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BOR - Papers in Press, published online ahead of print May 19, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026138
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BIOLOGY OF REPRODUCTION 71, 919–925 (2004)
DOI: 10.1095/biolreprod.103.026138
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Role of Messenger RNA Expression of Platelet Activating Factor and Its Receptor in Porcine In Vitro-Fertilized and Cloned Embryo Development1

So Hyun Lee3, Dae Young Kim4, Dong Hyun Nam3, Sang Hwan Hyun3, Gab Sang Lee3, Hye Soo Kim3, Chang-Kyu Lee5,6, Sung Keun Kang3,6, Byeong Chun Lee2,3,6, and Woo Suk Hwang3,5,6

Department of Theriogenology and Biotechnololgy,3 College of Veterinary Medicine, Seoul National University, Seoul, 151-742, Korea Division of Biological Science,4 Gachon Medical School, Incheon, 417-840, Korea School of Agricultural Biotechnology,5 Seoul National University, Seoul, 151-742, Korea The Xenotransplantation Research Center,6 Seoul National University Hospital, Seoul, 110-744, Korea


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Platelet activating factor (PAF) is known as an autocrine growth/survival factor in mammalian preimplantation embryos. This study investigated the expression of porcine PAF receptor (PAFr) mRNA and its role in porcine in vitro fertilized (IVF) or somatic cell nuclear transfer (SCNT) embryo development. The expression of PAFr mRNA in IVF or SCNT blastocysts was shown by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. Semiquantitative RT-PCR and Southern blot analysis demonstrated that PAFr mRNA was expressed during preimplantation embryo development, it was highly expressed through the 2-cell to 8-cell embryo stage, and it decreased at the morula stage. PAFr mRNA expression was detected steadily in IVF embryos, whereas it was varied at the 2-cell, 4-cell, and blastocyst stages in SCNT embryos. To determine the role of PAF in IVF and SCNT embryo development, embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with different concentrations of PAF (0, 0.037, 0.37, 3.72, or 37.2 nM). The PAF supplement significantly increased the rate of blastocyst formation in SCNT embryos, but not in IVF embryos. The PAF supplement for the entire 168 h of culture showed significantly higher blastocyst formation in SCNT embryos. Upregulation of PAFr mRNA by PAF in SCNT embryos indicated that the embryotrophic effect of PAF was mediated through its functional receptors in SCNT embryos. In conclusion, the present study demonstrated that PAFr mRNA was expressed in porcine IVF and SCNT embryos, and that PAF supplement improved the developmental competence of SCNT embryos through its specific receptors.

IVF, PAF, PAF receptor, porcine embryo, SCNT


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Successful births of cloned [1–3] and transgenic cloned pigs [4–6] for xenotransplantation by somatic cell nuclear transfer (SCNT) have raised to a tremendous degree the value of cloned pigs in the field of biotechnology and medicine. However, the production of cloned pigs is still inefficient, with only around 1% of the embryos transferred surviving to term [7]. Studies have been performed to improve the developmental competence of porcine in vitro-produced embryos [8–13] and have demonstrated that many factors are involved in vitro embryo development and viability after transfer. These include growth factors, oxygen, energy substrates, amino acids, and albumin [14, 15]. Therefore, it is expected that components in culture medium are one of the important factors affecting embryo viability of porcine SCNT embryos.

Platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a unique phospholipid mediator that may act as one of the autocrine growth/survival factors for mammalian preimplantation embryos [16] via G protein-linked receptor. Embryo-derived PAF has shown to be produced by mouse [17], sheep [18], rabbit [19], and human [20] embryos. It has been demonstrated that PAF is an important embryotrophic factor for mouse embryos [21, 22]. Addition of PAF to the culture medium reduced levels of apoptosis within inner cell masses (ICMs) in mouse embryos [21]. Ryan et al. [22] demonstrated a specific and direct influence of exogenous PAF on the oxidative metabolism of glucose and lactate in preimplantation mouse embryos, suggesting an autocrine role for embryo-derived PAF in early pregnancy. In the pig, limited studies have reported the roles of PAF. The presence of PAF was demonstrated in pig spermatozoa, uterine fluid [23], and in endometrial and embryonic tissues at the peri-implantation stage [24]. The PAF content in boar spermatozoa was related with farrow rate, number of piglets born, and number born alive [25]. However, the role of PAF and its receptor expression in porcine preimplantation embryos are not known.

Accordingly, the present study investigated 1) the cloning and temporal expression of PAF receptor (PAFr) mRNA, 2) the role of PAF in porcine in vitro fertilized (IVF) and SCNT embryo development, and 3) regulation of PAFr mRNA expression in SCNT embryos.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All chemicals were obtained from Sigma-Aldrich Corp. (St. Louis, MO) unless otherwise stated. This study was conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, published by the Consortium for Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, 1st ed, 1988.

Collection of Oocytes and In Vitro Maturation

Ovaries retrieved from prepubertal gilts at a local slaughterhouse were transported to the laboratory in 0.9% (v/v) NaCl solution at 30 to 35°C within 2 h of slaughter. Follicular fluid and cumulus-oocyte complexes (COCs) from follicles 5 to 6 mm in diameter were aspirated using an 18-gauge needle attached to a 5-ml disposable syringe. Compact COCs were selected and cultured in Hepes-buffered North Carolina State University (NCSU)-23 medium [15] supplemented with 10 ng/ml epidermal growth factor (EGF), 4 IU/ml eCG (Intervet, Boxmeer, The Netherlands), 4 IU/ ml hCG (Intervet), 10% (v/v) porcine follicular fluid (pFF), and 0.57 mM cysteine. The pFF was aspirated from 3- to 7-mm follicles from prepubertal gilt ovaries. After centrifugation at 1600 ;ts g for 30 min, supernatants were collected and filtered sequentially through 1.2-µm and 0.45-µm syringe filters (Gelman Sciences, Ann Arbor, MI). Prepared pFF was then stored at –20°C until used. Each group of 50 COCs was cultured in 500 µl of medium placed in a CO2 incubator maintained at 39°C in a humidified atmosphere of 5% CO2 and 95% air. After culturing for 22 h, COCs were washed three times and cultured in eCG- and hCG-free NCSU-23 medium for another 20 h. At the end of maturation culture, COCs were then transferred to Hepes-buffered NCSU-23 medium containing 0.5 mg/ ml hyaluronidase for 1 min, and the cumulus cells were subsequently removed by gentle pipetting.

In Vitro Oocyte Fertilization and Embryo Culture

Frozen Landrace boar semen thawed at 39°C for 1 min was selected by the swim-up technique [26]. At 42 h of in vitro maturation, oocytes freed from cumulus cells were washed in modified Tris-buffered medium (mTBM) containing 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2·2H2O, 20 mM Tris, 11 mM glucose, 5 mM sodium pyruvate, and 0.1% (w/v) BSA, and coincubated with 2 x 106 spermatozoa/ml in 50-µl droplets (20 oocytes per drop) of mTBM covered with mineral oil in 5% CO2 in air at 39°C for 6 h. After IVF, oocytes were cultured in 25-µl microdrops (five to seven oocytes per drop) of NCSU-23 medium supplemented with 4 mg/ ml fatty acid-free BSA covered with mineral oil, in 5% O2, 5% CO2, and 90% N2 at 39°C for 7 days. The number of oocytes that cleaved and developed to the blastocyst stage were visualized (via light microscopy) at 48 h and 168 h, respectively.

Primary Culture of Porcine Fetal Fibroblasts

Fetal fibroblasts were isolated from slaughterhouse-derived fetuses at approximately 40 days of gestation. The head of the fetus was removed using iris scissors, and soft tissues such as liver and intestine were discarded by scooping them out with two watchmaker forceps. After washing twice with PBS (Life Technologies, Rockville, MD), the carcass was minced with a surgical blade on a 100-mm culture dish (Becton Dickinson, Lincoln Park, NJ). The minced fetal tissues were dissociated in Dulbecco modified Eagle medium (DMEM) (Life Technologies) supplemented with 0.1% (w/v) trypsin and 1 mM EDTA (Life Technologies) for 1 to 2 h. Trypsinized cells were washed once by centrifugation at 300 x g for 10 min and subsequently seeded into 100-mm plastic culture dishes. Seeded cells were subsequently cultured for 6 to 8 days in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) (Life Technologies), 1 mM sodium pyruvate, 1% (v/v) nonessential amino acids (Life Technologies), and 10 µg/ml penicillin-streptomycin solution at 39°C in a humidified atmosphere of 5% CO2 and 95% air. After removal of unattached clumps of cells or explants, attached cells were further cultured until confluent, subcultured at intervals of 5 to 7 days by trypsinization for 5 min using 0.1% trypsin and 0.02% EDTA, and stored after two passages in a freezing medium of liquid nitrogen at –196°C. The freezing media consisted of 80% (v/v) DMEM, 10% (v/v) dimethylsulfoxide (DMSO), and 10% (v/v) FBS. Prior to SCNT, cells were thawed and subsequently cultured for 3 to 4 days until they were 80% confluent, and then subjected to serum starvation. For serum starvation, cells were cultured for 3 days in serum-starved DMEM supplemented with 0.5% FBS, and individual cells were retrieved from the monolayer by trypsinization for 30 sec and subsequently used for SCNT.

SCNT and Embryo Culture

After 42 h of in vitro maturation, a cumulus-free oocyte was held with a holding micropipette (110 µm inner diameter) and the zona pellucida (ZP) was partially dissected with a fine, glass needle to make a slit near the first polar body. The first polar body and adjacent cytoplasm presumably containing the metaphase-II chromosomes were extruded by squeezing them with the same needle. Oocytes were enucleated in Hepes-buffered NCSU-23 supplemented with 0.3% BSA and 7.5 µg/ml cytochalasin B (CB). After enucleation, oocytes were stained with 5 µg/ml Hoechst 33342 (bisbenzimide) for 5 min and observed with an inverted microscope equipped with epifluorescence. Oocytes still containing DNA materials were excluded from experiments. Trypsinized fetal fibroblast single cells with a smooth surface were transferred into the perivitelline space of enucleated oocytes. The couplets were equilibrated with 0.26 M mannitol solution containing 0.5 mM Hepes, 0.1 mM CaCl2, and MgCl2 for 4 min, and transferred to a chamber containing two electrodes overlaid with fusion and activation solution. Couplets were fused and activated simultaneously with a single DC pulse of 2.0 kV/cm for 30 µsec using a BTX Electro-Cell Manipulator 2001 (BTX, Inc., San Diego, CA). Activated oocytes were washed three times with NCSU-23 supplemented with 4 mg/ ml fatty acid-free BSA, and placed in 25-µl microdrops (five to seven oocytes per drop) of NCSU-23 under mineral oil and cultured at 39°C, 5% CO2, 5% O2, and 90% N2. The reconstructed embryos were cultured for 7 days after activation, and cleavage and blastocyst formation were monitored under a stereomicroscope at Days 2 and 7, respectively.

Total Cell Number Counts

Blastocysts at Day 7 were incubated with 5 µg/ml bisbenzimide (Hoechst 33342) for 30 min on a warm plate, mounted on slides with a coverslip placed over them, and examined under an inverted microscope (Nikon Corp., Tokyo, Japan) equipped with epifluorescence (wavelength, 350 to 390 nm).

RT-PCR Amplification of PAFr mRNA

In order to clone PAFr, 10 blastocysts derived from IVF or SCNT were washed in three changes of PBS and transferred into 0.2 ml of 4 M guanidium isothiocyanate (GITC) lysis solution containing 1% ß-mercaptoethanol. For positive control, 100 mg of porcine liver [27] was finely minced with a surgical blade and then transferred into 1 ml TRIzol Reagent (Life Technologies). Total RNA was extracted by thiocyanate extraction and dissolved in 12 µl of RNase-free water as described by Szafranska et al. [28]. The concentration of total RNA was determined by the absorbance at 260 nm using a Beckman DU 600 spectrophotometer (Beckman Instruments, San Ramon, CA). Total RNA (1 ng from 10 IVF or SCNT embryos or 1 µg from liver tissue) was subjected to reverse transcription-polymerase chain reaction (RT-PCR). Reverse transcription was carried out at 37°C for 60 min. Individual RT reactions (15 µl each) consisted of 5 mM MgCl2, 1x RT buffer, 2.5 µM oligo(dT), 1 mM deoxynucleotide triphosphate (dNTP), and 50 IU murine leukemia virus reverse transcriptase (Amersham Pharmacia Biotechnologies, Oakville, ON, Canada). Porcine ß-actin was used as an internal positive control for PCR. The primers for ß-actin were designed based published sequences (GenBank accession number U07786). The primers were sense, 5'-CCATGTACGTGGCCATCCAGGCTGT-3' and antisense, 5'-ATCTGCTGGAAGGTGGACAGCGAG-3'. To clone the complete coding region of the PAFr gene, one set of primers amplifying both PAFr transcripts 1 and 2 was designed based on the published sequence of the porcine PAFr gene coding sequence (accession number AF124054). The primers were sense, 5'-CTCCAGCCCATAAGGATGGAGCCA-3'; antisense, 5'-CCATGAAGGAGAAGCCTCTGGGCCT-3'. The cDNA (5 µl) was amplified in 50 µl of PCR reaction containing 1.25 units hot start Taq polymerase (Qiagen, Hilden, Germany) and its buffer, 1.5 mM MgCl2, 2 mM dNTP, and 25 pmol specific primers. The PCR amplification was carried out for one cycle with denaturing at 95°C for 15 min, and 35 subsequent cycles with denaturing at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 90 sec, and a final extension at 72°C for 15 min. Amplified PCR products were subjected to Southern blot analysis. Ten microliters of PCR products were fractionated on a 1.5% agarose gel, and stained with ethidium bromide. The PCR products were transferred to a nylon membrane and hybridized with a digoxigenin-labeled 343-base pair (bp) cDNA probe for porcine PAFr (accession number AF124054, 407 to 749 bp) following the manufacturer's recommended procedure (Roche, Penzberg, Germany). After washing, the membranes were exposed to Kodak Omat x-ray film (Eastman Kodak Co., Rochester, NY). The PCR product purified from the gel with an agarose gel extraction kit (Qiagen, Hilden, Germany) was cloned into pCRTopo cloning vector (Invitrogen, San Diego, CA). Sequence analysis was performed to further confirm the identity of amplified cDNA using an automated DNA sequence analyzer (ABI 3100, Applied Biosystem, Foster City, CA).

Quantification of PAFr mRNA Expression

In order to determine the temporal expression of PAFr during preimplantation, the same number of cells (forty 1-cell embryos, twenty 2-cell embryos, ten 4-cell embryos, five 5- to 8-cell embryos, three embryos at the morula stage, and 1.5 blastocysts) were collected based on our total cell number counting and a previous report [29]. Total RNA was extracted, dissolved in 8 µl of RNase-free water as described by Szafranska et al. [28], and subjected to a 15-µl RT reaction. For quantification, internal primers for PAFr were designed and semiquantitative PCR amplification was performed. The primers were sense, 5'-GCTCAGTGTCCTTCCTGGCTGTCATC-3'; antisense, 5'-ACCGTGCAGACCATCCAGAGTGCCCG-3'. The cDNA (5 µl) was amplified in a 50-µl PCR reaction containing 1.25 units hot start Taq polymerase and its buffer, 1.5 mM MgCl2, 2 mM dNTP, and 25 pmol specific primers. The PCR amplification was carried out for one cycle with denaturing at 95°C for 15 min, and 35 subsequent cycles with denaturing at 95°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 90 sec, and a final extension at 72°C for 15 min. Amplified PCR products were subjected to Southern blot analysis with the digoxigenin-labeled 343-bp cDNA probe, and the photographs were scanned and quantified using the Scion image analysis program (Scion Corp., Frederick, MD). Each treatment was replicated five times.

Preparation of PAF for Medium Supplement

An approximately equimolar mixture of hexadecyl and octadecyl isoforms of PAF (1-O-octadecyl/hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) was used in PAF supplementation in embryo culture medium. Stock solution (10 mg/ml) was dissolved in chloroform, aliquoted, and stored at –20°C until further use. For treatment, PAF was dissolved in NCSU-23 medium containing 4 mg/ml BSA by vigorous vortexing for 3 min, and the desired concentration of PAF was achieved by serial dilution with NCSU-23 medium [21].

Treatment of Exogenous PAF

All embryos produced by SCNT or IVF were randomly allotted into each experimental treatment (five to eight embryos/treatment). To determine the physiological role of PAF, IVF, and SCNT, embryos were cultured in NCSU-23 medium supplemented with different concentrations of PAF (0, 0.037, 0.37, 3.72, or 37.2 nM) for 7 days. In another experiment, SCNT embryos were cultured in NCSU-23 medium for 48 h in the presence or absence of the optimal concentration of PAF (3.72 nM) and then transferred to fresh medium with or without PAF for an additional 120 h. The rate of cleavage and blastocyst formation was monitored at 48 h and 168 h after culture, respectively. The total cell number was counted in blastocysts at Day 7.

Regulation of PAFr mRNA by Exogenous PAF Treatment

To investigate whether PAF regulates PAFr mRNA expression, SCNT embryos were treated with 3.72 nM PAF for 168 h. Total RNA was extracted from 10 embryos and subjected to semiquantitative PCR and Southern blot analysis. Relative PAFr mRNA expression levels were represented as the ratio of PAFr to ß-actin. The cDNA (5 µl) for ß-actin was amplified in the same conditions as PAFr. The data are represented as the percentage change relative to control (untreated SCNT embryos). The experiments were repeated three times with different samples.

Statistical Analysis

All data were analyzed by one-way ANOVA and the protected least significant different (LSD) test using general linear models in a Statistical Analysis Systems (SAS, Cary, NC) program to determine differences among experimental groups. Significant difference among the treatment was determined when the P value was less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of PAFr mRNA in Porcine IVF and SCNT Blastocysts

Using one set of primers, the complete coding region of the PAFr gene was amplified by RT-PCR amplification. As shown in Figure 1 (upper panel), the predicted PCR products (1068 bp) were obtained and validated as PAFr by hybridization with a specific probe for PAFr cDNA (Fig. 1, lower panel) in IVF and SCNT blastocysts. The possibility of genomic DNA or cross-contamination was ruled out, because no PCR products were observed and detected in negative controls (without template, Tm–; and without reverse transcriptase in the RT reaction, RT–) by ethidium bromide staining and Southern blot analysis. For positive control, an expected 1068-bp PCR product was amplified and validated in porcine liver tissue. Sequencing analysis further confirmed the identity of the amplified PCR products as PAFr (data not shown).



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  		FIG. 1. Detection of PAFr mRNA by RT-PCR amplification and Southern blot analysis. First-strand cDNAs from the porcine liver (positive control), IVF, and SCNT blastocysts were amplified using one set of PCR primers derived from porcine PAFr. The expected products were observed on an ethidium bromide-stained gel (top panel). The PCR products were transferred onto a nylon membrane and hybridized with a digoxigenin-labeled 403-bp cDNA probe (bottom panel)

Temporal Expression of PAFr mRNA During In Vitro Embryo Development

In order to quantify the expression level of PAFr mRNA during embryo development, semiquantitative RT-PCR and Southern blot analysis were performed. To determine the conditions under which PCR amplification for PAFr mRNA was in the logarithmic phase, 5 µl of cDNA from 10 IVF or SCNT blastocysts were amplified using different numbers of PCR cycles (30, 35, and 40). A linear relationship between PCR products and amplification cycles was observed in PAFr mRNA (data not shown). Thirty-five cycles were employed for quantification of PAFr mRNA. As shown in Figure 2 and Table 1, PAFr mRNA was detected during all developmental stages of IVF embryos; it was especially highly expressed in 2-cell to 8-cell embryos, it decreased at the 4-cell and morula stages, and began to rise again at the blastocyst stage. In SCNT embryos, PAFr mRNA was detected during the developmental stage, but its expression was varied at the 2-cell and 4-cell embryo and blastocyst stages (Fig. 2, Table 1).



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  		FIG. 2. Temporal expression of PAFr mRNA during preimplantation development of porcine IVF and SCNT embryos. Total RNA prepared from porcine IVF (upper panel) and SCNT embryos (lower panel) at different developmental stages was subjected to RT-PCR and Southern blot analysis with a digoxigenin-labeled 403-bp cDNA probe. Lane 1, 1-cell stage embryos; lane 2, 2-cell stage embryos; lane 3, 4-cell stage embryos; lane 4, 5- to 8-cell stage embryos; lane 5, morulae; lane 6, blastocysts


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  		TABLE 1. Expression pattern of PAFr mRNA in porcine IVF and SCNT embryos at various developmental stages.*

Effect of Different Concentrations of PAF on the Development of IVF and SCNT Embryos

In IVF embryos, adding PAF had no effect on cleavage rates, blastocyst formation, and total cell numbers in blastocysts (Table 2). In SCNT embryos, the presence of 0.37 and 3.72 nM PAF in the culture medium significantly (P < 0.05) increased the formation of blastocysts (23.1% and 26.4%, respectively) in comparison to the control group (9.5%) (Table 3). No difference was observed in cleavage rates and total cell numbers in blastocysts among experimental groups.


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  		TABLE 2. Effect of various concentrations of PAF supplement on the development of porcine IVF embryos


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  		TABLE 3. Effect of various concentrations of PAF supplement on the development of porcine SCNT embryos

Effect of Different Supplement Timing of PAF

Because the embryotrophic effect of PAF was observed only in SCNT embryos, the subsequent experiment was performed with only SCNT embryos. As shown in Table 4, culturing SCNT embryos with 3.72 nM PAF for the entire 168 h significantly improved development to the blastocyst stage (23.6%) compared to other experimental groups (10.1% to 18.6%). Exposure to PAF during only the first 48 h or during the latter 120 h did not improve the developmental competence of SCNT embryos (13.5% or 18.6% vs. 10.1%, respectively). No difference in cleavage rates and total cell number were observed among the experimental groups.


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  		TABLE 4. Effect of different timing of supplementation with PAF in culture medium on the development of porcine SCNT embryos

Regulation of PAFr mRNA Expression by PAF Treatment in SCNT Blastocysts

In order to investigate whether exogenous PAF regulates its receptor, the level of PAFr mRNA expression in PAF-treated and control blastocysts were compared (Fig. 3). The relative expression level of PAFr gene in IVF blastocysts was significantly higher (4.5-fold) than in untreated SCNT blastocysts (P < 0.05). Supplementing PAF in in vitro culture medium of SCNT embryos significantly increased the PAFr mRNA expression (a 2.0-fold increase vs. IVF blastocysts, P < 0.01; and a 9.1-fold increase vs. untreated SCNT embryos, P < 0.001).



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  		FIG. 3. Representative gel photograph of RT-PCR analysis of PAFr mRNA in PAF-treated SCNT blastocysts (upper panel). Total RNA was extracted from 10 blastocysts from each group and PAFr mRNA (403 bp) levels were normalized against ß-actin mRNA (673 bp). Lane 1, negative control; lane 2, untreated IVF blastocysts; lane 3, untreated SCNT blastocyst; lane 4, PAF-treated SCNT blastocysts. Data were expressed as percentage change relative to untreated IVF blastocysts and represent the mean ± SE of three different experiments (lower panel). a, P < 0.05 vs. IVF control; b, P < 0.05 vs. untreated SCNT blastocysts


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In order to investigate the role of PAF in porcine preimplantation embryos, the present study investigated the expression of PAFr mRNA and the effect of PAF supplement on embryo development. The results demonstrated that PAFr mRNA is expressed throughout the preimplantation developmental stage in both IVF and SCNT embryos. However, in SCNT embryos, PAFr mRNA expression was weaker than that of IVF embryos, and was especially retarded at the 2-cell, 4-cell embryo and blastocyst stages. When added to culture medium, exogenous PAF increased the rate of blastocyst formation and upregulated its receptor mRNA expression in SCNT embryos.

The evidence for the effect of PAF via its receptor, G-protein linked receptor, during embryo development was reported in studies using antibody [30] or antagonist [17, 31]. The PAF and its receptor have shown to be the biomarker of embryonic viability [32], implantation [24, 31], and sperm motility [25]. In view of the reduced secretion of embryo-derived PAF after in vitro culture [33], PAF may be a limiting factor in maintaining embryo viability during culture in vitro. The expression of PAFr and the effect of PAF have been demonstrated in porcine peri-implantation embryos and endometrium [24]. In the present study, the expression of PAFr mRNA in IVF and SCNT embryos was detected by RT-PCR amplification and confirmed by Southern blot and sequence analysis. The pattern of PAFr mRNA expression was also examined during the in vitro preimplantation embryo development stage. The amount of total RNA extracted from different development stages of IVF or SCNT embryos was adjusted by total cell number based on our total cell number counting and a previous report [29]. In IVF embryos, the PAFr mRNA was highly expressed through 2-cell to 8-cell embryos and decreased at the morula stage before beginning to rise again at the blastocyst stage. The PAFr mRNA expression was detected steadily in IVF embryos, while its expression in SCNT embryos was retarded at the 2-cell, 4-cell, and blastocyst stages, similar to the findings in mouse preimplantation embryos [34]. In contrast, a different pattern of PAFr mRNA expression was reported in another study with mouse embryos [35]. A different PAFr expression pattern in IVF embryos found between the study by Stojanov and O'Neill [35] and the present study may be due to species difference, experimental conditions, protocols for RNA extraction and detection of mRNA expression, or number of pooled embryos. One of the possible reasons for the different expression level and pattern of PAFr mRNA between IVF and SCNT embryos may be due to incomplete activation or aberrant epigenetic reprogramming of SCNT embryos. To support this idea, it has been reported that expression patterns of essential genes in in vitro-produced embryos is altered by culture conditions [36, 37], SCNT procedure [38], or in vitro production system [39]. In our preliminary results, we measured the expression level of PAFr mRNA in SCNT and parthenogenetic embryos derived from a different activation protocol (treatment of embryos with 7.5 µg/ml CB at 2 h after electric activation). It has been demonstrated that exposure of SCNT embryos to CB improved the rate of blastocyst formation and increased the number of total cells in blastocysts [40]. As a result, CB treatment induced a steady expression of PAFr mRNA in all stages of embryos derived from SCNT embryos. In particular, CB treatment induced a consistent expression of PAFr mRNA at the 4-cell stage of SCNT. However, the possibility cannot be excluded that the difference may be intrinsic to embryos themselves; that is, IVF and SCNT embryos may be programmed to express sets of gene with different patterns and levels of expression.

In vivo, autocrine, paracrine, or endocrine factors (or a combination of these) support embryo development and survival. Recent studies have demonstrated the presence and the importance of growth factors, including PAF, during the development of porcine embryos [24, 41] and the epithelium of the oviduct [42]. Based on PAFr expression in porcine IVF and SCNT embryos, we hypothesized that PAF supplementation would improve the developmental competence of IVF and SCNT embryos. Under the present experimental conditions, addition of PAF had no significant effect on IVF embryo development (Table 2). Consistent with our results, exogenous EGF supplement did not improve porcine IVF embryo development [13, 42]. In contrast, PAF supplement significantly increased during blastocyst development of SCNT embryos (Table 3). The supplement timing of PAF affected the developmental competence of SCNT embryos. Culturing SCNT embryos with PAF for an entire 168 h significantly improved their development to the blastocyst stage, while PAF supplementation during only the first 48 h or the latter 120 h did not improve the developmental competence of SCNT embryos. In contrast to blastocyst formation, PAF supplement did not increase the total cell number in blastocysts derived from IVF and SCNT embryos (Tables 2 and 3). Our IVF embryo development results in PAF-supplemented culture medium were different from some mouse embryo studies with PAF supplementation [21], in which PAF improved embryo development and increased the cell number during in vitro culture. In line with our results, it has been demonstrated that addition of exogenous growth factors in mouse embryo culture media was attributable to blastocyst formation in vitro, but did not increase the total cell number in blastocysts [43, 44]. Early embryo autocrine factors are known to act as survival factors, protecting some cell types from apoptosis [21, 45, 46] and improving embryo viability as evidenced by enhanced pregnancy rates in mice [16, 47]. These results suggest that PAF may improve embryo viability by inhibiting apoptosis in blastomeres. Further study will be needed to prove this hypothesis. The precise reasons for different effects of PAF supplementation on IVF and SCNT embryo development are not currently known. SCNT embryos were shown to have a lower rate of blastocyst formation and number of total cells in blastocysts compared to those found in IVF-derived embryos [48, 49]. In our results, a lower rate of blastocyst formation and a smaller number of total cells were also observed in SCNT embryos compared to IVF embryos (Tables 2 and 3). For these reasons, it has been suggested that SCNT embryos may have a deficiency in unknown factors that are essential to embryo growth [48]. In the present study, the amount of embryo-derived PAF may not be sufficient and thus, supplementing exogenous PAF may improve SCNT embryo development, suggesting that PAF may be one of several essential factors for SCNT embryo development. In contrast, IVF embryos may produce a sufficient amount of PAF for embryo development and thus, exogenous supplementation of PAF may not have any additional effect on embryo development. The measurement of the amount of embryo-derived PAF in culture medium will need to support this idea.

Embryos respond in a specific manner to exogenous PAF during the different developmental stages via the expression of functional PAFr molecules [43]. In this study, the expression of PAFr mRNA was upregulated in SCNT blastocysts cultured with PAF, suggesting that the embryotrophic effect of PAF in SCNT embryos was mediated through its specific receptor. In humans, PAF positively regulates the PAFr transcript [50], whereas there is no correlation between embryo-derived PAF and its receptor expression in mouse in vitro culture [34].

In conclusion, the present study demonstrated that PAFr mRNA was expressed in porcine IVF and SCNT embryos during preimplantation embryo development, and that supplementation of PAF improved in vitro developmental competence of SCNT embryos through its specific receptors.


   ACKNOWLEDGMENTS
 
We thank Dr. Barry D. Bavister for editing the manuscript.


   FOOTNOTES
 
1 Supported by grants M10310060000-03B4606-00000 from the Korean Ministry of Science and Technology and SC14031 from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Korean Ministry of Science and Technology. The authors acknowledge a graduate fellowship provided by the Ministry of Education through the BK21 program. Back

2 Correspondence: Byeong Chun Lee, Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea. FAX: 82 2 884 1902; firstlee{at}snu.ac.kr Back

Received: 2 December 2003.

First decision: 18 December 2003.

Accepted: 21 April 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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