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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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Effects of bGH treatment on IGFBP expression and various hormone concentration.(A) Circulating plasma concentration (mean ±SEM) of IGF-I in blood samples collected at different time intervals in Veh and bGH treated animals. Bars with different letters above them are significantly different (p<0.05). (B) Serum concentration of LH before and after GnRH administration to induce ovulation in buffalo cows treated with Veh (solid triangle) or bGH (solid circle). Concentration of LH was determined in blood samples collected at various time points before and after administration of GnRH to elicit endogenous LH surge. (C) Concentration of IGF-I (ng/ml) measured in the follicular fluid collected from follicles before (−2 h) and 22 h post peak LH surge of Veh or bGH treated animals. Bars with different letters above them are significantly different (p<0.05). (D) Mean ± SEM concentrations of E2 and P4 in follicular fluid collected from follicles before and at 22 h post peak LH surge of Veh or bGH treated animals. Bars with different alphabets within each hormone indicate significantly different concentration (p<0.05). (E) Protein expression of IGFBP2, IGFBP3 and Cleaved IGFBP3 in the ovarian follicular fluid. Protein lysates (100 µg) prepared from follicular fluid collected from the preovulatory follicle before (−2 h) and 22 h post peak LH surge of Veh or bGH treated animals were resolved on 10% SDS PAGE, transferred onto PVDF membrane and immunoblot analysis was performed using anti-IGFBP2 or anti-IGFBP3 antibody. The positions of 36 kDa IGFBP2 and 44–40 kDa IGFBP3 doublet bands are indicated by arrows and 30 kDa bands of IGFBP3 proteolytic fragment is highlighted in the boxed area. Densitometric values were determined and are indicated as mean ± SEM below the bands. The image of Ponceau-S stained membrane is depicted to indicate that equal amount of protein was loaded in each lane. (F) Densitometric analysis of immunoblots for 30 kDa IGFBP3 proteolytic fragment: mean ± SEM of relative amount of 30 kDa IGFBP3 proteolytic fragment expressed as intensity of Ponceau-S stained bands in each group (n = 3 animals/group). Bars with different letters are significantly different (p<0.05).
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pone-0020754-g008: Effects of bGH treatment on IGFBP expression and various hormone concentration.(A) Circulating plasma concentration (mean ±SEM) of IGF-I in blood samples collected at different time intervals in Veh and bGH treated animals. Bars with different letters above them are significantly different (p<0.05). (B) Serum concentration of LH before and after GnRH administration to induce ovulation in buffalo cows treated with Veh (solid triangle) or bGH (solid circle). Concentration of LH was determined in blood samples collected at various time points before and after administration of GnRH to elicit endogenous LH surge. (C) Concentration of IGF-I (ng/ml) measured in the follicular fluid collected from follicles before (−2 h) and 22 h post peak LH surge of Veh or bGH treated animals. Bars with different letters above them are significantly different (p<0.05). (D) Mean ± SEM concentrations of E2 and P4 in follicular fluid collected from follicles before and at 22 h post peak LH surge of Veh or bGH treated animals. Bars with different alphabets within each hormone indicate significantly different concentration (p<0.05). (E) Protein expression of IGFBP2, IGFBP3 and Cleaved IGFBP3 in the ovarian follicular fluid. Protein lysates (100 µg) prepared from follicular fluid collected from the preovulatory follicle before (−2 h) and 22 h post peak LH surge of Veh or bGH treated animals were resolved on 10% SDS PAGE, transferred onto PVDF membrane and immunoblot analysis was performed using anti-IGFBP2 or anti-IGFBP3 antibody. The positions of 36 kDa IGFBP2 and 44–40 kDa IGFBP3 doublet bands are indicated by arrows and 30 kDa bands of IGFBP3 proteolytic fragment is highlighted in the boxed area. Densitometric values were determined and are indicated as mean ± SEM below the bands. The image of Ponceau-S stained membrane is depicted to indicate that equal amount of protein was loaded in each lane. (F) Densitometric analysis of immunoblots for 30 kDa IGFBP3 proteolytic fragment: mean ± SEM of relative amount of 30 kDa IGFBP3 proteolytic fragment expressed as intensity of Ponceau-S stained bands in each group (n = 3 animals/group). Bars with different letters are significantly different (p<0.05).

Mentions: The data on expressions of PR and COX-2, regarded as marker of the ovulating follicle, is presented in Fig. S1D&E. Circulating plasma level of IGF-I in Veh and bGH treated animals is presented in Fig. 8A. A significant (p<0.05) increase in IGF-I level was observed within 12 h of bGH injection and the higher level was sustained throughout the observation period. The occurrence of endogenous surge of LH after sequential treatment with PGF2α and GnRH was essentially identical between the two groups of animals (Fig. 8B). The IGF-I concentration in follicular fluid prior to peak LH surge was 168.4±45.4 ng/ml and increased to 238.5±59.8 ng/ml in Veh treated animals, while concentration increased significantly (p<0.05) to 407.9±24.5 ng/ml in bGH treated animals (Fig. 8C). The follicular fluid E2 and P4 concentrations were similar between two groups except that the P4 concentration at 22 h time point was marginally lower in the bGH treated animals (Fig. 8D).


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)

Effects of bGH treatment on IGFBP expression and various hormone concentration.(A) Circulating plasma concentration (mean ±SEM) of IGF-I in blood samples collected at different time intervals in Veh and bGH treated animals. Bars with different letters above them are significantly different (p<0.05). (B) Serum concentration of LH before and after GnRH administration to induce ovulation in buffalo cows treated with Veh (solid triangle) or bGH (solid circle). Concentration of LH was determined in blood samples collected at various time points before and after administration of GnRH to elicit endogenous LH surge. (C) Concentration of IGF-I (ng/ml) measured in the follicular fluid collected from follicles before (−2 h) and 22 h post peak LH surge of Veh or bGH treated animals. Bars with different letters above them are significantly different (p<0.05). (D) Mean ± SEM concentrations of E2 and P4 in follicular fluid collected from follicles before and at 22 h post peak LH surge of Veh or bGH treated animals. Bars with different alphabets within each hormone indicate significantly different concentration (p<0.05). (E) Protein expression of IGFBP2, IGFBP3 and Cleaved IGFBP3 in the ovarian follicular fluid. Protein lysates (100 µg) prepared from follicular fluid collected from the preovulatory follicle before (−2 h) and 22 h post peak LH surge of Veh or bGH treated animals were resolved on 10% SDS PAGE, transferred onto PVDF membrane and immunoblot analysis was performed using anti-IGFBP2 or anti-IGFBP3 antibody. The positions of 36 kDa IGFBP2 and 44–40 kDa IGFBP3 doublet bands are indicated by arrows and 30 kDa bands of IGFBP3 proteolytic fragment is highlighted in the boxed area. Densitometric values were determined and are indicated as mean ± SEM below the bands. The image of Ponceau-S stained membrane is depicted to indicate that equal amount of protein was loaded in each lane. (F) Densitometric analysis of immunoblots for 30 kDa IGFBP3 proteolytic fragment: mean ± SEM of relative amount of 30 kDa IGFBP3 proteolytic fragment expressed as intensity of Ponceau-S stained bands in each group (n = 3 animals/group). Bars with different letters are significantly different (p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3119055&req=5

pone-0020754-g008: Effects of bGH treatment on IGFBP expression and various hormone concentration.(A) Circulating plasma concentration (mean ±SEM) of IGF-I in blood samples collected at different time intervals in Veh and bGH treated animals. Bars with different letters above them are significantly different (p<0.05). (B) Serum concentration of LH before and after GnRH administration to induce ovulation in buffalo cows treated with Veh (solid triangle) or bGH (solid circle). Concentration of LH was determined in blood samples collected at various time points before and after administration of GnRH to elicit endogenous LH surge. (C) Concentration of IGF-I (ng/ml) measured in the follicular fluid collected from follicles before (−2 h) and 22 h post peak LH surge of Veh or bGH treated animals. Bars with different letters above them are significantly different (p<0.05). (D) Mean ± SEM concentrations of E2 and P4 in follicular fluid collected from follicles before and at 22 h post peak LH surge of Veh or bGH treated animals. Bars with different alphabets within each hormone indicate significantly different concentration (p<0.05). (E) Protein expression of IGFBP2, IGFBP3 and Cleaved IGFBP3 in the ovarian follicular fluid. Protein lysates (100 µg) prepared from follicular fluid collected from the preovulatory follicle before (−2 h) and 22 h post peak LH surge of Veh or bGH treated animals were resolved on 10% SDS PAGE, transferred onto PVDF membrane and immunoblot analysis was performed using anti-IGFBP2 or anti-IGFBP3 antibody. The positions of 36 kDa IGFBP2 and 44–40 kDa IGFBP3 doublet bands are indicated by arrows and 30 kDa bands of IGFBP3 proteolytic fragment is highlighted in the boxed area. Densitometric values were determined and are indicated as mean ± SEM below the bands. The image of Ponceau-S stained membrane is depicted to indicate that equal amount of protein was loaded in each lane. (F) Densitometric analysis of immunoblots for 30 kDa IGFBP3 proteolytic fragment: mean ± SEM of relative amount of 30 kDa IGFBP3 proteolytic fragment expressed as intensity of Ponceau-S stained bands in each group (n = 3 animals/group). Bars with different letters are significantly different (p<0.05).
Mentions: The data on expressions of PR and COX-2, regarded as marker of the ovulating follicle, is presented in Fig. S1D&E. Circulating plasma level of IGF-I in Veh and bGH treated animals is presented in Fig. 8A. A significant (p<0.05) increase in IGF-I level was observed within 12 h of bGH injection and the higher level was sustained throughout the observation period. The occurrence of endogenous surge of LH after sequential treatment with PGF2α and GnRH was essentially identical between the two groups of animals (Fig. 8B). The IGF-I concentration in follicular fluid prior to peak LH surge was 168.4±45.4 ng/ml and increased to 238.5±59.8 ng/ml in Veh treated animals, while concentration increased significantly (p<0.05) to 407.9±24.5 ng/ml in bGH treated animals (Fig. 8C). The follicular fluid E2 and P4 concentrations were similar between two groups except that the P4 concentration at 22 h time point was marginally lower in the bGH treated animals (Fig. 8D).

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
Related in: MedlinePlus