Expression of Messenger Ribonucleic Acid for the Antiapoptosis Gene ...

0013-7227/01/$03.00/0 Endocrinology Copyright © 2001 by The Endocrine Society

Vol. 142, No. 6 Printed in U.S.A.

Expression of Messenger Ribonucleic Acid for the Antiapoptosis Gene P11 in the Rat Ovary: Gonadotropin Stimulation in Granulosa Cells of Preovulatory Follicles* SANG-YOUNG CHUN, HYUN-WOOK BAE, WAN-JU KIM, JEONG-HO PARK, SHEAU YU HSU, AND AARON J. W. HSUEH Hormone Research Center (S.-Y.C., H.-W.B., W.-J.K.), Chonnam National University, Kwangju 500 –757, Republic of Korea; and Division of Reproductive Biology (S.Y.H., A.J.W.H.), Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317 ABSTRACT P11, a member of the S100 family of calcium-binding proteins, has been shown to interact with BAD (Bcl-xL/Bcl-2-associated death promoter) in the yeast two-hybrid protein-protein interaction assay. Because overexpression of P11 dampens the proapoptotic activity of BAD in transfected cells, we tested the possibility that the expression of this antiapoptotic protein may be regulated by gonadotropins and other survival factors in the ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of P11 messenger RNA (mRNA) during prepubertal development in the theca cells of preantral and early antral follicles. Treatment of immature rats with PMSG did not affect P11 expression, whereas treatment of PMSG-primed rats with an ovulatory dose of human (h)CG stimulated ovarian P11 mRNA within 6 –9 h in the granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time-dependent stimulation of

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URING FOLLICLE development in the mammalian ovary, only a few follicles reach the ovulatory stage, whereas the majority of them undergo apoptosis (1–3). Studies on the hormonal control of follicle cell death demonstrate that deprivation of pituitary-derived gonadotropins in vivo results in follicle atresia, indicating that gonadotropins are required to prevent follicle apoptosis (4). In addition to gonadotropins, intrafollicular regulators, such as epidermal growth factor (EGF) (5), insulin-like growth factor I (IGF-I) (6), basic fibroblast growth factor (bFGF) (5, 7), and interleukin-1␤ (IL-1␤) (8), have been shown to suppress follicle cell apoptosis in vitro. Although the extracellular signals regulating follicle cell demise have been extensively studied, the intracellular and molecular events that control follicle atresia are largely unknown. An abundance of evidence has been accumulating to document that the final series of intracellular events that triggers apoptosis, regardless of cell lineage, involves changes in the expression of a common subset of genes including Bcl-2 and caspase family members (9, 10). The Bcl-2 family of proteins Received November 8, 2000. Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317. E-mail: [email protected]. * This work was supported by Korean Research Foundation Grants KRF-99 – 015-DP0361 and HRC-98k1– 0405, Republic of Korea (to S.Y.C.), and by NIH Grant HD-31566 (to A.J.H.).

P11 by gonadotropins. In addition, treatment of cultured preovulatory follicles with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-stimulated P11 mRNA, whereas treatment with forskolin, an adenylate cyclase activator, but not the protein kinase C activator, 2-O-tetradecanol-phorbal-13-acetate, mimicked the LH action, suggesting the role of adenylate cyclase activation in P11 expression. Treatment with other follicle survival factors, including the epidermal growth factor, the basic fibroblast growth factor, and interleukin1␤, could also stimulate P11 expression in cultured preovulatory follicles. These results demonstrate the expression of P11 mRNA in theca cells of different-sized follicles and in granulosa cells of preovulatory follicles following gonadotropin stimulation, and suggest that P11 may mediate, at least partially, the survival action of gonadotropins during the ovulatory process. (Endocrinology 142: 2311– 2317, 2001)

consists of anti- and proapoptotic members, and the balance between the anti- and proapoptotic Bcl-2 family of proteins determines the fate of the cell (2, 11). In the ovary, alterations in the expression of bcl-2, bax, and bcl-x genes have been shown to be associated with follicle atresia (12, 13). Moreover, targeted overexpression of Bcl-2 in ovarian cells leads to decreased follicle cell apoptosis and tumorigenesis in transgenic mice (14), whereas targeted deletion of the bcl-2 gene in mutant mice decreases the number of oocytes and primordial follicles at birth (15). Most recently, the ovarian expression of myeloid cell leukemia-1 (Mcl-1), an antiapoptotic Bcl-2 family protein, has been shown to be stimulated by gonadotropins in the rat (16). The expression of Bcl-xL/ Bcl-2-associated death promoter (BAD), a proapoptotic member of the Bcl-2 family, is also found in the granulosa cells of rat ovary, and BAD overexpression in granulosa cells leads to apoptosis (17). Collectively, these observations indicate that a group of anti- and proapoptotic Bcl-2 family of proteins is important in the regulation of ovarian follicle cell apoptosis. Recent studies further indicate interactions between the Bcl-2 family of proteins and signal transduction molecules activated by extracellular signals. BAD possesses a unique structural feature in that it does not have the C-terminal transmembrane domain found in most Bcl-2 family proteins, thus implying that BAD in the cytoplasm may interact with intracellular proteins in addition to its dimerization with

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other members of the Bcl-2 family (18). Indeed, phosphorylated BAD has been shown to bind to 14 –3-3, a group of proteins involved in intracellular signaling and cell cycle progression (19). Of interest, in the yeast two-hybrid screens using an ovarian fusion complementary DNA (cDNA) library, a BAD phosphorylation mutant not capable of binding 14 –3-3 preferentially interacts with P11 (20), a survival gene induced by the nerve growth factor (NGF) in the PC12 cells (21). P11, also known as 42C or calpactin I light chain, belongs to the S100 family of calcium-binding proteins (21). Furthermore, overexpression of P11 suppresses apoptosis induced by BAD (20), suggesting that P11 may be one of the intracellular signal molecules interacting with the Bcl-2 family of proteins to control apoptosis. Because the P11 gene has been identified in an ovarian cDNA library (20), we examined the changes in the expression of P11 mRNA during follicle development in the immature rat ovary. The present study demonstrates that P11 mRNA was induced by gonadotropins in the granulosa cells of preovulatory follicles both in vivo and in vitro through a protein kinase A pathway. Materials and Methods Hormones and reagents Ovine LH (LH-S-26; 2,300 IU/mg) was obtained from the National Hormone and Pituitary Distribution Program, NIDDK, NIH (Baltimore, MD). hCG, PMSG, forskolin, 2-O-tetradecanol-phorbol-13-acetate (TPA), and EGF were purchased from Sigma (St. Louis, MO). MDL12,330A and chelerythrine (CE) chloride were purchased from Calbiochem (La Jolla, CA). Recombinant human IGF-I, ␤-NGF, and recombinant murine Il-1␤ were purchased from R&D Systems Inc. (Minneapolis, MN). The purified recombinant human activin A was kindly donated by Dr. Yuzuru Eto (Ajinomoto Inc., Kawasaki, Japan).

Animals Immature female rats of the Sprague Dawley strain were purchased from Daehan Laboratories (Chungbuk, Korea). They were housed in groups of five with controlled temperature and photoperiod (10-h darkness, 14-h light, lights on from 0600 –2000 h). The animals had ad libitum access to food and water. Rats, ranging in age from 3–21 days, were killed by cervical dislocation and their ovaries were removed for RNA analysis. Ovaries were also collected from immature (26-day-old; BW 60 – 65 g) rats at various times after treatment with 10 IU PMSG to induce follicle growth. Some rats received a single ip injection of 10 IU hCG to induce ovulation, and ovaries were obtained at different time intervals for Northern blot and in situ hybridization analyses. In pilot experiments, these rats responded optimally to an ovulatory dose (10 IU) of hCG 2 days after 10 IU PMSG treatment based on their ovulation rate (34.4 ⫾ 5.5 ova per rat; n ⫽ 9).

Culture of preovulatory follicles Preovulatory follicles (⬎800 ␮m in diameter) were isolated from ovaries collected 48 –52 h after PMSG injection, and a follicle culture was performed as previously described (7). Fifteen to twenty follicles were cultured in glass vials containing 400 ␮l Eagle’s MEM (Life Technologies, Inc., Grand Island, NY) supplemented with penicillin, streptomycin, l-glutamine, and 0.1% BSA (wt/vol, Fraction V, Sigma) in the absence or presence of different hormones. Cultures were maintained in serumfree conditions for up to 24 h at 37 C under 5% CO2-95% O2. At the end of incubation, follicles were snap-frozen for RNA isolation.

Northern blot analysis Total RNA from ovaries or cultured follicles was isolated using Tri Reagent solution (Sigma). Ten to twenty micrograms of total RNA were fractionated by electrophoresis on a 1% agarose gel containing formal-

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dehyde and were transferred to nylon membranes by capillary blotting with 10⫻ sodium citrate-sodium chloride (SSC). After UV cross-linking and prehybridization, membranes were hybridized overnight at 42 C in a solution containing 50% formamide, 5⫻ SSC, 1 mm EDTA, 250 ␮g/ml denatured salmon sperm DNA, 500 ␮g/ml yeast transfer RNA, and a total of 2 ⫻ 106 cpm of a 32P-labeled full-length rat P11 cDNA probe (20). After hybridization, membranes were washed twice for 5 min at room temperature in 2 ⫻ SSC and 0.1 SDS, followed by washing for 1 h at 65 C in 0.5 ⫻ SSC and 0.1% SDS. Membranes were then exposed using Kodak RX film (Eastman Kodak Co., Rochester, NY) for 1–3 days at ⫺80 C. For normalization of data, blots were stripped by boiling in 0.1⫻ SSC and 0.5% SDS twice for 20 min before reprobing with a cDNA probe for rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). The intensities of hybridization signals were subsequently measured using a phosphorimager (Bio-Rad Laboratories, Inc., Hercules, CA), and normalized with the GAPDH RNA levels as internal controls.

In situ hybridization analysis Ovaries were fixed at 4 C for 6 h in 4% paraformaldehyde in PBS, followed by immersion in 0.5 m sucrose in PBS overnight. Cryostat sections (14-␮m thick) were mounted on poly-l-lysine (Sigma)-coated microscope slides, fixed in 4% paraformaldehyde in PBS, and stored at ⫺80 C until analyzed. The hybridization procedure was essentially the same as previously described (16). In brief, sections were pretreated serially with 0.2 m HCl, 2⫻ SSC, pronase (0.125 mg/ml), 4% paraformaldehyde, and acetic anhydride in triethanolamine. Hybridization was carried out at 52⫺55 C overnight in a mixture containing 35S-labeled rat P11 complementary RNA (cRNA) probe (2 ⫻ 108 cpm/ml), 50% formamide, 0.3 m NaCl, 10 mm Tris-HCl, 5 mm EDTA, 1 ⫻ Denhardt’s solution, 10% dextran sulfate, 1 ␮g/ml carrier transfer RNA, and 10 mm dithiothreitol. Posthybridization washing was performed under stringent conditions that included ribonuclease A (25 ␮g/ml) treatment at 37 C for 30 min and a final stringency of 0.1 ⫻ SSC. Slides were dipped into NTB-2 emulsion (Eastman Kodak Co.), exposed at 4 C, and developed after 2 weeks. The slides were stained with hematoxylin and eosin and examined under the light microscope with bright- and dark-field illumination.

Results Ovarian P11 mRNA expression during prepubertal development and following gonadotropin stimulation

Developmental changes in P11 mRNA levels in the ovary were determined by Northern blot analysis using the fulllength P11 cDNA probe. As shown in Fig. 1A, a 0.8-kb P11 transcript of similar size as found in PC12 cells (22) was detected in ovaries of 3-day-old rats and the hybridization signal markedly increased during development. The levels of ovarian P11 expression were 2.1- and 3.7-fold higher in 6- and 21-day-old rats, respectively, than in 3-day-old rats (Fig. 1B). To characterize gonadotropin regulation of P11 mRNA expression in the ovary, the total RNA extracted from ovaries at different times after gonadotropin treatment was analyzed by Northern blotting. As shown in Fig. 2A, PMSG treatment of 26-day-old rats for 6 h caused a 34% increase (n ⫽ 3) in ovarian P11 mRNA expression. Treatment with hCG of PMSG-primed rats, however, markedly stimulated ovarian P11 mRNA expression within 6 –9 h. Quantitative analysis of ovarian P11 signals (Fig. 2B) showed a 3.7-fold increase at 6 h after hCG treatment. To determine the cell types expressing P11 mRNA in gonadotropin-treated ovaries, antisense and sense cRNA probes for P11 were generated for in situ hybridization analysis. The ovaries of 26-day-old rats (control) contained numerous preantral follicles (PaF) and some early antral follicles (AnF; Fig. 3A). In these ovaries, P11 expression was


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FIG. 1. Developmental expression of rat ovarian P11 mRNA. A, Aliquots of total RNA (20 ␮g) isolated from ovaries on the indicated postnatal days were assayed for P11 mRNA levels by Northern blotting using a rat P11 cDNA probe. The expression of GAPDH was used as an internal standard. B, Quantitative estimation of ovarian P11 mRNA levels during development. The 0.8-kb P11 transcript was quantified using a phosphorimager and normalized for GAPDH RNA levels in each sample. Results are expressed relative to ovarian P11 mRNA levels found at 3 days of age. Each data point represents the mean ⫾ SEM from three independently performed experiments.

FIG. 2. Changes in P11 mRNA levels in ovaries of PMSG/hCG-treated immature rats. A, Aliquots of total RNA (20 ␮g) isolated from ovaries at the indicated time intervals after PMSG (upper panel) and PMSG/hCG stimulation (lower panel) were assayed for P11 mRNA levels by Northern blotting using a rat P11 cDNA probe. The expression of GAPDH was used as an internal standard. B, Quantitative estimation of ovarian P11 mRNA levels after hCG stimulation. The 0.8-kb P11 transcript was quantified using a phosphorimager and normalized for GAPDH RNA levels in each sample. Results are expressed relative to ovarian P11 mRNA levels found at 0 h after PMSG/hCG treatment. Each data point represents the mean ⫾ SEM from three independently performed experiments.

confined to theca cells of preantral and early antral follicles (Fig. 3, B and D; arrowhead). P11 signals were also detected in atretic follicles (AtF) with thinner granulosa cells showing pyknotic bodies (Fig. 3, A and B). At 12 h after PMSG treatment, the ovaries contained follicles at varying stages of growth with an increased number of antral follicles (AnF; Fig, 3E). P11 signals were still confined to theca cells, but not granulosa cells, of preantral and growing antral follicles (Fig. 3, F and H; arrowhead). Some atretic follicles also expressed P11 mRNA (Fig. 3, E and F). At 6 h after hCG treatment, the ovaries contained a large number of preovulatory follicles (Fig. 3I). High levels of P11 mRNA were detected in the granulosa cells of preovulatory follicles (PoF), but not of growing antral follicles (Fig. 3, J and L). P11 signals were also detected in the theca cells of follicles of different sizes (Fig. 3J; arrowhead). Ovarian sections hybridized with the sense P11 riboprobe showed only background hybridization (Fig. 3; C, G, and K).

Stimulation of P11 mRNA expression following treatment of cultured preovulatory follicles with gonadotropins and intraovarian survival factors as well as a protein kinase A activator

To study the regulation of P11 mRNA expression by different survival factors and intracellular regulators, preovulatory follicles obtained from ovaries of rats primed for 2 days with PMSG were incubated in a serum-free condition. In this follicle culture model, LH, EGF, bFGF, IGF-I, and IL-1␤ have been shown to suppress spontaneous follicle cell apoptosis (23). Northern blot analysis revealed that LH (200 ng/ml) treatment resulted in a transient stimulation of P11 mRNA in cultured preovulatory follicles, reaching a peak at 6 h (3.7-fold increase) and declining at 12 h after treatment (Fig. 4). To determine the signaling pathway for P11 mRNA stimulation by LH, preovulatory follicles were incubated for 6 h

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FIG. 3. Localization of P11 mRNA in PMSG/hCG-treated ovaries. Ovarian sections from rats at 26 days of age (Cont; left column) treated with PMSG for 6 h (middle column), or with PMSG/hCG for 6 h (right column) were hybridized with 35S-labeled P11 cRNA probes. Photomicrographs were taken under bright (A, E, and I) and darkfield (B–D, F–H, and J–L) illumination. Arrowheads (B, D, F, and H) and the asterisk indicate signals in the theca cells and atretic follicles, respectively. Sections hybridized with the P11 sense probe showed only background signals (C, G, and K). PaF, Preantral follicles; AnF, antral follicles; AtF, atretic follicles; PoF, preovulatory follicles; Gc, granulosa cells; Oo,oocyte. A–C, E–G, and I–K, ⫻40; D, H and L, ⫻100.

in the presence of LH with or without 10 ␮m MDL, an inhibitor of adenylate cyclase, or 10 ␮m chelerythrine (CE), an inhibitor of protein kinase C. In addition, some follicles were treated for 6 h with either 10 ␮m forskolin (an activator for protein kinase A) or 200 nm phorbol ester TPA (an activator for protein kinase C). As shown in Fig. 5, forskolin treatment stimulated the P11 gene expression thus mimicking the action of LH, whereas treatment with TPA had no effect. Moreover, inclusion of MDL markedly decreased LHstimulated P11 expression whereas the addition of CE did not affect the action of LH. Follicles incubated with MDL or CE alone did not show any effect. To test the role of intraovarian survival factors, P11 mRNA expression was evaluated after incubation of preovulatory follicles in the presence of LH (200 ng/ml), EGF (200 ng/ml),

bFGF (200 ng/ml), or IL-1␤ (100 ng/ml). Inclusion of EGF, bFGF, or IL-1␤ during a 9-h culture stimulated the P11 gene (Fig. 6). Maximum stimulation was detected 6 h after treatment (3.0- to 4.0-fold increases) with the same levels found in follicles treated with LH. In contrast, other intraovarian regulators including stem cell factor, NGF, activin, and IGF-I did not alter P11 mRNA expression (data not shown). Discussion

P11, a hormone-inducible survival factor, regulates apoptosis by interacting with hypophosphorylated BAD to dampen the proapoptotic action of BAD (20). In this study, we examined the developmental and gonadotropin regulation of P11 mRNA expression in the rat ovary. Additionally,


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FIG. 4. Stimulation of P11 mRNA expression by LH in preovulatory follicles cultured in vitro. Preovulatory follicles, obtained from the ovaries of PMSG-primed immature rats, were cultured in serum-free conditions under 5% CO2-95% O2 at 37 C in the presence of LH. Total RNA was extracted from follicles collected at the indicated time intervals after LH (200 ng/ml) stimulation. Twenty micrograms of follicular total RNA were then analyzed for P11 mRNA levels by Northern blotting using a rat P11 cDNA probe. The expression of GAPDH was used as an internal standard. Data are representative of two independently performed experiments.

FIG. 5. Effect of adenylate cyclase activation on P11 mRNA expression. Preovulatory follicles were incubated in serum-free conditions in the absence (Control; C) or presence of forskolin (FSK; 10 ␮M), MDL (10 ␮M), TPA (200 nM), and chelerythrine (CE; 10 ␮M) with or without LH (200 ng/ml). Ten micrograms of follicular total RNA were analyzed for P11 mRNA levels by Northern blotting using a rat P11 cDNA probe. The expression of GAPDH was used as an internal standard. Data are representative of two independently performed experiments.

we employed an in vitro follicle culture model to evaluate the hormonal regulation and signaling pathway of P11 mRNA expression. The present results indicate that ovarian P11 mRNA expression was increased during prepubertal development in theca cells of growing follicles and was induced after LH/hCG treatment in granulosa cells of preovulatory follicles. A recent report demonstrates that Mcl-1, an antiapoptotic Bcl-2 family protein capable of binding to the hypophosphorylated BAD, is also expressed in theca cells during prepubertal development (16). Interestingly, LH/hCG treatment induced a transient expression of P11 mRNA in the granulosa cells of preovulatory follicles by activating the protein kinase A pathway. This gonadotropin induction of P11 mRNA is consistent with the concept that P11 is a hormonally regulated and tissue-specific cell survival gene (21). Because LH/hCG rescues preovulatory follicles from death by activating the cAMP pathway (3, 23), P11 induced by LH/hCG may play a role in gonadotropin-mediated follicular survival. EGF, bFGF and IL-1␤, intraovarian hormones known to suppress follicle cell apoptosis (23), were also able to stimulate P11 mRNA expression in cultured preovulatory follicles. P11, also known as 42C or calpactin I light chain, has been shown to play a role in cell survival. P11 has been identified as an early inducible gene following NGF stimulation in the rat PC12 cell line (21). Overexpression of P11 stimulates neurite outgrowth and promotes PC12 cell survival in the

FIG. 6. Effect of intraovarian survival factors on P11 mRNA expression in cultured preovulatory follicles. Preovulatory follicles were incubated in serum-free conditions in the absence (Control; C) or presence of EGF (200 ng/ml), bFGF (200 ng/ml), or IL-1␤ (IL-␤, 100 ng/ml) for up to 9 h. Follicles cultured in the presence of LH (200 ng/ml) for 6 h were used as a positive control. Ten micrograms of follicular total RNA were analyzed for P11 mRNA levels by Northern blotting using a rat P11 cDNA probe. The expression of GAPDH was used as an internal standard. Data are representative of two independently performed experiments.

absence of NGF (22), indicating a role for P11 in apoptosis regulation. Although P11 belongs to the S-100 family of small calcium binding proteins (24), it does not have the ability to bind calcium ions due to amino acid deletions and substitutions in calcium binding motifs (25, 26). Instead, P11 is present in some cells as a monomer or as a heterotetramer bound to annexin II (27, 28). Of interest, induction of apoptosis by BAD can be attenuated through the interaction with the hormone-inducible P11 (20), implying the importance of P11 in the regulation of apoptosis by interacting with the Bcl-2-associated apoptotic machinery. The present observation that LH/hCG induces a rapid and transient expression of P11 mRNA in the granulosa cells of preovulatory follicles raises the possibility that P11 may play a role in the prevention of follicular cell apoptosis through interactions with the Bcl-2 family. The mature ovarian follicles deprived of the preovulatory surge of gonadotropins, undergo atresia with apoptotic degeneration of their granulosa cells (29, 30). In addition, activation of the cAMP pathway is important in the gonadotropin suppression of preovulatory follicle atresia (23). Our present observation, showing the transient stimulation of P11 mRNA in the granulosa cells of preovulatory follicles by LH through a cAMP-dependent pathway, indicates that P11, like Mcl-1 (16), may be one of the intracellular signaling molecules mediating extracellular survival signals in the granulosa cells of preovulatory follicles. Because some degree of apoptosis has been found in ovarian surface epithelium during ovulation, the survival role of P11 in the ovary is likely to be cell type-specific.

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As in other cell systems, a common subset of Bcl-2 family proteins including death promoters such as BAD (17), Bok (31), and BOD/Bim (32), and survival factors such as Bcl-2 (12) and Mcl-1 (16), is present in the ovary, and the fate of the granulosa cells is determined by the balance of these opposing activities. The survival of the granulosa cells of preovulatory follicles may occur through up-regulation of the survival factors and/or removal of the cell death promoters. Because BAD, a death promoter, interacts with P11 (20), our results raise the possibility that induction of P11 mRNA expression in the granulosa cells of preovulatory follicles by LH/hCG may be an important mechanism underlying the antiatretogenic action of gonadotropin in the ovary. It is possible that by inducing P11 overexpression in the granulosa cells of preovulatory follicles, LH/hCG is able to regulate the rheostat of apoptosis in favor of granulosa cell survival, and thus successful ovulation. Similar to P11 mRNA expression, inhibitor of apoptosis proteins (IAPs), a novel family of intracellular proteins that suppress apoptosis, is also expressed in the granulosa cells of preovulatory follicles following LH/hCG stimulation (33). The relative importance of P11 expression and the interactions of P11 with IAPs and/or intracellular regulators of apoptosis in the survival of the granulosa cells of preovulatory follicles remains to be determined. Apoptosis of atretic follicles in rodents is confined to granulosa cells. Interestingly, in the present study, P11 mRNA was also expressed in theca cells in follicles of different sizes. The constitutive expression of P11 mRNA in theca cells may be responsible for the scarcity of theca cell degeneration in rats. Alternatively, P11 may be involved in other as yet undetermined physiological processes such as theca cell differentiation. Because theca cells undergo differentiation in immature follicles of neonatal rats (34), our observation showing increased levels of P11 mRNA during prepubertal development suggests the involvement of P11 in theca cell development. Unexpectedly, granulosa cells of some follicles at the advanced stage of atresia also expressed high levels of P11 mRNA. At the present time, the reason(s) for the expression of P11 mRNA in granulosa cells of these atretic follicles is not known. In addition to apoptosis regulation, P11 could have other roles in the regulation of follicular development by interacting with unknown factors. P11 has been shown to inhibit the release of arachidonic acid by interacting with cytoplasmic phospholipase A2 in HeLa and BEAS-2B cells (35). In the ovary, prostaglandins and leukotrienes converted from arachidonic acid play an important role in ovulation (36, 37). These eicosanoids increase in rat ovarian follicles during the first several hours of ovulation induction and then decline to preovulatory levels at 6 –12 h preceding ovulation (38, 39). Thus, it may be possible that P11, produced during the 6 –9 h period after LH/hCG treatment, plays a role in follicle rupture by interacting with phospholipase A2, resulting in the modulation of eicosanoid activities. Studies are currently underway to identify the P11 interacting proteins in ovarian cells by using the yeast two-hybrid system. Indeed, P11 has been shown to interact with the FSH receptor as demonstrated by the yeast two-hybrid screening of the human ovarian cDNA library (40).

In summary, the present study has demonstrated that P11 mRNA is expressed in the rat ovary in a stage- and cellspecific manner during follicle development. P11 mRNA is expressed in theca cells regardless of follicle size and in granulosa cells of preovulatory follicles following gonadotropin stimulation. P11 appears to be an intracellular protein important in the decision of granulosa cell fate and may play a critical role as a cell survival factor during the ovulatory process. Acknowledgment We thank The National Hormone and Pituitary Distribution Program (NIDDK, NIH) for the ovine LH preparation.

References 1. Hsueh AJW, Billig H, Tsafriri A 1994 Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 15:707–724 2. Hsu SY, Hsueh AJW 2000 Tissue-specific Bcl-2 protein partners in apoptosis: an ovarian paradigm. Physiol Rev 80:593– 614 3. Johnson AL 2000 Granulosa cell apoptosis: conservation of cell signaling in an avian ovarian model system. Biol Signals Recept 9:96 –101 4. Braw RH, Bar-Ami S, Tsafriri A 1981 Effect of hypophysectomy on atresia of rat preovulatory follicles. Biol Reprod 25:989 –996 5. Tilly JL, Billig H, Kowalski KI, Hsueh AJW 1992 Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol 6:1942–1950 6. Chun S-Y, Billig H, Tilly JL, Furuta I, Tsafriri A, Hsueh AJW 1994 Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor I. Endocrinology 135:1845–1853 7. Chun S-Y, Eisenhauer KM, Minami S, Billig H, Perlas E, Hsueh AJW 1996 Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor. Endocrinology 137:1447–1456 8. Chun S-Y, Eisenhauer KM, Kubo M, Hsueh AJW 1995 Interleukin-1␤ suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. Endocrinology 136:3120 –3127 9. Williams GT, Smith CA 1993 Molecular regulation of apoptosis: genetic controls on cell death. Cell 74:777–779 10. Steller H 1995 Mechanisms and genes of cellular suicide. Science 267:1445–1449 11. Oltvai ZN, Milliman CL, Korsmeyer SL 1993 Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609 – 619 12. Tilly JL, Tilly KI, Kenton ML, Johnson AL 1995 Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropinmediated inhibition of apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xl messenger ribonucleic acid levels. Endocrinology 136:232–241 13. Johnson AL, Bridgham JT, Jensen T 1999 Bcl-X(long) protein expression and phosphorylation in granulosa cells. Endocrinology 140:4521– 4529 14. Hsu SY, Lai R J-M, Finegold M, Hsueh AJW 1996 Targeted overexpression of Bcl-2 in ovaries of transgenic mice leads to decreased follicle apoptosis, enhanced folliculogenesis and decreased germ cell tumorigenesis. Endocrinology 137:4837– 4843 15. Ratts VS, Flaws JA, Kopl R, Sorenson CM, Tilly JL 1995 Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology 136:3665–3668 16. Leo CP, Hsu SY, Chun S-Y, Bae H-W, Hsueh AJW 1999 Expression of the Bcl-2 family survival protein myeloid cell leukemia-1 (Mcl-1) in the rat ovary and its stimulation by gonadotropins. Endocrinology 140:5469 –5477 17. Kaipia A, Hsu SY, Hsueh AJW 1997 Expression and function of a proapoptotic Bcl-2 family member Bcl-xl/Bcl-2-associated death promoter (BAD) in rat ovary. Endocrinology 138:5497–5504 18. Yang E, Zha J, Jockel J, Boise LH, Thompson GB, Korsmeyer SJ 1995 Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 80:285–291 19. Zha J, Harada H, Yang E, Jockel J, Kormeyer SJ 1996 Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14 –3-3 not Bcl-XL. Cell 87:619 – 628 20. Hsu SY, Kaipia A, Zhu L, Hsueh AJW 1997 Interference of BAD (Bcl-XL/ Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14 –3-3 isoforms and P11. Mol Endocrinol 11:1858 –1867 21. Masiakowski P, Shooter EM 1988 Nerve growth factor induces the genes for two proteins related to a family of calcium-binding proteins in PC12 cells. Proc Natl Acad Sci USA 85:1277–1281 22. Masiakowski P, Shooter EM 1990 Changes in PC12 cell morphology induced


23. 24. 25. 26. 27. 28. 29. 30. 31.

32.

by transfection with 42C cDNA, coding for a member of the S100 protein family. J Neurosci Res 27:264 –269 Hsueh AJ, Eisenhauer K, Chun SY, Hsu SY, Billig H 1996 Gonadal cell apoptosis. Recent Prog Horm Res 51:433– 455 Kligman D, Hilt DC 1988 The S100 protein family. Trends Biochem Sci 13:437– 443 Gerke V, Weber K 1985 The regulatory chain in the p36-kd substrate complex of viral tyrosine-specific protein kinases is related in sequence to the S-100 protein of glial cells. EMBO J 4:2917–2920 Glenney J 1986 Phospholipid-dependent Ca2⫹ binding by the 36-kDa tyrosine kinase substrate (calpactin) and its 33-kDa core. J Biol Chem 261:7247–7252 Gerke V, Weber K 1984 Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calciumdependent binding to non-erythroid spectrin and F-actin. EMBO J 3:227–233 Erikson E, Tomasiewicz HG, Erikson RL 1984 Biochemical characterization of a 34-kilodalton normal cellular substrate of pp60v-src and an associated 6-kilodalton protein. Mol Cell Biol 4:77– 85 Tsafriri A, Braw RH 1984 Experimental approaches to atresia in mammalian ovulation. Oxf Rev Reprod Biol 6:226 –265 Hirshfield AN 1989 Rescue of atretic follicles in vitro and in vivo. Biol Reprod 40:181–190 Hsu SY, Kaipia A, McGee E, Lomeli M, Hsueh AJW 1997 Bok is a proapoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members. Proc Natl Acad Sci USA 94:12401–12406 Hsu SY, Lin P, Hsueh AJW 1998 BOD (Bcl-2-related ovarian death gene) is an

33.

34. 35.

36. 37. 38. 39. 40.

2317

ovarian BH3 domain-containing proapoptotic Bcl-2 protein capable of dimerization with diverse antiapoptotic Bcl-2 members. Mol Endocrinol 12:1432–1440 Li J, Kim J-M, Liston P, Li M, Miyazaki T, Mackenzie AE, Korneluk RG, Tsang BK 1998 Expression of inhibitor of apoptosis proteins (IAPs) in rat granulosa cells during ovarian follicular development and atresia. Endocrinology 139:1321–1328 Hirshfield AN 1991 Development of follicles in the mammalian ovary. Int Rev Cytol 124:43–101 Yao X-L, Cowan MJ, Gladwin MT, Lawrence MM, Angus W, Shelhamer JH 1999 Dexamethasone alters arachidonate release from human epithelial cells by induction of P11 protein synthesis and inhibition of phospholipase A2 activity. J Biol Chem 274:17202–17208 Richards JS 1994 Hormonal control of gene expression in the ovary. Endocr Rev 15:725–751 Espey LL 1994 Current status of the hypothesis that mammalian ovulation is comparable to an inflammatory reaction. Biol Reprod 50:233–238 Tsafriri A, Lindner HR, Zor U, Lamprecht SA 1972 Physiological role of prostaglandins in the induction of ovulation. Prostaglandins 2:1–10 Espey LL, Tanaka N, Okamura H 1989 Increase in ovarian leukotrienes during hormonally induced ovulation in the rat. Am J Physiol 256:E753–E759 Cohen B, Dias J 1999 Identification of follicle-stimulating hormone receptor interacting proteins by yeast two-hybrid analysis. 32nd Annual Meeting of the Society for the Study of Reproduction, Washington State University, Pullman, Washington, p 278