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BOR - Papers in Press, published online ahead of print October 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022137
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BIOLOGY OF REPRODUCTION 70, 488–494 (2004)
DOI: 10.1095/biolreprod.103.022137
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Search for the Bovine Homolog of the Murine Ped Gene and Characterization of Its Messenger RNA Expression During Bovine Preimplantation Development1

Trudee Fair2,3,4, Alfonso Gutierrez-Adan5, Madeline Murphy4, Dimitrios Rizos3,4, Finian Martin4, Maurice P. Boland3,4, and Patrick Lonergan3,4

Department of Animal Science and Production,3 University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland The Conway Institute for Biomedical and Biomolecular Research,4 University College Dublin, Belfield, Dublin 4, Ireland Departmento de Reproducción Animal,5 INIA, Ctra. de la Coruña Km. 5.9, Madrid 28040 Spain


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mice, a gene (Ped: preimplantation embryo development) that regulates preimplantation embryonic growth, including cleavage rate and embryo survivability, has been described. The objective of the current study was to identify the bovine homolog of the Ped gene and to characterize the mRNA expression pattern of this gene during bovine preimplantation embryo development. The NCBI GenBank/EBI expressed sequence tags (EST) databases were searched for bovine ESTs that were homologous to the murine Ped gene, and the resulting ESTs were aligned and assembled into a contiguous sequence. The homology of the sequence to the murine Ped gene was confirmed. Primers were designed for the sequence, and the mRNA expression pattern was characterized during bovine preimplantation embryo development in vivo and in vitro. In vitro-produced bovine zygotes were cultured either in vitro, in synthetic oviduct fluid, or in vivo in the ewe oviduct for 1–7 days and processed for quantitative real-time polymerase chain reaction (PCR). Transcript abundance increased at each stage of development. However, the expression levels were consistently higher in in vivo-cultured embryos at all stages, with in vivo-cultured embryos showing a 9-fold increase in relative transcript abundance during culture from the zygote to the blastocyst stage in contrast to just under a 4-fold increase during the same culture period in vitro. The mRNA expression pattern of the gene was investigated in early- and late-cleaving two-cell embryos collected at 25, 28, 32, and >=36 h postinsemination (pi). Transcript relative abundance was highest in those embryos that had cleaved by 28 hpi and decreased almost 3-fold thereafter. In conclusion, we have identified a potential bovine homolog of the murine Ped gene. We have characterized the mRNA expression pattern of this gene during preimplantation embryo development in cattle and shown that a greater relative abundance of the gene transcript is associated with embryos of higher quality (in vivo cultured) and greater developmental potential (early cleaving).

early development, embryo, gene regulation, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The time of the first cleavage division after insemination in vitro is highly correlated with the ability of the bovine embryo to reach the blastocyst stage; those oocytes cleaving earliest after IVF are more likely to reach the blastocyst stage than their later-cleaving counterparts [1–3]. This phenomenon is common to many species (hamster [4], buffalo [5], human [6, 7]). In mice, a gene that regulates preimplantation embryonic growth, including cleavage rate, known as the Ped (preimplantation embryo development) gene, has been identified [8]. The Ped gene is located at the Q region of the mouse major histocompatibility complex (MHC) [9, 10]. Two functional alleles of the Ped gene have been defined: Ped fast and Ped slow [8]. The protein product of the Ped gene is the Qa-2 antigen, which is responsible for the Ped gene phenotype [11]. Embryos that express Qa-2 protein cleave at a fast rate, whereas those that do not cleave at a slower rate. Further studies have shown that survival to birth, birth weight, and weaning weight are also influenced by the Ped gene [10, 12].

As bovine embryos exhibit similar Ped gene phenotypes and fast-cleaving embryos are more developmentally competent, we were interested in investigating if a homolog of the murine Ped gene exists in cattle. Therefore, the objective of the present study was to identify the sequence of the Ped gene in cattle, to describe the mRNA expression pattern of the gene during preimplantaion embryo development both in vivo and in vitro, and to investigate if differential expression of the Ped gene was associated with fast- and slow-cleaving phenotypes in bovine embryos.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sequence Assembly

The bovine homolog of the murine Ped gene was determined by searching for bovine expressed sequence tags that showed sequence homology with the murine Ped gene (accession no. X05389) using the Blast search tool in the National Center for Biotechnology Information (NCBI) GenBank/EBI expressed sequence tag (EST) databases. The resulting bovine ESTs were aligned using the NCBI Blast engine for local alignment. The alignment of all ESTs was compared with each other until one complete sequence had been assembled. The assembled sequence was subsequently aligned with the murine Ped gene to confirm that it was homologous.

Tissue Preparation

Bovine uterine tissue was collected by dissection from slaughtered cows. The tissue was transported to the laboratory in PBS at 37°C where it was further dissected into 1-cm3 volumes (200–500 mg) and placed in Eppendorf tubes and snap frozen at -196°C and stored at -80°C. Mammary glands were removed from pregnant mice that had been killed by cervical dislocation, snap frozen, and stored at -196°C.

Western Blots

The expression of the protein product of the Ped gene, the Qa-2 antigen, by bovine tissue was investigated using Western blot analysis. The tissue was chopped on ice in lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCL, 0.5 mM dithiothreitol [DTT], 0.2 mM PMSF, 1 mM Na3VO4, and protease inhibitor cocktail [Sigma, St. Louis, MO]) and incubated on ice for 15 min. The cytosolic protein component was recovered in the supernatant following centrifugation of homogenized lysed tissue in cytosolic extraction buffer (10 mM Tris-HCL pH 7.5, 300 mM sucrose, 1 mM PMSF, 1 µM Na2VO3, and protease inhibitor cocktail). The nuclear protein component was recovered in the supernatant following centrifugation of the resuspended pellet in nuclear extraction buffer (20 mM HEPES pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 1 mM Na3VO4, and protease inhibitor cocktail). The protein concentration of the lysates was determined using the Bradford assay. Following denaturation, the protein extracts were loaded on to a 12% SDS-polyacrylamide gel. The proteins were transferred on to Opitran-reinforced nitrocellulose 0.2 mM (Schleicher & Schuell, Einbeck, Germany) using the Bio-Rad Wet Blot Transfer Cell apparatus (Bio-Rad, Hercules, CA) using 1x transfer buffer (20 mM Tris-HCL pH 8.5, 150 mM glycine, 0.01% SDS, and 20% methanol) at 200 V for 1 h at 4°C. The blot was subsequently blocked with 1x TBS Tween containing 5% milk protein for 1 h at room temperature. The hybridizations were carried out using a 1:100 dilution of a monoclonal murine Qa-2 specific antibody (clone 69H1-9-9, Mouse IgG2a, kappa; eBioscience, San Deigo, CA). The hybridized filters were washed with TBS Tween (25 mM Tris-HCL pH 7.6, 150 mM NaCl, and 0.5% Tween-20) and hybridized with polyclonal goat anti-mouse IgG coupled with horseradish peroxidase using a dilution of 1:10 000 at room temperature for 1 h. The proteins were detected by enhanced chemiluminescence (1:1 luminol enhancer solution:stable peroxidase buffer solution [Supersignal, West Dura, Western Blotting Kit; Pierce, Rockford, IL]) and exposed to Fuji RX film (Fujifilm, Tokyo, Japan) for 6 min.

Zygote Production

Bovine presumptive zygotes were produced following in vitro maturation (IVM) and fertilization (IVF). Briefly, cumulus oocyte complexes (COCs) were obtained by aspirating follicles from bovine ovaries collected after slaughter. After four washes in PBS supplemented with 36 µg/ml pyruvate, 50 µg/ml gentamycin, and 0.5 mg/ml BSA (Sigma), groups of up to 50 COCs were placed in 500 µl maturation medium in four-well dishes (Nunc, Roskilde, Denmark) and cultured for 24 h at 39°C under an atmosphere of 5% CO2 in air with maximum humidity. The maturation medium was TCM-199 supplemented with 10% (v/v) fetal calf serum (FCS) and 10 ng/ml epidermal growth factor. For IVF, COCs were washed four times in PBS and then in fertilization medium before being transferred in groups of up to 50 into four-well dishes containing 250 µl of fertilization medium per well (Tyrode medium with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg/ml fatty acid-free BSA, and 10 µg/ml heparin-sodium salt (184 units/mg heparin; Calbiochem, San Diego, CA). Motile spermatozoa were obtained by centrifugation of frozen-thawed semen (Dairygold A.I. Station, Mallow, Ireland) on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient (2.5 ml 45% Percoll over 2.5 ml 90% Percoll) for 8 min at 700 x g at room temperature. Viable spermatozoa, collected at the bottom of the 90% fraction, were washed in Hepes-buffered Tyrode and pelleted by centrifugation at 100 x g for 5 min. Spermatozoa were counted in a hemocytometer and diluted in the appropriate volume of fertilization medium to give a concentration of 2 x 106 spermatozoa/ml. A 250-µl aliquot of this suspension was added to each fertilization well to obtain a final concentration of 1 x 106 spermatozoa/ml. Plates were incubated for approximately 20 h at 39°C under an atmosphere of 5% CO2 in air with maximum humidity. Semen from the same bull was used for all experiments.

Culture of Two-Cell Embryos

At approximately 20 h postinsemination (hpi), presumptive zygotes produced following IVM/IVF were denuded by gentle vortexing and transferred to synthetic oviduct fluid medium (SOF; 25 zygotes in 25 µl of medium under mineral oil) and cultured in an atmosphere of 5% CO2, 5% O2, and 90% N2 at maximum humidity. Dishes were examined at 25, 28, 32, 36, and 42 hpi, and two-cell embryos were removed at each time point, snap frozen in liquid nitrogen, and stored at -80°C. These time points have been previously shown to represent good cutoffs between developmentally competent and incompetent embryos [3]. A quantitative analysis of the putative Ped gene homolog was carried out on replicates of pools of 10 two-cell embryos collected at 25, 28, 32, and >=36 hpi from three different fertilizations.

Culture of Zygotes to the Blastocyst Stage

At approximately 20 hpi, presumptive zygotes produced following IVM/IVF were denuded by gentle vortexing and randomly divided in two groups and cultured either in vitro in SOF as described previously or in vivo in the ewe oviduct. Fetal calf serum (10%, v/v) was added to the SOF culture dishes at 48 hpi. For in vivo culture, zygotes were surgically transferred by midventral laparotomy to the ligated ewe oviduct (approximately 100 per oviduct) at 20 hpi [13, 14]. Embryos were recovered from both systems at approximately 30 hpi, 2, 3, 4, 5, 6, or 7 days pi. In the case of in vivo-cultured embryos, the oviduct was surgically removed from the ewe, transported to the laboratory, and flushed from the infundibulum to the uterotubal junction twice with 10 ml of PBS at 37°C. On recovery, embryos were examined for stage of development and snap frozen in liquid nitrogen. A quantitative analysis of the putative Ped gene homolog was carried out on four replicates of pools of 10 embryos at each stage of development. The replicates were generated from culture in two ewes and two IVM/IVF/IVC setups for each time point.

All animal experiments were performed in accordance with Institutional Animal Care and Use Committee guidelines and in adherence with guidelines established in the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the Society for the Study of Reproduction.

RNA Extraction and Reverse Transcription

Total RNA was extracted from each pool of embryos and digested with DNase I, using the Strataprep Total RNA microprep kit (Stratagene, La Jolla, CA, #400752) according to the manufacturer's instructions. Reverse transcription was performed using the Superscript II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA, #18064-014) supplemented with 200 ng of random primers (Invitrogen, #48190-011) according to the manufacturer's instructions under the following conditions: 10 min at room temperature, 50 min at 42°C, and 10 min at 72°C. Afterward, sterile H2O was added to bring the final volume of cDNA to 40 µl.

Quantitative Real-Time PCR

Polymerase chain reaction (PCR) was performed using a Rotorgene 2000 Real Time Cycler (Corbett Research, Sydney, Australia) and SYBR Green (Molecular Probes, Eugene, OR) as a double-stranded DNA-specific fluorescent dye. The PCR reaction mixture (25 µl) contained 2.5 µl 10x buffer, 3 mM MgCl2, 2 U Taq Express (MWGAG Biotech, Ebersberg, Germany), 100 µM of each dNTP, and 0.2 µM of each primer. In addition, the double-stranded DNA dye, SYBR Green I (1:3000 of 10 000x stock solution) was included in each reaction. The PCR protocol included an initial step of 94°C (2 min), followed by 40 cycles of 94°C (15 sec), 56–59°C (30 sec), and 72°C (30 sec). Fluorescent data were acquired at 85°C. The melting protocol consisted of holding at 40°C for 60 sec and then heating from 50 to 94°C, holding at each temperature for 5 sec while monitoring fluorescence. Product identity was confirmed by ethidium-bromide-stained 2% agarose gel electrophoresis. As negative controls, tubes were always prepared in which RNA or reverse transcriptase was omitted during the reverse transcription reaction.

The comparative CT method was used for quantification of expression levels (ABI Prism Sequence Detection System User Bulletin No. 2 [PE Applied Biosystems, Foster City, CA], 11–14). The quantification was normalized to the endogenous control Histone H2a. Fluorescence was acquired in each cycle in order to determine the threshold cycle or the cycle during the log-linear phase of the reaction at which fluorescence rose above background for each sample. Within this region of the amplification curve, each difference of one cycle is equivalent to a doubling of the amplified product of the PCR. According to the comparative CT method, the {Delta}CT value was determined by subtracting the H2a CT value for each sample from the Ped CT value of the sample. Calculation of {Delta}{Delta}CT involved using the highest sample {Delta}CT value (i.e., the sample with the lower target expression) as an arbitrary constant to subtract from all other {Delta}CT sample values. Fold changes in the relative mRNA expression of target was determined by using the formula 2-{Delta}{Delta}CT (ABI Prism Sequence Detection System User Bulletin No. 2 [PE Applied Biosystems], 11–14).

Primer sequences, annealing temperature, the approximate sizes of the amplified fragments, and the GenBank accession number are shown in Table 1.


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  		TABLE 1. Details of primers used for RT-PCR

Verification of PCR Products

The identities of the amplicon were confirmed by appropriate restriction digests of PCR products (data not shown). Furthermore, PCR products generated by reverse transcription (RT)-PCR, using oligo dT primers that had identical sequences to those used in real-time PCR, were subsequently sequenced using the Big Dye Sequencing Kit V1.1 (Applied Biosystems, #4337450). The resulting sequence was identified as homologous to several alleles of the bovine major histocompatibility class I complex (MHC I), including, for example, the 4221.1 gene (accession no. AJ010865, length 1090 base pairs [bp]) using BLAST searches of NCBI GenBank/EBI databases.

Statistical Analysis

Data on mRNA expression were analyzed using the SigmaStat (Jandel Scientific, San Rafael, CA) software package. One-way repeated-measures ANOVA (followed by multiple pairwise comparisons using Student-Newman-Keuls method) were used for the analysis of differences in mRNA expression assayed by quantitative RT-PCR. Differences of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Bovine ESTs that showed sequence homology with the murine Ped gene (accession no. X05389, length 1431 bp) were aligned using the NCBI Blast engine for local alignment. A contiguous sequence of 841 bp was assembled (Fig. 1a) and subsequently aligned with the murine Ped gene using the NCBI Blast engine for local alignment to confirm that the two sequences were homologous (Fig. 1b). The sequence was subsequently compared to sequences held in the (NCBI) GenBank/nonredundant database and showed 97% homology with the bovine MHC class I 4221.1 gene (accession no. AJ010865). The protein product of the murine Ped gene (accession no. CAA28977) was aligned with the predicted amino acid sequence of the bovine MHC I 4221.1/putative Ped gene (accession no. CAA09381) using the Clustawl alignment tool, and the conserved regions were identified using the Genedoc multiple-sequence alignment editor and shading utility (Fig. 2). There was a high degree of conservation between the two proteins. The murine Qa-2 antigen has a molecular mass of 40 kDa. Following Western blot analysis using a mouse monoclonal antibody to detect the mouse Qa-2 antigen, protein bands corresponding to ~40 kDa were detected in the cytosolic and nuclear extracts of the murine mammary tissue (positive control); however, the band was quite faint in the murine nuclear extract, and a band corresponding to 17 kDa was also detected in the murine cytosolic extract. A protein band corresponding to ~40 kDa was detected in the cytosolic extract of bovine nonpregnant uterine tissue (Fig. 3).



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  		FIG. 1. A) Contiguous nucleotide sequence assembled from bovine ESTs homologous to the murine Ped gene (accession no. X05389). B) Graphical representation of the alignment of (1) murine Ped gene (accession no. X05389) with (2) the assembled nucleotide sequence using the BLAST 2 sequences tool of the NCBI GenBank/EBI database search engine



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  		FIG. 2. Alignment of the murine Ped (Q7) gene product (accession no. CAA28977) with the predicted amino acid sequence of the bovine MHC I 4221.1/Ped gene (accession no. CAA09381) generated using the Clustawl alignment tool and the Genedoc multiple-sequence alignment editor and shading utility. The highly conserved regions are indicated with dark shading



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  		FIG. 3. Western blot analysis of Qa-2 antigen expression in mouse mammary cytosolic (lane 1) and nuclear (lane 2) tissue extracts and cattle uterine cytosolic (lanes 3 and 5) and nuclear (lanes 4 and 6) extracts, using the mouse monoclonal Qa-2 antibody (clone 69H1-9-9)

Putative Ped mRNA expression was compared between fast- and slow-cleaving bovine two-cell embryos. The results are summarized in Table 2. The relative abundance of the putative Ped transcript was highest in those embryos that had cleaved by 25 hpi (early cleaving) and lowest in those cleaving >=36 hpi (slow cleaving); the other time points (28 and 32 hpi) were intermediate.


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  		TABLE 2. Comparison of Ped mRNA relative abundance between early- and late-cleaving bovine two-cell embryos cultured in vitro

Verification of PCR Products

Putative Ped transcript abundance was compared between in vivo- and in vitro-cultured embryos; only embryos at the same stage for age were compared (Day 1: 2-cell; Day 2: 4-cell; Day 3: 8-cell; Day 4: 16-cell; Day 5: early morula; Day 6, compact/late morula; Day 7: blastocyst). The results are summarized in Table 3. In general, there was an increase in transcript abundance at each stage of development from the zygote to the blastocyst stage. There was a 9-fold increase in relative transcript abundance during in vivo culture from the zygote to the blastocyst stage; in contrast, relative transcript abundance increased only 4-fold during the same culture period in vitro. The relative transcript abundance was consistently lower in the in vitro-cultured embryos at all stages of preimplantation development compared to those cultured in vivo; the differences were greatest during the later stages of development, increasing from a 1.5-fold difference at the four-cell stage to an almost 4-fold difference at the blastocyst stage. The in vivo-cultured embryos showed a significant increase in transcript abundance from the 16-cell stage of culture onward; in contrast, the differences in the in vitro-cultured embryos did not become significant until the blastocyst stage.


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  		TABLE 3. Comparison of Ped mRNA relative abundance between bovine embryos cultured either in vivo or in vitro during the preimplantation development period


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we have identified a potential homolog of the murine Ped gene in cattle and characterized the mRNA expression pattern of this transcript during the preimplantation stages of bovine embryo development both in vivo and in vitro. Using alignment tools, we have clearly demonstrated that this transcript is homologous with the murine Ped gene and the bovine MHC I 4221.1 gene. Furthermore, we have shown that both the nucleotide and the amino acid sequences of these two genes are homologous. The protein product of the murine Ped gene, the Qa-2 antigen, is a cell surface molecule with a molecular mass of approximately 40 kDa [15]. We have clearly shown that the Qa-2 antigen is detectable in bovine uterine tissue. A smaller, unidentified protein band was also detected in the murine control tissue; however, as this band was not detected in the bovine tissue, it was not investigated further.

Studies in mice have demonstrated that the Ped gene influences the rate of preimplantation development [16, 17] and subsequent embryo survival (for review, see [18]). Two alleles of the Ped gene have been defined: Ped fast and Ped slow. The product of the Ped gene, the Qa-2 antigen, controls the rate of mouse embryonic cleavage during the preimplantation stages of development. The presence of the Qa-2 antigen in mouse embryos is directly correlated with the Ped fast phenotype (fast cleaving), and the absence of the Qa-2 antigen correlates with the Ped slow phenotype (slow cleaving) [9, 19]. Studies from our laboratory have clearly shown that a faster rate of cleavage is associated with higher developmental potential and higher embryo quality in cattle [2]. The present study goes further, clearly identifying an association between increased putative Ped transcript abundance with a faster rate of cleavage, reporting an almost 3-fold difference in transcript abundance between fast-cleaving (those cleaving by 28 hpi) and slow-cleaving (those cleaving after 32 hpi) two-cell bovine embryos. The faster rates of cleavage have been associated with higher cell numbers in both bovine and murine blastocysts [3, 20]. However, this does not appear to affect implantation or pregnancy rates in either species, as both Ped fast and Ped slow mouse embryos were reported to have an equal chance of survival to midgestation [21] and similar pregnancy rates were recorded following transfer of fresh bovine blastocysts originating from early- and late-cleaving zygotes [3]. However, Day 7 bovine blastocysts originating from fast-cleaving zygotes showed increased cryotolerance in terms of survival postthawing than their later-cleaving counterparts [2].

The Ped fast phenotype has been shown to confer a developmental advantage over the Ped slow phenotype. Embryos with the Ped fast allele have a higher chance of surviving to term, and the pups have a higher birth weight and weaning weight compared with those with the Ped slow allele, that is, those that lack Qa-2-encoding genes [10, 12, 21]. In the present study, mRNA expression of the potential bovine Ped homolog was detected in in vivo- and in vitro-cultured embryos at all stages of preimplantation development from the zygote to the blastocyst stage. While embryos from the two culture treatments showed a progressive increase in transcript expression throughout these stages of preimplanatation development, transcript abundance was lower in in vitro embryos at all stages of development, such that there was in excess of a 9-fold increase in transcript expression from the two-cell to the blastocyst stage in in vivo embryos compared to just under a 4-fold increase during the same period of development in vitro. It is interesting to note that the first significant increase in mRNA expression levels was detected at the 16-cell embryo stage in the in vivo-cultured group in contrast to the blastocyst stage in the in vitro-cultured group; the 16-cell stage of development follows the establishment and major activation of the bovine embryonic transcriptome ([22]; for review, see [23]). It would appear that the expression of the embryonic transcript is delayed or reduced in the in vitro-cultured embryos. Differences in expression levels of genes between in vitro- and in vivo-cultured bovine embryos have been reported previously; it is well known that the culture environment can have a dramatic influence on the abundance of various developmentally important gene transcripts [24–27]. Depending on the transcript, differences in abundance may be apparent by as little as 10 h after the initiation of culture [27]. Although blastocysts derived from culture in vivo or in vitro expressed the transcript, there was a 3-fold difference in abundance between in vivo- and in vitro-derived blastocysts. This may be linked to the reported differences in pregnancy rate following transfer of such embryos [28].

As in cattle, data from human IVF records provide clear evidence of the existence of the Ped gene phenotype in the human population [29]. The human leukocyte antigen (HLA)-G has been proposed as a possible human homolog of the Ped gene. HLA-G-positive embryos were shown to have faster cleavage and higher pregnancy rates than HLA-G-negative embryos [30]. More recently, it was reported that implantations following IVF occurred only in women showing soluble HLA-G molecules in embryo culture supernatants [31]. However, a similar study failed to detect soluble HLA-G molecules in embryo culture supernatants [32]; therefore, further studies are necessary. It is widely believed that the Qa-2 and HLA-G proteins act to protect the fetus from rejection by maternal immune system by blocking against maternal natural killer cells during implantation and placentation [18]. The analysis of the complete DNA sequence of the human MHC, the HLA complex, has led to the suggestion that although not strict genetic orthologues, Qa-2 and HLA-G are functional homologs as the result of convergent evolution [33, 34].

In conclusion, the existence of the Ped gene phenotype in cattle and its association with the differential expression of an MHC I gene strongly suggests that a homolog of the murine Ped gene is functional in the bovine species.


   ACKNOWLEDGMENTS
 
Mrs. Mary Wade is thanked for excellent technical assistance.


   FOOTNOTES
 
1 Supported by the Health Research Board under the Postdoctoral Fellowship Scheme (grant PD01/2000), the Spanish Ministerio de Ciencia y Tecnología (grant AGL2003/05783), and the Science Foundation Ireland. Back

2 Correspondence: Trudee Fair, Department of Animal Science and Production, University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland. FAX: 353 1 628 8421; trudee.fair{at}ucd.ie Back

Received: 11 August 2003.

First decision: 2 September 2003.

Accepted: 8 October 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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