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Mol. Hum. Reprod. Advance Access originally published online on April 7, 2007
Molecular Human Reproduction 2007 13(6):361-371; doi:10.1093/molehr/gam014
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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

Ping Zheng1, Rita Vassena1 and Keith E. Latham1,2,3

1 The Fels Institute for Cancer Research and Molecular Biology, Temple University Medical School, 3307 North Broad Street, Philadelphia, PA 19140, USA 2 The Department of Biochemistry, Temple University Medical School, 3307 North Broad Street, Philadelphia, PA 19140, USA

3 Correspondence address. Tel: +1-215-707-7577; Fax: +1-215-707-1454; E-mail: klatham{at}temple.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Acknowledgements
 References
 
Glucose plays a fundamental role during oogenesis and embryogenesis, satisfying the metabolic demands of oocytes and embryos, providing for stored energy reserves in the form of glycogen and supporting nucleotide biosynthesis via the pentose phosphate pathway. Glucose also contributes to the production of amino acids, glycosylated proteins and extracellular components. A detailed understanding of the molecular mechanisms that mediate and regulate glucose uptake and metabolism at different stages of oogenesis and preimplantation embryogenesis could greatly benefit the development of improved methods for in vitro oocyte maturation and in vitro embryo production. Although these processes have been examined in a variety of rodent and agricultural species, detailed information has not yet been described for non-human primates. In this study, we examined the expression of the genes encoding glucose transporters, glucose metabolism enzymes and potential regulators of glucose metabolism in rhesus monkey oocytes and embryos. The data reveal stage-specific regulation of expression of specific types of glucose transporters, stage-specific changes in expression of genes related to different pathways of glucose metabolism and temporal changes in the expression of mRNAs related to insulin signaling. Additionally, the data reveal significant differences in expression of some of these genes in cultured embryos as compared with flushed embryos and between oocytes and embryos obtained following different hormonal stimulation and oocyte maturation protocols.

Key words: glucose metabolism/oocyte/preimplantation embryo/rhesus monkey/insulin


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 Acknowledgements
 References
 
Glucose is an essential nutrient for most mammalian cells. Glucose is employed as an energy source via either glycolysis or the tricarboxylic acid (TCA) cycle. Additionally, it can be metabolized via the pentose phosphate pathway (PPP) to produce NADPH and ribose-5-phosphate, which can then either re-enter the glycolytic pathway or be employed for nucleotide biosynthesis. Glucose can also be stored in the early mouse embryo as glycogen (Houghton et al., 1996) and can be employed for the synthesis of amino acids. Further, glucose can be used for protein glycosylation, which can regulate protein function and stability (Lis and Sharon, 1993) and also for the formation of extracellular matrix macromolecules (Heilig et al., 1995).

Glucose has come to be perceived as detrimental for the in vitro culture of preimplantation embryos, and some medium formulations have either omitted or reduced glucose concentrations, while providing alternative energy sources in the form of three or five carbon sugars (Schini and Bavister, 1988a,b; Seshagiri and Bavister, 1989; Martin and Leese, 1995). The inhibitory effect of glucose, however, appears to be dependent upon the context of other solutes in the medium such as phosphate (Seshagiri and Bavister, 1989; Biggers and McGinnis, 2001), and indeed glucose is available to preimplantation embryos in vivo (Harris et al., 2005). Other oocyte and embryo culture data have documented the importance of glucose for oogenesis and oocyte maturation (Herrick et al., 2006a, b; Zheng et al., 2001), and glucose is beneficial to cultured embryos, particularly at later stages of development (Leese et al., 1993; Martin and Leese, 1995, 1999; Wirtu et al., 2004). Glucose transporters are necessary for early development (Heilig et al., 2003; Jensen et al., 2006), and their expression is regulated by insulin (Carayannopoulos et al., 2000; Navarrete et al., 2004a; Benomar et al., 2006), which promotes development via this mechanism as well as through its mitogenic activity (Navarrete et al., 2004b). Under diabetic or hypoinsulinemic conditions, hyperglycemia can lead to down-regulation of certain glucose transporters and subsequent inhibition of development (Moley et al., 1998a,b; Chi et al., 2000; Keim et al., 2001), whereas insulin-like growth factor-1 (IGF1) stimulation can enhance glucose transporter expression and promote development, as well as suppressing apoptosis (Fladeby et al., 2003; Oropeza et al., 2004; Russo et al., 2004). These observations indicate that glucose is required by the early embryo, not only as an energy source, but also as a key substrate for other regulatory and biosynthetic pathways as well. Any perturbation in glucose uptake and metabolism could thus result in the interruption of oocyte maturation or embryo development.

Understanding the molecular mechanisms that mediate and regulate glucose uptake and metabolism at different stages of oogenesis and preimplantation embryogenesis and how in vitro oocyte maturation (IVM) and embryo culture affect the expression of genes related to these processes could greatly assist in the development of improved methods for in vitro oocyte maturation and in vitro embryo production. Such knowledge may be employed to determine specific embryonic demands at different stages and to determine how certain culture conditions may perturb development. Although the importance of glucose for oocytes and embryo culture has been documented in a variety of rodent and agricultural species and correlated with patterns of gene expression at different stages, detailed information has not yet been described for non-human primates. Due to ethical, legal and logistical constraints on experimentation with human embryos, the continued development of non-human primate models is important. Through the analysis of non-human primate embryos, crucial differences in embryonic gene expression patterns between phylogenetic categories of mammals can be determined and thus employed to enhance our understanding of the unique culture requirements of human and non-human primate embryos. Additionally, healthy, high-quality non-human primate embryos can be employed for experimental purposes without the same ethical concerns that restrict studies in human embryos.

We established a novel resource to facilitate the rapid, quantitative analysis of gene expression patterns in rhesus monkey oocytes and preimplantation stage embryos as a model for better understanding how gene expression may be affected in human embryos by different procedures. The resource, designated as the Primate Embryo Gene Expression Resource (PREGER; http://www.preger.org), encompasses a large and diverse set of over 200 quantitatively amplified cDNA libraries corresponding to oocytes and embryos obtained through a range of different maternal hormonal stimulation, oocyte maturation and embryo culture protocols. The resource can provide detailed, quantitative information related to temporal patterns of gene expression, effects of hormonal stimulation and oocyte maturation protocols and effects of different in vitro culture systems (Zheng et al., 2004b). In this study, the expression of mRNAs related to glucose transport, glucose metabolism regulation of glucose metabolism and insulin signaling was examined. Our data reveal stage-specific changes in the expression of these mRNAs in oocytes and embryos, along with significant effects of hormonal stimulation, IVM and embryo culture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 Acknowledgements
 References
 
Oocytes and embryos
This study employed the PREGER (www.preger.org) (Zheng et al., 2004a,b, 2005a,b, 2006). This resource encompasses a collection of reverse transcribed and polymerase chain reaction (RT–PCR)-amplified cDNA libraries corresponding to > 200 samples of rhesus monkey oocytes and preimplantation stage embryos. The isolation and culture of oocytes and embryos during the construction of the PREGER sample set was described in detail elsewhere (Zheng et al., 2004b). Briefly, three types of oocytes were used in these studies. In vivo matured oocytes were obtained from large follicles (3–7 mm) of females stimulated with both follicle-stimulating hormone (FSH) and human chorionic gonadotrophin (hCG). In vitro matured oocytes were from large follicles (3–7 mm) of females stimulated with FSH only or from non-stimulated females (small follicles, 0.45–2 mm) at random stages of the menstrual cycle, and were matured as described (Zheng et al., 2004b). Samples of oocytes were collected at the germinal vesicle (GV) and metaphase II (MII) stages. Embryos were obtained by in vitro fertilization (IVF) of MII oocytes from each of the above three sources of oocytes, followed by culture in HECM9 or G1/G2 sequential media (Gardner and Lane, 1997). Morulae and blastocysts were also obtained by natural conception and flushed from the reproductive tract for lysis. Between 3 and 13 samples of one to four oocytes or embryos were obtained for each stage. 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 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 {alpha}-amanitin (24 µg ml–1) from the pronucleate stage onward in HECM9 culture were included to evaluate transcriptional dependence of mRNA expression. Details concerning the array, diversity, origin of samples and sensitivity and quantitative reliability of the quantitative amplification and dot blotting method have been described previously (Zheng et al., 2004b).

The care and housing of rhesus monkeys (Macaca mulatta) at the Wisconsin National Primate Research Center (WNPRC, from where oocytes and embryos were obtained) have been described (Resko et al., 1982). The WNPRC is fully accredited (Association for Assessment and Accreditation of Laboratory Animal Care, AAALAC). Animals were cared for and all experiments conducted according to recommendations of the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act (USA) with its subsequent amendments.

QADB Assay, cDNA probes, hybridization and data analysis
The PREGER sample collection is based on the RT and exponential amplification of entire cDNA populations using conditions that preserve the quantitative representation of sequences within the population (Brady and Iscove, 1993; Iscove et al., 2002). Cells are lysed in RT buffer supplemented with non-ionic detergent, followed by oligo(dT) annealing to mRNAs and immediate processing through the RT step, thereby avoiding any need for RNA purification. The amplified cDNA is applied to filters by dot blotting to produce blots containing the entire array of samples (Quantitative Amplification and Dot Blotting, QADB). The blots are then hybridized and the hybridization signals quantified. The quantitative validity and sensitivity of the QADB method have been extensively documented (Rambhatla et al., 1995; Latham et al., 2000; Zheng et al., 2004b).

The cDNA probes used in this study were obtained by RT–PCR (Table 1). The identities of the amplified cDNAs were confirmed by sequencing. Blot preparation, hybridization and quantitative analysis were performed as described previously (Rambhatla et al., 1995; Latham et al., 2000; Zheng et al., 2004b). Data were expressed as the mean CPM bound values 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 using the two-tailed t-test assuming equal variance.


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  		Table 1: Primers employed to obtain cDNA probes

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 Acknowledgements
 References
 
The overall goal of this study was to investigate the ontogeny and regulation of genes that support glucose transport and metabolism by examining the expression of mRNAs encoding glucose transporters, glucose metabolism enzymes and components of the insulin response pathway. Additionally, we investigated whether these processes vary with oocyte/embryo quality, hormonal stimulation and IVM and embryo culture. The genes included in the analysis, their functions and regulatory relationships are summarized in Fig. 1 and Table 2.


Figure 1
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  		Figure 1: Schematic representation of functional relationships between genes studied.

 

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  		Table 2: Genes assayed and their functions

 
Expression of glucose transporters
Two glucose transport systems mediate glucose uptake. One is the sodium-coupled glucose transporter (sodium-glucose co-transporter, SGLT) and the other is mediated by the sodium-independent facilitative glucose transporters (GLUTs) (Wood and Trayhurn, 2003). GLUTs comprise a family of structurally related, membrane-spanning glycoproteins, of which 13 members have been identified and designated as SLC2A1–12 and HMIT (Uldry and Thorens, 2004).

The expression of seven GLUT mRNAs was detected in rhesus monkey oocytes and preimplantation embryos (Fig. 2). The mRNAs for four of these genes (SLC2A1, SLC2A3, SLC2A4 and SLC2A6) yielded the strongest hybridization signals, whereas SLC2A5, SLC2A8, and SLC1A12 mRNAs showed variable, low expression signals throughout the development. The SLC2A3 mRNA dramatically increased in abundance at the eight-cell stage in an {alpha}-amanitin sensitive manner, indicating embryonic transcription. The SLC2A1 mRNA was also transcribed at a low-level beginning at the eight-cell stage. Although the abundance of SLC2A4 and SLC2A6 mRNAs increased at the eight-cell stage relative to the pronuclear or two-cell stages, their expression at the eight-cell stage was unaffected by {alpha}-amanitin, indicating that these mRNAs were largely maternal in origin and may undergo polyadenylation and recruitment for translation (this leads to enhanced efficiency of RT and increased detection; Rambhatla et al., 1995).


Figure 2
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  		Figure 2: Temporal expression patterns of glucose transporter mRNAs in rhesus monkey oocytes and preimplantation embryos. Graphs show the relative abundances for GV and MII stage oocytes and embryos from the pronucleate through hatched blastocyst stages produced by IVF of oocytes from hCG-stimulated females and then cultured in HECM9. GV, germinal vesicle stage oocyte; MII, MII-stage oocyte; PN, pronucleate 1-cell stage embryo; 2C, 2-cell stage; 8C, 8-cell stage; 8–16C {alpha}Am, 8–16-cell stage cultured in {alpha}-amanitin; EB, early blastocyst; XB, expanded blastocyst; HB, hatched blastocyst. Expression data for the mRNAs encoding the indicated proteins are expressed as the mean CPM bound and the SEM indicated. The y-axes are adjusted according to level of expression. Statistically significant differences in gene expression corresponding to some of the major increases or decreases in expression are denoted by the brackets (for comparisons between stages at the ends of the brackets). Letters ‘a’ through ‘d’ indicate statistically significant differences, P < 0.05, 0.01, 0.001 and 0.0001, respectively.

 
Expression of enzyme genes involved in three glucose metabolism pathways
During glycolysis, glucose is metabolized to pyruvate, which then enters the TCA cycle under aerobic conditions, or is converted to lactate under anaerobic conditions. The mRNAs encoding glycolysis enzymes, including phosphofructokinase muscle type and liver type (PFKM and PFKL, respectively), aldolase (ALDOA1), phosphoglycerate kinase (PGK1), enolase (ENO1) and pyruvate kinase muscle type and liver type (PKM2 and PKLR, respectively) were examined (Fig. 3). The PFKL mRNA was not detected in oocytes or embryos. The other glycolysis-related mRNAs displayed abundant expression in oocytes and embryos. Three of these (ALDOA1, PGK1, and ENO1) were predominantly expressed in embryos from the eight-cell stage onward in an {alpha}-amanitin sensitive manner. The PFKM and PKLR mRNAs were expressed throughout development. The PKM2 mRNA decreased in abundance during oocyte maturation and appeared variably increased in abundance thereafter.


Figure 3
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  		Figure 3: Temporal expression patterns of mRNAs encoding enzymes of glucose metabolism in rhesus monkey oocytes and preimplantation embryos. Stages shown and abbreviations are the same as described for Fig. 2. The y-axes are adjusted according to level of expression. Statistically significant differences in gene expression corresponding to some of the major increases or decreases in expression are denoted by the brackets as described for Fig. 2. Data for PGK1 and PDHA1 were published previously (Zheng et al., 2004b) and are reproduced here for comparative purposes.

 
Under anaerobic conditions, pyruvate is converted to lactate by lactate dehydrogenase (LDHA). The LDHA mRNA was detected at a low abundance in oocytes and embryos through the two-cell stage. At the eight-cell stage, LDHA mRNA increased dramatically in abundance in an {alpha}-amanitin sensitive manner, indicating embryonic LDHA gene transcription. PDHA1 is a key enzyme gene in the TCA cycle (Fig. 3). The PDHA1 mRNA was abundantly expressed in GV stage oocytes, decreased in abundance during oocyte maturation and was re-expressed at the eight-cell stage in an {alpha}-amanitin sensitive manner.

The mRNAs for two PPP genes, PGD and TALDO1, were examined (Fig. 3). The TALDO1 mRNA was abundantly expressed as a maternal transcript in oocytes and early embryos, with reduced but stable expression from the eight-cell stage onward. The PGD mRNA was expressed throughout development.

Expression of the genes involved in glucose metabolism regulation
Insulin stimulates glucose uptake by regulating the transporter activities at both the transcriptional and post-translational levels. For instance, insulin stimulates the transcription of SLC2A8 as well as the translocation of SLC2A4 from cytoplasm to plasma membrane where it mediates glucose uptake (Carayannopoulos et al., 2000; Benomar et al., 2006). The initiation of insulin signaling requires the binding of the ligand(s) to the insulin receptor (IR), resulting in the activation of diverse signaling pathways, including phosphoinositide-3-kinase (PI3K)-dependent and PI3K-independent pathways. In the former pathway, insulin stimulates the phosphorylation of IR substrate (IRS) proteins. Upon phosphorylation, IRS activates PI3K, which generates phosphatidylinositol 3,4,5-trisphosphate to regulate many proteins including Ser/Thr kinase PDK1. PDK1 activates several down-stream protein kinases, such as AKT1 and AKT2 (Chang et al., 2004; Watson et al., 2004). In addition to IR, the IGF1 receptor (IGF1R) can be activated by insulin or IGF1 to stimulate glucose uptake via these same intracellular signal transduction pathways (Shefi-Friedman et al., 2001).

IGF1 mRNA expression was significantly increased at the eight-cell stage in an {alpha}-amanitin sensitive manner, although the overall hybridization signals were quite low and was variably increased in hatched blastocysts compared with earlier stages (Fig. 4). The IGF1R mRNA was abundant in oocytes and early stage embryos, but decreased dramatically upon the formation of early blastocysts. The INSR, IRS1 and IRS2 mRNAs were expressed in oocytes and throughout preimplantation development. The gene for the catalytic subunit of PI3K, PIK3CA, displayed a maternal expression pattern, with its mRNA abundance decreasing by the morula stage. Similar to PIK3CA, the AKT1/AKT2 cDNA probe revealed down-regulation in morula and blastocyst stages, although the overall amount of transcript was low.


Figure 4
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  		Figure 4: Temporal expression patterns of mRNAs encoding insulin signaling pathway and ER stress response genes in rhesus monkey oocytes and preimplantation embryos. Stages shown and abbreviations are the same as described for Fig. 2. The y-axes are adjusted according to level of expression. Statistically significant differences in gene expression corresponding to some of the major increases or decreases in expression are denoted by the brackets as described for Fig. 2.

 
Oxygen concentration influences the glucose metabolism. Hypoxia-induced factor-1A (HIF1A) mediates the protective responses of cells under low oxygen stress. HIF1A mRNA was abundantly expressed in GV stage oocytes and decreased in abundance during oocyte maturation (Fig. 4). The HIF1A mRNA displayed a trend toward increased expression in blastocysts, but this increase was not statistically significant (Fig. 4).

The HSPA5 and HSP90B1, two genes that can be induced by glucose starvation, were expressed abundantly in oocytes and preimplantation embryos. The HSPA5 mRNA increased in abundance at the 8-cell stage, whereas the HSP90B1 mRNA displayed maternal expression in oocytes, was down-regulated at maturation and then increased again in abundance with further development (Fig. 4).

Effects of hormonal stimulation and culture on mRNA expression
To assess the possible relationships between glucose metabolism and oocyte/embryo quality, the expression of each mRNA was compared at each developmental stage among three groups of oocytes/embryos (oocytes/embryos from non-stimulated, FSH stimulated and FSH + hCG stimulated females, corresponding to the oocytes/embryos with low, medium and highest quality; Zheng et al., 2004b). These comparisons revealed many differences for individual genes for various stages conditions (Table 3). The majority of these effects were limited to single stages, and often a significant difference was observed at one stage, but not at the next stage. This indicates that the effects of different hormonal stimulation protocols and in vitro maturation are complex and affect a wide variety of genes at different stages. Prolonged effects were seen for four genes. There was an elevated expression of PIK3CA mRNA in MII stage oocytes from non-stimulated females and fertilized pronuclear stage embryos derived from these oocytes, relative to oocytes and embryos from hCG-stimulated females and matured in vivo (hCG:NS ratio = 0.3 and 0.23, respectively). The TALDO1 mRNA displayed increased expression in MII stage oocytes from non-stimulated females and corresponding pronucleate stage embryos, relative to in vivo matured oocytes (hCG:NS ratio = 0.37 and 0.27, respectively). The SLC2A6 mRNA displayed reduced expression for vitro matured oocytes from FSH-stimulated females and significantly reduced expression for pronuclear stage embryos derived from these oocytes, relative to both in vivo matured oocytes/embryos (FSH + hCG stimulation) and those from non-stimulated females (hCG:FSH ratios = 2.29 and 1.81; FSH:NS ratios = 0.64 and 0.39, respectively). This mRNA was also increased in expression in two-cell stage embryos from the non-stimulation protocol relative to the other two protocols (hCG:NS ratio = 0.35, FSH:NS ratio = 0.13). The SLC2A3 mRNA displayed reduced expression over the period from the eight-cell stage through the morula stage in embryos derived from IVM oocytes of FSH stimulated females (hCG:FSH ratio = 3.65 and 5.63, respectively). Thus, there were prolonged and significant effects of these treatments for these four genes.


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  		Table 3: Effects of hormonal stimulation protocol on gene expression

 
The expression of each mRNA was also compared between in vivo produced hatched blastocysts flushed from the reproductive tract immediately prior to lysis and hatched blastocysts developing in culture (Table 4). There were significant effects of culture on the expression of a minority of these genes. Among the genes examined with detectable expression in blastocyst (total 27), six of them (22%) showed a significant difference in mRNA expression between the two groups of blastocysts. Five of these (SLC2A1, SLC2A3, PGK1, IRS1 and HSPA5) were elevated in cultured embryos and one (PKM2) was reduced in culture. Other potential differences of 2-fold or more (HSP90B1, IGF1, IRS2, LDHA, PFKM, PGD, PIK3Ca, SLC2A12 and TALDO1) did not reach statistical significance (P > 0.05).


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  		Table 4: Comparisons of mRNA expression between flushed and cultured hatched blastocysts

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 Acknowledgements
 References
 
Previous studies revealed the importance of glucose metabolism in mammalian oocyte maturation and preimplantation embryo development (Downs et al., 1998; Martin and Leese, 1999; Zheng et al., 2001; Herrick et al., 2006a). Certain types of glucose transporters were identified in oocytes and preimplantation embryos of some species and the expression and activities of several glucose transporters were found regulated by insulin stimulation (Dan-Goor et al., 1997; Carayannopoulos et al., 2000; Augustin et al., 2001; Navarrete et al., 2004a). However, similar knowledge is missing for primates. By using the PREGER resource, we systematically examined the expression of the genes involved in glucose metabolism and its regulation in rhesus monkey oocytes and preimplantation embryos. Our data reveal stage-specific expression of major types of glucose transporters responsible for glucose uptake, a stage-specific transition in relative expression of genes for different glucose metabolism pathways and expression of genes to support responsiveness to insulin stimulation. Our data also reveal significant effects of hormonal stimulation and in vitro maturation protocols, as well as embryo culture on the expression of some of these genes, adding further evidence for the possibility of short- and long-term effects of IVM and embryo culture on human embryos produced through assisted reproduction.

Developmental transitions in glucose utilization
Glucose is a necessary energy substrate for oocyte maturation in the presence of cumulus cells (Khurana and Niemann, 2000; Zheng et al., 2001; Roberts et al., 2004; Preis et al., 2005). Studies on porcine, bovine and murine oocytes and embryos have shown that glucose supports oocyte maturation and pronucleus formation via the pentose phosphate pathway (PPP). Manipulation of the PPP by chemicals can change the maturation and developmental potentials of the oocytes (Downs et al., 1998; Comizzoli et al., 2003; Herrick et al., 2006a). Embryos subsequently switch to a glycolytic metabolism or TCA cycle as the development progresses (Houghton et al., 1996; Chi et al., 2002). Previous studies reported a beneficial role of glucose on rhesus monkey oocyte IVM that could not be replaced by lactate or pyruvate (Zheng et al., 2001), also pointing to a switch in energy substrate preference. Our data for the rhesus monkey indicate that this switch in energy substrate preference is driven by stage-specific changes in gene expression. The mRNAs encoding two major PPP enzymes, PGD and TALDO1, are predominantly expressed as maternal transcripts, whereas the genes involved in glycolysis and TCA cycle display low expression until embryonic genome activation, at which time expression of these mRNAs increases dramatically. This early bias in favor of glucose metabolism via the PPP may reflect early metabolic demands for glucose unrelated to ATP production, such as nucleotide biosynthesis.

Alterations in the expression of glucose transporter genes
Comparisons among different species reveal both similarities and differences in expression of glucsose transporters in oocytes and preimplantation embryos. For examples, Slc2a1–5 and Slc2a8 were reported in bovine oocytes and embryos (Augustin et al., 2001). Slc2a1 was observed in human oocyte and throughout preimplantation development (Dan-Goor et al., 1997). We show here that in rhesus monkey oocytes and embryos, the major types of glucose transporter mRNAs expressed are SLC2A1, SLC2A3, SLC2A4 and SLC2A6. In mouse, the mRNA transcripts of Slc2a1–5 and Slc2a8–10 showed hybridization signal in the microarray study of mouse oocytes and embryos, and in some cases temporal patterns somewhat different from the rhesus monkey (Fig. 5A). The temporal expression patterns for genes encoding enzymes involved in glucose metabolism (Fig. 5B) also displayed subtle differences between mouse and monkey. The differences between species indicate possible differences in glucose uptake and its regulation by exogenous cues. The species differences also highlight the importance of further development of non-human primate models to help understand early development in the human.


Figure 5
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  		Figure 5: Gene expression patterns in developing mouse embryos. Data are reproduced here from the published microarray dataset produced by Zeng et al. (2004). Graphs depict the average fluorescence intensities from four hybridized arrays per stage.

 
Three of the most highly expressed glucose transporters (SLC2A1, SLC2A3 and SLC2A4) in rhesus monkey oocytes and preimplantation embryos displayed significant increases in expression beginning at the eight-cell stage, around the time of embryonic genome activation. The increase in expression of glucose transporter mRNAs likely contributes to an increase in glucose uptake and metabolism at these later stages of rhesus monkey embryo development, given that human embryos display an increase in glucose metabolism at these stages (Leese et al., 1993). Studies in other species, including human, have reported the increased consumption of glucose by morula and blastocysts (Martin and Leese, 1995; Jones et al., 2001; Lane et al., 2001). The mRNA expression data indicate that glucose availability is likely advantageous to these later stages in the rhesus monkey as well.

The expression of SLC2A4 mRNA is significantly altered in MII stage oocytes by IVM, and SLC2A6 mRNA expression was also marginally decreased by IVM (P = 0.054). In embryos derived by IVM of oocytes from FSH stimulated females, the SLC2A6 mRNA was reduced during the early portion of preimplantation development and the SLC2A1 and SLC2A3 mRNAs were reduced in abundance at later stages. Thus, there is a disruption in the expression of the most prevalent glucose transporter mRNAs following IVM. This may lead to alterations in glucose uptake, which could affect blastomere apoptotic index, developmental competence and long-term physiology of the embryo (Moley et al., 1998a,b; Chi et al., 2000, 2002; Keim et al., 2001). Additionally, because insulin can affect expression or localization of SLC2A4 and SLC2A8 (Carayanopoulos et al., 2000; Chang et al., 2004; Benomar et al., 2006), a shift in the relative proportion of glucose transporters could affect the response to insulin stimulation following IVM.

Alterations in the expression of insulin signaling pathway genes
Our data provide evidence that rhesus monkey oocytes and embryos are likely insulin responsive and that insulin stimulation may promote glucose uptake and metabolism. The IRS1, IRS2, PIK3CA and AKT1/AKT2 mRNAs were all detected in rhesus monkey oocytes and embryos, consistent with the presence of the PI3K-dependent signaling pathway. Studies in mice likewise revealed the presence of a functional PI3K/AKT pathway throughout mammalian preimplantation development (Riley et al., 2005, 2006). Inhibiting this pathway leads to reduced insulin-stimulated glucose uptake, a significant delay in blastocyst hatching and an induction of apoptosis in blastocysts (Riley et al., 2005, 2006). Insulin signaling controls glucose uptake by regulating the expression and translocation of glucose transporters. Two glucose transporters SLC2A4 and SLC2A8 are known to be sensitive to insulin stimulation, which increases SLC2A8 mRNA expression and promotes translocation of SLC2A4 from cytoplasm to plasma membrane (Carayanopoulos et al., 2000; Chang et al., 2004; Benomar et al., 2006). In vitro studies revealed that insulin stimulation, expression and activity of glucose transporters and oocyte and preimplantation embryo quality are correlated (Chi et al., 2000; Heilig et al., 2003; Jensen et al., 2006). We show here that SLC2A4 and SLC2A8 mRNAs are both expressed in rhesus monkey oocytes and embryos. The abundant transcription of SLC2A4 throughout rhesus monkey preimplantation development provides one avenue by which glucose uptake can be regulated by insulin.

We also note species differences between mouse and monkey in the expression of insulin signaling genes. Most notably, the temporal pattern of expression of IGF1 is rather different between the two species, with this mRNA being dramatically up-regulated in the developing monkey embryos, but more uniformly expressed during mouse development (Fig. 5C). Thus, the relative role of IGF1 during development may differ for the two species.

We find an elevated expression of PIK3CA mRNA in matured oocytes from non-stimulated females and fertilized pronuclear stage embryos derived from these oocytes. In the mouse, PIK3CA mRNA is expressed in a temporal pattern similar to that shown here for the rhesus, with the expression diminishing to background level upon zygote genome activation (two-cell stage) (Fig. 5C). Interestingly, the PIK3CA protein is nevertheless detectable throughout the morula and blastocyst stages in the mouse (Riley et al., 2005). The over-expression of PIK3CA mRNA in ovulated oocytes/embryos from non-stimulated monkeys, coupled with a prolonged stability of the PIK3CA protein, raises the possibility that abnormal expression and activation of PIK3CA-dependent signaling pathways may contribute to the exceptionally low developmental potential of rhesus monkey embryos obtained by IVM and IVF of oocytes from non-stimulated females.

Expression of hypoxia and glucose starvation response genes
Perturbation of glucose metabolism, for example by glucose starvation or hypoxia, leads to endoplasmic reticulum (ER) stress characterized by the accumulation of mis-folded proteins in the ER. However, mammalian cells react to the ER stress by up-regulating proteins with protective function. Some of the examples include glucose-regulated proteins (GRP) and HIF1A. GRPs are a family of ER resident proteins induced at the transcription level under the stress-inducing conditions. HIF1A is a transcription factor capable of mediating the response to hypoxia by regulating the transcription of genes involved in glycolysis and angiogenesis. In the rhesus monkey (Fig 3), HIF1A and two GRP genes, HSPA5 (GRP78) and HSP90B1 (GRP94), were abundantly transcribed in oocytes and fertilized embryos through the blastocyst stage. This indicates that oocytes and early stage embryos may have the ability to respond to adverse metabolite conditions.


   Summary
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
Acknowledgements
 References
 
The data presented here provide a detailed examination in a non-human primate model system of the expression of 27 genes related to glucose uptake, metabolism and the regulation of these processes. The results reveal temporal transitions in the expression of genes related to different metabolic pathways, along with greater insight into the similarity and differences in expression between different species, including human. More significantly, the data reveal significant effects of hormonal stimulation, IVM and embryo culture on the expression of some of these genes, including those encoding glucose transporters and the key signaling protein PIK3CA. The results raise the possibility that procedures such as IVM and embryo culture, which are essential components of assisted reproduction technologies, may alter the metabolic demands of early embryos, as well as their responses to exogenous factors such as insulin.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Acknowledgements
References
 
This work was supported by a grant from the National Center for Research Resources (NIH/NCRR, RR15253). We thank Bela Patel for her excellent technical assistance. We also thank Catherine VandeVoort for expert comments on the manuscript.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Acknowledgements
 References
 
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