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Regular Article |
a The Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, Scotland, United Kingdom
b The Scottish Agricultural College, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, Scotland, United Kingdom
ABSTRACT
DNA damage and other forms of stress are believed to be important factors in reducing the efficiency of in vitro embryo transfer techniques in farm animals. The expression of mRNAs from stress-responsive genes such as gadd153 (CHOP-10, ddit3) may provide a means of assessing the quality of embryos produced in vitro. Treatment of bovine granulosa cell cultures with the DNA-damaging agents, methyl methane-sulphonate (MMS) or sodium arsenite, induced the expression of an mRNA, which hybridized with the hamster gadd153 cDNA. Part of the corresponding bovine cDNA was amplified by nested polymerase chain reaction (PCR), cloned, and sequenced. Using a sensitive reverse transcriptase-PCR assay we have investigated the expression of gadd153 and ß-actin in blastocyst-stage bovine embryos treated with MMS or sodium arsenite. Both agents produced an increase in the ratio of gadd153 mRNA relative to ß-actin. These results show that there are changes in gene expression in blastocyst-stage bovine embryos in response to genotoxic stress, suggesting that an increase in gadd153 mRNA is a useful marker of DNA damage and metabolic stress in preimplantation embryos.
conceptus, gene regulation, implantation/early development, IVF/ART, stress
INTRODUCTION
The culture of mammalian embryos in vitro exposes them to a variety of stresses that are not normally encountered in vivo. In ruminant species, prolonged periods of culture in vitro are associated with an increase in fetal mortality and an increased risk of abnormalities in those fetuses that do survive [1, 2]. Culture of a preimplantation embryo in vitro has wide-ranging effects on subsequent fetal and postnatal growth [3], altering the allometric coefficients for key organs, including heart, liver, kidneys, and skeletal muscle [4, 5]. Many culture systems used to support the development of fertilized cattle oocytes to the blastocyst stage rely on serum supplements, coculture cells, or both. Even with the most efficient systems a proportion of zygotes fail to develop and show changes in the patterns of developmentally important genes [6, 7], reflecting a stress response to suboptimal culture conditions [8]. Studies of mouse embryos have shown that the stress imposed by in vitro culture can cause permanent damage that compromises subsequent development [9, 10].
One of the critical components during the early stages of development is the genome; DNA must be replicated accurately, otherwise errors or deletions will be copied in each subsequent round of cell division and spread throughout the fetus as it develops. When damage occurs in genes that are not vital to blastocyst development per se, it need not result in early embryonic death. Instead, the consequences of damage to a gene that is silent in the blastocyst may become apparent only when it is required later in development and the remoteness of its occurrence will often preclude identification of the underlying cause. Thus, in the context of embryo production in vitro, any detectable early response system that reacts to the occurrence of DNA damage would provide a valuable method of screening embryo culture systems for risks of subsequent aberrant gene expression.
Despite the central importance of DNA replication, little is known about the response of the preimplantation ruminant embryo to DNA damage. Mammalian cells possess an elaborate system to protect them against agents that modify DNA. The RNAs from a number of genes are specifically up-regulated when cells in culture are exposed to DNA-damaging agents such as methyl methane-sulphonate (MMS) [11]. One of these is the growth arrest and DNA damage gene gadd153 (also known as CHOP-10 or ddit3), which codes for a DNA-binding protein. Dimers formed between gadd153 and other DNA-binding proteins interact with specific sites in the promoters of a variety of genes. These interactions control the transcription of the target genes. This regulation is both positive and negative, reducing the expression of some genes [12, 13] and increasing the expression of others [14]. We have shown previously that the gadd153 gene is expressed in the mouse blastocyst [15] and its mRNA is increased when the blastocyst is exposed to DNA-damaging agents [16]. The objectives of the present research were to identify the bovine homologue of the rodent gene and characterize its expression in bovine granulosa cells and embryos following exposure to two DNA damaging agents, an alkylating agent, MMS, and a metabolic inhibitor, sodium arsenite.
MATERIALS AND METHODS
Granulosa Cell Culture
Granulosa cells were isolated from bovine follicles obtained from the ovaries of heifers at slaughter. The follicles were aspirated, the oocytes recovered, and the remaining follicular liquid centrifuged for 5 min at 350 x g. The granulosa cells were recovered from the pellet and transferred to tissue culture dishes containing Medium 199 (Life Technologies, Paisley, UK) supplemented with 10% v/v steer serum (Globepharm Ltd., Esher, Surrey, UK). The cultures were incubated at 38°C in an atmosphere of 5% CO2 in air for 72 h. After 48 h, the medium was refreshed and the cells were treated with either 100 µg/ml MMS or 50 µM sodium arsenite (both from Sigma, Poole, Dorset, UK) for 4–6 h. Total RNA was isolated using Tri-reagent (Sigma) in accordance with the manufacturer's instructions.
Northern Analysis
Five-microgram samples of total RNA were separated on 1.2% (w/v) denaturing agarose gels. The gels were stained with ethidium bromide to confirm that equal amounts had been loaded before the RNA was transferred to a nylon membrane (Boehringer-Mannheim, Mannheim, Germany). Probe templates were labeled with [
-32P]dCTP using a Megaprime labeling kit (Amersham Pharmacia Biotech, Bucks, UK). After hybridization the blots were washed in 1x saline-sodium citrate (SSC) + 1% SDS at 65°C and autoradiographed on x-ray film.
Cloning the Bovine gadd153 Homologue from Granulosa Cells
One microgram of total RNA from MMS-treated granulosa cells was reverse transcribed in 20 µl of 1x reverse transcriptase buffer (Life Technologies) containing 20 µM dNTPs, 10 mM dithiothreitol (DTT), 2.5 µM HT11C adapter primer (5'-AAGCT(11)-3') and 200 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT; Superscript, Life Technologies). The mixture was incubated at 37°C for 1 h before the reaction was terminated by heating to 95°C for 5 min. Two nested PCR reactions were carried out in a 50-µl volume of 1x PCR buffer (Life Technologies) containing 20 µM dNTPs, 2.5 µM primers (Table 1), 1.5 mM MgCl2, 3 µl of template, and 1 U of Taq DNA polymerase. Both reactions were carried out for 35 cycles, each comprising denaturation at 94°C for 30 sec; annealing at 50°C for 45 sec, and extension at 72°C for 45 sec. The PCR product was purified from a 1% agarose gel and cloned into the SrfI site of the pCR-Script SK(+) plasmid using the pCR-Script Amp SK(+) Cloning Kit (Stratagene, Cambridge, UK). The plasmid insert was sequenced using an ABI 377 DNA sequencer.
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Bovine Embryo Culture
The oocytes were recovered and matured in the presence of granulosa cells for 24 h. After fertilization in vitro the zygotes were cocultured on granulosa cell layers in Medium 199 (Life Technologies) supplemented with 10% v/v steer serum [3, 17]. The same batch of serum was used for all experiments. The culture was continued until Day 7, when the embryos had reached the blastocyst stage of preimplantation development. On Day 7, groups of 10 blastocysts were transferred to fresh Medium 199 supplemented with 10% v/v steer serum. MMS or sodium arsenite dissolved in PBS was then added to give final concentrations of 100 µg/ml MMS or 50 µM sodium arsenite. PBS only was added to the control groups.
RNA Isolation from Embryos
Groups of 10 embryos were dissolved in 100 µl of guanidine isothiocyanate solution containing 4 µg rRNA (Boehringer-Mannheim) and 5 pg of rabbit -globin mRNA (Life Technologies). The samples were layered onto 1 ml of 5.7 M CsCl2 solution and centrifuged at 228000 x g for 4 h at 20°C (Beckman TL100 centrifuge using a TL100-2 rotor). The buffer and CsCl2 solutions were removed by aspiration, the RNA pellets were redissolved in diethyl pyrocarbonate (DEPC)-treated water, precipitated with 2.5 M ammonium acetate/100% ethanol, washed twice with 70% ethanol, air-dried, and dissolved in DEPC-treated water [18].
Semi-Quantitative RT-PCR
The reverse transcription mixture contained the RNA from 10 embryos, 1x RT buffer, 200 µM dNTPs, 10 mM DTT, 500 pg of HT11C adapter primer, and 200 U of M-MLV RT (Superscript; Life Technologies) in a final volume of 20 µl. The mixture was incubated at 37°C for 1 h and heated to 95°C for 5 min to terminate the reaction. Separate PCR reactions were set up for each gene studied using the primers shown in Table 1. For the amplification of gadd153, the mixture contained 6 µl of the RT mix and 10 pmol of gene-specific primers; the reactions for ß-actin contained 2 µl of the RT mix and 10 pmol of the primers and reactions for the amplification of -globin used 2 µl of the RT mix and 20 pmol of primers. All of the reactions were in 1x PCR buffer (Life Technologies) containing 4 µM dNTPs and 0.25 µCi of [-32P]dCTP (Amersham Pharmacia Biotech) made up to a final volume of 50 µl. The reactions were initiated by the addition of 1 U of Taq DNA polymerase (Life Technologies) and the PCR was carried out for 33 cycles (gadd153 and actin) or 25 cycles (-globin). The protocol consisted of denaturation at 94°C for 30 sec, annealing at 59°C for 45 sec, and extension at 72°C for 45 sec. Samples of the reaction mixture were separated on 4% polyacrylamide gels. Diluted samples of the total reaction mixture were applied to strips of filter paper and counted alongside the dried gels using a proportional wire counter (Instant Imager, Packard, Bucks, UK). The extent of the reaction was calculated from the fraction of radioactivity incorporated. Semi-log plots of product vs. cycle number were linear, showing that all of the reactions were in the exponential part of the progress curve. There was a linear relationship between the amount of template added and the amount of product formed [15, 16]. Embryos were treated in pools of 10. Duplicate RT and PCR reactions were carried out on the RNA that had been extracted from the embryos. The experiment was repeated three times and the average values from each individual experiment were used in the final calculations with n = 3. Data were analyzed using an unpaired Student t-test.
RESULTS
The upper panel of Figure 1 shows a typical example of the hybridization of the hamster gadd153 cDNA to a Northern blot of RNA prepared from bovine granulosa cells. The probe hybridized to a single message that was induced by up to 20-fold in cells treated with 100 µg/ml MMS or 50 µM sodium arsenite. At the same time, the treatments caused a small decrease in ß-actin mRNA levels (Fig. 1, middle panel) showing that the increase in gadd153 is not due to an overall increase in mRNA levels. The single bovine gadd153 mRNA was approximately 1000 base pairs (bp) in length, similar to the message described in hamster, human, and mouse cells.
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Attempts to amplify the cDNA corresponding to the bovine gadd153 gene using PCR primers based on the hamster or mouse sequences [12] were unsuccessful. Multiple alignments of the known gadd153 sequences were carried out using the CLUSTAL V programme [19]. Two regions corresponding to the bases 300 to 350 and 480 to 510 on the mouse CHOP-10 sequence [12] were found to be nearly identical in all species. Primers based on these sequences (Table 1) were used in a nested amplification of cDNA from MMS-treated bovine granulosa cells. A combination of the CP1 and HT11C primers was used in the first reaction. A portion of the product was then used as template in the second reaction using the CP2 and the HT11C primers. The single product hybridized strongly to the hamster gadd153 cDNA. This PCR product was isolated, cloned into the plasmid pCR Script SK(+), and sequenced. This partial sequence of the bovine gadd153 gene (shown in Fig. 2) was used to design a specific reverse primer (BR1). PCR using a combination of CP2 and BR1 primers with cDNA from bovine granulosa cells or embryos as template produced a single product with the anticipated size of 180 bp. This product hybridized strongly with the hamster gadd153 cDNA (Fig. 3a).
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DNA damage and oxidative stress were created in bovine blastocysts by treating them for 4 h with 100 µg/ml MMS or 50 µM sodium arsenite. These short treatments did not cause gross changes in embryo morphology; however, there was some darkening of the embryos treated with sodium arsenite. Typical examples of the products of RT-PCR carried out under semiquantitative conditions using RNA prepared from control and treated blastocysts are shown in the two panels on the left-hand side of Figure 3b. The relative amounts of target RNA in different samples were determined by comparing the ratio of PCR products generated by amplification of the target and a structurally unrelated endogenous gene in separate reactions [20]. A second aliquot of the cDNA was amplified with primers specific for ß-actin mRNA as shown on the right-hand side of Figure 3b. Because this mRNA is present in all embryonic cells it is a convenient internal standard. Figure 4a shows that the gadd153 to ß-actin ratio was significantly increased, by about twofold (P < 0.05) in the group treated with MMS. There was a similar, albeit nonsignificant increase in the ratio for embryos treated with sodium arsenite.
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A visual inspection of the gels suggested that ß-actin mRNA levels had fallen following inhibitor treatments (right-hand side of Fig. 3b). Therefore -globin mRNA was added to the embryonic RNA as an external standard and a third amplification was carried out using primers specific for the globin cDNA [18]. The relative ratios of gadd153: -globin and ß-actin:-globin are independent of nonspecific changes in gene expression and reflect the abundance of the mRNA in each group of embryos. Figure 4b shows that the total abundance of gadd153 mRNA was significantly increased in the sodium arsenite-treated blastocysts. There was a similar increase in the MMS-treated blastocysts, although this failed to achieve significance. At the same time the ratio of ß-actin to -globin PCR products fell (Fig. 4c). This change was significant (P < 0.01) in respect of the cDNA from MMS-treated embryos but not significant for cDNA from those treated with sodium arsenite. The effect of MMS and sodium arsenite was similar in both the blastocyst groups and the granulosa cells, with each exhibiting an increase in the relative abundance of gadd153 mRNA and a corresponding fall in the ß-actin mRNA.
DISCUSSION
These results show that the bovine blastocyst can mount an active response to DNA damage through an increase in the expression of the gadd153 gene. This is similar to the increase of about twofold in expression of the murine homologue, CHOP-10, in the mouse blastocyst exposed to MMS or sodium arsenite [16]. Both MMS and sodium arsenite are rather general DNA-damaging agents with more than one site of action. MMS is an alkylating reagent that reacts principally with the ring nitrogens of purine bases. Sodium arsenite reacts with vicinal thiols, blocking the action of several important enzymes, depleting ATP levels, and subjecting the embryo to oxidative stress. Therefore, these agents are both extreme examples of the types of stress encountered by the embryo in culture. The induction of gadd153 suggests that it can be used as a marker for the evaluation of embryos and their environments both in vivo and in vitro.
The relative increase in gadd153 may be similar to the induction of HSP70 mRNA when bovine and murine embryos are exposed to culture stress [6, 7, 21]. The heat shock proteins play an essential role in the endoplasmic reticulum, folding proteins prior to export. The accumulation of unfolded proteins is believed to be one of the signals that induces gadd153 expression [22, 23] and the induction may represent a change in posttranslational processing of proteins. However, this is not the only system that can increase gadd153 expression. Recent studies have suggested that there is a second pathway involving the transactivator, ATF-2, which links gadd153 expression directly to the amino acid supply [24]. If this pathway is functional in the embryo then it is likely to be important in regulating responses to nutrients in the medium.
The choice of the endogenous reference mRNA is particularly important in the interpretation of the RT-PCR data. Whereas the expression of ß-actin by the mouse embryo is unaffected by MMS and sodium arsenite treatment [16], there was a significant reduction in the abundance of its mRNA in the treated bovine embryo, suggesting that there are other effects on gene expression in the bovine embryo. Because mild culture stress does not produce an overall reduction in transcription [7] there may be a biphasic response with toxic agents such as MMS and sodium arsenite causing a generalized reduction in transcription through the inhibition of RNA synthesis. A fall in total transcription in nonviable embryos may be responsible for the correlation between mRNA content and developmental capacity observed in bovine embryos [25].
Analysis of gadd153 expression in the coculture cells may also provide useful information. Because the granulosa cells share the same medium as the embryo yet remain in the culture dish, they are readily available on a relatively large scale at all stages of the in vitro production process. The changes in gadd153 expression can be measured in the granulosa cells, avoiding the destruction of the eggs or embryos that are available for further analyses or the establishment of pregnancy. Furthermore, the cells offer an improvement in sensitivity because stress increases gadd153 expression from a lower background (compare Figs. 1 and 3). However, because these are terminally differentiated cells that do not produce the same products or have the same metabolic requirements as the embryo, it must be emphasized that there may be important differences in some of the responses. The changes in gadd153 expression in the granulosa cells are nevertheless important in their own right, as stress may also affect their function, causing them to produce factors that influence the development of the embryo. Stress may also affect the maturing oocyte through the intimate links that exist between the granulosa cells and the oocyte prior to ovulation or during conventional in vitro maturation of oocyte-cumulus complexes. Stress-sensitive genes such as gadd153 may therefore be useful as markers of the prefertilization events that influence subsequent development [26] and provide a means to assess in vitro maturation conditions.
The expression of gadd153 (CHOP-10) is not essential for normal development of the mouse blastocyst and transgenic animals with a targeted deletion in the gene are apparently normal [27]. In embryonic cells such as the F9 embryonal carcinoma cell, induction of gadd153 expression precedes the initiation of programmed cell death or apoptosis [16]. The product of the gadd153 gene may therefore be a marker for a protective system that eliminates damaged or defective cells and the endogenous expression may be due to the presence of apoptotic cells that have been shown to be present in the bovine blastocyst [28]. However, the induction of gadd153 is not irreversible; the stimulation of growth-arrested cells with serum is sufficient to prevent the up-regulation of gadd153 expression [29]. Therefore, it may be that, unwittingly or perversely, the use of culture media containing serum facilitates the survival of damaged blastocysts while failing to repair inherent defects in their longer-term developmental capability. Because blastocyst survival rather than normality has been used as the sole criterion for the optimization of nearly all bovine embryo culture systems, the importance of cell death in protecting against defects in future development has tended to be overlooked. If conditions are chosen to maximize the number of embryos that reach blastocyst stage, the induction of stress-mediated apoptosis may be suppressed, with damaged embryos inadvertently allowed to progress. There are a number of reports of fetal abnormalities in ruminant species following the culture of preimplantation embryos in vitro, the most common being fetal oversize and an increase in the incidence of hydramnios [30]. The further study of markers such as gadd153 will provide information on the effect of culture stress on subsequent developmental abnormalities in the ruminant embryo.
ACKNOWLEDGMENTS
The excellent technical assistance of Mary E. Staines is gratefully acknowledged.
FOOTNOTES
1 This work was supported by the Scottish Office Agriculture, Environment and Fisheries Department. N.F.-R. was the recipient of a Boyd Orr Research Centre studentship. 
2 Correspondence: William D. Rees, The Rowett Research Institute, Greenburn Rd., Bucksburn, Aberdeen AB21 9SB, Scotland, UK. FAX: 44 1224 715349; wdr{at}rri.sari.ac.uk
Accepted: December 12, 2000.
Received: June 8, 2000.
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