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Embryo; |
The Fels Institute for Cancer Research and Molecular Biology,3 Department of Biochemistry,4 Temple University, Philadelphia, Pennsylvania 19140
ABSTRACT
Correct cell cycle regulation is especially challenging at the start of life. Ovulated oocytes must maintain meiotic arrest until fertilization, and then complete meiosis and initiate a series of modified cell divisions without growth. Moreover, myriad key developmental events, such as chromatin remodeling and transcriptional activation of the genome, are coordinated with each other via the cell cycle, particularly passage through the DNA synthesis phase (S Phase). We examined here the expression of more than 30 mRNAs related to cell cycle regulation in rhesus monkey oocytes and embryos and compared the expression of these mRNAs between oocytes and embryos of different developmental potentials. We find that the maternally inherited stores of cell cycle regulatory mRNAs are especially susceptible to disruption in cases of diminished oocyte and embryo quality in the rhesus monkey. In comparison to published mouse array data, we also observed striking species differences in the temporal expression patterns of many of these genes, suggesting that mechanisms of cell cycle control may differ and that the responses of oocytes and embryos to external insults may likewise differ.
assisted reproductive technology, cell cycle, cleavage arrest, embryo, gene expression, gene regulation, maternal mRNAs, oocyte quality, rhesus monkey
The development from a single-cell zygote to fertile adult requires many rounds of cell division. During each division, cells complete an ordered series of events that collectively form the cell cycle. This cycle includes accurate duplication of the genome during the DNA synthesis phase (S phase) and segregation of complete sets of chromosomes to each of the daughter cells in M phase. Components of the cell cycle control system are cyclin-dependent protein kinases (CDKs), whose activity depends on association with regulatory units called cyclins. Activation of S-phase cyclin-CDK complexes initiates S phase, whereas activation of M-phase cyclin-CDK complexes triggers mitosis. The coordinated action of multiple protein kinases is required for the transition between the different phases of the cell cycle and for successful cell growth and division. Two enzymes complexes, the SKP1/cullin/Fbox complex (SCF) and the anaphase-promoting complex (APC), are also crucial components of the cell cycle control system. They induce the proteolysis of specific cell cycle regulators by ubiquitination.
Timing is important during embryogenesis. During the early preimplantation period, a number of dramatic and key developmental processes must be coordinated with each other temporally, including degradation of maternal mRNAs, DNA replication, chromatin remodeling, and transcriptional activation of the embryonic genome. Early studies primarily in the mouse pointed to the possible existence of a "cleavage clock" as a key regulator of timing in the early embryo [1]. More recent studies, however, indicated that many of these early processes are actually regulated by passage through S phase or by other cell cycle-dependent mechanisms. For example, passage through the first S phase is apparently required for activating embryonic gene transcription, and passage through the second S phase appears to be essential for establishing a transcriptionally repressive chromatin state wherein enhancers are necessary for gene activation [2–4]. Moreover, cell cycle regulators may play a role in controlling maternal mRNA translational recruitment [5].
These observations indicate that the mechanisms that regulate passage through the cell cycle likely control key processes in the early embryo and thus play a key role in orchestrating these processes and ensuring that they occur with the correct timing. Defects in this temporal regulation would be expected to be deleterious to the embryo. Consistent with this expectation, embryos of lesser developmental potential display prolonged cell cycles during cleavage [6] or even undergo cell cycle arrest [7–10]. One of the most striking examples of this is the two-cell block in cultured mouse embryos, wherein disruptions arise in the activities of key cell cycle regulators, particularly under unfavorable culture conditions [8, 11–19]. Hamster [6], bovine [20], and human [21–26] embryos that display timely cleavage divisions also display enhanced developmental potentials compared with those that cleave more slowly. Uniparental embryos and cloned embryos, which also have limited developmental potentials, display delayed cleavage divisions and fewer cells at the blastocyst stage [27–30]. In the rhesus monkey, embryos derived by fertilization of ooctyes from prepubertal females or oocytes collected during the nonbreeding season undergo cleavage arrest [31]. Collectively, these observations indicate that perturbations in cell cycle progression are a common feature of developmentally compromised embryos of many species.
In cases where developmental arrest is observed, arrest is most commonly seen around the time of major genome activation event, when the embryonic genome undergoes a dramatic surge in transcriptional activity [32]. This includes the two-cell block in mouse embryos and cleavage arrest in rhesus monkey embryos at the eight-cell stage [10, 33, 34]. Cleavage arrest at the time of genome activation reflects the coordination that is needed between degradation of maternal mRNAs and expression of embryonically encoded cell cycle genes. The mRNAs encoding some cell cycle regulators are enriched in oocytes, whereas others increase in expression after genome activation [35]. This suggests that early transcripts encoded by the embryonic genome must be produced in order to sustain embryonic cleavage divisions. Early gene transcription also appears to be required to suppress apoptotic processes [36], which can be activated in response to various forms of stress [37–41]. The timely onset of expression of key cell cycle regulators is essential for normal development, and defects in such expression can lead to apoptosis and developmental arrest [42]. Overall, the correct expression of cell cycle regulators during preimplantation development is critical in order to sustain development and permit the embryo to respond to such factors as DNA damage, stress, and other adverse conditions by activating either survival and repair mechanisms or apoptotic processes.
In seeking to understand the molecular processes that restrict embryo developmental potential, it is therefore of interest to understand in detail the temporal regulation of key cell cycle regulatory genes and how the expression of these genes is altered in embryos of lesser developmental potential. To answer these questions in the rhesus monkey model, we took advantage of the Primate Embryo Gene Expression Resource (PREGER), which has been established to permit detailed quantitative mRNA expression studies in rhesus monkey oocytes and embryos [43–47]. PREGER contains a set of more than 200 samples representing oocytes and embryos of many stages and differing developmental potentials and representing the products of different protocols for hormonal stimulation, in vitro or in vivo oocyte maturation, and embryo culture [43]. In addition to the sample set, an online database of mRNA expression data is available at www.preger.org. PREGER is thus a permanent, renewable resource that permits cumulative studies of mRNA expression over time using a uniform, biologically diverse sample set, and it is available to any interested investigator without need for direct access to freshly isolated nonhuman primate (NHP) oocytes or embryos. The PREGER sample set also offers a valuable tool for NHP modeling of human embryos. The mRNA expression data obtained with PREGER provide insight into patterns of gene regulation during development, as well as the extent of disruption of these patterns by diverse procedures and in association with compromised oocyte and embryo quality. Such studies of gene expression can thus reveal specific molecular mechanisms or pathways that support normal development or that may be altered during abnormal embryogenesis, including effects on gene transcription and effects on the maternal mRNA population. The mRNA expression data can also support the formulation of testable hypotheses and can help guide the design of additional functional studies and studies at the protein level. The analysis of cell cycle regulatory genes presented here revealed a high incidence of aberrant expression of cell cycle regulatory mRNAs among oocytes and embryos of poor developmental potential.
We employed PREGER (www.preger.org). All procedures employed to obtain oocytes and embryos were conducted according to recommendations of the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act and its amendments. Details of oocyte and embryo isolation and culture are described in detail in our previous studies [43–48] and at the above web site.
The PREGER resource encompasses a large, biologically rich set of >170 samples representing rhesus monkey oocytes and embryos obtained through a variety of different protocols, including in vitro oocyte maturation, in vivo maturation, in vitro fertilization, in vivo fertilization, in vitro culture, and development in vivo. Between 3 and 13 samples of one to four oocytes or embryos were obtained for each stage or condition (note: because the entire mRNA population is uniformly amplified during the RT-PCR procedure, the amount of input mRNA within 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. Blastomeres displayed uniform granularity. A minimum of three females was employed to obtained 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 hamster embryo culture medium (HECM9) were included to evaluate transcriptional dependence of mRNA expression after the time of the major embryonic genome activation event. Of special interest for this study were comparisons between oocytes and embryos of different developmental potentials. Oocytes of differing developmental potentials were obtained either by in vivo maturation (fully grown oocytes, large antral follicles, high developmental potential) following stimulation with FSH and hCG, by in vitro maturation following stimulation with FSH only (fully grown oocytes, large antral follicles, intermediate developmental potential), or by in vitro maturation with no hormonal stimulation (fully grown oocytes, small antral follicles, low developmental potential).
Complementary DNA Probes and Hybridization
The PREGER resource was constructed using the quantitative amplification and dot blotting (QADB) method. This method entails the direct lysis of small numbers of oocytes or embryos in a reverse transcription (RT) buffer supplemented with nonionic detergent, thereby avoiding RNA losses associated with organic extractions. The mRNA within the cell is reverse transcribed and quantitatively amplified under conditions that preserve quantitative representation of sequence abundances. The cDNA is then available for many different purposes, including the production of dot blots for gene expression analysis by hybridization. Once the dot blots are prepared, they are hybridized to mRNA-specific probes, and the hybridization results are analyzed.
Complementary DNA probes were obtained by PCR (Table 1) from specific cDNA clones obtained from Open Biosystems (Huntsville, AL). Blot preparation, probe preparation, hybridization, and quantitative analyses were performed as described [43–45, 48]. Data were expressed as the mean counts per minute (CPM) bound value (±SEM) for each stage/condition of oocytes and embryos included in the analysis. For some analyses, the ratios of expression were calculated among the three classes of oocytes or embryos derived from them, and the mean ratio (±SEM) was calculated for each stage. Significance of differences was evaluated using the t-test.
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The genes analyzed were preselected for involvement in a single process (cell cycle regulation), and so the expression of any one gene in the pathway is unlikely to be independent of the expression of others in the pathway. So, too, the genes expressed at each developmental stage are not likely to be independent of the collection of genes expressed at the previous or subsequent developmental stages. The three hormonal treatment groups, however, constitute independent variables, and the expression of each gene has been tested in each of the three possible comparisons. To address the need for correction for multiple testing, we have noted those comparisons that achieve statistical significance after applying the Bonferroni correction for the performance of three tests (i.e., significance at P < 0.016) [49]. Because NHP oocytes and embryos are experimental samples that are both expensive and difficult to obtain, however, and because additional data could become available in the future to augment this data set, we also indicate those differences in expression passing the uncorrected marginal statistical significance value of P < 0.05.
The patterns of expression of 35 mRNAs related to cell cycle control were examined in rhesus monkey oocytes and embryos using the PREGER sample set. The expression of additional cell cycle-related mRNAs was examined in previous studies [45, 47] and, where appropriate, the results for these additional genes were included here for consideration, as indicated in the text. The mRNAs examined encoded cyclins, spindle/centrosome proteins, transcriptional regulators, proapoptotic proteins, and proteins controlling DNA replication. Expression patterns were evaluated both temporally and with respect to differences in oocyte and embryo developmental potential.
Expression of mRNAs Encoding Cyclins and CDKs
Cyclin and the CDKs are the principal regulators of cell cycle progression. These proteins comprise the cores of the complexes that drive the irreversible progression past cell cycle checkpoints, which must be successfully negotiated for cells to divide. We examined the expression of mRNAs encoding seven cyclins and three CDKs (Table 1). None of the CDK mRNAs was detected (CDC2/CDK1, CDK2, and CDK4) in the monkey by this method. Among the cyclin mRNAs, two (CCND1 and CCND2) were not detected (Fig. 1 and Table 1). CCNA1 mRNA expression was low from GV stage, transiently increased at eight-cell and morula stages (P < 0.05), and decreased again thereafter. The CCNA2 mRNA was expressed as a maternal mRNA in oocytes and embryos through the two-cell stage, and at a lower but constant level from the eight-cell stage onward (P < 0.01). The CCNB2 mRNA yielded the strongest hybridization signal of any in this group of genes at GV stage. This expression declined upon oocyte maturation and remained low thereafter. The CCNE1 and CCNE2 mRNAs displayed lowest expression in oocytes and early embryos, with expression increasing dramatically in hatching blastocysts (P < 0.05). With exception of CCNA2, the cyclin mRNAs were all -amanitin sensitive at the 8- to 16-cell stage, indicating active transcription following embryonic genome activation (P < 0.05).
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Expression of mRNAs Encoding Cyclin Kinase Inhibitors (CDKIs)
Cyclin-dependent kinase inhibitors (CDKIs) bind to and inhibit the activity of CDKs. They suppress growth, and some CDKIs may be tumor suppressors [50, 51]. In embryos, CDKIs suppress cleavage [12]. Two CDKI mRNAs (CDKN1A and CDKN1B) were examined previously [45]. The CDKN1A mRNA was not detected, and the CDKN1B mRNA displayed very low expression in oocytes and embryos through the eight-cell stage, with a dramatic increase as blastocyst stage was attained. In this study, the CDKN2A mRNA also was not detected. Two additional members of this group yielded detectable signals for the monkey (Fig. 2). The CDC25A mRNA yielded a moderately strong hybridization signal throughout development, with predominantly maternal expression, declining to a minimum value at the morula stage, and a subsequent re-expression increasing from early to hatched blastocyst stages. The CDKN3 mRNA yielded a strong signal for GV-stage oocytes, which diminished upon maturation (P < 0.0001) and remained very low until the morula through blastocyst stages.
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Expression of mRNAs Encoding Transcriptional Regulators of Cell Cycle Progression
The E2F1, E2F2, and E2F3 proteins direct the transcription of genes needed for progression through the G1/S checkpoint [52–55]. These proteins also interact with the retinoblastoma (RB) protein, a negative regulator of cell cycle progression [56]. Neither E2F4 nor E2F5 interact with RB, and E2F6 lacks transcriptional activation activity and thus may be a transcriptional repressor [55]. Transcription factor Dp1 (TFDP1) associates with E2F proteins to regulate transcription [57, 58]. PA2G4 is a DNA-binding protein that is expressed highly between G1 and mid-S phase. PA2G4 encodes a DNA-binding protein that is present in the nucleus during interphase and early prophase but is otherwise in the cytoplasm, and appears to be involved in the control of cell replication [59]. Of the E2F family members tested, the E2F4 mRNA was not detected (Fig. 3). The E2F1, E2F2, and E2F6 mRNAs all showed predominantly embryonic expression, with low hybridization signals in the oocyte and early embryo, followed by significant upregulation at the eight-cell stage (P < 0.05). The E2F6 mRNA produced the strongest and most consistently detected hybridization signals. The RB1 mRNA was not detected. The mRNA encoding the E2F accessory protein, TFDP1, was similarly upregulated at the eight-cell stage. The PA2G4 mRNA was expressed throughout development. The PA2G4 mRNA increased in apparent abundance during oocyte maturation (P < 0.0001), signaling possible maternal mRNA polyadenylation (this would enhance its ability to support oligo(dT)-primed reverse transcription [60]) and remained -amanitin insensitive through the 8- to 16-cell stage, gradually increasing in abundance thereafter.
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Expression of mRNAs Related to DNA Replication
An earlier study reported the expression patterns of genes related to DNA repair in the rhesus monkey oocyte and embryo [45]. We examined here three additional mRNAs related to DNA replication (Fig. 4). The minichromosome maintenance (MCM) complex, which includes MCM2, is loaded onto DNA at replication origins at the start of S phase and is needed for elongation during DNA replication [61]. The chromatin licensing and DNA replication factor 1 (CDT1) protein is required for this loading, and this activity is inhibited by another protein called Geminin [62]. The MCM2 mRNA was expressed most highly in oocytes and persisted throughout development, with a slight decrease in abundance (Fig. 4). The CDT1 mRNA was expressed highly in the monkey GV-stage oocyte, declined dramatically upon oocyte maturation (P < 0.0001), and then increased in expression from the eight-cell stage onward. The Geminin (GMNN) mRNA was also expressed prominently as a maternal mRNA in the rhesus monkey, with declining abundance from the morula stage onward.
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Expression of mRNAs Related to Spindle and Centromere Functions
Key components of the cell cycle are the successful assembly and alignment of chromosomes on the mitotic spindle, formation of the spindle, and subsequent chromosome segregation. We examined here the expression of 14 mRNAs related to these processes (Fig. 5). The expression of two additional related mRNAs, PLK1 and PLK3, was described in a previous study [45]. DCTN3, INCENP, and KIF2C were not detected in rhesus monkey oocytes and embryos. The majority of these mRNAs were maternally expressed (AURKA, CENPC, CENPE, KIF23, PLK4, PRC1, and PTTG1), as was previously reported for the PLK1 and PLK3 mRNAs [45]. Only two of these (KIF23 and PRC1) displayed -amanitin-sensitive expression (P < 0.001) at the 8- to 16-cell stage, indicative of active embryonic transcription. The AURKB and KIF22 mRNAs were detectable in oocytes but were more prominently expressed at later stages (P < 0.05). The KIFC2 and KIF4A mRNAs were expressed at very low abundances in oocytes and displayed increasing expression during cleavage, with the rise in KIF4A abundance preceding that for the KIFC2 mRNA. Interestingly, different members within gene families often displayed opposing mRNA expression profiles, for example, comparing AURKA with AURKB and comparing KIF22 and KIF23 with KIFC2 and KIF4A.
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Expression of mRNAs Related to Apoptosis
We examined the expression of three proapoptotic mRNAs encoding the proteins BCL2-associated athanogene (BAG1), baculoviral IAP repeat-containing 5 (BIRC5, also known as survivin), and FAS-associated factor 1 (FAF1) that function with the cell cycle (Fig. 6 and Table 2). The three mRNAs showed similar patterns, having abundant expression at the GV stage and a significant decrease upon oocyte maturation (P < 0.0001), followed by increased expression from the eight-cell stage onward. FAF1 had lowest expression among the three. BAG1 mRNA displayed -amanitin-sensitive expression at the 8- to 16-cell stage (P < 0.01).
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Effects of Hormonal Stimulation Protocol on mRNA Expression
The oocytes obtained from nonstimulated (NS) females have very poor developmental competence once fertilized, with the majority of embryos arresting during cleavage [63]. Oocytes from FSH (in vitro maturation)-stimulated and FSH + hCG (in vivo maturation)-stimulated females fare much better during cleavage [63]. To determine whether this difference in developmental potential could reflect differences in expression of cell cycle regulatory genes, we compared gene expression between oocytes of the three classes and fertilized embryos derived from them, combining data from the genes analyzed here along with previously reported data for cell cycle regulatory genes. Of a total of 71 genes examined, expression of 56 was detected. Of these, 30 displayed significant differences (P < 0.05) among oocytes and embryos of the three classes (Table 3). We observed a very striking overall difference between the three classes of metaphase II (MII) stage oocytes, with 26 displaying significant (P < 0.05) differences between MII stage oocytes of FSH + hCG-stimulated females compared with the other two classes, 18 of these differing in both FSH and NS oocytes, 2 differing only in FSH oocytes, and 6 differing only in NS oocytes. Even with the correction for multiple testing (P < 0.016), we observed 12 significant differences between FSH + hCG and NS oocytes and 7 significant differences between FSH + hCG and FSH oocytes. The overwhelming effect (19 of 26 [P < 0.05] and 11 of 12 [P < 0.016] affected mRNAs) was an increased apparent abundance of the affected mRNAs in NS alone or both NS and FSH oocytes. Of the 12 mRNAs differing between FSH + hCG and NS MII stage oocytes at the level of P < 0.016, 7 were contained with two categories (cyclins and centromere + spindle proteins). Of the seven mRNAs differing significantly between the FSH + hCG and FSH oocytes, six were contained in these two categories, the one exception being BRCA1.
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Following fertilization, pronucleate-stage embryos produced with oocytes from FSH-stimulated females displayed many fewer (eight, P < 0.05; and two, P < 0.016) significant differences, but five of these were cases of lower apparent abundance in FSH embryos. Two of the latter five were unaffected in MII-stage FSH oocytes, and three of the five had displayed elevated abundances in MII-stage FSH oocytes. In contrast to the more normalized mRNA expression pattern in FSH pronucleate-stage embryos, embryos from NS oocytes continued to display significant disruptions in a large number (16, P < 0.05; and 7, P < 0.016) of mRNAs. Ten of these had displayed higher apparent abundances than their FSH + hCG counterparts in oocytes, and continued to do so in PN-stage embryos. As with the MII stage, the majority of these affected mRNAs fell in the cyclin and centromere/spindle protein categories.
At the two-cell stage, embryos from FSH oocytes displayed significant alterations in five mRNAs relative to their FSH + hCG counterparts, three of which appeared to be the result of aberrant mRNA stabilization. By contrast, two-cell embryos from NS oocytes displayed alterations in 16 mRNAs relative to FSH + hCG embryos (P < 0.05), and three other mRNAs (CDT1, KIF22, and FBXW2) were detected in FSH + hCG embryos but not detected in NS embryos. Seven of the mRNAs displaying elevated signals in NS MII-stage oocytes displayed reduced signals in NS two-cell stage embryos. The 19 affected mRNAs displayed a more uniform distribution among the six categories included in Table 3, so that the specificity toward cyclins and centromere/spindle protein categories appeared lessened. Of the five mRNAs displaying significant differences between FSH + hCG and NS embryos at the more stringent significance level of P < 0.016, however, two were in the centromere/spindle protein category, again pointing to the prominence of this category. Another two were in the DNA damage-sensing category (BRCA1 and MDM2), and one (E2F6) was in the transcription regulation category. The BRCA1 mRNA was thus overexpressed in NS embryos at both the one-cell and two-cell stages.
The period spanning oocyte maturation, fertilization, and preimplantation development is notable for unique modes of cell cycle regulation. After oocyte maturation, the mammalian oocyte remains arrested at the second meiotic metaphase until fertilization. Fertilization then leads to a series of mitotic cell cycles that are uniquely characterized by multiple rounds of DNA replication and cell division without growth, collectively known as cleavage. Severe disruptions in the cell cycle can lead to spontaneous oocyte activation and parthenogenesis, failures in oocyte activation after fertilization, DNA replication/repair defects, mitotic errors, and developmental arrest during cleavage. Additionally, the cell cycle likely serves as a key choreographer within early embryos, coordinating myriad molecular events over time. Even without developmental arrest, delays in cell cycle progression are correlated with poor oocyte and embryo quality.
We report here a detailed examination of the expression patterns of 35 cell cycle regulatory mRNAs in rhesus monkey oocytes and embryos, which combined with previously published data, brings the total number of cell cycle regulatory mRNAs examined to 71. The genes analyzed here affect all of the major aspects of cell cycle regulation, including cell cycle progression, DNA replication and repair, cell cycle arrest related to apoptosis, mitotic spindle formation, and chromosome segregation, as well as transcription factors that regulate genes that participate in these processes. We find that, indeed, aberrant regulation of cell cycle-related genes is a prominent feature of oocytes and embryos of compromised developmental potential. Of the 56 mRNAs for which expression was detected, we found significant aberrations related to oocyte and embryo quality in the expression of more than one half (n = 30) at the significance level of P < 0.05, and more than one fifth (n = 12) at the more stringent level of significance (P < 0.016). The vast majority of these (26 of 30) display significant differences in MII-stage oocytes, 20 being altered in oocytes of moderately reduced potential (those from FSH-stimulated females) and 24 of 30 being altered in oocytes of severely compromised potential (those from NS females; 9 and 12, respectively, at the more stringent level of P < 0.016), and 17 displaying either increased or decreased hybridization signals in both kinds of oocytes (eight at the significance level of P < 0.016). The affected mRNAs were distributed across a range of functional categories, including cyclins, spindle/centromere proteins, transcription regulatory proteins, APC-SCF complex, and DNA damage sensors. Two categories in particular, cyclins and centromere/spindle proteins, appeared to be affected to a greater degree than the other categories in MII-stage oocytes and fertilized embryos.
The most prevalent pattern of effect among all of the affected mRNAs was increased hybridization signals in MII-stage oocytes from FSH and NS females. Because these mRNAs are produced and deposited in the oocyte as maternal transcripts during the final stages of oogenesis, the increased hybridization signals relative to the oocytes from FSH + hCG females (in vivo matured, highest quality) most likely reflect precocious mRNA polyadenylation and recruitment for translation (polyadenylation can promote more efficient oligo(dT)-primed reverse transcription, thereby enhancing apparent hybridization signal [60]). This phenomenon was previously reported for maternal mRNAs [44] and was particularly notable for oocytes from NS females among the maternal mRNAs examined. The striking feature of the results presented here for cell cycle-related mRNAs is that this category of maternal mRNAs appears to be especially sensitive to this effect, both in terms of proportion of affected mRNAs and that the mRNAs are strongly affected in both FSH and NS oocytes. It appears, therefore, that disruptions in the regulation of maternal mRNAs that control the cell cycle may be a prominent feature of compromised oocyte quality.
We also previously reported that many of the maternal mRNAs that are precociously recruited in MII-stage oocytes are eliminated during development to the two-cell stage, thereby creating a deficiency in mRNA expression around the time of cleavage arrest [44]. We indeed observed that pattern here for eight of the affected mRNAs (CDC25A, CDT1, AURKB, KIF22, CENPE, PLK4, and FBXW2); three of these regulate the G1 to S transition, and four of these regulate chromosome segregation. These observations suggest that entry into S phase and DNA replication, as well as progress through mitosis and correct chromosome segregation, may be particularly susceptible to disruption in embryos that are derived from oocytes of diminished quality, thus contributing to the observed cleavage arrest. Thus, a variety of endogenous and exogenous factors may affect genomic integrity in primate oocytes and embryos. The resulting developmental arrest would thus serve the crucial function of eliminating these cells before implantation, thereby conserving maternal resources.
Given the central importance of correct cell cycle regulation to the early mammalian embryo, one might expect that the expression of these genes would be highly conserved across species. To examine this question in detail, we compared the temporal expression profiles obtained here with rhesus monkey samples to previously reported mouse array expression data [64]. The analysis presented here provides the first comparative study of cell cycle regulatory gene expression between nonhuman primate and rodent models. Although direct quantitative comparisons of expression levels between specific stages for the two species are constrained by differences in methodology and developmental kinetics, such comparisons nevertheless can reveal differences or similarities in arrays of genes expressed and differences or similarities in broad temporal patterns of expression over the preimplantation period. We find that there is considerable variation between the two species. Nine of the rhesus monkey genes selected for analysis failed to yield detectable hybridization signals. Among these, at least four (DCTN3, KIF2C, CDK4, and RB1) yielded moderate to strong signals (i.e., >500 units) on mouse arrays (Fig. 7). Additionally, of the genes analyzed here, nine (CCNA2, CCNE1, CDKN3, AURKA, KIF22, KIFC2, KIF4A, PTTG1, and BIRC5) yielded highly divergent temporal expression patterns, and two mRNAs (CDKN3 and CCNA1) differed dramatically in relative abundances (strong signals with rhesus monkey; very weak signals with mouse array). Thus, a conservative estimate is that of the 26 cell cycle-related mRNAs for which expression was detected in this study, more than one half (n = 15) display highly divergent patterns of expression between the two species. Accordingly, it does not appear that the naive expectation that cell cycle regulatory genes would be expressed similarly among different classes of mammals is supported. This suggests that cell cycle regulatory mechanisms may also differ considerably between primate and rodent species and that the oocytes and embryos of the two species may respond quite differently to external insults. The results presented here thus offer a compelling justification for the continued development and application of nonhuman primate reproductive models as an aid to understanding better human oocyte and embryo biology and as an aid to developing improved methods for assisted reproduction that enhance outcome while minimizing potential risks to the long-term health of the offspring.
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The mRNA expression data presented here provide novel information about disruptions in the expression of genes controlling the cell cycle in oocytes and embryos of compromised developmental potential. Further detailed studies of cell cycle regulatory proteins, chromosome ploidy during cleavage, and other functional studies in the rhesus monkey should provide additional insight into how the nonhuman primate embryo correctly regulates this important class of molecules and how this may be disrupted in response either to endogenous or exogenous factors, and either in vivo or in vitro.
ACKNOWLEDGMENTS
We thank Bela Patel, Malgorzata McMenamin, and Ann Marie Paprocki for their technical assistance. We also thank R. Dee Schramm for his contribution to the development of the PREGER resource.
FOOTNOTES
1Supported by research resource grants from the National Centers for Research Resources (RR15253) at the National Institutes of Health. 
Correspondence: 2Keith E. Latham, 3307 North Broad St., Philadelphia, PA 19140. FAX: 215 707 1454; e-mail: klatham@temple.edu
Received: 8 August 2007.
First decision: 3 September 2007.
Accepted: 11 September 2007.
REFERENCES
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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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