Pharmacological inhibition of mTORC1 prevents over-activation of the primordial follicle pool in response to elevated PI3K signaling

Abstract

The majority of ovarian primordial follicles must be preserved in a quiescent state to allow for the regular production of gametes over the female reproductive lifespan. However, the molecular mechanism that maintains the long quiescence of primordial follicles is poorly understood. Under certain pathological conditions, the entire pool of primordial follicles matures simultaneously leading to an accelerated loss of primordial follicles and to premature ovarian failure (POF). We have previously shown that loss of Pten (phosphatase and tensin homolog deleted on chromosome ten) in mouse oocytes leads to premature activation of the entire pool of primordial follicles, subsequent follicular depletion in early adulthood, and the onset of POF. Lack of PTEN leads to increased phosphatidylinositol 3-kinase (PI3K)-Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling in the oocytes. To study the functional and pathological roles of elevated mTORC1 signaling in the oocytes, we treated the Pten-mutant mice with the specific mTORC1 inhibitor rapamycin. When administered to Pten-deficient mice prior to the activation of the primordial follicles, rapamycin effectively prevented global follicular activation and preserved the ovarian reserve. These results provide a rationale for exploring the possible use of rapamycin as a drug for the preservation of the primordial follicle pool, and the possible prevention of POF.

Figure 1. Enhanced Akt-rpS6 activation and in vitro inhibition of rpS6 activation in OoPten ^−/− oocytes by rapamycin.

(A) Comparison of Akt-rpS6 signaling in OoPten ^−/− and OoPten ^+/+ oocytes. Oocytes were isolated from ovaries of mice at postnatal day 12–14 and immunoblotting was performed as described in Materials and Methods. 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 OoPten ^−/− oocytes compared with OoPten ^+/+ oocytes. Levels of total rpS6, Akt, and β-actin were used as internal controls. (B) Activation of rpS6 in OoPten ^−/− oocytes is dependent on mTORC1 signaling. Oocytes were isolated from ovaries of OoPten ^−/− mice at PD 12–14 as described in Materials and Methods. 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 OoPten ^−/− 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 OoPten ^−/− oocytes is dependent on both PI3K and mTORC1 signaling. Levels of total Akt, rpS6, and β-actin were used as internal controls.

Figure 2. Preservation of the primordial follicle pool in OoPten ^−/− ovaries by rapamycin treatment.

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