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Embryo |
The Fels Institute for Cancer Research and Molecular Biology3
the Department of Biochemistry,4Temple University School of Medicine, Philadelphia, Pennsylvania 19140
The Wisconsin National Primate Research Center,5 University of Wisconsin-Madison, Madison, Wisconsin 53715
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While preimplantation embryogenesis has been studied in detail in such species as mouse and rabbit, very little is known about preimplantation embryogenesis in primates. Studies have been limited in nonhuman primate species due to cost and availability of embryos. While significantly more data have been generated from spare human embryos or lower quality embryos produced in the course of assisted reproduction, the overall pace of such research is nevertheless hindered by ethical and legal constraints. Moreover, many of the experimental studies that have been undertaken in nonprimate species have not been undertaken in a nonhuman primate model due to a poor foundation of basic molecular knowledge (e.g., lack of knowledge of gene-expression patterns). To circumvent these limitations, it is essential that novel resources and tools be developed to permit detailed studies in nonhuman primate oocytes and embryos in a way that maximizes the amount of information that can be obtained from the available samples and preferably provides the opportunity for unlimited sharing of material among investigators. In this way, our knowledge and understanding of primate embryogenesis could be developed to a degree on par with that available for other species.
We have developed the Non-Human Primate Embryo Gene Expression Resource (PREGER), which can be used by any investigator to study primate preimplantation embryogenesis, without a need to obtain embryos. The resource, which is based on our previously established and extensively tested methods for quantitative amplification of mRNA populations from single embryos, consists of a system of molecular tools, amplified cDNA libraries, and an on-line database that will facilitate detailed studies of gene expression, broad dissemination of resulting data, and collaborative interactions. The mission of PREGER is to share such materials with interested investigators, allowing them to produce relevant data for their research programs at greatly reduced cost and effort, and to share the resulting data and discoveries worldwide to facilitate the advancement of primate embryology.
In the analysis presented here, we address the relative conservation of gene-expression patterns between rhesus and mouse. The major genome activation event occurs between the six-cell and eight-cell stages in the rhesus monkey embryo [14], but at the two-cell stage in the mouse embryo [2, 3]. Both species, however, display a limited capacity for gene transcription as early as the one-cell (mouse) or two-cell (rhesus) stage. It can be hypothesized that housekeeping genes will follow similar patterns of expression between different mammalian species while displaying appropriate differences related to the timing of the major genome activation event. To examine the relationship between the major genome activation event and the temporal patterns of individual mRNA expression and to evaluate the overall similarity of these relationships between rhesus monkey and mouse, we analyzed the temporal patterns of expression of five housekeeping mRNAs that have been studied previously in mouse embryos. We also examined the temporal expression patterns of mRNAs encoding two basal transcription factors and a third transcription factor that is related to a response to stress. We find that, in several respects, gene-expression patterns are similar between the two species. We also observed, however, differences in temporal expression patterns between the two species, which do not appear explicable on the basis of a simple shift in the timing of genome activation. These include different contributions to the maternal mRNA pool in oocytes and differences in the up- or down-regulation after genome activation. The existence of such differences between species provides a clear illustration of the value of undertaking detailed studies in a primate model and the value of a research resource such as PREGER for facilitating such analyses.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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. 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.
For collection of in vivo-matured oocytes, monkeys received twice daily i.m. injections of 30 IU recombinant human FSH (rhFSH; Organon Inc., West Orange, NJ) for 7 days, beginning on Days 1–3 of the menstrual cycle (Day 1 = first day of menstruation). Recombinant hCG (1000 IU; Ares Advanced Technology, Randolph, MA) was injected (i.m.) on Treatment Day 8 for induction of oocyte maturation. Oocytes were aspirated laparoscopically into Tyrode lactate (TL)-Hepes medium (37°C) containing 0.1 mg/ml polyvinyl alcohol and 10 IU/ml heparin 27–32 h following injection of hCG. Oocytes were retrieved from aspirates using an EM Con filter (Veterinary Concepts, Spring Valley, WI). Cumulus masses were treated with 0.1% hyaluronidase to facilitate recovery of oocytes. Oocytes were cultured in modified [15] CMRL-1066 medium (Connaught Medical Research Laboratories, Invitrogen, Carlsbad, CA) containing 20% bovine calf serum (Hyclone, Logan, UT) in microdrops under mineral oil at 37°C in a humidified atmosphere of 5% CO2 in air for 4–8 h before insemination.
Immature (germinal vesicle stage), but fully grown, oocytes for in vitro maturation were obtained from both FSH-primed and nonstimulated monkeys. FSH-primed monkeys received rhFSH as described above but did not receive hCG before follicular aspiration [16, 17]. Oocytes were retrieved from monkeys laparoscopically on the morning following the last day of rhFSH treatment. Nonstimulated monkeys displaying normal menstrual cycles were ovariectomized during a nonspecified period of the menstrual cycle. Excised ovaries were immediately transported to the laboratory in TL-Hepes medium at 37°C, and oocytes were recovered from antral follicles following repeated puncture of the ovaries with a 20-gauge needle and mincing of the ovarian tissue with a scalpel blade. Fully grown immature oocytes, enclosed by at least three layers of cumulus cells, were cultured in modified CMRL-1066 medium containing human gonadotropins (5 µg/ml hFSH and 10 µg/ml hLH) and 20% bovine calf serum in microdrops under mineral oil at 37°C in a humidified atmosphere of 5% CO2 in air, as described previously [18, 19]. Oocytes were cultured for 28–30 (FSH-primed monkeys) or 34–36 h (nonstimulated monkeys) before insemination.
In Vitro Fertilization/Embryo Culture
Sperm samples were collected from adult males by penile electrostimulation, and sperm capacitation and in vitro fertilization were done as described previously [20], with a few minor modifications [17]. Briefly, 10 x 106 washed sperm/ml were resuspended in 2 ml Tyrode albumin lactate pyruvate (TALP) medium and incubated at 37°C in 5% CO2 in air for 1–10 h. Sperm were treated for 30–45 min with 1 mM each of dibutyryl cyclic AMP (dbcAMP) and caffeine to induce hyperactivation. Hyperactivated sperm (300 000/ml) were then co-incubated with oocytes for 12–16 h in TALP medium containing 1 mM each of dbcAMP and caffeine in microdrops under mineral oil at 37°C in a humidified atmosphere of 5% CO2 in air. Sperm and remaining cumulus cells were then removed manually by pipetting through a finely pulled glass pipette, and oocytes were examined for evidence of fertilization. Diploid zygotes were cultured in one of two sequential culture systems employing either HECM-9 medium [21] with 5% serum added after 48 h or in G1 medium followed by G2 after 48 h [22]. Embryos were cultured in 5% CO2, 5% O2, and 90% N2 at 37°C in microdrops under mineral oil and placed into fresh media every other day until processed for reverse transcription-polymerase chain reaction (RT-PCR).
Recovery of In Vivo-Produced Embryos
In vivo-produced morula- and blastocyst-stage embryos were obtained by uterine lavage of naturally cycling monkeys, as described previously [23, 24]. Briefly, females were mated with fertile males during a normal menstrual cycle. Blood samples were collected daily beginning 7 days after the first day of menstruation and continued until the day of the LH surge was determined by radioimmunoassay. Uterine lavage was performed 6–8 days after the day of the LH surge. Morula- and blastocyst- stage embryos were processed immediately for RT-PCR.
Embryo Quality and Source
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. Wherever possible, a minimum of three females were employed to obtain samples for each stage and hormonal stimulation protocol, as indicated in Table 1. For samples obtained from hCG-stimulated females and employed for the analyses presented here, three or more females were obtained for all stages, except that two females were employed for samples at the two-cell stage and two for the hatched blastocyst-stage embryos cultured in HECM9. The samples for embryos cultured in G1/G2 media were generally obtained from single females for a given stage.
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Quantitative RT-PCR
The molecular components of PREGER were constructed using a quantitative RT-PCR approach that has been used extensively in our laboratory, known as the QADB method (quantitative amplification and dot blotting) [25–37]. This method represents a combination of the RT-PCR protocol of Brady and Iscove [38] coupled to a system of procedures that permit quantitative analysis of gene expression by dot-blot hybridization.
The quantitative aspects of the QADB method have been documented previously. The RT-PCR method was designed to maintain quantitative representation of sequences within the cDNA population during amplification by limiting the length of the first cDNA strand, and thus minimize selection against mRNAs of long length [38]. Reamplification of cDNA through additional rounds of PCR exerts a minimal effect on results [27]. By calibrating blots using published values for total mRNA content and actin mRNA content, we were able to obtain estimates of mRNA copy number in studies of the mouse embryo [25]. Those estimates have been very close to estimates produced by other methods, such as Northern blotting [e.g., 25, 36]. The temporal patterns of expression revealed using the QADB method are consistent with those observed by other methods, such as semiquantitative RT-PCR, Northern blotting, and Western blotting (after accounting for differences in frequency of sampling, the number of stages assayed, and the quantitative resolution of the other assays), or patterns inferred from results of other approaches, such as antisense oligonucleotide inhibition (e.g., see results for Hprt, actin, tPA, G protein 
S, q, i2, 13, 11, and 14 isoforms, c-myc, Pgk1, U2afbp-rs, Bcl2, Bax, Bclx, Bad, Bclw, Caspase2, Na+/K+-ATPase 1 subunit; [25, 30–32, 34–36]). The temporal expression profiles obtained with the QADB method are typically smooth and reproducible, with small standard errors, indicating quantitative reliability of the method. Using the QADB method, we were the first to show that the Xist RNA is first transcribed at the two-cell stage in the mouse [34], an observation that was confirmed 6 and 7 yr later by other laboratories using other quantitative methods, such as real-time PCR [39–41]. This observation of early Xist RNA transcription was accomplished using the QADB method even though the Xist RNA was present at a very low level (500 copies per embryo or less), attesting to the sensitivity and quantitative reliability of the QADB method. Other studies of X-linked gene and imprinted gene expression have discerned the expected quantitative differences (as small as twofold) in expression between nuclear transfer and fertilized control embryos [e.g., 34, 35, 37]. We have shown that the data produced by the QADB method are reproducible, with standard deviations of less than 5% for actin mRNA in aliquots representing single-embryo equivalents from a combined pool of 20 blastocyst- stage mouse embryos [32]. We have also shown that the QADB assay is linear over at least three orders of magnitude (R2 = 0.992) [32]. Taken together, these previous observations indicate that the QADB method is a reliable, sensitive, efficient, and quantitative approach, ideally suited to the task of quantitative gene-expression studies in samples of preimplantation mammalian embryos. Additionally, the QADB method has the unique advantage over other methods of analysis in that it can produce materials that can be freely distributed among laboratories, thus satisfying a key requirement for development of a research resource.
All procedures were performed as described thoroughly elsewhere [25, 26, 38; see also website www.preger.org]. Briefly, oocytes or embryos were treated with acid Tyrode to remove the zona pellucida and then lysed in buffer that consisted of RT buffer supplemented with nonionic detergent, RNase inhibitors, and other reaction components. Oligo(dT) priming was used to initiate the RT reaction. First-strand synthesis was intentionally limited to facilitate linear amplification of the 3' terminal portions of the entire mRNA population during the PCR procedure, without loss of representation of long mRNAs or mRNAs with complex secondary structures. Terminal transferase in the presence of excess dATP was employed to add a poly(dA) stretch to the 3' end of the first strand. For PCR amplification, a primer of 
60 base pairs containing a 3' terminal stretch of 23 T residues was used to prime the PCR reaction in both directions. The initial PCR reactions were amplified through 50 cycles, adding fresh Taq polymerase after 25 cycles, as described [25, 26]. The initial PCR reactions were then divided into smaller aliquots and these were frozen at –70°C. Secondary PCR reactions were then performed to amplify 1 µl of each original PCR reaction. These secondary PCR reactions contained a small amount of 32P-dCTP to permit later quantification of the amount of DNA bound to each dot on the dot blots. All samples to be analyzed together on a given set of blots were coamplified using a common master PCR mix to ensure appropriate normalization after dot blotting. Dot blots were prepared as described [25, 26], bound DNA quantified by phosphorimaging, and blots then permitted to undergo radioactive decay before hybridization.
The cDNA Probes, Blot Hybridization, and Data Analysis
The cDNA probes were obtained by RT-PCR from human cell RNA. Primers employed and the amplified cDNA regions are listed in Table 1. The identities of amplified cDNAs were confirmed either by using diagnostic restriction digests or DNA sequencing. Gel-purified cDNA fragments were radiolabeled by the random primer method [42]. Labeled probes were hybridized to blots as described [25, 26]. After hybridization, blots were washed four times at 65°C in Church wash (40 mM sodium phosphate, pH 7.2, 1% SDS) and then subjected to phosphorimaging along with paper discs containing small quantities of 32P to permit conversion of data from phosphorimager units to counts per minute (cpm). Any residual 32P remaining from the blot preparation process was subtracted from the probe hybridization signals, and the data were then normalized to correct for differences in the amounts of DNA bound to each dot. Data were expressed as the mean (± SEM) cpm bound value for each stage/condition of oocytes and embryos included in the analysis. The significance of differences between stages and conditions was evaluated using a t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Over 160 samples of oocytes and embryos of various stages and produced by various protocols were obtained and displayed satisfactory PCR amplified products, as judged by agarose gel electrophoresis. This extensive collection of samples was designed to permit PREGER to address multiple experimental objectives, an essential feature of a shared resource. One objective was to be able to ascertain temporal expression patterns during oocyte maturation and preimplantation development. For this purpose, samples of germinal vesicle-stage oocytes, in vivo-matured oocytes (not inseminated), and in vitro-produced embryos cultured in HECM9 sequential media were employed. A second objective of PREGER was to permit initial evaluations of the effects of different culture systems on gene expression and of the similarity between cultured and in vivo developing embryos. For this purpose, embryos cultured in the G1/G2 sequential media and embryos isolated directly from the reproductive tracts of spontaneously cycling females were included for comparison with embryos cultured in HECM9 sequential media. A third objective was to evaluate the effects of in vitro versus in vivo oocyte maturation and in vitro maturation of fully grown oocytes from large versus small follicles on gene expression in oocytes and embryos derived from these different types of oocytes (which display different degrees of developmental competence). Results related to two of these objectives are described in this article and in the accompanying article. In this article, we focus our discussion on temporal expression patterns and effects of the culture system. For most stages/ conditions, three or more samples of 1–4 oocytes/embryos (from more than one female) were obtained, but in some cases, only 1–2 samples were available. It should be noted that, because the entire mRNA population is uniformly amplified during the PCR procedure, the amount of input mRNA (i.e., the range of 1–4 embryos) does not affect the quantitative representation of sequences within the amplified material. A summary of samples included in the current PREGER sample set is given in Table 2. Additional samples can be added to this sample set to enhance further the utility of PREGER.
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Expression of Housekeeping Genes in Rhesus Monkey Oocytes and Embryos
The temporal pattern of expression of actin was similar between rhesus monkey and mouse [25]. In both species, the actin mRNA exists at a low abundance in oocytes and early embryos and then increases in abundance during the latter portion of the preimplantation period. In the mouse, the actin mRNA increase is noticeable between the four- and eight-cell stages, with a dramatic and progressive increase with development to the blastocyst. In the rhesus monkey embryo, transcriptional accumulation of embryonically encoded actin mRNA initiates between the eight- cell and morula stages, with significant increases in morulae relative to the small amount of maternal mRNA remaining in -amanitin-treated eight-cell embryos (P < 0.05), and further significant increases upon development to the blastocyst stage (P < 1 x 10–5) (Fig. 1). Thus, in both species, the increase in actin mRNA expression occurs at 1–2 cell divisions after the major genome activation event. One difference between the two species is that the ratio of expression in blastocyst versus oocyte was much less in the rhesus monkey, about 2.4-fold as compared with 34-fold or greater in the mouse [25]. There was no discernible effect of embryo culture condition on actin expression and no apparent difference between cultured and in vivo developing blastocysts.
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The PGK1 mRNA displayed very low expression before the morula stage in the rhesus monkey (Fig. 1). Expression in the eight-cell stage rhesus embryo was low, but -amanitin-sensitive, indicating transcription dependence (P < 0.02). This expression was much lower relative to the blastocyst stage in the rhesus as compared with the mouse [34], revealing a possible species-dependent difference in the overall degree of maternal endowment of the oocyte with PGK1 mRNA. At the morula stage, PGK1 mRNA abundance increased dramatically in the rhesus monkey embryo in comparison with the eight-cell and earlier stages (P < 0.02). By the hatched blastocyst stage, PGK1 mRNA content was nearly 15-fold greater than in eight-cell stage embryos (P < 4 x 10–5). Transcription at the eight-cell stage in the rhesus monkey thus resembled transcriptional activation of Pgk1 at the late two-cell stage in the mouse [34]. There was no consistent effect of embryo culture medium on expression. Expression appeared to be reduced in embryos developing in vivo versus cultured embryos, but this was not statistically significant.
PDHA1 mRNA expression was highly similar between the two species. In the rhesus monkey, a large decrease in apparent abundance occurred during oocyte maturation (compare GV with MII stages) (Fig. 1) (P < 4 x 10–9). Gene transcription (-amanitin sensitive) was clearly evident at the eight-cell stage (P < 0.02) and expression increased rapidly to a high level that persisted throughout preimplantation development (P < 0.01 for morulae and P < 0.0001 for blastocysts, compared with eight-cell embryos). The large decrease in expression during oocyte maturation, the transcriptional activation (at the two-cell stage in mouse), and the high, sustained expression were evident in the mouse embryo as well, although expression did decline at the blastocyst stage in the mouse [34]. There was no apparent effect of embryo culture system on PDHA1 mRNA expression and no difference between cultured and in vivo-produced embryos.
The temporal pattern of HSC70 mRNA expression resembled closely that of PDHA1, but displayed a much less dramatic decrease upon oocyte maturation. Transcription of the HSC70 gene was evident as a significant increase in mRNA abundance at the morula stage as compared with the maternal mRNA remaining in -amanitin-treated embryos (P < 0.05) (Fig. 1). Hsc70 gene transcription is observed at the time of genome activation in the mouse as well [43]. No apparent effect of culture system or culture versus in vivo production on HSC70 mRNA expression was seen.
The temporal patterns of expression of the HPRT mRNA displayed clear species differences. In the mouse, the Hprt mRNA is expressed at a low level in the oocyte and early embryo, begins to be transcribed at the late two-cell stage, and its mRNA undergoes a dramatic increase in abundance thereafter [25]. In the rhesus monkey, a nearly reciprocal pattern was observed, with abundant expression in the oocyte and early embryo and much lower expression from the morula stage onward (Fig. 1). Apparent abundance was greater in MII-stage rhesus oocytes versus GV-stage oocytes, indicating possible polyadenylation and recruitment during maturation (P < 0.01). Treatment of eight-cell stage embryos with -amanitin produced about a two-fold decrease in mean expression value, but this reduction was not statistically significant (P = 0.13), indicating that a considerable quantity of maternally inherited HPRT mRNA remained at the eight-cell stage. Moreover, expression in - amanitin-treated embryos remained well above that observed at later stages (P < 0.01). Previous studies in human embryos revealed a similar decrease in expression of the HPRT embryo during preimplantation development [44], indicating that this represents a basic difference between murine and primate species. There was no significant effect of different culture media on HPRT mRNA expression, but expression was slightly greater for in vivo-grown embryos than cultured embryos (P < 0.03).
Transcription Factor mRNAs
To begin to explore the primate embryo's ability to undertake and regulate gene transcription, we next examined the expression of mRNAs encoding two basal transcription factors, TBP and its associated factor CA150, and one transcription factor that is involved in cellular response to stress, ATF6 (Fig. 2). The expression pattern of the TBP mRNA in rhesus monkey embryos displayed noticeable similarities with the expression of TBP mRNA in murine embryos. In the mouse, TBP mRNA is expressed highly in the GV-stage oocyte and undergoes an apparent decrease in abundance upon maturation [32]. There is a moderate, -amanitin-insensitive increase in abundance through the early two-cell stage before TBP gene transcription, possibly reflecting polyadenylation of maternal transcripts. The TBP gene is transcribed at the late two-cell stage and TBP mRNA abundance then persists during development to the blastocyst stage. In rhesus monkey oocytes and embryos, a decrease was again observed upon oocyte maturation (P < 5 x 10–5). There was an apparent rise during the two-cell stage before genome activation, but this was somewhat variable and did not reach statistical significance (P = 0.09). Expression at the eight-cell stage was -amanitin insensitive. TBP mRNA abundance decreased further after the morula stage, and then increased between the early and hatched blastocyst stages (P < 0.01). Expression appeared insensitive to the embryo culture system and similar between in vitro- and in vivo-produced embryos.
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The CA150 mRNA was also expressed abundantly in the oocyte and early stage embryos, and was -amanitin insensitive at the eight-cell stage. The CA150 mRNA decreased in abundance between the MII and early blastocyst stages, and then remained reduced thereafter. There was no discernible effect of the embryo culture system.
The ATF6 mRNA encodes an endoplasmic reticulum (ER)-associated factor that is activated and released to enter nuclei under ER stress conditions, whereupon it activates the transcription and expression of chaperones and other proteins involved in protein folding [45]. The ATF6 mRNA displayed an apparent decrease in abundance during oocyte maturation and development to the two-cell stage (P < 0.05; with the caveat that the two-cell embryos were obtained from two females). Hybridization signals were insensitive to -amanitin treatment at the eight-cell stage and expression remained relatively constant throughout the remaining culture period. Expression of this mRNA appeared to be reduced in hatched blastocysts grown in vivo compared with those produced in vitro, but this difference was not statistically significant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Another component of the PREGER is an on-line web site (www.preger.org) that will provide access to all expression data generated, along with a variety of other information, including methods and protocols, relevant literature citations, links to a murine gene-expression database that is also being developed using the QADB method, and links to a variety of other databases and related web sites. Users of the resource will submit their data to this website for inclusion in the PREGER database. Because the QADB method permits repeated analyses on a common set of samples, it will thus be possible to maintain a cumulative database for both mouse and rhesus monkey embryo gene- expression data. This should improve our understanding not only of primate embryo development but also of what molecular mechanisms may be shared or divergent between primates and rodents. Further links to other data produced in other species such as the cow should expand this capability further.
The data presented here reveal compelling similarities between rhesus monkey and mouse gene-expression patterns and yet also reveal interesting and noteworthy differences. This indicates that, while rodent gene-expression data may be relevant for understanding the function and regulation of some genes in primate embryos, for many other genes, such understanding will not be achievable by simply imposing murine data on a different time scale that accounts for differences in timing of events such as genome activation. This illustrates clearly the need for a resource such as PREGER to make possible informative, novel studies in a suitable nonhuman primate model.
In several instances, we observed differences between rhesus monkey and mouse with respect to mRNA representation in the maternal mRNA pool of oocytes and the subsequent fate of that maternal mRNA. The actin mRNA appeared to be more abundant in rhesus monkey as opposed to mouse oocytes relative to later stages, and the PGK1 mRNA appeared to be less abundant. Other mRNAs (e.g., PDHA1 and TBP) were regulated very similarly between the two species. The HPRT gene displayed the most dramatic difference, with nearly reciprocal temporal patterns of expression. The differences observed indicate that primates and rodents may have different strategies for providing certain housekeeping functions to the oocyte and early embryo (i.e., mRNA versus protein deposition) or simply differing requirements for those gene products.
PREGER should prove a valuable resource for basic embryological studies. In addition, PREGER should prove a valuable asset for understanding early human embryo development by complementing studies undertaken with human embryos. PREGER should allow human embryo researchers to circumvent limitations in available embryo number and quality. Through its array of samples, PREGER can produce data that are relevant to a variety of issues in assisted reproduction, such as effects of culture, effects of in vitro maturation, and suitability of oocytes from small antral follicles for supporting normal development. Additionally, because experimental studies can be undertaken in the rhesus monkey embryo that cannot be undertaken with human embryos, PREGER should provide the ability to establish the necessary foundation of gene-expression data to support more incisive experimental studies.
Future Expansion of PREGER
The set of 160 samples currently included in the PREGER permit a wide variety of comparisons to be made, e.g., with respect to temporal patterns of expression, effects of culture system on gene expression, and effects of in vitro versus in vivo oocyte maturation on gene expression. The PREGER is designed to be a cumulative resource, and as such, this current set of samples can be expanded over time to incorporate a larger number of samples for each stage and condition. Oocytes and embryos obtained through other protocols (e.g., incorporating exogenous LH into the ovarian stimulation protocol to evaluate its effects on gene expression [46–48] or using different in vitro-maturation protocols [17]), samples of inner cell masses and embryonic stem cells and other types of samples should increase the utility of PREGER. Additionally, it will be possible for other scientists to produce sets of experimentally manipulated embryos and link these to the PREGER sample set. Because the QADB method is fully quantitative, it may be possible to calibrate the data and obtain estimates of mRNA copy number, as is done in the mouse studies [25]. Additionally, the quantitative nature of the QADB method coupled with an expanding sample set will permit extensive comparisons between different stages and types of samples, with appropriate statistical evaluations of results.
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FOOTNOTES |
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2 Correspondence: Keith E. Latham, 3307 North Broad Street, Philadelphia, PA 19140. FAX: 215 707 1454. klatham{at}temple.edu
Received: 1 October 2003.
First decision: 3 November 2003.
Accepted: 5 January 2004.
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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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  L. Hao, R. Vassena, G. Wu, Z. Han, Y. Cheng, K. E. Latham, and C. Sapienza The Unfolded Protein Response Contributes to Preimplantation Mouse Embryo Death in the DDK Syndrome Biol Reprod, May 1, 2009; 80(5): 944 - 953. [Abstract] [Full Text] [PDF]
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 J. K. Nyholt de Prada, Y. S. Lee, K. E. Latham, C. L. Chaffin, and C. A. VandeVoort Role for cumulus cell-produced EGF-like ligands during primate oocyte maturation in vitro Am J Physiol Endocrinol Metab, May 1, 2009; 296(5): E1049 - E1058. [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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N. R Mtango and K. E Latham Differential Expression of Cell Cycle Genes in Rhesus Monkey Oocytes and Embryos of Different Developmental Potentials Biol Reprod, February 1, 2008; 78(2): 254 - 266. [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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P. Zheng, R. D. Schramm, and K. E. Latham Developmental Regulation and In Vitro Culture Effects on Expression of DNA Repair and Cell Cycle Checkpoint Control Genes in Rhesus Monkey Oocytes and Embryos Biol Reprod, June 1, 2005; 72(6): 1359 - 1369. [Abstract] [Full Text] [PDF]
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P. Zheng, B. Patel, M. McMenamin, E. Moran, A. M. Paprocki, M. Kihara, R. D. Schramm, and K. E. Latham Effects of Follicle Size and Oocyte Maturation Conditions on Maternal Messenger RNA Regulation and Gene Expression in Rhesus Monkey Oocytes and Embryos Biol Reprod, April 1, 2005; 72(4): 890 - 897. [Abstract] [Full Text] [PDF]
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 M. F. Pera and A. O. Trounson Human embryonic stem cells: prospects for development Development, November 15, 2004; 131(22): 5515 - 5525. [Abstract] [Full Text] [PDF]
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P. Zheng, B. Patel, M. McMenamin, A. M. Paprocki, R. D. Schramm, N. G. Nagl Jr, D. Wilsker, X. Wang, E. Moran, and K. E. Latham Expression of Genes Encoding Chromatin Regulatory Factors in Developing Rhesus Monkey Oocytes and Preimplantation Stage Embryos: Possible Roles in Genome Activation Biol Reprod, May 1, 2004; 70(5): 1419 - 1427. [Abstract] [Full Text] [PDF]
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