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Gene expression profiling of preovulatory follicle in the buffalo cow: effects of increased IGF-I concentration on periovulatory events.

Rao JU, Shah KB, Puttaiah J, Rudraiah M - PLoS ONE (2011)

Bottom Line: Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes.The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR).These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.

ABSTRACT
The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

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Classification of differentially expressed genes into various canonical pathways by Ingenuity pathway analysis (IPA).The pathway analysis indicates that a large number of differentially expressed genes belonged to canonical pathways such as PI3K/AKT signaling; IGF-I signaling and coagulation system. The orange line represents score for the likelihood [−log (B–H P<0.05)] that genes belonging to a specific signaling pathway category affected at 1 and 22 h post peak LH surge. The stacked bars indicate the percentage of genes distributed according to regulation i.e. green (down), red (up) and open bars (No overlap with dataset) in each canonical pathway.
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pone-0020754-g003: Classification of differentially expressed genes into various canonical pathways by Ingenuity pathway analysis (IPA).The pathway analysis indicates that a large number of differentially expressed genes belonged to canonical pathways such as PI3K/AKT signaling; IGF-I signaling and coagulation system. The orange line represents score for the likelihood [−log (B–H P<0.05)] that genes belonging to a specific signaling pathway category affected at 1 and 22 h post peak LH surge. The stacked bars indicate the percentage of genes distributed according to regulation i.e. green (down), red (up) and open bars (No overlap with dataset) in each canonical pathway.

Mentions: Ingenuity pathway analysis (IPA) was used to classify the differentially expressed genes into different function and disease categories. Of the 450 and 111 differentially expressed genes at 1 and 22 h, respectively, 290/66 genes were determined to be network eligible of which 240/59 genes were determined to be function/pathway or list eligible. Under the list of biological category, the largest number of down regulated genes was associated to cell to cell signaling and interaction, cellular growth and proliferation. Gene classification according to canonical signaling pathways revealed that genes associated with IGF-I signaling, coagulation system and PI3K/AKT signaling were the categories containing largest number of genes differentially regulated post LH surge as per the IPA terminology (Fig. 3 and Fig. S5). Genes most significantly affected post peak LH surge, when classified into molecular and cellular functions revealed top 15 functions to be cell to cell signaling and proliferation related as shown in Fig. S3. The top few genes most dramatically up regulated with a fold change greater than 5.5 (p<0.05) at 1 h post peak LH surge were PLK2 (7.98), ADAMTS1 (5.69) and CTGF (5.56) genes. There was also increased expression of other genes involved in the regulation of cell to cell signaling and interaction. The top few genes which displayed the most dramatic down regulation were RASL11B (−3.52), ASPN (−2.73) and FKHR (−2.11). Whereas, the genes such as CTGF (5.29) and ASPN (−3.89) also had stronger regulation at 22 h post peak LH surge. From IPA, 35 networks (p<0.05) were identified with 20 networks each having 10 or more focus genes among the differentially expressed genes. At 1 h, the top gene network identified was cell-to-cell signaling and interaction, cell cycle and tissue development and morphology. (Network 1; score 40, 25 focus molecules; Fig. 4), while at 22 h the significant gene network identified was cell death, cell-to-cell signaling and interaction and endocrine system disorders. (Network 2; score 35, 20 focus molecules; Fig. 5).


Gene expression profiling of preovulatory follicle in the buffalo cow: effects of increased IGF-I concentration on periovulatory events.

Rao JU, Shah KB, Puttaiah J, Rudraiah M - PLoS ONE (2011)

Classification of differentially expressed genes into various canonical pathways by Ingenuity pathway analysis (IPA).The pathway analysis indicates that a large number of differentially expressed genes belonged to canonical pathways such as PI3K/AKT signaling; IGF-I signaling and coagulation system. The orange line represents score for the likelihood [−log (B–H P<0.05)] that genes belonging to a specific signaling pathway category affected at 1 and 22 h post peak LH surge. The stacked bars indicate the percentage of genes distributed according to regulation i.e. green (down), red (up) and open bars (No overlap with dataset) in each canonical pathway.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3119055&req=5

pone-0020754-g003: Classification of differentially expressed genes into various canonical pathways by Ingenuity pathway analysis (IPA).The pathway analysis indicates that a large number of differentially expressed genes belonged to canonical pathways such as PI3K/AKT signaling; IGF-I signaling and coagulation system. The orange line represents score for the likelihood [−log (B–H P<0.05)] that genes belonging to a specific signaling pathway category affected at 1 and 22 h post peak LH surge. The stacked bars indicate the percentage of genes distributed according to regulation i.e. green (down), red (up) and open bars (No overlap with dataset) in each canonical pathway.
Mentions: Ingenuity pathway analysis (IPA) was used to classify the differentially expressed genes into different function and disease categories. Of the 450 and 111 differentially expressed genes at 1 and 22 h, respectively, 290/66 genes were determined to be network eligible of which 240/59 genes were determined to be function/pathway or list eligible. Under the list of biological category, the largest number of down regulated genes was associated to cell to cell signaling and interaction, cellular growth and proliferation. Gene classification according to canonical signaling pathways revealed that genes associated with IGF-I signaling, coagulation system and PI3K/AKT signaling were the categories containing largest number of genes differentially regulated post LH surge as per the IPA terminology (Fig. 3 and Fig. S5). Genes most significantly affected post peak LH surge, when classified into molecular and cellular functions revealed top 15 functions to be cell to cell signaling and proliferation related as shown in Fig. S3. The top few genes most dramatically up regulated with a fold change greater than 5.5 (p<0.05) at 1 h post peak LH surge were PLK2 (7.98), ADAMTS1 (5.69) and CTGF (5.56) genes. There was also increased expression of other genes involved in the regulation of cell to cell signaling and interaction. The top few genes which displayed the most dramatic down regulation were RASL11B (−3.52), ASPN (−2.73) and FKHR (−2.11). Whereas, the genes such as CTGF (5.29) and ASPN (−3.89) also had stronger regulation at 22 h post peak LH surge. From IPA, 35 networks (p<0.05) were identified with 20 networks each having 10 or more focus genes among the differentially expressed genes. At 1 h, the top gene network identified was cell-to-cell signaling and interaction, cell cycle and tissue development and morphology. (Network 1; score 40, 25 focus molecules; Fig. 4), while at 22 h the significant gene network identified was cell death, cell-to-cell signaling and interaction and endocrine system disorders. (Network 2; score 35, 20 focus molecules; Fig. 5).

Bottom Line: Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes.The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR).These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.

ABSTRACT
The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

Show MeSH