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p27^kip1 (Cyclin-Dependent Kinase Inhibitor 1B) Controls Ovarian Development by Suppressing Follicle Endowment and Activation and Promoting Follicle Atresia in Mice

Department of Medical Biochemistry and Biophysics (S.R., P.R., C.D., L.L., K.J., W.T., Y.S., K.L.), Umeå University, SE-901 87 Umeå, Sweden; Qilu Hospital (L.L.), Shandong University, Jinan, 250012 Shandong, China; National Cancer Institute (C.B., P.K.), Mouse Cancer Genetics Program, National Cancer Institute-Frederick, Frederick, Maryland 21702-1201; and Department of Inflammation (S.L.P.), Autoimmunity and Transplantation Research, Roche Palo Alto, LLC, Palo Alto, California 94304

Address all correspondence and requests for reprints to: Kui Liu, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden. E-mail: kui.liu{at}medchem.umu.se.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In humans, the molecular mechanisms underlying ovarian follicle endowment and activation, which are closely related to the control of female reproduction, occurrence of menopause, and related diseases such as premature ovarian failure, are poorly understood. In the current study, we provide several lines of genetic evidence that the cyclin-dependent kinase (Cdk) inhibitor 1B (commonly known as p27^kip1 or p27) controls ovarian development in mice by suppressing follicle endowment and activation, and by promoting follicle death. In p27-deficient (p27^–/–) mice, postnatal follicle assembly was accelerated, and the number of endowed follicles was doubled as compared with p27^+/+ mice. Moreover, in p27^–/– ovaries the primordial follicle pool was prematurely activated once it was endowed, and at the same time the massive follicular death that occurs before sexual maturity was rescued by loss of p27. In early adulthood, however, the overactivated follicular pool in p27^–/– ovaries was largely depleted, causing premature ovarian failure. Furthermore, we have extensively studied the molecular mechanisms underlying the above-mentioned phenotypes seen in p27^–/– ovaries and have found that p27 controls follicular development by several distinct mechanisms at different stages of development of the ovary. For example, p27 controls oocyte growth by suppressing the functions of Cdk2/Cdc2-cyclin A/E1 in oocytes that are arrested at the diplotene stage of meiosis I. This function of p27 is distinct from its well-known role as a suppressor of cell cycle progression. In addition, we have found that p27 activates the caspase-9-caspase-3-caspase-7-poly (ADP-ribose) polymeraseapoptotic cascade by inhibiting Cdk2/Cdc2-cyclin A/B1 kinase activities in follicles, thereby inducing follicle atresia. Our results suggest that the p27 gene is important in determining mammalian ovarian development. This study therefore provides insight into ovary-borne genetic aberrations that cause defects in folliculogenesis and infertility in humans.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
FEMALE GERM CELLS in prenatal mouse ovaries are found in the form of clusters (syncytia) (see schematic diagram in Fig. 1A), which are connected by intercellular bridges as a result of incomplete cytokinesis (1). By the time mice are a few days old, individual oocytes are enclosed in primordial follicles with a few flattened pregranulosa cells and become arrested at the diplotene stage of meiosis I (Fig. 1A). This process is accompanied by widespread apoptosis of oocytes, leading to the endowment of finite numbers of primordial follicles in the ovaries (Fig. 1A) (2). The molecular mechanisms that control ovarian primordial follicle assembly have not been thoroughly investigated. Studies in mice and sheep have revealed that during follicle formation, somatic cells invade the clusters of germ cells, and syncytial breakdown occurs just before primordial follicles are assembled (Fig. 1A) (3, 4), suggesting that pregranulosa cells have an active role in the formation of follicles (5).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Schematic Diagram of Follicle Formation and Endowment, Primordial Follicle Activation, and Follicle Atresia during the Initial Wave of Postnatal Ovarian Development in Mice A, Female germ cells in prenatal mouse ovaries are in the form of clusters (syncytia), which are surrounded by somatic cells. By the time mice are a few days old, somatic cells invade the clusters of germ cells, and syncytial breakdown occurs just before primordial follicles are assembled, which is accompanied by widespread apoptosis of oocytes leading to the endowment of finite numbers of primordial follicles in the ovaries. B, Once formed, the pool of primordial follicles serves as the source of developing follicles. During primordial follicle activation in mammals, the rapid growth of oocytes is a major event. During the phase of oocyte growth, granulosa cells proliferate from one layer of a few flattened pregranulosa cells in primordial follicles to three layers of cuboidal granulosa cells by the time oocyte growth is almost complete. Most of the granulosa cell proliferation and differentiation that is stimulated by FSH occurs after the oocyte has almost stopped growing, however, which is not shown in this schematic diagram. C, During the initial wave of postnatal ovarian development in mice, large quantities of oocytes/follicles are removed from the nongrowing follicle reservoir, a phenomenon similar to the rapid loss of oocytes/follicles before puberty in human ovaries. The diagram has been adapted from published diagrams of Bristol-Gould et al. (36 ) and Liu et al. (24 ).

In addition, during the initial wave of postnatal ovarian development in mice, large quantities of oocytes/follicles are depleted from the nongrowing follicle reservoir (6, 10, 11) (Fig. 1C). Compared with the numerous primordial follicles that have died and disappeared, the number of primordial follicles recruited into the growing population is much lower (10, 11). This phenomenon is similar to the rapid loss of oocytes/follicles before puberty in human ovaries (6). The reproductive life of the female is terminated when the pool of primordial follicles is exhausted (2, 6). However, the mechanisms that govern the activation of primordial follicles and the rapid death of nongrowing follicles have not been fully elucidated.

Previous studies from our and other research groups have demonstrated that a component of the phosphatidylinositol 3 kinase (PI3K) pathway, Foxo3a, serves as a suppressor of follicular activation and growth (12, 13). We have shown that when Foxo3a is overexpressed in oocytes of primary follicles, oocyte growth and follicular development are retarded (13). We presumed that one of the causes might be the retained expression of the cyclin-dependent kinase (Cdk) inhibitor 1B, commonly known as p27^kip1 or p27, in the nuclei of oocytes (13). This has led us to hypothesize that p27 may suppress early follicular development. p27 itself is a negative regulator of cell cycle progression and a tumor suppressor (14). In addition, p27 has been shown to be essential for ovulation and luteinization in mice (15, 16, 17). However, the role and related mechanisms of p27 in controlling early follicular development and oocyte growth have not been investigated. In the current study, we have used wild-type and p27-deficient (p27^–/–) mice and provide several lines of evidence to demonstrate that 1) p27 suppresses follicle endowment/formation and activation; 2) p27 induces follicle atresia that occurs before sexual maturity; and 3) the overactivated follicles in p27^–/– ovaries are depleted in early adulthood, causing premature ovarian failure (POF). The molecular mechanisms underlying these phenotypes in p27^–/– ovaries were also extensively investigated. We propose that p27 is a key molecule in manipulation of ovarian development in mammals.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Dynamic Expression Patterns of p27 Protein in Perinatal Mouse Ovaries
Unexpectedly, we found that in perinatal mouse ovaries that precede follicle formation, p27 is expressed in somatic cells but not in oogonia/oocytes. At embryonic d 14.5 (E14.5) (Fig. 2A), E18.5 (data not shown), and postnatal day (PD) 1 (Fig. 2B), p27 expression was not detected in dividing oogonia or oocytes by immunostaining (Fig. 2, A and B, yellow arrows), but could be seen in somatic cells surrounding germ cells (Fig. 2, A and B; red arrows). As shown in the inset of Fig. 2B (white arrow), germ cells were identified by staining of germ cell nuclear antigen 1 (GCNA1). At PD4, when the primordial (type 2) follicles had already been formed, p27 expression was initiated in the oocyte nuclei of primordial follicles (Fig. 2C, yellow arrow) and growing transient (type 3a) follicles (Fig. 2C, yellow arrowhead, and inset). Expression of p27 was also observed in pregranulosa cells (Fig. 2C). In growing primary (type 3b) follicles (Fig. 2C, white arrow) and secondary (type 4) follicles (Fig. 2D, white arrows), p27 was found to be expressed in both oocyte nuclei and in granulosa cells. In further developed follicles from 23-d-old mice, where oocytes are partially grown, however, p27 was no longer found to be expressed in oocyte nuclei (Fig. 2D, yellow arrow) but was still expressed in granulosa cells (Fig. 2D, white arrowhead). The specificity of p27 immunostaining was verified on ovarian sections from p27^–/– mice (15), where only minimal background staining was seen in granulosa cells (Fig. 2E, white arrows), but not in oocytes (Fig. 2E, yellow arrows).

View larger version (53K): [in this window] [in a new window]   Fig. 2. Expression of p27 in Embryonic and Postnatal Mouse Ovaries A–D, Ovaries from E14.5, 1-, 4-, and 23-d-old wild-type mice were isolated, fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections of 5-µm thickness (E14.5 and PD1) or 8-µm thickness (PD4 and 23) were prepared and immunostained for the presence of p27 as described in Materials and Methods. Signals appear as reddish to reddish-brown color using AEC as substrate. The ages of the mice are stated individually for each ovarian section. Germ cells were identified using an antibody against GCNA1 (B, arrow in inset). E, Immunostaining of p27 in ovarian sections from p27–/– mice as a negative control. F, Expression of p27 in oocytes that were larger or smaller than 25 µm. Oocytes were isolated from 17- to 20-d-old female C57/BL6J mice, sorted using a cell dispersing screen with 25-µm opening, and lysed as described in Materials and Methods. Levels of ß-actin were used as an internal control. The experiments were repeated three times, and representative results are shown. For each experiment and each genotype in panel F, 15–20 mice were used.

Elevated Postnatal Ovarian Follicle Endowment in p27^–/– Mice
To determine the functional roles of p27 that is expressed by somatic cells surrounding oocytes in perinatal ovaries during follicular formation and endowment, we used p27^–/– mice. We first quantified the oocyte numbers at PD1 and follicle numbers at PD8 by counting oocytes in serial sections of whole ovaries from p27^–/– and p27^+/+ mice, to determine whether the lack of p27 in embryonic and postnatal ovaries influences the endowment of finite numbers of follicles in mice. As shown in Table 1, at PD1 the GCNA1-positive oocytes in p27^–/– ovaries were approximately double compared with those in p27^+/+ovaries. The follicles in PD8 p27^–/– ovaries were also approximately double compared with those in p27^+/+ovaries (Table 1 and Fig. 3). These data indicate that more follicles are endowed in p27^–/– ovaries. When the number of follicles at PD8 was compared with the number of GCNA1-positive oocytes at PD1, a similar rate of oocyte survival (57%) was determined in p27^–/– and p27^+/+ ovaries. Thus, we presume that the increased number of follicles in p27^–/– mice may be caused by elevated mitosis in female germ cells. The numbers of follicles in p27^+/+ ovaries counted in this study are comparable to those in a previous report by Pepling and Spradling (4).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Numbers of Ovarian Follicles in p27–/–and p27+/+ Mice Ovaries from 8-, 18-, 23-, and 35-d-old p27–/– and p27+/+ littermates were embedded in paraffin and serial sections of 8-µm thickness were prepared and stained with hematoxylin, after which total numbers of primordial (type 2) and activated follicles (as the sum of type 3a, type 3b, and types 4–7 follicles) per ovary (mean ± SD) were counted, as described in Materials and Methods. A, Numbers of all follicles, primordial follicles, and activated follicles per ovary in p27+/+ mice at different ages. B, Numbers of all follicles, primordial follicles, and activated follicles per ovary in p27–/– mice at different ages. For each genotype and each age, ovaries from three to six mice were used. The figures are derived from Table 2.

View larger version (82K): [in this window] [in a new window]   Fig. 4. Histological Analysis of Postnatal Ovaries from p27–/– and p27+/+ Littermates Ovarian sections of 5-µm thickness (PD1) or 8-µm thickness (PD2, PD8, PD35, and 12-wk) were prepared and stained with anti-GCNA1 antibody (A–C) or anti-VASA antibody (D) to identify oocytes, or stained with hematoxylin for morphological observation (E–J), as described in Materials and Methods. All experiments were repeated at least four times, and representative images of ovaries are shown. CL (I, arrows): corpora lutea.

Averted Follicle Atresia before Sexual Maturity in p27^–/– Mice
During the initial wave of postnatal follicular development in mice, large numbers of follicles disappear from the nongrowing follicle pool before the onset of sexual maturity, as a result of follicle atresia (10, 11). In agreement with previous reports, we found that in wild-type mice, the total number of follicles per ovary declined sharply from PD8 (1863) to PD35 (706) (Table 1 and Fig. 3A). During this period, the numbers of activated follicles in p27^+/+ ovaries were found to be relatively constant: 539 per ovary at PD8 and 380 at PD35 (Table 2 and Fig. 3A). The number of primordial follicles in p27^+/+ ovaries decreased dramatically, from 1325 per ovary at PD8 to 326 at PD35 (Table 2 and Fig. 3A), either by atresia, or by entering the growing follicle pool, which undergoes atresia at a later stage. The number of all follicles in PD35 p27^+/+ ovaries indicates a follicle survival rate of 38% relative to PD8 (Table 1).

Even so, in p27^–/– mice the total numbers of follicles per ovary at PD18, PD23, and PD35 were close to that at PD8, which did not show any significant decrease (Table 1 and Fig. 3B). The number of follicles in p27^–/– mice at PD35 was 3979, which was almost 6 times than that in PD35 p27^+/+ ovaries (706) (Table 1). As also demonstrated in Fig. 3B, in p27^–/– mice follicle loss occurring before sexual maturity was prevented, indicating that the premature activation of primordial follicles in p27^–/– mice may have rescued them from atresia.

Studies on the Molecular Mechanisms Underlying Enhanced Follicle Formation in p27^–/– Mice
Elevated Kinase Activity Associated with Cdk2-Cyclin A in Ovaries of Newborn p27^–/– Mice.
Based on the findings that lack of Cdk4 (20) or Cdk6 (Ref. 21 and our unpublished data) in mice does not cause any apparent defect in ovarian follicle formation and activation, we hypothesized that the major target of p27, Cdk2 (22), may be the positive regulator that drives the overformation of follicles in p27^–/– mice. We first measured the kinase activities associated with Cdk2, Cdc2, cyclin A, cyclin B1, and cyclin E in newborn (PD0) p27^+/+ and p27^–/– ovaries, which is a developmental stage that precedes follicle formation. As shown in Fig. 5, we found that kinase activities associated with Cdk2 and cyclin A were elevated in newborn p27^–/– ovaries relative to those in PD0 p27^+/+ ovaries. Kinase activities associated with Cdc2 (Fig. 5), cyclin B1, and cyclin E1 (data not shown) were at similar levels in PD0 p27^–/– and p27^+/+ ovaries. These results suggest that the increased kinase activity of the Cdk2-cyclin A complex may be one of the forces that drive the accelerated follicle formation and endowment. The cellular localization of the elevated Cdk2-cyclin A kinase activity remains to be determined.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Studies on the Molecular Mechanisms of Follicle Formation in p27–/– Mice Kinase activities associated with Cdk2, cyclin A, and Cdc2 in ovaries of newborn (PD0) p27–/– and p27+/+ mice. Ovaries from PD0 p27–/– and p27+/+ mice were collected and homogenized, and ovarian lysates (150 µg) were used for immunoprecipitation with indicated antibodies. Kinase activity assay was performed using histone H1 as substrate, as described in Materials and Methods. The experiments were repeated three times, and representative images are shown. For each experiment and each genotype, 20–30 mice were used. IP, Immunoprecipitation.

View larger version (43K): [in this window] [in a new window]   Fig. 6. Studies of Follicle Activation in p27–/– Mice A, Kinase activities associated with Cdk2, cyclin A, cyclin E1, Cdc2, and cyclin B1 in isolated oocytes of p27–/– and p27+/+ mice. Oocytes from ovaries of 8- to 12-d-old p27–/– and p27+/+ mice were isolated and lysed, and 50 µg of oocyte lysates were used for immunoprecipitation with indicated antibodies. Kinase activity assay was performed using histone H1 as substrate, as described in Materials and Methods. The experiments were repeated three times, and representative images are shown. For each experiment, 10–15 p27–/– mice and 20–25 p27+/+ mice were used. B, Suppression of follicle activation by Roscovitine in cultured PD2 ovaries of p27–/– mice. Ovaries of PD2 p27–/– mice (where primordial follicles have formed) were cultured without or with treatment of the Cdk2/Cdc2 inhibitor Roscovitine (20 µM), as described in Materials and Methods. After the 3-d culture period, the ovaries were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections of 8-µm thickness were stained with hematoxylin for morphological observation of follicular activation. All experiments were repeated at least four times, and representative images are shown. C and D, Normal expression of p27 and Foxo3a in Foxo3a–/–and p27–/– oocytes, respectively. Oocytes from ovaries of 8- to 12-d-old Foxo3a–/– or p27–/– mice and their wild-type control mice were isolated. Western blots were performed to measure protein levels of Akt, p-Akt (serine 473), Foxo3a, and p-Foxo3a (threonine 32) in p27–/– and p27+/+ oocytes, and levels of p27 expression in Foxo3a–/– and Foxo3a+/+ oocytes, as described in Materials and Methods. Levels of ß-actin are shown as internal controls. The experiments were repeated three times. For each experiment and each genotype, five to 10 mice were used. Representative images are shown. E–K, Comparison of primordial follicle activation in ovaries of p27–/–, Foxo3a–/–, and p27–/–Foxo3a–/– DKO mice. Ovaries from 13- and 23-d-old p27–/–, Foxo3a–/–, and p27–/–Foxo3a–/– DKO mice were isolated, fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections of 8-µm thickness were prepared and stained with hematoxylin for morphological observation, as described in Materials and Methods. Primordial follicles (E–G, arrows) and activated or transient follicles (E–G, arrowheads) are indicated. The genotypes and ages of mice are given in the figure. K, Percentages of primordial follicles per ovary (mean ± SD) in ovaries of 13-d-old wild-type, p27–/–, Foxo3a–/–, and p27–/–Foxo3a–/– DKO mice. Follicles were counted as described in Materials and Methods (three ovaries from three individual mice were used for each genotype). Lowercase letters (a, b, c, and d) indicate significant differences (P < 0.01). IP, Immunoprecipitation.

Follicle Activation Is Suppressed in p27^–/– Ovaries upon Inhibition of Both Cdk2 and Cdc2.
To evaluate the importance of Cdk2 and Cdc2 in the overactivation of primordial follicles in p27^–/– ovaries, we cultured 2-d-old p27^–/– ovaries (where primordial follicles had formed) with Roscovitine (20 µM), a substance that specifically inhibits both Cdk2 and Cdc2. We found that Roscovitine could largely block the activation of primordial follicles in cultured PD2 p27^–/– ovaries (Fig. 6B, arrows). Therefore, it is likely that in oocytes of p27^–/– primordial follicles, although Cdc2 kinase activity is not elevated, kinase activities associated with both Cdk2-cyclin A/E1 and Cdc2-cyclin A/E1 complexes are important for the accelerated follicular activation.

p27 and Foxo3a Suppress Primordial Follicle Activation Independently of Each Other.
Recently, activation of the oocyte PI3K pathway, including the activation of Akt and suppression of Akt substrate Foxo3a, has been suggested to be important for initiation and further development of follicles (13, 18, 24). Foxo3a is an Akt substrate that can be phosphorylated and suppressed by Akt (25, 26). Previous reports have indicated that loss of Foxo3a in mice leads to early activation of primordial follicles (12), and overexpression of Foxo3a in mouse oocytes facilitates the nuclear localization of p27 in oocytes, which is thought to be partly responsible for the retarded oocyte and follicular development in the transgenic mice (13). Thus, we endeavored to study whether p27 and Foxo3a functions in an up- or downstream cascade that suppresses follicle activation.

We found that the expression of p27 and Foxo3a appeared to be normal in ovaries of 8- and 13-d-old Foxo3a^–/– (27) and p27^–/– mice, respectively, as detected by Western blot and immunohistochemistry (data not shown). Moreover, in oocytes isolated from 8- to 12-d-old mice where overactivation of primordial follicles has been seen in both Foxo3a^–/– and p27^–/– ovaries, the expression/phosphorylation levels of Akt and Foxo3a in p27^–/– oocytes were similar compared with those in p27^+/+ oocytes (Fig. 6C); and the p27 expression in Foxo3a^–/– oocytes was also at a similar level as compared with Foxo3a^+/+ oocytes (Fig. 6D).

We then crossed p27^–/– mice with Foxo3a^–/– mice to generate double knockout (DKO) mice that lack both p27 and Foxo3a, and compared the ovarian phenotypes of p27^–/– mice, Foxo3a^–/– mice, and p27^–/–Foxo3a^–/– DKO mice. By looking at PD13, which is a time when p27^–/– ovaries still have some primordial follicles (Fig. 6E, arrows), we found that in Foxo3a^–/– ovaries, in agreement with the previous report (12), there were more activated transient follicles, which appeared to have somewhat enlarged oocytes surrounded by flattened pregranulosa cells (Fig. 6F, arrowheads) that distinguish them from primordial follicles (Fig. 6F, arrows). In p27^–/– ovaries, however, transient follicles with enlarged oocytes but with flattened pregranulosa cells were rarely seen: the enlarged oocytes in activated follicles were always accompanied by elevated numbers of cuboidal granulosa cells (Fig. 6E, arrowheads). These results indicate that p27 deficiency, but not Foxo3a deficiency in pregranulosa cells, triggers pregranulosa cell proliferation and differentiation during follicle activation. In ovaries of PD13 p27^–/–Foxo3a^–/– DKO mice, the majority of the follicles were activated, with apparently enlarged oocytes that were surrounded by mostly cuboidal granulosa cells (Fig. 6G, arrowheads); typical primordial follicles were rarely seen (Fig. 6G).

By counting follicle numbers in serially sectioned ovaries, we found that in PD13 Foxo3a^–/– ovaries, primordial follicles represented approximately 30% of all follicles (Fig. 6K), which was a significantly lower rate than the primordial follicle rate in wild-type or p27^–/– ovaries (Fig. 6K). Moreover, in PD13 DKO ovaries the proportion of primordial follicles was less than 10%, which was significantly lower than that in p27^–/– and Foxo3a^–/– mice (Fig. 6K). These data indicate that the simultaneous loss of p27 and Foxo3a synergistically accelerates follicle initiation. Thus, we propose that p27 and Foxo3a suppress follicle activation independently of each other.

The hypothesis that Foxo3a and p27 may function through distinct pathways to suppress follicle activation and growth was also supported by the finding that in Foxo3a^–/– ovaries, the activated follicles (Fig. 6F, arrowheads) did not appear to grow as rapidly as those in p27^–/– ovaries: at PD23, the Foxo3a^–/– ovaries did not appear to be apparently enlarged (Fig. 6I) as the p27^–/–ovaries (Fig. 6H). The PD23 DKO ovaries, however, were found to have numerous developing preantral follicles and were larger than both Foxo3a^–/– and p27^–/– ovaries (Fig. 6, H–J), confirming the notion that concurrent loss of Foxo3a and p27 synergistically accelerates follicle initiation and growth. At PD35, clusters of activated primordial follicles with apparently enlarged oocytes were seen in Foxo3a^–/– ovaries (supplemental Fig. S2 published as supplemental data on The Endocrine Society’s Journals Online web site; PD35, arrows), suggesting that the overactivated oocytes in Foxo3a^–/– ovaries undergo rapid growth at this stage. Ovaries of both p27^–/– and p27^–/–Foxo3a^–/– DKO mice also showed numerous developing follicles at PD35 (supplemental Fig. S2). At the age of 12 wk, however, Foxo3a^–/– ovaries were depleted of follicle structures (supplemental Fig. S2), which is a similar phenomenon to that of the p27^–/– ovaries (Fig. 4J).

Studies on the Molecular Mechanisms Underlying Averted Follicle Atresia in p27^–/– Mice
Elevated Cdk2/Cdc2-Cyclin A/B1 Kinase Activities in p27^–/– Ovaries Prevent Follicle Death through Suppression of the Caspase Pathway.
It was notable that as compared with p27^+/+ ovaries, granulosa cells in p27^–/– ovaries did not undergo hyperproliferation, as shown by in vivo bromodeoxyuridine (BrdU) incorporation assay (Fig. 7A, arrows). This is distinct from the prolonged proliferation of luteinizing granulosa cells of p27^–/– ovaries and p21^–/–; p27^–/– double null ovaries (28), and is also distinct from the case in hyperplastic thymus and spleen of p27^–/– mice (15), indicating that p27 does not play a major role in controlling granulosa cell proliferation. To determine the molecular mechanism that prevents follicle atresia before sexual maturity in p27^–/– mice, we used 18-d-old p27^–/– and p27^+/+ ovaries in which no dramatic follicle loss has occurred (Fig. 3). Our data showed that in p27^–/– ovaries, there was an overall increase in kinase activities associated with Cdk2, Cdc2, cyclin A, and cyclin B1, but not with cyclin E1 (Fig. 7B), implying that the kinase activities of Cdk2/Cdc2-cyclin A/B1 complexes may be involved in preventing the death of ovarian follicles in p27^–/– ovaries. It seems to us that at different developmental stages and cell populations of the ovary, Cdc2, cyclin B1, and cyclin E1 activities are differently regulated by the loss of p27.

View larger version (47K): [in this window] [in a new window]   Fig. 7. Elevation of Kinase Activities Associated with Cdk2/Cdc2-cyclin A/B1 Complexes Prevent Follicle Atresia through Suppression of the Caspase Pathway A, BrdU incorporation assay indicated that proliferation of granulosa cells was not accelerated in p27–/– ovaries. In vivo BrdU incorporation assay was performed with 18-d-old p27–/– and p27+/+ mice, as described in Materials and Methods. Reddish signals indicate proliferating cells. Similar BrdU incorporation was seen in granulosa cells of ovaries from p27–/–and p27+/+ mice. The experiments were repeated three times and representative images are shown. For each experiment and each genotype, one mouse was used. B, Kinase activities associated with Cdk2, Cdc2, cyclin A, cyclin B1, and cyclin E1 complexes in ovaries of p27–/– and p27+/+ mice. Ovaries from 18-d-old p27–/– and p27+/+ mice were homogenized and 300 µg of ovarian lysate was used for immunoprecipitation with the antibodies indicated. Kinase activity assays were performed using histone H1 as substrate, as described in Materials and Methods. All experiments were repeated three times and representative images are shown. For each experiment and each genotype, five to 10 mice were used. C, Suppressed activation of caspase-9, -3, and -7, and PARP in p27–/–ovaries. Ovaries from 18-d-old p27–/– mice and p27+/+ mice were collected and used for Western blot, as described in Materials and Methods. Levels of cleaved caspase-9, -3, and -7, and cleaved PARP, indicating apoptosis in p27–/–and p27+/+ ovaries at PD18, are shown. Levels of p27 and ß-actin in the ovaries were used as internal controls. The experiments were repeated three times, and for each experiment, five to 10 mice were used. Representative images are shown. IP, Immunoprecipitation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The endowment of a definite number of primordial ovarian follicles and the activation of some of these follicles at chosen times, leading to further development and eventual release of mature oocytes via ovulation, are essential for the propagation of all mammalian species. The end of female reproductive life occurs when the pool of primordial follicles is exhausted (2, 6). In the current study, we have provided several lines of evidence that implicate the Cdk inhibitor p27 as an essential molecule for controlling ovarian development via suppression of follicle endowment and activation, and provoking follicle death in mice.

Somatic Cell-Expressed p27 in Perinatal Ovaries Suppresses Follicle Formation and Endowment in Mice
In this study, we have provided evidence that p27 expressed in somatic cells surrounding oocytes in perinatal ovaries suppresses follicular formation and endowment (Fig. 8A). In perinatal ovaries before primordial follicle formation, p27 is expressed mainly in somatic cells surrounding female germ cells, but not in oogonia/oocytes. The functional role of p27 was elucidated by the finding that primordial follicles were formed earlier in p27^–/– ovaries, and postnatal follicle endowment is doubled in p27^–/– mice compared with that in p27^+/+ mice. Based on our data that in wild-type ovaries before follicle formation, somatic cells surrounding germ cells do not appear to be proliferating as measured by in vivo BrdU incorporation assay (data not shown), we suggest that p27 may suppress the proliferation of these somatic cells at this stage and may also regulate the migration of somatic cells during their invasion into the germ cell clusters. The loss of p27 in somatic cells may therefore lead to enhanced proliferation and/or increased mobility of these cells, which may result in accelerated formation of primordial follicles.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Suggested Functions of p27 in Controlling Mouse Ovarian Development A, In mouse ovaries before follicle formation, somatic cell-expressed p27 may suppress follicular endowment and follicle formation by inhibiting germ cell mitosis and also by suppressing migration/proliferation of somatic cells that will invade germ cell syncytia to form primordial follicles. B, Once a definite number of primordial follicles have been endowed, p27 expressed in oocyte nuclei and pregranulosa cells suppresses the activation of primordial follicles. C, Meanwhile, p27 expressed in oocyte nuclei, pregranulosa cells, and granulosa cells promotes follicle atresia and causes the rapid loss of follicles that occurs before sexual maturity.

To identify the molecules that accelerate follicle formation in p27^–/– mice, we measured the kinase activity of the major target of p27, Cdk2 (22). Our data suggest that in neonatal p27^–/– ovaries, the kinase activities associated with Cdk2-cyclin A are elevated and may be involved in driving the accelerated follicle formation in these mice. Further experiments are needed to determine whether the elevated Cdk2-cyclin A kinase activity in newborn (PD0) p27^–/– ovaries is located in oocytes or in somatic cells, or both. Equally importantly, the relationship between p27 and other molecules that have been suggested to have roles in follicle formation, such as neurotrophins and neurotrophin receptors (33, 34), synaptonemal complex protein-1 (35), activin (36), and factor in the germline (37), is worthy of further study.

p27 Expressed by Oocyte Nuclei and Pregranulosa Cells in Primordial Follicles Suppresses Follicle Activation in Mice
As illustrated in Fig. 8B, in this study we have provided evidence that in primordial follicles, p27 molecules expressed in oocyte nuclei and pregranulosa cells function as suppressors of oocyte growth and pregranulosa cell proliferation/differentiation, respectively. In p27^–/– mice, the pool of primordial follicles was prematurely activated by early oocyte growth and proliferation of the few flattened pregranulosa cells into cuboidal granulosa cells. Thus, our data support the notion that follicular activation is caused by the release from the inhibitory mechanism, which involves p27, that maintains the primordial follicles in a resting state, as previously hypothesized (6). Moreover, we have shown that premature activation of the primordial follicle pool in p27^–/– mice results in depletion of functional follicles in early adulthood (i.e. in 12-wk-old mice), which is a symptom similar to that of POF in humans. Thus, the p27^–/– mice may be a useful model for the study of ovarian failure, in which follicles are depleted due to overactivation.

Previous studies have suggested that oocyte growth is a major event during primordial follicle activation in mammals (9). At the same time, p27 has always been considered to be a suppressor of cell cycle progression (14). It is thus interesting to study how the p27-Cdk system regulates cell growth, rather than cell division, using growing oocytes as a model system. In the current study, we have shown that in oocytes of primordial and early growing follicles in p27^–/– mice, the kinase activities associated with Cdk2-cyclin A/E1 appears to be elevated. Further in-depth studies are necessary, however, to uncover the mechanisms by which p27 functions as a suppressor of cell (oocyte) growth.

Although it is apparent that both Foxo3a and p27 function as suppressors of primordial follicle activation, our data from the current study have indicated that, unlike in other cell types, p27 and Foxo3a do not appear to function in an upstream/downstream fashion in regulating follicle activation in mice. In DKO mice that lack both p27 and Foxo3a, synergistically accelerated follicle activation was seen, indicating that the mechanisms of suppression of follicle activation by p27 or Foxo3a are independent of each other. Additionally, large numbers of transient follicles with enlarged oocytes that were surrounded by a few flattened pregranulosa cells were observed in Foxo3a^–/– mice (Ref. 12 and the current study). In p27^–/– mice, however, enlarged oocytes in activated follicles were always surrounded by increased numbers of cuboidal granulosa cells. These results indicate that p27, but not Foxo3a, is the factor that suppresses the proliferation and differentiation of pregranulosa cells in primordial follicles. The in-depth mechanisms, including a possible cross talk between p27-mediated cascade and PI3K/Akt/Foxo3a signaling pathway in follicular activation, are currently under investigation in our laboratory. Moreover, our preliminary data showing that both p27 and Foxo3a are expressed in oocyte nuclei of human primordial and primary follicles suggest that these molecules may have a role in regulating follicle activation in humans.

p27 Promotes Postnatal Follicle Death via the Caspase-Dependent Pathway
In mice, a large proportion of follicles disappear from the nongrowing follicle pool during the first wave of follicle development (10, 11). However, in p27^–/– ovaries this rapid follicle loss is largely prevented, indicating that p27 is a key molecule in the promotion of follicle atresia before sexual maturity, as illustrated in Fig. 8C. The prevented follicle loss in p27^–/– ovaries may be achieved by the premature activation of the primordial follicle pool. We there propose that p27 expressed in primordial and early primary follicles functions as a key regulator that determines the fate of follicles of either being activated and recruited into the growing population at chosen times, or being removed by atresia.

By performing kinase assays, we showed that Cdk2/Cdc2-cyclin A/B1 complex-associated kinase activities were elevated in p27^–/– ovaries, which may outweigh the death signals in p27^–/– ovaries. Moreover, caspases are known to be involved in mediating follicle atresia (29, 30, 31, 32). Our data demonstrated that the activation of the caspase-9-caspase-3-caspase-7-PARP cascade was suppressed in p27^–/– ovaries, suggesting that the elevated kinase activities associated with Cdk2/Cdc2-cyclin A/B1 complexes that were caused by p27 loss may prevent follicle atresia via suppressing the activation of caspases -9, -3, and -7. Thus, we can designate p27 as a possible upstream enhancer for caspase activation, which induces follicle atresia.

In conclusion, the current study has provided several lines of evidence that define the previously unknown function of p27 to be a suppressor of ovarian follicle endowment/formation and activation, and an enhancer of ovarian follicle atresia. Together with previous reports that p27 is important for luteal cell differentiation (15, 16, 28), we propose that the deregulation, or malfunctioning, of the ovarian p27-mediated cascade may lead to defects in follicular development, which may cause disturbed ovarian function and pathological changes in the ovary. The findings in this study may provide some useful knowledge in the search for genetic aberrations of the ovary that lead to defects in follicle development in human diseases, such as POF.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mouse Lines
C57BL/6J mice were obtained from Charles River Laboratory (Sulzfeld, Germany). The p27^–/– mice have been described previously (15), and had been backcrossed to C57BL/6J for 10 generations before we performed the experiments. Generation of Foxo3a^–/– mice was described previously (27). In the current study, the Foxo3a^–/– mice have been backcrossed to C57BL/6J for seven generations. Breeding pairs of p27^+/– or p27^–/– male mice with p27^+/– female mice, or Foxo3a^+/– or Foxo3a^–/– male mice with Foxo3a^+/– female mice were used to obtain littermates for comparison. To obtain p27^–/– Foxo3a^–/– DKO mice, an initial breeding with p27^–/– males x Foxo3a^+/– females was set up to obtain pups for further breeding, and breeding pairs of p27^+/–Foxo3a^+/– females x p27^–/–Foxo3a^–/– males or p27^+/– Foxo3a^–/– females x p27^–/–Foxo3a^–/– males were used to obtain p27^–/–Foxo3a^–/– female pups. The mice were housed under controlled environmental conditions with free access to water and food. Illumination was on between 0600 and 1800 h. E0.5 refers to the day that a vaginal plug was found. The ethics committee of Umeå University approved all experimental protocols.

Reagents, Antibodies, Immunostaining, Western Blots, and Kinase Activity Assays
Rabbit polyclonal antibodies to p27, phospho-Akt (serine 473), cleaved caspase-3, cleaved caspase-6, cleaved caspase-7, cleaved caspase-9, cleaved PARP, and cleaved lamin A were obtained from Cell Signaling Technologies (Beverly, MA). Rabbit polyclonal antibodies against Foxo3a and phospho-Foxo3a (threonine 32) were from Upstate Biotechnology, Inc. (Lake Placid, NY). BrdU and mouse monoclonal antibodies against ß-actin and BrdU were purchased from Sigma-Aldrich Sweden AB (Stockholm, Sweden). A rat monoclonal antibody to GCNA1 was kindly provided by Dr. George C. Enders (University of Kansas, Lawrence, KS). Goat polyclonal antibody against human VASA was purchased from R&D Systems (Minneapolis, MN). Cdk2/Cdc2-specific inhibitor Roscovitine was obtained from Calbiochem (San Diego, CA). The ABC Staining System for immunohistochemistry was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Immunostaining was performed according to the instructions of the manufacturer. Western blot analysis was carried out according to the instructions for different antibodies from the suppliers and visualized using the ECL Plus Western Blotting Detection System (Amersham Biosciences, Uppsala, Sweden). For immunoprecipitations and kinase assays using histone H1, antibodies to Cdk2, Cdc2, cyclin A, cyclin B1, and cyclin E1, all conjugated to agarose beads, were obtained from Santa Cruz Biotechnology, and kinase assays were performed as previously described (38).

Quantification of Oocytes and Follicles
Ovaries were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. For quantification of germ cells in ovaries of PD1, serial sections of 5-µm thickness were prepared from an ovary, and all sections were stained for GCNA1 to visualize germ cells (19). Thus, the germ cell numbers counted represent the GCNA1-positive oocytes. The total germ cell number of an ovary was calculated as the sum of the number of germ cells from each individual section.

To count the numbers of ovarian follicles in 8- to 35-d-old mice, paraffin-embedded ovaries were serially sectioned at 8-µm thickness and stained with hematoxylin for morphological analysis, as previously described (13). Ovarian follicles at different stages of development, including primordial (type 2), transient (type 3a, i.e. follicles with enlarged oocytes but flattened pregranulosa cells), primary (type 3b), secondary (type 4), preantral, early tertiary, and tertiary follicles (types 5–7) were counted in all sections of an ovary, based on the well-accepted standards established by Pedersen and Peters (39). In each section, follicles that contained oocytes with clearly visible nuclei were scored as previously described (13, 40). The total number of follicles in an ovary was calculated as the sum of the numbers of follicles from all sections. Judging from careful morphological analysis, the incidence of counting the same oocyte or germ cell twice or missing an oocyte/germ cell is negligible. All quantification of oocytes and germ cells and all morphological analyses were performed with a Zeiss AX10 microscope (Carl Zeiss, Thornwood, NY).

Isolation of Oocytes from Postnatal Mouse Ovaries
Isolation of oocytes, separation of small oocytes from partially grown oocytes using a cell dispersing screen with 25-µm opening, and lysis of oocytes were performed as previously described (18). Red blood cells were removed using a hypotonic buffer containing 144 mM NH₄Cl and 17 mM Tris-HCl (pH 7.2).

Culture of Postnatal Ovaries
Ovarian cultures were performed as previously described (34), with minor modifications. PD2 p27^–/– ovaries were removed aseptically, and the whole ovary was cultured in a Cell Strainer (40 µm pore size) (BD Biosciences, Stockholm, Sweden) in 1 ml -MEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 28 µM ascorbic acid and 0.3% (wt/vol) BSA, with or without kinase inhibitors. The cultured ovaries were incubated in a humidified incubator (5% CO₂, 37 C) with one third of the medium exchanged for fresh medium every day for the duration of the culture period. For fixation, the ovaries were washed once in PBS and fixed overnight in 4% paraformaldehyde and embedded as mentioned above for morphological analysis.

Preparation of Ovarian Extracts
Ovaries were dissected free of fat and adhering tissues, and extracts were prepared on ice by homogenizing in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 20 mM NaF, 20 mM ß-glycerophosphate, 1 mM EDTA, 6 mM EGTA (pH 8.0), 1% Nonidet P-40, 1 mM dithiothreitol, 5 mM benzamidine, 1 mM phenylmethylsulfonylfluoride, 250 µM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µg/ml pepstatin, followed by centrifugation at 14,000 rpm for 20 min at 4 C. The supernatants were collected and protein concentrations were determined using the bicinchoninic acid protein assay.

Statistical Analysis
All experiments were repeated at least three times. For counting of oocytes and follicles, three to six mice per group were used, and data were analyzed with Student’s t test. A difference was considered to be significant when P < 0.01.

	ACKNOWLEDGMENTS

 
We thank Dr. Melissa Pepling for constructive discussions.

	FOOTNOTES

 
This work was supported by the Swedish Research Council, the Swedish Cancer Foundation, and the Swedish Lions Cancer Research Foundation in Norrland.

Present address for C.B.: Oncodesign, 20 rue Jean Mazen, BP 27627, 21076 Dijon Cedex, France.

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 12, 2007

Abbreviations: BrdU, Bromodeoxyuridine; cdk, cyclin-dependent kinase; DKO, double knockout; E14.5, embryonic d 14.5; PARP, poly (ADP-ribose) polymerase; PD1, postnatal d 1; PI3K, phosphatidylinositol 3 kinase; POF, premature ovarian failure.

Received for publication April 4, 2007. Accepted for publication June 5, 2007.

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