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Mol. Hum. Reprod. Advance Access originally published online on October 20, 2009
Molecular Human Reproduction 2009 15(12):765-770; doi:10.1093/molehr/gap092
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© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

This article appears in the following Molecular Human Reproduction issue: Special Issue: The ovary: from basic research to clinic [View the issue table of contents]

Disruption of Tsc2 in oocytes leads to overactivation of the entire pool of primordial follicles

Deepak Adhikari1,6,{dagger}, Gilian Flohr2,{dagger}, Nagaraju Gorre1, Yan Shen1, Hairu Yang1, Eva Lundin3, Zijian Lan4, Michael J. Gambello5 and Kui Liu1

1Department of Medical Biochemistry and Biophysics, Umeå University SE-901 87, Umeå, Sweden 2 Hogeschool Leiden, Zernikedreef 11, 2333 CK Leiden, The Netherlands 3Medical Biosciences/Pathology, Umeå University SE-901 87, Umeå, Sweden 4 University of Louisville Health Sciences Center, Louisville, KY, USA 5 University of Texas Health Science Center at Houston, Department of Pediatrics, Houston, Texas, USA

6 Correspondence address. E-mail: deepak.adhikari{at}medchem.umu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
To maintain the length of reproductive life in a woman, it is essential that most of her ovarian primordial follicles are maintained in a quiescent state to provide a continuous supply of oocytes. However, our understanding of the molecular mechanisms that control the quiescence and activation of primordial follicles is still in its infancy. In this study, we provide some genetic evidence to show that the tumor suppressor tuberous sclerosis complex 2 (Tsc2), which negatively regulates mammalian target of rapamycin complex 1 (mTORC1), functions in oocytes to maintain the dormancy of primordial follicles. In mutant mice lacking the Tsc2 gene in oocytes, the pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in oocytes. This results in depletion of follicles in early adulthood, causing premature ovarian failure (POF). Our results suggest that the Tsc1–Tsc2 complex mediated suppression of mTORC1 activity is indispensable for maintenance of the dormancy of primordial follicles, thus preserving the follicular pool, and that mTORC1 activity in oocytes promotes follicular activation. Our results also indicate that deregulation of Tsc/mTOR signaling in oocytes may cause pathological conditions of the ovary such as infertility and POF.

Key words: follicular activation/oocytes/Tsc/mTOR signaling/premature ovarian failure


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
To maintain the lengthy female reproductive life, the majority of primordial follicles must be preserved in a quiescent state for later use (Hirshfield, 1991; McGee and Hsueh, 2000). In order to produce mature ova, primordial follicles are recruited from the reservoir of dormant follicles into the growing follicular pool, through a process termed follicular activation (McGee and Hsueh, 2000). The activation of primordial follicles is a highly regulated process, and its underlying mechanisms are not fully revealed (Liu et al., 2006, 2007; Adhikari and Liu, 2009; Reddy et al., 2009). Menopause, or ovarian senescence occurs when the pool of primordial follicles has become exhausted (Faddy et al., 1992; Broekmans et al., 2007; Hansen et al., 2008).

Our earlier studies revealed that PTEN (phosphatase and tensin homolog deleted on chromosome 10), a negative regulator of phosphatidylinositol 3 kinase (PI3K), functions in oocytes as a suppressor of follicular activation (Reddy et al., 2008). Inhibition with rapamycin, the specific inhibitor of the mammalian serine/threonine kinase mammalian target of rapamycin (mTOR), indicated a role for mTOR as well as P13K in follicle activation (Reddy et al., 2008). Signaling events through mTOR, which regulate cell growth and proliferation in many types of cells, are closely related to the PI3K signaling pathway (Wullschleger et al., 2006; Guertin and Sabatini, 2007). mTOR modulates important processes such as protein synthesis, ribosome biogenesis and autophagy (Sarbassov et al., 2005; Wullschleger et al., 2006; Guertin and Sabatini, 2007). In human cells, mTOR complex 1 (mTORC1) activity is negatively regulated by a heterodimeric complex consisting of two protein molecules: tuberous sclerosis complex 1 (TSC1, or hamartin) and tuberous sclerosis complex 2 (TSC2, or tuberin). TSC1 and TSC2 are products of two distinct tumor suppressor genes: TSC1 and TSC2, respectively. These two genes are the genetic loci that are mutated in the autosomal dominant tumor syndrome tuberous sclerosis complex (TSC), which is characterized by numerous benign tumors (Kwiatkowski et al., 2002; Crino et al., 2006; Yang and Guan, 2007). The TSC1–TSC2 complex suppresses the activation of mTORC1 through a GTPase activating protein domain located in TSC2. The function of TSC1 is to stabilize TSC2 and protect it from ubiquitination and degradation (Chong-Kopera et al., 2006).

To determine whether the Tsc/mTORC1 signaling in oocytes takes part in the regulation of follicular activation, in this study, we deleted the Tsc2 gene from mouse oocytes in primordial and developing follicles. Deletion of Tsc2 caused premature activation of all primordial follicles around the time of puberty, due to elevated mTORC1 activity in oocytes. This eventually led to follicular depletion in early adulthood, causing premature ovarian failure (POF). Thus, the Tsc1–Tsc2 complex mediated suppression of mTORC1 activity in oocytes is indispensable for sustaining the dormancy of primordial follicles, which is essential for preserving the female reproductive lifespan.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 Acknowledgements
 References
 
Mice
Tsc2loxP/loxP mice (Hernandez et al., 2007) with a mixed 129S4/SvJae and C57BL/6J genomic background were crossed with transgenic mice carrying growth differentiation factor 9 (Gdf-9) promoter-mediated Cre recombinase that had a C57BL/6J background (Lan et al., 2004; Reddy et al., 2008). After multiple rounds of crossing, we obtained homozygous mutant female mice lacking Tsc2 in oocytes (referred to as OoTsc2–/– mice). Control mice that do not carry the Cre transgene are referred to as OoTsc2+/+ mice. All comparisons were made between littermates. The mice were housed under controlled environmental conditions with free access to water and food. Illumination was on between 0600 and 1800 h. Experimental protocols were approved by the regional ethical committee of Umeå University, Sweden.

Reagents, antibodies and immunological detection methods
Rabbit polyclonal antibodies to phospho-rpS6 (S240/4), Tsc2/tuberin and phospho-S6K1 (T389), and also rabbit monoclonal antibody to S6K1 were obtained from Cell Signaling Technologies (Beverly, MA, USA). Mouse monoclonal antibody to rpS6 was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Mouse monoclonal antibody to β-actin was 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 Plus Western Blotting Detection System (Amersham Biosciences, Uppsala, Sweden).

Histological analysis of ovarian tissues
Histological analysis of ovary was performed as previously described (Reddy et al., 2008). Briefly, ovaries were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Paraffin-embedded ovaries were serially sectioned at 8-µm thickness and stained with hematoxylin for morphological observation, and all sections from each ovarian block were examined. One or both ovaries from more than three mice of each genotype were used for each time point.

Isolation of oocytes from post-natal mouse 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 dissolved 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 µg/ml streptomycin, with frequent agitation and pipetting. After the tissues had mostly been digested by 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 37°C 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 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 160 g for 5 min at room temperature. Red blood cells were subsequently removed using a hypotonic buffer containing 144 mM NH4Cl 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 β-glycerophosphate, 1 mM EDTA, 6 mM EGTA (pH 8.0), 1% NP-40, 1 mM DTT, 5 mM benzamidine, 1 mM PMSF, 250 µM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 µg/ml pepstatin, followed by centrifugation at 14 576 g for 20 min at 4°C. 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 Acknowledgements
 References
 
Generation of mutant mice with oocyte-specific deletion of Tsc2
We generated mutant mice in which the Tsc2 gene was deleted in oocytes of primordial and developing follicles (OoTsc2–/– mice) by crossing Tsc2loxP/loxP mice (Hernandez et al., 2007) with transgenic mice expressing Gdf-9 promoter-mediated Cre recombinase (Lan et al., 2004). Western blot result showed that the expression of the Tsc2 protein (tuberin) was completely absent in OoTsc2–/– oocytes (Fig. 1), indicating a successful deletion of the Tsc2 gene from oocytes.


Figure 1
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  		Figure 1 Oocyte-specific deletion of Tsc2 in mice. Western blots showing the absence of Tsc2 (tuberin) protein expression in oocytes of OoTsc2–/– mice. Oocytes were isolated from ovaries of 12–14-day-old OoTsc2+/+ and OoTsc2–/– mice as described in Materials and Methods. For each experiment, material from three to five mice was used per lane. For each lane, around 20 µg of protein was loaded. Level of β-actin was used as an internal control. The experiments were repeated three times and representative images are shown.

 
Shortened reproductive lifespan of OoTsc2–/– female mice
We found that the OoTsc2–/– females had a normal vaginal opening at the age of 5–6 weeks (which is the appropriate age). However, during the examined period from 7 to 27 weeks of age, OoTsc2–/– females were found to produce at most 2 litters of normal size, then they became infertile in young adulthood (i.e. after 12–13 weeks of age) (Fig. 2).


Figure 2
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  		Figure 2 Subfertility in mice lacking Tsc2 in oocytes. Comparison of the cumulative number of pups per OoTsc2–/– female (n = 6, dashed line) and per OoTsc2+/+ female (n = 6, solid line). All OoTsc2–/– females became infertile in young adulthood after Week 12–13.

 
Enhanced mTORC1 activity in OoTsc2–/– oocytes
We found that loss of Tsc2 led to enhanced mTORC1 activity in OoTsc2–/– oocytes, as indicated by the elevated phosphorylation of mTORC1 substrate S6K1 (p70 S6 kinase 1) (Fig. 3, p-S6K1, T389). Such elevated p-S6K1 had apparently led to an increase in activity, as the phosphorylation of the substrate of S6K1, rpS6 (ribosomal protein S6), was dramatically elevated in OoTsc2–/– oocytes (Fig. 3, p-rpS6, S240/4). These data showed that the mTORC1–S6K1–rpS6 signaling were elevated in OoTsc2–/– oocytes.


Figure 3
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  		Figure 3 Studies of mTORC1 signaling in OoTsc2–/– and OoTsc2+/+ oocytes. Comparison of Tsc2/mTORC1 signaling in OoTsc2–/– and OoTsc2+/+ oocytes. Oocytes were isolated from ovaries of mice at PD12–14 and western blot was performed as described in Materials and Methods. mTORC1 signaling in OoTsc2–/– oocytes was enhanced, as indicated by elevated levels of phosphorylated S6K1 (p-S6K1, T389) and phosphorylated rpS6 (p-rpS6, S240/4). Levels of total S6K1, rpS6 and β-actin were used as internal controls. The experiments were repeated three times. For each experiment, material from three to five mice was used per lane. Representative images are shown.

 
Accelerated activation of the entire pool of primordial follicles followed by premature follicular depletion in OoTsc2–/– mice
To learn how the elevated mTORC1–S6K1–rpS6 signaling in oocytes may lead to shortened reproductive lifespan in OoTsc2–/– mice, we compared the post-natal follicular development in OoTsc2–/– mice to that in OoTsc2+/+ mice. We found that the pool of primordial follicles was formed normally in OoTsc2–/– mice, and no apparent morphological difference was observed in post-natal day (PD) 13 ovaries of OoTsc2–/– (Fig. 4B) and OoTsc2+/+ (Fig. 4A) mice, where ovaries of both mutant and control mice had clusters of primordial follicles (Fig. 4A and B, inset, arrows). At PD23, the OoTsc2–/– ovaries (Fig. 4D) appeared somewhat larger than the OoTsc2+/+ ovaries (Fig. 4C). By this age, all primordial follicles had been activated with enlarged oocytes in OoTsc2–/– ovaries (Fig. 4D, inset, red arrows). However, in OoTsc2+/+ ovaries, cluster of primordial follicles were seen (Fig. 4C, inset, arrows). At PD35, OoTsc2–/– ovaries appeared larger, and contained many activated follicles with enlarged oocytes (Fig. 4F, inset, red arrows), whereas the control OoTsc2+/+ ovaries were much smaller (Fig. 4E) and contained clusters of primordial follicles (Fig. 4E, inset, arrows). All types of activated follicles, including transient follicles (containing enlarged oocyte surrounded by flattened pregranulosa cells), type 3b, type 4, type 5 and type 6 follicles were seen in OoTsc2–/– ovaries. Thus, with Tsc2 deleted in oocytes, the entire pool of primordial follicles was activated by PD23.


Figure 4
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  		Figure 4 Activation of the entire pool of primordial follicles leading to follicle depletion and POF at young adulthood in OoTsc2–/– mice. (A–F) Morphological analysis of ovaries from OoTsc2–/– and OoTsc2+/+ littermates at PD13, PD23 and PD35. Ovaries from13-, 23- and 35-day-old OoTsc2+/+ and OoTsc2–/– mice were embedded in paraffin, and serial sections of 8-µm thickness were prepared and stained with hematoxylin. At PD13, similar ovarian morphologies were seen in sections from OoTsc2+/+ (A) and OoTsc2–/– (B) ovaries. Cluster of primordial follicles were seen in both OoTsc2+/+ (A, inset, arrows) and OoTsc2–/– (B, inset, arrows) ovaries at PD13. At PD23, OoTsc2–/– ovaries were larger (D) than OoTsc2+/+ ovaries (C). No primordial follicles could be seen by PD23 in OoTsc2–/– ovaries, and almost all the primordial follicles were activated with apparently enlarged oocytes surrounded by flattened granulosa cells (D, inset, red arrows). In OoTsc2+/+ ovaries primordial follicles could be readily observed at PD23 (C, inset, arrows). At PD35, OoTsc2–/– ovaries were much larger (F), with all the follicles activated (F, inset, red arrows) than OoTsc2+/+ ovaries (E), which still had cluster of primordial follicles (E, inset, arrows). (G–H) Morphological analysis of ovaries from OoTsc2–/– and OoTsc2+/+ littermates at 4 months of age. At 4 months of age, almost all follicles had degenerated and healthy follicular structures had completely disappeared in OoTsc2–/– ovaries (H). Two degenerating oocytes were shown (H, inset, arrows). As a control, OoTsc2+/+ mice had normal ovarian morphology (G). CL was seen showing that OoTsc2+/+ mice had been ovulating (G). The experiments were repeated at least four times, and for each time and each age, ovaries from one mouse of each genotype were used. CL, corpus luteum.

 
By 4 months of age, no healthy follicular structure could be identified in OoTsc2–/– ovaries (Fig. 4H), and only unhealthy dying oocytes were observed (Fig. 4H, inset, arrows). In contrast, the control OoTsc2+/+ mice contained healthy follicles and corpus luteum (CL) (Fig. 4G). Thus, premature activation of the primordial follicle pool led to follicle depletion and POF in OoTsc2–/– mice in early adulthood.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 Acknowledgements
 References
 
In this study, by using a mouse model with oocyte-specific deletion of the Tsc2 gene, we showed that the tumor suppressor Tsc2 plays an essential physiological role in oocytes to preserve the female reproductive lifespan by suppressing activation of primordial follicles. We demonstrated that deletion of Tsc2 in oocytes leads to premature activation of the entire pool of primordial follicles. We also showed that the driving force underlying the overactivation of primordial follicles in OoTsc2–/– mice is the elevated intra-oocyte mTORC1–S6K1–rpS6 signaling. We observed all types of activated follicles, including transient, types 3b, 4, 5 and 6 follicles in OoTsc2–/– ovaries. However, even though more primordial follicles were prematurely recruited into the growing follicular pool in OoTsc2–/– mice, the litter sizes of OoTsc2–/– mice do not seem to be apparently different from those of OoTsc2+/+ mice.

In our parallel study with a mouse model carrying oocyte-specific deletion of Tsc1 (referred to as OoTsc1–/– mice), we found a similar phenotype in that the entire pool of primordial follicles was prematurely activated in young adulthood (Adhikari et al., 2009). These studies indicate that the tumor suppressors Tsc1 and Tsc2 do play physiological roles in oocytes of primordial follicles to suppress their activation. On the basis of the current study and our parallel study with the Tsc1 in mice (Adhikari et al., 2009), we confirm that the Tsc1–Tsc2 complex mediated suppression of mTORC1 activity in oocytes is indispensable for maintenance of the dormancy of primordial follicles, thus preserving the follicular pool.

Nevertheless, one major unsolved question is that how the expressions and functions of Tsc1 and Tsc2 in oocytes are regulated, in order to orchestrate follicular activation throughout the female reproductive life.

It was hypothesized more than a decade ago that dormant primordial follicles may be under constant inhibitory influences of local origin to remain quiescent (Wandji et al., 1996). Our earlier reports have shown that PTEN in oocytes functions as a suppressor of follicular activation (Reddy et al., 2008). It is clear now that Tsc2 and Tsc1 (Adhikari et al., 2009) in oocytes are also parts of the inhibitory mechanisms. Other recognized inhibitory molecules include the cyclin-dependent kinase (Cdk) inhibitor p27kip1 (p27, or Cdkn1b) (Rajareddy et al., 2007), which functions in both oocytes and pregranulosa cells to suppress follicular activation; and Foxo3a, which is a downstream transcription factor of the PTEN/PI3K pathway that functions in oocytes to suppress follicular activation (Castrillon et al., 2003; Reddy et al., 2008).

In women, reproductive lifespan and menopausal age are determined by their ovarian reserve, i.e. the size and persistence of the primordial follicle pool. Our results from the current study and several recent reports using genetically modified mouse models imply that deregulation of signaling events in oocytes, such as the Tsc1–Tsc2/mTORC1 signaling (Adhikari et al., 2009), the PTEN/PI3K signaling (Reddy et al., 2008, 2009) and the p27-Cdk system (Rajareddy et al., 2007), may contribute to defects in primordial follicle development in humans, which may result in pathological conditions of the ovary, such as POF and infertility. The possible involvement of the above-mentioned molecules in human POF, however, needs to be investigated. In this sense, our work may have both physiological and clinical implications.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
Acknowledgements
 References
 
This work was supported by grants from the Swedish Research Council (K.L.), the Swedish Cancer Foundation (K.L.), the Young Researcher Award of Umeå University, Sweden (K.L.), the Lions Cancer Research Foundation in Norrland, Sweden (K.L.), and the Novo Nordisk Foundation, Denmark (K.L.), and the Cutting-Edge Research Grant from the County Council of Västerbotten, Sweden (E.L.).


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
References
 
We thank Dr Austin Cooney (Baylor College of Medicine, TX, USA) for kindly providing the Gdf-9-Cre mice.


   Footnotes
 
{dagger} These authors equally contributed to this article. Back


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 Introduction
 Materials and Methods
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 Discussion
 Funding
 Acknowledgements
 References
 
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Submitted on October 9, 2009; resubmitted on October 16, 2009; accepted on October 17, 2009.


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D. Adhikari, W. Zheng, Y. Shen, N. Gorre, T. Hamalainen, A. J. Cooney, I. Huhtaniemi, Z.-J. Lan, and K. Liu
Tsc/mTORC1 signaling in oocytes governs the quiescence and activation of primordial follicles
Hum. Mol. Genet., February 1, 2010; 19(3): 397 - 410.
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