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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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Standardization of experimental animal model for characterization of periovulatory events during the LH surge.(A) Schematic representation of the experimental protocol employed for collection of follicular contents before and at different time points after peak LH surge. Follicular fluid, granulosa cells and follicle wall of the ovulatory follicle were harvested from the ovaries collected at the indicated time points for carrying out various analyses. (B) Circulating concentration of P4 during different days of estrous cycle for collection of follicular contents before and at different time points post peak LH surge. Serum P4 level was determined in blood samples collected daily beginning on day 1 of estrous cycle and at 12 h intervals after administration of PGF2α and GnRH treatments. For different days of estrous cycle, values from all animals recruited for this experiment (n = 23) were pooled and represented as mean ± SEM. (C) Mean ± SEM serum concentration of LH and FSH during the preovulatory period. (D) Follicular fluid concentration (mean ± SEM) of E2 and P4 at different time points before and after peak LH surge (n = >5 animals/time point). For each hormone, bars with different superscripts are significantly different (p<0.05).
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pone-0020754-g001: Standardization of experimental animal model for characterization of periovulatory events during the LH surge.(A) Schematic representation of the experimental protocol employed for collection of follicular contents before and at different time points after peak LH surge. Follicular fluid, granulosa cells and follicle wall of the ovulatory follicle were harvested from the ovaries collected at the indicated time points for carrying out various analyses. (B) Circulating concentration of P4 during different days of estrous cycle for collection of follicular contents before and at different time points post peak LH surge. Serum P4 level was determined in blood samples collected daily beginning on day 1 of estrous cycle and at 12 h intervals after administration of PGF2α and GnRH treatments. For different days of estrous cycle, values from all animals recruited for this experiment (n = 23) were pooled and represented as mean ± SEM. (C) Mean ± SEM serum concentration of LH and FSH during the preovulatory period. (D) Follicular fluid concentration (mean ± SEM) of E2 and P4 at different time points before and after peak LH surge (n = >5 animals/time point). For each hormone, bars with different superscripts are significantly different (p<0.05).

Mentions: Experiments (Experiment 1 and 2) in buffalo cows described in this study were cleared by the institutional animal ethics committee, Indian Institute of Science, Bangalore, India. The validation of experimental model system employed for studying gene expression profiling of the ovulating follicle has been provided as Data S1. The details of protocol employed in this experiment are represented in Figure 1A. Briefly, to monitor various hormone concentrations, blood samples were collected from the experimental non lactating buffalo cows. On day 7 of the cycle, Juramate (500 µg, i.m.) was administered to animals to induce luteal regression and initiation of follicular phase. GnRH (100 µg, i.m.) was administered 36 h post Juramate injection to induce gonadotropin surge. Serum samples were subjected to assay of P4 and gonadotropin concentration to verify CL regression and to determine the timing of LH surge. Ovaries were collected at slaughter before, 3, 10 and 24 h post GnRH injection (n = 4 animals or more/time point), following confirmation of time of peak LH surge, these time points correspond to −2, 1, 10 and 22 h post peak LH surge. These time points were chosen with a view to catalogue gene expression changes in the preovulatory follicle at the earliest time point post peak LH surge (i.e., 1 h) and at a later time point (i.e., 22 h) the time point closest to the time of ovulation. Ovaries containing preovulatory follicles were processed for retrieval of GCs and the follicle wall, as reported previously [38].


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)

Standardization of experimental animal model for characterization of periovulatory events during the LH surge.(A) Schematic representation of the experimental protocol employed for collection of follicular contents before and at different time points after peak LH surge. Follicular fluid, granulosa cells and follicle wall of the ovulatory follicle were harvested from the ovaries collected at the indicated time points for carrying out various analyses. (B) Circulating concentration of P4 during different days of estrous cycle for collection of follicular contents before and at different time points post peak LH surge. Serum P4 level was determined in blood samples collected daily beginning on day 1 of estrous cycle and at 12 h intervals after administration of PGF2α and GnRH treatments. For different days of estrous cycle, values from all animals recruited for this experiment (n = 23) were pooled and represented as mean ± SEM. (C) Mean ± SEM serum concentration of LH and FSH during the preovulatory period. (D) Follicular fluid concentration (mean ± SEM) of E2 and P4 at different time points before and after peak LH surge (n = >5 animals/time point). For each hormone, bars with different superscripts are significantly different (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020754-g001: Standardization of experimental animal model for characterization of periovulatory events during the LH surge.(A) Schematic representation of the experimental protocol employed for collection of follicular contents before and at different time points after peak LH surge. Follicular fluid, granulosa cells and follicle wall of the ovulatory follicle were harvested from the ovaries collected at the indicated time points for carrying out various analyses. (B) Circulating concentration of P4 during different days of estrous cycle for collection of follicular contents before and at different time points post peak LH surge. Serum P4 level was determined in blood samples collected daily beginning on day 1 of estrous cycle and at 12 h intervals after administration of PGF2α and GnRH treatments. For different days of estrous cycle, values from all animals recruited for this experiment (n = 23) were pooled and represented as mean ± SEM. (C) Mean ± SEM serum concentration of LH and FSH during the preovulatory period. (D) Follicular fluid concentration (mean ± SEM) of E2 and P4 at different time points before and after peak LH surge (n = >5 animals/time point). For each hormone, bars with different superscripts are significantly different (p<0.05).
Mentions: Experiments (Experiment 1 and 2) in buffalo cows described in this study were cleared by the institutional animal ethics committee, Indian Institute of Science, Bangalore, India. The validation of experimental model system employed for studying gene expression profiling of the ovulating follicle has been provided as Data S1. The details of protocol employed in this experiment are represented in Figure 1A. Briefly, to monitor various hormone concentrations, blood samples were collected from the experimental non lactating buffalo cows. On day 7 of the cycle, Juramate (500 µg, i.m.) was administered to animals to induce luteal regression and initiation of follicular phase. GnRH (100 µg, i.m.) was administered 36 h post Juramate injection to induce gonadotropin surge. Serum samples were subjected to assay of P4 and gonadotropin concentration to verify CL regression and to determine the timing of LH surge. Ovaries were collected at slaughter before, 3, 10 and 24 h post GnRH injection (n = 4 animals or more/time point), following confirmation of time of peak LH surge, these time points correspond to −2, 1, 10 and 22 h post peak LH surge. These time points were chosen with a view to catalogue gene expression changes in the preovulatory follicle at the earliest time point post peak LH surge (i.e., 1 h) and at a later time point (i.e., 22 h) the time point closest to the time of ovulation. Ovaries containing preovulatory follicles were processed for retrieval of GCs and the follicle wall, as reported previously [38].

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