Molecular Human Reproduction, Vol. 5, No. 6, 541-547,
June 1999
© 1999 European Society of Human Reproduction and Embryology
Search for a human homologue of the mouse Ped gene
1 Department of Biology, 414 Mugar Hall, Northeastern University, Boston, MA 02115, 2 Institute for Reproductive Medicine and Science, Saint Barnabas Medical Center, West Orange, NJ 07052 USA
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Abstract |
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The Ped gene influences the rate of cleavage division of preimplantation mouse embryos and subsequent embryonic survival. The mouse Ped gene product is a major histocompatibility complex (MHC) class Ib protein called Qa-2. Studies from many human in-vitro fertilization (IVF) clinics suggest that the mouse Ped gene has a human homologue because embryos fertilized at the same time have different cleavage rates, and those embryos that cleave at a faster rate are more likely to result in a viable pregnancy. Candidates for the human homologue of the mouse Ped gene include the MHC class Ib genes HLA-E, HLA-F, and HLA-G. The presence of mRNA for these three genes was tested in 108 spare day 3 human preimplantation embryos from 25 couples by using reverse transcription–polymerase chain reaction (RT–PCR). Of the 86 embryos tested for HLA-E mRNA, 72 were positive (84%), and of the 88 embryos tested for HLA-G mRNA, 39 were positive (44%). None of the 17 embryos tested for HLA-F mRNA were positive (0%). Studies of expression of HLA-G protein were undertaken to ascertain whether HLA-G was attached to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage similar to that found in Qa-2 protein. Treatment of JEG-3 cells, an HLA-G expressing cell line, with phospholipase C did not result in removal of HLA-G showing that HLA-G, unlike Qa-2, is not GPI linked to the cell surface. The pros and cons of HLA-E, HLA-F, and HLA-G as candidates for the human Ped gene are discussed.
cleavage rate/GPI-anchored proteins/HLA-class Ib/human preimplantation embryo/Ped gene
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Introduction |
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Major histocompatibility complex (MHC) genes are critical in many aspects of reproduction, including fertility rate (Lerner et al., 1988
), spontaneous abortion (Ober and Van der Ven, 1997), protection of the embryo from NK cell attack (King et al., 1997), and embryo survival and development (Warner et al., 1998a,b,c). It has been shown that the rate of development of preimplantation mouse embryos is influenced by an MHC class Ib protein, Qa-2, the product of the mouse Ped (preimplantation embryo development) gene (Warner et al., 1987, 1998a,Warner et al., b,c; Xu et al., 1994; Cai et al., 1996). The mouse Ped gene has two functional alleles, fast and slow, defined by the rate of cleavage during the preimplantation period (Verbanac and Warner, 1981). In addition, embryos with the Ped fast allele have an overall reproductive advantage compared with their Ped slow counterparts (Warner et al., 1991, 1993; Exley and Warner, 1998).
The Qa-2 protein is a 40 kDa protein found in both membrane bound and secreted forms (reviewed in Stroynowski and Tabaczewski, 1996). There are two membrane bound forms of Qa-2 that are distinguishable by their mode of attachment to the cell membrane, either via a glycosylphosphatidylinositol (GPI) linkage or via a transmembrane domain. In most mouse tissues, including lymphocytes and preimplantation embryos, Qa-2 is GPI-linked to the embryonic cell surface and can be cleaved off the cell surface by the enzyme phospholipase C (Tian et al., 1992). Removal of Qa-2 protein from the surface of the embryos slows their rate of cleavage division.
Qa-2 protein is encoded by four genes that are located in the Q region of the MHC: Q6, Q7, Q8, and Q9. Mice with the presence of these four genes express the fast Ped phenotype whereas mice with the slow Ped phenotype have a deletion of all four genes. Recent studies have shown that only the Q7 and/or Q9 genes are transcribed in Ped fast preimplantation embryos, suggesting that only the Q7 and/or Q9 genes are responsible for the mouse Ped phenotype (Wu et al., 1998).
Using the mouse as a model system, we have initiated a search for a human homologue of the mouse Ped gene. In a recent review (Warner et al., 1998a), data from many in-vitro fertilization (IVF) clinics showed that embryos from pools of oocytes fertilized simultaneously are not necessarily synchronized in their rate of development, suggesting that there is a Ped gene in humans which influences the rate of cleavage of human preimplantation embryos. In addition, those embryos that divide more rapidly are more likely to lead to a pregnancy (Bolton et al., 1989; Ziebe et al., 1997; Sakkas et al., 1998). These observations suggest that the Ped phenotype is present in the human population. The phenotype may be derived from either parent (Janny and Ménézo, 1994).
Because the mouse Ped gene product, Qa-2 protein, is encoded by two MHC class Ib genes, our search for the human Ped gene homologue was initiated by the analysis of expression of the three best-studied human MHC class Ib genes, HLA-E, HLA-F, and HLA-G (Geraghty et al., 1987; 1990; Ulbrecht et al., 1992; Shawar et al., 1994; LeBouteiller, 1994; 1997; LeBouteiller and Lenfant, 1996; Clark, 1997; Hammer et al., 1997; O'Callaghan and Bell, 1998). A previous study reported HLA-G mRNA expression in preimplantation embryos (Jurisicova et al., 1996), but there are no previous reports of HLA-E or HLA-F mRNA expression in human preimplantation embryos. In this paper we report the analysis of HLA-E, HLA-F, and HLA-G mRNA expression in human preimplantation embryos using reverse transcription–polymerase chain reaction (RT–PCR). In addition, we assayed the type of membrane linkage of HLA-G to the cell surface to ascertain whether HLA-G is linked to the cell surface by a GPI linkage, similar to the mouse Ped gene product, Qa-2 protein.
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Materials and methods |
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Human embryos
Spare human preimplantation embryos (n = 108) were donated by couples (n = 25) attending the IVF programme of the Institute for Reproductive Medicine and Science of Saint Barnabas (IRMS, West Orange, NJ, USA). The embryos were provided in a `double-blind' mode where neither the provider (C.B. at IRMS, who did not have access to clinical data) nor the recipient (W.C. at Northeastern University) knew the identity of the patients. The protocols were approved by the Internal Review Board of Saint Barnabas Medical Center. Day 3 embryos were used in all the studies. Each embryo was lysed in 100 µl of denaturing solution and 0.72 µl of ß-mercaptoethanol from a Stratagene Micro RNA isolation kit (Stratagene, La Jolla, CA, USA). The embryo lysates were frozen at –70°C and shipped on dry ice from the IRMS to Northeastern University for RT–PCR analysis.
PCR primers
The primers used in this study for each cDNA of interest are summarized in Table I. Unless otherwise indicated, primers were designed with OLIGO 5.0 Primer Analysis Software (National Bioscience, Plymouth, MN, USA). To ensure that the product detected resulted from amplification of cDNA rather than contaminating genomic DNA, all primers were chosen to cross intron/exon boundaries.
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RNA isolation from single human embryos
Total RNA was extracted from the frozen samples using a Micro RNA isolation kit (Stratagene). Briefly, the frozen samples were thawed, and 1 µl of pAW109 RNA (106 copies) was added as an internal control. The remaining details were as described by the manufacturer, except that the samples were centrifuged at 11 800 g for 45 min instead of 5 min after the addition of isopropanol and glycogen, and the RNA pellet was air-dried or dried at 50°C for 5 min. To each embryonic RNA pellet, 7.25 µl RNase-free dH2O, 0.20 µl 0.1M dithiothreitol (DTT), 1.0 µl 50 µM random hexamers (Perkin Elmer, Branchburg, NY, USA), 0.05 µl RNase inhibitor (20 IU/µl) were added. The mixture was vortexed, and then 20 µl of autoclaved mineral oil was added to the surface of the solution. Finally, the solution was heated to 70°C for 6 min to dissolve the RNA, and cooled to 30°C for 1 minute.
cDNA synthesis, PCR and nested PCR.
cDNA was synthesized from half the total RNA from each embryo, and the reverse transcription (RT) reaction was carried out in a 20 µl volume using a kit from Perkin Elmer. RNA was added with 3 µl of 10 mM MgCl2, 2 µl 10x PCR buffer (100 mM Tris–HCl, pH 8.3, 900 mM KCl), 2 µl of each 10 mM dNTP, 1 µl of RNase Inhibitor, 1 µl of Oligo d(T), and 1 µl of MuLV-reverse transcriptase. The reaction mixture was allowed to stand at room temperature for 5 min and then a wax bead (Perkin Elmer) was added to prevent evaporation. The RT reaction was performed at 37°C for 1 h and 95°C for 5 min.
PCR was carried out in a 50 µl volume: 20 µl of the RT mixture, 3 µl of PCR buffer, 1 µl of each primer, 1 µl of Taq polymerase (5 IU/µl), and 23.5 µl of water. The outer primer pairs, such as HLA-G Tm and HLA-G Put-2 (Table I
), were used in the first round of the PCR reaction. To increase the specificity and the sensitivity of the RT–PCR amplifications, nested or hemi-nested PCR was performed using an inner primer pair (nested) or one inner primer in conjunction with one of the outer primers (hemi-nested). Because of the possibility of RNA splicing variants (Le Bouteiller and Lenfant, 1996), the primers were designed to maximize the likelihood of detecting the major known RNA transcripts for each of the genes.
PCR reactions were carried out in a thermal cycler (Perkin Elmer) according to one of the following three cycling protocols depending on the primer set as described below: (i) cDNA product was denatured at 95°C for 1 min, followed by 40 cycles of PCR, which included denaturation at 95°C for 1 minute, annealing at 61°C for 2 min, and extension at 72°C for 3 min; (ii) one cycle of 95°C for 5 min, followed by 35 cycles of 95°C for 1 min, 54°C for 1 min, 72°C for 1 min; or (iii) one cycle of 95°C for 5 min, 10 cycles of 95°C for 1 min, 50°C for 1 min, 72°C for 1 min, followed by 35 cycles of 95°C for 1 min, 59°C for 1 min, 72°C for 1 min. Most PCR primers were designed specifically to have melting temperatures compatible with protocol 1. Only the ß2m inner primer pair required the alternate conditions of protocol 2, and the HLA-E outer primer set required the conditions of protocol 3. The amplified DNA product was analysed on a 6% polyacrylamide gel stained with ethidium bromide, and verified by restriction digestion and sequencing.
Subcloning and sequencing of the PCR products
The final PCR products were analysed on a 0.8% agarose gel. Products of the expected size were purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA), and cloned into a pCRII vector using the Original TA cloning kit (Invitrogen, San Diego, CA, USA).
Double-stranded DNA was isolated from independent clones using a QIAprep 8 Miniprep kit (Qiagen), and denatured by the alkaline-denaturation method (Tonneguzzo et al., 1988). DNA sequencing was performed using a Sequenase Version 2.0 DNA sequencing kit (US Biochemicals, Cleveland, OH, USA) and 
-[35S]-dATP (specific activity 1000µCi/mmole) (Amersham Life Science, Arlington Heights, IL, USA). PCR primers were used as primers for sequencing. The sequencing reactions were carried out according to the manufacturer's instructions and analysed on a 6% polyacrylamide/urea gel.
Mice
C57BL/6 mice, bred in our laboratory in an American Association for the Accreditation of Laboratory Animal Care (AAALAC) approved facility, were used as a source of splenic T cells. All protocols followed the NIH guidelines for the use and care of laboratory animals.
Cell lines
JEG-3 cells (ATCC, Rockville, MD, USA) served as positive control for detecting HLA-G expression, and were cultured in minimum Eagle's medium supplemented with 10% fetal bovine serum (FBS) (heat-inactivated at 56°C for 30 min.) and 0.1% antibiotics/antimycotics (Gibco). HSB-2 cells (ATCC, Rockville, MD, USA) served as a positive control for detecting HLA-E and HLA-F mRNA. HSB-2 cells were cultured in minimum essential medium (MEM) modified for suspension culture supplemented with 10% FBS and 0.1% antibiotics/antimycotics (Gibco, Gaithersburg, MD, USA).
Monoclonal antibodies
A hybridoma cell line producing a mouse immunoglobulin (Ig)G anti-Qa-2 monoclonal antibody (1–12–1) was grown and used as described previously (Sharrow et al., 1989; Xu et al., 1994). A hybridoma cell line producing a mouse IgM anti-HLA-G monoclonal antibody (1B8) was kindly provided by Dr McMaster (University of California, San Francisco, CA, USA), and was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 0.1% antibiotics/antimycotics (McMaster et al. 1995).
Phosphatidylinositol-phospholipase C (PI–PLC) treatment and FACScan analysis
T cells were isolated from C57BL/6 mouse spleen cells by Ficoll–Hypaque and nylon wool, and suspended in RPMI 1640 medium containing 10% FBS and 0.02% NaN3 at 106 cells/ml (Stiernberg et al., 1987). Trypsin-treated JEG-3 cells were suspended at a concentration of 106 cells/ml in MEM containing 10% FBS and 0.02% NaN3. Purified PI–PLC from Bacillus thuringiensis (ICN, Costa Mesa, CA, USA) was incubated with the mouse T cells or the human JEG-3 cells at 37°C for 1–3 h at the concentrations indicated below. Mock treatment was performed by culturing the cells at 37°C in growth medium without PI–PLC.
After incubation, the cells were washed three times with PBSAZ [phosphate-buffered saline (PBS) + 0.1% NaN3 + 1% bovine serum albumin (BSA)]. The cells were resuspended in PBSAZ, and primary antibody, 1B8 for HLA-G protein and 1–12–1 for Qa-2 protein, was incubated with cells at 4°C at the proper dilution. After 1 h, the cells were washed once with PBSAZ and incubated with secondary antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Fc of IgG or FITC-conjugated goat anti-mouse µ chain of IgM (both from ICN) at the proper dilution for 1 h. Then the cells were washed again with PBSAZ, and fixed with 3% formaldehyde/PBSAZ. Stained cells (5000 cells/sample) were analysed for immunofluorescence using a FACScan flow cytometer and Beckton-Dickinson Lysis II software.
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Results |
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RT–PCR assay of HLA-E, HLA-F, and HLA-G mRNA in human preimplantation embryos
A total of 108 single preimplantation embryos from 25 couples was examined in this study. All the embryos were collected on day 3 after fertilization. Each embryo was assayed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a positive control, and for ß2m (the light chain non-covalently associated with all functional MHC class I molecules) as a second control. The RT–PCR conditions and the fidelity of the products were tested in JEG-3 cells for HLA-G and in HSB-2 cells for HLA-E and HLA-F. All RT–PCR products were correct both from restriction enzyme fragment analysis and from DNA sequencing results. GAPDH was detected in 98% (106/108) of the embryos, and ß2m was detected in 91% (98/108) of the embryos (Table II
). Only embryos with detectable GAPDH and ß2m mRNA (97/108) were considered viable and were included in subsequent analyses. Figure 1 is an example of the RT–PCR results from individual embryos analysed for HLA-E, HLA-F, and HLA-G. A summary of the results is shown in Table II.
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PI–PLC treatment of HLA-G protein on JEG-3 cells
Since previous studies from several groups indicated that Qa-2 proteins could be released from mouse T cells by PI–PLC (Stiernberg et al., 1987
; Stroynowski et al., 1987; Waneck et al., 1988; Tian et al., 1992), Qa-2 proteins on mouse T cells were used as a positive control. Figure 2 shows that the presence of Qa-2 proteins on the cell surface decreased drastically after PI–PLC treatment. This result demonstrates that 0.2 unit of PI–PLC per 106 T cells is sufficient to cleave the Qa-2 proteins off the T cell surface.
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Subsequently, a similar experiment was carried out for the HLA-G protein on JEG-3 cells to monitor the change in HLA-G expression on the cell surface after PI–PLC treatment. Figure 3
shows that the HLA-G proteins were completely resistant to PI–PLC hydrolysis in that virtually identical profiles were obtained from mock-treated and PI–PLC treated groups. We also analysed the effect of various levels of PI–PLC and various incubation periods, and our data showed that the cell surface HLA-G proteins are unaffected by PI–PLC treatment regardless of the level of enzyme used or the time of incubation. The resistance to cleavage by PI–PLC suggests that the HLA-G protein is not attached to the cell membrane by a GPI linkage.
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Discussion |
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Using the mouse as a model system our laboratory has identified a gene, Ped, that influences the rate of cleavage division of preimplantation mouse embryos and subsequent embryonic survival (reviewed in Warner et al., 1998a
,b,c). The effect of the Ped gene on embryo survival makes it interesting to search for its human homologue. Because the mouse Ped gene product is a MHC class Ib protein (Qa-2), we initiated our search for its human homologue by analysing expression of the three best-studied human MHC class Ib genes, HLA-E, HLA-F, and HLA-G.
Among these three human MHC class Ib genes, HLA-E, HLA-F, and HLA-G, the best studied is HLA-G. It has been shown that the HLA-G gene shares many structural similarities with the Q7 and Q9 genes, and HLA-G was originally suggested to be the human homologue of the mouse Qa-2 protein (the Ped gene product) (Geraghty et al., 1987; Jurisicova et al., 1996; Stroynowski and Tabaczewski, 1996). However, it has recently become clear that HLA-G and Qa-2 protein differ from each other in several respects. First, the HLA-G gene lacks an interferon response sequence (IRS) in the 5' regulatory region while Qa-2 protein has an IRS that mediates up-regulation of Qa-2 protein by 
-interferon. Second, Qa-2 and HLA-G differ from each other in their tissue distribution (Wei and Orr, 1990; Schmidt and Orr, 1993). Third, HLA-G, unlike Qa-2, is quite polymorphic (LeBouteiller and Lenfant, 1996). Fourth, a newly described mouse MHC class Ib gene, named the blastocyst MHC gene, has been suggested to be the mouse homologue for HLA-G (Sipes et al., 1996). Fifth, the types of nine amino acid peptides bound by Qa-2 are different from those bound by HLA-G (O'Callaghan and Bell, 1998). Finally, the work in this paper shows that HLA-G is different from Qa-2 because it cannot be cleaved off the cell surface with PI–PLC implying that it is not linked to the cell surface by a GPI linkage (Figure 2). Therefore, although one published report has suggested that a fast rate of human embryonic development is associated with expression of HLA-G mRNA (Jurisicova et al., 1996), it seems most likely that the gene encoding HLA-G is linked to a putative human Ped gene rather than encoding the Ped gene itself.
Both the results reported in this paper (Table II) and a previous study (Jurisicova et al., 1996), show that ~40% of the embryos donated from IVF patients express mRNA for HLA-G. However, it has not yet been determined if the embryos negative for HLA-G mRNA are failing to transcribe a HLA-G gene that is present, or if the HLA-G gene is missing from some human embryos. It will be interesting to determine the percentage of HLA-G positive embryos, both at the mRNA and DNA levels, in the normal population and compare it to results obtained from IVF patients. This should elucidate whether the lack of the HLA-G gene and/or its expression results in reproductive disadvantage.
Our studies have demonstrated for the first time that HLA-E mRNA is present in most day 3 human embryos (Table II and Figure 3). However, many of the same arguments mitigating against HLA-G being the human Ped gene are similar for HLA-E. Examination of the DNA sequence of the HLA-E gene suggests that, like HLA-G, there are no sites in the HLA-E protein to mediate GPI-linkage to the cell membrane. HLA-E protein also has a very different tissue distribution pattern from Qa-2 protein. Moreover, recent studies have suggested that HLA-E is the human homologue of mouse Qa-1, not Qa-2 (reviewed in Long, 1998). Therefore, HLA-E may not be the human Ped gene product.
Although neither HLA-G nor HLA-E may be the human Ped gene product, studies suggest that HLA-G and HLA-E play a critical role in reproduction and embryonic development. Several groups have shown that HLA-G and HLA-E are involved in natural killer (NK) cell recognition of targets (reviewed in Clark, 1997; King et al., 1997; LeBouteiller, 1997; O'Callaghan and Bell, 1998). HLA-E has recently been shown to bind conserved leader sequence peptides from MHC class Ia molecules that are recognized by NK cells (O'Callaghan et al., 1998). Expression of HLA-G inhibits NK cells expressing CD94/NKG2 receptors (O'Callaghan and Bell, 1998). Thus, the relative expression of HLA-E and/or HLA-G may protect the developing embryo from NK-mediated cell lysis.
Finally, it also seems unlikely that HLA-F encodes the human Ped gene because mRNA for HLA-F is not detectable in day 3 human embryos (Table II and Figure 3). Thus, none of the three candidate genes tested for mRNA expression in human embryos seem likely to encode the Ped phenotype. However, because the present paper reports results on mRNA levels, a final conclusion about the candidacy of HLA-E, HLA-F, and HLA-G for the human Ped gene awaits the measurement of protein levels for each of these gene products.
Interestingly, the MHC class Ia protein HLA-C has been reported to be linked to the cell membrane by a GPI anchor (Davis et al., 1997) and to possibly be involved in reproduction because it is expressed on the extravillous trophoblast (King et al., 1997). A recent article (Hughes et al., 1999) has pointed out that the classification of MHC class I proteins into class Ia and class Ib is arbitrary and may impede the identification of homologues between species. For instance, the cottontop tamarin, a New World Primate, has a class Ia gene that is homologous to the class Ib gene HLA-G. Therefore, even though HLA-C is a MHC class Ia gene, it may be the human homologue of the mouse Ped gene. It is compelling to examine both mRNA and protein expression of HLA-C in human preimplantation embryos to test this hypothesis. Also, since two genes, Q7 and Q9, have been identified as contributing to the Ped phenotype in the mouse, it is likely that more than one MHC class I gene will contribute to the Ped phenotype in humans.
The HLA complex encodes 30–50 MHC class Ib genes, most with unknown function. The identification of the human Ped gene most likely awaits sequencing information from the HLA complex that will be available from the Human Genome Project during the next few years (Collins et al., 1998). To this end, a high-density sequence tagged site (STS) map of the human HLA complex has recently been published (Janer and Geraghty, 1998). This study, and former papers (Geraghty et al., 1992, Watanabe et al., 1997), have reported large-scale variability and deletions in the HLA complex among individuals. Deletion polymorphisms have been reported near HLA-E and near HLA-G (Janer and Geraghty, 1998), making these likely spots in which to continue the search for the human homologue of the mouse Ped gene.
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Acknowledgments |
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We thank Melissa Amendola for care of the mice. The work on mice and human cell lines was supported by NIH grant HD31505. We thank the team of embryologists at the IRMS and Doctors Richard Scott and Paul Bergh for support of this study. The work on spare human embryos was funded by the Institute for Reproductive Medicine and Science of Saint Barnabas.
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Notes |
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3 To whom correspondence should be addressed
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Submitted on December 21, 1998; accepted on March 10, 1999.
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