Variation of the expression of <i>FSHR</i> mRNA in the granulosa cells transfected by reconstructed pcDNA3.0-FOXL2 plasmids and cultured with activin A, GDF9 and follistatin.

<p>(A), the total cellular extracts were subjected to Western blot analysis. Chicken β-actin (42 kDa) was used as the loading control. Control 1, negative control, without cellular extracts; Control 2, the granulosa cells cultured with the basal medium, no transfection with pcDNA3.0 blank vector or the reconstructed plasmid pcDNA3.0-FOXL2 vector, indicating the endogenous FOXL2 was expressed. pcDNA3, the cells were transfected only by pcDNA3.0 blank vector, also indicating the endogenous FOXL2. pcFOXL2, the cells were transfected by the reconstructed plasmid pcDNA3.0-FOXL2 vector, indicating a transient expression of a recombinant FOXL2 protein. (B), Variation of the expression of <i>FSHR</i> mRNA in the cells responding to the expression of FOXL2, cultured in presence or absence of activin A, GDF9 or FST to the basal medium as indicated. Control 1, the granulosa cells cultured with the basal medium, no transfection with pcDNA3.0 blank vector or the reconstructed plasmid pcDNA3.0-FOXL2 vector, indicating the endogenous <i>FSHR</i> mRNA was expressed. Control 2, the cells were transfected only by pcDNA3.0 blank vector, also indicating the endogenous <i>FSHR</i> mRNA expression. Control 3, expression of <i>FSHR</i> mRNA in the cells transfected by the pcDNA3.0-FOXL2 plasmid. Control 1–3, absent of any of the activin A, GDF9 and follistatin, Control 4, the cells cultured with the same basal medium to control 1–3, but present of the activin A(10 ng/ml), GDF9 (100 ng/ml) and follistatin (100 ng/ml) as indicated. pcDNA3.0, the cells were transfected by pcDNA3.0 blank vector and cultured same to the control 4; FOXL2, the cells were transfected by the reconstructed plasmid pcDNA3.0-FOXL2 vector. Each treatment was performed in triplicate at 38.5°C in 5% CO₂ for 24 h. The values on the bar graphs are the mean ± SEM of 10 hens (n = 10) form a representative experiment. For each treatment, bars with different letter superscripts above them are significantly different in expression levels of <i>FSHR</i> mRNA in the cultured granulosa cells (<i>P <</i> 0.05).