Molecular Human Reproduction, Vol. 5, No. 2, 116-124,
February 1999
© 1999 European Society of Human Reproduction and Embryology
In-vitro fertilization and culture of mouse embryos in vitro significantly retards the onset of insulin-like growth factor-II expression from the zygotic genome
Human Reproduction Unit, Department of Physiology, University of Sydney, Royal North Shore Hospital of Sydney, St Leonards, NSW 2065, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study, the effect of in-vitro fertilization (IVF) and culture of mouse embryos in vitro on the normal expression of insulin-like growth factor-II (IFG-II) ligand and receptor was examined. The expression of IGF-II increased in a linear fashion at least up to the 8-cell stage of development. IGF-II expression in embryos collected fresh from the reproductive tract was significantly (P < 0.001) greater than in embryos fertilized in the reproductive tract and cultured in vitro (in-situ fertilized: ISF), and its expression was further reduced (P < 0.001) in IVF embryos at all development stages tested. The expression of IGF-II was significantly (P < 0.001) lower when embryos were cultured individually in 100 µl drops compared with culture in groups of 10 in 10 µl drops of medium. The addition of platelet activating factor to culture medium partially overcame this density-dependent decline of expression. Culture of ISF and IVF zygotes also caused the onset of new IGF-II mRNA transcription from the zygotic genome to be significantly (P < 0.001) retarded, until at least the 8-cell stage of development. This effect was greater (P < 0.05) for IVF than for ISF embryos. Neither IVF nor culture had any obvious effect on IFG-II/mannose-6-phosphate receptor (IGF-IIr) mRNA expression.
embryo/growth factor/insulin-like growth factor-II/in-vitro fertilization/platelet activating factor
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors. This hypothesis is supported by: (i) the relative autonomy of preimplantation embryo growth; (ii) the observation that the rate of embryo development in vitro is density dependent, with embryos growing in relatively small volumes having faster development rates than embryos developing in larger volumes and those in groups developing better than individual embryos (Paria and Dey, 1990
; Lane and Gardner, 1992; O'Neill, 1997); (iii) the synthesis by the preimplantation embryo of a number of growth factors and their receptors, including platelet activating factor (PAF) (O'Neill, 1985; Collier et al., 1988; Roudebush et al., 1997), insulin-like growth factor-I (IGF-I) (Rappolee et al., 1990; Doherty et al., 1994), insulin-like growth factor-II (IGF-II) (Heyner et al., 1989; Harvey and Kaye, 1991; Rappolee et al., 1992), growth hormone (Pantaleon et al., 1997), transforming growth factor-
(TGF-) (Rappolee et al., 1988); and (iv) evidence of trophic actions of a range of growth factors on embryo growth, metabolism and cell-cycle progression in vitro (Harvey and Kaye, 1992; O'Neill, 1997).
The density dependence of successful embryo development shows that the released autocrine factors are limited by dilution. Zygotes, produced by in-vitro fertilization (IVF), were markedly more sensitive to the adverse effects of culture at low embryo concentrations than equivalent zygotes fertilized in the reproductive tract. It was suggested (O'Neill, 1997) that this might be due to lower levels of production or release of the putative growth factors by IVF zygotes. It was shown that zygotes fertilized in the reproductive tract released ~7-fold more PAF than did IVF zygotes, while fresh 2-cell embryos released 3-fold and 21-fold as much PAF as in-situ fertilized (ISF) and IVF zygotes respectively (O'Neill, 1997). Furthermore, the actions of the autocrine/survival factors, including PAF, were required to act by the 2-cell stage of development (O'Neill, 1998).
The addition of PAF to medium, however, was only capable of partially reversing the effects of IVF and culture in dilute culture, suggesting a requirement for other growth factors. IGF-I (30 ng/ml) and IGF-II (1 ng/ml) could also partially compensate for the adverse effects of low embryo concentration on IVF zygote development. Epidermal growth factor (EGF) had no effect over the range 0.2–2000 ng/ml. In one study (Ho et al., 1994), it was shown that culturing embryos in medium with a high sodium concentration (125 mM) reduced the expression of IGF-I and IGF-II ligand and receptor mRNA compared with culture in low sodium (85 mM). However, the effect of IVF on the normal pattern of expression of these growth factors has not been reported. Since PAF alone could not completely compensate for the adverse effects of limiting dilution and from the evidence of the trophic action of other growth factors, it seems likely that the production or release of other growth factors is also disrupted by IVF. In this study we examine the effects of medium type, IVF and embryo culture concentration on the ontogeny of expression of IGF-II and IGF/mannose-6-phosphate receptor (IGF-IIr) in the mouse embryo and show that onset of transcription of ligand from the zygotic genome is markedly delayed by IVF or culture in vitro.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Embryo culture and collection medium
Oocytes and embryos were collected in HEPES-buffered synthetic human tubal fluid medium (HEPES-HTF) (Quinn et al. 1985
) and cultured under standard conditions using modified human tubal fluid medium (mod-HTF) (Quinn, 1995). Fertilization was performed in HTF (Quinn et al., 1985). All components of medium were tissue culture grade from Sigma Chemical Co. (St Louis, MO, USA). Unless otherwise stated all medium was supplemented with 3 mg bovine serum albumin/ml (BSA, Fraction V; CSL Ltd, Melbourne, Victoria, Australia).
It is known that zygotes cultured in vitro develop more poorly when cultured at relatively low embryo densities compared with more crowded conditions (Paria and Dey, 1990; Lane and Gardner, 1992; O'Neill, 1997; 1998). It is conceivable that these results were artefacts of the ionic composition of the media used. Thus, in some experiments the effect of culture of zygotes in mod-HTF was compared with kSOM medium [medium that has been optimized for the support of early embryo development (Lawitts and Biggers, 1993)].
Embryos
Random-bred Swiss albino mice (University of Sydney, NSW, Australia), 8–10 weeks old, were superovulated by intraperitoneal (i.p.) injection of 10 IU equine chorionic gonadotrophin (eCG, Folligon; Intervet International, Boxmeer, Holland) followed 48 h later by an i.p. injection of 10 IU human chorionic gonadotrophin (HCG; Chorulon, Intervet). Animals were either left unmated or paired overnight with males of proven fertility and the presence of a copulation plug indicated day 1 of pregnancy. Cumulus masses or embryos were flushed from the reproductive tract with HEPES-HTF. Embryos at various stages of development were recovered by flushing the oviducts or uterus of mated animals (21, 42, 52, 66, 90 and 114 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/ml of hyaluronidase (Sigma) in HEPES-HTF. They were then thoroughly washed in five changes of HEPES-HTF. IVF was performed as previously described (Singh et al., 1995) with modification. Two males, 15–20 weeks of age and of proven fertility, were used for each IVF procedure. Following cervical dislocation the epididymides were removed and placed into 1 ml of pre-equilibrated HTF in a Petri dish. The epididymides were punctured with a sterile needle and the spermatozoa gently squeezed out into medium. The dish was placed into an incubator at 37°C with 5% CO2 in air for 40 min to allow spermatozoa to disperse. Cumulus masses containing oocytes were then collected 15–17 h after HCG and extensively washed in HEPES-HTF. Groups of ~30 oocytes with their associated cumulus masses were placed into 5 ml plastic tubes (Falcon; Becton Dickinson Labware, Lincoln Park, NJ, USA) containing 1 ml of mod-HTF. Following dispersal of the spermatozoa, they were thoroughly mixed and the concentration was assessed using a haemocytometer. Motile spermatozoa (0.5x106) were added to each tube containing oocytes. The fertilization status of each oocytes was assessed at 5–6 h after insemination by visualization of pronuclei. All fertilized oocyte were extensively washed in HEPES-HTF to remove spermatozoa and cumulus cells and then pooled. They were then transferred to culture in either mod-HTF or kSOM according to the experimental design.
Embryo culture at different concentrations
Two different culture vessels were used to achieve differing volumes of culture: (i) for 10 µl volumes: 60-well (Nunc, Naperville, IL, USA) HLA plates; (ii) for 100 µl volumes: 96-well flat-bottom plates (Flow Laboratories, McLean, VA, USA). Each type of plate was equally suitable for culture (O'Neill, 1997). The medium was overlayed by ~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: (i) one embryo in 10 µl volume of mod-HTF; (ii) 10 embryos in 10 µl volume of mod-HTF; and (ii) one embryo in 100 µl volume of mod-HTF.
Cell counts
Cells were counted by visualization of cell nuclei after staining with 4 µg/ml Hoechst dye (33342 bisbenzimide; Sigma). Embryos were left in this solution for 45–60 min and then prepared as wet mounts. The stained nuclei were visualized using mercury lamp UV illumination and epifluorescence on a Nikon (Tokyo, Japan) Optiphot microscope with Nikon filter block UV-1A.
Preparation of PAF
PAF (1-o-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine; an approximately equimolar mixture of hexadecyl and octadecyl isoforms of PAF; Sigma) was stored as a stock solution of 10 mg/ml in chloroform at –20°C. Aliquots were placed in sterile siliconized glass tubes and dried under N2. The PAF was solubilized by the addition of mod-HTF with 3 mg BSA/ml, followed by vigorous vortexing for 3 min and then allowed to stand for 1 h at 37°C with gentle mixing. The desired concentration of PAF (18.6 nM) was achieved by serial dilution in mod-HTF.
Immunofluorescence
Embryos were recovered fresh from the reproductive tract or removed from culture at various developmental stages and washed extensively in flush medium prior to fixation in 2% formaldehyde in phosphate-buffered saline (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 non-specific binding. Embryos were incubated in primary antibody (goat anti-human IGF-II; R&D Systems, Minneapolis, MN, USA) at 25°C for 24 h, followed by rabbit anti-goat IgG (FITC labelled; Zymed Laboratories, San Francisco, CA, USA) at 25°C for 1 h. Embryos were viewed with an epifluorescent microscope (Nikon) and 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 isotope non-immune 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, USA), using the same exposure conditions for all images. Every experiment incorporated several negative control treatments: incubation of embryos in non-immune IgG (Southern Biotechnology Associates, Birmingham, Alabama, USA); no primary antibody; no secondary antibody; and non-fluorescent secondary antibody.
RNA extraction
Oocytes (stripped of granulosa cells in HEPES-HTF medium containing 300 IU/ml of hyaluronidase; Sigma) and 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(TM) Reagent (Life Technologies, Gaithersburg, MD, USA) containing 50 µg of carrier RNA (transfer yeast RNA; Sigma). RNA was extracted according to the manufacturer's instructions. Isolated RNA was treated with DNase (RQ1 Dnase; Promega Corporation, Madison, WI, USA) to eliminate possible contamination with genomic DNA. The RNA pellet was resuspended in 20 µl of resuspension solution [RS; 40 mM Tris-HCL, pH 7.9, 10 mM NaCl, and 6 mM MgCl2 (Doherty et al., 1994)] containing 1 unit per 10 µl of RQ1 DNase (Promega) and incubated at 37°C for 30 min. Following the addition of a second equal volume of RS, RNA was re-extracted. The pellet was dissolved in double-autoclaved Milli-Q water in the presence of RNase Inhibitor (Promega) (final concentration 1 U/ml). The RNA was either immediately subjected to RT–PCR, or stored at –70°C.
RT–PCR
RNA from the equivalent of 20 embryos or oocytes was 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) or random hexamers (Perkin-Elmer, Foster City, CA, USA) in 20 µl of reaction mix (Perkin-Elmer). The RT reaction was then terminated by heating at 98°C for 5 min and cooling to 5°C.
10 µl of RT reaction volume was used for 50–60 cycles of PCR in a final 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, 0.4 µM each of specific PCR primers and 1.5 U AmpliTaq DNA polymerase (all reagents supplied by Perkin-Elmer) using Hybaid Thermal Reactor (Hybaid Ltd, Teddington, Middlesex, UK).
All RT–PCR reactions had two routine controls: (i) some samples were prepared without reverse transcriptase, to control for false positive PCR amplification of contaminating DNA; and (ii) water substituted for the test sample, to control for extraneous contamination. Treatment of randomly chosen samples with RNase I (Promega) prior to RT was used as another internal control.
PCR reaction products were analysed by electrophoresis on 2% agarose gel stained with ethidium bromide to visualize PCR product on a UV transilluminator. Fragments were verified by size and restriction enzyme mapping.
Primers were obtained from Fisher Biotech (Perth, WA, Australia). ß-Actin was used as a positive control for all embryo samples. The sequence of oligonucleotide primers and the product size were as follows: IGF-II 5'-GGCCCCGGAGAGACTCTGTGC, 3'-GCCCACGGGGTATCTGGGGAA (Rappolee et al., 1992), 255 bp; IGF-IIr 5'-TGTACACTCTTCTTCTGGCA, 3'-AGAGATGTTGATGTAGAAGACAGG (Rappolee et al., 1992), 186 bp; ß-actin 5'-CGTGGGCCGCCCTAGGCACCA, 3'-TTGGCCTTAGGGTTCAGGGGG (Tokunaga et al., 1986), 243 bp.
Statistics
All statistical analyses were performed with version 7.5 SPSS statistical programme. Analysis of differences in the expression of IGF-II detected by quantitative immunofluorescence and the analysis of cell numbers were performed by two-way analysis of variance (ANOVA). Dichotomous outcomes, such as the expression of a given mRNA, were analysed by logistic regression analysis. When the data took the form of a two-by-two contingency table analysis was by 
2-test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The ability of kSOM to support embryo development was not different from mod-HTF (P > 0.05) when judged either by the proportion of zygotes that developed to the blastocyst stage or by the number of cells present in blastocysts (Table I
). Decreasing the embryo concentration (from 10 embryos/10 µl to one embryo/10 µl) significantly (P < 0.001) reduced the proportion of zygotes developing to blastocyst stage during culture. The number of cells present in blastocysts was lower (P < 0.05) for embryos cultured at concentration of one embryo/10 µl compared to embryos cultured in groups of 10 embryos/10 µl regardless of media used (Table I). Two-way ANOVA showed that there was no interaction effect between media type and embryo density. Thus, the effect of embryo concentration is not apparently an artefact of the type of medium used and is therefore likely to be due to the dilution of released trophic factors below optimal concentrations.
|
The IGF-II ligand was detected in embryos by immunofluorescence at all development stages tested for fresh, ISF and IVF embryos (at least 50 embryos from five different experiments for each treatment at each time point) (Figures 1A and 2
). Fluorescence intensity increased in a linear fashion up to the 8-cell stage of development (the last stage at which quantitative measures where made) (Figure 1A). The amount of IGF-II detected in fresh embryos, however, was significantly (P < 0.001) greater than in ISF embryos at equivalent developmental stages. ISF embryos expressed significantly (P < 0.001) more IGF-II than those produced by IVF (Figure 1A). The concentration at which embryos were cultured also significantly (P < 0.001) influenced the expression of IGF-II ligand (Figure 1B). For both IVF and ISF embryos, there was significantly more IGF-II detected at all stages tested when embryos were cultured at 10 embryos/10 µl of medium compared with one embryo/100 µl. When compared directly, the amount of IGF-II detected in IVF embryos cultured at a concentration of 10 embryos/10 µl was equivalent to that detected in ISF embryos cultured at a density of one embryo/100 µl (Figure 1B).
|
), compared with those fertilized by in-situ fertilization (ISF) (
) or IVF (
) and then cultured in vitro. The results are the mean and SD of at least 50 embryos from five different experiments. (B) The effect of the site of fertilization (IVF, ISF) and different embryo concentration on the pattern of IGF-II polypeptide expression. Four types of embryos were tested: IVF embryos cultured at a concentration of 10 embryos in 10 µl (10/10) of medium (
) and ISF embryos cultured at a concentration of 10/10 (
|
The effect of embryo concentration on the expression of IGF-II ligand suggests that there may be other factors released from the embryos which regulate its expression. PAF is released by embryos before they begin to express IGF-II, from the zygotic genome. To determine whether PAF is an autocrine mediator which influences IGF-II synthesis, medium was supplemented with PAF (18.6 nM) and the effect on IGF-II expression examined (Figure 3
). In both 4-cell and 8-cell IVF embryos cultured at a density of one embryo/100 µl, the IGF-II levels were enhanced (P < 0.005) in the presence of PAF. This increase in expression was more pronounced in 4-cell compared with 8-cell embryos. In the presence of PAF IGF-II ligand expression did not achieve (P < 0.005) the high levels observed in equivalent stage embryos collected fresh from the reproductive tract.
|
To assess whether these differences in the pattern of IGF-II protein expression were exerted at the transcriptional level, the effect of IVF and culture in vitro on the pattern of expression of IGF-II mRNA was studied with RT–PCR (Figure 4
). The RNA was extracted from groups of 10 embryos and the presence or absence of amplification of the IGF-II mRNA tested in each group. The results are expressed as the proportion of groups in which transcript was detected (Table II). IGF-II mRNA was expressed in all groups of oocytes (14 h post HCG). In zygotes [21 h post HCG, at which time there is extensive degradation of maternal transcripts (Piko and Clegg, 1998)] no IGF-II transcripts were detected. Expression of IGF-II transcripts was again detected in the late 2-cell and early 4-cell (both 52 h post HCG) stage and persisted until at least the blastocyst stage (90 h post HCG). The onset of this expression was inhibited by -amanitin (results not shown) indicating it resulted from new transcription from the zygotic genome, as previously described (Bachvarova et al. 1989).
|
|
By contrast, the expression of IGF-II mRNA was significantly retarded when both ISF and IVF zygotes were cultured in vitro (P < 0.001). The majority of ISF zygotes had begun to express IGF-II mRNA by the 8-cell stage (66 h post HCG). The retardation of expression in IVF zygotes was greater (Table II
, P < 0.05) than that observed for ISF embryos, with mRNA not detected in the majority of embryo groups until the morula stage (90 h post HCG) and persisted until at least the blastocyst stage (114 h post HCG) (Table II). When IVF embryos were cultured in kSOM the pattern and timing of expression of IGF-II mRNA was not different from that observed in mod-HTF (Table III).
|
The IGF-II/mannose-6-phosphate receptor (IGF-IIr) mRNA was present in all development stages from unfertilized oocytes through to the blastocyst stage and this was unaffected by the method of fertilization (Table IV
) or the embryo culture concentration (results not shown). Culturing zygotes in the presence of the transcription inhibitor -amanitin resulted in a complete loss of expression of IGF-IIr transcripts by 66 h post HCG of development, a decline in expression being first evident by 42 h post HCG (Table V). Thus, the persistent expression of the IGF-IIr transcripts in the early mouse embryo requires its transcription from the zygotic genome. This zygotic transcription is not qualitatively affected by fertilization or culture in vitro (Table IV).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Fertilization and culture of the early embryo in vitro is associated with a marked reduction in its viability in vitro and pregnancy potential following embryo transfer (Bolton et al., 1991
). This phenomenon is common to all mammalian species studied in detail and is a significant limitation to the success of assisted reproduction technology. Some of this may be due to an increase in chromosomal defects (Delhanty et al., 1997) although experiments in rats showed that it was the process of fertilization and culture in vitro of the zygote that caused retarded development, but that the retardation was not manifested until later in the preimplantation phase (Vanderhyden and Armstrong, 1988). Media widely used in IVF can be regarded as minimal essential media, that is, they allow some embryo survival but do not promote optimal development. Minimal essential media are widely used in cell biology to define important cellular processes, and they were used in the current study to help define the regulation of autocrine growth factor expression. For clinical use, more optimal media have been recently described which include the addition of amino acids (Gardner and Lane, 1993; Lane and Gardner, 1997), growth factors (O'Neill et al., 1989; Paria and Day, 1990) and sequential use of carbohydrate sources (Gardner, 1994, Lane and Gardner, 1997)
There is a body of evidence showing synthesis of a number of growth factor ligands by human preimplantation embryos. These include IGF-I (Smotrich et al., 1996), IGF-II (Hemmings et al., 1992; Lighten et al., 1997a), EGF (Chia et al., 1995), TGF- (Hemmings et al., 1992; Chia et al., 1995), PAF (O'Neill et al., 1985a,b) and platelet-derived growth factor-A (Osterlund et al., 1996). Lighten et al. (1997a) reported the presence of mRNA transcripts for both IGF-II ligand and receptor at all stages tested (oocyte, 2-cell, 8-cell and blastocyst stages). Semi-quantitatively analysis of the expression of IGF-I and -II ligand and receptors, and insulin receptor (Liu et al., 1997), showed that the intensity of gene expression was positively correlated with the morphology of embryos. O'Neill et al. (1987) showed that PAF released by human embryos produced by IVF was variable, with a significant positive association between PAF release and the viability of embryos, while the addition of PAF to media enhanced embryo viability (O'Neill et al., 1989). Both IGF-II and -TGF ligands were released into media by human embryos in culture (Hemmings et al., 1992). The levels of both significantly increased from day 5 of culture. IGF-I ligand and receptor proteins were detected in 8-cell IVF human embryos (Smotrich et al., 1996). In contrast, Lighten et al. (1997a) did not detect transcripts for IGF-I ligand at any developmental stage of human IVF embryos tested (oocyte, 2-cell, 8-cell and blastocyst stages), but did find mRNA transcripts for IGF-I receptor at all developmental stages tested. Lighten et al. (1997b) reported a beneficial effect of exogenous IGF-I on the development of human embryos cultured in vitro. Taken together, these reports for human embryos suggest that provision of autocrine growth/survival factors is a limiting factor for embryo development and viability.
The absence of good control data from normal human embryos fertilized in the reproductive tract prevents definitive conclusions in this regard. However, the similarities in outcomes of mouse (O'Neill, 1997, 1998) and human embryos (Bolton et al., 1989, 1991) produced by IVF suggests that the mouse IVF model is suitable for investigation of the causes of these aberrations in autocrine factor expression.
The current study shows, for the first time, that IVF and culture cause a profound retardation of transcription of the IGF-II ligand from the zygotic genome. This evidence, together with previous observations of reduced PAF released from embryos produced by IVF and the ability of exogenous growth factors to partially rescue embryo viability, suggests that autocrine growth factor starvation is an important limiting factor for embryo survival after IVF. It also shows that IVF and culture in vitro acts as a functional multi-knock-out model, which is useful for helping to elucidate the normal functions of these autocrine growth factors.
This report shows that IGF-II is present in the oocyte and early zygote as maternal transcripts, and is rapidly lost (probably due to degradation of maternal stores of mRNA) after fertilization. By the early 2-cell stage, IGF-II mRNA was not present in any group of embryos examined. In fresh embryos, transcripts were again detected in the latter stages of the 2-cell cycle. This expression was clearly due to new transcription from the zygotic genome since it was completely inhibited by the presence of -amanitin (an inhibitor of RNA polymerase II). There was an additive adverse effect of IVF and culture in vitro on the transcription of this mRNA from the zygotic genome, resulting in a marked delay in the onset of detectable transcription. This was reflected in markedly lower levels of detectable protein in the embryos as assessed by immunofluorescence. The mRNA expressed in these embryos, although delayed, was from new transcription since it also was inhibited by -amanitin (results not shown).
While IVF retarded the expression of IGF-II, this was further exacerbated by culture at low embryo concentrations. This indicates that a released factor limited by dilution plays a role in the onset of expression of IGF-II. It was previously demonstrated that PAF release was reduced by IVF, and development at limiting concentrations was improved by addition of PAF to culture medium (Stoddart et al., 1996; O'Neill, 1997). Those observations are now extended to demonstrate that exogenous PAF significantly up-regulated the level of IGF-II protein in cultured embryos. It was noted, however, that under such conditions the amount of IGF-II expressed by the embryo did not achieve the levels expressed by embryos collected fresh from the reproductive tract. Thus, the presence of PAF in medium at a concentration at least as great as the embryo may experience in situ, while enhancing expression of IGF-II, was not sufficient to completely mimic the normal functions of the reproductive tract. This result suggests that other unidentified factors limited by dilution or IVF are necessary for the full expression of IGF-II, or that factors associated with IVF and culture prevent PAF from fully exerting its actions on IGF-II expression.
It has been shown previously (Ho et al. 1994) that the ionic composition of culture medium could reduce overall rates of RNA synthesis, and IGF-II mRNA synthesis in particular. However, in this study neither the effect on the development rate, the number of cells per blastocyst, nor the time of onset of transcription of IGF-II ligand were improved when embryos were cultured in kSOM compared to culture in mod-HTF. Furthermore, the observation that embryo development was limited by dilution in kSOM, just as it was in mod-HTF, indicates that the release of the range of putative autocrine trophic factors is not improved by the optimized ionic composition of kSOM.
Ho et al. (1994) also found that expression of IGF-IIr mRNA was affected by culture in vitro. We observed that qualitative expression of IGF-IIr mRNA occurred at all embryo stages tested, and this was the same irrespective of the method of fertilization, or the culture concentration. The persistence of expression of the IGF-IIr mRNA through the 1-cell to 2-cell transition makes it difficult to ascertain whether the presence of these transcripts in later stages of preimplantation development is a consequence of the persistence of maternal transcripts or due to new transcription from the zygotic genome. Evidence that the presence of these transcripts was reduced by the late 2-cell stage when embryos were cultured in -amanitin shows for the first time that new transcription of this gene from the zygotic genome during the 2-cell stage is required for its presence throughout the preimplantation phase. At a qualitative level, the onset of this IGF-IIr gene transcription was not found to be adversely affected by IVF. It is possible that there may have been some quantitative effects which were not detected in this study.
It can be considered that the wave of transcription from the zygotic genome that occurs at the 2-cell stage in mice, together with the marked degradation of maternal RNA and protein, marks the shift from maternal to embryonic control of embryo development. General inhibition of this new transcription by -amanitin blocks embryo development (Crosby et al., 1988). The results of this study show that aspects of the initiation of this zygotic transcription may be compromised by IVF and culture in vitro. The observation that IGF-IIr mRNA was apparently still being transcribed at the late 2-cell stage in IVF embryos suggests that the retardation in initiation of transcription was not generalized or absolute, but may be specific to some genes, including IGF-II. The IGF-II gene shows epigenetic imprinting with the maternal allele being silent and the paternally inherited allele being transcriptionally active (DiChiara et al., 1991). An effect on imprinted genes may have longer term implications for fetal development (DeChiara et al., 1991) and perhaps disease status in later life (Ward, 1997). It is noteworthy, however, that the IFG-IIr gene is also imprinted (Barlow et al., 1991) but its quantitative expression was not affected by IVF. The relative effect of IVF and culture on imprinted and non-imprinted gene expression warrants further study.
The synthesis or release of two putative autocrine growth/survival factors (PAF and IGF-II) produced by the early embryo are now shown to be profoundly adversely affected by fertilization in vitro. The ability of PAF to at least partially correct the defect in IGF-II synthesis suggests that this may be one mechanism of PAF's trophic action on the embryo. PAF has been shown to act by the 2-cell stage on a defined G-protein-linked receptor, which causes transient elevations in intracellular calcium concentrations in the embryo (Roudebush et al., 1997). Perhaps the calcium secondary messenger, acting via calmodulin or calcineurin, is necessary for the initiation of IGF-II transcription. The actions of the embryonic autocrine growth factors were shown to be essential by the 2-cell stage of development (O'Neill, 1998). The deprivation up to this stage caused subsequent cell death in the preimplantation embryo, a significant amount of which was caused by apoptosis. IGF-II stimulates growth and metabolism of early embryos (Harvey and Kaye, 1992) and is a well-known survival factor, protecting a range of cell types from apoptosis (Ueda and Ganem, 1996). PAF has also been reported to stimulate growth and metabolism of early embryos (Ryan et al., 1989; O'Neill, 1997) and acts as a survival factor in some cell types (Toledano et al., 1997).
The previous (O'Neill, 1997) failure to demonstrate an additive effect of IGF-II and PAF on embryo development in vitro may be explained by the current results. If a major role for PAF is in the initiation of IGF-II synthesis, then supplementation of medium with IGF-II may make PAF's action redundant. It places the action of PAF as a potential autocrine link between fertilization [which initiates PAF synthesis (Wells and O'Neill, 1994)] and initiation of transcription of some specific genes (including IGF-II). If such separate, yet redundant, action is a general feature of the many putative embryonic autocrine growth factors, it may help to explain the observations that mice with targeted mutations of some of the putative autocrine growth factors (DeChiara et al., 1990; Liu et al., 1993) have generally been without significant phenotype during the preimplantation phase.
This study provides further support for a role of IGF-II as an important growth/survival factor for the preimplantation mouse embryo. It shows that the aberrations in the production of growth factors caused by IVF or culture in vitro may be a significant cause of poor embryo viability. It also shows that the mouse IVF model may be useful for the study of the normal physiological role of growth factors in normal embryo development.
|
Acknowledgments |
|---|
We thank Dr K.Sturm, Z.Dung and Dr C.Moran for assistance with development of the RT–PCR technique, K.O'Neill for assistance with the manuscript and Dr J.Cheng for advice on statistical analysis. This study was supported by the Northern Sydney Area Health Service Grant Scheme.
|
Notes |
|---|
1 To whom correspondence should be addressed
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bachvarova, R., Cohen, E., DeLeon, V. et al. (1989) Amounts and modulation of actin mRNA in mouse oocytes and embryos. Development, 106, 561–565.[Abstract]
Barlow, D.P., Stoger, R., Herrmann, B.G. et al. (1991) The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature, 349, 84–87.[Medline]
Bolton, V.N., Hawes, S.M., Taylor, C.T. et al. (1989) Development of spare human preimplantation embryos in vitro: an analysis of the correlations among gross morphology, cleavage rates, and development to the blastocyst. J. In Vitro Fertil. Embryo Transfer, 6, 30–35.[ISI][Medline]
Bolton, V.N., Wren, M.E. and Parsons, J.H. (1991) Pregnancies after in vitro fertilization and transfer of human blastocysts. Fertil. Steril., 55, 830–832.[ISI][Medline]
Chia, C.M., Winston, R.M. and Handyside, A.H. (1995) EGF, TGF-alpha and EGFR expression in human preimplantation embryos. Development, 121, 299–307.[Abstract]
Collier, M., O'Neill, C., Ammit, A.J. et al. (1988) Biochemical and pharmacological characterisation of human embryo-derived platelet activating factor. Hum. Reprod., 3, 993–998.
Crosby, I.M., Gandolfi, F. and Moor, R.M. (1988) Control of protein synthesis during early cleavage of sheep embryos. J. Reprod. Fertil., 82, 769–775.[Abstract]
DeChiara, T.M., Efstratiadis, A. and Robertson, E.J. (1990) A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature, 345, 78–80.[Medline]
DeChiara, T.M., Robertson, E.J. and Efstratiadis, A. (1991) Parental imprinting of the mouse insulin-like growth factor II gene. Cell, 64, 849–859.[ISI][Medline]
Delhanty, J.D., Harper, J.C., Ao, A. et al. (1997) Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum. Genet., 99, 755–760.[ISI][Medline]
Doherty, A.S., Temeles, G.L. and Schultz, R.M. (1994) Temporal pattern of IGF-I expression during mouse preimplantation embryogenesis. Mol. Reprod. Dev., 37, 21–26.[ISI][Medline]
Gardner, D.K. (1994) Mammalian embryo culture in the absence of serum or somatic cell support. Cell. Biol. Int., 18, 1163–1179.[ISI][Medline]
Gardner D.K.and Lane, M. (1993) Embryo culture systems. In Trounson, A., Gardner, D.K. (eds), Handbook of In Vitro Fertilization. CRC Press, Boca Raton, pp. 85–114.
Giddings, S.J., King, C.D., Harman, K.W. et al. (1994) Allele specific inactivation of insulin 1 and 2, in the mouse yolk sac, indicates imprinting. Nature Genet., 10, 1125–1129.
Harvey, M.B. and Kaye, P.L. (1991) IGF-2 receptor are first expressed at 2-cell stage of mouse development. Development, 111, 1057–1060.
Harvey, M.B. and Kaye, P.L. (1992) IGF-2 stimulates growth and metabolism of early mouse embryos. Mech. Dev., 38, 169–173.[ISI][Medline]
Hemmings, R., Langlais, J., Falcone, T. et al. (1992) Human embryos produce transforming growth factors activity and insulin-like growth factor II. Fertil. Steril., 58, 101–104.[ISI][Medline]
Heyner, S., Smith, R.M. and Schultz, G.A. (1989) Temporally regulated expression of insulin and insulin-like growth factors in early mammalian development. BioEssays, 11, 171–176.[ISI][Medline]
Ho, Y., Doherty, A.S. and Schultz, R.M. (1994) Mouse preimplantation embryo development in vitro: effect of sodium concentration in culture media on RNA synthesis and accumulation and gene expression. Mol. Reprod. Dev., 38, 131–141.[ISI][Medline]
Lane, M. and Gardner, D.K. (1992) Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. Hum. Reprod., 7, 558–562.
Lane, M.and Gardner, D.K. (1997) Differential regulation of mouse embryo development and viability by amino acids. J. Reprod. Fertil., 109, 153–64.[Abstract]
Lawitts, J.A. and Biggers, J.D. (1993) Culture of preimplantation embryos. Methods Enzymol., 225, 153–164.[ISI][Medline]
Lighten, A.D., Hardy, K., Winston, R.M.L. et al. (1997a) Expression of the insulin-like growth factors and their receptors in human preimplantation embryos. Mol. Reprod. Dev., 47, 134–139.[ISI][Medline]
Lighten, A.D., Moore, G.E., Winston, R.M.L. et al. (1997b) Role of maternal IGF-I in the development of the human preimplantation embryo. J. Reprod. Fertil., 20, (Abstract) 5.
Liu, H.C., He, Z.Y., Mele, C.A. et al. (1997) Expression of IGFs and their receptors is a potential marker for embryo quality. Am. J. Reprod. Immunol., 38, 237–245.
Liu, J.P., Baker, J., Perkins, A.S. et al. (1993) Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell, 75, 59–72.[ISI][Medline]
O'Neill, C. (1985) Partial characterisation of the embryo-derived platelet activating factor in mice. J. Reprod. Fertil., 75, 375–380.[Abstract]
O'Neill, C. (1997) Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro. Biol. Reprod., 56, 229–237.[Abstract]
O'Neill, C. (1998) Autocrine mediators are required to act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro. Biol. Reprod., 58, 1303–1309.
O'Neill, C., Gidley-Baird, A.A., Pike, I.L. et al. (1985a) Maternal blood platelet physiology and luteal phase endocrinology as a means of monitoring pre and post implantation embryo availability following in-vitro fertilisation. J. In Vitro Fertil. Embryo Transfer, 2, 59–65.[Medline]
O'Neill, C., Pike, I.L., Porter, R.N. et al. (1985b) Maternal recognition of pregnancy prior to implantation — methods of monitoring embryonic viability in-vitro and in-vivo. Ann. NY Acad. Sci., 442, 429–439.[ISI][Medline]
O'Neill, C., Gidley-Baird, A.A., Pike, I.L. et al. (1987) Use of a bioassay for embryo-derived platelet-activating factor as a means of assessing quality and pregnancy potential of human embryos. Fertil. Steril., 47, 969–975.[ISI][Medline]
O'Neill, C., Ryan, J.P., Collier, M. et al. (1989) Supplementation of IVF culture media with platelet activating factor (PAF) increased the pregnancy rate following embryo transfer. Lancet, ii, 769–772.
Osterlund, C., Wramsby, H. and Pousette, A. (1996) Temporal expression of platelet-derived growth factor (PDGF)-A and its receptors in human preimplantation embryos. Mol. Reprod Dev., 2, 507–512.
Pantaleon, M., Whiteside, E.J., Harvey, M.B. et al. (1997) Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos: A role for GH in early embryogenesis? Proc. Natl. Acad. Sci. USA, 94, 5125–5130.
Paria, B.C. and Dey, S.K. (1990) Preimplantation embryo development in vitro: Cooperative interactions among embryos and the role of growth factors. Proc. Natl. Acad. Sci. USA, 87, 4756–4760.
Piko, L. and Clegg, K.B. (1998) Quantitative changes in total RNA, total Poly(A), and ribosomes in early mouse embryos. Dev. Biol., 89, 362–378.
Quinn, P. (1995) Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate. J. Assist. Reprod. Gen., 12, 97–105.[ISI][Medline]
Quinn, P., Warnes, G.M., Kerin, J.F. et al. (1985) Culture factors affecting the success rate of IVF and embryo transfer. Ann. NY Acad. Sci., 442, 195–199.[Abstract]
Rappolee, D.A., Sturm, K.S. and Schultz, G.A. (1990) The expression of growth factor ligands and receptors in preimplantation embryos. In Heyner, S. and Wiley, L.M. (eds), Early Embryo Development and Paracrine Relationships. Alan R.Liss, New York, pp. 11–25.
Rappolee, D.A., Sturm, K.S., Behrendtsen, O. et al. (1992) Insulin-like growth factor II acts through an endogenous growth pathway regulated by imprinting in early mouse embryos. Genes Dev., 6, 939–952.
Rappolee, R.A., Brenner, C.A., Schultz, R. et al. (1988) Developmental expression of PDGF, TGF- and TGF-ß genes in preimplantation mouse embryos. Science, 241, 1823–1825.
Roudebush, W.E., LaMarche, M.D., Levine, A.S. et al. (1997) Evidence for the presence of the platelet-activating factor receptor in the CFW mouse preimplantation two-cell-stage embryo. Biol. Reprod., 57, 575–579.[Abstract]
Ryan, J.P., Spinks, N.R., O'Neill, C. et al. (1989) Platelet activating factor (PAF) production by mouse embryos in-vitro and its effects on embryonic metabolism. J Cell Biochem., 40, 387–395.[ISI][Medline]
Singh, J., O'Neill, C. and Handelsman, D.J. (1995) Induction of spermatogensis by androgens in gonadotrophin-deficient (hpg) mice. Endocrinology, 136, 5311–5321.[Abstract]
Smotrich, D.B., Stillman, R.J., Widra, E.A. et al. (1996) Immunocytochemical localisation of growth factors and their receptors in human pre-embryos and Fallopian tubes. Hum. Reprod., 11, 184–190.
Stoddart, N.R., Wild, A.E. and Fleming, T.P (1996) Stimulation of development in vitro by platelet-activating factor receptor ligands released by mouse preimplantation embryos. J. Reprod. Fertil., 108, 47–53.[Abstract]
Tokunaga, K., Taniguchi, H., Yoda, K. et al. (1986) Nucleotide sequence of a full-length cDNA for mouse cytoskeletal ß-actin mRNA. Nucleic Acids Res., 14, 2829
Toledano, B.J., Bastien, Y., Noya, F. et al. (1997) Platelet-activating factor abrogates apoptosis induced by cross-linking of the surface IgM receptor in a human B lymphoblastoid cell line. J. Immunol., 158, 3705–3715.[Abstract]
Ueda, K. and Ganem, D. (1996) Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II. J. Virol., 70, 1375–1383.[Abstract]
Vanderhyden, B.C. and Armstrong, D.T. (1988) Decreased embryonic survival of in-vitro fertilized oocytes in rats is due to retardation of preimplantation development. J. Reprod. Fertil., 83, 851–857.[Abstract]
Ward, A. (1997) Beckwith–Wiedemann syndrome and Wilms' tumour. Mol. Hum. Reprod., 3, 157–168.
Wells, X.E. and O'Neill, C. (1994) Detection and preliminary characterization of two enzymes involved in biosynthesis of platelet-activating factor in mouse oocytes, zygotes and preimplantation embryos: dithiothreitol-insensitive cytidinediphospho-choline:1-o-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase and acetyl-coenzyme A:1-o-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase. J. Reprod. Fertil., 101, 385–391.[Abstract]
Submitted on June 18, 1998; accepted on October 22, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. O'Neill The potential roles for embryotrophic ligands in preimplantation embryo development Hum. Reprod. Update, May 1, 2008; 14(3): 275 - 288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. P. Fleming, W. Y. Kwong, R. Porter, E. Ursell, I. Fesenko, A. Wilkins, D. J. Miller, A. J. Watkins, and J. J. Eckert The Embryo and Its Future Biol Reprod, October 1, 2004; 71(4): 1046 - 1054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
| ||||||||||||||||||||||||||||