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

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 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