-G5 secretion or membrane-bound HLA-G1 cleaved from the A possible explanation for the variance between HLA-G mRNA cell surface. However, two other reports using different ELISA and protein expression in the early embryos is that the HLA-G systems do not support these findings. Van Lierop et al. (23) used protein at this stage may be of maternal origin. It is known that the mAb G233 (raised against mouse L-cells transfected with both ␤ fertilized oocyte transcription is silenced in the early stages of human 2m and HLA-G) as a capture Ab and Ab 56B (raised embryo development and ϳ90% oocyte maternal mRNAs degrade against the ␣2 domain of HLA-G) as the reporter. Although they 8384 HLA-G mRNA ISOFORM EXPRESSION IN HUMAN EMBRYOS

FIGURE 6. Immunofluorescence labeling for HLA-G protein in different developmental stages of human preimplantation embryos and ICM. Embryos and ICM were stained using MEM-G/9 (an HLA-G-specific mAb) and donkey anti-mouse IgG conjugated with FITC. Negative control embryos were stained with mouse IgG and donkey anti-mouse IgG conjugated with FITC. The nuclei were labeled with 4Ј,6Ј-diamidino-2-phenylindole. could detect recombinant HLA-G and native HLA-G in tropho- truncated forms may be expressed inside the cell, there is no ev- blast culture supernatants and amniotic fluid, they were unable to idence for them reaching the cell surface. Because no mAbs spe- detect soluble HLA-G in follicular fluid or embryo culture super- cific for these isoforms are currently available, it is not possible to Downloaded from natants from eight-cell, blastocyst, and late blastocyst stages. More determine whether they are expressed on the surface of human recently, Noriko et al. (24) have used an ELISA based on the Abs embryos and have a functional role. 87G and MEMG/9 and were similarly unable to detect soluble As mentioned above, only soluble HLA-G5 secretion has been HLA-G in 106 culture supernatants from day 3 embryos and blas- measured in human embryos. However, the soluble HLA-G6 iso- tocysts. The combinations of Abs used in both studies should de- form has been reported to be secreted into the circulation of tect both HLA-G1 and soluble HLA-G5. women throughout pregnancy and to be produced exclusively by http://www.jimmunol.org/ Our results are more consistent with the latter findings as they extravillous cytotrophoblast cells (30). In the current study, show that the majority of cleavage stage embryos do not express HLA-G6 mRNA was not detected until the blastocyst stage, and HLA-G5 mRNA. It is only at the morula and expanded blastocyst therefore, it will be of great interest to determine whether HLA-G6 stages that it is detectable and, even then, in no more than 20% of protein is secreted by human embryos and at what stage. the embryos. These results also bring into question the specificity In later gestation, HLA-G is expressed predominantly by extra- of the mAbs used, particularly MEMG/9, which we found to stain villous cytotrophoblast cells but not in general by fetal cells, which almost all embryos despite the absence of HLA-G1 and -G5 tran- express classical HLA-A and B class I Ags. It is not known when scripts. Although the numbers studied are low due to the small this differential expression of HLA first occurs, but the most likely by guest on September 23, 2021 numbers of human embryos available for research, as mentioned time is at blastocyst formation when fetal cells first develop as the above, it is possible that HLA-G in 2-8 stage embryos is not from ICM, while trophoblast cells develop from the trophectoderm. The de novo transcription and translation but from stores in the oo- immunofluorescence staining shown here and in other studies (4, cytes, which are expressed in the early stages of embryo develop- 5) shows the trophectoderm to be HLA-G positive. However, it is ment. Thus, HLA-G expressed at this stage may be more a marker difficult to distinguish whether the ICM expresses HLA-G using of oocyte rather than embryo quality. this method. Therefore, we isolated the ICM from blastocysts us- A consistent finding in the current study was that at all stages of ing immunosurgery and analyzed HLA-G expression. We found embryo development G3 and G4 were the predominant mRNA that the inner cell mass still contained HLA-G mRNA, but only the isoforms expressed. Ulbrecht et al. (25) report a similar finding in G3 and G4 isoforms were detectable. This was consistent with transfectants of the six different isoforms, with the amount of tran- finding that the ICM did not stain with MEM-G/9, which only scripts of G2, G3, G4, G5, and G6 exceeding by far that of G1. recognizes G1 and G5. These results support the concept of a There is considerable debate about the function of the truncated down-regulation of HLA-G in the ICM as differentiation occurs, isoforms and in particular whether they are expressed on the cell with perhaps the more abundant HLA-G3 and -G4 transcripts be- surface. Riteau et al. (26) have reported that HLA-G2, -G3, and ing the slowest to degrade. -G4 can be expressed on the surface of transfected cells, thereby If HLA-G was not down-regulated initially in the inner cell protecting these cells from lysis by both NK and CTL effector mass, then all the cells of the developing fetus would be HLA-G cells, and in this way may contribute to fetal survival (27) How- positive, which is not the case. However, it has been shown re- ever, we (28) and others (25, 29) have found that although these cently that fetal erythroid progenitor cells and endothelial cells label with a mAb5A6G7 (raised against a peptide sequence specific for HLA-G5 and -G6) from as early as day 32 of pregnancy (31). Therefore, it must be assumed that the HLA-G gene is reac- tivated in these distinct cell populations as they differentiate. It will be of great interest to also determine at what stage of development and in which fetal and placental tissues up-regulation of classical class I MHC HLA-A, -B, and -C Ags occurs.

Acknowledgments FIGURE 7. HLA-G mRNA isoforms in the ICM of human blastocyst. We thank Drs. Enda McVeigh and Karen Turner and the patients and staff The lanes are marked as follows: M, 1-kb DNA marker; G1, HLA-G1; G2, of the Oxford Fertility Unit for providing the human embryos for this study HLA-G2; G3, HLA-G3; G4, HLA-G4; G5, HLA-G5; G6, HLA-G6; PG, and Professor Helen Mardon and Janet Carver for their assistance with Pan HLA-G; and -ve, negative control. imaging. The Journal of Immunology 8385

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