Role of PI3K-Akt-mTOR and AMPK signaling pathways in FSH-induced <i>PRLR</i> and <i>StAR</i> mRNAs expression in cultured granulosa cells of prehierarchical (6–8 mm) follicles.

<p>(A and C) Changes in relative mRNA levels of <i>PRLR</i> (A) and <i>StAR</i> (C) after culture of granulosa cells for 4 h in the absence or presence of 10 ng/ml FSH together with PI3K inhibitor (20 μM LY294002) or mTOR inhibitor (10 μM Rapamycin). (B and D) Relative <i>PRLR</i> (B) and <i>StAR</i> (D) mRNAs levels in granulosa cells cultured for 4 h in the absence or presence of 10 ng/ml FSH, or together with AMPK activator (1 mM AICAR). Relative expression level was normalized to <i>18S rRNA</i>. Data are expressed as fold differences ± SEM of three independent experiments using tissues from different hens and are compared to control cells. ^*, <i>P</i> < 0.05 compared to control cells; ^#, <i>P</i> < 0.05 compared to cells only stimulated by 10 ng/ml FSH.</p

Abundance of ERK2 protein in chicken developing follicles and its role in FSH-induced <i>PRLR</i> and <i>StAR</i> expression in cultured granulosa cells of 6–8 mm follicles.

<p>(A) Top panel: Representative western blot analysis of phosphorylated and total ERK2 in the stroma and walls of prehierarchical (< 2, 2–4 and 4–6 mm) follicles as well as in theca and granulosa cell layers separated from the 6–8 mm and F3-F1 follicles. β-actin was used as loading control. Bottom panel: Quantitative analysis of relative protein abundance of phosphorylated ERK2 by densitometry using Image Lab software (Version 4.1, Bio-Rad laboratories). Relative protein abundance was normalized to total ERK2. Data are expressed as fold differences ± SEM compared to an appropriate tissue (n = 4 hens). Bars with different letters are significantly different at <i>P</i> < 0.05. (B and C) Changes in relative mRNAs levels of <i>PRLR</i> (B) and <i>StAR</i> (C) in granulosa cells treated without or with 10 ng/ml FSH, or together with the MEK inhibitor (1 μM PD0325901) after a 4-h culture. Relative mRNA expression level was normalized to <i>18S rRNA</i>. Data are expressed as fold differences ± SEM of three independent experiments using tissues from different hens and are compared to control cells. ^*, <i>P</i> < 0.05 compared to control cells; ^#, <i>P</i> < 0.05 compared to cells only stimulated by 10 ng/ml FSH.</p

Primer pairs for real-time quantitative PCR.

<p>Primer pairs for real-time quantitative PCR.</p

Role of PKA and PKC signaling pathways in FSH-induced <i>PRLR</i> and <i>StAR</i> mRNAs expression in cultured granulosa cells of prehierarchical (6–8 mm) follicles.

<p>(A and C) Changes in relative mRNA levels of <i>PRLR</i> (A) and <i>StAR</i> (C) after culture of granulosa cells for 4 h in the absence or presence of 10 ng/ml FSH in combination with PKA activator (10 μM Forskolin) or inhibitor (20 μM H89). (B and D) Changes in relative mRNA levels of <i>PRLR</i> (B) and <i>StAR</i> (D) in granulosa cells treated without or with 10 ng/ml FSH in combination with PKC activator (20 nM PMA) or inhibitor (10 μM GF109203X). Relative expression level was normalized to <i>18S rRNA</i>. Data are expressed as fold differences ± SEM of three independent experiments using tissues from different hens and are compared to control cells. ^*, <i>P</i> < 0.05 compared to control cells; ^#, <i>P</i> < 0.05 compared to cells only stimulated by 10 ng/ml FSH.</p

Effects of manipulation of several intracellular signaling pathways on basal and FSH-induced ERK2 phosphorylation in cultured granulosa cells of prehierarchical (6–8 mm) follicles.

<p>Top panel: Representative western blot analysis of phosphorylated and total ERK2 in granulosa cells treated without or with 10 ng/ml FSH in combination with PKA activator or inhibitor (10 μM Forskolin or 20 μM H89, respectively), or PKC activator or inhibitor (20 nM PMA or 10 μM GF109203X, respectively), or PI3K inhibitor (20 μM LY294002) or mTOR inhibitor (10 μM Rapamycin), or AMPK activator (1 mM AICAR). β-actin was used as loading control. Bottom panel: Quantitative analysis of relative protein abundance of phosphorylated ERK2 by densitometry using Image Lab software (Version 4.1, Bio-Rad laboratories). Relative protein abundance was normalized to total ERK2. Data are expressed as fold differences ± SEM of three independent experiments using tissues from different hens and are compared to control cells. ^*, <i>P</i> < 0.05 compared to control cells; ^#, <i>P</i> < 0.05 compared to cells only stimulated by 10 ng/ml FSH.</p

Expression pattern of <i>PRLR</i> mRNA in chicken developing follicles.

<p>(A) Abundance of <i>PRLR</i> mRNA in the stroma and walls of prehierarchical (< 2, 2–4, 4–6 and 6–8 mm) and preovulatory (9–12 mm, F5 and F4) follicles. (B) Relative <i>PRLR</i> mRNA levels in theca and granulosa cell layers isolated from the three largest preovulatory follicles (namely F3-F1; F3 < F2 < F1). Relative expression level was normalized to <i>18S rRNA</i>. Data are expressed as fold differences ± SEM compared to either the stroma or F3 granulosa cells (n = 4 hens). Different lowercase letters indicate a significant effect of follicular developmental stage, whilst different uppercase letters indicate a significant effect of cell type (granulosa versus theca cell layer). <i>P</i> < 0.05 was accepted as statistically significant.</p

Detection of apoE in the plasma of control and transgenic (apoE-shRNA1) cloned pigs.

<p>(A) Immunoblots showing the detection of apoE protein in plasma of control (lanes 1–3) and apoE-shRNA1 transgenic (lanes 4–8) cloned pigs. (B) The intensity (mean ± SEM) of the apoE bands in equal volumes of plasma samples was assessed by densitometric analysis. Mean band intensity between groups was compared by ANOVA. Mean band intensity between groups was compared by ANOVA (*<i>P</i><0.05).</p

Sequence of oligonucleotide primers.

<p>Sequence of oligonucleotide primers.</p

ApoE knockdown with synthetic siRNAs in cultured porcine cells.

<p>(A) Synthetic siRNAs sequences (siRNA1, siRNA2 and siRNA3) targeting the porcine apoE mRNA. (B) Effect of the siRNAs on apoE transcripts levels in cultured porcine granulosa cells. The siRNAs were introduced into the cells by lipofection. Control cells were treated with the lipofection agent alone. Cells were harvested 48 h after treatment and apoE mRNA levels were analyzed by qRT-PCR. Values were normalized to the abundance of GAPDH mRNA. The inhibitory effect of each siRNA was compared to the control group. Values are shown as percent of the control value, as the means ± SEM (n = 3 replicates). Bars that do not share a common superscript are statistically different (<i>P</i><0.05).</p

Structure and integration of the apoE-shRNA1 expression vector in transfected porcine fibroblasts.

<p>(A) The expression of the apoE-shRNA1 sequence is under the control of the U6 promoter and linked to GFP and neomycin resistance markers. (B) GFP expression in surviving cells that were selected for neomycin resistance indicating the stable integration of the apoE-shRNA1 expression vector.</p