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Biology of Reproduction 64, 696-705 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

In Vitro Fertilization Causes Epigenetic Modifications to the Onset of Gene Expression from the Zygotic Genome in Mice1

T. Stojanov a, and C. O'Neill2,a

a Human Reproduction Unit, Department of Physiology, University of Sydney, Royal North Shore Hospital of Sydney, St. Leonards, NSW 2065, Australia

ABSTRACT

The effect of in vitro fertilization (IVF) and culture of mouse preimplantation embryos in vitro on the onset of expression of insulin-like growth factor 1 (IGF-1) ligand and receptor, insulin ligand and receptor, alpha-transforming growth factor ({alpha}-TGF) ligand, PAF:acetylhydrolase 1b (Pafah1b; {alpha}1, {alpha}2, and ß subunits of the enzyme), and the transcription requiring complex proteins (TRC) was examined. The IGF-1 ligand was detected in preimplantation embryos by immunofluorescence at all developmental stages tested. However, IVF and culture significantly reduced the amount of protein detected in the 8-cell embryo and blastocyst (P < 0.001), and this was due to a delayed onset of expression of the mRNA for IGF-1 ligand from the zygotic genome. The expression of the {alpha}1 subunit of Pafah1b was first detected at the 2-cell stage in fresh embryos, but expression was significantly retarded (P < 0.001) when IVF and ISF (in situ-fertilized) zygotes were cultured in vitro. In vitro fertilization or ISF did not delay the onset of expression of TRC nor mRNA for the IGF-1 receptor, insulin receptor, {alpha}2 or ß subunit of Pafah1b, nor did they effect {alpha}-TGF protein synthesis. Thus, IVF causes epigenetic modification in the normal pattern of expression of some but not all genes involved in normal embryo growth and survival.

fertilization, gene regulation, growth factors, platelet-activating factor, insulin, IVF/ART

INTRODUCTION

Embryos produced by in vitro fertilization and then cultured in vitro have significantly reduced viability when compared with development in vivo. The causes are likely to be multifactorial but it was shown that lower levels of production or release of autocrine growth factors (GFs) by in vitro-fertilized (IVF) zygotes made a significant contribution to the reduction of viability [1]. A role for autocrine or endogenous growth factors was suggested by observations that embryo development was less successful when embryos were cultured in relatively large volumes of medium [1–3], and evidence shows that this was due to dilution of embryo-derived diffusible autocrine factors [1, 2, 4].

The early mouse embryo expresses a range of GFs and their receptors. Platelet-activating factor (PAF) synthesis has been reported as early as the 1-cell embryo stage [5, 6], while new synthesis of insulin-like growth factor 1 (IGF-1) ligand [7, 8], insulin-like growth factor 2 (IGF-2) ligand and receptor [9–11], and growth hormone (GH) receptor [12] are expressed at the 2-cell stage; alpha-transforming growth factor ({alpha}-TGF) [13], platelet-derived growth factor (PDGF) [13], GH [12], and interleukins [14] were detected at later stages of preimplantation embryo development.

The production or release of several growth factors is delayed by IVF. O'Neill [1] showed that mouse zygotes fertilized in the reproductive tract (in situ fertilized; ISF) released approximately seven times more PAF than did IVF zygotes, while 2-cell embryos collected from the reproductive tract released 3 times and 21 times as much PAF as 2-cell embryos resulting from culture of ISF and IVF zygotes, respectively. The effect of IVF and culture of embryos in vitro on the pattern of expression of the PAF-receptor gene was also examined and compared with the pattern of expression of embryos collected fresh from the reproductive tract [15]. When zygotes (irrespective of the method of fertilization) were cultured in vitro, the onset of transcription of the PAF-receptor from the zygotic genome was markedly delayed by culture in vitro. Furthermore, 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, suggesting a faster rate of mRNA degradation after IVF.

In one study [16], it was shown that culturing embryos in medium with a high sodium concentration (125 mM) reduced the expression of IGF-1 and IGF-2 ligand and receptor mRNA compared with culture in low sodium (85 mM). Another study [17] examined the effects of medium type, IVF and embryo culture concentration on the ontogeny of expression of IGF-2 ligand and receptor in the mouse embryo and showed that onset of transcription of ligand from the zygotic genome was markedly delayed following IVF or culture in vitro.

The intracellular form of PAF-acetylhydrolase (Pafah1b) may form an intracellular receptor and/or signal transducer [18] for PAF and is essential for normal embryo development [19]. We previously reported [15] the absence of ß and {alpha}2 subunits of Pafah1b mRNA in preimplantation mouse embryos. However, using a different set of primers for Pafah1b, these transcripts were recently detected in early mouse embryos [20]. Furthermore, deletion of the ß subunit of Pafah1b by homologous recombination leads to early embryonic lethality [19], suggesting an important role for this enzyme in mediating or modulating PAF's actions in embryo development. In view of the conflicting reports on the expression of the Pafah1b and its apparently important role in normal embryo development, we have reassessed the expression of the three subunits ({alpha}1, {alpha}2, and ß) of the enzyme in the preimplantation embryo and examined the effect of IVF on their expression.

The changes in expression of a range of embryonic factors following IVF suggest that modifications in the normal pattern of onset of transcription from the zygotic genome may occur. The zygotic genome is thought to become transcriptionally permissive during the late zygote stage [21, 22], with the first major round of transcription occurring in the G1 phase of the second cell cycle [22]. The onset of transcription coincides with a sharp decline in DNA methylation [23, 24], is dependent upon cAMP-dependent kinase activity [21, 22] and is independent of DNA replication [25, 26]. The first major round of transcription includes the synthesis of a group of proteins known as the transcription-requiring complex (TRC), which is a group of proteins of approximately 70 kDa of unknown function [27]. These proteins serve as convenient markers of the activation of this first round of transcription. As such they can be used to determine whether IVF adversely affects the onset of this first round of transcription.

We used qualitative reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and radioactive pulse-chase methodology to determine whether IVF and/or culture in vitro delayed the onset of expression of some landmarks of transcription from the zygotic genome. This study shows that IVF causes epigenetic modification to the onset of zygotic gene expression of IGF-1 ligand and the {alpha}1 subunit of Pafah1b but had no obvious effect on other transcription products.

MATERIALS AND METHODS

Embryo Culture and Collection Media

Oocytes and embryos were collected in Hepes-buffered synthetic human tubal fluid medium (Hepes-HTF) [28]. To support development of zygotes through the 2-cell block, embryos were cultured in modified-synthetic HTF medium (modified-HTF) [29]. Fertilization was performed in HTF [28]. 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 BSA/ml (Fraction V; CSL Ltd., Melbourne, Victoria, Australia). In some experiments the effect of culture of zygotes in modified-HTF was compared with kSOM medium (medium that has been optimized for the support of early embryo development [30]).

Embryos

In most experiments random-bred Swiss albino mice (Quackenbush strain, 8–10 wk old) or in one experiment C57bl/6 (6–8 wk old) (both strains supplied by Laboratory Animal Services, University of Sydney, NSW, Australia) were superovulated by i.p. injection of 10 IU (5 IU for C57bl/6) equine chorionic gonadotropin (Folligon; Intervet International, Boxmeer, Holland) followed 48 h later by an i.p. injection of 10 IU (5 IU for C57bl/6) 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 were of three types: those collected fresh from the reproductive tract immediately prior to testing (fresh); those produced by fertilization in the reproductive tract but subsequently cultured in vitro (ISF); and those fertilized by IVF and cultured in vitro. Fresh embryos of desired stages of development were recovered by flushing the oviducts or uterus of mated animals (14, 21, 42, 52, 66, and 90 h post-hCG injection). The ISF zygotes were collected from the oviducts 20–21 h after hCG. They were freed of any remaining cumulus cells by brief exposure to 300 IU/ml hyaluronidase (Sigma) in Hepes-HTF and then thoroughly washed in five changes of Hepes-HTF. In vitro fertilization was performed as previously described using cumulus-intact oocyte complexes and epididymal sperm [31]. 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 zygotes into either modified-HTF (control) or modified-HTF containing 11 µg/ml of {alpha}-amanitin (Sigma), an inhibitor of RNA polymerase II.

Spermatozoa and Cumulus Cells

Motile spermatozoa were collected by diluting freshly collected epididymal sperm 10x in modified-HTF. They were then gently aspirated into a Pasteur pipette, passed through a 3-ml modified-HTF medium layer (in a 5-ml plastic tube; Falcon, Lincoln Park, NJ) to the bottom of the tube and carefully layered under the medium. After 60 min incubation at 37°C the upper 1.5 ml of the modified-HTF layer containing viable spermatozoa was carefully removed, transferred into 15-ml centrifuge tubes (Falcon), and centrifuged at 100 x g for 6 min. The supernatant was discarded, and the sperm pellet was washed three times in Dulbeccos PBS. The spermatozoa concentration was assessed and the desired number of cells was used for RNA extraction. Similarly, cumulus cells freed of oocytes by brief exposure to 300 IU/ml hyaluronidase were extensively washed by repeated centrifuging (100 x g, 6 min) and redilution in PBS at least three times. The concentration was assessed and the desired number of cells was used for RNA extraction.

Embryo Culture at Different Concentrations

Two different culture vessels were used to achieve differing volumes of culture: 1) for 10 µl volumes, 60-well (Nunc, Naperville, IL) HLA plates; and 2) for 100 µl volumes, 96-well flat-bottom plates (Flow Laboratories, McLean, VA), as previously described [1]. The medium was overlayed by approximately 2 mm of heavy paraffin oil (BDH Laboratory Supplies, Poole, UK). In the case of the 60-well plate this involved overlaying the entire plate; for the other plate, individual wells were covered with oil. Culture plates were equilibrated in the culture incubator for at least 4 h prior to use. Embryos were cultured at three different concentrations: 1) 1 embryo in a 10-µl volume of modified-HTF; 2) 10 embryos in a 10-µl volume of modified-HTF; and 3) 1 embryo in a 100-µl volume of modified-HTF.

Detection of TRC Synthesis

Zygotes produced by both fertilization methods were cultured and viewed at 1-h intervals by phase-contrast microscopy. When cleavage to the 2-cell stage occurred, embryos were immediately radiolabeled for 2 h in modified-HTF containing 1 mCi/ml L-[35S]methionine (1175 Ci/mmol; New England Nuclear Corporation, Boston, MA). Radiolabeled embryos were washed free of extracellular [35S]methionine with Hepes-HTF and treated with 50 mM Tris-HCl, pH 7.4, containing 2% Triton X-100 and 0.3 M KCl for 10 min at 25°C [22]. The TRC proteins remain insoluble under these conditions, whereas most of the radiolabeled proteins become soluble [32]. Embryos were transferred into 4 µl of sample buffer (10 mM Tris-HCl, pH 8.5; 2.5% SDS; 10% glycerol; 2.5% 2-mercapthoethanol) and analyzed by one-dimensional gel electrophoresis on a 10% Tris-glycine gel (Novex, San Diego, CA) using Tris-glycine SDS running buffer (LC-2675-4, Novex). The gel was fixed with 40% methanol and 10% acetic acid in PBS for 30 min. The fixed and dried gel was then analyzed by phosphorimaging (Molecular Dynamics, Sunnyvale, CA) for the presence of labeled proteins. Kaleidoscope prestained standards (Bio-Rad, Hercules, CA) were used to determine the size of labeled proteins. In some experiments the transcription dependence of labeling was tested by culturing embryos in modified-HTF containing 11 µg/ml of {alpha}-amanitin.

Immunofluorescence

Embryos were recovered fresh from the reproductive tract or removed from culture at various developmental stages and washed extensively in protein-free Hepes-HTF prior to fixation in 2% formaldehyde (Sigma) in PBS at 25°C for 1 h. They were permeabilized with 1% Tween 20 in PBS containing 0.1% BSA at 4°C for 60 min. Sheep heat-inactivated serum in PBS (30% v/v) with 2% BSA at 25°C for 30 min was used to block nonspecific binding. Embryos were incubated in primary antibody (IGF-1:anti-human IGF-1, polyclonal goat IgG: R&D Systems, Minneapolis, MN; {alpha}-TGF:anti-TGF{alpha} mouse monoclonal IgG1: Santa Cruz Biotechnology, Santa Cruz, CA) at 25°C (IGF-1) or 4°C ({alpha}-TGF) for 24 h, followed by secondary fluorescent antibody (IGF-1:rabbit anti-goat IgG, fluorescein isothiocyanate [FITC] labeled, Zymed Laboratories, San Francisco, CA; {alpha}-TGF:goat anti-mouse IgG, FITC labeled: Santa Cruz) at 25°C for 30 min. Whole embryos placed on a microscope slide in 25 µl of anti-fade media (50% v/v glycerol in PBS). A coverslip was gently pressed onto vaseline strips placed on two sides of the slide until the embryos bulged slightly. Excessive fluid was removed with tissue and coverslip sealed around edges with varnish. Embryos were then viewed with an epifluorescent microscope (Nikon, Tokyo, Japan) fitted with a DPlanApo 40 UV objective (40x; Olympus, Tokyo, Japan), standard mercury lamp, and UV-1A filter block. The fluorescence intensity was measured using a Nikon P1 photometer, with the same conditions of the microscope and photometer setting for all embryos in each experimental replicate. The mean value of nonimmune isotype IgG control for each experiment was subtracted from experimental readings to obtain the value of fluorescence intensity for each embryo. Photographs were taken with Kodak Tri-X pan 400 print film (Eastman Kodak Company, Rochester, NY), using the same exposure conditions for all images. Every experiment incorporated several negative control treatments: incubation of embryos in nonimmune IgG (Southern Biotechnology Associates, Birmingham, AL), no primary antibody, no secondary antibody, and nonfluorescent secondary antibody.

Detection of Gene Expression

The mRNA for IGF-1 ligand and receptor, insulin, insulin receptor, and Pafah1b (ß, {alpha}1, and {alpha}2 subunits) in tissue (liver, brain, endometrium), oocytes, and embryos, and the mRNA for Pafah1b ({alpha}1 subunits) in spermatozoa and cumulus cells were detected by RT-PCR. The results are expressed as the proportion of test samples in which transcripts were detected.

All RT-PCR assays were performed and validated as previously described [15], and the following controls were always undertaken: 1) Mouse ß-actin was used as a positive control for the efficiency of all RNA extractions. The ß-actin primer pair was designed so it spanned the first intron (87 base pairs [bp] in length) of the rodent ß-actin gene [33]; contaminating genomic DNA could be detected using these primers. 2) To control for false-positive PCR amplification of contaminating genomic DNA, some samples did not include reverse transcriptase. 3) Water was added instead of sample to test for contamination with extraneous DNA. 4) Some samples were randomly treated with RNase I (Promega Corp., Madison, WI) prior to RT, confirming the RNA origin of positive RT-PCR reactions.

Extraction of RNA

Fifty to 200 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 transfer RNA; Sigma). Alternatively, 100 mg of mouse tissue (controls: liver, brain, or endometrium tissue) was finely minced with a surgical blade and then transferred into TRIzol Reagent. In the case of cumulus cells and spermatozoa, 103 cells were transferred in a minimal volume of PBS into TRIzol Reagent. Total RNA from samples was extracted and treated with DNAse as previously described [15]. The yielded RNA pellet was dissolved in double-autoclaved Milli-Q water in the presence of RNase inhibitor (Promega) (final concentration: 1 U/ml) and then either immediately subjected to RT-PCR or stored at -70°C.

Messenger RNA for the Pafah1b subunits from oocytes and embryos was obtained by a nonextraction protocol as previously described [15]. Briefly, 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 a minimum volume of culture media into a 0.2-ml thin-wall Eppendorf tube with 15.5 µl of reaction mix (identical to mix used for RT-PCR). 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. The whole sample volume containing embryonic RNA was either immediately subjected to RT-PCR, or stored at -70°C.

Reverse Transcription-PCR

The RNA extracted from the equivalent of 20 oocytes or embryos was placed in a thin-wall 0.6-ml eppendorf tube overlaid with 50 µl of paraffin liquid (BDH) or the whole sample containing embryonic RNA obtained by nonextraction preparation was placed in a 0.2-ml thin-wall tube and reverse transcribed by incubating at 42°C for 30 min with 2.5 U murine leukemia virus reverse transcriptase primed with 2.5 µM oligo(dT) (Perkin-Elmer, Foster City, CA) or 0.75 µM specific 3' primer for extracted and nonextracted samples, respectively, in 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 PCR reaction in a final 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 a specific primer pair using a Hybaid Thermal Reactor (Hybaid Limited, Teddington, Middlesex, UK) for extracted samples or Corbett Thermal Reactor (Corbett Research) for nonextracted preparations.

The PCR reaction products were analyzed by electrophoresis on a 4% agarose gel stained with ethidium bromide to visualize PCR product on a UV transilluminator. Fragments were verified by size and also confirmed by sequencing of the PCR product, using an ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer) and performed at Supamac (Redfern, NSW, Australia). Primers were obtained from Fisher Biotech (Perth, WA, Australia). The size of the PCR product and the sequences of primers are shown in Table 1.


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  		TABLE 1. Oligonucleotide primers used for RT-PCR

Statistics

All statistical analyses were performed with the version 9 SPSS statistical program. Analyses of differences in the expression of IGF-1 ligand detected by quantitative immunofluorescence were performed by one- and two-way ANOVA, and comparisons of multiple means were performed by the least significant difference test. Dichotomous outcomes, such as the expression of a given mRNA, were analyzed by logistic regression with individual treatments at each timepoint tested by chi-square analysis where necessary or Fishers exact test.

RESULTS

Effect of the Method of Fertilization on the Duration of the First Cell-Cycle and on Onset of Zygotic Transcription

To determine whether the method of fertilization had any effect on the time that the zygotic genome was activated, expression of the marker of the onset of transcription (TRC) was measured throughout the 2-cell stage, following fertilization of oocytes in vitro or in vivo. The time of first detectable expression was measured relative to the time zygotes cleaved. However, onset of zygotic gene expression may be independent of the first cell cycle [34]. To validate this timing strategy, it was therefore necessary to determine initially whether IVF had any effect on the rate of progression to first mitosis. Following fertilization by either method, oocytes were viewed at 1-h intervals by phase-contrast microscopy. The time when two pronuclei were first observed for each individual fertilized oocyte was noted, and the time from then until cleavage to the 2-cell stage occurred was recorded. Zygotes produced by ISF took 15.8 ± 1.1 h (mean ± SD, n = 222, four replicates) and by IVF, 16.9 ± 1.2 h to progress from the pronuclear stage to the 2-cell stage. The interval between each observation of the fertilized oocyte was 1 h. Thus, within the limit of sensitivity of this assay methodology the small difference was not considered to be significant. The time from pronuclei formation to the time of cell division was the same following IVF and ISF. The time of zygotic gene expression was therefore measured relative to the time of cleavage to the 2-cell stage.

To test for the expression of TRC, zygotes produced by ISF were cultured and viewed at 1-h intervals by phase-contrast microscopy. Embryos were "picked-off" as they cleaved to 2-cell embryos (0 h after cleavage) and were binned into groups of 10 in 10-µl drops of modified-HTF and assayed for the presence of TRC at 0.5, 2.5, 4.5, and 6.5 h postcleavage. It was found that by 4.5 h all ISF embryos expressed TRC (Fig. 1). The IVF embryos were tested for expression of TRC at 4.5 h postcleavage. All IVF embryos (four replicates) expressed TRC at this time (Fig. 1), showing that expression was not delayed compared to its time of first expression in ISF embryos [22, 27]. Expression of TRC was not detected in ISF and IVF embryos following culture in the presence of {alpha}-amanitin (three different replicates for each treatment), confirming the transcriptional dependence of expression (Table 1).



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  		FIG. 1. Representative pattern of TRC synthesis in 2-cell embryos. Lane 1: In situ-fertilized 2-cell embryos 4.5 h post-2-cell cleavage cultured in the presence of {alpha}-amanitin (AM); lanes 2–5: ISF embryos 0.5, 2.5, 4.5, and 6.5 h post-2-cell cleavage, respectively; lane 6: 2-cell IVF embryo 4.5 h post-2-cell cleavage. Each lane represents TRC synthesis in five embryos. M, Kaleidoscope prestained standard

Effect of IVF on Expression of Later Gene Products

Insulin-like growth factor-1 ligand and receptor Using a polyclonal antibody directed against the IGF-1 protein, staining was detected in embryos at all developmental stages tested (2-cell, 4-cell, 8-cell, and blastocyst) for the three treatments: fresh, ISF, and IVF embryos. Measurements were made on at least 30 embryos (from each of four experiments) for each treatment (three) and at each time point (four) (Figs. 2 and 3).



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  		FIG. 2. Expression of IGF-1 in mouse preimplantation embryos. Mouse embryos were prepared for indirect immunofluorescence using polyclonal antibody to human IGF-1. A) Fresh 2-cell, B) fresh 8-cell, C) fresh blastocyst, D) fresh blastocyst incubated in nonimmune IgG, E) IVF 2-cell, F) IVF 8-cell, G) IVF blastocyst, and H) IVF blastocyst incubated in nonimmune IgG

The amount of IGF-1 detected in embryos collected fresh from the reproductive tract was similar for the 2-cell and blastocyst stage (P > 0.05) but significantly decreased for the 4- and 8-cell stage (P < 0.05) (Fig. 3). In ISF and IVF embryos the amount of immunodetectable IGF-1 protein was similar for the 2-cell, 4-cell, and blastocyst stage (P > 0.05) but was lower for the 8-cell stage (P < 0.05) with the intensity of immunofluorescence also significantly lower than for freshly collected 8-cell embryos (P < 0.001) (Fig. 3). By the blastocyst stage, the level of IGF-1 increased (P < 0.05) but was still significantly less than in corresponding fresh blastocysts (P < 0.001) (Fig. 3). There was no difference (P > 0.05) (Fig. 3) in the level of IGF-1 protein expression between ISF and IVF embryos.



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  		FIG. 3. Effect of fertilization and culture in vitro on the pattern of IGF-1 protein expression detected with quantitative immunofluorescence. Three types of embryos were tested: fresh embryos collected from reproductive tract of female () compared with those fertilized by ISF and then cultured at a concentration of 1:10 (one embryo in 10 µl of medium) ({blacktriangleup}) and IVF embryos cultured at a concentration of 1:10 ({block}). Each point is the mean and SD for at least 30 embryos. *Significantly different from corresponding IVF and ISF embryo developmental stages

To determine whether the density at which embryos were cultivated influenced the level of expression of IGF-1 (which tests for the action of diffusible autocrine factors), oocytes fertilized by IVF were cultured at a density of one embryo in 10 µl of medium and compared with those cultured at a density of one embryo in 100 µl. There was no difference (P > 0.05) in the level of expression from the 2-cell through to the blastocyst stage under these conditions (results not shown).

In embryos collected fresh from the reproductive tract, mRNA for IGF-1 ligand was found at all developmental stages tested, from 2-cell to blastocyst stage embryos (Table 2 and Fig. 4). The pattern of expression in ISF embryos was similar to freshly collected embryos with IGF-1 ligand found at the 2-, 4-cell, and blastocyst stage. However, at the 8-cell stage (66 h post-hCG, Table 2) the message was detected only in 1 of 13 groups of embryos (P < 0.001). By contrast, the incidence of expression of IGF-1 transcripts was significantly reduced in IVF embryos from the 2-cell (42 h post-hCG) through to the morula stage (90 h post-hCG) of development compared to fresh and ISF embryos (P < 0.001; Table 2 and Fig. 4). The expression of IGF-1 mRNA in ISF and IVF embryos was not detected at the 2-cell (42 h post-hCG) and 4-cell stages (52 h post-hCG) following their culture in the presence of {alpha}-amanitin (Table 2), confirming that the presence of the transcript at this time was due to new transcription from the zygotic genome.


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  		TABLE 2. The pattern of expression of IGF-1 mRNA in embryos collected fresh from the reproductive tract compared with those fertilized by IVF or ISF and then cultured in vitro in modified-HTF, modified-HTF + {{alpha}}-amanitin, or kSOM (values represent number of experiments with mRNA present/total number of experiments)



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  		FIG. 4. Representative expression pattern of mRNA transcripts for IGF-1 ligand. Liver tissue (L); 2-cell, 42 h post-hCG (2c); 4-cell, 52 h post-hCG (4c); 8-cell, 66 h post-hCG (8c); blastocyst, 90–114 h post-hCG (BI); all embryos collected fresh from the reproductive tract (fresh) or produced by IVF and cultured in vitro (IVF). M, Molecular weight size markers (PhilX 174 DNA/HeaIII). Complementary DNA reverse transcribed from 10 embryos (5 embryos for ß-actin) was used for the RT-PCR (50 cycles) analysis in each lane. Expected size of RT-PCR amplification product: IGF-1, 210 bp. ß-Actin was used as a parallel positive control for the efficiency of the RT-PCR (243 bp). Negative control samples did not include reverse transcriptase (-ve control)

When IVF embryos were cultured in kSOM, the pattern and timing of expression of IGF-1 mRNA was not different from that observed in modified-HTF (Table 2), showing that the reduced onset of zygotic gene expression was not an artifact of a particular medium but was a general effect. The results suggest that the stable levels of IGF-1 protein expression in the 2-cell and 4-cell stage embryos occurs due to the maintenance of maternal protein and mRNA. By the 8-cell stage, however, continued expression of protein required new transcription of RNA from the zygotic genome. Following IVF and culture, this transcription from the zygotic genome was delayed, causing the decline of IGF-1 protein in the 8-cell stage.

The IGF-1 receptor mRNA was first detected at 66 h after hCG (8-cell stage) in embryos collected fresh from the reproductive tract (Table 2). There was no effect of the method of fertilization or culture on the pattern of expression.

The enzyme Pafah1b The intracellular form of the PAF catabolic enzyme PAF:acetylhydrolase (Pafah1b) is a trimer. Primers were produced against the murine sequences for the ß (Lis 1, 45 kDa), {alpha}1 (29 kDa), and {alpha}2 subunits (30 kDa) of the enzyme. Validation of these primers was performed using RNA extracted from mouse brain tissue. Tissue samples gave a positive signal, and the identity of the product was confirmed by size (Table 1) and sequencing analysis.

Transcripts for ß and {alpha}2 subunits were detected in all developmental stages from unfertilized oocytes through to the blastocyst stage (Table 3). Culturing zygotes in {alpha}-amanitin resulted in a complete loss of expression of both these subunits by 66 h post-hCG (8-cell stage) (Table 3). Thus, the persistent expression of those transcripts in the early mouse embryo required a contribution from gametic RNA stores and transcription from the zygotic genome. Due to the overlap in expression of RNA from these two sources, it was not possible in this study to determine when transcription from the zygotic genome first occurred. The transcription was not qualitatively affected by fertilization or culture in vitro (Table 3).


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  		TABLE 3. The pattern of expression of Pafah1b ß}, {{alpha}2, {{alpha}}1 subunits (45 kDa, 30 kDa, and 29 kDa subunits, respectively) mRNA in embryos collected fresh from the reproductive tract compared with those fertilized by IVF and then cultured in vitro in modified-HTF or modified-HTF + {{alpha}}-amanitin (values represent number of experiments with mRNA present/total number of experiments)

The expression of Pafah1b {alpha}1 subunit was first detected in 2-cell embryos (42 h post-hCG) and persisted until at least the blastocyst stage (Table 3 and Fig. 5). The onset of expression was inhibited by {alpha}-amanitin (Table 3), indicating it required new transcription from the zygotic genome. The expression of {alpha}1 subunit was significantly retarded when IVF and ISF zygotes were cultured in vitro (P < 0.001; Table 3 and Fig. 5), with mRNA not detected in the majority of embryo groups until the blastocyst stage (90–114 h post-hCG) (Table 3 and Fig. 5).



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  		FIG. 5. Representative expression pattern of mRNA transcripts for Pafah1b {alpha}1 subunit (29 kDa). Brain tissue (B); unfertilized oocyte, 14 h post-hCG (Oo); 1-cell fertilized zygote, 21 h post-hCG (1c); 2-cell, 42 h post-hCG (2c); 8-cell, 66 h post-hCG (8c); blastocyst, 90–114 h post-hCG (BI), all embryos collected fresh from the reproductive tract (fresh) or produced by IVF and cultured in vitro (IVF). M, Molecular weight size markers (PhilX 174 DNA/HeaIII). Complementary DNA reverse transcribed from 10 embryos (5 embryos for ß-actin) was used for the RT-PCR (50 cycles) analysis in each lane. Expected size of RT-PCR amplification product: Pafah1b {alpha}1 subunit, 293 bp. ß-Actin was used as a parallel positive control for the efficiency of the RT-PCR (243 bp). Negative control samples did not include reverse transcriptase (-ve control)

This failure to detect Pafah1b {alpha}1 subunit in oocytes and zygotes conflicts with the results of an earlier study [20]. To determine whether the negative results in oocytes and variable results in early stage embryos for the {alpha}1 subunit was a consequence of limited sensitivity of the RT-PCR technique, further tests and optimization of the technique were undertaken. A second, different set of RT-PCR primers (Table 1) for this gene, designed against the sequence reported for the mouse [35], was used. These primers were tested against mouse brain tissue RNA to confirm their ability to amplify the mRNA coded by the {alpha}1 subunit gene. Tissue samples gave a positive signal (Fig. 6) and the identity of the product was confirmed by size and sequencing analysis. The RT-PCR assay readily detected the {alpha}1 subunit in as few as 10 2-cell embryos (42 h post-hCG) (Figs. 5 and 6) collected fresh from the reproductive tract, but increasing the number to 100 failed to produce a positive result for oocytes, zygotes, and in 2-cell embryos produced by IVF (Fig. 6). The mRNA for ß-actin acted as a positive control in all experiments, and was detected in all samples tested. The Pafah1b {alpha}1 subunit was detected in five cumulus cells and mouse spermatozoa (Fig. 6). To determine whether these results were due to strain differences, the study was repeated with oocytes and embryos from C57bl/6 animals. These gave the same pattern of Pafah1b {alpha}1 subunit expression as was observed with Swiss albino mice and the expression was again retarded following IVF (Table 3).



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  		FIG. 6. Representative expression pattern of mRNA transcripts for Pafah1b {alpha}1 subunit (29 kDa) using an alternative set of primers. Brain tissue (B); 2-cell, 42 h post-hCG (2c); unfertilized oocyte, 14 h post-hCG (Oo); 1-cell fertilized zygote, 21 h post-hCG (1c); all embryos collected fresh from the reproductive tract; 2-cell, 42 h post-hCG, produced by IVF and cultured in vitro (2cIVF). Complementary DNA reverse transcribed from 10 embryos (2c), 100 oocyte/embryos (Oo, 1c, 2cIVF), or 5 embryos for ß-actin was used for the RT-PCR (50 cycles) analysis in each lane. Complementary DNA reverse transcribed from RNA from five cumulus cells (CC), 100 spermatozoa (Sperm). M, Molecular weight size markers (PhilX 174 DNA/HeaIII). Expected size of RT-PCR amplification product: Pafah1b {alpha}1 subunit, 134 bp. ß-Actin was used as a parallel positive control for the efficiency of the RT-PCR (243 bp). Negative control samples did not include reverse transcriptase (-ve control)

Insulin and {alpha}-TGF Insulin mRNA was not detected at any stage of development from unfertilized oocytes through to the blastocyst stage, in either fresh or cultured embryos irrespective of the method of fertilization (Table 4). The insulin-receptor transcript was first consistently observed at 66 h post-hCG (8-cell stage), and its expression was unaffected by the method of fertilization or by embryo culture concentration (results not shown).


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  		TABLE 4. The pattern of expression insulin and insulin receptor mRNA in embryos collected fresh from the reproductive tract compared with those fertilized by IVF or ISF and then cultured in vitro (values represent number of experiments with mRNA present/total number of experiments).a

The expression of {alpha}-TGF protein was measured in 2-cell, 8-cell, and blastocyst stage embryos by immunofluorescence. Two- and 8-cell embryos did not show staining above the level of isotype control; blastocysts showed staining above the levels of isotype control antibodies (2.69 + 0.11 units, mean + SEM, n = 59) (Fig. 7) when collected fresh from the reproductive tract. Embryos produced by IVF and cultured at low concentrations (of one embryo/100 µl) for 114 h exhibited a similar amount of staining for {alpha}-TGF (2.79 ± 0.13 units, n = 59; P > 0.05) as their fresh counterparts (Fig. 7).



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  		FIG. 7. Expression of {alpha}-TGF protein in mouse preimplantation embryos. Mouse embryos were prepared for indirect immunofluorescence using polyclonal antibody to mouse {alpha}-TGF. A) Fresh blastocyst, B) fresh blastocyst incubated in nonimmune IgG, C) IVF blastocyst, and D) IVF blastocyst incubated in nonimmune IgG

DISCUSSION

Despite extensive trials of culture conditions and media design, embryos produced by IVF have significantly reduced viability compared with corresponding embryos fertilized in the reproductive tract [36]. Carefully controlled experiments show that this was primarily caused by the process of fertilization in vitro itself, rather than an effect of handling of zygotes or ovulation induction [37]. One defined cause of reduced viability is an aberration in the autocrine stimulation of embryo development and survival by diffusible factors [1]. This effect was manifested by a significantly greater sensitivity of IVF zygotes to culture at low embryo density or large medium volumes [1] and by the ability of the addition of several putative autocrine trophic factors (PAF, IGF-1, and IGF-2) to enhance embryo development.

Despite evidence that the action of autocrine trophic factors were required by the 2-cell stage, there was no evidence that partial deprivation of these factors influenced the duration of the embryo's first or second cell cycle, suggesting that their actions were not as classical GFs [36]. Using a different method of timing the cell cycle, the current study confirmed that IVF and culture had no influence on the time from pronuclei formation to the first mitosis. This confirms previous observations [37] that the aberrations in development caused by IVF are manifested at later stages.

This study shows for the first time immunolocalization of IGF-1 in the mouse embryo (Fig. 2). Cleavage stage embryos displayed uniform staining throughout their blastomeres. By the blastocyst stage there was an accumulation of IGF-1 staining in the inner cell mass (ICM), with relatively less expression in the trophectoderm. It was previously demonstrated [38] that maternally derived IGF-1 ligand was internalized, from the 8-cell stage, through receptor-mediated endocytosis. In this current study, the accumulation of IGF-1 in the ICM of IVF embryos was similar to that observed in fresh embryos. This suggests that the expression detected was not primarily the internalization of maternal IGF-1, but rather reflected the localization of the embryo's own IGF-1.

The capacity of exogenous trophic factors to enhance the viability of IVF embryos [1, 39] suggests that their production by embryos after IVF is a limiting factor. Evidence supporting this is the observation of reduced release of PAF [4] and the delayed transcription from the IGF-2 ligand gene and lower levels of immunodetectable IGF-2 protein in embryos produced by IVF [17]. The current study extends these findings to show that embryo-derived IGF-1 expression is also reduced following IVF in this model. This occurred at the level of gene expression and protein expression as detected by quantitative immunofluorescence. There was, however, no detectable influence of IVF on the expression of {alpha}-TGF in embryos. The normal levels of {alpha}-TGF are consistent with the observation that supplementation of medium with epidermal growth factor (EGF, which acts like TGF-{alpha}) over the range 0.2–2000 ng/ml had no beneficial effects on the viability of mouse IVF embryos [1]. These results are consistent with the observation that supplementation of culture medium with IGF-1 (like PAF and IGF-2) enhanced viability of IVF embryos [1]. In contrast to IGF-2, the expression of IGF-1 protein was adversely affected by culture in vitro after normal fertilization in the reproductive tract. There was no additive adverse effect of IVF. There was also no additional effect of reducing the density (concentration) of embryos in culture. These results argue that, unlike IGF-2 [17], the expression of IGF-1 was not influenced by other diffusible autocrine factors released by the embryo.

The detection of the expression of mRNA for IGF-1 ligand during preimplantation development of mouse embryos confirms previous observations [7]. The negative effect of IVF and culture on the onset of new IGF-1 transcription was reflected in lower levels of detectable protein in the IVF 8-cell embryos compared with fresh embryos. Although it was not possible to detect mRNA for IGF-1 ligand by the 8-cell stage of IVF embryos, IGF-1 protein was still detected, albeit at lower levels than in embryos collected from the reproductive tract. This expression probably reflects the continued presence of protein produced from maternal stores of mRNA. It reflects a greater stability of the protein than is the case for mRNA. This stability may assist the embryo in overcoming some of the adverse effects of the epigenetic change in IGF-1 gene expression. The animal model used here has previously been shown to result in embryos with reduced viability after IVF [1, 4]. This reduction could be partially ameliorated by addition of IGF-1 to media [1]. The observation in this study, that IGF-1 ligand expression is reduced shows that IGF-1 is an important autocrine growth and survival factor for the early mouse embryo. It was previously shown [1] that most loss of embryo viability in this IVF model occurred after the 8-cell stage, and it is at this time that the IGF-1 receptor gene expression occurs. It seems likely, therefore, that the autocrine action for IGF-1 occurs after the 8-cell stage. The action of IGF-1 maybe through multiple receptors, including the insulin, IGF-1, and IGF-2 receptors [40–43]. Qualitative changes in IGF-1 ligand expression demonstrated in this study and the positive correlation with the embryo phenotype [44] proffer IGF-1 as one possible indicator of embryo viability.

The evidence that IVF and culture caused a retardation of IGF-1 ligand production and the ability of exogenous growth factors to partially rescue embryo viability [1] suggest that those factors are important limiting stimulants for embryo survival after IVF. Studies of growth kinetics using gene knockout mice have been inconclusive, however. For instance, mouse embryos carrying null mutations of the genes encoding IGF-1 and -2, and the IGF-1 receptor, alone or in combination, are without any apparent adverse effect on developmental potential of embryos during the preimplantation period [45]. However, it will be of interest to examine the phenotype of the embryos following IVF and culture in vitro, a circumstance where other participants of the embryo's autocrine drive might be expected to be limiting.

Although aberrant expression of trophic factors was readily observed due to their functional effects, it seemed plausible that these effects were simply a manifestation of a general derangement in the embryo's transcriptional machinery following IVF. This hypothesis was not supported by this study. Apparently normal ontogeny of expression of {alpha}-TGF, Pafah {alpha}2, and ß subunits was observed. A marker of activation of the first round of transcription (TRC expression) was also not delayed.

It was noted that addition of exogenous trophic factors such as PAF, IGF-2, or IGF-1 was only partially successful in rescuing the viability of IVF embryos [1]. This may indicate that there are other factors outside of autocrine regulation of embryo development that are adversely affected by IVF, or that autocrine regulation is deranged not only in the production and release of the autocrine factors but also in the ability of embryos to respond to these factors. Expression of specific receptors is necessary for cells to respond to ligands. For the three trophic factors reported to be affected by IVF in this model (IGF-1, PAF, and IGF-2), specific receptors exist. The current study confirmed [11] the presence of transcripts for IGF-1 receptor and insulin receptor from 8-cell stage and showed that there were no qualitative differences in the expression of IGF-1 or insulin receptor genes [17] following IVF or culture in vitro, and we previously showed [17] that IGF-2 receptor gene expression was qualitatively unaffected.

We have previously demonstrated [15] that expression of the G-protein PAF-receptor gene is delayed following IVF. However, deletion of this gene by gene-targeting creates mice without any adverse reproductive phenotype, questioning whether this receptor is important in PAF signalling in reproductive processes [46]. By contrast, deletion of the ß subunit of Pafah1b caused early embryo lethality [19]. Pafah1b has structural homology with G-proteins [18] but exerts PAF:acetylhydrolase rather than GTPase activity. It may act as an intracellular receptor or signal transducer for PAF, although formal proof of this is awaited. We previously reported [15] a failure to detect expression of this enzyme in the mouse preimplantation embryo using RT-PCR with primers designed against the bovine gene. However, it was recently shown that Pafah1b {alpha}1, {alpha}2, and ß subunit mRNA was expressed in mouse oocytes and zygotes [20]. The expression of the ß subunit increased after fertilization and this appears to be due to stabilization of maternal transcript by selective polyadenylation in the cytoplasm [20]. There was no detection of RNA inherited from sperm. We therefore repeated these studies using primers designed against the mouse gene. Under these circumstances we detected all three subunits of the enzyme in the 2-cell embryo. It was confirmed [20] that the ß and {alpha}2 subunits were also detected in oocytes and zygotes. It was shown for the first time that they were expressed throughout the preimplantation phase. In the case of {alpha}1 subunits, however, we found a different result from Cahana and Reiner [20]; there being no expression in the oocyte and zygote, with its first expression detected at the 2-cell stage. We found this pattern of expression to be the same for both Swiss albino and C57bl/6 embryos. We also used two different sets of RT-PCR primers for this gene with the same outcome. The assay was sensitive, with the {alpha}1 subunit being readily detected in 10 2-cell embryos. It was found that mRNA for this subunit was detected in cumulus cells and in sperm. It was detected in as few as five cumulus cells. We did not have access to the same mouse strain as Cahana and Reiner [20], and it is therefore possible that the different results may reflect strain differences. Alternatively, the detection of this transcript in oocytes and zygotes may have been a consequence of carryover of sperm or cumulus cells, or other somatic cells. In this, and in previous studies [1, 4, 15, 17], we have chosen to use a random outbred strain of mice to reflect more faithfully the circumstances in clinical populations. Some mouse strains seem to have high viability rates after IVF and culture, and it is likely that some of the epigenetic changes observed in this study do not occur in those strains. A screen of gene expression in various mouse strains of differing viability after IVF may allow the identification of those genes that are essential to normal early embryo development.

Although the {alpha}1 subunit transcript was detected in sperm, the failure to detect the transcript in zygotes (even when using RNA from 100 zygotes) suggests that it was not carried over to the oocyte at the time of fertilization or was rapidly degraded. This demonstrated that expression of the {alpha}1 subunit required new transcription from the zygotic genome, while IVF delayed expression of this gene. It is likely that this delayed expression has functional implications. It has been demonstrated that a dimer of the {alpha} subunits form the catalytic unit of Pafah1b, while the ß subunit has a regulatory role [47–49]. The {alpha} subunits can form homo- and heterodimers, giving three different catalytic forms: {alpha}1 {alpha}1, {alpha}1 {alpha}2, or {alpha}2 {alpha}2. It has been suggested that differential expression of {alpha} subunits may cause changes in cell function [50]. For example, it was shown that a change from migratory to nonmigratory behavior of neuronal cells was associated with a switch in expression from {alpha}1 {alpha}1 or {alpha}1 {alpha}2 dimers to {alpha}2 {alpha}2 dimers. Furthermore, the ß subunit suppresses the actions of {alpha}1 {alpha}1 and {alpha}1 {alpha}2 dimers but enhances the actions of {alpha}2 {alpha}2 dimers [51]. Thus, the delayed expression of {alpha}1 subunit genes in IVF embryos may cause different functional forms of Pafah1b in IVF embryos to result.

The qualitative methods used in this study clearly show changes in the first onset of expression of some zygotic genes that may be important for development. It also seems likely that the expression of some genes could be adversely affected quantitatively but without a qualitative change in their ontogeny of expression. The use of quantitative RT-PCR and protein assays will allow these different patterns of epigenetic modification to be defined in future studies.

This study shows that IVF and culture in vitro caused no obvious delay in the normal onset of the first round of transcription from the zygotic genome. However, there was a delay in transcription of some specific genes. Several of these genes code for putative autocrine growth factors for the early embryo. The aberrations in the production of GFs and their receptors caused by IVF or culture in vitro may be a significant cause of poor embryo viability. The study shows that IVF and culture have different effects on a variety of genes expressed soon after the onset of activation of the zygotic genome. These differential effects indicate that IVF and culture prevent some signalling pathways for the activation of transcription but not others.

FOOTNOTES

First decision: 19 June 2000.

1 This study was supported by the Northern Sydney Area Health Service Grant Scheme. Back

2 Correspondence. FAX: 61 2 9926 6343; chriso{at}med.usyd.edu.au Back

Accepted: October 4, 2000.

Received: May 8, 2000.

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