ovarian follicular fate ( i.e. development / ovulation versus atresia ) in the mammals is determined by the 
 fate of the cells within the follicles and is regulated by complex interactions of endocrine 
 ( gonadotropin and steroid hormones ) and locally produced ( growth factors and cytokines ) factors ( hirshfield , 1991 ) . although follicular atresia can occur at all the 
 stages of follicular development , a high incidence of granulosa cell apoptosis , a well recognized 
 cellular basis for this degenerative process , is predominantly observed at the early antral stage of 
 follicular development during the ovarian cycle ( kim et al . , 
 1998 ) . fas antigen ( fas ) , a member of the tnf receptor family , and its ligand fas ligand ( fasl ) , have been 
 identified as cell death mediator and factor , respectively in a variety of cell types ( watanabefukunaga et al . , 1992 ; suda 
 et al . , 1993 ) . their involvement in the induction of granulosa and luteal cell apoptosis has 
 been demonstrated in several mammalian species ( quirk et al . , 
 1995 ; hakuno et al . , 1996 ; kondo et al . fas 
 and fasl expression is up - regulated in granulosa cells undergoing apoptosis during early stage of 
 follicular development ( kim et al . , 1998 ) and p53-mediated 
 granulosa cell apoptosis is associated with the activation of fas / fasl system during follicular atresia 
 ( kim et al . , 1999 ) . in addition , follicle stimulating hormone 
 ( fsh ) is an important negative regulator of the fas / fasl system and a key cell survival factor during 
 the follicular development and the role of the fas / fasl system in granulosa cell apoptosis appears more 
 prominent at early than at late stages of follicular maturation . interferon gamma ( ifn- ) is a member of a family of heterogeneous regulatory proteins having antiviral , 
 immunomodulatory , antiproliferative , and cytodifferentiation regulatory activities ( pestka et al . , 1987 ) . it increases adrenal steroidogenesis ( blalock and harp , 1981 ) , increases iodide uptake by thyroid cells 
 ( friedman et al . , 1982 ) , and suppresses hcg - induced testosterone production by leydig cells ( orava et al . , 1989 ) . at physiological concentrations , ifn- 
 inhibits granulosa cell differentiation and steroidogenesis ( gorospe et 
 al . , 1988 ; xiao and findlay , 1992 ) . in addition , ifn- 
 increases fas mrna expression in mouse lymphoma cells and bam3 cells ( watanabefukunaga et al . , 1992 ) and fas content in human granulosa / luteal cells ( quirk et al . , 1995 ) . apoptosis in ovarian surface epithelial cells 
 is mediated by increased fas expression induced by ifn- ( quirk et al . , 
 1997 ) . moreover , ifn- induces fas - mediated apoptosis in differrentiated mouse granulosa 
 cells which could be potentiated by the presence of tnf ( quirk et al . , 
 1998 ) . although the importance of ifn- in undifferentiated or poorly differentiated 
 granulosa cells is well established , the presence of this cytokine in the ovary throughout follicular 
 development has not been investigated . in addition , the regulation of fas system in undifferentiated 
 granulosa cells by ifn- , subsequent fas - mediated apoptotic response as well as their modulation by 
 gonadotropin in vivo remain unclear . the purpose of the present study was to examine the role of ifn- in regulation of fas expression and 
 apoptosis in granulosa cells at different stages of follicular development and granulosa cell 
 differentiation in vitro . in addition , we assessed the presence of ifn- in the ovary 
 during follicular maturation and studied the relationship between the presence of ifn- , fas / fasl system 
 and apoptosis in vivo . [ p]-ddatp and dntps were obtained from amersham pharmacia biotech ( piscataway , nj ) . horse radish peroxidase ( hrp)-conjugated goat anti - rabbit secondary antibody was purchased from 
 bio - rad laboratories ( hercule , ca ) . rabbit anti - rat interferon- ( ifn- ) was obtained from biosource 
 international ( camarillo , ca ) . biotinylated 16-dutp , in situ cell death detection 
 ( fluorescein ) kit , proteinase - k and terminal deoxynucleotidyl transferase ( tdt ) were purchased from 
 boehringer - mannheim ( indianapolis , in ) . recombinant rat ifn- was from genzyme ( cambridge , ma ) . 
 fetal bovine serum ( fbs ) , minimal essential medium ( mem ) , non - essential amino acids ( naa ) , 
 penicillin and streptomycin were from gibco / brl ( burlington , on , canada ) . fitc - conjugated mouse 
 anti - hamster igg , hamster anti - mouse fas monoclonal antibody ( clone jo2 ) and hamster igg were 
 obtained from pharmingen ( san diego , ca ) . avian myeloblastosis virus ( amv ) reverse transcriptase , 
 oligo(dt)15 primer , rrnasin ribonucleotide inhibitor and taq dna polymerase were from 
 promega ( madison , wi ) . rabbit igg , rabbit peroxidase kits , rabbit polyclonal anti - mouse fas ( sc-716 ) 
 and anti - rat fasl ( sc-834 ) antibodies , and neutralization peptides ( fas ; sc-716p , fasl ; sc-834p ) 
 were from santa cruz biotechnology ( santa cruz , ca ) . agarose , bovine serum albumin ( bsa ; fraction 
 v ) , diethylstilbesterol ( des ) , equine chorionic gonadotropin ( ecg ) , methyl green , normal goat serum , 
 phenylmethylsulfonyl fluoride ( pmsf ) and veronal acetate were purchased from sigma chemical co. ( st 
 louis , mo ) . to obtain ovaries consisting mainly of follicles synchronized at the preantral / early antral and 
 medium / large antral stages in immature ( 21 - 23 days old ) sprague - dawley rats ( charles river canada ; 
 quebec , canada or samtako bio - korea ; osan , korea ) , des ( 1mg / day , sc , for 3 consecutive days and 
 sacrificed 24 hr after last injection ) and ecg ( 15 iu , ip and sacrificed 48 hr thereafter ) were 
 administered , respectively . a 14 h light:10 h dark photo - cycle was maintained with 
 lights - on at 0600 h. all procedures were performed in accordance with protocols approved by the 
 dong - a university animal care and use committee . following removal of connective tissues , the 
 ovaries were washed in pbs ( ph 7.4 ) to remove excess blood and were either immediately fixed in 10% 
 neutral buffered formalin ( ph 7.4 ) for histological processing or used for granulosa cell isolation 
 by follicle puncture ( rao et al . , granulosa cells ( 2.510 ) from ovaries of des- and ecg - primed rats were plated for 6 hr 
 ( 5% co2 , 37 ) in 60 mm falcon plastic culture dishes ( becton dickins , lincoln park , nj ) 
 in mem supplemented with 2% fbs , 1x naa , penicillin ( 100 u / ml ) and streptomycin ( 100 g / ml ) . the 
 medium was then replaced with mem supplemented with 0.1% bsa , 1x naa , penicillin ( 100 u / ml ) and 
 streptomycin ( 100 g / ml ) in subsequent culture . non - viable ( floating ) cells were removed at this 
 medium - replacement step . for induction of fas mrna and protein expression , granulosa cells were cultured in the presence of 
 recombinant rat ifn- ( 0 - 1,000 u / ml of the medium ) for 18 hr . floating and attached cells harvested 
 by centrifugation and trypsinization respectively , were combined and washed with ice - cold pbs , 
 pending protein and rna extraction , and flowcytometric analysis . for immunocytochemistry and tunel , 
 sterilized cover glasses ( 2222 mm ) were added onto the bottom of the culture dishes prior to cell 
 plating . at the end of the culture period , the cover glasses were removed , washed briefly with 
 ice - cold pbs and processed for immunocytochemistry .   to assess the influence of ifn- on fas expression and subsequent granulosa cell apoptotic response , 
 cells pretreated for 18 hr with / without the cytokine were incubated for an additional 6 h with 
 either agonistic fas monoclonal antibody ( hamster anti - mouse fas mab , igg ; 1 g / ml ) or hamster igg 
 ( control ; 1 g / ml ) . the effect of the treatment was assessed by phase - contrast microscopy , tunel and 
 dna fragmentation analysis . paraffin embedded whole ovarian sections ( 4 - 5 m ) were used for ifn- , fas and fasl 
 immunohistochemistry . the deparaffinized and hydrated sections were quenched in 
 h2o2 ( 0.3% ; 30 min ) and rinsed thoroughly with pbs ( 315 min ) . the 
 sections were blocked with normal goat serum in pbs [ 1.5% ; room temperature ( rt ) , 1 hr ] , and 
 then incubated ( rt , 45 min ) with rabbit polyclonal anti - rat ifn- ( 0.8 g / ml ) or rabbit 
 polyclonal anti - mouse fas ( 0.3 g / ml ) , or rabbit polyclonal anti - rat fasl antibodies ( 0.3 g / ml ) 
 in pbs containing normal goat serum ( 1.5% ) . the sections were incubated at rt with 
 biotin - conjugated goat anti - rabbit igg ( 1:200 ; 1 hr ) , avidin - biotin - peroxidase complex ( santa 
 cruz rabbit peroxidase kit ; 1 hr ) and dab solution ( 1 - 5 min ) , with nuclei lightly counterstained 
 with hematoxylin . for negative controls , rabbit igg ( 1 g / ml ) instead of the primary antibodies 
 was added to the reaction . in this experiment , antibody specificity was confirmed by antibody 
 preabsorption test , using recombinant rat ifn- ( 2 - 3 g ) and fas and fasl neutralizing peptides 
 ( 1 g ) in the respective primary antibody reaction . for localization of fas protein in cultured granulosa cells , the cells were fixed for 10 min in 
 ice - cold paraformaldehyde in pbs ( 2% ; ph 7.4 ) . after several washes with pbs , the cells were 
 incubated ( 20 min ) in pbs containing normal goat serum ( 10% ) to suppress nonspecific igg 
 binding , washed with pbs ( 35 min ) and incubated for 1h with rabbit anti - mouse antibody [ 0.1 
 g / ml in pbs containing normal goat serum ( 1.5% ) ] . following a pbs wash , the cells were 
 incubated ( 30 min in the dark ) with fluorescein - conjugated goat anti - rabbit antibody [ 1 g / ml 
 pbs with normal goat serum ( 1.5% ) ] , mounted with 90% glycerol in pbs and observed under 
 fluorescence microscope . ( 1992 ) was used to localize apoptotic cells in paraffin whole ovarian sections 
 mounted on positively charged slides ( probeon plus ; fisher scientific ltd , ottawa , on ) . briefly , 
 the sections were deparaffinized , hydrated , treated with proteinase - k ( 10 g / ml . , 30 min . ; 37 ) . endogenous peroxidase activity was removed by immersing the sections in methanol containing 
 h2o2 ( 0.3% , 30 min . , the sections were soaked in the tdt buffer 
 [ tris - hcl ( 25 mm ) , sodium cacodylate ( 200 mm ) , cobalt chloride ( 5 mm ) , bsa ( 250 g / ml ) , ph 6.6 ; 
 15 min ) , incubated in 50 l of tdt buffer containing tdt ( 10 u ) and biotinylated 16-dutp ( 1 
 nmol ) [ 60 min . , 37 ] , reacted with avidin - biotin - peroxidase complex ( santa cruz rabbit 
 peroxidase kit ; 30 min ) and subsequently with dab solution ( 1 - 5 min ) , as per manufacturer s 
 instruction . if required , the nuclei were counterstained with methyl green ( 5% ; in 0.1 m veronal 
 acetate , ph 4.0 ) . tdt enzyme or biotinylated 16-dutp in the labelling reaction were omitted in 
 the negative control slides . for in situ detection of apoptosis in cultured 
 cells , cells were fixed ( 10 min , 0 ) in fresh paraformaldehyde ( 4% in pbs ) and an in 
 situ cell death detection ( fluorescein ) kit ( boehringer mannheim ; quebec , canada ) 
 was used , as per manufacturer s instructions . total dna was extracted from cultured granulosa cells ( floating cells + trypsinized attached 
 cells ) according to the modified procedure of gross - bellard 
 et al . ( 1973 ) , as previously described ( li et al . , 1998 ) . for biochemical assessment of dna fragmentation , dna was 
 radiolabeled at the 3-ends according to an established method ( tilly et al . , 1993 ) . briefly , one microgram of dna was incubated ( 37 ; 60 min ) with 
 50 l of the labelling buffer [ tris - hcl ( 25 mm ) , sodium cacodylate ( 200 mm ) , cobalt chloride ( 5 
 mm ) , bsa ( 250 g / ml ) , ph 6.6 ] containing terminal deoxynucleotidyl transferase ( tdt ; 25 u ) and 
 [ p]-ddatp ( 5 mci , 3,000 ci / mmol ) . the labeled dna were resolved on 1.8% 
 agarose gel ( 3.5 h , 60 v ) in tae buffer . the gel was dried and subsequently exposed to x - ray 
 film at -70.   total rna was isolated from cultured granulosa cells using the qiagen rneasy mini rna isolation 
 kit ( valencia , ca ) . briefly , the cells attached to the culture dishes were lyzed with lysis 
 solution ( buffer rlt ) provided by the manufacturer , and the lysate was passed through the qiagen 
 qia shredder ( valencia , ca ) . to ensure the extracted rna for pcr was free of dna , two micrograms of total rna was reverse - transcribed ( 42 , 15 min ) with oligo ( dt)15 
 primers ( 0.5 g/g rna ) and avian myeloblastosis virus ( amv ) reverse transcriptase ( 15 u/g rna ) 
 in a 20 l reaction mixture containing 10x reverse transcription buffer [ tris - hcl ( 100 mm ) ( ph 
 8.8 ) , kcl ( 500 mm ) , triton x-100 ( 1% ) ] , mgcl2 ( 25 mm ) , dntp mixture ( 10 mm ) and 
 rrnasin ribonucleotide inhibitor ( 2 u ) . the reaction was terminated by inactivating the reverse 
 transcriptase ( heating at 99 , 5 min ) . gene - specific oligonucleotide primers ( 46.1% gc content ) were designed based on published rat fas 
 cdna sequence [ 22 ; genbank accession no . d26112 ] and synthesized using a beckman oligo 1000 m dna 
 synthesizer ( fullerton , ca ) . primers for rat -actin ( nudel et 
 al . , 1983 ) were obtained from clontech ( palo alto , ca ) . the primers used for pcr 
 amplification for fas were 5'-tgcacctcgtgtggacttgaag-3'[479 - 500 ; forward primer ] and 
 5'-ccagtcttcccgtgagattgatac-3 ' [ 888 - 865 ; reverse primer ] . polymerase chain reactions were 
 carried out with a pct-100 programmable thermal controller ( mj research , inc . , watertown , ma ) . the pcr reaction mixture included the oligonucleotide primers ( 5 pmol each ) , and taq dna 
 polymerase ( 0.5 u ) for each reaction . the total volume was brought up to 50 l with 1x pcr 
 buffer [ tris - hcl ( 50 mm ) , kcl ( 20 mm ) , mgcl2 ( 3 mm ) , dmso ( 0.5% ) ] and dntp ( 20 m ) . amplification was carried out for 35 cycles under the annealing temperature of 60 ( 45 sec ) , 
 primer extention at 72 ( 1 min ) and denaturation at 94 ( 30 sec ) . the amplified dna was 
 electrophoresed on 2% agarose gel , and the gel was stained with ethidium bromide for 
 visualization under uv light . at initial stage of this experiment , the amplified fas product was 
 confirmed by southern hybridization ( sambrook et al . , 
 1989 ) , using the rat fas cdna probe . the intensities of the bands were 
 densitometrically scanned and each fas amplified product was normalized by -actin .   harvested granulosa cells ( 210 cells ) were resuspend in ice - cold wash buffer 
 [ nan3 ( 0.1% ) , fbs ( 0.5% ) in pbs ] and centrifuged ( 500 g ; 5 min , 4 ) . the cells 
 were immediately incubated in primary antibody solution [ hamster anti - mouse fas ( 0.5 g / ml ) in 
 wash buffer ) ] at 4 for 30 min . , washed ( 500 g , 25 min in wash buffer ) , and incubated ( 30 
 min . , 4 in dark ) , with fitc - conjugated mouse anti - hamster igg ( 1.0 g / ml ) . the cells were then 
 washed twice with the wash buffer , filtered through a 35 m nylon mesh to remove cell clumps and 
 aliquoted for appropriate cell concentration ( final concentration , 110 cells in 500 
 l ) for flow cytometric analysis ( coulter epics profile ii , hialeah , fl ) . as negative controls , nonspecific binding in 
 flowcytogram was determined in the absence of primary antibody . pcr and flowcytometry data presented herein were expressed as the meansem of three separate 
 experiments . the presence and relative abundance of ifn- protein in the follicles during development were 
 screened by immunohistochemistry , using a rat specific ifn- antibody ( fig . 1a ) exhibit immunoreactivity for ifn- , however , the intense and aggregated 
 signals were detected in granulosa cells ( arrows ) as well as oocyte ( including its nucleus ) . in 
 contrast to its intense immunoreactivity in the cytoplasm , the nuclei of oocytes of secondary and 
 tertiary preantral follicles showed considerably lower immunostaining . 1b ) , granulosa cells in preantral follicles exhibited 
 immunopositivity for ifn- , although with lesser intensity and aggregated characteristics than those 
 in primordial and early stage of preantral follicles . 1d ) antral follicles was much less than that seen in the 
 preantral follicles . generally , the immunereactivity in theca cells was minimal in the antral 
 follicles .   a , secondary ( preantral ) follicles ; b , preantral follicle ; c , medium antral follicle ; d , 
 large antral follicle . gc , granulosa cells ; tc , theca cells ; it , interstitial cells ; cc , 
 cumulus cells ; o , oocyte ; n nucleus ; at , antrum . arrows indicate intense and aggregated 
 immunoreactivity for ifn-. magnification : 400. scare bar : 50 m . based upon the immunohistochemical evidence for the presence of inf- at different stage of 
 follicular development in vivo ( fig . 1 ) , the 
 potential role of ifn- in the regulation of fas mrna and protein in relatively undifferentiated 
 ( des - primed ) and differentiated ( ecg - primed ) granulosa cells was investigated in 
 vitro , using rt - pcr ( fig . 3 ) . in cultured des - primed granulosa 
 cells , ifn- up - regulated fas mrna in a concentration - dependent manner ( fig . high concentration ( > 100 u / ml ) of ifn- ( 1,000 u / ml ) resulted in the 
 loss of effectiveness of the cytokine in inducing fas mrna and protein expression ( fig . 2 ) . a , analysis of fas mrna expression by rt - pcr ; b , densitometric quantification of fas mrna 
 expression obtained from panel a. * , p<0.05 ; * * * , p < 
 0.001 ; # # # , p<0.001 vs. control group ( 0 u / ml ) . insert in each flowcytogram shows representative fluorescence microphotograph of fas 
 immunocytochemistry ( arrows and arrowheads indicate immunoreactivity in cytoplasm and 
 membrane , respectively . numbers 1 and 2 in each flowcytogram 
 indicate nonspecific ( partially overlapped with low intensity of fas - positive portion ) and 
 specific fas - positive ( high fluorescence intensity ) portion , respectively . although cultured ecg - primed granulosa cells also expressed fas mrna , its abundance was considerably 
 lower than that in des - primed cells . in addition , while fas mrna also increased in a 
 concentration - dependent manner in the presence of ifn- , significantly higher concentrations of the 
 cytokine ( 100 - 1,000 u / ml , p<0.01 ) were required to elicit a significant increase 
 in fas expression than those in des - primed cells ( fig . the 
 fas protein was localized in the des- and ecg - primed granulosa cells after ifn- treatment and the 
 expression levels were subjected to fas antibody - conjugated immuno - flowcytometric analysis ( fig . 3 ) . as shown in the inserts within each representative 
 flowcytogram ( fig . 3 ) , fas protein was localized in both the 
 cellular membrane ( arrow head ) and the cytoplasm [ peri - nuclear region ( arrow ) ] . in addition , intense 
 and aggregated immuno - fluorescence was observed in both des- ( fig . 3 ) granulosa cells 
 after ifn- exposure . whereas a significant population of the control group in the des - primed 
 granulosa cells exhibited the high intensity of immunofluorescence for endogenous fas 
 immunoreactivity [ fig . 3a , portion 2 ( 184.3% ) ] , the ecg - primed 
 control granulosa cells showed relatively smaller population with high immunofluorescence intensity 
 for fas [ fig . ifn- treatment ( 100 
 u / ml ) of des - primed granulosa cells significantly increased high intensity ( 337.5% ) portion of fas 
 immunofluorescene ( fig . however , treatment with 
 high concentration of ifn- ( 1,000 u / ml ) resulted in a lower number of cells exhibiting 
 immunofluorescence positivity for fas in high intensity ( 252.3% ) portion than those with 100 u / ml 
 but higher than that of the control group . in ecg - primed cells , however , only small increases in 
 immunofluorescence were detected with ifn- at concentrations of 100 and 1,000 u / ml when compared to 
 controls ( fig . 3e and 3f ) . the functional role of ifn--induced fas expression in induction of apoptosis were tested in 
 undifferentiated ( des - primed ) and differentiated ( ecg - primed ) granulosa cells in vitro 
 was determined by assessing their the ability to undergo apoptosis following a challenge 
 with an agonistic effect of fas mab . while typical cellular morphology and apoptosis in des - primed 
 granulosa cells are illustrated in fig . 4 , differences in these 
 features between des- and ecg - primed groups are summarized in table 
 1 . in des - primed ( undifferentiated ) granulosa cells , ifn- had minimal effects on 
 cellular morphology ( fig . however , addition of fas mab to the cell 
 cultures resulted in the appearance of apoptotic cellular morphology and characteristics ( fig . 4c & 4 g ) , which could be enhanced with pretreatment with 
 ifn- ( fig . 4d & 4h ) . in ecg - primed ( differentiated ) 
 granulosa cells , however , ifn- pretreatment failed to potentiate the pro - apoptotic effects of 
 agonistic fas mab , which were observed prominently in des - primed cells ( table 1 ) . left and right panels illustrate cellular morphology ( phase - contrast microscopy ) and 
 in situ apoptotic cell detection ( tunel ) , respectively . a and e , 
 control group [ ifn- vehicle ( medium ) ( 24 hr ) + igg ( 6 hr ) ] ; b and f , ifn- group [ ifn- ( 24 
 hr ) + igg ( 6 hr ) ] ; c and g , fas mab group [ ifn- vehicle ( medium ) ( 24 hr ) + fas mab ( 6 hr ) ] ; 
 d and h , ifn- and fas mab group [ ifn- ( 24 hr ) + fas mab ( 6 hr ) ] . arrowheads in left and right panels indicate floating cells and 
 tunel - positive cells , respecttively . magnification : 400. inserts in right panels show 
 representative apoptotic dna fragmentation pattern in each treatment group . - : rare , + : occasional , + + : frequent , + + + : extensive , + + + + : very extensive to further examine the colocalization of ifn- , fas and fasl expression and occurrence of 
 apoptosis in vivo , follicles at very early stage of antral formation ( estimated 
 between preantral and early antral stages ) were analyzed serially by immunohistochemistry and 
 in situ tunel method ( fig . 5 ) . immunoreactivity for ifn- was distributed in the granulosa but not the thecal layers ( fig . likewise , the most intense and aggregated form of fas 
 immuno - positive signals were detected in the loosely attached granulosa cells ( fig . although fasl immunoreactivity could be colocalized in fas - positive 
 cells , he most aggregated and intense ( arrows ) immunoreactivity was present in granulosa cells 
 mainly lining the antrum ( fig . 5c ) . similarly , apoptotic 
 ( tunel - positive ) granulosa cells were also detected in antral granulosa cells ( fig . a , ifn- ; b , fas ; c , fasl ; d , tunel . gc , tc and it represent granulosa cells , theca cells and 
 interstitial cells , respectively . in the present study , we have localized rat ifn- in the rat ovary during the follicular development and 
 tested its role on granulosa cell death via induction of the fas / fasl system in granulosa cells at 
 different stage of differentiation . we have found that the responsiveness of granulosa cells to ifn- in 
 terms of fas receptor expression was significantly compromised with follicular maturation . the 
 immunoreactivity of ifn- was most intense in early stages of folliculogenesis , such as in preantral 
 follicles . up - regulation of fas by ifn- and the subsequent increased responsiveness to an apoptotic 
 death signal ( e.g. fas activation by agonistic fas mab ) were more evident in granulosa cells from the 
 preantral follicles than those from antral follicles previously exposed to gonadotropins . the importance of intraovarian interaction between immune cells ( lymphocytes and macrophages ) and 
 granulosa cells in ovarian physiology via the synthesis and actions of cytokines ( e.g.tnf and il ) has 
 been studied extensively ( adashi , 1992 ; terranova et al . , 1993 ) . although the modulatory roles of ifn- in granulosa cell 
 differentiation and steroidogenesis have been demonstrated in vitro ( gorospe et al . , 1998 ; xiao and 
 findlay , 1992 ) , ifn- is 
 detectable in the follicular fluids ( grasso et al . , 1988 ) and 
 lymphocytes present in the ovary have been suggested to be an important contributor to the follicular 
 ifn- ( adashi , 1992 ) . immune cells ( lymphocytes / macrophages ) are 
 believed to be mainly present in atretic antral follicles ( best et al . , 
 1996 ) , , 1994a ; brannstrom et al . , 1994b ) 
 where they remove damaged and dead cells by secreting cytotoxic cytokines and phagocytosis . the presence 
 of immune cells during early stage of follicular development has not been reported and the possibility 
 that immune cells are the only the source of ifn- in preantral follicles can not be excluded . in this 
 study , ifn- was localized predominantly in granulosa cells and oocytes throughout the follicular 
 development but more abundantly in those at the early follicular ( preantral ) stage . it has been 
 demonstrated that tnf- and fas ligand are expressed in granulosa cells and oocytes ( chen et al . although we have not studied ifn- gene expression in ovarian cells in the current 
 study , it is possible that oocytes and granulosa cells in the preantral follicles may be an important 
 source of ifn- in the ovary and that this cytokine plays an autocrine and/or paracrine role in 
 regulating the expression and actions of other cytokines involved in cell removal process in the 
 degrading follicles . ifn- is an important modulator of granulosa cell differentiation during the early stage of follicular 
 development ( gorospe et al . , 1988 ; xiao and findlay , 1992 ) , although its functional role in differentiated granulosa 
 cells in late follicular development ( i.e. , preovulatory follicles ) remains to be further established . granulosa cell apoptosis is frequently observed in early antral ( penultimate ) stage of follicular 
 development , it is only assumed that up - regulation of fas receptor by ifn- in granulosa cells of 
 preantral follicles is prerequisite for fasl ligation and that the latter could be activated by 
 intra - ovarian factor(s ) following antrum formation . it has been suggested that ifn- facilitate 
 apoptosis in luteinized granulosa cells by decreasing endogenous igf activity via stimulation of igfbps 
 rather than of fas activation ( cataldo et al . , 1998 ) . however , the 
 concentrations of ifn- used in above studies could have been pharmacological and cytotoxic to the 
 cells . quirk et al ( 1995 ) demonstrated fas - mediated apoptosis in 
 human granulosa - luteal cells and suggested that ifn- up - regulates fas mrna and protein expression and 
 that pretreatment of cells with ifn- alone or in combination with hcg facilitates fas mab - induced 
 cytotoxicity . therefore , it is unlikely that the physiological concentration of ifn- could induce fas 
 expression in fully differentiated luteinized ( or luteinizing ) granulosa cells . recently , quirk et al ( 1998 ) showed that combined ifn- and tnf treatment 
 was more effective in inducing fas expression and potentiating fas mab - induced apoptosis in cultured 
 granulosa cells ( mostly from large preovulatory follicles in ecg - primed mice ) than by either ifn- or 
 tnf alone . the results suggest that the mechanism(s ) of induction of apoptosis and responsiveness 
 against apoptotic insult ( e.g. ifn- ) in differentiated granulosa cells might be much more complex and 
 resistant than those of undifferentiated granulsoa cells , as also seen in the present study . the possible mediatory role of the fas / fasl system in granulosa and luteal cell apoptosis during 
 follicular atresia and luteal regression , respectively have been investigated in several mammalian 
 species ( quirk et al . , 1995 ; previous reports have demonstrated that increased granulosa cell fas and fasl protein 
 content is associated with apoptosis at the penultimate ( early ) stage of follicular development 
 in vivo ( kim et al . , 1998 ) , and that 
 p53-mediated granulosa cell apoptosis involves the activation of the fas / fasl system ( kim et al . , 1999 ) . these studies also suggested that fsh is a key 
 inhibitory factor on granulosa cell death as well as the involvement of the fas / fasl system in 
 follicular atresia . the nature of the regulation of fas receptor expression throughout the follicular 
 development , however , remains unclear . although the regulation of fas receptor expression by ifn- has 
 been demonstrated in human granulosa - luteal cells in vitro ( quirk et al . , 1995 ) , the potential regulatory role of ifn- in fas mrna and protein 
 expression in undifferentiated granulosa cells during early stages of folliculogenesis has not been 
 thoroughly investigated . in the present studies , we have studied the possible role of ifn- in the 
 regulation of granulsoa cell fas expression during follicular development . while increased fas 
 expression in granulsoa cells from preantral or early antral follicles could be induced with low 
 concentrations of ifn- ( 10 - 100 u / ml ) , higher concentrations ( 1,000 u / ml ) were required in ecg - primed 
 cells . our present immuno - flowcytometric analysis also confirmed that undifferentiated ( des - primed ) 
 granulsoa cells were more responsive to ifn- in the induction of fas receptor than cells pretreated 
 with gonadotropin . these findings are not only consistent with the studies by quirk et al ( 1995 ) which showed increased expression of fas protein in 
 gonadotropin - primed human granulosa cells following exposure to very high concentration ( > 10,000 
 u / ml ) of ifn- in vitro , but extended their observations to suggest a role for the 
 cytokine in the ovary at physiological concentrations . it is well established that fsh is an important cell survival factor in the mammalian ovarian follicle 
 ( chun et al . whereas stimulation of immature rats with ecg resulted in suppressed basal 
 granulosa cell fas / fasl levels and apoptosis , decreased atresia and enhanced follicular growth , 
 withdrawal of gonadotropic support with the injection of a neutralizing antibody in 
 vivo during early antral development led to increased granulosa cell fas / fasl expression 
 and apoptotic cell death , and eventual follicular demise ( kim et al . , 
 1998 ) . in the present studies , we have demonstrated that while ifn- effectively up - regulated 
 fas expression in granulosa cells from preantral and early antral follicles and sensitized them to the 
 pro - apoptotic action of agonistic fas antibody , cells isolated from large antral follicles previously 
 exposed to the gonadotropin in vivo were more differentiated but less responsive to 
 ifn-. these findings suggest that the anti - apoptotic role of fsh in the granulosa cells may in part 
 involve its modulatory influence on ifn- action , and are consistent with the concept that ovarian 
 follicles beyond the  penultimate  stage of development are protected physiologically from pro - apoptotic 
 insult and destined to ovulate ( chun et al . , 1996 ; boone et al . , 1997 ) . the mechanism(s ) by which the gonadotropin 
 modulates ifn--induced fas expression granulosa cells is unclear . it has been demonstrated that fas 
 expression is regulated by the newly identified fas - regulatory gene tdag51 , the expression of which is 
 pkc - dependent ( wang et al . , 1998 ) . since ifn- , like tnf ( sancho tello and terranova , 1991 ) and il-3 ( farrar et al . , 1985 ) , appears capable of activating pkc ( ostrowski et al . , 1988 ) , it is possible that the reduced responsiveness of 
 differentiated granulosa cells to ifn- might be due to pka activation by fsh . in addition , activation 
 of pka pathway is known to inhibit pkc - dependent mechanism(s ) in a variety of biological systems 
 including the granulosa cell ( shinohara et al . , 1985 ) . in this 
 context , differential responsiveness of granulosa cells at different cytodifferentiation to ifn- may be 
 related to possible interactions between signal transduction pathways of ifn- and gonadotropin . it is 
 possible that the efficient fas gene expression in undifferentiated granulosa cells may involve 
 activation of pkc pathway by ifn-. fas receptor signaling is a complex process and continues to capture considerable attention and research 
 efforts . fas activation initiated by ligation and oligomerrization results in recruitment of fadd 
 ( fasassociated death domain - containing protein ) ( chinnaiyan et al . , 
 1995 ) . fadd then activates procaspase8 ( muzio et al . , 
 1996 ) , which appears to be the first step of a proteolytic cascade that activates other 
 caspases such as caspases3 , 6 , and 7 ( hirata et al . , 1998 ) ifn- significantly increased the population of granulosa cells exhibiting high 
 fluorescence intensity of fas immunoreactivity . while this increase might merely represent an increase 
 in the cell population with enhanced fas expression , it is tempting to speculate that the high intensity 
 of fluorescence reflects the oligomerization of fas receptor proteins , and that fas oligomerization in 
 response to ifn- in undifferentiated granulsoa cells may be more efficient and functional than in 
 differentiated granulosa cells . whether this indeed is the case requires further investigation and 
 confirmation . in this context , intense and aggregated immunoreactive fas protein has consistently been 
 detected in cytoplasm as well as cellular membrane of cultured granulosa cells of the present study . these results support our previous observation suggesting the presence of aggregated fas immunostaining 
 in the granulosa cells of atretic early antral follicles in the ovary ( kim et al . , 1998 ) . moreover , recent studies have indicated that fas is also localized in the 
 peri - nuclear region of the cytoplasm and that fas - trafficking from golgi apparatus to membrane is 
 p53-dependent and required for fas - mediated apoptosis ( bennett et al . , 
 1998 ) . in light of our recent demonstration of the role of p53 in the regulation of fas 
 expression in rat granulosa cells ( kim et al . , 1999 ) and their 
 intense fas immunostaining following ifn- challenge , investigations into whether this cytokine plays a 
 role in fas trafficking in the granulosa cell would provide important clues on the cellular mechanism 
 involved in the control of fas - mediated granulsoa cell apoptosis during early folliculogenesis . in summary , we have demonstrated that ifn- plays a pivotal role in regulating granulosa cell 
 differentiation and apoptosis by potentiating fas function , a process modulated by gonadotropin ( i.e. 
 fsh ) . the role of ifn- is most prominent in undifferentiated granulosa cells during early stage of 
 follicular development .
abstractfas ligand ( fasl ) and its receptor fas have been implicated in granulosa cell apoptosis during 
 follicular atresia . 
 although interferon - gamma ( ifn- ) is believed to be involved in the regulation 
 fas expression in differentiated granulosa or granulosa - luteal cells , the expression of this 
 cytokine and its role in the regulation of the granulosa cell fas / fasl system and apoptosis during 
 follicular maturation have not been thoroughly investigated . in the present study , we have examined 
 the presence of ifn- in ovarian follicles at different stage of development by immunohistochemistry 
 and related their relative intensities with follicular expression of fas and fasl , and with 
 differences in granulosa cell sensitivity to fas activation by exogenous agonistic anti - fas 
 monoclonal antibody ( fas mab ) . although ifn- immunostaining was detectable in oocyte and granulosa 
 cells in antral follicles , most intense immunoreactivity for the cytokine was observed in these 
 cells of preantral follicles . 
 intense immunoreactivity for ifn- was most evident in granulosa cells 
 of atretic early antral follicles where increased fas and fasl expression and apoptosis were also 
 observed . whereas low concentrations of ifn- ( 10 - 100 u / ml ) significantly increased fas expression 
 in undifferentiated granulosa cells ( from preantral or very early antral follicles ) in 
 vitro , very higher concentrations (  1,000 u / ml ) 
 were required to up - regulate of fas in 
 differentiated cells isolated from ecg - primed ( antral ) follicles . 
 addition of agonistic fas mab to 
 cultures of granulosa cells at the two stages of differentiation and pretreated with ifn- ( 100 
 u / ml ) elicited morphological and biochemical apoptotic features which were more prominent in cells 
 not previously exposed to the gonadotropin in vivo . 
 these findings suggested that 
 ifn- is an important physiologic intra - ovarian regulator of follicular atresia and plays a pivotal 
 role in regulation of expression of fas receptor and subsequent apoptotic response in 
 undifferentiated ( or poorly differentiated ) granulosa cells at an early ( penultimate ) stage of 
 follicular development .