Reproduction in Domestic Animals

Death Ligand and Receptor Pig Ovarian Follicle 269Death Ligand and Receptor System RegulatesFollicular Atresia in Pig OvaryDeath ligands, which are grouped the tumour necrosisfactor (TNF) family, are synthesized as type-II membraneproteins (Ashkenazi and Dixit 1998). Most deathligands can be cleaved from the cell membrane tobecome a soluble form and act as trimers. Deathreceptors, which are classified as type-I membraneproteins, constitute a subfamily within the TNF receptorsuperfamily (TNFRsf) that has a cytoplasmic deathdomain (DD) necessary for the activation of apoptosis(Hengartner 2000). These receptors are trimerized andthen bind to death ligands, which are a trigger forapoptosis. Cell death ligand–receptor systems known inmammals include the Fas ligand (FasL) and Fas (CD95,APO-1 or TNFRsf6), TNFa and TNFa receptors(TNFRs), and TNFa-related apoptosis-inducing ligand(TRAIL, Apo2L) and TRAIL receptors (Nagata 1997;Ashkenazi and Dixit 1998; Wallach et al. 1999). In mostcases, the cell death receptor-mediated apoptotic signallingpathway is as follows: (1) Cell death ligands,binding with cell membranes or existing as a solubleform, bind to the extracellular domain of trimerized celldeath receptors, each of which contains an intracellularDD. (2) The DD of the death receptor binds with theDD of the adaptor proteins (TNF receptor 1-associateddeath domain protein: TRADD and Fas-associateddeath domain protein: FADD) through homophilicinteraction (Inoue et al. 2007). (3) An initiator caspase(procaspase-8) binds to FADD through homophilicinteraction with the death effector domain (DED; theresulting complex is called the ‘death-inducing signallingcomplex’: DISC) (Medema et al. 1997). (4) Dimerizationof procaspase-8 induces auto-proteolytic cleavageand activation (Boldin et al. 1995). (5) The activatedcaspase-8 subsequently activates downstream caspaseseither directly (‘type I’) or via mitochondrial perturbation(‘type II; mitochondrion-dependent’) (Matsui et al.2003). (6-a) In type I apoptotic cells, caspase-8 directlyactivates the effector enzyme, caspase-3. (7-a) Trancatedcaspase-3, active form, activates endogenous endonucleasesthat results in apoptosis. (6-b) In type II apoptoticcells, caspase-8’s activation leads to the release ofcytochrome c from mitochondrion, which results in theinteraction of procaspase-9 with apoptotic proteaseactivatingfactor 1 (Apaf1) [formation of cytochromec-Apaf1-caspase-9 complex (apoptosome)]. (7-b) Activatedcaspase-9 cleaves procaspase-3. (8-b) Active caspase-3activates the endonucleases that results inapoptosis (Nagata 1997; Ashkenazi and Dixit 1998;Matsui et al. 2003) (Fig. 1).FasL–Fas and other cell death ligand–receptor systems ingranulosa cellThe FasL–Fas system is one of the most studiedparadigms of instructive apoptosis, with strong apoptosis-inducingactivity (Wallach et al. 1999). The apoptosissignal triggered and mediated by FasL and Fas isas follows: (1) FasL binds to the extracellular domainof Fas. (2) The intracellular DD of Fas interacts withthe adaptor protein (FADD) through DD. (3) FADDDiscFasLFasFADDActivated caspase-8ApoptosisProcaspase-8: Death domain (DD): Death effector domain (DED): Enzymatic domainCell membraneFig. 1. Intracellular signalling in TNF family. When cell death ligandbinds to the extracellular region of cell death receptor, adoptor (Fasassociateddeath domain protein: FADD) binds to the intracellularregion of receptor through the homophilic interaction of the deathdomains (DD). Then, initiator caspase (procaspase-8) binds to FADDthrough the homophilic interaction of the death effector domain(DED). Dimerized procaspase-8 is auto-cleaved, and then truncatedcaspase-8 (active caspase-8) induces activation of down-stream caspases(ex. caspase-3), which results in apoptosisinteracts with procaspase-8 through their DED and theactive caspase-8 is formed. (4) A caspase cascade isactivated and eventually apoptosis is induced. TheFasL–Fas system is the most characterized apoptosissignalling machinery in granulosa cells (Inoue et al.2006). In many species, both FasL and Fas areexpressed in granulosa cells. In murine ovaries, FasLand Fas mRNA and protein are expressed in granulosacells of healthy and atretic follicles (Sakamaki et al.1997). Moreover, treatment of female mice with Fasactivatingantibody promoted granulosa cell apoptosisand follicular atresia, suggesting a pro-apoptotic functionof FasL–Fas signalling in vivo. In rat ovaries, FasLand Fas protein occur in granulosa cells, which tend tobe more abundant in atretic follicles than healthyfollicles (Hakuno et al. 1996). In human females, thegranulosa cells of antral follicles express Fas during theearly stages of atresia, the levels of Fas expressionincrease as atresia progresses, and granulosa FasLprotein also increases with atresia in antral follicles(Cataldo et al. 2000). In bovine ovaries, FasL mRNAlevels are higher in granulosa cells from atretic folliclesthan those from healthy follicles, and Fas mRNAexpression is stronger in granulosa cells of atretic thanhealthy follicles (Porter et al. 2000, 2001). Moreover,CD45, a pan-leucocyte marker, was not detected inbovine granulosa cells, indicating that immune cellswere not a source of FasL or Fas in the granulosa celllayer (Hu et al. 2001). Trace levels of FasL and FasmRNA and protein were detected in granulosa cells ofhealthy follicles but the levels increased during atresiain porcine ovaries (Inoue et al. 2006). These expressionpatterns of FasL and Fas strongly indicate the importantrole of FasL–Fas signalling in follicular atresia.Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag