Normal follicular development in <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> mice.

<p>Morphological analysis of ovaries from 13- and 23-day-old, and 16-week-old <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> mice, <i>Pten^loxP/loxP</i>; <i>GCre+</i> mice, and control <i>Pten^loxP/loxP</i> mice. Ovaries were embedded in paraffin and sections of 8-µm thickness were prepared and stained with hematoxylin. Note the overactivation of primordial follicles in <i>Pten^loxP/loxP</i>; <i>GCre+</i> ovaries (C, F, and I, arrows) and the normal follicular development and CL in <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> ovaries (B, E, H and K), which is comparable to the control <i>Pten^loxP/loxP</i> ovaries (A, D, G, and J). CL, corpora lutea.</p

Characterization of <i>Pten</i> deletion by western blot and PCR.

<p>(A) Oocytes were prepared and lysed for western blot as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006186#s4" target="_blank"><i>Materials and Methods</i></a>. PTEN expression was found to be completely absent in <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> oocytes. For each lane, 150 oocytes were used. β-actin was used as internal control. (B) PCR analysis showing the complete deletion of <i>Pten</i> exon 5 (<i>Pten</i> Δ5) in one allele of the genomic DNA of pups from <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> females.</p

Generation of mutant mice with oocyte-specific deletion of <i>Pten</i>.

<p>A schematic representation of deletion of <i>Pten</i> exon 5 in oocytes of primary and further developed follicles by using the <i>Zp3</i> promoter-mediated Cre transgenic mice. The developmental stages at which the <i>Gdf-9</i> promoter and the <i>Zp3</i> promoter become active are indicated above the illustration of follicles in the figure.</p

Enhanced Akt signaling in oocytes of <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> mice.

<p>Oocytes were prepared from ovaries of 3–4 week old mice that were treated with PMSG, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006186#s4" target="_blank"><i>Materials and Methods</i></a>. Signaling studies in <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> oocytes showed elevated levels of p-Akt (Ser473), p-Akt (Thr308), and p-Tsc2 (Thr1462) as compared to <i>Pten^loxP/loxP</i> oocytes. Levels of total Akt, Tsc2, and β-actin were used as internal controls. 100–150 oocytes were used for each lane. All experiments were repeated at least three times and representative results are shown.</p