Enhanced Akt-rpS6 activation and <i>in vitro</i> inhibition of rpS6 activation in Oo<i>Pten</i>^−/− oocytes by rapamycin.

<p>(<b>A</b>) Comparison of Akt-rpS6 signaling in Oo<i>Pten</i>^−/− and Oo<i>Pten</i>^+/+ oocytes. Oocytes were isolated from ovaries of mice at postnatal day 12–14 and immunoblotting was performed as described in <i>Materials and Methods</i>. Loss of PTEN led to enhanced PI3K signaling as indicated by an increase in phosphorylated Akt (p-Akt). The level of phosphorylated rpS6 (p-rpS6) was also increased in Oo<i>Pten</i>^−/− oocytes compared with Oo<i>Pten</i>^+/+ oocytes. Levels of total rpS6, Akt, and β-actin were used as internal controls. (<b>B</b>) Activation of rpS6 in Oo<i>Pten</i>^−/− oocytes is dependent on mTORC1 signaling. Oocytes were isolated from ovaries of Oo<i>Pten</i>^−/− mice at PD 12–14 as described in <i>Materials and Methods</i>. Treatment of oocytes with the mTORC1-specific inhibitor rapamycin (Rapa, 50 nM) for 2 h was found to largely suppress levels of phosphorylated rpS6 (p-rpS6), but did not affect the level of phosphorylated Akt (p-Akt). As a control, treatment of Oo<i>Pten</i>^−/− oocytes with the PI3K-specific inhibitor LY294002 (LY, 50 µM) for 2 h also largely suppressed levels of phosphorylated rpS6 (p-rpS6), but it also suppressed the level of phosphorylated Akt (p-Akt). This suggests that activation of rpS6 in Oo<i>Pten</i>^−/− oocytes is dependent on both PI3K and mTORC1 signaling. Levels of total Akt, rpS6, and β-actin were used as internal controls.</p

Normal follicular development in Oo<i>Rptor</i>^−/− mice.

<p>Morphological analysis of ovaries from Oo<i>Rptor</i>^−/− and Oo<i>Rptor</i>^+/+ littermates at PD35 and at 16 weeks of age. Ovaries from Oo<i>Rptor</i>^−/− and Oo<i>Rptor</i>^+/+ mice were embedded in paraffin, and sections 8 µm thick were prepared and stained with hematoxylin. The overall development of follicles (arrows) and corpora lutea (CL) in Oo<i>Rptor</i>^−/− mice was found to be normal (B, D, F, and H) compared to Oo<i>Rptor</i>^+/+ mice (A, C, E, and G).</p

PI3K–Akt signaling in Oo<i>Rptor</i>^−/− and Oo<i>Rptor</i>^+/+ oocytes.

<p>Oocytes were isolated from the ovaries of Oo<i>Rptor</i>^−/− and Oo<i>Rptor</i>^+/+ mice at PD12–14, and western blots were performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110491#s4" target="_blank">Materials and Methods</a>. Levels of phosphorylation of Akt at S473 and T308 are elevated in Oo<i>Rptor</i>^−/− oocytes compared to Oo<i>Rptor</i>^+/+ oocytes, and this indicates that PI3K–Akt signaling in Oo<i>Rptor</i>^−/− oocytes is enhanced. Levels of total Akt and β-actin were used as internal controls.</p

Fertility is not altered in Oo<i>Rptor</i>^−/− females.

<p>Fertility curve comparing the average cumulative number of pups per Oo<i>Rptor</i>^−/− (red line) and Oo<i>Rptor</i>^+/+ (black line) female. All Oo<i>Rptor</i>^−/− females are fertile indicating that loss of mTORC1 from oocytes does not affect the fertility of female mice.</p

Fertility measurement of the female mice directly injected with bpV(HOpic).

<p>(<b>A</b>) Weekly comparison of the cumulative number of pups for the low dose bpV(HOpic)-injected mice (n = 2, blue bars), high dose-injected mice (n = 2, red bars) and control mice (n = 2, black bars). All mice had been bred with CD-1 strain males. (<b>B</b>) Weekly comparison of the cumulative number of pups produced by breeding the F1 mice. Breeding between F1 males and F1 females produced by low dose-injected females (n = 2, red bars), breeding between F1 males and F1 females produced by high dose-injected females (n = 2, green bars) and breeding between F1 males and F1 females produced by PBS-injected females (n = 2, black bars). n = number of breeding pairs used.</p

Fertility measurement of the first and second generation progeny mice.

<p>The F1 female and F1 male mice that were obtained by embryonic transfer were bred with B6/C57J male and B6D2F1 female mice, respectively. Fertility was also checked by breeding F1 males and F1 females. During the testing period, the mice regularly produced normal-sized F2 generation litters at normal intervals. To determine the fertility of the second generation mice, F2 females were bred with F2 males. n =  number of breeding pairs used.</p

Enhanced follicular development by transient treatment of neonatal mouse ovaries with the PTEN inhibitor bpV(HOpic).

<p>(<b>A</b>) Comparison between the sizes of treated and control ovaries transplanted under the kidney capsules. One ovary from a PD3 mouse was cultured for 24 h with 1 µM bpV(HOpic) and another ovary was cultured without bpV(HOpic) and then transplanted under the capsule of each kidney of the same ovariectomized recipient as described in <i>Materials and Methods</i>. Ovaries that were treated with bpV(HOpic) before transplantation grew bigger than the non-treated control ovaries. K represents kidney tissue from the recipient, O represents the transplanted ovary, and the ovarian border is outlined by dashed circles. Scale bar = 1 mm. (<b>B</b>) Morphological analysis of treated and control ovaries excised from the kidney capsules. Ovaries from PD3 mice were cultured for 24 h with or without 1 µM bpV(HOpic) before transplantation under each kidney capsule of the same ovariectomized recipient as described in <i>Materials and Methods</i>. One day after the transplantation, recipient mice were treated daily with 2 IU of pregnant mare serum gonadotropin for 18 days. Fourteen hours before being killed, the mice were treated with 5 IU of human chorionic gonadotropin. Ovaries were excised from the kidney capsules and embedded in paraffin, and serial sections of 8 µm thickness were prepared and stained with hematoxylin. A larger number of antral follicles were observed in the bpV(HOpic)-treated ovaries (arrows) than in the control ovaries. The experiments were repeated at least 4 times, and 5 mice were used each time. Scale bar = 250 µm.</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

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

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