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The Fels Institute for Cancer Research and Molecular Biology,3
Department of Biochemistry,4 Temple University School of Medicine, Philadelphia, Pennsylvania 19140
The Wisconsin National Primate Research Center,5 University of Wisconsin, Madison, Wisconsin 53715
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cell cycle control, DNA repair, embryo, gene expression, gene regulation, oocyte, primate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA repair likely constitutes an essential component of the overall process of creating an embryonic genome. Damage to the maternally inherited genome may arise in the oocyte during the long delay between entry into meiosis and fertilization. Damage to the paternally inherited genome may arise due to mutagenic events during spermatogenesis, which occur with greater frequency as compared with oogenesis [3, 4]. Endogenous nucleases may exist in sperm [5], and induce nicks or double-strand breaks in paternal DNA either before or after fertilization. Oxidative DNA damage or damage as a result of exposure of gametes to DNA modifying agents may also present a problem to the early embryo [6, 7]. The DNA damage arising from these processes would need to be repaired, ideally before the first round of DNA replication, to minimize the mutation load of the developing embryo.
The need to ensure genome integrity at the start of life must be balanced against the need to prevent excessive cell loss if an embryo is to survive. Sensing mechanisms that detect DNA damage can trigger cell cycle arrest, leading to cell death through apoptosis (see review in [8]). Cell death during the first few cell cycles would greatly diminish chances for embryo survival, and indeed it appears that mammalian embryos may be inhibited from undergoing apoptosis until late cleavage or blastocyst stages [9–12].
One way to reconcile the conflicting needs of the embryo would be for the oocyte to be endowed with a supply of DNA repair enzymes that can repair DNA damage initially, together with a supply of antiapoptotic proteins to prevent early apoptosis. An early role for ooplasmic factors in preventing apoptosis has been suggested [13], but early expression of DNA repair-related genes has not been examined in detail, particularly in primates. Deficient expression of maternal stores of these DNA repair factors could affect preimplantation development or long-term embryo viability, or both. Some DNA repair factors exist in the oocyte [14, 15] and may be responsible for incorporating DNA by transgenesis, following microinjection into MII stage oocytes [16, 17], via application to permeabilized sperm [18], or following microinjection into fertilized zygotes [19]. These factors also direct DNA repair in normal fertilized embryos [20–22]. The early embryo can regulate DNA repair gene expression. In preimplantation stage rat embryos sired by males exposed to cyclophosphamide, genes that participate in nucleotide excision repair, mismatch repair, and recombination repair display increased mRNA expression, whereas those encoding components of the base excision repair family and recombination repair family decrease in mRNA expression [23]. Acquisition of a dosage threshold for radiation-induced malformations between the one-cell and two-cell stages indicates activation of DNA repair mechanisms [24]. The Ddit3 (Gadd153) gene is induced in mouse embryos exposed to genotoxic stress [7]. These observations indicate that newly fertilized embryos of at least some species possess the ability to sense, respond to, and repair at least some types of DNA damage, particularly in the incoming sperm DNA.
As development proceeds, the embryo may acquire a greater ability to respond to DNA damage by activating and regulating DNA repair and proapoptotic genes in a manner similar to that typically observed in somatic cells. Induced mutations for several genes involved in DNA repair lead either to impaired ability to form blastocysts or inner cell masses (e.g., Flap endonuclease Fen1, Atr [25, 26]), increased apoptosis in blastocysts (e.g., Poly(ADP-ribose)polymerase 1 [Parp1] combined with Xrcc5 [Ku80] or Atm deficiency [27, 28]), or to embryonic lethality during or shortly after embryo implantation (e.g., Xrcc1 [29, 30]; apurinic/apyrimidinic endonuclease [Apex/Ref1 deficiency] [31]; and Rev3 deficiency [32]). Mutations in some DNA repair genes lead to defects during later life, including lethality, sterility, or predispositions to cancer (e.g., exonuclease I; Atm; Brca2; or Xrcc1 mutation alone) [33–35]. The failure of the latter to cause early developmental arrest indicates that some DNA repair mechanisms either are not expressed or are dispensable for early embryogenesis, but may become active later during development.
To understand to what degree preimplantation embryos can respond to and repair DNA damage, and to what extent preimplantation embryos may be selectively sensitive to certain forms of DNA damage, detailed information about the timing of expression of DNA repair genes is needed. Additionally, ascertaining whether in vitro manipulation, embryo culture, or parental/gamete chemical exposure activate DNA repair or cell cycle checkpoint genes could be helpful for developing improved methods for basic embryo research, applied reproductive biology, and assisted reproduction in clinical medicine. We report here the analysis of the expression of DNA repair and cell cycle checkpoint control genes in oocytes and embryos of a nonhuman primate model species, the rhesus monkey, along with an examination of the effects of in vitro embryo culture on these genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The studies undertaken here employed the Primate Embryo Gene Expression Resource (PREGER) (www.preger.org) [36–38]. This resource encompasses a collection of reverse transcribed and polymerase chain reaction (RT-PCR)-amplified cDNA libraries corresponding to more than 170 samples of rhesus monkey oocytes and preimplantation stage embryos. The isolation and culture of the oocytes and embryos during the construction of the PREGER sample set has been described in detail [36]. Oocytes contained in the PREGER sample set were obtained from gonadotropin-stimulated monkeys treated with hCG (metaphase II stage) or without hCG (germinal vesicle stage). For the samples employed in the studies reported here, embryos were obtained either by natural conception (morula/blastocysts) or by in vivo oocyte maturation followed by in vitro fertilization and in vitro embryo culture in HECM9 or G1/G2 sequential media [39]. Between 3 and 13 samples of one to four oocytes or embryos were obtained for each stage. It should be noted that because the entire mRNA population is uniformly amplified during the RT-PCR procedure, the amount of input mRNA (i.e., the range of one to four embryos) does not affect the quantitative representation of sequences within the amplified cDNA population. The embryos collected for inclusion in the PREGER sample set were of high quality and healthy in appearance, with blastomeres displaying uniform granularity. Fragmented embryos were avoided. A minimum of three females were employed to obtain samples for each stage, with the exception of the two-cell stage, for which two females were employed. Samples of eight-cell and morula-stage embryos treated with the RNA polymerase II inhibitor 
-amanitin from the pronucleate stage onward in HECM9 culture were included to evaluate transcriptional dependence of mRNA expression after the time of embryonic genome activation. Details concerning the array, diversity, and origin of samples, and the sensitivity and quantitative reliability of the quantitative amplification and dot blotting method have been described previously [36] and in other references available at our Web site (www.preger.org).
The general care and housing of rhesus monkeys (Macaca mulatta) at the Wisconsin National Primate Research Center have been described previously [40, 41]. The Wisconsin National Primate Research Center is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and animal protocols and experiments were approved by the Graduate School Animal Care and Use Committee and the University of Wisconsin-Madison. The animals were maintained according to recommendations of the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act with its subsequent amendments.
Complementary DNA Probes and Hybridization
The cDNA probes used in this study were obtained by RT-PCR (Table 1 ), or from other sources as indicated. The identities of amplified cDNAs were confirmed either by using diagnostic restriction digests or DNA sequencing. Additionally, cDNAs harboring the full-length open reading frames of wild-type human TP53 (p53), CDKN1 (p21), MTBP, and MDM2 were generated by restriction endonuclease digestion of plasmids (kindly provided by Dale Haines) containing these sequences. Blot preparation, probe preparation, hybridization, and quantitative analyses were performed as described [36, 42, 43]. Data were expressed as the mean cpm bound value for each culture condition and stage of oocytes and embryos included in the analysis. Statistical differences in hybridization signals obtained for different stages or conditions were evaluated by the t-test.
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Limitations of Analysis
Because human cDNAs were employed here as probes, interspecies variation might affect hybridization signals. Of the genes for which no hybridization signals were obtained (see below), Macaca sequence data were available for two (TOP3A and CDKN1A), and revealed 91%–95% identity with the published human sequences. For three mRNAs that produced low hybridization signals, available Macaca sequence data revealed 94%–99% identity with the published human sequences. These values were very similar to the range of sequence identities (91%–99% identity) found between published human and Macaca sequences for more highly expressed mRNAs, such as ATM, ATR, APEX1, RFC3, MSH3, UBE2B, RAD51, and XRCC1. Thus, while interspecies sequence diversity might affect the strength of hybridization signals, this effect should generally be very small. One exception would be for mRNAs that bear long, 3' untranslated regions, which could display greater sequence divergence.
While the PREGER sample set encompasses more than 170 samples, these samples are distributed over a diverse array of experimental conditions. For nearly all stages and conditions, a minimum of three independent samples were obtained, and oocytes or embryos were derived from at least three different females [36]. The PREGER sample collection continues to expand as the resource continues to be developed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We first examined gene expression in oocytes and in vitro developing embryos cultured in HECM9 medium to obtain a basic understanding of the levels of expression, timing of expression, and the completeness with which oocytes and embryos of various stages might express essential members of each functional category. Of the 48 mRNAs examined, we observed detectable expression for 41. The data for these 41 mRNAs are shown in Figures 2–4, representing genes that are involved in sensing DNA damage, repairing DNA damage, and controlling the cell cycle, respectively.
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Expression of mRNAs Encoding DNA Damage Sensor Proteins
The first requirement in a cell for repairing damaged DNA is for the cell to detect the damage so that it may activate the appropriate machinery to mediate repair. We examined the expression of mRNAs encoding components of some of the major DNA damage sensing pathways (Fig. 2). The ATR mRNA was detectable at a low abundance in oocytes and embryos before the eight-cell stage, and then increased in a transcription-dependent (i.e., -amanitin-sensitive) manner. The ATM mRNA appeared to be more abundant than the ATR mRNA. The ATM mRNA displayed greater abundance as a maternal transcript, and was not -amanitin sensitive at the 8- to 16-cell stage. Both mRNAs, however, displayed increased expression during subsequent development to the blastocyst stage. The ATM and ATR proteins are structurally related proteins that play an important role in coupling DNA damage-sensing proteins to cell cycle regulators such as CHEK1 and CHEK2. Of interest, probes for CHEK1 and CHEK2 mRNAs yielded similar expression profiles, but a nearly 200-fold stronger hybridization signal for CHEK1 as compared with CHEK2. The TREX1 mRNA, encoding a protein that associates with ATR, was marginally elevated in eight-cell embryos relative to -amanitin treated embryos (P = 0.053), and was then significantly elevated by the morula stage.
A majority of the mRNAs examined that encode proteins related to sensing single-strand break (SSB) damage was expressed in the oocyte (e.g., ATR, BRCA1, HUS1, RAD17, RFC3, RFC4, RAD1, and RPA2; Fig. 2). The mRNA encoding RAD9A, however, was not detected in oocytes, but was up-regulated significantly at the eight-cell stage, and the probe encoding RFC5 was not detected at any stage. A majority of the SSB-sensing protein mRNAs that were examined displayed increases in expression beginning at the eight-cell stage, and this was sensitive to -amanitin treatment in most cases. The mRNA encoding the BRCA1 protein, which exists in a large multiprotein complex [52] and participates in the repair of a variety of forms of DNA damage, was abundantly expressed throughout development.
As with the SSB-sensing proteins, a majority of the mRNAs encoding proteins involved in maintaining the replication fork (e.g., ATR, BRCA1, PCNA, BLM, and TOPBP1) were expressed in the oocyte, but the mRNA-encoding RFC1 displayed little or no hybridization signal in the oocyte (Fig. 2). The mRNA encoding MRE11A, which is involved in detecting double-strand breaks, produced low hybridization signals throughout development.
Some of the mRNAs assayed above displayed apparent increases in abundance during either oocyte maturation or progression to the pronucleate stage. Because the major genome activation event occurs at the six- to eight-cell stage, this most likely reflects an effect at the level of polyadenylation of maternal transcripts, which could enhance oligo(dT) priming during reverse transcription. The PREGER sample set does not include -amanitin treated samples of pronucleate stage embryos needed to discern the transcription-dependence of such increases.
Expression of mRNAs Encoding DNA Damage Repair Proteins
DNA mismatch repair (MMR) genes participate either in mismatch repair or in meiotic recombination. The MMR genes that participate in meiosis include MLH1, MLH3, PMS2, MSH4, and MSH5 [53]. MMR genes participating in mismatch repair include MLH1, MLH3, PMS2, MSH2, MSH3, and MSH6 [53]. MutS, a heterodimer of MSH2 and MSH6, is predominantly responsible for recognizing DNA base/base mismatches and insertion-deletion loops, while MutSß, a heterodimer of MSH2 and MSH3, also corrects insertion-deletion loops [54]. All of the mRNAs examined that encode MMR proteins were expressed in oocytes and embryos (Fig. 3). The MLH1 mRNA, however, decreased in apparent abundance during oocyte maturation and remained expressed at a low level throughout development to the blastocyst stage. The MSH2 mRNA was expressed throughout development, with a variable and transient increase in expression at the eight-cell and morula stages. The MSH3 mRNA increased significantly in abundance during embryogenesis. The hybridization signals for MSH3 mRNA from the eight-cell stage onward were, interestingly, at least 40-fold greater than the signals for MSH6, suggesting that MSH3 was vastly overexpressed in proportion to MSH6. The PMS2 mRNA was expressed at a low level throughout development, but without the initial high level of expression in oocytes observed for the MLH1 mRNA.
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A majority of the mRNAs examined that encode base excision repair proteins were expressed in oocytes and embryos (Fig. 3). The mRNA encoding OGG1, however, was not detected. Additionally, the mRNA encoding UNG was largely absent from the morula stage onward. All three (ATR, XPC, and ERCC2) of the mRNAs examined that code for nucleotide excision repair (NER) proteins were expressed. Both the XPC and ERCC2 mRNAs were expressed in oocytes and early embryos and then declined in expression (Fig. 3).
Among the mRNAs examined that encode proteins for the repair of single-strand DNA breaks, both RPA2 and APTX were transcriptionally induced by the eight-cell stage (Figs. 2 and 3). RPA2 in fact appeared to be induced as early as the two-cell stage, although -amanitin treated two-cell embryos were not obtained to confirm this.
Among the mRNAs examined that encode double-strand break repair proteins, most (G22P1, RAD54L, and RAD51) were expressed in the oocyte, decreased in abundance during oocyte maturation, and then increased in abundance again at the eight-cell stage (Fig. 3). This contrasts somewhat with the low level of expression of MRE11A mRNA (Fig. 2). The mRNA encoding RAD50 was not detected at any stage. Of the two mRNAs specifically related to postreplication DNA repair, the UBE2A mRNA was expressed predominantly as a maternal mRNA and was gradually down-regulated during blastocyst formation. In contrast, the UBE2B mRNA increased steadily in abundance from the eight-cell stage onward.
Expression of Genes Encoding Cell Cycle Control Genes
Cell cycle arrest at G1/S and G2/M checkpoints provides cells with an opportunity to repair DNA damage either before commencing DNA replication or before committing to mitotic division. The proteins that sense DNA damage operate via CHEK1 and CHEK2 to mediate cell cycle arrest through specific pathways (Fig. 1). The proteins that participate in the G1/S phase checkpoint include CDKN1B (also known as P27/KIP), MDM2, MTBP, and TP53. All four of these mRNAs displayed comparatively low levels of expression in the oocyte and early embryo. The CDKN1B and MDM2 mRNAs were then up-regulated as development proceeds (Fig. 4). The TP53 hybridization signal at the early blastocyst stage was also significantly increased over earlier stages, but this mRNA remained expressed at a low abundance (Fig. 4). The polo-like kinases (PLK1 and PLK3) provide important regulation of the G2/ M phase transition [55]. PLK1 is required for correct spindle function during mitosis and meiosis [56–59]. Both PLK mRNAs were expressed as maternal transcripts and continued to be expressed throughout development. The PLK1 mRNA appeared several times more abundant than the PLK3 mRNA.
Effects of Culture on Expression of DNA repair and Cell Cycle Checkpoint Genes
Aberrant expression of DNA repair or cell cycle checkpoint genes could impair embryo development or lead to increased rates of apoptosis. To determine whether in vitro culture could affect the expression of these genes, we compared expression between cultured blastocysts and blastocysts flushed from the reproductive tract of spontaneously mated females (Table 3 and annotated with arrows in Fig. 1). No significant difference was observed between embryos grown in G1/G2 medium versus HECM9 medium, so the data for all cultured embryos were combined for comparison with flushed embryos. Of the 41 mRNAs for which expression was detected, cultured and flushed blastocysts differed significantly in the expression of 14 mRNAs. Of these, 13 mRNAs were significantly elevated (1.41-fold to 10-fold) in cultured blastocysts and 1 mRNA (MSH2) was significantly reduced. Most (7 of 14) of these mRNAs display increased expression during embryogenesis compared with that of oocytes (annotated expression pattern E, Table 3). In addition to these 13 mRNAs, 7 other mRNAs displayed a trend toward increased expression, but with P values between 0.05 and 0.1; most of these were expressed predominantly as maternal transcripts (annotated expression pattern M, Table 3). Thus, more than one-quarter of the detected mRNAs were significantly elevated in cultured embryos and nearly half (20/41) of the mRNAs overall displayed signs of increased expression in cultured embryos. (It should thus be noted that the prominent increase in ERCC2 mRNA expression at the HB stage [Fig. 3] is due in large part to the effect of culture.)
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several aspects of the mRNA expression data suggest that, while the rhesus embryo expresses many of the genes needed for DNA repair, its ability to repair DNA may in fact be highly limited. For example, the limited expression of CHEK2 mRNA may severely limit response to certain kinds of DNA damage that require the G2/M phase checkpoint. The double-strand DNA break repair pathway appears particularly dependent on CHEK2 activity. The ability to undertake double-strand break repair and base excision repair may also be limited by the combined effects of little or no expression of the NBS1, MRE11A, and RAD50 mRNAs for double-strand break repair and the OGG1 mRNA for base excision repair.
It is interesting to note that homozygous mutations for Atr in mice leads to early embryonic lethality, whereas homozygous Atm mutants are viable [26, 35]. The expression of ATM mRNA as a maternal transcript could lessen the early impact of embryonic ATM gene deficiency.
An additional limitation in the ability of the embryo to respond to DNA damage by cell cycle arrest is apparent in the lack of detectable expression of the CDKN1A (p21) mRNA. CDKN1A is a target gene of TP53, and may mediate TP53-dependent cell cycle arrest or apoptosis [60]. Deficiency for CDKN1A compromises the ability of fibroblasts to arrest their cell cycle in response to DNA damage [61].
Mismatch repair may be compromised in rhesus monkey embryos due to the apparent overexpression of the MSH3 mRNA. In somatic cells, overexpression of MSH3 can cause a deficiency of mismatch repair by reducing the presence of the MutS complex via sequestration of MSH2 and degradation of MSH6 [54, 62]. The low level of MLH1 mRNA may also limit mismatch repair capability.
Most of the mRNAs encoding proteins required for maintaining the replication fork, for repairing single-strand breaks, and for postreplication repair are expressed. The mRNAs encoding proteins needed for nucleotide excision repair are likewise expressed. These four pathways of DNA repair can operate through ATR and CHEK1, for which mRNAs are also expressed. Of interest, however, the RFC5 mRNA, which encodes a component of the replication complex that recruits DNA polymerases to primed DNA and also binds to single-strand DNA during repair of breaks, was not detected. Additionally, two topoisomerase mRNAs (TOP3A and TOP3B) were not detected at any stage, and the probe for BLM, which interacts with topoisomerases [63], yielded comparatively low hybridization signals. These three proteins participate in the resolution of a recombination intermediate containing a double Holliday junction [64] during somatic recombination [65]. The early embryo may therefore be limited in its ability to perform DNA repair requiring recombination.
The pronounced effect of in vitro embryo culture on the expression of DNA repair genes in blastocysts is surprising. At first glance, this result could be taken to indicate that in vitro culture might enhance the likelihood of the embryo incurring DNA damage and then activating the expression of appropriate repair genes. It appears, however, that a reciprocal explanation may prevail, in which in vitro culture actually diminishes the ability to undertake DNA repair. For example, in vitro culture reduces expression of the MSH2 mRNA, which could exacerbate the effects of elevated expression of MSH3 mRNA and thus further diminish mismatch repair capability. The TOPBP1 protein is down-regulated by UV irradiation to release MIZ1 and promote growth arrest via CDKN1A (p21/CIP) [66]. An increase in TOPBP1 expression may thus inhibit G1 checkpoint control. However, an increase in TOPBP1 expression may also reduce the onset of apoptosis [67]. Increased RPA expression can lead to endoreduplication [68]. Overexpression of MDM2 may inhibit TP53 function and subsequent regulation of the G1/S checkpoint [69]. In addition to effects on DNA repair activity, overexpression of other proteins (e.g., RFC1, RAD17, PLK1, PLK3, and CDKN1B) in the absence of actual DNA damage could lead to cell cycle arrest and potentially apoptosis. Why the expression of such genes is dysregulated in in vitro cultured embryos remains to be determined. The fact that this occurs, however, raises possible questions about the utility and potential adverse effects of prolonged in vitro culture of human embryos.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Keith E. Latham, 3307 N. Broad Street, Philadelphia, PA 19140. FAX: 215 707 1454; klatham{at}temple.edu
Received: 16 December 2004.
First decision: 6 January 2005.
Accepted: 4 February 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit mRNA abundance in preimplantation mouse embryos using a rapid, quantitative RT-PCR approach. Mol Reprod Dev 1995 41:314-324[CrossRef][Medline]This article has been cited by other articles:
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  I. Adriaens, J. Smitz, and P. Jacquet The current knowledge on radiosensitivity of ovarian follicle development stages Hum. Reprod. Update, May 1, 2009; 15(3): 359 - 377. [Abstract] [Full Text] [PDF]
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 R. G. Sturmey, J. A. Hawkhead, E. A. Barker, and H. J. Leese DNA damage and metabolic activity in the preimplantation embryo Hum. Reprod., January 1, 2009; 24(1): 81 - 91. [Abstract] [Full Text] [PDF]
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 Y. S. Lee, K. E. Latham, and C. A. VandeVoort Effects of in vitro maturation on gene expression in rhesus monkey oocytes Physiol Genomics, October 8, 2008; 35(2): 145 - 158. [Abstract] [Full Text] [PDF]
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 T. S. Barton, B. Robaire, and B. F. Hales DNA Damage Recognition in the Rat Zygote Following Chronic Paternal Cyclophosphamide Exposure Toxicol. Sci., December 1, 2007; 100(2): 495 - 503. [Abstract] [Full Text] [PDF]
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N. R. Mtango and K. E. Latham Ubiquitin proteasome pathway gene expression varies in rhesus monkey oocytes and embryos of different developmental potential Physiol Genomics, September 11, 2007; 31(1): 1 - 14. [Abstract] [Full Text] [PDF]
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P. Zheng, R. Vassena, and K. E. Latham Effects of in vitro oocyte maturation and embryo culture on the expression of glucose transporters, glucose metabolism and insulin signaling genes in rhesus monkey oocytes and preimplantation embryos Mol. Hum. Reprod., June 1, 2007; 13(6): 361 - 371. [Abstract] [Full Text] [PDF]
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