materials : anti - fas antibody , its blocking peptide and 5 different kinds 
 of antibodies were purchased commercially ( supplementary table 1 ) . vectastain elite abc 
 staining kit was obtained from vector laboratories ( burlingame , ca , u.s.a . ) . animals and tissue sampling : animal handling and experimental procedures 
 were approved by the committee for laboratory animals care and use at kitasato university 
 school of veterinary medicine ( approval number , 12006 ) . female rats of wistar - imamichi 
 strain that were 3~4 months old , 250~350 g in weight and showing a regular 4-day estrous 
 cycle were used . pseudopregnancy was induced by mechanical stimuli of vagina , and the first 
 day of the diestrous smear was designated as day 1 of pseudopregnancy ( psp1 ) . pregnancy was 
 induced by mating with a fertile male , and the day of fertilization ( the estrous smear with 
 sperm ) was designated as day 1 of pregnancy ( prg1 ) . the duration of pregnancy was 23 days , 
 and therefore , prg23 corresponded to day 0 of postpartum ( pp0 ) . during 09:00~11:00 hr of the day , the ovaries of cycling rats were harvested on the 
 estrus , based on the finding that prl surge occurring in the proestrous afternoon is a 
 potential inducer of apoptotic changes and macrophage infiltration in cycls [ 9 , 22 ] . pseudopregnant animals were sacrificed on psp6 ( active luteal phase ) , psp12 ( functionally 
 regressed phase ) and psp15 . pregnant rats were sacrificed on prg6 , prg15 , prg19 , prg21 and 
 prg23 ( just before delivery or just delivering ) . postpartum rats were done on pp0 ( just 
 after delivery ) , pp3 and pp6 . immunohistochemsitry : expression of fas , fasl , cleaved caspase-3 ( active 
 form ) , myeloperoxidase ( mpo , a neutrophil marker ) or cd68 ( a monocyte / macrophage marker ) in 
 the cls was analyzed by immunohistochemistry . tissues were serially sectioned ( 35 m in thickness ) , 
 deparaffinized and examined . endogenous peroxidase activity was quenched by pretreatment 
 with 0.3% h2o2 in methanol for 30 min . all the following procedures 
 used 0.01 m phosphate buffered saline ( pbs ) ph 7.5 as the standard aqueous buffer for fas , 
 mpo and cd68 staining . for staining of fasl and cleaved caspase-3 , 0.01 m tris buffered 
 saline ( tbs ) ph 7.5 was used instead , and for retrieval of these two antigens , tissue 
 sections were treated with microwave in 10 mm citrate buffer for 10 min followed by 
 treatment with 0.1 mm glycine in tbs for 30 min . tissue sections were incubated with each 
 specific antibody , whose dilution at use was described in supplementary table 1 , at 4c 
 overnight . antigen / antibody complexes were visualized with the vectastain abc staining kit 
 and 3,3-diaminobenzidine tetrahydrochrolide as peroxidase substrate . negative controls were 
 performed with non - immunized mouse igg . to confirm the specificity of the anti - fas antibody 
 against fas especially , an equal volume of the antibody with fas / apo1 blocking peptide 
 solution ( 0.5 mg / ml ) all 
 slides were counterstained with hematoxylin . as signals of fas immunoreaction were not always present evenly throughout the tissue but 
 often localized densely to several focal sites , the number of all signals was counted 
 throughout cross - sectioned area of each cl and was divided by the area ( mm ) . much care was taken for cycls examination to differentiate into four stages ( generations ) 
 based on their histological features [ 21 , 30 ] and to exclude the newest cls that just began to be 
 formed in the morning of the harvest day . as for pscls and prcls , five to 9 different ovarian 
 specimens of each group were investigated , and the numbers of cl evaluated were indicated in 
 figure legends . statistical analysis : differences of the means among groups were analyzed by tukey - kramer s multiple comparison 
 test . immunohistochemical localization of fas in cycls of the estrous rat : fas 
 immunostaining was first validated in the ovary harvested in the estrous morning . in many cycls , fas immunoreactions were evidently and densely detected at multiple focal sites with 
 tissue damage and accumulation of inflammatory cells ( fig . ( b ) pre - absorption of the antibody with the blocking peptide 
 completely abolished the immunoreaction . ( c ) immunoreactive fas - positive cell types 
 include large ( black arrowheads ) and small ( white arrowheads ) luteal cells , capillary 
 endothelial cells ( blue arrowheads ) and blood derived granulocytes and macrophages 
 ( green arrowheads ) . positive immunoreactions for fasl ( d ) , fas ( e ) and cleaved 
 caspase-3 ( f ) , the most of which were densely scattered in inflammatory - like and 
 fibrotic sites ( closed dashed lines in e ) in the cycl . larger magnification of 
 portions of central zone of d and f was available in supplementary fig . 1a and 1b , 
 respectively , to identify the immunopositive cell types . associated immunostaining for 
 fas ( g ) , mpo ( h ) and cd68 ( i ) was also abundant to inflammatory - like and fibrotic 
 sites ( surrounded by the dashed line in g ) . magnification of rectangles with dashed 
 lines in h and i was available in supplementary fig . scale 
 bars , 50 m ( a , b ) , 25 m ( c ) , 100 
 m ( d  i ) . ) . the reaction was less frequently , but evidently scattered in the 
 remaining , seemingly intact sites . the positive cell types 
 in cycls included large and small steroidogenic cells , capillary endothelial cells and 
 leukocytes ( granulocytes and macrophages ) , many of which lost the integrity of plasma and 
 nuclear membranes ( fig . ( b ) pre - absorption of the antibody with the blocking peptide 
 completely abolished the immunoreaction . ( c ) immunoreactive fas - positive cell types 
 include large ( black arrowheads ) and small ( white arrowheads ) luteal cells , capillary 
 endothelial cells ( blue arrowheads ) and blood derived granulocytes and macrophages 
 ( green arrowheads ) . positive immunoreactions for fasl ( d ) , fas ( e ) and cleaved 
 caspase-3 ( f ) , the most of which were densely scattered in inflammatory - like and 
 fibrotic sites ( closed dashed lines in e ) in the cycl . larger magnification of 
 portions of central zone of d and f was available in supplementary fig . 1a and 1b , 
 respectively , to identify the immunopositive cell types . associated immunostaining for 
 fas ( g ) , mpo ( h ) and cd68 ( i ) was also abundant to inflammatory - like and fibrotic 
 sites ( surrounded by the dashed line in g ) . magnification of rectangles with dashed 
 lines in h and i was available in supplementary fig . scale 
 bars , 50 m ( a , b ) , 25 m ( c ) , 100 
 m ( d  i ) . adjacent sections of cycls were used to investigate whether the detected fas signals 
 associated or not spatially with fasl and cleaved caspase-3 localization . 1e ) at 
 focal sites , probably along the distribution of intra - luteal blood capillaries . fasl 
 immunoreactions were present in blood - derived , non - luteal cells and steroidogenic cells 
 ( supplementary fig . intra - tissue distribution 
 of immunoreactive cleaved caspase-3 overlapped in large part with that of fas , with their 
 dominant presence at focal inflammatory - like sites ( fig . large magnification revealed the cell types possessing the executor caspase as 
 steroidogenic cells , capillary endothelial cells and granulocytes ( supplementary fig . the spatial association of a set of fasl / fas / cleaved caspase-3 
 was also evident in granulosa cells of an atretic follicle present in the same ovarian 
 specimen ( data not shown ) . a previous finding that neutrophils and monocytes / macrophages were the most abundant 
 leukocytes in rat cls   led us next to determine 
 their distribution in relation with that of fas signal . 1 g ) also 
 exhibited accumulated signals of mpo - positive cells ( fig . in addition to 
 disappearance of signals after antibody neutralization , these associations of apoptotic and 
 phagocytotic systems increase the validity of the current immunological detection of fas and 
 cells that would undergo fas - mediated cell death and their engulfment . fas immunolocalization in regressing pscl and prcl : next , we determined 
 fas expression in regressing pscl and prcl that had once been functionally activated . in the 
 ovary harvested on psp15 , fas signals were accumulated to the focal , inflammatory - like 
 regions of degenerating pscl ( fig . immunoreactive fas 
 signals on psp15 located frequently at focal sites of inflammatory - like reactions 
 ( marked by the dashed lines ) in regressed pscl ( a ) . the signals were present on 
 steroidogenic cells ( black arrowhead ) , endothelial cells ( blue arrowhead ) and 
 granulocytes ( green arrowheads ) ( b ) . fas signals ( highlighted with black arrowheads ) 
 ( c ) as well as mpo - positive cells ( d ) and cd68-positive cells ( e ) were fairly evenly 
 scattered throughout the prcl tissue sampled on pp0 . fas signals were present on 
 endothelial cells ( blue arrowhead ) and migratory cells ( green arrowheads ) , but not 
 steroidogenic cells with intact nuclear structure ( black arrowheads ) ( f ) . signals of 
 cleaved caspase-3 in a portion of prcl were predominant in endothelial cells ( blue 
 arrowheads ) and absent in steroidogenic cells ( black arrowheads ) ( g ) . scale bars , 100 
 m ( a , c , d , e ) , 12.5 m ( b , f ) , 20 
 m ( g ) . ) , similarly to that in cycl . the immunoreactive cell types were 
 mostly steroidogenic cells with irregular shapes of nuclear structures , almost identical to 
 that in cycls ( fig . immunoreactive fas 
 signals on psp15 located frequently at focal sites of inflammatory - like reactions 
 ( marked by the dashed lines ) in regressed pscl ( a ) . the signals were present on 
 steroidogenic cells ( black arrowhead ) , endothelial cells ( blue arrowhead ) and 
 granulocytes ( green arrowheads ) ( b ) . fas signals ( highlighted with black arrowheads ) 
 ( c ) as well as mpo - positive cells ( d ) and cd68-positive cells ( e ) were fairly evenly 
 scattered throughout the prcl tissue sampled on pp0 . fas signals were present on 
 endothelial cells ( blue arrowhead ) and migratory cells ( green arrowheads ) , but not 
 steroidogenic cells with intact nuclear structure ( black arrowheads ) ( f ) . signals of 
 cleaved caspase-3 in a portion of prcl were predominant in endothelial cells ( blue 
 arrowheads ) and absent in steroidogenic cells ( black arrowheads ) ( g ) . scale bars , 100 
 m ( a , c , d , e ) , 12.5 m ( b , f ) , 20 
 m ( g ) . in contrast to cycls and pscls , prcls harvested on pp0 did not involve focal 
 inflammatory - like reactions . 3a ) were scattered throughout the tissue and mpo - positive 
 neutrophils ( fig . 2f ) . hypertrophied steroidogenic cells with intact nuclei 
 structure were negative for fas . the immunoreactions for cleaved caspase-3 in prcls were 
 dominant in endothelial cells , positive but to a lesser extent in leukocytes , and very faint 
 in steroidogenic cells ( fig . age - related dynamics of the number of fas - positive cells in cls : fas 
 expression was morphometrically evaluated by counting the numbers of fas - positive cells in 
 three types of cls under different functional and structural states ( table 1table 1.age-related changes in the number of fas - positive cells in 3 types of clcyclpsclprclstage 19.3 + / 2.1 ( 18)psp66.0 + / 3.4 ( 11)prg67.7 + / 1.9 ( 17)226.9 + / 3.9 ( 34)1228.1 + / 7.1 ( 20)1517.3 + / 3.4 ( 9)310.1 + / 2.7 ( 17)1515.8 + / 4.6 ( 23)193.3 + / 1.0 ( 16)410.0 + / 2.7 ( 30)216.4 + / 2.7 ( 17)2328.3 + / 4.7 ( 17)pp323.4 + / 4.1 ( 27)62.8 + / 1.3 ( 9)fas antigen was counted in distinct ages of cycl , pscl and prcl and displayed as the 
 number ( mean + /sem ) of positive cells per defined area ( /mm ) of cl 
 sections . the number in parentheses showed each number of cls examined that were 
 obtained from 59 ovarian specimens . different alphabetical letters in the superscript 
 within the same row mean significant differences among the stages of each cls 
 ( p<0.05 ) . ) . fas antigen was counted in distinct ages of cycl , pscl and prcl and displayed as the 
 number ( mean + /sem ) of positive cells per defined area ( /mm ) of cl 
 sections . the number in parentheses showed each number of cls examined that were 
 obtained from 59 ovarian specimens . different alphabetical letters in the superscript 
 within the same row mean significant differences among the stages of each cls 
 ( p<0.05 ) . in cycls , multiple focal sites with inflammatory - like and degenerative reactions were 
 evident in the stage 2 of cls , where fas expression was marked ( fig . 1a , 1e and 1 g ) . the number of fas - positive cells in the newest 
 cls ( stage 1 ) was 9.3  2.1 /mm , and it showed a 2.9 fold - increase 
 ( p<0.05 ) in the stage 2 . the number decreased in the stage 3 as the 
 inflammatory - like reaction turned to resolution . it remained low in the last stage as the 
 resolution of inflammation - like reaction and fibrotic replacement were almost completed . this stage - related change in fas expression is similar to those of inflammation - associated 
 infiltration of neutrophils and macrophages described in our previous report . in pscls , the number of fas - positive cells increased from 6.0  3.4 /mm on psp6 
 to 28.1  7.1 on psp12 ( p<0.05 ) ( table 1 ) . the value decreased on psp15 to the level insignificantly different from 
 both of those on psp6 and psp12 . in prcls , the number of fas expressing cells was 7.7  1.9/mm on prg6 and increased 
 significantly on prg15 ( table 1 ) when p4 
 secretory function was further potentiated . the value once declined to the basal level on 
 prg19 and prg21 and increased drastically on pp0 ( a 4.4 fold - increase from prg21 , 
 p<0.05 ) . it remained elevated on pp3 and decreased to the basal level 
 on pp6 ( p<0.01 ) . we here have specified and localized the cells expressing immunoreactive fas in naturally 
 regressing cls of rats . histological evidence supports the good spatial association of those 
 fas - positive cells with apoptotic ( fasl and active casapse-3 ) and phagocytotic ( macrophages 
 and neutrophils ) machineries . the comparative study has revealed that prcl shows the unique 
 features in steroidogenic cell fas expression and occurrence of inflammatory - like reaction , 
 both of which are different from those in cycl and pscl . several reports on fas immunohistochemistry of rat and mouse ovaries have been so far 
 available , but all of those prepared the negative control groups by replacement with 
 non - specific antibody or the omission of the primary antibody . the currently prepared 
 negative control ( the antibody neutralization ) is the most appropriate one , warranting the 
 more reliability of fas detection . rat cl cells possessing fas immunoreactivity in 
 vivo include steroidogenic cells and vascular endothelial cells mainly , and 
 granulocytes additionally . in the term of the cell 
 types , our data are largely consistent with previous reports on fas immunohistochemistry of 
 rat and mouse cycls [ 15 , 29 , 30 ] . intra - tissue distribution 
 and aging - related changes of fas expression are , however , distinct from those in previous 
 reports . frequent and focal occurrence of fas - expressing cells at multiple sites and the 
 temporal increase in fas - positive cells number are common characteristics in cycls and 
 pscls . this feature of spatial distribution of fas is well consistent with and strongly 
 supported by morphologic assessment of apoptosis in naturally regressing cycls [ 14 , 15 , 17 ] and pscls [ 12 , 
 13 ] using the terminal deoxynucleotidyl transferase 
 nick end labeling ( tunel ) method . one important finding of this study is the probable lack of fas expression specifically in 
 steroidogenic cells of prcls . this might coincide with the previously reported [ 5 , 25 ] and 
 currently confirmed fact that the effecter caspase-3 was present in steroidogenic cells at 
 the lesser amount than that in endothelial cells . furthermore , not a few studies in 
 vivo have presented evidence for vague or modest apoptotic signs of dna 
 fragmentation in steroidogenic cells of peri - parturient prcls [ 5 , 11 , 20 , 25 , 33 ] . evidence in vitro that rat prcl isolated before and 
 after parturition represented dna fragmentation following serum deprivation [ 1 , 11 ] does not 
 mean its actual occurrence in vivo . taken with the consideration of evident 
 fas expression in steroidogenic cells in cycl and pscl , an unknown mechanism associated with 
 further activation of prcl in the second half of pregnancy   may protect steroidogenic cells specifically from fas expression . action of 
 sole p4 is unlikely responsible , because steroidogenic cells expression of fas occurred in 
 pscl . previous studies have shown enhanced expression and activity of caspase-3 and its 
 up - stream caspases-2 , -8 and -9 in rat prcl tissue at term [ 25 , 33 ] . it may be possible that a non - fas 
 death signal , tumor necrosis factor ( tnf )  shown to be expressed in steroidogenic cells in 
 prcl   causes caspase-3 activation , but not reaching 
 to dna degradation in vivo . we found marked presence of cytoplasmic vacuole 
 and seemingly intact nuclei in steroidogenic cells of prcl previously   and here again . prcl regression is likely to involve 
 cell - type - dependent different modes of cell death . spatio - temporally matched appearance of fas - positive cells in regressing cls strongly 
 suggests fas expression as a critical regulatory component of regression . previous reports 
 with a variety of cls models [ 5 , 6 , 15 , 17 , 28 ] demonstrated that fasl , 
 rather than fas , was more actively regulated and thus was more responsible for 
 fasl / fas - mediated apoptotic signaling during regression . however , our quantitative data of 
 fas expression correlated temporally with tunel - sensitive apoptosis in cycl [ 9 , 22 ] and prcl 
 [ 11 , 20 , 
 25 , 33 ] , 
 suggesting an active role of fas p4 suppressed fas mrna expression in steroidogenic cells from rat   and bovine   cls in vitro . in vivo this steroid inhibited 
 apoptosis induction in postpartum rat prcl [ 10 , 11 ] , but facilitated prl - induced apoptosis in rat cycl 
 [ 8 , 26 ] . in 
 our experiments of modulation of p4 synthesis or action by local administration of p4 , ru486 
 or trilostane in vivo , there were no alterations in fas expression in cycl , 
 pscl and prcl ( data not shown ) . the discrepancies between in vivo and 
 in vitro studies and among different cls models suggest the complex 
 mechanism elaborated through the synthesis and action ( sensitivity ) of p4 . they may also be 
 related partly to the differential indices of determining fas expression between previous 
 studies [ 19 , 23 ] ( rt - pcr analysis on purified steroidogenic cells ) and ours ( morphometric 
 counting of all cell types positive for fas ) . distribution of fasl was similar to that of fas in regressing cycls , but the cell types 
 expressing fasl are not sufficiently solved in this study . [ 17 , 18 ] 
 documented cd3-positive t lymphocytes as an effecter of prl - stimulated fasl expression and 
 its induction of fas - mediated apoptosis of steroidogenic cells in a rat cycl model . previous 
 studies with other types of rat cls [ 5 , 28 , 30 ] and this 
 study ( supplementary fig . 1a ) , however , strongly 
 suggest that fasl - expressing cells are not such lymphocytes exclusively . to clarify the 
 source , expressional regulation and functional interaction with fas of fasl are essential to 
 further understand fas - mediated cell death occurring in regressing cl . similar distribution of accumulated neutrophils / macrophages and fas expression in any types 
 of regressing cls is significant . these data support the general concept that phagocytes are 
 actively recruited into impaired region(s ) of regressing cls and contribute to tissue 
 destroy and remodeling through secreting cytotoxic tnf and interferon 
  , killing and engulfing cells and debris , and secreting extracellular matrix 
 proteases [ 14 , 15 , 24 ] . accumulation of phagocytes at 
 multiple foci in cycl and pscl represents a typical inflammatory - like feature , whereas their 
 distribution evenly throughout prcl tissue does a distinct (  non - focal  and  mild  ) 
 inflammatory feature . this is , as far as we know , the first indication of the spatial nature 
 of fas expression and immune cells distribution in regressing cl and of cl type - dependent 
 difference . furthermore , the occurrence of fas - positive cell in prcl is also similar to the 
 temporal alteration in infiltrated neutrophils and macrophages ( our unpublished data ) , 
 suggesting a shared local regulation of fas expression and phagocytes mobilization . our 
 effort is now made to study some unique features exhibited by rat prcl in the latter half of 
 pregnancy and postpartum . in summary , this study reveals a part of the nature of fas expression in rat cl . findings 
 on fas expression by multiple cell types in a spatio - temporally regulated manner provide 
 further insight into the luteolytic mechanism involving fas - related cell death and 
 phagocytes - associated inflammatory - like response . their differences among the cl types 
 within a species make us recognize again the diverse regulatory mechanism of mammalian cl . 
 in many experimental models as well as cls , different methods of apoptosis detection and 
 data evaluation have produced so variable results and conclusions . the cell death mechanism during cl regression should be searched 
 further and again with the aid of more advanced knowledge and methodology on apoptosis and 
 other modes of cell death [ 7 , 16 ] .
though fas / fas ligand ( fasl ) system - dependent apoptosis is considered to be the primary 
 form of cell death in regressing corpus luteum ( cl ) , the cellular identity and regulation 
 of expression of the ligand and receptor molecules are not fully understood . here , we 
 focused on immunohistochemical determination of fas expression during natural regression 
 with comparison of three different types of rat cls . 
 detected fas was in good spatial 
 association with cleaved caspase-3 and fasl proteins and with macrophages and neutrophils . 
 in cls of the cycle and pseudopregnancy , fas - positive cell types included large and small 
 luteal ( steroidogenic ) cells and capillary endothelial cells mainly , and blood - derived 
 immune cells occasionally . 
 fas signals were abundant at multiple focal inflammatory - like 
 sites . 
 in contrast , fas signals in cl of pregnancy did not localize in steroidogenic 
 cells , but almost exclusively in endothelial cells and granulocytes . 
 the signals scattered 
 evenly throughout the cl tissue as phagocytes also did . in all cls types , 
 the numbers of 
 fas - expressing cells increased transiently after functional inactivation and at the early 
 phase of structural regression . 
 this observation revealed spatio - temporally regulated 
 expression of fas that was highly associated with apoptotic and phagocytotic systems and 
 type - dependent differences in fas expression and phagocytes dynamics in naturally 
 regressing cl of rats .