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Abstract
BackgroundBovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classify them by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in terms of healthy and atretic conditions by investigating mRNA localization of identified genes.MethodsGlobal gene expression profiles of F1 (10.7 +/- 0.7 mm) and F2 (7.8 +/- 0.2 mm) were analyzed by hierarchical cluster analysis and expression profiles of 16 representative genes were confirmed by QPCR analysis. In addition, localization of six identified transcripts was investigated in healthy and atretic follicles using in situ hybridization. The healthy or atretic condition of examined follicles was classified by progesterone and estradiol concentrations in follicular fluid.ResultsHierarchical cluster analysis of microarray data classified the follicles into two clusters. Cluster A was composed of only F2 and was characterized by high expression of 31 genes including IGFBP5, whereas cluster B contained only F1 and predominantly expressed 45 genes including CYP19 and FSHR. QPCR analysis confirmed AMH, CYP19, FSHR, GPX3, PlGF, PLA2G1B, SCD and TRB2 were greater in F1 than F2, while CCL2, GADD45A, IGFBP5, PLAUR, SELP, SPP1, TIMP1 and TSP2 were greater in F2 than in F1. In situ hybridization showed that AMH and CYP19 were detected in granulosa cells (GC) of healthy as well as atretic follicles. PlGF was localized in GC and in the theca layer (TL) of healthy follicles. IGFBP5 was detected in both GC and TL of atretic follicles. GADD45A and TSP2 were localized in both GC and TL of atretic follicles, whereas healthy follicles expressed them only in GC.ConclusionWe demonstrated that global gene expression profiling of F1 and F2 clearly reflected a difference in their follicular status. Expression of stage-specific genes in follicles may be closely associated with their growth or atresia. Several genes identified in this study will provide intriguing candidates for the determination of follicular growth.
Highlights
Bovine follicular development is regulated by numerous molecular mechanisms and biological pathways
It is well documented that increased expression of luteinizing hormone (LH) receptor (LHR) in granulosa cells (GC) and specific changes of intrafollicular factors such as the insulin-like growth factor (IGF) and inhibin-activin-follistatin systems play a critical role in E2 production in the dominant follicle (DF) [6,7]
Messenger RNA expression for chemokine ligand 2 (CCL2), GADD45A, IGF binding protein 5 (IGFBP5), plasminogen activator urokinase receptor (PLAUR), secreted phosphoprotein 1 (SPP1), selectin P (SELP), tissue inhibitor of matrix metalloprotease-1 (TIMP1) and thrombospondin 2 (TSP2) was greater in the F2 than in the F1 (P < 0.05)
Summary
Bovine follicular development is regulated by numerous molecular mechanisms and biological pathways. Some studies compared the gene expression profiles between DF and SF around the time of follicular selection. They showed that DF up-regulates genes regulating E2 synthesis, anti-apoptosis, cell proliferation and gene transcription. Recent studies found that 93 mostly novel genes were differently expressed in the GC of newly selected DF compared with SF and/or growing cohort follicles whereas most of these genes were down-regulated in the GC of preovulatory follicles during final maturation before the LH surge [15,17]. Ndiaye et al identified a subset of novel genes down-regulated in preovulatory follicles after human chorionic gonadotropin (hCG) stimulation compared with DF, which may contribute to ovulation and luteinization [11]
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