Role of apoptosis-related factors in follicular atresia

Follicular atresia is the term used for the fate of follicles which undergo degenerative changes before rupturing during ovulation. Recent studies suggest that granulosa cell apoptosis play a major role in follicular atresia. The factors which lead the cell to apoptosis and which protect the cell death, still remain complicated and more studies are needed to elucidate the whole process. Here in this review, we aimed to simplify the factors and mechanisms taking place in granulosa cell apoptosis, to make the process more understandable.

val factors (Quirck et al., 2004).To date, many apoptosisrelated factors have been implicated in follicular atresia, including death ligands and receptors, Bcl-2 family proteins, Nodal, caspases, growth factors, gonadotropins, and calcium.In this report, we will overview these factors one by one.

Proapoptotic regulators in the cell death receptorligand system
Death receptors constitute a subgroup within the tumor necrosis factor (TNF) receptor family.They are located on the cell surface, anchored to the cell membrane, are trimerized and have cytoplasmic death domains (DDs) which are necessary to induce apoptosis (Park et al., 2005;Ashkenazi and Dixit, 1998;Wallach et al., 1999).Fas receptor, TNF receptor, and TNF related apoptosis inducing ligand receptors (TRAILr) are the members of TNF receptor family that are found to have roles in follicular atresia in mammalian ovaries (Park et al., 2005).
In most of the cases, the cell death receptor-mediated apoptosis takes place as follows: 1) The cell death ligand binds to the extracellular domain of cell death recepor (Fas ligand-Fas L for Fas receptor [CD 95, APO-1, TNFR sf 6], TNF α for TNF α receptors, and TNF α related apoptosis inducing ligand [TRAIL] for TRAIL receptors).
In type 1 apoptotic cells, caspase 8 directly activates the effector enzyme caspase 3, active form of which activates endogenous endonucleases and causes apop-tosis.In type 2 apoptotic cells, activation of procaspase 8 leads to the release of cytochrome c from the mitochon-drion.Cytochrome c binds to the apoptosis activating factor (Apaf 1) and causes activation of procaspase 9 (the complex formed by cytochrome c, Apaf 1, and cas-pase 9 is called apoptosome).Activated caspase 9 cleaves procaspase 3 and causes its activation, leading to activa-tion of endonucleases, and apoptosis is the result of the pathway (Ashkenazi and Dixit, 1998;Nagata, 1997;Matsui et al., 2003).

Fas-Fas L system
Fas and Fas L are the best characterized apoptotic signalling machinery in the granulosa cells of many species, including humans.In human females, Fas is expressed in granulosa cells of atretic antral follicles and its level increases as atresia progresses (Quirk et al., 1995;Kondo et al., 1996).
When Fas L binds to the extracellular domain of Fas, the intracellular DD of the receptor interacts with FADD through its DD.FADD and procaspase 8 interact through their DEDs and procaspase 8 becomes activated.This leads to activation of a caspase system and eventually apoptosis is induced as shown in the Figure 1 (Inoue et al., 2006).

TNF α-TNF receptor system
TNF is produced by granulosa cells and the oocyte, and it is another important regulator of follicular development and atresia (Jiang et al., 2003).When it binds to the TNF receptor 1, it stimulates apoptosis via its DD and when it binds to TNF receptor 2 which lacks DD, it acts as a survival factor (Matsuda-Minehata et al., 2006).
Increased mRNA expression of TNF receptor asssocia- et al. 55 ted DD (TRADD) which transmits the death signal from death receptor 4 and/or 5 to intracellular apoptosis inducing pathways in granulosa cells, was demonstrated only in atretic follicles, showing that the TRAIL receptor system induces apoptosis in granulosa cells during atresia in porcine ovaries (Wada et al., 2002).

Caspases
Caspases are a family of intracellular cysteine proteases which have roles in both initial and final stages of apoptosis in almost all types of vertebrate cells (Johnson and Bridgham, 2002).As discussed earlier, procaspase 3 activates endogenous endonucleases and causes apoptosis, while procaspase 9 is activated via cytochrome c and Apaf 1, and the complex named apoptosome formed by these three, activates procaspase 3 in type 2 cells.In humans and mice, it showed that antibody raised against the activated form of procaspase 3 reacted strongly with the granulosa cells of degenerating antral follicles.The studies in caspase 3 deficient mice showed that caspase 3 is required for granulosa cell apoptosis and therefore necessary for the process of follicular atresia (Matikainen et al., 2001).Caspase 9 deficient mice were found to contain numerous developing follicles that failed to complete the process of atresia due to the failure of granulosa cell apoptosis (Johnson and Bridgham, 2002).

Nodal
Nodal is a member of transforming growth factor β (TGF β) family whose members act through cell surface serine / threonine kinase receptor complexes.It was shown that Nodal is a critical regulator of early vertebrate development and involved in the induction of dorsal mesoderm, anterior paterning, and formation of left-right asymmetry (Iannaccone et al., 1992;Brennan et al., 2002;Eimon and Harland, 2002).Proapoptotic and growth inhibitory effects of Nodal in ovarian granulosa cells have also been reported (Wang et al., 2006).Like the other members of the TGF β family, Nodal has type 1 (ALK 4 and ALK 7) and type 2 receptors (Activin type 2 receptors ActR2A and ActR2B) (Oktem and Oktay, 2008).Nodal exerts its function by binding to and bringing together on the cell surface type 1 and 2 receptors to form a ternary ligand-receptor complex (Massague, 1998).Then, type 2 receptor phosphorylates type 1 receptor, which activates Smads, the intracellular signalling members of TGF β family, by phosphorylation.Smads 2 and Smad 3 are the ones that respond to Nodal.The phosphorylated Smads are released from the receptors and form complexes with common partner Smad and Smad 4 and translocate into the nucleus to regulate the transcription of target genes (Wang and Tsang, 2007).Nodal and its type 1 receptor ALK 7, are expressed in a cell type specific and follicular stage-dependent manner during folliculogenesis.Nodal immunoreactivity is the strongest in the preantral follicles Ligand binds to its receptor on the cell membrane 2)Intracellular death domain of the receptor binds to death domain of adaptor protein 3) Procaspase 8 binds to adaptor protein and become activated,procaspase 8 and FADD together called DISC( death inducing signalling complex) 4-5) In type I cells activated caspase 8 activates caspase 3 which activates endogenous endonucleases and causes apoptosis of the cell.6-7) In type II cells, activated caspase 8 causes mitochondrial release of cyt c which together with Apaf 1(apoptosis activating factor 1) activates procaspse 9 (cyt c,Apaf 1 and procaspase 9 together are called apoptosome) 8) Activated caspase 9 activates caspase 3and leads to apoptosis.9)cFLIPinhibits activation of caspase 8 10-11) Akt,which is inhibited by Nodal,improves cFLIP effect 12-13-14) XlAP (X linked inhibitor of apoptosis protein) which is inhibited by nodal and activated by FSH,inhibits caspase 3 15) Mitochondrial release of cyt c is inhibited by Bcl 2 and activated by Bax proteins 16) Nodal inhibits Bcl 2 and improves Bax protein's effect on mitochondrial release of cyt c.

Figure 1.
(1) Ligand binds to its receptor on the cell membrane, (2) intracellular death domain of the receptor binds to death domain of adaptor protein, (3) procaspase 8 binds to adaptor protein and becomes activated procaspase 8 and FADD together called death inducing signalling complex (DISC), (4 and 5) type I cells activated caspase 8 activates caspase 3 which activates endogenous endonucleases andcauses apoptosis of the cell, (6 and 7) type II cells activated caspase 8 causes mitochondrial release of cyt c which together with apoptosis activationg factor 1 (Apaf 1) activates procaspase 9 (cytochrome c (cyt c) Apaf 1 and procaspase 9 together are called apoptosome), (8) activated caspase 9 activates caspase 3 and leads to apoptosis, (9) cFLIP inhibits activation of caspase 8, (10 and 11) Akt, which is inhibited by Nodal improves cFLIP effect, (12, 13 and 14) X linked inhibitor of apoptosis is protein (XIAP) which is inhibited by Nodal and activated by FSH, inhibits caspase 3, (15) mitochondrial release of cyt c is inhibited by Bcl2 and activated by Bax protein, and (16) Nodal inhibits Bcl2 and improves Bax protein effection mitochondrial release of cyt c.
when compared with the later stages of development, whereas ALK 7 was mainly detected in the interstitial cells at the preantral stage.Nodal was found to be expressed in theca cells, but ALK 7 was only present in granulosa cells.During the development through the penultimate stage, the granulosa cells have the capability of under-going apoptosis due to the presence of ALK 7, but fail to do so due to low levels of its ligand (Wang et al., 2006).
When the gonadotropin support decreases the antral follicle destined for atresia begins to express increased levels of Nodal and also shows colocalization of both the ligand and its receptor in the granulosa cells.This allows us to accept that increased granulosa cell Nodal expression may be a physiological signal for induction of atresia (Wang and Tsang, 2007).It was also shown in studies that Nodal or ALK 7 activation downregulates the X linked inhibitor of apoptosis protein (XlAP) which acts as a direct inhibitor of caspases 3, 7, and 9 (Wang et al., 2006;Asselin et al., 2001;Deveraux et al., 1998).
Overexpression of Nodal and ALK 7 activation can significantly decrease the ratio of acitvated Akt protein which is an important antiapoptotic factor in granulosa cells (Wang et al., 2006;Asselin et al., 2001).Inactivation of Akt increases the mitochondrial release of Smac-Omi and cytochrome c, thus leading to activation of caspases and eventually granulosa cell apoptosis (Wang and Tsang, 2007).
One of the most studied members of the Bcl-2 family proteins is Bax, which is proapoptotic as previously mentioned.Its role has been emphasized by many studies.It has been shown that Bax deficient mice have abnormal follicles with an excessive number of granulosa cells (Perez et al., 1999).In humans and other species it was found that the atretic follicles and the granulosa cells going to apoptosis had increased levels of Bax expression at the mRNA and protein levels (Kim and Tilly, 2004).Moreover, Bax protein was abundantly expressed in granulosa cells of early atretic follicles, while it was found to be scarce in amount or undetectable in healthy follicles (Kugu et al., 1998).It was also shown that apoptosis could be induced by microinjection of recombinant Bax protein in oocytes (Kim and Tilly, 2004).
Interaction between pro-and antiapoptotic members of Bcl-2 family proteins in the mitochondrion determines whether pathways of apoptosis will be activated or not (Zinkel et al., 2006).The exact mechanism of apoptosis inhibition is not well-known, but the antiapoptotic members of Bcl-2 family are supposed to inhibit the mitochondrial release of some apoptotic molecules like cytochrome c.
Many studies have shown the importance of Bcl-2 in follicular development and growth.The number of follicles was shown to decrease in Bcl-2 deficient mice (Ratts et al., 1995) and overexpression of Bcl-2 was correlated with decreased follicular apoptosis and atresia, and increased susceptibility for germ cell tumorigenesis (Hsu Gumus et al. 57 et al., 1996;Morita and Tilly, 1999).

Antiapoptotic regulators in the cell
The antiapoptotic members of Bcl-2 family was discussed in the Bcl-2 family proteins.

cFLIP
Cellular FLICE-like inhibitory protein (cFLIP), also named as CASH, Casper, CLARP, FLAME, I-FLICE, MRIT or usurpin (Goltsev et al., 1997;Han et al., 1997;Hu et al., 1997;Inohara et al., 1997;Irmler et al., 1997;Shu et al., 1997;Srinivasula et al., 1997), is a homlog of procaspase 8 (FLICE) and is one of the intracellular proteins interfering with the apoptotic effects of death ligands.It was discovered in several viruses as viral FLIP (vFLIP) which contains two DEDs that interact with FADD to avoid the host's apoptotic response (Thome et al., 1997).In mammalian cells, homologue of vFLIP was discovered and named as cFLIP (Irmler et al., 1997).cFLIP was defined to have two forms, short and long ones, cFLIP S and cFLIP L , respectively.cFLIP S is similar to vFLIP in structure and has two DEDs, whereas cFLIP L contains an additional pseudoenzymatic domain which is similar to the enzymatic domain of procaspase 8, but lacks enzymatic activity.Therefore, cFLIP L blocks the death ligand inducible apoptosis by competing with procaspase 8 and interfering the activation of caspase 8 (Thome and Tschopp, 2001).The researches in this subject showed that granulosa cells of healthy follicles had highly expressed cFLIP L mRNA and proteins, whereas the atretic ones had decreased levels (Goto et al., 2004).The mRNA levels of cFLIP S in granulosa cells are low and showed no changes among the stages of follicular development (Matsuda-Minehata et al., 2005, 2006, 2007).
cFLIP acts as a survival promoting factor in granulosa cells and not only inhibits Fas-signalling, but also can inhibit TNF α and TRAIL signalling (Manabe et al., 2008;Cheng et al., 2007;Manabe et al., 2003).

Phosphotidyl inositide-3 kinase and Akt pathway
The phosphotidyl inositide-3 kinase (PI 3 K)/Akt pathway has an important role in regulating granulosa cell apoptosis (Asselin et al., 2001).Activation of PI 3 K results in activation of broad spectrum of downstream kinases one of which is Akt (Matsuda-Minehata et al., 2006).Akt is a serine/threonine phosphokinase and is an important antiapoptotic factor.Bcl-2 associated death promoter (Bad), caspase 9, and forkhead transcription factors (FOXO) are some of the targets of Akt and they are proapoptotic (Datta et al., 1999).
When growth factor receptors are stimulated by their ligands, first activation of PI 3 K occurs, leading to activation of downstream kinases including Akt.Growth factor-Akt, phosphorylates and thereby regulates proteins that function to maintain the basic needs of the cell like transportation and oxidation of glucose (Cross et al., 1995;Kohn et al., 1996), or attenuates apoptotic pathways by its substrates Bad and procaspase 9.
Akt causes upregulation of the expression of cFLIP which results in inhibition of apoptosis (Suhara et al., 2001;Panka et al., 2001), and also suppresses the mitochondrial release of death proteins like Smac, Omi or cytochrome c (Wang and Tsang, 2007).

Growth factors
Insulin like growth factors (IGFs) play an important role in follicular development and the granulosa cell apoptosis.There are two forms of it, namely, IGF 1 and IGF 2.
Researches have shown us that mice lacking IGF 1 are sterile and have arrested follicular development at the preantral and early antral stages leading to ovulation failure (Baker et al., 1996;Zhou et al., 1997).
IGF 1 was shown to activate PI 3 K/Akt pathway by phosphorylation in rat and bovine granulosa cells (Matsuda-Minehata et al., 2006), and thereby prevented apoptosis.Although, IGF 1 has an essential role in ovarian follicular development in many species, and IGF 2 is more abundant in humans (Geisthovel et al., 1989;Zhou and Bondy, 1993;Thierry van Dessel et al., 1996).
IGF believed to play its role in follicular development by stimulating proliferation, increasing responsiveness to gonadotropins mainly FSH, and by the way increasing estradiol secretion (Monniaux and Pisselet, 1992;Campbell et al., 1995;Glister et al., 2001).It was also shown that follicle-stimulating hormone (FSH) receptor expression was reduced in preantral IGF 1 null follicles and mostly restored to wild type levels after two weeks of exogeneous IGF 1 supplementation.FSH receptor expression and aromatase activity found to be decreased in IGF deficient mice, and IGF 1 administration restored normal FSH expression (Zhou et al., 1997).
Progression through the cell cycle is necessary for IGF to prevent apoptosis (Quirck et al., 2004).IGF, basic fibroblast growth factor (bFGF), and EGF were shown to decrease Fas L induced apoptosis of cultured bovine granulosa cells (Quirk et al., 2000), and this protective effect of IGF 1 is mediated through the PI 3 K/Akt pathway (Hu et al., 2004).When the granulosa cells were treated with PI 3 K inhibitor LY294002, the protective effect of IGF 1 against Fas L induced apoptosis was shown to be blocked (Quirck et al., 2004).
IGF is found to be bound to IGF binding proteins (IGFBP), which have six subgroups, in the body fluids.Although, circulating IGFBPs prolong the half life of IGF, they mostly inhibit its functions (Ui et al., 1989;Bicsak et al., 1990;Adashi et al., 1992).IGFBPs were shown to be expressed in the ovary (Adashi et al., 1985;Giudice, 1992).IGFBP 4 and IGFBP 5 are produced by rat granu-losa cells and FSH treatment decreases their secretion (Erickson et al., 1992a, b;Adashi et al., 1990).Dominant and subordinate follicles differ in their IGFBP contents (Monget et al., 1996).Their presence is controlled at the level of synthesis (Armstrong et al., 1998) by the gonadotropins and by the presence of proteases which breakdown low molecular weight IGFBP in the healthy antral follicles of cows (Rivera andFortune, 2001, 2003a, b), humans (Conover et al., 2001), and mice (Conover et al., 2002).
Growth differentiation factor 9 (GDF 9) is another growth factor found to be important in granulosa cell apoptosis.It exerts its function by activating the IP 3 K/Akt pathway.It was observed that when the intracellular GDF 9 decreased by intraoocyte injection of its inhibitor GDF 9 antisense morpholino, caspase 3 activation increased, leading to granulosa cell apoptosis (Craig et al., 2007).
EGF, TGFα, and bFGF, as well as their receptors, have been found in the ovary (Hsu and Hammond, 1987;Khan-Dawood, 1987;Kudlow et al., 1987) and shown to inhibit spontaneous onset of apoptotic DNA cleavage in cultured granulosa cells (Tilly et al., 1992a, b).EGF suppression of granulosa cell apoptosis is mediated by the stimulation of progesterone production and the regulation of intracellular free calcium concentration (Luciano et al., 1994).

Gonadotropins
Follicular development of primordial to secondary follicles does not need gonadotropin support.FSH is required for the follicular growth from the time of postantrum formation till ovulation (Craig et al., 2007).Decrease of circulating gonadotropins by hypophysectomy or blockage of LH/FSH surge were shown to cause massive atresia of preovulatory follicles on the day of proestrus (Ingram, 1953;Braw and Tsafriri, 1980).FSH treatment of hypophysectomized immature rats, decreased granulosa cell apoptosis in vivo (Billig et al., 1994).Prevention of apoptotic cell death in early antral and preovulatory follicles by FSH or hCG treatment was also shown in cultured follicles (Chun et al., 1994;Eisenhauer et al., 1995a, b).
Gonadotropins can induce the expression of survival molecules like Bcl-2, FLIP, and XlAP (Kim and Tilly, 2004;Hsu and Hsueh, 2000;Perez et al., 1999;Kugu et al., 1998;Hsu et al., 1996;Craig et al., 2007;Krysko et al., 2008).In mid-to late-follicular stages, FSH increases XlAP expression and activates PI 3 K/Akt pathway, leading to suppression of the release of mitochondrial death proteins (Wang et al., 2003).In mid-to late-follicular stages when the FSH levels decrease, colocalization of Nodal and its receptor ALK 7 in granulosa cells takes place, and triggering of the downsteram events including Smad 2 activation, Akt inhibition, and XlAP downregulation take place (Wang and Tsang, 2007).
A preovulatory follicle undergoes the final stage of maturation if it is stimulated by the luteinizing hormone (LH) surge.Studies have shown that granulosa of rodents and primates withdraw from the cell cycle after the LH surge (Robker and Richards, 1998a, b;Chaffin et al., 2001), and become resistant to apoptosis (Quirck et al., 2004;Porter et al., 2000).
Progesterone receptor (PR) is a potential mediator of the changes in granulosa cell proliferation and survival induced by the LH surge.Before the LH surge, PR expression is very low in preovulatory follicles, but begins to increase in the granulosa cells after the LH surge in cows (Cassar et al., 2002;Jo et al., 2002), and in primates (Chaffin et al., 1999).When PR antagonist RU 486 is used, to test the antiapoptotic effect of PR, it was observed that granulosa cells reentered the cell cycle, and regained susceptibility for the Fas L induced apoptosis (Quirck et al., 2004).
Gonadotropin withdrawal may also cause increased p53 protein expression which is an antiproliferative transcription factor that regulates the expression of several genes involved in mitosis and apoptosis.Overexpression of p53 results in granulosa cell apoptosis (Wang et al., 2006;Kim et al., 1999;Hughes and Gorospe, 1991;Wang et al., 2002;Mussche and D'Herde, 2001;Orisaka et al., 2006;Zwain and Amato, 2001).

Estradiol
Estradiol is an important intraovarian growth, differentiation, and survival factor (Rosenfeld et al., 2001).It stimulates the proliferation of granulosa cells and prevents apoptosis (Quirck et al., 2004).Treatment of immature hypophysectomized rats with diethylstilbestrol (DES) caused stimulation of development of large numbers of healthy, multilayered preantral follicles (Richards, 1980).Subsequent removal of DES resulted in apoptosis in the granulosa cell layer of antral and preantral follicles.No increase in DNA breakdown was detected in the primordial or primary follicles (Billig et al., 1993).
Estradiol enhances the ability of FSH to induce expression of LH receptors by which follicular growth and differentiation was shown to be promoted in cattle (Oktem and Oktay, 2008).Estradiol was also shown to increase IGF 1 in porcine granulosa cells (Hsu CJ, Hammond, 1987).But in the bovine granulosa cells, it was shown that treatment with anti IGF receptor antibody which effectively blocked the protective effect of IGF 1 against apoptosis, did not prevent the protective effect of estradiol (Quirck et al., 2004).In the research, estradiol treatment was found to be effective in preventing Fas L induced apoptosis, and this protective effect occurs if only progression through the cell cycle takes place.
Cell cycle progression is mediated by a family of cyclin dependent kinases (cdk) which are activated by binding with specific cyclin proteins.Estradiol also increases expression of cyclin D2 in rat granulosa cells (Robker and Richards, 1998a, b).

Progesterone
Progesterone was shown to prevent apoptosis in immature rat granulosa cells, although these cells did not contain nuclear progesterone receptor (PR) (McMurray et al., 2000;Tibbetts et al., 1999).Researches have shown us that PR is expressed by granulosa cells just prior to the ovulation (Natraj and Richards, 1993;Park and Mayo, 1991).So, we can assume that it inhibits apoptosis of the granulosa cells isolated during the periovulatory period (Svensson et al., 2000).
Recent evidence demonstrate that progesterone controls apoptosis by maintaining low basal intracellular calcium ion levels via membrane initiated events (Peluso et al., 2001a, b;Peluso, 2003).The exact mechanism of how the progesterone maintains low intracellular calcium ion levels in the granulosa cells is not known but cGMP dependent protein kinase (protein kinase G) plays an important role in other cells like the cardiac muscle cell, smooth muscle cell or endothelial cells.Protein kinase G, stimulates Ca +2 influx by closing calcium channels and blocks inositol triphospate (IP 3 ) receptor mediated Ca +2 release from cellular stores in these cell types (Carvajal et al., 2000).Prevention of apoptosis by protein kinase G activators and attenuation of antiapoptotic action of progesterone by protein kinase G antagonists are the pharmacological evidence for us to assume that progesterone exerts its antiapoptotic effects via protein kinase G pathway (Peluso, 2003;Hubbard and Greenwald, 1981;Hubbard, 1980).

Calcium
Calcium is a signalling agent involved in cell growth and differentiation.Transient intracellular Ca +2 rise was shown to induce apoptosis in quail granulosa explants (D'Herde andLeybaert, 1997, 1998).Bcl-2 was demonstrated to suppress apoptosis by a mechanism that is linked to intracellular Ca +2 to compartmantalization (McConkey, 1996).Abnormal Ca +2 elevations cause fragmentation of the DNA and other indicators of apoptosis due to the loss of the balance between anti-and proapoptotic proteins (Fissore et al., 2002;Gordo et al., 2002).DNA fragmentation promotes cytochrome c release from the respiratory chain into the cytosol and caspase cascade becomes activated (Kluck et al., 1997;Vander Heiden and Thompson, 1999).

CONCLUSION
There are many researches ongoing to elucidate the exact mechanism of granulosa cell apoptosis in vivo and factors that cause it.Solving this problem is crucial for us to prevent premature ovarian failure, and to increase the success rates in in vitro fertilization trials.The mediators which found to be increased in the atretic granulosa cells, like Fas, Nodal, and activated procaspase 3 may be targeted by the new researches; and the inhibiton of these in the selected patient population of suffering poor ovarian capacity may lead to more follicular development, enabling us to obtain more oocytes in the in vitro fertilization programmes.The antiapoptotic factors like XlAP, Akt protein, IGF 1 and 2, and cFLIP can also be used for prevention of apoptosis in the same patient group.On the other hand, germ cell tumorigenesis which is increased by Bcl-2 overexpression, can be inhibited by producing inhibitors of Bcl-2, or recombinant proapoptotic molecules.
Figure:Ligand binds to its receptor on the cell membrane 2)Intracellular death domain of the receptor binds to death domain of adaptor protein 3) Procaspase 8 binds to adaptor protein and become activated,procaspase 8 and FADD together called DISC( death inducing signalling complex) 4-5) In type I cells activated caspase 8 activates caspase 3 which activates endogenous endonucleases and causes apoptosis of the cell.6-7) In type II cells, activated caspase 8 causes mitochondrial release of cyt c which together with Apaf 1(apoptosis activating factor 1) activates procaspse 9 (cyt c,Apaf 1 and procaspase 9 together are called apoptosome) 8) Activated caspase 9 activates caspase 3and leads to apoptosis.9)cFLIPinhibits activation of caspase 8 10-11) Akt,which is inhibited by Nodal,improves cFLIP effect 12-13-14) XlAP (X linked inhibitor of apoptosis protein) which is inhibited by nodal and activated by FSH,inhibits caspase 3 15) Mitochondrial release of cyt c is inhibited by Bcl 2 and activated by Bax proteins 16) Nodal inhibits Bcl 2 and improves Bax protein's effect on mitochondrial release of cyt c.