mTORC1 Signaling in Oocytes Is Dispensable for the Survival of Primordial Follicles and for Female Fertility

The molecular mechanisms underlying reproductive aging and menopausal age in female mammals are poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) is a central controller of cell growth and proliferation. To determine whether mTORC1 signaling in oocytes plays a direct role in physiological follicular development and fertility in female mice, we conditionally deleted the specific and essential mTORC1 component Rptor (regulatory-associated protein of mTORC1) from the oocytes of primordial follicles by using transgenic mice expressing growth differentiation factor 9 (Gdf-9) promoter-mediated Cre recombinase. We provide in vivo evidence that deletion of Rptor in the oocytes of both primordial and further-developed follicles leads to the loss of mTORC1 signaling in oocytes as indicated by loss of phosphorylation of S6K1 and 4e-bp1 at T389 and S65, respectively. However, the follicular development and fertility of mice lacking Rptor in oocytes were not affected. Mechanistically, the loss of mTORC1 signaling in Rptor-deleted mouse oocytes led to the elevation of phosphatidylinositol 3-kinase (PI3K) signaling that maintained normal follicular development and fertility. Therefore, this study shows that loss of mTORC1 signaling in oocytes triggers a compensatory activation of the PI3K signaling cascade that maintains normal ovarian follicular development and fertility.


Introduction
In mammals, the pool of primordial follicles serves as the source of developing follicles and fertilizable ova for the entire reproductive lifespan of the organism [1]. At any given time, only a limited number of primordial follicles are recruited into the growing follicle pool through follicular activation and the majority of primordial follicles remain in a dormant state. Menopause, also known as ovarian senescence, occurs when the pool of primordial follicles has been virtually exhausted [2][3][4]. The duration of fertility and the timing of menopause are thus determined by the size and persistence of the primordial follicle pool [1][2][3][4].
Conventional knockout of the Rptor gene in mice -which codes for the specific and essential mTORC1 component Raptor -is embryonic lethal [13]. To directly explore the role of mTORC1 in ovarian follicular development and fertility in vivo, we generated mice lacking Rptor specifically in the oocytes of both primordial and further-developed follicles by using transgenic mice expressing growth differentiation factor 9 (Gdf-9) promoter-mediated Cre recombinase. We found that deletion of Rptor specifically in the oocytes leads to loss of mTORC1 signaling. However, follicular development and fertility in mice lacking Rptor in their oocytes were not affected by the loss of mTORC1 signaling. Interestingly, PI3K signaling was found to be elevated upon the loss of mTORC1 signaling in Rptor-deleted oocytes, and this activity is presumed to maintain the follicular development and fertility in these mice.

Generation and validation of mutant mice with oocytespecific deletion of Rptor
To study how mTORC1 in oocytes regulates the activation and development of primordial follicles, we generated mutant mice in which the Rptor gene was deleted specifically in the oocytes of primordial and further-developed follicles (referred to as OoRptor 2/2 mice). This was achieved by crossing Rptor loxP/loxP mice [14] with transgenic mice carrying Gdf-9 promoter-mediated Cre recombinase [15,16] (Fig. 1A). To determine the efficiency of deletion of Rptor in oocytes, we performed western blot analysis on oocytes collected from postnatal day (PD)12-14 OoRptor 2/2 and OoRptor +/+ mice. We found that expression of Raptor protein was completely abolished in growing OoRptor 2/2 oocytes (Fig. 1B) indicating successful deletion of the Rptor gene from the oocytes. To further validate that the loss of Rptor in oocytes leads to loss of mTORC1 signaling in OoRptor 2/2 oocytes, we examined the phosphorylation of its well-known substrates S6K1 and 4e-bp1 [12,17]. As shown in Fig. 1B, phosphorylation of S6K1 and 4e-bp1 at T389 and S65, respectively, was effectively abolished in the OoRptor 2/2 oocytes indicating that mTORC1 signaling is suppressed in the mutant oocytes.
Loss of mTORC1 signaling in oocytes does not affect the fertility of female mice We found that the OoRptor 2/2 females sexually matured and had a normal vaginal opening at the age of 5-6 weeks. To determine whether the loss of mTORC1 signaling from oocytes influences the fertility of OoRptor 2/2 mice, we housed OoRptor 2/2 and OoRptor +/+ mice with wild-type males. We found that the fertility of OoRptor 2/2 females was comparable to that of OoRptor +/+ females during the testing period from 6 weeks to 30 weeks of age (Fig. 2). These results show that loss of mTORC1 signaling in oocytes does not affect the fertility of female mice.

PI3K-Akt signaling is enhanced in OoRptor 2/2 oocytes
In recent years, the PI3K-Akt signaling cascade in oocytes has been shown to have important roles in controlling the activation and development of ovarian follicles and fertility [16,[18][19][20]. To explore the molecular mechanisms underlying the normal fertility of OoRptor 2/2 mice, we investigated PI3K signaling in OoRptor 2/2 oocytes. We found that the activity of Akt is enhanced in OoRptor 2/2 oocytes as indicated by the hyperphosphorylation of Akt at S473 and T308 (Fig. 3). This demonstrated that the loss of mTORC1 signaling leads to the hyperactivation of PI3K-Akt signaling in OoRptor 2/2 oocytes. Elevated PI3K-Akt signaling leads to normal follicular development in OoRptor 2/2 mouse ovaries To investigate whether ovarian follicular development in OoRptor 2/2 mice is normal due to the elevated PI3K-Akt signaling, we studied the morphology of ovaries collected from OoRptor 2/2 and OoRptor +/+ mice at PD35 and at 16 weeks of age. At PD35, follicles at various developmental stages ranging from primordial to preovulatory were found in OoRptor 2/2 ovaries ( Fig. 4B and D), and this was comparable to OoRptor +/+ ovaries ( Fig. 4A and C). In addition, we found healthy corpora lutea along with all types of follicles in OoRptor 2/2 ovaries at 16 weeks of age ( Fig. 4F and H), and this was also comparable to OoRptor +/+ ovaries ( Fig. 4E and G). These results show that the loss of mTORC1 signaling in OoRptor 2/2 oocytes leads to elevated PI3K-Akt signaling and that this is sufficient for normal follicle development.

Discussion
In this study, we used a mouse model with an oocyte-specific deletion of Rptor to show that Raptor in oocytes is essential for maintaining mTORC1 signaling in oocytes. Follicular develop-ment and fertility in mice lacking Rptor in their oocytes were not affected by the loss of mTORC1 signaling, but PI3K signaling was found to be elevated upon the loss of mTORC1 signaling in Rptordeleted oocytes. Due to the elevated PI3K-Akt signaling, ovarian follicular development and fertility were found to be normal in mice lacking Rptor in the oocytes of both primordial and furtherdeveloped follicles. Therefore, we conclude that loss of mTORC1 signaling in oocytes triggers a compensatory activation of the PI3K-Akt signaling cascade that maintains normal ovarian follicular development and fertility.
In our earlier study, we showed that constitutively enhanced oocyte PI3K-Akt signaling by loss of Pten (Phosphatase and tensin homolog) in primordial oocytes, which is the upstream negative regulator of PI3K-Akt signaling, causes global activation of all primordial follicles and premature ovarian failure (POF) [16]. In contrast, oocyte-specific deletion of Pdk1 (3-phosphoinositidedependent protein kinase-1), which plays a major role in phosphorylating and activating Akt and S6K1 [21], leads to the premature loss of primordial follicles and POF by suppressing Akt-S6K1 signaling. Interestingly, concurrent loss of Pdk1 and Pten in oocytes reverses the global activation of the primordial follicle pool caused by loss of Pten [19]. However, the global activation of primordial follicles in oocyte-specific Pten mutant mice (OoPten 2/2 ) is not completely prevented by treatment with rapamycin in vivo [22], which is a well-known pharmacological inhibitor of mTORC1 [8]. Similarly, phosphorylation of Akt is not altered when wild-type oocytes are treated with rapamycin in vitro [19]. However, our in vivo results demonstrate that loss of mTORC1 signaling in oocytes triggers a compensatory activation of the PI3K-Akt signaling cascade and that this is required to maintain normal ovarian follicular development and fertility.
Deletion of Tsc1 (tuberous sclerosis complex 1 or hamartin) in oocytes (OoTsc1 2/2 ), which is a negative regulator of mTORC1, also leads to premature activation of the entire pool of primordial follicles and subsequent POF due to the enhanced mTORC1 signaling in oocytes. Such over-activation of primordial follicles is rescued when OoTsc1 2/2 mutant mice are treated with rapamycin in vivo [23,24]. Together with the current paper, our studies indicate that the mTORC1 signaling may not be indispensable for physiological activation of primordial follicles.
In this study, compensatory activation of the PI3K-Akt signaling cascade was observed when Raptor was missing from the oocytes, and this activity appears to be essential to maintain normal physiological follicular development and fertility in OoRptor 2/2 females. Such compensatory activation of PI3K-Akt signaling has been seen in mice with both adipocyte-specific and skeletal muscle-specific ablation of Rptor [25,26]. Our results demonstrate that activation of PI3K-Akt signaling in the absence of mTORC1 signaling in oocytes is required to compensate for this loss and to support physiological development of ovarian follicles and female fertility. Although we observed the elevation of PI3K signaling in the absence of mTORC1 signaling, it is possible that other unidentified factors might contribute to the compensation of the Raptor deletion. Our results suggest the dual inhibition of both mTORC1 and PI3K pathways, which is commonly used to treat certain types of malignancies, might have adverse effect on follicular survival and female fertility.

Mice
Rptor loxP/loxP mice [14] in a C57BL/6J genomic background were crossed with transgenic mice carrying Gdf-9 promotermediated Cre recombinase [15,16] that also had a C57BL/6J background. After multiple rounds of crossing, we obtained homozygous mutant female mice lacking Rptor in their oocytes (OoRptor 2/2 mice). Control mice that do not carry the Cre transgene are referred to as OoRptor +/+ mice. The mice were housed under controlled environmental conditions with free access to water and food. Illumination was on between 0600 and 1800. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the University of Gothenburg and were carried out in accordance with the approved guidelines.

Reagents, antibodies, and immunological detection methods
Rabbit monoclonal antibody (EP539Y) to Raptor was purchased from Abcam. Rabbit polyclonal antibodies to phospho-S6K1 (T389), phospho-4E-BP1 (S65), and phospho-Akt (S473) as well as rabbit monoclonal antibodies to S6K1 and 4e-bp1 were obtained from Cell Signaling Technologies (Beverly, MA, USA). Mouse monoclonal antibody to phospho-Akt (T308) was purchased from BD Bioscience (Franklin Lakes, NJ, USA). Mouse monoclonal antibodies to b-actin and paraformaldehyde were purchased from Sigma-Aldrich Sweden AB (Stockholm, Sweden). Western blots were carried out according to the instructions of the suppliers of the different antibodies and visualized using the ECL Prime western blotting detection system (Amersham Biosciences, Uppsala, Sweden). Paraffin and hematoxylin were purchased from Histolab, Sweden.

Histological analysis
Ovaries were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. The paraffin-embedded ovaries were serially sectioned at 8-mm thickness and rehydrated followed by staining with hematoxylin for morphological observation. Ovarian  Ovarian morphology was determined based on images taken with a light microscope (Zeiss Axio Scope A1 upright microscope). One or both ovaries from more than three mice of each genotype were used for each time point.

Isolation of oocytes from postnatal mice ovaries
Mice were sacrificed by decapitation, and the ovaries were dissected free of fat and connective tissue using a dissection microscope. The ovaries were then minced with a pair of dissection scissors before being incubated in 0.05% collagenase in Dulbecco's modified Eagle's medium-F12 (DMEM/F12; Invitrogen) supplemented with 4 mg/mL bovine serum albumin (BSA), 100 units/mL penicillin, and 100 mg/mL streptomycin. The solution was mixed with frequent agitation and pipetting. After the tissues had mostly been digested by the collagenase, usually within 45-60 min, EDTA was added to this mixture to a final concentration of 40 mM and the mixture was incubated at 37uC with frequent pipetting for another 15-20 min until clusters of granulosa cells or other cells were completely dispersed. The mixture of cells and oocytes was then washed once and cultured in a 6 cm or 10 cm tissue culture dish with the above-mentioned serum-free DMEM/F12 medium for 12 h to allow the granulosa cells and other ovarian cells to attach to the plastic. The unattached oocytes and red blood cells were then recovered by collection of the supernatant and centrifugation at 1300 rpm for 5 min at room temperature. Red blood cells were subsequently removed using a hypotonic buffer containing 144 mM NH 4 Cl and 17 mM Tris-HCl (pH 7.2). After several washes, oocytes were collected by centrifugation. They were then lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 20 mM NaF, 20 mM b-glycerophosphate, 1 mM EDTA, 6 mM EGTA (pH 8.0), 1% NP-40, 1 mM DTT, 5 mM benzamidine, 1 mM . Normal follicular development in OoRptor 2/2 mice. Morphological analysis of ovaries from OoRptor 2/2 and OoRptor +/+ littermates at PD35 and at 16 weeks of age. Ovaries from OoRptor 2/2 and OoRptor +/+ mice were embedded in paraffin, and sections 8 mm thick were prepared and stained with hematoxylin. The overall development of follicles (arrows) and corpora lutea (CL) in OoRptor 2/2 mice was found to be normal (B, D, F, and H) compared to OoRptor +/+ mice (A, C, E, and G). doi:10.1371/journal.pone.0110491.g004 mTORC1 Signaling in Oocyte Development PLOS ONE | www.plosone.org PMSF, 250 mM sodium orthovanadate, 10 mg/mL aprotinin, 10 mg/mL leupeptin, and 1 mg/mL pepstatin followed by centrifugation at 14,000 rpm for 20 min at 4uC. The supernatants were collected and protein concentrations were measured using the bicinchoninic acid (BCA) protein assay, and equal amounts of proteins were used for western blot.