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. 2010 Feb 5:8:11.
doi: 10.1186/1477-7827-8-11.

Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles

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Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles

Ken-Go Hayashi et al. Reprod Biol Endocrinol. .

Abstract

Background: Bovine 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.

Methods: Global 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.

Results: Hierarchical 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.

Conclusion: We 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.

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Figures

Figure 1
Figure 1
Hierarchical cluster analysis of 76 differentially expressed genes in largest (F1) and second-largest follicles (F2). These genes were enhanced between at least two follicles by more than 20-fold. Red scale indicates relative higher expression level and green scale indicates relative lower expression level. The expression levels were transformed to log2 values. Dendrograms of sample axis (above matrix) and gene axis (to the left of matrix) represent overall similarities in gene expression profiles. Five follicles were classified into two major clusters (A and B). The follicles divided into cluster A were all F2 and the follicles divided into cluster B were all F1. The cluster A was characterized by highly expression of 31 genes, whereas the cluster B was predominately expressed 45 genes.
Figure 2
Figure 2
QPCR analysis of representative eight genes (CCL2, GADD45A, IGFBP5, PLAUR, SELP, SPP1, TIMP1 and TSP2) in F1 and F2. These genes were highly expressed in F2 (cluster A) compared with F1 (cluster B) in microarray analysis. The expression of mRNA was normalized to the expression of GAPDH measured in the same RNA preparation. The black bar and the white bar indicate the F1 and the F2, respectively. Data are shown as the mean ± SEM. Different letters denote significant differences (P < 0.05).
Figure 3
Figure 3
QPCR analysis of representative eight genes (AMH, CYP19, FSHR, GPX3, PlGF, PLA2G1B, SCD and TRB2) in F1 and F2. These genes were highly expressed in F1 (cluster B) compared with F2 (cluster A) in microarray analysis. The expression of mRNA was normalized to the expression of GAPDH measured in the same RNA preparation. The black bar and the white bar indicate the F1 and the F2, respectively. Data are shown as the mean ± SEM. Different letters denote significant differences (P < 0.05).
Figure 4
Figure 4
Localization of GADD45A, IGFBP5 and TSP2 mRNA in healthy and atretic follicles. These genes were expressed more in F2 than in F1 in QPCR analysis. (A, C, E, G, I and K) DIG-labeled anti-sense cRNA probes were used. (B, D, F, H, J and L) DIG-labeled sense cRNA probes were used. Seven-micrometer sections of bovine follicles were hybridized with each probe. GADD45A (A, B, C and D) and TSP2 (I, J, K and L) mRNA were found in both granulosa cells (GC) and theca layer (TL) of atretic follicle, whereas it was localized in only GC of healthy follicle. IGFBP5 mRNA (E, F, G and H) was localized in GC and TL of atretic follicle but not found in healthy follicle. Scale bar = 20 μm.
Figure 5
Figure 5
Localization of AMH, CYP19 and PlGF mRNA in healthy and atretic follicles. These genes were expressed more in F1 than in F2 in QPCR analysis. (A, C, E, G, I and K) DIG-labeled anti-sense cRNA probes were used. (B, D, F, H, J and L) DIG-labeled sense cRNA probes were used. Seven-micrometer sections of bovine follicles were hybridized with each probe. AMH (A, B, C and D) and CYP19 (E, F, G and H) mRNA was localized in granulosa cells (GC) of healthy as well as atretic follicles. PlGF mRNA (I, J, K and L) was found in GC and theca layer of healthy follicle but not atretic follicle. Scale bar = 20 μm.

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