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Physiol. Genomics 31: 1-14, 2007. First published June 5, 2007; doi:10.1152/physiolgenomics.00040.2007
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Received 23 February 2007; accepted in final form 17 May 2007.
Physiological Genomics 31:1-14 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Ubiquitin proteasome pathway gene expression varies in rhesus monkey oocytes and embryos of different developmental potential

Namdori R. Mtango 1 and Keith E. Latham 1,2

1 The Fels Institute for Cancer Research and Molecular Biology
2 Department of Biochemistry, Temple University, Philadelphia, Pennsylvania


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protein degradation via the ubiquitin-proteasome pathway (UPP) plays a key role in diverse aspects of cell physiology and development. In the early embryo, the UPP may play an important role in the transition from maternal to embryonic control of development. Disruptions in the UPP could thus compromise embryo developmental potential. Additionally, species-specific requirements may dictate diverse patterns of regulation of the UPP components. To investigate the expression of UPP components in a nonhuman primate embryo model, to compare expression between a primate and nonprimate species, and to determine whether disruption of this pathway may contribute to reduced developmental potential, we examined the expression of >50 mRNAs encoding UPP components in rhesus monkey oocytes and embryos. We compared this expression between the rhesus monkey and mouse embryo and between rhesus monkey oocytes and embryos of high, intermediate, and low developmental potential. We report here the temporal patterns of UPP gene expression in oocytes and during preimplantation development, including striking differences between the rhesus monkey and mouse. We also report significant differences in UPP gene expression correlating with oocyte and embryo developmental competence and associated with altered regulation of maternally inherited mRNAs encoding these proteins.

in vitro maturation; oocyte maturation; evolution; cleavage; preimplantation; gene expression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
MAMMALIAN PREIMPLANTATION embryo development encompasses four major events following fertilization: degradation of oocyte transcripts, transcriptional activation of the zygotic genome during cleavage, compaction, and establishment of the inner cell mass and trophectoderm lineages. The reprogramming of gene expression from maternal to embryonic pattern, however, is not limited to just transcriptional production of new mRNAs but also requires turnover of maternally inherited proteins, production of newly synthesized proteins, and the correct folding of these proteins to permit binding to cofactors and appropriate biochemical activity.

Protein folding involves molecular chaperones, such as heat shock proteins. Incorrect folding or aberrant accumulation of proteins can be dangerous to the cell, and in most cells powerful protein quality control mechanisms prevent this. Mechanisms by which cells recognize and remove unfolded proteins includes molecular chaperones, which bind to the hydrophobic patch created during misfolding and attempt to repair the defective protein by folding it and at the same time by covering the patches. When this fails, protein proteolysis occurs (1). Additionally, proteolytic mechanisms support the stage-dependent elimination of maternally inherited proteins (13).

Intracellular proteolysis occurs via two pathways: a lysosomal pathway and a nonlysosomal ATP-dependent pathway [ubiquitin proteasomal pathway (UPP)]. The UPP degrades most cell proteins, including regulatory proteins, and begins with the covalent linking of proteins to multiple molecules of the polypeptide ubiquitin (UB) (21). This modification marks the protein for rapid degradation by the proteasome, a 26S (200 kDa) complex that contains a 20S (673 kDa) proteasome or multicatalytic protease complex as the key proteolytic component, and a 19S complex containing several ATPases and a binding site for UB chains. The 19S particle "caps" each extremity of the 20S proteasome and unfolds the protein substrates to inject them into the 20S proteasome and stimulate proteolysis (26, 46).

UB is a small protein that is composed of 76 amino acids with molecular mass of 8.5 kDa. It is heat stable and folds into a compact globular structure. It is found throughout the cell (thus giving rise to its name) and can exist either in free form or as part of a complex with other proteins. In the latter case, UB is attached (conjugated) to proteins through a covalent bond between the glycine at the COOH-terminal end of UB and the side chains of lysine on the proteins. Single UB molecules can be conjugated to the lysine of these proteins, or more commonly, UB-chains can be attached. Conjugation is a process that depends on the hydrolysis of ATP. The attachment of UB to a target protein requires the action of four enzymes, called E1 (UB-activating enzymes), E2 (UB-conjugating enzymes), E3 (UB ligases), and E4 (UB elongation enzymes), which work sequentially in a cascade. There are at least four classes of E3 ligases: HECT-type, RING-type, PHD-type, and U-box containing (52). The E3 ligases are the only enzymes that are subjected to regulation; however, balance in the UB system is also achieved through a set of deubiquitinylating isopeptidases that cleave UB substrates (8, 9, 20, 22). The selectivity of UB conjugation is determined by specific degradation signals (degrons) in short-lived proteins, including the degrons that give rise to the N-end rule (4, 5, 19). Many proteins are recognized following posttranslational modification (e.g., phosphorylation) or association with an ancillary protein (e.g., HSC or HPV-E6).

The UPP serves a broad range of functions in cellular physiology, primarily by controlling levels of specific proteins. Such a regulatory role is very important. By regulating protein degradation, cells can quickly eliminate a protein that in turn regulates another function (like a transcription factor that is needed to express a particular gene). UB is involved in many cell processes such as a cell cycle progression (18, 27, 38), DNA repair (32), protein degradation and recycling (7, 10, 14, 25), protein synthesis and processing (16), regulation of transcription (11, 24, 52), membrane trafficking (12, 23), cell signaling (35), apoptosis (33), stress responses (15, 39), and diseases (2, 17). The UPP also contributes to correct sperm function, the control of mitochondria inheritance, oocyte maturation, fertilization, nuclear remodeling, and correct proteome remodeling during embryonic development (13, 28–30, 34, 36, 37, 41–45, 49).

In view of the importance of UPP in regulating cell function, it is useful to asses the ontogeny of expression of this pathway in oocytes and embryos and to ascertain how disruptions in the UPP may correlate with altered developmental potential. A newly established primate embryo gene expression resource (PREGER) provides a large sample set representing nonhuman primate (rhesus monkey) oocytes and embryos of distinct developmental stages and diverse developmental competence and is an ideal tool for rapid, quantitative analysis of gene expression. Using this resource, we report here temporal patterns of UPP gene expression in oocytes and during preimplantation development, striking differences between the rhesus monkey and mouse, and significant differences in UPP gene expression correlating with oocyte and embryo developmental competence.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oocytes and embryos.
The studies undertaken here employed the Primate Embryo Gene Expression Resource (PREGER) (www.preger.org) (53–55). The resource contains a collection of reverse transcribed and polymerase chain reaction (RT-PCR)-amplified cDNA libraries corresponding to >170 samples of rhesus monkey oocytes and preimplantation-stage embryos. The isolation and culture of the oocytes and embryos during the construction of the PREGER sample set have been described in detail (53). Oocytes contained in the PREGER sample set were obtained from monkeys treated with follicle-stimulating hormone (FSH) only, or FSH followed by human chorionic gonadotropin (hCG), and matured either in vitro or in vivo, respectively. The sample collection also contains oocytes obtained without hormonal stimulation (nonstimulated, denoted as NS). Embryos were obtained from these three categories of oocytes, and also by natural conception (morula/blastocysts) as described (53). Between 3 and 13 samples of one to four oocytes or embryos were obtained for each stage. The embryos included in the PREGER sample set were all high quality and healthy in appearance (53). Samples of eight-cell and morula-stage embryos treated with the RNA polymerase II inhibitor {alpha}-amanitin from the pronucleate stage onward in HECM9 culture were included to evaluate transcriptional dependence of mRNA expression. Details concerning the array, diversity, and origin of samples, and the sensitivity and quantitative reliability of the quantitative amplification and dot-blotting method have been described previously (53) and in other references available at our web site (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, the Animal Welfare Act, and its amendments.

The PREGER sample collection was created using a well-established method for reverse transcription (RT) and exponential cDNA amplification that maintains the quantitative representation of the original mRNA population (6, 31). The cells are lysed in a modified RT buffer, followed by oligo(dT) annealing and processing through the RT step. This approach avoids RNA loss normally associated with RNA purification. After amplification, aliquots of each sample library are spotted onto filters by dot blotting as described (quantitative amplification and dot blotting). It should be noted that, 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. Once the dot blots are prepared, they are hybridized to mRNA-specific probes and the hybridization results analyzed.

Complementary DNA probes and hybridization.
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 (53–56). Data were expressed as the mean (± SE) cpm bound value 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 (± SE) was calculated for each stage. Significance of differences was evaluated using the t-test (P < 0.05 considered significant).


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  		Table 1. Primers employed for obtaining cDNA probes

 
Correction for the performance of multiple tests.
Our analysis encompasses the expression of 53 genes across a series of developmental stages and three hormonal stimulation treatments. However, there are not a large number of independent variables. The genes were preselected for involvement in a single biochemical pathway (Fig. 1), 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 do constitute independent variables, and the expression of each gene has been tested in each of the three possible comparisons. Because nonhuman primate oocytes and embryos are experimental samples that are both expensive and difficult to obtain, we have chosen to include all genes whose difference in expression passes the uncorrected marginal statistical significance value of P < 0.05 (see Table 3). However, we have also 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 (3).


Figure 1
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  		Fig. 1. Summary of the ubiquitin-proteasomal pathway. UB, ubiquitin; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; DUBs, deubiquitinating enzymes.

 
Murine gene expression data.
Expression data for murine homologs were extracted from the microarray data deposited in the Gene Expression Omnibus repository by Zeng et al. (50), which were obtained originally using the Affymetrix MOE 430A and 430B chips. The stages represented were germinal vesicle (GV) stage oocytes and embryos at the one-cell, two-cell, eight-cell, and blastocyst stages. These data were expressed as the mean (± SE) Affymetrix array hybridization signal.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We examined the patterns of expression of 53 genes involved in the UPP. The functional categories of these genes included UB and ubiquitin-like proteins (UBL), E1, E2, E3, proteasome proteins, and deubiquitinating enzymes, and molecular chaperones (Table 2 and Fig. 1). Of the 53 genes examined, we detected the expression of 47 in our sample set of rhesus monkey oocytes and embryos.


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  		Table 2. Descriptions of ubiquitin proteasome pathway protein functions

 
Expression of mRNAs encoding UB and UBL proteins.
The expression of 10 mRNAs encoding UB and UBL proteins was examined in the rhesus monkey. Nine of these mRNAs were detected in oocytes and embryos, with the UBD mRNA being undetected (Fig. 2). At the GV stage, UBB yielded the strongest hybridization signal indicating its mRNA was likely the most abundant of the group, followed in order by UBA80, UBC, SENP8, UBL7, NEDD8, and finally SUMO1, UBA52, and UBAP1 showing the weakest signals. Interestingly, most of these mRNAs significantly decreased in abundance during maturation from GV-stage oocyte to the metaphase II (MII) stage. Thereafter, most of these mRNAs were expressed throughout cleavage and blastocyst formation, with some (NEDD8, SUMO1, UBA80, UBA52) increasing in abundance, and others either diminishing in abundance (UBL7) or simply remaining at low abundance (SENP8). Only the UBA80 mRNA displayed {alpha}-amanitin sensitive expression at the 8- to 16-cell stage (P < 0.05), indicating that the maternal supply of these mRNAs persists well into cleavage. At the blastocyst stage, UBA80 yielded the strongest hybridization signals, followed by UBB and UBC as the next most abundant members.


Figure 2
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  		Fig. 2. Temporal expression patterns of mRNAs encoding ubiquitin (UB) and UB-like (UBL) proteins in rhesus monkey and mouse oocytes and embryos. Monkey graphs show the relative levels of expression for GV and MII stage oocytes and pronucleate through hatched blastocyst-stage embryos produced by in vitro fertilization of oocytes from human chorionic gonadotropin (hCG)-stimulated females and then cultured in vitro in HECM9. GV, germinal vesicle stage oocyte; MII, metaphase II stage oocyte; PN, pronucleate 1-cell stage embryo; 2C, 2-cell stage embryo; 8C, 8-cell stage embryo; 8–16C {alpha}Am, 8- to 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 SE is indicated. 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). a–d, P < 0.05, 0.01, 0.001, and 0.0001, respectively. Mouse data are reproduced here from the published microarray dataset produced by Zeng et al. (50). Graphs depict the average fluorescence intensities from 4 hybridized arrays per stage.

 
Similarities in expression patterns were seen between the rhesus monkey and the mouse. The UBB, UBA80, and UBC mRNAs were the most abundant overall throughout development in both species. The UBA52, UBL7, UBA80, and UBC mRNAs displayed qualitatively similar temporal expression profiles, although quantitatively the relative strengths of hybridization signals were not always in accordance between the two species. The SENP8 mRNA displayed the same dramatic decrease in abundance after oocyte maturation in both species; however, this decrease was not recapitulated for all mRNAs (e.g., UBB, NEDD8, and UBL7). The UBAP1 gene appeared to be transiently induced in two-cell mouse embryos but was not transcribed at the equivalent stage (8- to 16-cell stage) in the rhesus monkey corresponding to the time of embryonic genome activation.

Expression of mRNA encoding E1.
E1 catalyzes the initial step in UB-isopeptide bond formation, forming a thioester bond with the carboxy-terminal glycine of UB in an ATP-dependent process. This activated UB is then transferred to the lysine of target proteins via the E2/E3 conjugation cascade. E1 is a critical component for the initiation of in vitro conjugation reactions. In this category only mRNA encoding UBE1 was examined in the rhesus monkey (Fig. 3). The UBE1 mRNA was detectable at a low abundance in oocytes and embryos before the morula stage, was not {alpha}-amanitin sensitive at the 8- to 16-cell stage, but displayed increased expression during subsequent development to the blastocyst stage. The mouse embryo displayed a somewhat different temporal profile, with maximum expression in the one-cell embryo, and abundance decreasing with development to the 8-cell stage.


Figure 3
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  		Fig. 3. Temporal expression patterns of mRNA encoding E1. Data are presented as in Fig. 2.

 
Expression of mRNAs encoding E2.
The next step in the ubiquitination pathway is the transfer of the activated UB from E1 to the active site of E2 by another thioester bond. We detected the expression of mRNAs encoding nine E2s in the rhesus monkey series (Fig. 4). The genes yielding strongest hybridization signals in the GV oocyte were BRCA1, UBC9, UBE2R2, and UBE2A. Two of these mRNAs, UBC9 and UBE2R2, diminished in abundance during oocyte maturation. The BRCA1 and UBE2B mRNAs displayed the most consistent expression during cleavage and blastocyst formation, although neither mRNA appeared to be {alpha}-amanitin sensitive at the 8- to 16-cell stage. The UBC9 mRNA also displayed moderately strong hybridization signals during development, but with greater variability than these other two mRNAs. Several of the E2 family mRNAs were expressed at low abundances (CDC34, UBE2C, UFDIL, UFD2), although UBE2C displayed a transient and variable elevation at the early blastocyst stage. Several mRNAs (UFDIL, UBE2R2, UBE2A) were downregulated during cleavage. The UFD2 mRNA was transiently elevated at the eight-cell stage.


Figure 4
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  		Fig. 4. Temporal expression patterns of mRNAs encoding proteins E2. Data are presented as in Fig. 2.

 
The temporal expression profiles for CDC34, UBC9, UBE2A, and UBE2C mRNAs were quite similar between the rhesus monkey and mouse, including the transient elevation of the UBE2C mRNA just after the time of the major genome activation event. Other mRNAs in this family, however, display differences in their temporal expression profiles. The BRCA1 mRNA seemed to increase slightly over the course of cleavage in the rhesus monkey, whereas its expression diminished during cleavage in the mouse. Whereas the UBE2R2, UFDIL, and UFD2 mRNAs declined in abundance during development to the blastocyst stage in the rhesus monkey embryo, their abundances tended to increase in the developing mouse embryo. The BRCA1, UBC9, and UBE2A mRNA appeared to be among the most abundantly expressed members, overall, in both species; however, the two species differed in the relative abundances of the CDC34, UBE2B, UBE2R2, and UFDIL mRNAs.

Expression of mRNAs encoding E3.
The E3 ligases are responsible for substrate selection and the transfer of the ubiquitin to a lysine residue of the substrate proteins targeted for degradation, as well as UB chain elongation (48). The family of putative E3 ligases is vast. We detected the expression of 17 representative members of each of the four classes of E3 ligases in the rhesus monkey series: HECT-type (SMAD2, SMURF2), RING-type (ANAPC1, ANAP2, ANAPC3, ANAPC5, ANAPC10, ANAPC13, BRACA1, CUL1, CUL2, CUL3, CUL4, CUL5, RBX1, SKP1A, SKP2), PHD-type, and U-box containing (STUB1) (Fig. 5). Some of these mRNAs display enhanced expression as maternal transcripts (ANAPC1, ANAPC10, CUL1, SMAD2), and others display enhanced expression in the embryo (ANAPC2, ANAPC5, BTRC, CUL2, CUL4, ITCH3, SKP1A, SKP2, STUB1). Of the 17 mRNAs examined, the strongest hybridization signals at the GV-stage oocyte were obtained for ANAPC1, ANAPC10, SKP1A, RBX1, FBXW2, and SMAD3. With respect to regulation of expression during oocyte maturation, some of the 17 mRNAs diminished during oocyte maturation (CUL1, CUL2, CUL5, RBX1, SKP1A, SMAD3), whereas others displayed apparent increase in abundance (ANAPC10, ITCH3, SKP2, SMAD2), most likely reflecting polyadenylation and recruitment for translation [note: polyadenylation can result in enhanced efficiency of oligo (dT) priming during reverse transcription, yielding greater apparent abundance; (40)]. Two of the mRNAs were essentially eliminated from the embryo soon after oocyte maturation and fertilization (CUL2, SMAD2). Only the ANAPC10, CUL2, and CUL4 mRNAs displayed {alpha}-amanitin sensitive expression at the 8- to 16-cell stage, indicating that the majority of these mRNAs persist as maternal transcripts well into cleavage.


Figure 5
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  		Fig. 5. Temporal expression patterns of mRNAs encoding E3. Data are presented as in Fig. 2.

 
The ANAPC1, ANPC15, BTRC, CUL1, ITCH3, RBX1, SKP2, and STUB1 mRNAs displayed qualitatively similar expression profiles between the rhesus monkey and mouse. Somewhat contrasting expression profiles were observed for ANAPC10, CUL2, CUL5, and SKP1A mRNAs. The ANAPC1, CUL4, and ITCH3 mRNAs yielded some of the strongest hybridization signals in the rhesus monkey, but relatively weaker signals compared with other genes in the mouse. Similarly, The Cul1, Cul5, Rbx1, Skp1a, and Skp2 mRNAs appeared to be comparatively well expressed in the mouse but more weakly expressed in the rhesus monkey relative to other transcripts.

Expression of mRNAs encoding proteasome proteins.
The ubiquitinated proteins are passed to the proteasome, where they are degraded. We detected the expression of five mRNAs encoding components of the proteasome in the rhesus monkey (Fig. 6). All of these mRNAs displayed an increased expression as preimplantation development progressed, although the mRNA for ATAD1 yielded a generally low hybridization signal only slightly above background. In the mouse, by contrast, the Atad1 and Atad2 mRNAs appeared to be expressed at similar abundances initially, and the Atad2 mRNA decreased markedly in abundance during development.


Figure 6
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  		Fig. 6. Temporal expression patterns of mRNAs encoding proteins related to the proteasome. Data are presented as in Fig. 2.

 
Expression of mRNAs encoding deubiquitinating enzymes.
Deubiquitinating enzymes help to regulate the overall process of proteolysis via the UPP. These are UB-specific peptidases that has important regulatory role at the level of protein turnover by preventing degradation of proteins through the removal of conjugated UB. Of the five genes in this category examined, four mRNAs were detected in the rhesus monkey (Fig. 7). The strongest hybridization signal was obtained for USP2, which was expressed as a maternal transcript most likely polyadenylated at the one- to eight-cell stages, and USP10, also expressed as a maternal transcript that declined in abundance during development to the eight- to 16-cell stage. Surprisingly, none of these mRNAs displayed strong hybridization signals from the morula through expanded blastocyst stages. A similar pattern was recapitulated for the mouse, with a dramatic downregulation for three of the four genes, and only the Usp10 mRNA being expressed at later times.


Figure 7
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  		Fig. 7. Temporal expression patterns of mRNAs encoding DUBs. Data are presented as in Fig. 2.

 
Expression of mRNAs encoding molecular chaperones.
We examined the expression of two molecular chaperones that function with the UPP, HSPA8 (formerly HSC70), and HSP90AA1 (Fig. 8). The HSP90AA1 mRNA was abundantly expressed in the rhesus monkey GV-stage oocytes and then downregulated at oocyte maturation. The HSPA8 mRNA was expressed at a low abundance in rhesus monkey oocytes and early embryos. The HSPA8 gene was transcriptionally activated at the eight-cell stage, and the HSP90AA1 gene was activated at the morula stage.


Figure 8
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  		Fig. 8. Temporal expression patterns of mRNAs encoding ancillary proteins/molecular chaperones related to ubiquitin pathway. Data are presented as in Fig. 2.

 
Effects of oocyte quality on gene expression.
Given the importance of the UPP pathway to the overall health and physiology of the cell, we next determined whether the expression of UPP genes varied according to developmental competence. The PREGER sample collection contains oocytes obtained through three different hormonal stimulation protocols, which yield embryos of different developmental competence, ranging from low (NS, no hormonal stimulation, fully grown oocytes from small antral follicles, matured in vitro), intermediate (FSH, FSH stimulation only, in vitro maturation), and high (HCG, FSH, and hCG stimulation, in vivo maturation), along with embryos derived from these three categories of oocytes by in vitro fertilization and embryo culture (53). Thus, the PREGER collection is ideally suited to determine how gene expression is affected by protocols yielding oocytes and embryos of different developmental competences.

We observed statistically significant differences (P < 0.05) in gene expression amongst these three categories of oocytes and embryos for 14 of the 47 (30%) mRNAs for which expression was detected (Table 3). The stage at which the greatest effect was seen was the MII oocyte. At this stage, the overwhelming effect (9 of the 14 affected genes) was increased expression in oocytes from FSH and/or NS cycles, and seven of these mRNAs were altered in both FSH and NS oocytes relative to hCG oocytes. We observed decreased expression of three mRNAs in MII stage oocytes from either FSH or NS protocols relative to the hCG protocol. The BRCA1 mRNA was notably elevated in NS embryos at both the PN and two-cell stage, but not at the MII oocyte stage, indicating a different pattern of regulation from the other affected mRNAs, possibly at the level of relative mRNA stability. Four of these mRNAs displayed elevated abundances in pronucleate stage embryos from NS cycles relative to those from hCG cycles, and these were not altered in embryos from FSH cycles. Four other mRNAs were reduced in pronucleate stage embryos from FSH cycles relative to those from hCG cycles. At the two-cell stage, we observed elevated expression in five of the mRNAs in embryos from NS cycles compared with hCG cycles and reduced expression of three other mRNAs. Of the five mRNAs that were elevated in two-cell NS embryos, one remained expressed near background. At the more stringent significance level (P < 0.016, used to address possible effects of multiple testing, see MATERIALS AND METHODS), four genes displayed differences amongst the three classes, with all four differing at the MII stage. Two displayed reduced expression in FSH oocytes relative to hCG oocytes, and two displayed increased expression in NS oocytes. One gene (BRCA1) displayed increased expression in NS pronucleate and two-cell stage embryos. When the ratios of FSH/hCG and NS/hCG (Fig. 9) are compared, it is clear that for the four genes showing high expression and overexpressed in NS two-cell embryos, the FSH embryos are quite similar to hCG embryos. Moreover, much less difference was seen comparing two-cell embryos from FSH and hCG cycles for all of the mRNAs, with only two mRNAs displaying reduced expression and one displaying increased expression.


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  		Table 3. Effect of hormonal stimulation protocol on gene expression in oocytes and embryos

 

Figure 9
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  		Fig. 9. Average ratios of expression of UBL7, UBA80 RBX1, and BRCA1 mRNAs comparing oocytes/embryos from nonstimulated females to follicle-stimulating hormone (FSH)+hCG-treated females ({circ}) or oocyte/embryos from FSH-treated females to FSH+hCG-treated females (•). The ratios were calculated for each of the 4 genes and at each of the stages indicated, and the averages of the 4 ratios were plotted for each stage (± SE).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The UPP serves essential functions for the survival and normal physiology of cells of all types. The UPP may have a unique role to play in the early embryo, by supporting the regulated, stage-specific elimination of oocyte-specific proteins as the embryonic pattern of gene expression becomes established. This need must be balanced, however, with a need for the embryo to sustain its cellular functions, while relying upon a reservoir of maternal proteins and mRNAs before transcriptional activation of the embryonic genome. Thus, the correct regulation of proteolytic processes in the embryo would be expected to be a key for maintaining maximum developmental viability. Environmental or physiological factors that damage cellular components or interfere with this regulation would thus seriously compromise cellular functions.

We show here that, indeed, nearly one-third of the UPP mRNAs analyzed display alterations in apparent abundance that correlates with reduced embryo viability. For most of the affected genes (7 of 9 showing increased expression P < 0.05), expression is elevated in both FSH and NS oocytes compared with hCG oocytes. We previously reported that many maternal transcripts display elevated hybridization signals at the MII oocyte stage, followed by greatly reduced signals at the two-cell stage, consistent with precocious polyadenylation and translational recruitment, followed by accelerated degradation and elimination (55). We did not observe the identical pattern for most of the genes assayed here. Only two of the mRNAs that yielded elevated signals in NS MII-stage oocytes yielded reduced signals in NS two-cell embryos. This indicates that the UPP-related mRNAs may be less susceptible to the same disruptive process that leads to the early destruction of many mRNAs. We observe instead that five of the mRNAs are elevated in abundance in NS two cell-stage embryos. The disruptions in expression in two-cell FSH embryos were much less severe than that seen for NS embryos. A similar difference in the expression of maternal mRNAs between FSH and NS embryos was seen previously (55). This difference between FSH and NS embryos correlates with the greater developmental competence seen for FSH oocytes compared with NS oocytes. Thus, it does not appear that in vitro maturation alone substantially impairs the expression of most of the genes involved in the UPP.

It is interesting that far greater alterations in UPP gene expression correlating with developmental potential are observed at the MII oocyte stage rather than in cleaving embryos. This suggests that the differences in developmental potential may result from the degradation of proteins in the oocyte rather than during cleavage. As development proceeds, the UPP mRNA abundances may become closer to normal in NS and FSH embryos, but this may be inadequate to restore the maternal proteins that have been eliminated from the oocyte. This would indicate that, just as the process of correct temporal recruitment of maternal mRNAs for translation appears crucial for maximum viability, so too is the correct regulation of maternal protein degradation. Detailed studies of oocyte protein content for oocytes obtained through the different protocols may thus reveal specific protein constituents that confer high developmental potential.

We previously reported significant differences in arrays and patterns of gene expression between the rhesus monkey and mouse, for a housekeeping gene (HPRT) (53), DNA methyltransferase genes (47), and WNT genes (56). The comparisons here reveal many additional differences between these two species, with respect to temporal patterns of expression and relative levels of expression. Among the UB/UBL class, the UBB, NEDD8, UBAP1, and UBL7 mRNAs diverged in their temporal profiles, and UBAP1 in particular displayed a stage-specific transient induction only in the mouse. The E1 enzyme UBE1 displayed somewhat opposing temporal profiles for the two species. The E2 class BRCA1, UBE2R2, UFDIL, and UFD mRNAs displayed opposing temporal profiles, and the CDC34, UBE2B, UBE2R2, and UFDIL mRNAs displayed very different relative abundances between the species. Many similar differences were seen for the E3 category. The ATAD1 mRNA encoding a proteasome component and the USP10 mRNA encoding a deubiquitinating enzyme were also expressed quite differently between the two species. Thus, many differences were observed between species affecting all of the categories of genes examined here. This suggests that proteolysis may be regulated differently between the two species. Moreover, this observation further highlights the value of studying a nonhuman primate embryo model to understand better gene regulation and the cell biology of human embryos.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Centers for Research Resources Grant RR-15253.


   ACKNOWLEDGMENTS
 
We thank Bela Patel, Malgorzata McMenamin, and Ann Marie Paprocki for technical assistance. We also thank R. Dee Schramm for his contribution to the development of the PREGER resource. We thank Prof. Carmen Sapienza for comments on the manuscript and advice related to statistical analysis.


   FOOTNOTES
 
Address for reprint requests and other correspondence: K. E. Latham, 3307 No. Broad St., Philadelphia, PA 19140 (e-mail: klatham{at}temple.edu)

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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