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a Human Reproduction Unit, Department of Physiology, University of Sydney, Royal North Shore Hospital of Sydney, St. Leonards, New South Wales 2065, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amanitin sensitive. The results indicated that mRNA for this receptor is a maternal transcript that was degraded during the zygote–2-cell stage. New expression of the receptor transcript required activation of the zygotic genome. Fertilization of embryos in vitro caused this transcript not to be expressed in the zygote. Culture of zygotes (irrespective of their method of fertilization) caused expression from the zygotic genome to be retarded by more than 24 h. This retardation did not occur if culture commenced at the 2-cell stage. The transcripts for the subunits of intracellular PAF:acetylhydrolase were not detected in oocytes or at any stage of embryo development examined, despite their being readily detected in control tissue. This study confirms the presence of the G protein-linked PAF receptor in the 2-cell embryo and describes for the first time its normal pattern of expression during early development. The adverse effects of in vitro fertilization (IVF) and embryo culture on the expression of this transcript may be a contributing factor for the poor viability of embryos produced in this manner. The reduced expression of PAF-receptor mRNA following IVF predicts that such embryos may have a deficiency in autocrine stimulation and also suggests that supplementation of growth media with exogenous PAF would be only partially beneficial. The effect of IVF and culture may also explain the conflicting literature.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Selective PAF-receptor antagonists inhibit this trophic action of PAF on the preimplantation embryo [1, 6]. Treatment of rodents during early pregnancy with a range of PAF-receptor antagonists reduced the incidence of successful implantation [9–13]. However, the failure of some investigators [14] to observe this effect, and the discovery by others [15] that the effective dose range of antagonists was very narrow, called into question the nature of the PAF receptor present in the early embryos.
One well-characterized receptor for PAF [16] is a member of the G protein-linked superfamily of plasma membrane receptors that possess 7 transmembrane spanning domains, an extracellular ligand-binding domain, and an intracellular effector domain. Transgenic overexpression of the receptor caused reduced fertility of an undefined nature [17], while its genetic knock-out was without obvious reproductive phenotype [18]. A second class of putative receptors has been proposed that is intracellular and implicated in neural function [19]. This receptor class has not yet been identified or characterized.
Using reverse transcription-polymerase chain reaction (RT-PCR), one study failed [20] to demonstrate the presence of mRNA for the G protein-linked receptor in human preimplantation embryos. This contrasts with a recent report [8] showing the presence of this mRNA in 2-cell mouse embryos. These conflicting reports arise despite evidence that PAF exerts trophic actions on both mouse [2, 6] and human [7] preimplantation embryos.
A further interesting aspect of the biology of PAF in the early embryo is that embryos produced by in vitro fertilization (IVF) release 7-fold less PAF than corresponding embryos produced by fertilization in the reproductive tract [3]. We have previously demonstrated that mouse embryos possess the biosynthetic pathways for PAF synthesis [21] and that a rate-limiting enzyme (lysoPAF:acetyltransferase) in this pathway was activated by fertilization [22]. It was also found that the early embryo possessed only a limited capacity for PAF degradation [21], suggesting that PAF-acetylhydrolase activity was limiting in the early embryo.
Based on a crystal structure analysis [23] of the intracellular form of PAF-acetylhydrolase, it was proposed that the intracellular form of the enzyme is a G protein-like trimer. The 45-kDa subunit has structural homology with p-21Ras and the -subunit of G protein, while the 30-kDa subunit is essential for normal neural cortical development. These observations raise the question whether that enzyme may form an intracellular receptor/signal transducer. In view of the conflicting reports on the expression of the G protein-linked PAF receptor, it was of interest to assess the expression of the PAF:acetylhydrolase enzyme in the embryo. Furthermore, a possible cause of the reduced release of PAF by IVF embryos would be the enhanced expression of PAF-acetylhydrolase enzymes resulting in enhanced turnover and thus reduced net synthesis.
The aim of the current study was 1) to confirm the status of expression of the G protein-linked PAF-receptor gene in the 2-cell embryo, 2) to examine its ontogeny of expression from fertilization and throughout the preimplantation phase, 3) to assess the effects of IVF and culture in vitro on the normal pattern of expression of this gene, and 4) to study the expression of genes coding for PAF-acetylhydrolase in preimplantation embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocytes and embryos were collected in Hepes-buffered synthetic human tubal fluid medium (Hepes-HTF) [24]. To support development of zygotes through the 2-cell block, embryos were cultured in modified synthetic human tubal fluid medium (modified-HTF) [3]. All components of media were tissue culture grade from Sigma Chemical Company (St. Louis, MO). Unless otherwise stated, all media were supplemented with 3 mg/ml BSA (fraction V; CSL Ltd., Melbourne, Victoria, Australia).
Embryos
Random-bred Swiss albino mice (University of Sydney, NSW, Australia), 8–10 wk old, were superovulated by i.p. injection of 10 IU eCG (Folligon; Intervet International, Boxmeer, Holland) followed 48 h later by an i.p. injection of 10 IU hCG (Chorulon, Intervet). Animals were either left unmated or paired overnight with males of proven fertility. The presence of a copulation plug indicated Day 1 of pregnancy.
Cumulus-oocyte masses or embryos were flushed from the reproductive tract with Hepes-HTF. Embryos of desired stages of development were recovered by flushing the oviducts or uterus of mated animals (21, 24, 27, 36, 42, 52, 66, and 90 h post-hCG injection). Oocytes fertilized in situ (ISF) were collected from the oviducts 20–21 h after hCG. They were freed of any remaining cumulus cells by brief exposure to 300 IU hyaluronidase (Sigma) in Hepes-HTF. They were then thoroughly washed in five changes of Hepes-HTF.
IVF was performed as previously described [25]. The fertilization status of each oocyte was assessed at 5–6 h after insemination by visualization of pronuclei. All fertilized oocytes were extensively washed in Hepes-HTF to remove sperm and cumulus cells and then pooled. In some experiments, the requirement for new transcription for mRNA detection was assessed by transferring 2-cell embryos into either modified-HTF (control) or modified-HTF containing 11 ng/µl of -amanitin (Sigma).
Embryos were cultured at a concentration of 10 embryos in 10-µl volumes of modified-HTF using 60-well HLA plates (Nunc, Naperville, IL). The entire culture plate was overlaid by approximately 2 mm of heavy paraffin oil (BDH Laboratory Supplies, Poole, UK). Culture plates were equilibrated in the culture incubator for at least 4 h prior to use.
Detection of Gene Expression
The mRNA for the PAF receptor in tissue (liver, endometrium), oocytes, and embryos and for PAF-acetylhydrolase enzymes in tissue (brain), oocytes, and embryos were detected by RT-PCR on extracted RNA. The mRNA for the PAF receptor in oocytes and embryos was also detected in whole oocyte/embryo homogenates [26] using RT-PCR and nested RT-PCR.
For all RT-PCR assays performed, four controls were always undertaken. 1) Mouse ß-actin was used as an internal positive control for the efficiency of all RNA extractions and RT-PCR reactions performed on those samples. The use of specific 3'-primer during the RT step (to increase RT-PCR sensitivity) for oocyte/embryo homogenates excluded the use of internal ß-actin control for such samples; however, ß-actin-positive controls were performed on identically treated material and run in parallel for every set of RT-PCR. The ß-actin primer pair was designed so that it spanned the first intron (87 base pairs [bp] in length) of the rodent ß-actin gene [27]. Thus contaminating genomic DNA could be detected using these primers. 2) Reverse transcriptase was omitted from some samples to control for false-positive PCR amplification of contaminating genomic DNA. 3) Water was added instead of sample to test for contamination with extraneous DNA. 4) Some samples were treated with RNase I (Promega Corp., Madison, WI) prior to RT, confirming the RNA origin of positive RT-PCR reactions.
PCR reaction products were analyzed by electrophoresis on 4% agarose gel stained with ethidium bromide to visualize PCR product on a UV transilluminator. Fragments were verified by size and restriction enzyme mapping. PCR product was precipitated, re-diluted in the enzyme buffer, and incubated with the desired digestion enzyme. Predicted fragments of the restricted PCR product were verified by size. The transcripts were also confirmed by sequencing of the PCR product, using ABI PRISM Dye terminator Cycle Sequencing Ready Reaction Kit from Perkin-Elmer (Foster City, CA) and performed at SUPAMAC (Redfern, NSW, Australia). Primers were obtained from Fisher Biotech (Perth, WA, Australia). The primers for PAF receptor were designed using the Prima computer program (ANGIS, Sydney, NSW, Australia). The size of the PCR product and the sequences of primers are shown in Table 1.
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RNA Extraction
Fifty to two hundred oocytes (stripped of cumulus cells) or embryos were thoroughly washed in five changes of Hepes-HTF and three changes of PBS and then transferred in a minimal volume of PBS into 0.8 ml of TRIzol Reagent (Life Technologies, Gaithersburg, MD) containing 50 µg of carrier RNA (yeast tRNA; Sigma). Alternatively, 100 mg of mouse tissue (controls: liver, endometrium, or brain tissue) was finely minced with surgical blade and then transferred into TRIzol Reagent. Samples were then homogenized by vigorous shaking by hand, incubated with chloroform, and centrifuged. The top-aqueous phase containing RNA was transferred into a new Eppendorf tube, and RNA was precipitated with isopropyl alcohol (BDH) rinsed in 75% (v:v) ethanol (BDH), centrifuged, and left to air dry. Isolated RNA was treated with DNase, to eliminate possible contamination with genomic DNA, by resuspending the RNA pellet in 20 µl of resuspension solution (RS; 40 mM Tris-HCl, pH 7.9, 10 mM NaCl, and 6 mM MgCl2) [28] containing 2 U of RQ1 DNase (Promega), and incubated at 37°C for 30 min. After addition of a second equal volume of RS, RNA was reextracted with phenol-chloroform. The RNA pellet was dissolved in double-autoclaved tissue culture grade water in the presence of RNase Inhibitor (Promega) (final concentration 1 U/ml). The concentration of extracted total RNA from tissue samples was measured using a Beckman DU 600 spectrophotometer (Beckman Instruments, Jan Ramon, CA) and adjusted to the required concentration. The RNA yield was then either immediately subjected to RT-PCR or stored at -70°C.
RT-PCR of Extracted RNA
RNA from the equivalent of 20 embryos or oocytes were placed in a thin-wall 0.6-ml Eppendorf tube overlaid with 50 µl of paraffin liquid (BDH) and reverse transcribed by incubating at 42°C for 30 min with 2.5 U MuLV Reverse Transcriptase primed with 2.5 µM oligo(dT) (Perkin-Elmer) in a 20 µl of reaction mix containing 5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1 mM each dNTP, and 1 U RNase inhibitor (all reagents supplied by Perkin-Elmer). The RT reaction was then terminated by heating at 98°C for 5 min and cooling to 5°C.
Ten microliters of RT reaction volume was used for test sample and 10 µl for internal ß-actin control in a final PCR reaction volume of 50 µl containing 1.6 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 0.2 mM each dNTP, 1.5 U AmpliTaq DNA polymerase (all reagents supplied by Perkin-Elmer), and 0.4 µM each of specific primer pairs using Hybaid Thermal Reactor (Hybaid Limited, Teddington, Middlesex, UK).
Nested RT-PCR
Detection of mRNA for the PAF receptor was also performed using nested primers. Oocytes (stripped of cumulus cells) and embryos were thoroughly washed in three changes of Hepes-HTF, and then up to 20 oocytes or embryos were transferred in minimum volume of culture medium into 0.2-ml thin-wall Eppendorf tubes with 15.5 µl of reaction mix (identical to mix used for non-nested RT-PCR of extracted RNA). Samples were immediately placed in a prewarmed PCR cycling machine (Corbett Thermal Reactor; Corbett Research, Mortlake, NSW, Australia), heated at 99°C for 1 min, and then placed on ice. A mixture of 1 µl of RNA RQ I DNase (Promega) to destroy genomic DNA and 0.5 µl of RNase inhibitor was added; the sample was incubated at 37°C for 30 min, followed by 2 min at 99°C, and placed again on ice. Whole sample volumes containing embryonic RNA were either immediately subjected to nested RT-PCR or stored at -70°C.
The whole sample containing embryonic RNA obtained by nonextraction preparation was reverse transcribed by incubating at 42°C for 30 min with 1.5 U MuLV Reverse Transcriptase (Perkin-Elmer) primed with either 2.5 µM oligo(dT), 2.5 µM random hexamers (all reagents supplied by Perkin-Elmer), or 2 µM specific outer 3'-primer. The RT reaction was terminated by heating at 99°C for 5 min and cooling to 5°C.
When RT was performed with specific outer 3'-PCR primer, 10 µl of RT reaction volume was used for 35 cycles of first-round PCR in a final PCR reaction volume of 50 µl (same as for RT-PCR). In the presence of oligo(dT) or random hexamers, 10 µl of RT reaction volume was used for the test sample and 10 µl for internal ß-actin control. An aliquot (6.5 µl) of the PCR product from the first round was added to a second PCR reaction volume (50 µl) primed with 0.4 µM of each specific inner 5'- and 3'-primer and was subjected to 35 cycles of second-round PCR using the Corbett thermal reactor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial experiments with embryos used RT-PCR with 50 cycles of amplification. RNA was extracted from the equivalent of 20 oocytes or embryos collected at various stages during the preimplantation phase (Table 2). The results are expressed as the proportion of oocyte or embryo groups tested in which the transcript was detected. PAF-receptor mRNA was not expressed in any group of oocytes and was expressed only in one group out of six freshly fertilized zygotes (21 h post-hCG). By the 2-cell stage (42 h post-hCG), expression was more frequent. From the 4-cell stage onward, PAF-receptor expression was consistently observed (Table 2). As an internal control, ß-actin mRNA was also assessed in all samples and was detected in all cases.
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To determine whether the negative and variable results in oocytes and early-stage embryos were a consequence of limited sensitivity of the technique, further validation and optimization of the RT-PCR technique were undertaken. Samples containing mouse liver total RNA in the range of 100–0.001 ng (total RNA content in mouse preimplantation embryos ranges from 0.24 ng for 2-cell embryos to 1.47 ng for blastocysts [30]) were compared using RT-PCR for the PAF receptor (50 cycles) and nested RT-PCR (35 cycles with outer primers and a further 35 cycles with inner primers) (Fig. 1). The primers used in RT-PCR acted as the inner primers for nested RT-PCR, and new outer primers were designed. The limit of detection was 0.1 ng for 50 cycles and 0.01 ng for nested-primer RT-PCR. This comparison was performed using specific 3'-primer for the RT reaction. To determine the type of primers for the RT reaction with the greatest sensitivity, oligo(dT), random hexamers, and specific 3'-primers were compared. Figure 2 shows that for 0.1 ng liver total RNA, specific 3'-primers gave a more sensitive assay compared to RT catalyzed with either oligo(dT) or random hexamers. This was the case for both nested RT-PCR and RT-PCR with 50 cycles of amplification (Fig. 2). Only nested RT-PCR catalyzed with 3'-specific primer detected 0.01 ng of liver total RNA (Fig. 2). Figure 3 shows a comparison of nested RT-PCR and RT-PCR of mouse oocytes, and also compares the sensitivity of specific 3'-primer and oligo(dT) for RT. The results showed that only nested RT-PCR, regardless of the primer used for RT, could detect mRNA for PAF receptor in mouse oocytes.
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Extracting the very small amounts of RNA present within the preimplantation embryo may result in the loss of RNA. Even small losses during the extraction procedure may result in artifactual results. Therefore the sensitivity of RT-PCR in embryos subjected to RNA extraction and that of direct RT-PCR of lysed embryos were compared. Nested RT-PCR was consistently more sensitive when performed on embryos without extraction (lysed embryos) as compared with extracted material, suggesting loss of some mRNA during the extraction procedures (results not shown).
Thus standard conditions for detecting the PAF receptor were the use of homogenized embryos/oocytes, with RT catalyzed with 3' gene-specific primers, and amplification of the resulting cDNA using nested primers with two rounds of amplification of 35 cycles each round.
When embryos and oocytes were analyzed using these standardized methods, similar results (Table 2, nested PCR; Fig. 4) were observed as for unnested RT-PCR, except that mRNA was consistently detected in oocytes and early zygotes. Expression of PAF-receptor mRNA was still variable in the late zygote and early 2-cell-stage embryo. These results show that in the periovulatory oocyte a relatively low concentration of mRNA for the PAF receptor is present, which could be detected only with the highly sensitive nested PCR technique. By the late 1-cell- to early 2-cell-stage, many embryos had lost this mRNA, but its expression increased from the 2-cell stage.
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The mRNA for ß-actin acted as a positive control and was detected at all developmental stages tested. In no case was a product formed with PAF-receptor primers in the absence of RT. However, if DNase treatment was not performed, amplification of genomic DNA was often observed in the absence of RT. Restriction analysis with BglI showed the restriction pattern predicted for the PAF receptor. Sequencing of the RT-PCR product differed from the published sequence only at nucleotide 693, where A was replaced with G. This change resulted in a difference in the amino acid sequence of the resulting protein, replacing valine with isoleucine.
Transcription from the zygotic genome becomes essential for normal development by the 2-cell stage. To examine whether the variable yet increasing expression of PAF-receptor mRNA from the 2-cell stage reflected the onset of new transcription from the zygotic genome, embryos were cultured in the presence or absence of -amanitin. At various times after exposure, embryos were tested for the presence of PAF-receptor or ß-actin mRNA (Table 3). Two-cell embryos (42 h post-hCG) cultured in vitro showed a progressive increase in the incidence of PAF-receptor mRNA over the 28 h after culture was initiated. Culture over the same period in the presence of -amanitin completely blocked the expression of this gene. By contrast, the expression of ß-actin persisted for a longer period; but by 28 h of culture in -amanitin, this transcript was also not detected.
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It was previously reported [20] that human embryos produced by IVF do not express mRNA for the PAF receptor. The effect of IVF and culture of zygotes in vitro on the pattern of expression of the PAF-receptor gene was examined and compared with the pattern of expression of embryos collected fresh from the reproductive tract using nested RT-PCR. When zygotes (irrespective of the method of fertilization) were cultured in vitro, the incidence of detection of PAF-receptor mRNA was low until the 8- to 16-cell stage (Table 4). Zygotes produced by IVF did not have detectable levels of PAF receptor at the zygote stage, but receptor mRNA was generally present in zygotes fertilized in vivo. When the culture of embryos commenced at the 2-cell stage, however, PAF-receptor transcript was routinely detected at all development stages after culture (Table 4). This contrasts with the situation for embryos collected fresh from the reproductive tract, in which PAF-receptor transcript was routinely detected by the late 2-cell to 4-cell stage (Table 2). Thus, both IVF and culture of zygotes adversely affected the qualitative expression of the receptor; but once expression from the zygotic genome had commenced, culture in vitro was without obvious effect.
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The intracellular form of the PAF catabolic enzyme PAF:acetylhydrolase is a trimer. Primers were designed for the mRNA of 45-kDa and 30-kDa subunits of the enzyme. Validation of these primers was performed using RNA extracted from mouse brain tissue. The RT-PCR reaction (50 cycles) detected transcripts for 45-kDa and 30-kDa subunits down to 0.1 and 0.001 ng of brain tissue RNA (Fig. 5), respectively. Sequencing of PCR products confirmed the correct identity of these transcripts (96% and 99% homology for the 45-kDa and 30-kDa subunits, respectively, compared to the published bovine sequences). No product was found when RT was not performed, or in the absence of brain RNA, confirming the specificity of the reaction.
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RNA from freshly ovulated oocytes; from embryos collected fresh from the reproductive tract at the 2-cell-, 8-cell-, and blastocyst-stages; and from embryos produced by IVF and cultured in vitro for 24 and 48 h, were screened with the primers for PAF:acetylhydrolase 45-kDa and 30-kDa subunits (Fig. 6). Embryo lysates were analyzed directly to minimize loss of RNA during extraction. The reactions were repeated with several different groups of embryos, and in no instances were any transcripts for these subunits detected. This was also the case when the embryo RNA concentration in the reaction was increased to the equivalent of 100 embryos. In all assays, positive controls included amplification of the mouse embryo mRNA for ß-actin and also amplification of 45-kDa and 30-kDa PAF:acetylhydrolase in mouse brain RNA in parallel assays. These positive controls confirmed the adequate function of the RT-PCR. In all cases, no product was found when RT was not performed or in the absence of RNA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of this study show for the first time that mRNA for the G protein-linked PAF receptor is present within the oocyte and zygote. This message was observed only at a low level and was not consistently detected unless nested RT-PCR was used. It remains to be determined whether such low levels of expression result in functional expression of the PAF receptor. Detection of this message became quite inconsistent by the late zygote–early 2-cell stage, suggesting that, like many other gametic transcripts [28, 31], it was degraded at this time. During the 2-cell stage of development there was a progressive increase in the incidence of detection of receptor mRNA. The ability of -amanitin to inhibit this increased expression of receptor mRNA indicates for the first time that it was a consequence of new transcription from the zygotic genome.
It was previously shown [8] that a consequence of PAF's action on the 2-cell embryo was a transient elevation of intracellular concentrations of calcium, which presumably acts as a secondary messenger. Intracellular calcium transients of similar magnitude enhanced early embryo development rates [32]. PAF supplementation of media enhanced embryo development [2, 3] and survival [5] rates and also increased embryo metabolism [4]. Careful timing studies [5] revealed that the action of PAF to enhance embryo development and survival was not required until the late 2-cell stage, while exposure to PAF during only the 1-cell and early 2-cell stage was without obvious beneficial effect. Such results may suggest that a functional PAF receptor is expressed only from the late 2-cell stage onward. If this is the case, it indicates that the low levels of receptor mRNA detected (only by nested RT-PCR) in the oocyte and zygote may not be functionally significant, at least as assessed by the effects of exogenous PAF on embryo development and survival.
The pattern of expression of mRNA for the PAF receptor was adversely affected by fertilization in vitro or culture of zygotes in vitro. The incidence of expression in zygotes after fertilization in vitro was lower than for corresponding zygotes collected fresh from the reproductive tract. The results do not show whether this was caused by a generalized increase in RNA degradation or was transcript specific, although ß-actin transcript was routinely detected in these samples.
Of interest was the observation that the onset of expression of PAF-receptor mRNA from the zygotic genome was markedly delayed in embryos that were cultured from the zygote stage. This occurred irrespective of the method of fertilization. It was previously shown that culture in vitro markedly reduced the release of PAF from embryos [33]. It will be of interest to determine whether the two processes—PAF release from embryos and expression of PAF-receptor gene from the zygotic genome—are related phenomena. Once PAF-receptor gene expression from the zygotic genome was initiated (late 2-cell stage), however, subsequent qualitative expression was not influenced by culture in vitro. This suggests that it may be the activation of this gene, rather than subsequent transcription, that is adversely affected by culture.
It was previously shown that supplementation of culture medium with PAF enhanced the development rates and viability of mouse [6] and human [7] embryos produced by IVF. This enhancement was limited, however, with treated embryos not achieving the development rates observed for embryos collected fresh from the reproductive tract. The current observation of retarded expression of PAF-receptor mRNA from the zygotic genome after culture may provide an explanation for the limited success [6, 33] of the PAF supplementation strategy. An attempt [20] to detect the PAF receptor in human embryos produced by IVF was unsuccessful. The current study provides two potential explanations for that result. Transcription from the zygotic genome occurs later in the human embryo (4-cell stage) [34] than in the mouse embryo. Thus, it is likely that the PAF-receptor mRNA from the zygotic genome may not normally be detected in the 2-cell human embryo. If human embryos behave similarly to mouse embryos after culture in vitro, it might also be expected that expression of this gene from the zygotic genome would be delayed beyond the normal time of expression. These considerations may be sufficient to explain a failure to detect expression of this gene in embryos produced by IVF. A consideration of the role of the PAF receptor in human embryo development may need to await the screening of embryos produced by natural conception.
Some technical factors regarding the detection of the PAF-receptor gene warrant consideration. The entire coding sequence for this protein is from one exon. The absence of introns creates the potential for technical artifacts when RT-PCR is used as the detection assay, since the cDNA produced following RT is indistinguishable from genomic DNA. This is not the case for genes containing introns, and that difference can be exploited to test that the DNA being amplified is in fact the target cDNA rather than genomic DNA. The correct detection of intronless genes requires that the sample be completely free of genomic DNA prior to RT. In the present experiments, this was achieved by extensive digestion with DNase prior to RT. A number of controls were included in each experiment to confirm the cDNA origin of the PCR product. Where specific 3'-primers were used for RT, it was necessary to run ß-actin-negative controls on parallel samples. This is not as desirable as running control within the same sample, but this disadvantage was offset by the increased sensitivity and reliability of 3'-primer RT in relation to other methods. Furthermore, for standard RT-PCR on extracted embryo samples, ß-actin was performed as an internal standard, with the same results. Controls always included 1) the absence of RT—in the case of genomic contamination a transcript would detected; 2) RT-PCR with water instead of tissue added—this controlled for extraneous DNA contamination; and 3) ß-actin primers, which were designed across exons—thus genomic contamination showed as 2 bands of differing size (243-bp cDNA; 330-bp genomic DNA). In some samples, embryo extracts were digested with RNase prior to RT; the absence of a PCR product confirmed that products were derived from cDNA.
The number of cycles of amplification performed in this study agrees well with other RT-PCR studies performed on preimplantation-stage embryos. Temeles et al. [35] determined that for the ß-actin gene, the amount of product showed an exponential increase as a function of cycle number after 45 cycles using equivalent of 5 embryos per reaction. Similarly, Hogan et al. [36] amplified the product for ß-actin from 10 to 30 embryo equivalents after 40 cycles of RT-PCR. For hypoxanthine phosphoribosyl transferase and adenosine phosphoribosyl transferase, - and ß-globins, Daniels et al. [37] used 32 + 30 cycles of nested RT-PCR. Doherty et al. [28] showed that when RNA from 2-cell mouse embryos (equivalent of 20 embryos) was used, product formation as a function of cycle number increased exponentially up to 60 cycles for insulin-like growth factor (IGF)-I. Several other studies used up to 60 cycles of RT-PCR amplification in studying the mRNA expression of various genes in mouse and human preimplantation embryos [20, 38, 39]. One study [8] demonstrated the presence of the PAF-receptor mRNA with as few as 30 cycles; however, 200 mouse embryos were used.
Primers designed against bovine 45-kDa and 35-kDa subunits for bovine intracellular PAF:acetylhydrolase (the nucleotide homology for the primer embraced regions is 93% and 92% for 45-kDa and 30-kDa subunits, respectively [40, 41]) were shown to successfully detect cDNA for these genes from 0.1 and 0.001 ng of mouse brain tissue total RNA extracts, respectively, an amount lower than the level expected in the single mouse preimplantation embryo [30]. Using the same primers, however, it was not possible to detect these gene products in any preimplantation embryo stage tested even when as many as 100 embryos per assay were used. It is concluded that this form of PAF:acetylhydrolase is not normally expressed in the early embryo and that this is not influenced by culture in vitro. This shows that this enzyme is unlikely to act as an intracellular receptor/transducer for PAF in the embryo, nor does its activity account for variations in the amount of PAF produced/released by the embryo.
This study confirms the presence of transcripts for the G protein-linked plasma membrane PAF receptor in the 2-cell embryo and shows for the first time the ontogeny of its expression during the normal preimplantation stage of development. It is also shown for the first time that IVF and/or culture of zygotes in vitro profoundly retards the onset of transcription of this gene from the zygotic genome. The intracellular trimeric PAF:acetylhydrolase gene is not transcribed in the preimplantation embryo, excluding the possibility that it may act as an intracellular receptor/transducer or that it acts to regulate net PAF synthesis. In view of this evidence confirming the presence of the G protein-linked PAF receptor in the mouse embryo, and the independent reports of the production and autocrine action of PAF on the early embryo of several species, it is of interest to note a recent description [18] of mice with the PAF receptor knocked out. These were without apparent adverse reproductive phenotype, making the role for PAF in reproduction questionable. This observation is reminiscent of findings for IGF-II and IGF-I, which also have well-defined trophic actions on the early embryo but show no relevant preimplantation-phase phenotype following gene knock-out (of either receptor or ligand). Indeed, it is surprising that many genes generally considered to have central roles in cell growth regulation are not shown by gene knock-out experiments to be essential for mammalian preimplantation development. A likely explanation is that in the normal in vivo setting there are several (or many) factors (autocrine, paracrine, and perhaps endocrine) that can serve to support embryo development and survival. They may act with varying degrees of redundancy. This scenario indicates that an understanding of the regulation of embryo development will require identification of each growth factor candidate and full characterization of the modes of action of each.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 612 992 66343; chriso{at}med.usyd.edu.au
Accepted: October 15, 1998.
Received: May 4, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 P. Sutovsky, W. Plummer, K. Baska, K. Peterman, J. R. Diehl, and M. Sutovsky Relative Levels of Semen Platelet Activating Factor-Receptor (PAFr) and Ubiquitin in Yearling Bulls With High Content of Semen White Blood Cells: Implications for Breeding Soundness Evaluation J Androl, January 1, 2007; 28(1): 92 - 108. [Abstract] [Full Text] [PDF]
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 N. Gopichandran and H. J Leese The effect of paracrine/autocrine interactions on the in vitro culture of bovine preimplantation embryos Reproduction, February 1, 2006; 131(2): 269 - 277. [Abstract] [Full Text] [PDF]
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C. O'Neill The role of paf in embryo physiology Hum. Reprod. Update, May 1, 2005; 11(3): 215 - 228. [Abstract] [Full Text] [PDF]
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S. H. Lee, D. Y. Kim, D. H. Nam, S. H. Hyun, G. S. Lee, H. S. Kim, C.-K. Lee, S. K. Kang, B. C. Lee, and W. S. Hwang Role of Messenger RNA Expression of Platelet Activating Factor and Its Receptor in Porcine In Vitro-Fertilized and Cloned Embryo Development Biol Reprod, September 1, 2004; 71(3): 919 - 925. [Abstract] [Full Text] [PDF]
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R. Levy, K. Elder, and Y. Menezo Cytoplasmic transfer in oocytes: biochemical aspects Hum. Reprod. Update, May 1, 2004; 10(3): 241 - 250. [Abstract] [Full Text] [PDF]
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 D. P. Lu, V. Chandrakanthan, A. Cahana, S. Ishii, and C. O'Neill Trophic signals acting via phosphatidylinositol-3 kinase are required for normal pre-implantation mouse embryo development J. Cell Sci., March 15, 2004; 117(8): 1567 - 1576. [Abstract] [Full Text] [PDF]
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D. P. Lu, Y. Li, R. Bathgate, M. Day, and C. O'Neill Ligand-Activated Signal Transduction in the 2-Cell Embryo Biol Reprod, July 1, 2003; 69(1): 106 - 116. [Abstract] [Full Text] [PDF]
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T. Stojanov and C. O'Neill In Vitro Fertilization Causes Epigenetic Modifications to the Onset of Gene Expression from the Zygotic Genome in Mice Biol Reprod, February 1, 2001; 64(2): 696 - 705. [Abstract] [Full Text]
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 M. Emerson, A. R. Travis, R. Bathgate, T. Stojanov, D. I. Cook, E. Harding, D. P. Lu, and C. O'Neill Characterization and Functional Significance of Calcium Transients in the 2-Cell Mouse Embryo Induced by an Autocrine Growth Factor J. Biol. Chem., July 14, 2000; 275(29): 21905 - 21913. [Abstract] [Full Text] [PDF]
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C. Wu, T. Stojanov, O. Chami, S. Ishii, T. Shimuzu, A. Li, and C. O'Neill Evidence for the Autocrine Induction of Capacitation of Mammalian Spermatozoa J. Biol. Chem., July 13, 2001; 276(29): 26962 - 26968. [Abstract] [Full Text] [PDF]
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