Isolation of genes associated with developmental competency of bovine oocytes

Theriogenology 68S (2007) S84–S90 www.theriojournal.com

Isolation of genes associated with developmental competency of bovine oocytes P.L. Pfeffer *, B. Sisco, M. Donnison, J. Somers, C. Smith AgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand

Abstract Eggs differ widely in their ability to develop into an embryo. To address this characteristic, the concept of developmental competency has been coined, defined as the ability or potential of an oocyte to undergo maturation, fertilization and development to blastocyst stages or live offspring. Developmental competency is acquired progressively during folliculogenesis and is linked to follicular size. In an effort to understand the molecular changes underlying differences in competency we compared oocytes derived from large follicles (5 mm) to those from small follicles (2 mm). We used an approach combining suppressive subtraction hybridization with a linear amplification step to identify genes upregulated in the more competent oocytes. Real-time RT-PCR quantification indicated highly significant upregulation for 10 genes. However, the observed changes did not exceed three-fold suggesting that the molecular causes for poor developmental capacity may be reliant on many small changes. In monovulatory species oocyte developmental competency is further modulated in a process termed follicular dominancy, whereby only one of a cohort of developing ovarian follicles continues to grow. In our second approach, we aimed to identify genes that may be involved in the choice of one follicle as becoming dominant and thus restricting the developmental competency to a single oocyte. This approach, focusing on granulosa cells, yielded a small set of five genes that could be verified to be reliable markers for dominant follicles. We have further analyzed one of these involving the activin/inhibin pathway. Lastly, in a third approach we are investigating the feasibility of using nuclear transfer (NT) to interrogate oocyte developmental competency. # 2007 Elsevier Inc. All rights reserved. Keywords: Oocyte; Developmental competency; Follicle; Dominancy; Nuclear transfer; Bovine

1. Introduction Within mammalian species, individual eggs differ widely in their ability to develop into an embryo. To address this characteristic, the concept of developmental competency has been coined, defined as the ability or potential of an oocyte to undergo maturation, fertilization and development to blastocyst stages or live offspring [1]. For human assisted reproduction technol-

* Corresponding author. Tel.: +64 7 838 5002; fax: +64 7 838 5628. E-mail address: [email protected] (P.L. Pfeffer). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.03.016

ogy, livestock production and scientific research purposes it would be invaluable to be able to predict the developmental competency of an oocyte, ideally in a non-invasive or at least in a non-destructive manner. Initial studies have highlighted that developmental competency is acquired progressively during folliculogenesis in oocyte populations [2]. Thus oocytes from preantral follicles are unable to resume meiosis after arresting in prophase I, whereas those from very small antral follicles (in the bovine <0.9 mm) are competent to progress to metaphase I, and those from larger antral follicles to metaphase II and beyond [3–7]. However, morphologically indistinguishable oocytes from larger

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antral follicles which are competent to complete nuclear maturation (meiosis) and fertilization show differences in their ability to develop to the blastocyst stage [3,5–9]. There are three main factors that could explain cytologically non-apparent differences in oocyte developmental potential: (1) DNA damage (including mitochondrial DNA [10]); (2) the epigenetic make-up of the oocyte is defective (e.g. incorrect imprinting); (3) the oocyte has not accumulated the correct (threshold) levels of cytoplasmic factors necessary to drive development until embryonic gene activation sets in (incomplete/aberrant ‘‘cytoplasmic maturation’’). Though methods exist to detect gross chromosomal DNA abnormalities, subtler genetic defects or epigenetic errors could not be detected on a more global scale in tiny samples until very recently [11,12]. Therefore most attention has been focused on cytoplasmic factors. Whereas the oocyte stores both cytoplasmic protein and transcripts, limited material has precluded analyses of the proteome. Fortunately RNA transcripts can be amplified enzymatically by PCR or linear amplification which have made the transcriptome (global gene expression profile) of the oocyte the most amenable arena to detect markers that predict developmental competency. We describe here three different approaches we have used for determining the factors that are associated with developmental competency in the cow. The most direct approach involves a subtractive hybridization methodology combined with an amplification step using bovine oocytes derived from follicles of different size. In a second approach that is restricted to monoovulatory species, we have compared gene expression in granulosa cells of dominant (greater viability) and subordinate follicles. Granulosa cells and oocytes interact reciprocally. Granulosa cells, which can be easily obtained, thus present an interesting non-invasive avenue for evaluating the viability of the oocyte. Lastly we have explored the possibility of using somatic cell nuclear transfer as a technique to probe the developmental potential of the host oocyte. 2. Direct isolation of gene transcripts enriched in developmentally competent oocytes A clear correlation has emerged between follicular size and developmental potential in cattle [3,5,6,9,13– 15]. The conclusion from these studies is that oocytes from follicles with a diameter smaller than 2 or 3 mm develop in vitro to the blastocyst stage at much lower rates than do those from follicles greater than 4 or 5 mm. From the 3 mm-follicle stage until embryonic

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genome activation at the eight-cell stage, oocytes/ embryos have been shown by 3H-uridine incorporation to be transcriptionally practically quiescent [3,16,17]. Thus if maternal RNA stores contribute to developmental competency, a comparison of oocyte transcripts from small versus large follicles should highlight those genes that are accumulated during, and potentially critical for, the achievement of competency. We and others have exploited these observations to isolate such genes [18–20]. In essence, two classes of methods exist. One is based on subtractive procedures (subtractive suppression hybridization (SSH) and differential display) where RNA from the two populations are mixed and those fragments that do not hybridize to each other (and thus are unique to one population) are enriched and isolated. The other class involves measuring the abundance of individual RNA transcripts separately in each sample (Northern blotting, RNAase protection, RT-PCR, SAGE, Microarrays). With the increasing availability of more complete livestock genome sequence information and cDNA arrays and standardization of microarray protocols, microarrays are becoming the preferred choice for global expression analysis. However, in two situations subtractive procedures may be preferable to microarrays. Firstly, for genomes that have not been adequately sequenced and secondly, for the identification of genes expressed at low abundance as these do not yield sufficiently strong signals on microarrays. The SSH technique incorporates a normalization based on the suppression PCR effect. Thus, less abundant messages are enriched up to 1000-fold [21]. We developed a method [19] combining linear amplification with SSH thereby avoiding the bias introduced when amplifying small amounts of starting material using PCR based methods [22]. RNA of oocytes from 1 to 2 mm follicles was subtracted from that derived from more competent follicles of at least 5 mm diameter. The success of this method was verified by the observation that 90% of the isolated clones were at least 30% more abundant in three batches of developmentally competent oocytes, with one novel gene showing a >200% enrichment. Furthermore, by measuring with real-time RT-PCR the absolute copy number per oocyte for these genes we could show that this procedure does not select only abundant messages. We isolated genes expressed at levels ranging from hundreds to tens of thousands of copies per oocyte [19]. The set of 10 maternally expressed genes identified in our study as being enriched in competent oocytes included genes coding for transcription factors, namely the homeodomain containing Oct4 and Msx1 and Zinc-

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Table 1 Difference in transcript number in oocytes derived from more competent (5 mm) relative to less competent (1–2 mm) follicles [19] Gene

Fold increase

Significance

1166 Cyclin A Dja4 GDF9 Msx1 NDFIP1 Oct4 SLBP Trappc Znf198

3.4 2.0 2.3 2.2 2.1 1.7 1.2 1.7 1.7 1.5

<0.01 <0.01 <0.01 <0.01 <0.01 <0.05 <0.05 <0.01 <0.01 <0.01

finger domain Znf198 proteins, the histone stem-loop binding protein SLBP, Cyclin A, the heat-shock protein 40/DNA-J like (Dja4), NEDD4-interacting protein 1 (NDFIP1), trafficking protein particle complex 3 (Trappc), the TGFb superfamily member GDF9 and unknown clone 1166 which appears to be the trailer for a Ras-related GTP-binding C transcript. The enrichment is shown in Table 1. The preponderance for genes involved in transcription and RNA stability (Oct4, Msx1, Znf198 and SLBP) highlights the importance of mRNA synthesis and turnover in oocyte competency. The isolation of a gene involved in cell cycle control (Cyclin A) is not unexpected considering that the oocyte needs to contain sufficient quantities of transcripts coding for cell cycle proteins to allow progression to embryonic gene activation which occurs only three cell divisions later. Interestingly, in the two other reported studies aimed at isolating oocyte competence factors, several additional cell cycle regulator genes were identified [18,20]. In the more recent of the two studies, two of the cell cycle regulator genes (PTTG1 and CCNB2) found to be associated with increased developmental competency based on follicular size were also significantly upregulated in earlier cleaving embryos [18]. Such earlier cleaving embryos have been demonstrated to be of higher developmental competence [23]. Our observations of higher levels of mRNA stores for GDF9 in competent oocytes are well in line with a recent report that has also identified this growth factor as being important for competency [24]. What is evident from the work of several groups [15,18,19] is that the changes in expression levels between competent and less competent oocytes seldom exceeds two-fold. Bearing in mind that the identified genes represent only a fraction of all genes expressed in the oocyte, this suggests that developmental competency may be a quantitative trait, being dependent on small changes in the RNA levels of many genes. It will

be interesting to see whether future work based on comprehensive microarrays covering the complete transcriptome will detect critical markers which show dramatic differences or whether it will provide further evidence that developmental competency is a subtle quantitative trait that is evident only upon global transcriptional analysis. 3. Genes associated with follicular dominance In monovular species such as cattle, a transient rise in systemic FSH nearly coincident with the time of ovulation (1 day after estrus) recruits a cohort of antral ovarian follicles smaller than 4 mm to grow [25,26]. Once the FSH levels decline, follicles continue to grow but within 48 h of the FSH peak, selection has occurred. One follicle of about 8–9 mm and generally slightly larger than the next-biggest follicles, has been chosen as the dominant follicle (DF) and it alone continues to grow [27,28]. In contrast, the rest of the follicles, termed subordinate follicles (SF), will undergo programmed cell death as the FSH levels decrease below a critical threshold. A complex relationship exists between follicular dominance and the developmental competency of the oocyte derived from dominant and subordinate follicles [13,29,30] suggesting that the DF has a repressive effect on subordinate oocyte competence. This repressive effect is masked somewhat by the beneficial effect of (DF-induced) mild atresia on oocyte competence [9]. The elucidation of the genes involved in establishing and maintaining follicular dominancy is thus likely to yield insight into what pathways are involved in the maintenance or suppression of oocyte competency. Considering that oocytes have passed into a transcriptionally largely quiescent stage at the time of follicular dominance, we focused our attention on granulosa cells which are known to influence and be influenced by the oocyte until ovulation [31]. We used suppressive subtraction hybridization to compare genes expressed in dominant and subordinate bovine follicles [32]. Inhibin-bA (INHBA), apolipoproteinE receptor2 (apoER2, LRP8), MAP kinase kinase kinase5 (MAP3K5/ask1) and carboxypeptidaseD (CPD) were isolated and verified to be reliable markers for dominant follicles using real-time RT-PCR. Before the time point where dominant follicles can be distinguished by virtue of their deviation in size and growth rate, transcripts for INHBA, ApoER2 and p450 aromatase (CYP19) were elevated specifically in the one to three largest follicles (Fig. 1) [32]. At Day 2.5 post-ovulation, near the time of DF selection, the mRNA expression profiles of

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Fig. 1. Genes expressed at significantly higher levels in granulosa cells of dominant follicles. Filled-in circles represent dominant follicles or, at Day 1.5, the largest follicles, one of which will be selected to become the future DF. Genes expressed at higher levels in dominant follicles are indicated.

MAP3K5 and CPD paralleled that of the other three genes, thus anticipating the clear molecular expression differences seen between the DF and the next largest follicle a day later. INHBA codes for the inhibin-bA peptide which can homodimerize to form activin A or heterodimerize with inhibin-bB or inhibin-a to generate activin AB or inhibin A, respectively. As inhibin A and activin A have opposite biological effects, the significance of an increase in the shared INHBA subunit mRNA is difficult to interpret without knowing the levels of the other two subunits. Similarly, expression of the activin antagonist follistatin (FST) has to be considered. We could show (B.S. and P.L.P.; unpublished data) that Inhibin-a (INHA) as well as FST are transcribed at higher levels in dominant follicles from Day 3.5 post-ovulation (Fig. 1). The third inhibin gene, Inhibin-bB, could only be detected at very low levels from Day 7 post-ovulation and thus is unlikely to play a prominent role. Overall we noted a 10-fold decrease in ‘‘activin tone’’ as measured by the ratio of INHBA to INHA plus FST transcripts in dominant but not subordinate follicles between 1.5 and 7 days post-ovulation (r = 0.93; P = 0.07). A decrease in activin tone with increasing follicular diameter has been recently observed in follicular fluid peptide measurements [33]. How would the higher expression levels of the inhibin genes in dominant follicles and decrease in activin tone over follicular development specifically in DF granulosa cells affect developmental competency of the oocyte? In human oocytes, mRNA for activin receptors was readily detected, opening up the possibility for paracrine signaling of activin from granulosa cells [34]. Notably, moderate granulosa

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expression of INHA was associated with normal oocyte and follicle maturation, whereas excessive INHA was associated with poor embryo quality as measured by grading based on degree of blastomere fragmentation and sizing [35]. The connection between dominance, activin signaling and oocyte competence will present a fascinating area for future research. Reassuringly, INHBA, ApoER2 and CPD expression were also found to be upregulated in dominant follicles in an independent screen comparing dominant with subordinate follicles [36]. These and more recent screens are highlighting an expanding set of genes upregulated either in the DF or SF, providing novel avenues for establishing links between follicular dominancy and oocyte developmental competency [32,36–39]. 4. Nuclear transfer and oocyte competency In a third approach we investigated the feasibility of using nuclear transfer (NT) to interrogate developmental competency. During NT (or ‘cloning’), fertilization can be completely bypassed and live offspring obtained by replacing the maternal (oocyte) genome with a diploid genome derived from a host cell (for reviews, see [40,41]). The fact that this procedure is possible means that cytoplasmic factors stored in the matured oocyte are able to reprogram the transcriptional status of the donor cell genome. Thus the efficiency of reprogramming, as measured for example by gene transcription at the blastocyst stage, could in principle be used to assay aspects of the developmental competency of the host oocyte [42–44]. We asked whether the NT treatment introduces more variability in gene expression than is seen for IVP embryos. To this end we used the same pool of slaughterhouse derived oocytes and minimized genetic effects by generating control IVP embryos using sperm from the bull whose skin fibroblasts were used for NT. Furthermore, by culturing NT embryos in parallel to IVP embryos under identical (zona free) conditions, effects specific to the NT procedure as opposed to gene expression differences arising due to embryo culture could be addressed. After optimizing our protocol so as to exclude technical variation, we measured by quantitative real-time RT-PCR the expression of eight genes in 16–20 individual embryos of each treatment [45]. We found that the level of embryo to embryo variation for each of the genes did not differ significantly between NT and IVP embryos. Indeed, the variability in expression levels appeared to be intrinsic to genes, with more highly expressed genes generally, but not always, displaying less variation in

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their levels. Furthermore, the total gene expression variability across all eight genes for individual embryos did not differ between NT and IVP embryos. Interestingly, in vivo embryos not only displayed different levels of expression but also showed much lower total gene expression variability than their NT or IVP counterparts suggesting that culture per se (inclusive of zona removal) may have a stronger influence on gene expression than does the nuclear transfer treatment [45]. We next examined gene transcription profiles in individual bovine in vitro produced (IVP) and NT halfsibling embryos on a more global scale by using blastocyst-specific microarrays [46]. These experiments, using four to five individual embryos for each treatment, showed that the NT procedure results in numerous changes in the expression profile, such that about 8% of the approximately 2000 different genes were consistently (P < 0.05) over- or under-expressed, but by only a small amount (between 1.5- and 2.6-fold). When subjected to accurate quantitative RT-PCR analysis in a much larger set of 20 embryos it became apparent that many (six of eight analyzed) of the gene expression changes were no longer significant. Where significant changes of the average expression levels were seen, these were small (<2-fold). Small or non-significant changes have also been observed in careful quantitative studies on various sets of candidate genes [45,47]. How does this relate to oocyte developmental competency? The absence of genes strongly (more than 2.6-fold) and consistently misregulated in NT blastocyst-stage embryos argues for a predominantly stochastic nature of reprogramming errors where expression of many genes may be affected in individual embryos but the set of genes affected differs from embryo to embryo [46]. Together with the aforementioned intrinsic gene expression variability in cultured embryos, this translates to a situation in which we do not yet possess transcriptional markers that are highly sensitive to the nuclear transfer treatment. Yet it is precisely the identification of these genes that is required in order to identify which and understand how ooplasmic factors remodel the donor (or in a more physiological situation, the paternal) genome so as to allow further development. Should such genes come to light, nuclear transfer may yet provide a unique assay for identifying oocyte factors that impinge specifically on post-fertilization competency. 5. Conclusions and future prospects The potential of an oocyte to undergo further development to ultimately give rise to a live offspring is

an issue that has always captured the attention of reproductive biologists yet the definition of such developmental competency in molecular terms has remained enigmatic. Results from several laboratories, including our own, using multiple approaches to detect molecular markers, have indicated potential candidate genes that could fulfill such a role. However, none of these markers are as yet of much practical use for two reasons. First, gene over or under-expression is not particularly dramatic which means that only with very accurate quantitative measurements can misexpression be detected on a background of normal gene-inherent variation in transcript levels. Second, considering the complexity of development and the number of genes required at each developmental step, it is clear that a characteristic such as developmental competency must be a quantitative trait depending on a very large number of loci (genes). As oocyte developmental competence can be destroyed by the loss of single genes, as shown in the mouse [48,49], it will be necessary to identify and be able to monitor most genes involved in competency before accurate predictions as to an oocyte’s potential can be made. Whereas microarrays are making the monitoring part more feasible, the identification of which genes to examine is still in its infancy. Lastly, it is worthwhile bearing in mind that some genes associated with developmental competency may be altered epigenetically but not transcriptionally in incompetent oocytes. Such epigenetic changes may lead to decreased viability at stages well beyond the blastocyst. Hence only with the use of a wide variety of assays and experimental approaches which expose alternate genes associated with developmental competency can we hope to eventually achieve success in the quest to define this elusive entity. Acknowledgements Work from our laboratory was supported by the New Zealand Foundation for Research, Science and Technology and AgResearch. References [1] Duranthon V, Renard JP. The developmental competence of mammalian oocytes: a convenient but biologically fuzzy concept. Theriogenology 2001;55:1277–89. [2] Eppig JJ. Coordination of nuclear and cytoplasmic oocyte maturation in eutherian mammals. Reprod Fertil Dev 1996;8: 485–9. [3] Fair T, Hyttel P, Greve T. Bovine oocyte diameter in relation to maturational competence and transcriptional activity. Mol Reprod Dev 1995;42:437–42.

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