Reproduction in Domestic Animals

Use of Microarray Technology for Reproduction in Cattle 361Global Gene Expression During OvarianFollicle DevelopmentFolliculogenesis starts during foetal life when millions ofprimordial germ cells surrounded by pregranulosa cellsdevelop and form a life-time supply of follicles. At as yetpoorly understood intervals, these follicles develop fromthe resting pool to grow and mature. During thisdevelopment, most follicles undergo atresia and die, witha small proportion developing to the later stages beyond5 mm in diameter and few developing to ovulatory sizeand fewer still going on to ovulate. These later stages offollicle development are the most susceptible to manipulationand have been the focus of many studies over theyears (reviewed in Mihm and Evans 2008).It is clear that antral follicle growth occurs insuccessive waves of development in cattle that are eachcharacterized by the growth of a cohort of follicles thatdevelop in parallel until the growth of one folliclerapidly increases and becomes dominant (this follicle isselected to become dominant) with the other folliclescease growing, begin to regress and undergo atresia(subordinate follicles). The hormonal changes andassociated regulation of growth, selection and atresiaof follicles has been well studied. After selection, theparameters that differentiate the dominant follicle fromsubordinate follicles are larger diameter, enhancedoestradiol production, more LH receptors in granulosacells, and an increase in IGF binding protein (IGFBP)protease activity in follicular fluid, low concentrations ofIGFBPs and increased concentrations of free IGF infollicular fluid (Fortune et al. 2004; Webb and Campbell2007; Mihm and Evans 2008). As with other areas ofbiomedical science, the importance of the expression ofgenes in controlling follicle development in cattle hasbeen studied one gene at a time (the candidate geneapproach) and has been highly revealing [reviewed by(Mihm and Bleach 2003)]. Alternatively, studying theexpression of many genes simultaneously has beenachieved using suppressive subtraction hybridization(Sisco et al. 2003), serial analysis of gene expression(SAGE) (Mihm et al. 2008) or microarrays (see next).Microarrays have been used to study the progressionof ovarian follicle development and have shown changesin gene expression associated with a decrease in FSHdependence and an increase in LH dependence asdominant follicles continue to develop after selection(Mihm et al. 2006). Continued development of thedominant follicle was associated with decreased mRNAexpression for genes known to be induced by FSH ingranulosa cells (FSHR, ESR2, INHA, ACVR1 andCCND2), increased mRNA expression for the LHreceptor (LHCGR) in granulosa cells and alterationsin mRNA expression of numerous genes potentiallyinvolved in survival and apoptosis of granulosa andtheca cells (SIVA, FADD, TIAF1, LASS4 andTNFSF8) (Mihm et al. 2006). In another study, microarrayswere used to characterize the global pattern ofgene expression in aberrant persistent dominant follicles(low fertility) and concluded that alteration of theexpression of genes involved in energy and proteinmetabolism (e.g. phosphofructokinase and fructose-1,6-bisphosphatase), amino acid transport (e.g. amino acidtransporter A2) and apoptosis (e.g. caspase-3, Bcl-2 andBcl-x) in granulosa cells may negatively impact on theirability to support a healthy oocyte and thereby contributeto decreased fertility (Lingenfelter et al. 2008).In a number of experiments, we have used bovinecDNA microarrays to ask what genes in granulosa andtheca cells contribute to selection of the dominant fromsubordinate follicles. We identified 261 genes ⁄ ESTs thatwere differentially expressed between dominant andsubordinate follicles and focused our attentions offamilies (ontologies) of genes. First, we examined genesinvolved in apoptosis and showed that dominant folliclesurvival was associated with genes involved in theinhibition of apoptosis (aromatase, LH receptor, oestradiolreceptor b, DICE-1 tumour suppressor protein andMCL-1) and the regression of subordinate follicles wasassociated with enhanced expression of genes involved inthe apoptotic pathways (Betaglycan, COX1, TNFalpha,caspase-activated DNase, DRAK-2, caspase 13,P58(IPK), Apaf-1, BTG-3 and TS-BCLL) (Evans et al.2004). We also identified 83 genes involved in signaltransduction (largely hormone receptors and intracellularsignalling molecules) that were differentially expressedbetween dominant and subordinate folliclesand after a number of additional experiments hypothesizedimportant roles for CAMkinase1 and EphA4 intheca cells and BCAR1 in granulosa cells for thedevelopment of dominant follicles and for betaglycanand FIBP in granulosa cells of regressing subordinatefollicles (Forde et al. 2008a). The fate of cells is regulatedin part by the transcription of new genes and we alsoexamined the relative expression of genes coding fortranscription factors in follicles during the development.We identified 34 genes that code for transcription factorsin granulosa and theca cells with good evidence tosuggest that genes for CEBP-b, SRF, FKHRL1,NCOR1 and Midnolin may be important in determiningthe fate of follicle cells (Zielak et al. 2007a). In summary,these studies used a microarray approach to identify alarge number of genes which indicates that ovarianfollicle development involves the concerted actions ofgroups of molecules with diverse functions. However, themicroarray approach also lead to the discovery of genesthat have not previously been thought to be involved infollicle development, for example, the prion protein(Forde et al. 2007) and completely novel (some as yetunidentified) genes (Zielak et al. 2007b).Global Gene Expression During OocyteMaturationPrior to embryonic genome activation, embryo developmentin cattle is driven by maternal mRNAs andproteins produced during the oocyte growth phase.Intrinsic oocyte developmental competence, as assessedby development to the blastocyst stage has beenpositively associated with a number of factors, includingfollicular environment (Lonergan et al. 2004) and thesite of oocyte maturation (Rizos et al. 2002). Numerousstudies have been carried out to investigate the affect ofthese factors on the oocyte transcriptome using a widerange of gene expression analysis techniques such asÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag