SRD Young Investigator Award Role of Cell-Death Ligand-Receptor System of Granulosa Cells in Selective Follicular Atresia In

Journal of Reproduction and Development, Vol. 57, No. 2, 2011, 10-198E

SRD Young Investigator Award

Role of Cell-Death Ligand-Receptor System of Granulosa Cells in Selective Follicular Atresia in Porcine Ovary Naoko INOUE1), Fuko MATSUDA2), Yasufumi GOTO2) and Noboru MANABE2) 1)Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601 and 2)Animal Resource Science Center, The University of Tokyo, Kasama 319-0206, Japan Abstract. In the mammalian ovary, more than 99% of follicles degenerate without ovulation and few oocytes ovulate and succeed to the next generation. Granulosa cell apoptosis plays a critical role in this process, follicular atresia. However, the molecular mechanisms responsible for the regulation of granulosa cell apoptosis have not been clarified. Death ligand and receptor systems are major apoptosis-inducing factors. This review describes the granulosa cell apoptosis via death ligand and receptor systems during follicular atresia in the porcine ovary. Key words: Apoptosis, Fas ligand (FasL)/Fas, Granulosa cell, Tumor necrosis factor α (TNFα)/TNF receptor (TNFR), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor (TRAILR) (J. Reprod. Dev. 57: 169–175, 2011)

he mammalian ovary contains a huge number of follicles at domestic ruminants [6]. Follicular atresia can occur at any time birth. However, the majority of follicles undergoes degenera- during the growth and development of follicles [7–9]. In the por- tive process and less than 1% of follicles survive and their oocytes cine ovary, apoptosis is induced in granulosa cells located in the ovulate. This degenerative process, called “follicular atresia”, has inner surface of granulosa layer, but not in cumulus cells, oocytes, long been understood to cause a loss of granulosa cells. Flemming or inner or extra theca layers in the early stages of atresia [10, 11]. [1] first observed apoptotic granulosa cells in 1885, but did not In the progressed stages, most granulosa cells undergo apoptosis, understand their physiological role. In the early 1970’s, John Kerr the basement membrane is broken, and apoptosis is induced in noticed that some hepatocytes develop a small rounded cytoplasm theca layers and cumulus cells. Thus, apoptosis is induced by the containing pyknotic chromatin [2]. He called this phenomenon neighboring granulosa cells. However, the molecular mechanism shrinkage necrosis and named the type of cell death “apoptosis”. of apoptotic signal transduction is not well understood. The present The histological features of apoptosis are : (i) condensation of the review focuses on the role of cell death ligand and receptor systems nuclear chromatin into a sharply circumscribed mass; (ii) swirling during the atresia of antral follicles in porcine ovaries. of nuclear and cellular outlines; (iii) fragmentation of the nucleus and cell, and production of membrane-bound apoptotic bodies; and Cell Death Ligand and Receptor Systems (iv) phagocytosis of apoptotic bodies by macrophages and cell death. In 1991, Tilly and Hughes suggested that granulosa cell apo- Death ligand and receptor systems are the most important apop- ptosis, at least in part, involved the induction of follicular atresia. totic signal transduction pathways (Fig. 1). When cells receive a They found granulosa cells with DNA fragmentation entering into cytotoxic signal from cell death ligands, apoptosis is initiated by apoptosis in atretic follicles [3, 4]. Since then, numerous research- the binding of ligands to specific receptors located on the cell mem- ers have attempted to confirm the primary trigger of apoptotic brane. Death ligands and receptors include tumor necrosis factor stimuli and the intracellular signal transduction pathway engaged in alpha (TNFα) and its receptors (TNFRs), FasL (also called Apo-1 granulosa cell apoptosis during follicular atresia. To date, many ligand or CD95 ligand) and Fas (Apo-1 or CD95), tumor necrosis apoptotic factors involved in follicular atresia, including cell death factor-related apoptosis-inducing ligand (TRAIL) and its receptors ligands and receptors, pro-and anti-apoptotic factors, growth fac- (TRAILRs), and so on. These death ligands belong to the TNF tors, and cytokines, have been identified. superfamily and are classified as type2 cell membrane proteins, In pigs, there are 5 million primordial follicles in postnatal ova- whose extracellular domain can also be released as a soluble form ries but less than 0.14% approximately 1,600 of all primary oocytes [12, 13]. Death receptors belong to the TNF receptor family and are ovulate [5]. Sows have a complete estrus cycle, a clearly defined classified as type1 cell membrane proteins. Death ligands mediate follicular phase and luteal phase, and many follicles of different cell death signaling via death receptors that contain a cytoplasmic stages and sizes because of a lack of follicular waves’ unlike 68-amino acid motif essential for the induction of apoptosis, called a death domain (DD) [14–16]. When the death ligand binds spe- Received: December 21, 2010 Accepted: December 21, 2010 cific receptors, the receptors are trimerized, and DDs are ©2011 by the Society for Reproduction and Development oligomerized. The oligomerization generates a homophilic interac- Correspondence: N Inoue (e-mail: [email protected]) tion with DD-containing adaptor proteins, such as Fas-associated 170 INOUE et al.

Fig. 1. Schema of cell death ligand and receptor signal transduction.

death domain (FADD). The adaptor protein in turn engages an ini- (NFκB)-inducing kinase (NIK) and N-terminal kinase (JNK), tiator caspase, such as caspase-8, leading to subsequent activation which promote cell survival and proliferation [18–21]. TNFR2 has of an effector caspase, such as caspase-3, that executes apoptotic no DD in its intracellular region and recruits TRAF2 directly via its cell death [17]. Some death receptors have decoy and/or survival- N-terminus (T2bs-N). TRAF2 induces strong NFκB and JNK acti- related roles not associated with the induction of apoptotic signal- vation [22]. Many types of cells co-express TNFR1 and TNFR2, ing. which co-operate to generate responses to TNFα. Kaipia et al. [23] detected an increase of apoptosis in antral fol- TNFα and TNFRs in Granulosa Cell Apoptosis licles of rat ovaries stimulated with TNFα and concluded that TNFα is a potential causative factor for follicular atresia. Prange- TNFα trrigers both pro-survival and pro-apoptotic signals (Fig. Kiel et al. [24] showed that TNF induced both cell proliferation and 1). A multifunctional cytokine, TNFα exerts its effects by binding apoptosis in primary cultured porcine granulosa cells. In healthy to either of two cellular receptors, TNFR type 1 (TNFR1) and type follicles, Nakayama et al. [25] detected intense staining for TNFα 2 (TNFR2). The effect induced differs depending on the combina- and TRAF2 and their mRNAs in the outer zone of the granulosa tion of receptor and adaptor protein. The major difference between layer, where many proliferating cells but no apoptotic cells were TNFR1 and TNFR2 is in their manner of signal transduction. observed. In atretic follicles, however, decreased or weak staining TNFR1 has a DD in the C-terminal region that transduces apoptotic for TNFα and TRAF2 and decreased expression of TNFR2 were signals. TNFα binds TNFR1, and recruits TNF receptor-related found in the granulosa layer, where many apoptotic cells were seen. protein (TRADD) via the DD. TRADD can recruit two adaptor These findings indicated that TNFα has an important role in the proteins, FADD and TNF receptor-associated protein 2 (TRAF2). induction of pro-survival/proliferating signals in granulosa cells FADD interacts with procaspase-8 and initiates apoptosis. On the during follicular atresia in porcine ovaries in vivo. More investiga- other hand, TRAF2 recruits cellular inhibitory proteins (cIAPs) that tions are needed to clarify the pro-apoptotic role of TNFα in have an anti-apoptotic role. TRAF2 also activates nuclear factor κB porcine granulosa cells. REVIEW: APOPTOSIS PATHWAY OF PORCINE GRANULOSA CELL 171

Fas Ligand and Fas in Granulosa Cell Apoptosis undergoing apoptosis in response to FasL [43]. These findings indi- cate that many anti-apoptotic and pro-survival factors were The FasL/Fas system is the best characterized death ligand/ expressed in healthy follicles. In early atretic follicles, the FasL receptor system (Fig. 1). Fas ligand and Fas induce apoptotic sig- expressed in the granulosa cells binds to Fas, which is expressed in naling in both normal cells and tumor cells. In mutant mice (lpr/lpr; the neighboring granulosa cell membrane, and subsequently, lymphoproliferation) lacking a functional Fas, abnormal ovarian FADD and procaspase-8 are recruited. Procaspase-8 is shortened follicular development and increased numbers of secondary folli- and activated. The activated caspase-8 transmits an apoptotic signal cles were observed [26]. In normal mice, the injection of an to effector caspases, such as procaspase-3. Finally, apoptosis is ini- agonistic anti-Fas antibody induced an increase in the number of tiated. The granulosa cell apoptosis seems to depend on a delicate atretic follicles. Moreover, studies in vitro showed that Fas medi- balance of endogenous anti-apoptotic/pro-survival factors and apo- ated apoptosis in cultured granulosa cells, luteal cells and ovarian ptotic signals via the FasL and Fas system. surface epithelial cells [27–30]. As described, FasL and Fas may be involved in follicular development and atresia. FasL and Fas are TRAIL and TRAILRs in Granulosa Cell Apoptosis expressed in mouse [31, 32], human [27], rat [33], and bovine [30] ovaries. However, their expression in vivo shows species-specific TRAIL, initially identified as a homolog of TNFα, binds to an differences. In atretic follicles of bovine ovaries, FasL has been unusually complex family of receptors: apoptosis signaling death detected in the theca layers, and Fas, in the granulosa layer [30]. In receptor (DR)-4 (also called TRAIL-R1), DR-5 (TRAIL-R2, rat ovaries, Fas was scattered in the granulosa cells of early and TRICK2, or KILLER). Decoy receptor (DcR)-1 (TRAIL-R3, TRID progressed atretic follicles, and no FasL was observed in the granu- or LIT) [44, 45], and DcR-2 (TRAIL-R4) [46, 47]. The latter two losa cells of healthy antral follicles [33]. In murine ovaries, FasL have a higher affinity for TRAIL than death receptors (DRs) and protein was located in granulosa cells and Fas protein was compete with DRs, and osteoprotegerin (OPG) [48, 49]. DR4 and expressed in oocytes [32]. The precise role of the FasL and Fas sys- DR5 contain an intracellular death domain (DD) necessary for tem in follicular selection has not been revealed yet. inducing apoptosis upon TRAIL-mediated receptor ligation. In To determine the role of FasL and Fas in follicular atresia, we contrast, neither DcR1 nor DcR2 mediates apoptosis, due to the examined the expression of FasL and Fas mRNA and protein by complete and partial absence of an intracellular DD, respectively RT-PCR and western blotting and the distribution of FasL and Fas (Fig. 1). TRAIL induces apoptotsis and its mRNA has been by immunohistochemistry in the porcine ovary [34]. Levels of detected in peripheral blood lymphocytes, spleen, thymus, prostate, FasL mRNA and protein were very low in the granulosa cells of ovary, small intestine, colon and placenta [50], but the biological healthy follicles, and increased during follicular atresia (Fig. 2A roles of TRAIL in ovarian tissues are not yet fully understood. and B). FasL was not detected in the granulosa cells of healthy fol- Notably, its roles in granulosa cell apoptosis have not been eluci- licles (Fig. 2C). Levels of Fas mRNA and protein were low in the dated. granulosa cells of healthy follicles, and increased during follicular To determine the role of TRAIL and its receptor in the regula- atresia (Fig. 2A and B). Staining for Fas was weak in the granulosa tion of porcine follicular atresia, we first examined levels of mRNA cells of healthy follicles, and found in scattered cells of the granu- and protein by RT-PCR and western blotting in granulosa cells and losa layers of atretic follicles (Fig. 2C). We investigated the effect the distribution of TRAIL and its receptor, DR4, by immuno-his- of FasL and Fas downstream cascades on granulosa cell apoptosis tochemistry in the ovary [51]. We demonstrated that the levels of during porcine follicular atresia. The mRNA for an adaptor protein, TRAIL mRNA and protein increased during follicular atresia (Fig. FADD, which has a critical role in intracellular signal transduction, 4A and B). TRAIL was expressed in granulosa cells and increased was expressed in granulosa cells at all stages [35, 36]. To confirm during follicular atresia [51]. DR4 was detected in granulosa cells, the activation of caspases during atresia, we used caspase-specific and showed no significant change during atresia (Fig. 4B) [51]. colorimetric peptidyl substrates. Key enzymes of the caspase cas- DR5 was also detected in granulosa cells of both healthy and atretic cade, caspase-8 and caspase-3, increased in activity during follicles, whereas DcR1 was found in granulosa cells of healthy follicular atresia (Fig. 3A and B). Moreover, activated caspase-3 follicles but not atretic follicles [52]. Then, we investigated was detected in granulosa cells but not cumulus cells of atretic fol- whether TRAIL induces apoptosis in primary cultured porcine licles (Fig. 3C). Recently, an anti-apoptotic factor, cellular FLICE- granulosa cells derived from healthy follicles. Apoptosis was inhibitory protein (cFLIP; also called CASH, Casper, CLARP, induced by TRAIL in a dose- and time-dependent manner in the FLAME, I-FLICE, MRIT or usuripin) was detected in healthy por- granulosa cells (Fig. 5A). As shown in Fig. 5B, when DcR1, which cine granulosa cells and inhibited the activation of procaspase-8 by lacks an intracellular death domain, was removed from the cell binding the DED of procaspase-8 or FADD in cultured granulosa membrane by PI-PLC treatment, a lower dose of TRAIL induced cells [37–40]. Over-expression of porcine cFLIP inhibited anti-Fas apoptosis [52, 53]. TRAIL has been reported to induce apoptosis in antibody-induced apoptosis in human tumor-derived granulosa various tumor cells but not in normal cells [46, 50, 54]. Preclinical cells (KGN) [41]. X-linked inhibitor of apoptosis protein (XIAP), studies in mice and nonhuman primates have shown that the admin- which suppresses caspase-3, -7 and -9 activities, was also strongly istration of TRAIL induces apoptosis in human tumors, but is not expressed in granulosa cells of healthy porcine follicles [42]. More- cytotoxic to normal organs or tissues [54, 55]. The susceptibility of over, Insulin-like growth factor-I (IGF-I), a well-established tumor cells to TRAIL and an apparent lack of activity in normal growth factor, prevented cultured bovine granulosa cells from cells have led to the proposed use of TRAIL in cancer treatment. 172 INOUE et al.

Fig. 2. Changes in levels of FasL and Fas mRNA and protein in the granulosa cells of healthy (H), early atretic (EA), and progressed atretic (PA) follicles. FasL and Fas mRNA levels were normalized to the GAPDH mRNA level, and the percent increase relative to healthy follicles was calculated (A). Levels of FasL and Fas proteins in granulosa cells were quantified by Western blotting. The increase relative to healthy follicles is shown (B). Data are the mean ± SEM. ** and ***, P<0.01 and 0.001, respectively, vs. healthy follicles. The distribution of the FasL and Fas proteins was demonstrated by immunohistochemistry (C). Porcine ovarian sections from healthy and progressed atretic follicles were immunohistochemically stained for FasL (green fluorescence) and Fas. Red fluorescence shows the nucleus. G, granulosa layer and T, theca layer. × 200. Modified from Inoue et al. [34].

Fig. 3. The activity of caspase-8 and -3 in granulosa cells of healthy (H), early atretic (EA), and progressed atretic (PA) follicles. The caspase activity was determined by the ApoAlert caspase-8, -3 colorimetric assay (A and B). Cell lysate was incubated with the colorimetric peptidyl substrate for caspase-8 (Ac-IETD-pNA) or caspase-3 (Ac-DEVD-pNA). The control was performed by treating the lysate with caspase inhibitor (Ac-DEVD- fmk) before incubation with the substrates. The significance of differences was determined with paired t-tests. Data are the mean ± SEM. *, P<0.05 vs. healthy and early atretic follicles. Porcine ovarian section from progressed atretic follicle was stained for active caspase-3 (green fluorescence) (C). Red fluorescence indicates the nucleus. The arrow indicates a cumulus cell. G, granulosa layer and T, theca layer. × 200.

However, a report that TRAIL induced apoptosis in normal human In the chicken ovary, TRAIL mRNA levels were elevated both in hepatocytes but not in hepatocytes of rats, mice, or rhesus monkeys atretic follicles and in prehierarchal follicles induced to undergo [56], suggests species-specific differences in the mode of action of atresia in vitro [57]. In the adult human ovary, mRNA of TRAIL TRAIL and its receptors. Recent findings partly support our results. and its receptors, DR4, DR5, DcR1 and DcR2, was expressed in REVIEW: APOPTOSIS PATHWAY OF PORCINE GRANULOSA CELL 173

Fig. 4. Changes in levels of TRAIL/DR4 mRNA and protein in the granulosa cells of healthy (H), early atretic (EA), and progressed atretic (PA) follicles. TRAIL mRNA levels (TRAIL mRNA/GAPDH mRNA ratio) in granulosa cells were determined, and the increase relative to healthy follicles was calculated (A). Levels of TRAIL and DR4 proteins in granulosa cells were quantified by Western blotting and the increase relative to healthy follicles was calculated (B). Data are the mean ± SEM. * and **, P<0.05 and 0.01, respectively, vs. each healthy sample. Modified from Inoue et al. [51].

Fig. 5. Apoptosis-inducing ability of TRAIL. Primary cultured granulosa cells were treated with TRAIL (0, 50, 100, 200 or 400 ng/ml) for 0–16 h. The rate of apoptosis (activated caspase-3-positive cells/PI-positive nuclei) was determined by an immunocytochemical technique, and the increase relative to the control was calculated (A). Modified from Inoue et al. [51]. Inhibitory effect of DcR1 on TRAIL-induced apoptosis (B). No apoptosis was induced by TRAIL treatment (0, 10, 50 or 100 ng/ml) in porcine granulosa cells without PI-PLC pretreatment (–). In the cells pretreated with PI-PLC (+), apoptosis was induced by TRAIL in a dose-dependent manner. *, P<0.001 vs. vehicle control. Modified from Wada et al. [53].

granulosa cells and oocytes. TRAIL efficiently induced apoptosis Conclusion in KGN cells [58]. TRAIL may regulate directly or indirectly the apoptosis of granulosa cells. Based on these results, TRAIL has Cell death ligand and receptor systems, TNFα/TNFR2, FasL/ apoptosis-inducing activity in porcine granulosa cells, and DcR1 Fas and TRAIL/TRAILRs, have been identified to play crucial inhibits TRAIL-induced apoptosis in the granulosa cells of healthy roles as initiators and/or inhibitors of apoptosis in granulosa cells of follicles. In healthy follicles, a sufficient amount of DcR1 binds porcine ovarian follicles during atresia. In healthy porcine follicles, with TRAIL, preventing binding to TRAIL-DRs. In early atretic TNFR2, DcR1 and anti-apoptotic factors, cFLIP and XIAP, pre- follicles, the amount of DcR1 on the cell membrane of granulosa vent granulosa cells from undergoing apoptosis (Fig. 6A). In atretic cells decreases. Consequently, TRAIL binds with cell-death recep- follicles, the binding of TRAIL/TRAILRs and/or FasL/Fas results tors (DR4 and DR5), and apoptotic signals are transferred to the in the recruitment of an adaptor protein, FADD, and an initiator granulosa cells. caspase, procaspase-8. Procaspase-8 is activated, and subsequently transduces signals downstream to effectors to perform granulosa 174 INOUE et al.

Fig. 6. Schema of apoptotic signaling in granulosa cells. In the healthy follicle, TNF-TNFR2, TRAIL-DcR1, and anti-apoptotic factors (ex. cFLIP and XIAP) inhibit the activation of caspases and subsequent apoptotic signal transduction (A). In the atretic follicle, FasL-Fas and/or TRAIL-DR4/ DR5 interaction causes the cleavage of procaspase-8, and subsequent apoptotic signaling is induced (B).

cell apoptosis (Fig. 6B). In fertile individuals, ovarian follicles 3. Hughes FM, Jr, Gorospe WC. Biochemical identification of apoptosis (programmed undergo cycles of growth and development induced by gonadotro- cell death) in granulosa cells: evidence for a potential mechanism underlying follicular pin. Steroid and peptide hormones, local ovarian growth factors, atresia. Endocrinology 1991; 129: 2415–2422. and anti-apoptotic/pro-apoptotic factors help to prevent granulosa 4. Tilly JL, Kowalski KI, Johnson AL, Hsueh AJ. Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology 1991; 129: 2799–2801. cell apoptosis. Identifying the precise mechanism by which cell 5. Black JL, Erickson BH. Oogenesis and ovarian development in the prenatal pig. Anat death ligand and receptor systems induce apoptosis should help us Rec 1968; 161: 45–55. to control follicular development and rescue more oocytes. 6. Evans AC. Characteristics of ovarian follicle development in domestic animals. Reprod Domest Anim 2003; 38: 240–246. 7. Dailey RA, Clark JR, Staigmiller RB, First NL, Chapman AB, Casida LE. Growth of Acknowledgments new follicles following electrocautery in four genetic groups of swine. J Anim Sci 1976; 43: 175–183. The authors wish to thank all of the members of the society for 8. Clark JR, Brazier SG, Wiginton LM, Stevenson GR, Tribble LF. Time of ovarian fol- licle selection during the porcine estrous cycle. Theriogenology 1982; 18: 697–709. Reproduction and Development for conferring the SRD Young 9. Foxcroft GR, Hunter MG. Basic physiology of follicular maturation in the pig. J Investigator Award. The authors are deeply grateful to Drs K Saka- Reprod Fertil Suppl 1985; 33: 1–19. maki, T Miyano, H Miyamoto, M Sugimoto, M Nakayama, S 10. Nakayama M, Manabe N, Nishihara S, Miyamoto H. Species specific differences in Wada and S Nishihara for their support and suggestions. This study apoptotic cell localization in granulosa and theca interna cells during follicular atresia in porcine and bovine ovaries. J Reprod Dev 2000; 46: 147–156. was supported by a Grant-in Aid for Young Scientists (B) 11. Manabe N, Myoumoto A, Tajima C, Fukumoto M, Nakayama M, Uchio K, Yamagu- (17780216) to NI from the Japan Society for the Promotion of Sci- chi M, Miyamoto H. Immunochemical characteristics of a novel cell death receptor ence (JSPS), by a Grant-in-aid for Creative Scientific Research and a decoy receptor on granulosa cells of porcine ovarian follicles. Cytotechnology 2000; 33: 189–201. (13GS0008) to NM from the Ministry of Education, Culture, 12. Tanaka M, Suda T, Takahashi T, Nagata S. Expression of the functional soluble form Sports, Science and Technology of Japan and by Grants-in-aid for of human fas ligand in activated lymphocytes. EMBO J 1995; 14: 1129–1135. Challenging Exploratory Research (18658105 and 21658092) and 13. Zauli G, Secchiero P. The role of the TRAIL/TRAIL receptors system in hematopoie- Scientific Research (B18380164, B22380148 and S16108003) to sis and endothelial cell biology. Cytokine Growth Factor Rev 2006; 17: 245–257. 14. Yonehara S, Ishii A, Yonehara M. A cell-killing monoclonal antibody (anti-Fas) to a NM from the JSPS. cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med 1989; 169: 1747–1756. References 15. Rouvier E, Luciani MF, Golstein P. Fas involvement in Ca(2+)-independent T cell- mediated cytotoxicity. J Exp Med 1993; 177: 195–200. 16. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the 1. Flemming W. Uber die bildung von richtungsfiguren in saugethiereiern beim unter- Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993; 75: 1169– gang graaf'scher follikel. Archiv Anat Entwgesch 1885: 221–244. 1178. 2. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide- 17. Trauth BC, Klas C, Peters AM, Matzku S, Moller P, Falk W, Debatin KM, Krammer ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239–257. PH. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Sci- REVIEW: APOPTOSIS PATHWAY OF PORCINE GRANULOSA CELL 175

ence 1989; 245: 301–305. 2007; 74: 1165–1170. 18. Stanger BZ, Leder P, Lee TH, Kim E, Seed B. RIP: a novel protein containing a death 41. Matsuda F, Inoue N, Goto Y, Maeda A, Cheng Y, Sakamaki K, Manabe N. cFLIP reg- domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell 1995; ulates death receptor-mediated apoptosis in an ovarian granulosa cell line by inhibit- 81: 513–523. ing procaspase-8 cleavage. J Reprod Dev 2008; 54: 314–320. 19. Hsu H, Xiong J, Goeddel DV. The TNF receptor 1-associated protein TRADD signals 42. Cheng Y, Maeda A, Goto Y, Matsuda F, Miyano T, Inoue N, Sakamaki K, Manabe cell death and NF-kappa B activation. Cell 1995; 81: 495–504. N. Changes in expression and localization of X-linked inhibitor of apoptosis protein 20. Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM. FADD, a novel death domain- (XIAP) in follicular granulosa cells during atresia in porcine ovaries. J Reprod Dev 2008; containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 54: 454–459. 1995; 81: 505–512. 43. Quirk SM, Cowan RG, Harman RM, Hu CL, Porter DA. Ovarian follicular growth 21. Boldin MP, Varfolomeev EE, Pancer Z, Mett IL, Camonis JH, Wallach D. A novel and atresia: the relationship between cell proliferation and survival. J Anim Sci 2004; protein that interacts with the death domain of Fas/APO1 contains a sequence motif 82 E-Suppl: E40–52. related to the death domain. J Biol Chem 1995; 270: 7795–7798. 44. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a 22. Rothe M, Pan MG, Henzel WJ, Ayres TM, Goeddel DV. The TNFR2-TRAF signaling death domain-containing receptor for TRAIL. Science 1997; 277: 815–818. complex contains two novel proteins related to baculoviral inhibitor of apoptosis pro- 45. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, teins. Cell 1995; 83: 1243–1252. Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ash- 23. Kaipia A, Chun SY, Eisenhauer K, Hsueh AJ. Tumor necrosis factor-alpha and its kenazi A. Control of TRAIL-induced apoptosis by a family of signaling and decoy second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles. Endo- receptors. Science 1997; 277: 818–821. crinology 1996; 137: 4864–4870. 46. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A. Induction of 24. Prange-Kiel J, Kreutzkamm C, Wehrenberg U, Rune GM. Role of tumor necrosis fac- apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine fam- tor in preovulatory follicles of swine. Biol Reprod 2001; 65: 928–935. ily. J Biol Chem 1996; 271: 12687–12690. 25. Nakayama M, Manabe N, Inoue N, Matsui T, Miyamoto H. Changes in the expres- 47. Marsters SA, Sheridan JP, Pitti RM, Huang A, Skubatch M, Baldwin D, Yuan J, sion of tumor necrosis factor (TNF) alpha, TNFalpha receptor (TNFR) 2, and TNFR- Gurney A, Goddard AD, Godowski P, Ashkenazi A. A novel receptor for Apo2L/ associated factor 2 in granulosa cells during atresia in pig ovaries. Biol Reprod 2003; 68: TRAIL contains a truncated death domain. Curr Biol 1997; 7: 1003–1006. 530–535. 48. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, 26. Sakamaki K, Yoshida H, Nishimura Y, Nishikawa S, Manabe N, Yonehara S. Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Involvement of Fas antigen in ovarian follicular atresia and luteolysis. Mol Reprod Dev Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill 1997; 47: 11–18. D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Boyle WJ. 27. Quirk SM, Cowan RG, Joshi SG, Henrikson KP. Fas antigen-mediated apoptosis in Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. human granulosa/luteal cells. Biol Reprod 1995; 52: 279–287. Cell 1997; 89: 309–319. 28. Quirk SM, Cowan RG, Huber SH. Fas antigen-mediated apoptosis of ovarian surface 49. Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C, Dul E, Appel- epithelial cells. Endocrinology 1997; 138: 4558–4566. baum ER, Eichman C, DiPrinzio R, Dodds RA, James IE, Rosenberg M, Lee JC, 29. Quirk SM, Porter DA, Huber SC, Cowan RG. Potentiation of Fas-mediated apoptosis Young PR. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem of murine granulosa cells by interferon-gamma, tumor necrosis factor-alpha, and 1998; 273: 14363–14367. cycloheximide. Endocrinology 1998; 139: 4860–4869. 50. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, 30. Vickers SL, Cowan RG, Harman RM, Porter DA, Quirk SM. Expression and activity Smith TD, Rauch C, Smith CA, Goodwin RG. Identification and characterization of a of the Fas antigen in bovine ovarian follicle cells. Biol Reprod 2000; 62: 54–61. new member of the TNF family that induces apoptosis. Immunity 1995; 3: 673–682. 31. Mori T, Xu JP, Mori E, Sato E, Saito S, Guo MW. Expression of Fas-Fas ligand system 51. Inoue N, Manabe N, Matsui T, Maeda A, Nakagawa S, Wada S, Miyamoto H. Roles associated with atresia through apoptosis in murine ovary. Horm Res 1997; 48 Suppl 3: of tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in gran- 11–19. ulosa cell apoptosis during atresia in pig ovaries. J Reprod Dev 2003; 49: 313–321. 32. Dharma SJ, Kelkar RL, Nandedkar TD. Fas and Fas ligand protein and mRNA in 52. Wada S, Manabe N, Inoue N, Nakayama M, Matsui T, Miyamoto H. TRAIL-decoy normal and atretic mouse ovarian follicles. Reproduction 2003; 126: 783–789. receptor-1 disappears in granulosa cells of atretic follicles in porcine ovaries. J Reprod 33. Kim JM, Yoon YD, Tsang BK. Involvement of the Fas/Fas ligand system in p53-medi- Dev 2002; 48: 167–173. ated granulosa cell apoptosis during follicular development and atresia. Endocrinology 53. Wada S, Manabe N, Nakayama M, Inoue N, Matsui T, Miyamoto H. TRAIL-decoy 1999; 140: 2307–2317. receptor 1 plays inhibitory role in apoptosis of granulosa cells from pig ovarian folli- 34. Inoue N, Maeda A, Matsuda-Minehata F, Fukuta K, Manabe N. Expression and cles. J Vet Med Sci 2002; 64: 435–439. localization of Fas ligand and Fas during atresia in porcine ovarian follicles. J Reprod 54. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Dev 2006; 52: 723–730. Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch 35. Manabe N, Inoue N, Miyano T, Sakamaki K, Sugimoto M, Miyamoto H. Ovarian DH. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand follicle selection in mammalian regulatory mechanisms of granulosa cell apoptosis in vivo. Nat Med 1999; 5: 157–163. during follicular atresia. In: Leung PK, Adashi E (eds.), The Ovary 2nd ed. Amsterdam: 55. Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA, Blackie C, Academic Press Elsevier Science; 2003; 369–385. Chang L, McMurtrey AE, Hebert A, DeForge L, Koumenis IL, Lewis D, Harris L, 36. Inoue N, Matsuda-Minehata F, Goto Y, Sakamaki K, Manabe N. Molecular charac- Bussiere J, Koeppen H, Shahrokh Z, Schwall RH. Safety and antitumor activity of teristics of porcine Fas-associated death domain (FADD) and procaspase-8. J Reprod recombinant soluble Apo2 ligand. J Clin Invest 1999; 104: 155–162. Dev 2007; 53: 427–436. 56. Jo M, Kim TH, Seol DW, Esplen JE, Dorko K, Billiar TR, Strom SC. Apoptosis 37. Goto Y, Matsuda-Minehata F, Inoue N, Matsui T, Maeda A, Manabe N. Porcine (Sus induced in normal human hepatocytes by tumor necrosis factor-related apoptosis- scrofa) cellular FLICE-like inhibitory protein (cFLIP): molecular cloning and compari- inducing ligand. Nat Med 2000; 6: 564–567. son with the human and murine cFLIP. J Reprod Dev 2004; 50: 549–555. 57. Johnson AL, Ratajczak C, Haugen MJ, Liu HK, Woods DC. Tumor necrosis factor- 38. Matsuda-Minehata F, Goto Y, Inoue N, Manabe N. Changes in expression of anti- related apoptosis inducing ligand expression and activity in hen granulosa cells. apoptotic protein, cFLIP, in granulosa cells during follicular atresia in porcine ovaries. Reproduction 2007; 133: 609–616. Mol Reprod Dev 2005; 72: 145–151. 58. Jaaskelainen M, Kyronlahti A, Anttonen M, Nishi Y, Yanase T, Secchiero P, Zauli 39. Matsuda-Minehata F, Inoue N, Goto Y, Manabe N. The regulation of ovarian granu- G, Tapanainen JS, Heikinheimo M, Vaskivuo TE. TRAIL pathway components and losa cell death by pro- and anti-apoptotic molecules. J Reprod Dev 2006; 52: 695–705. their putative role in granulosa cell apoptosis in the human ovary. Differentiation 2009; 40. Matsuda-Minehata F, Goto Y, Inoue N, Sakamaki K, Chedrese PJ, Manabe N. Anti- 77: 369–376. apoptotic activity of porcine cFLIP in ovarian granulosa cell lines. Mol Reprod Dev