Blastocyst quality was evaluated by the ratio of ICM/TCN.

<p>(<b>A</b>) The blastocysts derived from the oocytes matured in vitro without rh-AMH (A–C) or with rh-AMH (D–F), and treated with AdH1-SiRNA/NS (G–I) and AdH1-SiRNA/AMH (J–K) respectively were stained with OCT4 for ICM (A, D, G, J), DAPI for TCN (B, E, H, K) and merged images (C, F, I, L). Bars = 20 µm. (<b>B</b>) The ratio of ICM/TCN represents by mean±SD in each group with 6 replicates. * Indicates significant differences (p<0.05) between the groups.</p

Effect of different concentrations of rh-AMH supplemented into IVM medium on GDF9 and BMP15 mRNA expression and protein production from the oocytes.

<p>GDF9 and BMP15 mRNA in the oocytes were measured by RT-PCR, and GDF9 and BMP15 proteins in IVM-medium were measured by ELISA, respectively. Oocytes were harvested at 16–18 h after IVM. *Indicates significant differences (p<0.05) compared to the control group. Data were from 3 replicates.</p

Real-time PCR primer sequences.

<p>Real-time PCR primer sequences.</p

Effect of IVM medium supplemented with or without 100/ml rh-AMH on GDF9 and BMP15 mRNA expression and protein production from the oocytes following IVM at different time points.

<p>GDF9 and BMP15 mRNA expression in the oocytes were measured by RT-PCR, and GDF9, and BMP15 proteins in IVM-medium were measured by ELISA, respectively. Oocytes and IVM-medium supernatants were collected at 6, 12 and 18 h respectively during IVM. *Indicates significant differences (p<0.05) compared to the control group. Data were from 3 replicates.</p

Effect of AMH knockdown on GDF9 and BMP15 mRNA expression from mouse cumulus cells and oocytes.

<p>Cumulus cells and oocytes were harvested from COCs infected by AdH1-SiRNA/NS or AdH1-SiRNA/AMH during IVM, respectively. (A) Shows AMH mRNA expression in cumulus cells infected by AdH1-SiRNA/NS or AdH1-SiRNA/AMH; (B) Shows GDF9 and BMP15 mRNA expressions in oocytes from COCs infected by AdH1-SiRNA/NS or AdH1-SiRNA/AMH, respectively. *Indicates significant differences (p<0.05) compared to the control group. Data were from 3 replicates.</p

The effect of different concentrations of rh-AMH in IVM-medium on mouse oocyte maturation following IVM.

<p>Data showed with 6 replicates.</p><p>*There are no significant differences among groups.</p

Localization of AMH and AMHR-II by PCR and immunofluorescence staining.

<p>(<b>A</b>) RNA was isolated from oocytes and cumulus cells from 30 COCs that were at GV stage or at MII stage after IVM, respectively. One-microliter amounts of cDNA were used as templates for PCR. (<b>B</b>) COCs after 16–18 h of culture in vitro were stained with mouse isotype IgGs as negative controls of AMH (A) and AMHR-II (G), AMH (D), AMHR-II (J) respectively, followed by TRITC conjugated secondary antibodies and DAPI (B,E,H,K), and merged images (C,F,I,L). Bars = 50 µm.</p

Subsequent embryonic development of immature COCs matured in IVM-medium without rh-AMH (Control) or with 100 ng/ml rh-AMH, AdH1-SiRNA/NS, AdH1-SiRNA/AMH respectively and followed by in vitro fertilization (IVF).

<p>Data showed 3 replicates.</p><p>*Indicates significant difference compared to control group (P<0.05);</p><p>**indicates significant difference compared to AdH1-SiRNA/NS group (P<0.05).</p