Preservation of the primordial follicle pool in Oo<i>Pten</i>^−/− ovaries by rapamycin treatment.

<p>(<b>A</b>) Prevention of the primordial follicle over-activation in Oo<i>Pten</i>^−/− mice by treatment with rapamycin. Rapamycin (5 mg/kg body weight) was injected daily into Oo<i>Pten</i>^−/− mice from postnatal day (PD) 4 to PD 22, and the ovaries were collected at PD 23 for morphological analysis. Ovaries from rapamycin-treated Oo<i>Pten</i>^−/− mice appeared smaller (c) than the ovaries from vehicle-treated Oo<i>Pten</i>^−/− mice (a). Scale bar = 50 µm. Clusters of primordial follicles were seen in rapamycin-treated Oo<i>Pten</i>^−/− mice at PD 23 (d, arrows) whereas all primordial follicles were activated in vehicle-treated Oo<i>Pten</i>^−/− mice at PD 23 (b, arrows). Scale bar = 50 µm. (<b>B</b>) Average numbers of total and primordial follicles in Oo<i>Pten</i>^+/+, Oo<i>Pten</i>^−/− (vehicle-treated), and Oo<i>Pten</i>^−/− (rapamycin-treated) ovaries at PD 23. Proportions of primordial follicles ± SEM (relative to the total number of follicles) are also shown. The proportion of primordial follicles in rapamycin-treated Oo<i>Pten</i>^−/− ovaries was 20±4.1%, which was smaller than the proportion in the Oo<i>Pten</i>^+/+ ovaries (70±3.1%). Three mice were used for each experimental group. Rapa, rapamycin. (<b>C</b>) Comparison of the rpS6 and Akt phosphorylation levels in the ovaries of vehicle- and rapamycin-treated Oo<i>Pten</i>^−/− mice. Rapamycin (5 mg/kg body weight) was injected daily into Oo<i>Pten</i>^−/− mice from PD 4 to PD 22, the ovaries were collected at PD 23 and homogenized, and immunoblotting was performed as described in <i>Materials and Methods</i>. Rapamycin injection effectively suppressed the level of phosphorylated rpS6 (p-rpS6) without affecting the level of phosphorylated Akt (p-Akt) in the ovaries of Oo<i>Pten</i>^−/− mice. Levels of total rpS6, Akt, and β-actin were used as internal controls.</p

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

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

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

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