Patent Application: US-9006006-A

Abstract:
methods and compositions for enhancing development of a preimplantation mammalian embryo and for increasing the live birth potential of an in vitro fertilized mammalian embryo are disclosed . an in vitro method of activating the peroxisome proliferator activated receptor δ in a preimplantation mammalian embryo comprises culturing an embryo in an embryo culture medium , and upon or after commencement of expression of pparδ in the cells of the embryo , activating the pparδ by adding an amount of a pparδ ligand to said medium effective to bind to pparδ to deter apoptosis in the cells of the cultured embryo and / or increase proliferation of the cells of the cultured embryo .

Description:
oviduct - derived prostacyclin optimizes the development of preimplantation embryos and enhances their potentials of hatching , implantation and live birth . the signaling of prostacyclin in the preimplantation embryos is not clear . to explore the possibility that peroxisome proliferator activated receptor δ ( pparδ ) may mediate the effects of pgi2 in the preimplantation embryos ( e . g ., pgi2 signaling ), the expression of pparδ in preimplantation embryos was investigated , and it was activated by synthetic pparδ ligand . the preimplantation embryos &# 39 ; responses to synthetic pparδ ligand were assessed based on complete embryo hatching and apoptosis of embryonic cells . to reveal the role of pparδ in the development of preimplantation embryos , the development of pparδ knockout ( pparδ targeted ( pparδ −/−)) and wild type embryos were compared . it was found that preimplantation mouse embryos express pparδ , as determined by rt / pcr and western blot analysis , as described in more detail below . its activation by synthetic ligand enhanced embryo hatching and reduced apoptosis . synthetic pparδ ligand reduced apoptosis , increased proliferation , and enhanced embryo hatching in a concentration - dependent manner ( ed50 = 21 nm ). ablation of pparδ by gene targeting led to decreased cell proliferation and irreversible developmental delay in the preimplantation embryos . thus , preimplantation embryos express pparδ , which is critical to normal development and embryonic cell proliferation . the activation of pparδ by synthetic ligand enhances embryo hatching in - vitro . in light of these representative studies in a mouse model , it is proposed that mammalian embryos , including human , express pparδ . the expression of pparδ ensures orderly development of preimplantation embryos , and its activation by synthetic ligand enhances embryo hatching in vitro . source of reagents and institutional approval . unless specified otherwise , all reagents were purchased from sigma - aldrich co . ( st . louis , mo ., usa ). the research protocol was approved by the animal welfare committee of the university of texas health science center at houston . the care and the manipulation of mice were consistent with the guide for the care and use of laboratory animals published by the u . s . national institute for health ( nih publication no . 85 - 23 , revised 1996 ). harvest and culture of mouse embryos . mice were obtained from the following sources : c3h , 129s1 / svimj and c57bl6 / j , the jackson laboratory ( bar harbor , me ., usa ); icr and c57bl6 / nhd , harlan ( indianapolis , ind ., usa ); pparδ −/−, a gift from dr . r . evans ( the salk institute , la jolla , calif .). the wild type ( wt ) counter parts of pparδ −/− were bred by mating 129s1 / svimj and the offspring of c57bl6 / j × 129s1 / svimj . mice were kept in temperature and humidity controlled environment ( 07 : 00 light on , 19 : 00 light off ) with free access to food and water . the mouse embryos were harvested and cultured as described previously ( huang et al ., 2003 ). briefly , three - week old female mice were super - ovulated with intra - peritoneal injection of pmsg ( 5 iu ) followed by hcg ( 5 iu ) 42 - 46 hrs later . after hcg injection ( at 15 : 00 ), each female mouse was caged with one male mouse with proven fertility . unless specified otherwise , embryos ( at two - cell stage ) were harvested 44 - 48 hrs later and cultured ( 17 - 20 per group ) at 37 ° c . under 5 % co 2 in 4 - well plates ( nalge nunc international , naperville , ill ., usa ) containing 600 ul protein - free media . the htf media ( sage biopharma , bedminster , n . j ., usa ) was used during the first 48 hrs ; the αmem ( irvine scientific ), with earle &# 39 ; s salts and 2 mm glutamine , was used during the second 48 hrs . after 96 hrs of culture , each embryo was examined for the presence of zona pellucida . those completely free of the zona pellucida were counted as having completely hatched . the rate of complete hatching was calculated by dividing the number of completely hatched embryos by the number of total embryos . complete embryo hatching was used as an end point because it is a clear cut endpoint and correlates with the likelihood of implantation and viable pregnancy ( huang et al ., 2003 ; huang et al ., 2004b ). the concentration of the vehicle ( dmso ) was less than 1 : 10 , 000 ; control embryos received the same concentration of dmso . comparing the development of pparδ −/− and wt embryos in vivo . the progression of pparδ −/− and wt embryos in vivo was compared as follows . embryos were obtained from plugged mice 70 and 96 hrs after hcg injection . these time points were chosen because at 70 hrs after hcg injection , the majority of wt embryos develop into eight - cell or morula stage embryos , and , at 96 hrs after hcg injection , the majority of wt embryos advanced to blastocysts . the developmental stages of embryos obtained at 70 hrs after hcg injection were compared based on embryonic cell number , because a compacted eight - cell embryo and a morula - staged embryo cannot be differentiated reliably based on their morphology under phase contrast microscope . the developmental stages of embryos obtained 96 hrs after hcg injection were determined based on morphology under phase contrast microscope . the embryonic cell number was determined based on nuclear staining with 4 ′- 6 - diamidino - 2 - phenylindole ( dapi ), a dna - binding fluorescence dye , followed by examination with an immunofluorescence microscope under a uv filter ( axioplan 2 , carl zeiss , germany ). 5 - bromo - 2 ′- deoxy - uridine ( brdu ) uptake and the proliferation index . the proliferation of embryonic cells was determined based on their abilities to incorporate brdu . the assay was performed using a commercial kit ( brdu labeling and detection kit i , roche applied science , indianapolis , ind .) according to manufacturer &# 39 ; s protocols with some optimizations . one of the changes was the duration of incubation . preliminary experiments showed the optimal incubation time to differentiate the proportions of cells in the s - phase between pparδ −/− and wt embryos was 10 mins . for the comparison between control and l165 , 041 treated embryos , the optimal time was 6 minutes . the modified procedure is as follows . immediately after harvest , the embryos were incubated in 100 μl pre - equilibrated htf medium containing 10 μm brdu for 10 min at 37 ° c . under 5 % co 2 . following the incubation , the embryos were fixed in ice - cold glycine ( 50 mm ) in 70 % ethanol ( ph2 . 0 ) at − 20 ° c . for 30 mins . following rehydration / blocking in phosphate buffered saline ( pbs ) containing 1 % bsa , the embryos were incubated with diluted anti - brdu mouse monoclonal antibody ( 1 : 2 ) in the incubation buffer containing 1 % bsa and followed by another incubation with a diluted fitc - conjugated anti - rabbit igg antibody ( 1 : 4 ) in pbs containing 1 % bsa . both incubations were at 37 ° c . and lasted for 30 mins ; the embryos were washed in 200 μl pbs containing 1 % bsa after each incubation . after a final incubation in dapi ( 5 μg / ml ) for 90 mins at 25 ° c ., the embryos were mounted in 0 . 1 m tris hcl ( ph 8 . 5 ) containing 16 . 6 % elvanol ® 50 - 42 ( dupont wilmington , del ., usa ) and 2 . 5 % 1 , 4 - diazabicyclo -( 2 . 2 . 2 )- octane . cells that were in s - phase and , therefore , incorporated brdu were identified under a fitc filter . total cell number was determined based on dapi nuclear staining under a uv filter ( axioplan 2 , carl zeiss , germany ). the proliferation index ( percent of cells in s - phase ) was determined by dividing the number of brdu - positive cells by the number of total cells then multiplies by 100 %. terminal deoxynucleotidyl transferase mediated dutp nick end labeling ( tunel ) and dead cell index . the embryonic cells that undergo programmed cell death were identified by tunel assay using a commercial kit ( in situ cell death detection kit , roche applied science ) with some modifications . at each time point indicated , embryos were collected after culture or harvested from mice and fixed in 4 ° c . pbs containing 4 % buffered paraformaldehyde for 30 min . prior to tunel , the embryos were incubated at 37 ° c . with 5 % triton in pbs for 20 mins . the manufacturer &# 39 ; s protocol was modified . up to 20 blastocysts were incubated at 37 ° c . in 20 ul of tunel reaction solution ( 2 ul of tunel enzyme and 18 ul of 1 : 4 diluted labeling solution ) in a humidified chamber for 60 mins . the embryos were then incubated in dapi ( 30 ug / ml ) for 5 mins at 37 ° c . before mounted in 16 . 6 % elvanol ® 50 - 42 ( dupont , wilmington , del ., usa ) in 0 . 1 m tris hcl ph 8 . 5 . the apoptotic cells and the total cells were visualized using a fluorescence microscope ( axioplan 2 , carl zeiss , germany ) with fitc and uv filters , respectively . dnase - treated embryos were used as positive controls . the dead cell index ( percent of cells that were tunel positive ) was determined by dividing the number of tunel - positive cells by the number of total cells then multiplies by 100 %. rna extraction , rt / pcr and restriction enzyme digestion analysis . total rna was extracted from 20 blastocysts using a commercial kit ( rneasy , qiagen , chatsworth , calif ., usa ) and used for rt / pcr . the primers were selected based on mouse pparδ sequence from the gene bank ( nm — 011145 ). a blast analysis showed there was no published mouse sequence that shared homology with the sequence of the 334 bp amplicon . the rt was carried out at 42 ° c . for 30 mins using a primer ( 5 ′ ttctagagcccgcagaatgg ) based on the nucleotide sequence from exon 6 . the upstream ( 5 ′ gccaagaacatccccaacttc ) and downstream ( 5 ′ cctggatggcttctacctgg ) primers were from exons 5 and 6 , respectively . the pcr reaction consisted of 45 cycles of 94 ° c . for 15 sec , 60 ° c . for 1 min and 72 ° c . for 1 min and concluded with a 7 - min extension at 72 ° c . the amplicon with the expected molecular weight (˜ 330 base pair ) was eluted ( qiaex ii gel extraction kit ) and digested with the restriction enzyme sst1 ( invitrogen , carlsbad , calif .). the pcr products and the digested dna ( expected to yield two fragments : 95 and 239 bp ) were separated using a 2 % and 3 % agarose gel , respectively , and visualized with uv light . western blot analysis . western blot analysis was performed as described previously ( huang et al ., 2002 ) with some modifications . affinity purified , polyclonal antibody against a synthetic peptide based on mouse pparδ protein ( meqpqeetpearee , abcam inc ., cambridge , mass ., usa ) was used . briefly , the proteins in the cell lysate were electrophoresed on a 10 % acrylamide gel and transferred to a nitrocellulose membrane ( schleicher & amp ; schuell , inc ., keene , n . h ., usa ). the pparδ protein bound by the antibody was visualized using enhanced chemi - fluorescence ( amersham biosciences , piscataway , n . j ., usa ), whose signals were detected by a storm 860 laser scanner ( amersham biosciences ). total cell lysate from mouse liver was used as a positive control . the cell lysate of blastocysts was prepared as follows . sixty mouse blastocysts in 2 μl of media were transferred to 1 . 5 ml eppendorf tubes containing 30 μl of lysis buffer ( 150 mm nacl , 1 % np - 40 , 0 . 25 % sodium deoxycholate , 1 mm sodium orthovanadate , 1 mm egta and 1 mm sodium fluoride ), and protease inhibitors ( 1 mm 4 -( 2 - aminoethyl ) benzene sulfonyl fluoride hydrochloride , 0 . 8 μm aprotinin , 50 μm betastatin , 15 μm e - 64 , 20 μm leupeptin hemisulfate , 10 μm pepstatin a , calbiochem - novabiochem corp ., san diego , calif ., usa ). the mixture was vortexed for 5 secs , centrifuged for 10 secs and stirred on ice for another 30 mins . after two bursts of sonication ( sonifier 250 , branson co . danbury , conn ., usa ), one sec each , the lysate was mixed with 4 × protein loading dye . the supernatant of the mixture was used for western blot analysis . immunohistochemistry . the same antibody used in the western blot analysis was used to localize pparδ in the embryos . the immunohistochemistry was performed as described previously ( huang et al ., 2003 ). briefly , the embryos ( icr ) were fixed in ice - cold pbs containing 4 % buffered paraformaldehyde for 30 mins . after permeabilization with 1 % triton in pbs for 20 mins , the embryos were incubated at 37 ° c . with anti - pparδ antibody ( 32 μg / ml ) in pbs containing 5 % milk for 30 mins . embryos were then incubated at 37 ° c . in 2 . 5 μg / ml goat anti - rabbit igg antibody conjugated with fitc ( molecular probes , portland , oreg .) for 30 mins . embryos were washed four times in pbs , five minutes each , between the incubations . after a final incubation with dapi ( 30 ug / mg ) for 5 mins at 37 ° c ., the embryos were mounted as described above ( in the tunel section ) and examined under fitc and uv filters using a fluorescence microscope ( axioplan 2 , carl zeiss , germany ). unfertilized eggs and embryos from various developmental stages ( from one - cell embryos to blastocyst - staged embryos ) were examined . pparδ −/− blastocysts were used as negative controls . prism ® version 3 . 0 ( graphpad software inc . san diego , calif . ), with the hill slope set at 1 . 0 , was used to estimate the ed50 of l - 165041 . chi - square and student &# 39 ; s t - tests were used where appropriate ( instat ® version 3 . 05 , graphpad software inc .). a p & lt ; 0 . 05 was considered statistically significant . preimplantation mouse embryos express pparδ . rt / pcr and western blot analysis were first performed on blastocysts ( c3b6f1 ) to determine the expression of pparδ . the primers used for the pcr were from exons 5 and 6 , respectively ; the expected size of the amplicon was 334 bp . analysis of pcr products by agarose gel electrophoresis revealed two amplicons : one has the expected molecular weight (˜ 330 bp ), the other has a lower molecular weight (˜ 250 bp ) ( fig1 a ). the 330 bp amplicon was eluted from the gel and digested with a restriction enzyme ( sst1 ). analysis of the digested products by agarose gel electrophoresis revealed two fragments of expected sizes (˜ 95 and 240 bp ) as predicted by the cdna sequence of pparδ ( fig1 b ). western blot analysis on total cell lysate of blastocysts revealed a single band corresponding to pparδ protein from the liver ( fig1 c ). pparδ protein is detectable in eight - cell - staged and onward embryos . the same antibody used in the western blot analysis was used to localize pparδ protein in preimplantation embryos . immunohistochemistry was performed on unfertilized eggs and embryos ( icr , an outbred strain ) from various developmental stages . the results show the staining of pparδ was localized in the nuclei of embryonic cells . it had a granular pattern that mirrored the nuclear staining by the dna binding dye dapi . pparδ first became detectable in eight - cell embryos ( not shown ). in the blastocysts , both inner cell mass and trophectoderm showed paprδ staining ( fig2 ). as expected , pparδ −/− blastocysts showed no staining . synthetic pparδ ligand selectively enhances mouse embryos hatching . to confirm the biological function of pparδ , the response of embryos to three isotype - specific synthetic ppar ligands was determined . two - cell embryos ( c3b6f1 ) were cultured in media supplemented with each ligand for 96 hrs and complete embryo hatching was used as the endpoint . of the three synthetic ppar ligands , only l165 , 041 , which interacts specifically with pparδ , enhanced complete embryo hatching ( fig3 a ). l165 , 041 is 4 -[ 3 -( 4 - acetyl - 3 - hydroxy - 2 - propylphenoxy ) propoxy ] phenoxy - acetic acid . the addition of l165 , 041 enhanced embryo hatching in a concentration dependent manner ; the ed50 was estimated to be 21 nm ( fig3 b ). these results suggest that the pparδ in preimplantation embryos was functional and that its activation enhanced embryo hatching . response to synthetic pparδ ligand in the preimplantation embryos is developmental stage dependant . beginning at two - cell stage when the genome become activated , the embryos become progressively responsive to external stimuli . to determine when the embryos become responsive to synthetic pparδ ligand , the following experiment was performed . two - cell embryos ( c3h and c57bl6nhd mixed genetic background ) were randomly assigned to one of the three groups , where l165 , 041 was supplemented 24 , 48 and 72 hrs after the beginning of culture . at these time points , the majority of them developed into eight - cell -, morula - and blastocyst - staged embryos , respectively . the rates of complete hatching were compared among the groups after 96 hrs of culture . the present results show the groups receiving l165 , 041 during 24 - 96 hrs and 48 - 96 hrs ( corresponding to eight - cell stage onward and morula stage onward , respectively ) had similar rates of complete hatching as the group receiving l165 , 041 during 0 - 96 hrs ( fig4 ). although embryos receiving l165 , 041 during 72 - 96 hrs ( corresponding to blastocyst stage onward ) appeared to have a higher rate of complete hatching than the control embryos , the difference was not statistically significant ( p = 0 . 08 ) ( fig4 ). these results indicate that after 48 hrs of culture , the embryos became responsive to l165 , 041 . based on the developmental stages of embryos at this time , it can be concluded that embryos became responsive to l165 , 045 at morular stage and onward . this is consistent with the developmental stage - dependent expression of pparδ based on immunohistochemistry study . synthetic pparδ ligand decreases apoptosis and increases cell proliferation in preimplantation embryos . previous study indicates that successful embryo hatching in vitro depends on a sufficiently high number of cells ( montag et al ., 2000 ). initial studies showed that total cell number could be determined reliably in an embryo after 72 hrs of culture , but not after 96 hrs . therefore , in subsequent studies the rates of apoptosis and proliferation in embryos were compared after 72 hrs of culture . apoptotic cells and cells in the s - phase were determined using tunel assay and brdu incorporation , respectively ; dapi , a dna binding dye , was used to determine total embryonic cells . the results show that the number of cells in control and experimental embryos were comparable ( about 60 per embryo ) after 72 hrs of culture , but l165 , 041 - treated embryos had less dead cells and more cells in the s - phase ( fig5 ). these results suggest that given additional 24 hrs , when the complete embryo hatching is assessed , l165 , 041 - treated embryos are likely to have more live cells . pparδ is critical to the orderly progression of preimplantation embryos in vivo and in vitro . to explore the role of pparδ in the development of preimplantation embryos , the development of pparδ −/− and wt embryos in vitro and in vivo was compared . for the former , two - cell embryos were cultured and observed for 96 hrs ; for the latter , embryos were retrieved 70 and 96 hrs after hcg injection ( see material / method section for rationale ). the results show that pparδ deficiency hampered embryo development . the difference in development became clear after 48 hrs of culture , when 57 % wt embryos became either blastocysts or hatching blastocysts whereas 10 % pparδ −/− embryos advanced to blastocysts . the disparity remained at the end of 96 hrs : 21 % of wt embryos and 4 % of pparδ −/− embryos hatched completely ( fig6 ). it is worth mentioning that pparδ −/− embryos did not “ catch up ” with the wt embryos even after the duration of culture was extended to 120 hrs : during 96 - 120 hrs , some embryos remained unchanged whereas others became degenerated ( data not shown ). the contrast in in - vitro development between pparδ −/− and wt embryos also appeared in embryos harvested 70 and 96 hrs after hcg injection . at 70 hrs after hcg injection , the majority of wt embryos became eight - cell embryos , whereas the majority of pparδ −/− embryos were two - or four - cell embryos ( fig7 a ). similarly , at 96 hrs after hcg injection , 56 % of wt embryos and 22 % of pparδ −/− embryos advanced to blastocysts ( fig7 c ). as expected , wt embryos had significantly more cells than pparδ −/− embryos ( fig7 b and 7 d ). these results suggest that pparδ is critical to the orderly progression of preimplantation embryos . ablation of pparδ is associated with decreased embryonic cell proliferation but not increased apoptosis . to determine the extent to which different rate of cell proliferation contributed to the developmental disparity in pparδ −/− and wt embryos , brdu labeling in embryos harvested 70 and 96 hrs after hcg injection was performed . the results show that more wt embryos incorporated brdu than ppar −/− embryos and that wt embryos had higher proliferation indices . at 70 hrs post hcg injection , 92 % of the wt embryos and 4 % of the pparδ −/− embryos incorporated brdu . the proliferation indices of wt and pparδ −/− embryos were 45 % and 1 %, respectively ( fig8 a ). similarly , at 96 hrs post hcg injection , 100 % wt embryos incorporated brdu , compared with 57 % of the pparδ −/− embryos . the proliferation indices of wt and pparδ −/− embryos were 40 % and 18 %, respectively ( fig8 b ). to determine the extent to which apoptosis led to the developmental delay in pparδ −/− embryos , embryos were obtained 96 hrs after hcg injection , the tunel assay was performed , and then dead cell indices were compared . the results showed 28 of 61 ( 54 %) pparδ −/− embryos and one of the 58 ( 2 %) wt embryos examined were developmentally arrested ; they contained 15 or less cells per embryo . three of these embryos stained positive for tunel : one tunel positive cell each in two embryos , two tunel positive cells in the third embryo . the dead cell indices in embryos containing 16 or more cells were comparable : 2 . 2 ± 3 . 5 % versus 2 . 9 ± 3 . 9 % ( mean sd of 33 pparδ −/− embryos and 58 wt embryos , p = 0 . 33 ). these results suggest that ablation of pparδ in preimplantation embryos is associated with decreased cell proliferation but not increased apoptosis . it is shown here for the first time that preimplantation mouse embryos express pparδ . its activation by synthetic pparδ ligand enhanced embryo hatching and reduced apoptosis ; its ablation by genetic targeting led to an irreversible delay in embryo development due to decreased cell proliferation . the present findings corroborate an earlier report regarding the expression and the proposed biological functions of pparδ . in e8 . 5 day rat embryos ( the earliest developmental stage examined thus far ), ubiquitous pparδ message ( but not pparα or pparγ messages ) was revealed by in - situ hybridization ( braissant et al ., 1998 ). based on those findings , it was proposed that pparδ may participate in more basic cellular functions such as membrane lipid turnover or cell cycle progression . the present results go beyond the earlier reports , however , and show that lack of pparδ expression in preimplantation embryos is associated with decreased embryonic cell proliferation and irreversible developmental delay . although pparδ has been implicated in cell proliferation , its exact role is controversial . whereas over - expression of pparδ in cultured vascular smooth muscle cells promotes the proliferation of post - confluent cells ( julan et al ., 2005 ), pparδ +/− mutant mice respond more profoundly to proliferation stimuli than the wild type mice ( michalik et al ., 2001 ; tan et al ., 2001 ). an increase of cyclin a and cdk2 and a decrease of p57kip2 ( a cdk2 inhibitory protein ) have been proposed to be the mechanisms for the former ; it is not clear what causes the latter . in preimplantation mouse embryos , the ablation of pparδ is associated with a significant decrease in cell proliferation and the activation of pparδ is associated with a significant increase in cell proliferation . pparδ activation is likely to enable embryonic cells to overcome g1 - restriction point and facilitate their entry into the s - phase . the present experimental results showing that synthetic pparδ ligand increases cell proliferation in wt embryos are consistent with the growth promoting properties of pparδ in the former report . previous studies regarding pparδ activation and decreased apoptosis are more consistent . the anti - apoptosis properties of pparδ has been proposed to facilitate wound healing of skin ( michalik et al ., 2001 ; wahli 2002 ) and increase the resistance to hypertonic stress in kidney cells ( hao et al ., 2002 ). it may also play a central role in the tumorigenesis of colorectal cancer ( gupta et al ., 2000 ; cutler et al ., 2003 ). the present experimental results showing that synthetic pparδ ligand reduces apoptosis in wt embryos are consistent with the previous reports of anti - apoptosis properties of pparδ . the molecular mechanisms involving increased cell proliferation and decreased apoptosis in l165 , 041 - treated embryos remain to be determined . without wishing to be limited to a particular theory , it is suggested that the mechanisms may perhaps involve ligand dependent transcriptional activation and co - repressor bcl - 6 mediated transcriptional repression . liganded pparδ reportedly upregulates the expression of 14 - 3 - 3 , which in turn upregulates the expression of anti - apoptotic members of the bcl2 family ( liou et al ., 2004 ). recent evidence indicates that apo - pparδ sequesters co - repressor bcl - 6 and that , upon binding by the pparδ ligand , apo - pparδ releases bcl - 6 , which is now free to repress the transcription of other genes ( shi et al ., 2002 ). genes known to be repressed by bcl - 6 include cell cycle inhibitor such as p27kip1 ( shaffer et al ., 2000 ; kusam et al ., 2004 ). this transcriptional activation / repression coupling may also involve other cell cycle regulators . for example , increased cyclin a and cdk2 and decreased p57kip2 ( a cdk2 inhibitory protein ) expression reportedly lead to the proliferation of post - confluent cells over - expressing pparδ ( julan et al ., 2005 ). contrary to what was observed in pparδ ligand - treated wt embryos ( i . e ., reduced apoptosis but comparable total cell number ), pparδ −/− embryos did not have more apoptosis than wt embryos . pparδ −/− embryos had markedly decreased cell proliferation but no appreciable increase in apoptosis . there may be several possible reasons for this observation . firstly , at 96 hrs post hcg injection , 54 % of the pparδ −/− embryos ( compared with 2 % of wt embryos ) had less than 16 cells , a cell number that should have been achieved at 72 hrs after hcg injection . all but three of these severely delayed embryos showed tunel staining . without wishing to be limited to any particular theory , it is suggested that perhaps these severely delayed embryos underwent apoptosis earlier and that the dna damage may be so severe that they no longer stained positive by tunel assay . secondly , soluble factors from the oviduct may compensate for pparδ deficiency and maintain a “ normal ” rate of apoptosis in in vivo embryos . embryos co - cultured with human oviductal cells are know to have significantly less apoptosis ( xu et al ., 2000 ). thirdly , it is possible , although not likely , that functions of synthetic pparδ ligand such as l165 , 041 may be different from those of natural pparδ ligands . these findings provide new insights into the reproductive phenotypes of pparδ −/− mice . although pparδ ablation is frequently lethal , those surviving pparδ −/− mice are generally healthy and fertile ( barak et al ., 2002 ). one of the reproductive phenotypes of pparδ −/− mice is small litter size ( personal communication with dr . r . evans ). this has been attributed to abnormal placento - decidual contact leading to mid - gestational death ( barak et al ., 2002 ), i . e ., a postimplantation event . the present observations indicate that high attrition of pparδ −/− embryos during the preimplantation period may be another cause . pparδ −/− and wt females responded equally well to super - ovulation by pmsg ( number of eggs retrieved from 11 pparδ −/− and seven wt mice were 26 . 0 ± 17 . 2 and 33 . 1 ± 22 . 7 , mean ± sd , respectively ); pparδ −/− and wt males impregnate the females with comparable efficiency ( the fertilization rates of the above eggs were 93 % and 95 %, respectively ). however , irreversible developmental delay became evident in pparδ −/− embryos 70 hrs after ovulation was triggered by hcg ( fig7 a ). thus , loss of embryos during the preimplantation is another cause for the small litter of pparδ −/− mice . synthetic pparδ ligand may have potential application in human ivf . it has been previously reported that iloprost , a pgi2 analog , enhances embryo hatching ( huang et al ., 2003 ) and that iloprost - treated embryos yielded 28 % more live births over control embryos when transferred to gestational carriers ( huang et al ., 2004b ); pct / us2004 / 029167 ( huang et al . ), the disclosures of which are hereby incorporated herein by reference . since l165 , 041 ( a synthetic pparδ ligand ) enhances embryo hatching , it is likely that medium supplemented with synthetic pparδ ligand may also enhance the live birth potentials of embryos and increase ivf success . it is possible that pparδ and pgi2 may exert additive or even synergistic effects on the embryos . therefore , supplementing ivf media with both pgi2 analog and synthetic pparδ ligand may offer even better ivf success than the ligand alone or pgi2 analog alone . the results observed using the above - described mouse model are believed to be predictive , at least to some extent , of results that will be obtained with other mammalian embryos , including human . of course , the use of the pparδ ligand in human ivf requires further safety studies before therapeutic application . it is believed that all pparδ ligands , including the synthetic compound gw501516 ( 2 - methyl - 4 -(( 4 - methyl - 2 -( 4 - trifluoromethylphenyl )- 1 , 3 - thiazol - 5 - yl )- methylsulfanyl ) phenoxy - acetic acid ( calbiochem , u . s . a . )), have activity , to at least some extent , like that demonstrated with the representative ligand l165 , 041 . a series of further studies were carried out to investigate enhancement via the pparδ pathway of embryo hatching and implantation . except as noted below , the materials and methods employed were as described above . western blot analysis was performed as described previously ( huang et al ., 2004c ), using an affinity purified , polyclonal antibody ( abcam inc ., cambridge , mass .) against a mouse pparδ peptide ( meqpqeetpearee ). twenty - five mouse blastocysts in 2 μl of media were transferred to 1 . 5 ml eppendorf tubes containing 30 μl of lysis buffer ( 150 mm nacl , 1 % np - 40 , 0 . 25 % sodium deoxycholate , 1 mm sodium orthovanadate , 1 mm egta and 1 mm sodium fluoride ), and protease inhibitors ( 1 mm 4 -( 2 - aminoethyl ) benzene sulfonyl fluoride hydrochloride , 0 . 8 μm aprotinin , 50 μm betastatin , 15 μm e - 64 , 20 μm leupeptin hemisulfate , 10 μm pepstatin a , calbiochem - novabiochem corp ., san diego , calif .). the mixture was vortexed for 5 sec , centrifuged for 10 sec and stirred on ice for another 30 min , followed by two 1 - sec bursts of sonication ( branson co ., danbury , conn .). after being mixed with 4 × protein loading dye , the supernatant was used for western blot analysis . the lysate was electrophoresed on a 12 % gradient acrylamide gel and transferred to a nitrocellulose membrane ( schleicher & amp ; schuell , inc ., keene , n . h .). the pparδ protein bound by the antibody was visualized using enhanced chemi - fluorescence ( ge healthcare bio - sciences corp ., piscataway , n . j . ), whose signals were detected by a storm 860 laser scanner ( ge healthcare bio - sciences corp ). total cell lysate from mouse testes was used as a positive control . the cell lysate of blastocysts was prepared as follows . the immunohistochemistry was performed as described previously ( huang et al ., 2004c ). in brief , embryos were fixed in ice - cold pbs containing 4 % buffered paraformaldehyde for 30 min . after permeabilization with 1 % triton x - 100 in pbs for 20 min , the embryos were incubated at 37 ° c . with anti - pparδ antibody ( 32 μg / ml ) in pbs containing 5 % milk for 30 min . the embryos were then incubated at 37 ° c . in goat anti7 rabbit igg antibody conjugated with fitc ( 2 . 5 μg / ml , invitrogen ) for 30 min . between incubations , embryos underwent four 5 - min washes in pbs . after a final 5 - min incubation in hoechst 33258 ( 30 μg / ml ) at room temperature , the embryos were mounted and examined under fitc and uv filters . unfertilized eggs and embryos from various developmental stages ( from one - cell embryos to blastocyst - staged embryos ) were examined . the proliferation of embryonic cells was determined based on their abilities to incorporate brdu . the assay was performed using brdu labeling and detection kit i ( roche applied science , indianapolis , ind .) according to manufacturer &# 39 ; s protocols with modifications . in brief , embryos were incubated in 100 μl pre - equilibrated htf media containing 10 μm brdu for 6 min at 37 ° c . under 5 % co 2 , fixed in ice - cold glycine ( 50 mm ) in 70 % ethanol ( ph 2 . 0 ) at − 20 ° c . for 30 min , and incubated at 37 ° c . for 30 min with diluted anti - brdu mouse monoclonal antibody ( 1 : 2 ) buffer containing 1 % bsa . embryos were washed in 200 μl pbs containing 1 % bsa and incubated at 37 ° c . for 30 min with diluted fitc - conjugated anti - rabbit igg antibody ( 1 : 4 ) in pbs containing 1 % bsa . after washing , embryos were treated with hoechst 33258 ( 30 μg / ml ) for 5 min at 25 ° c ., and mounted in 0 . 1 m tris hcl ( ph 8 . 5 ) containing 16 . 6 % elvanol 50 - 42 ( dupont , wilmington , del .) and 2 . 5 % 1 , 4 - diazabicyclo -( 2 . 2 . 2 )- octane . cells that incorporated brdu were identified under a fitc filter ( axioplan 2 , zeiss , oberkochen , germany ). total cell number was determined based on hoechst 33258 nuclear staining under a uv filter . embryo transfer was performed as described previously ( huang et al ., 2004b ). briefly , embryos were transferred to gestational carriers ( icr female mice ) on day 2 . 5 of pseudopregnancy under a dissecting microscope ( olympus sz - pt , shinjuku - ku , tokyo , japan ). after anesthesia , each uterine horn was accessed via a 1 . 5 cm flank incision . with the proximal oviduct held by a pair of forceps , an opening was created at the distal end of the uterine horn on the anti - mesenteric side with a 30 gauge needle . the opening permitted the entry of the transfer pipette which had an inner diameter of 135 μm ( midatlantic diagnostics , inc ., mount laurel , n . j .). up to seven embryos in 1 . 5 μl transfer medium ( mem with 25 mm hepes and 1 % bsa ) were transferred to each horn . after each transfer , the contents of the pipette were examined under a stereomicroscope to identify retained embryos . to avoid the mixing of embryos as a result of embryo crossover ( dr . andreas zimmer , university of bonn , germany , mg118 list , the jackson laboratory ), each gestational carrier received only one kind of embryo . to maintain consistent transfer techniques , the embryo transfer was performed following the same protocol and by one individual ( j - c h ). seventy - two h after embryo transfer , the rates of implantation were determined based on a previously - described method with modifications ( paria et al ., 1993 ). briefly , 3 min before euthanasia , 0 . 1 ml of chicago blue ( 1 %) was injected via the tail vein of the gestational carrier . after the carrier was sacrificed , the uterine horns were opened and the gestation sacs counted . the implantation rate was expressed as percentages of gestation sacs over total embryos transferred . ninety - seven wild type and fifty - four pparδ −/− embryos were transferred to seven and four gestational carriers , respectively . blastocyst staged embryos expressed pparδ mrnas and proteins as determined by rt - pcr and western blots , respectively ( fig9 a and 9 b ). pparδ was detected in eight - cell staged and onward wt embryos but was not detected in pparδ −/− embryos ( fig9 c upper vs . lower panels ). to evaluate the role of pparδ in embryo development , we compared blastocyst hatching between pparδ +/+ and pparδ −/− embryos . two - cell staged embryos prepared from pparδ wt and null mice were cultured in vitro for 96 h . completely hatched embryos were counted . thirty percent ( 20 / 67 ) of the pparδ +/+ embryos had hatched completely at 96 h compared with 3 % ( 4 / 144 ) of pparδ −/− embryos ( fig1 ). iloprost ( 0 . 1 μm ) increased the number of completely hatched embryos to 50 % ( 37 / 74 ) in wt but had no effect on pparδ −/− embryos ( 3 / 144 ). l - 165041 , a specific pparδ ligand , increased the proportion of completely hatched embryos to 54 %, as compared with 30 % in untreated wt embryos . neither iloprost nor l - 165041 affected the hatching of pparδ −/− embryos . combined iloprost and l - 165041 treatment increased hatched embryos to 52 %, an enhancement which is not significantly greater than that of either ligand alone . these results suggest that iloprost and l - 165041 act via pparδ . experiments were then performed to determine the role of pparδ in embryo development . two - cell embryos from wt and pparδ −/− mice were cultured in media without pparδ ligands for 96 h . at 48 h , 72 h , and 96 h , blastocysts , morula and earlier - staged embryos ( designated & lt ; morula ) were counted . pparδ −/− embryos developed significantly more slowly than wt embryos at each time point . at 96 h , 29 % of the pparδ −/− embryos remained at stages earlier than morula and 28 % were hatching or hatched , whereas all wt embryos had reached the blastocyst stage and 85 % underwent hatching or had completely hatched ( table i ). it is worth mentioning that preliminary experiments showed that extending the culture to 120 h did not change the percentages of completely hatched embryos in either group ( not shown ). taken together , these data indicate that pparδ is fundamental to embryo development and hatching in vitro . embryo hatching is influenced by blastocyst cell numbers ( montag et al ., 2000 ) which cause thinning of zona pellucida thereby facilitating hatching . we hypothesized that the impaired hatching of pparδ −/− embryos is a result of decreased cell proliferation . to test this hypothesis , we compared the brdu uptake by wt and pparδ −/− embryos . the number of brdu - positive cells was greatly reduced in pparδ −/− embryos ( fig1 a ). cell numbers per embryos were also markedly reduced in pparδ −/− embryos ( fig1 b ). both iloprost and l - 165041 increased wt embryo hatching to a similar extent ( fig1 ). their stimulatory effects were abrogated by pparδ deletion ( fig1 ). l - 165041 increased the percentage of hatched embryos in a concentration - dependent manner with an ed50 value of 21 nm ( fig1 a ). neither wy14 , 643 , a pparα ligand , nor ciglitazone , a pparγ agonist , stimulated embryo hatching ( fig1 b ). l - 165041 was effective in stimulating embryo hatching when it was added to embryos at two - cell , eight - cell or morula stage ( fig1 c ). once a majority of embryos reached the blastocyst stage , the addition of l - 165041 to the cultured medium was no longer effective in stimulating hatching ( fig1 c ). it has been proposed that embryo hatching occurs when the cells in blastocysts reach a critical number ( montag et al ., 2000 ). therefore , we determined the extent to which l - 165041 enhanced embryonic cell proliferation . l - 165041 ( 10 μm ) increased the percentages of brdu - positive cells over the untreated control ( fig1 d ). taken together , these results indicate that exogenous pparδ ligands such as l - 165041 and iloprost accelerate hatching by stimulating the cell proliferation in embryos . to determine the extent to which pparδ is involved in implantation , two - cell embryos obtained from wt and pparδ −/− mice were cultured for 48 h , enumerated and transferred to receptive uteri of gestational carriers . gestation sacs were counted 72 h later . numbers of early and late blastocysts developed from two - cell staged pparδ −/− embryos were significantly lower than those developed from wt embryos ( fig1 a ). the implantation rate for pparδ −/− embryos was also significantly lower than that for wt embryos ( 28 % vs . 44 %, p & lt ; 0 . 05 ) ( fig1 b ). we next determined the extent to which pretreatment of two - cell embryos with l - 165041 influences implantation rate . two - cell embryos from wt mice were treated with l - 165041 for 48 h . embryos were enumerated and transferred to gestational carriers . implantation rate was evaluated 72 h later . although l - 165041 did not significantly enhance blastocyst formation ( fig1 c ), it significantly increased the implantation rate ( 64 % vs . 41 %, p & lt ; 0 . 05 ) ( fig1 d ). these results indicate that embryo pparδ plays an important role in implantation . exogenous pparδ ligands promote implantation without altering blastocyst formation . our study suggests that embryo development and hatching requires pparδ . two - cell embryos from pparδ −/− mice are capable of developing into blastocysts but the rate of blastocyst formation lags behind that of wt embryos . pparδ is especially fundamental to blastocyst hatching . compared with wt embryos , a significant number of two - cell pparδ −/− embryos failed to hatch after 96 h in culture . it is worth mentioning that extending the culture duration to 120 h did not change the outcome as the majority of the blastocysts became collapsed during this period . it has been proposed that embryo hatching is mediated by two major factors : ( 1 ) a crucial cell number increase in embryos ( montag et al ., 2000 ), which results in thinning of zona pellucida and ( 2 ) the digestion of zona by proteolytic enzymes ( sawada et al ., 1990 ). our results reveal that pparδ mediates the proliferation embryonic cells and thereby increases embryonic cell mass . compared with wt embryos , pparδ −/− embryos exhibit very low brdu incorporation . the cell numbers of pparδ −/− embryos were only about 30 % of those of wt embryos after 72 h in culture . pparδ is a nuclear receptor which binds a number of endogenous and synthetic ligands ( forman et al ., 1997 ). our previous study suggests that cox - 2 derived pgi2 in embryos may be an endogenous pparδ ligand . ligand - activated pparδ forms a heterodimer with retinoid x receptor ( rxr ) which binds ppar response elements and activates gene transcription ( kliewer et al ., 1994 ). a number of pparδ - mediated genes have been reported and two of these genes , 14 - 3 - 3ε and phosphoinositide dependent kinase - 1 ( pdk - 1 ), are involved in protecting cells from apoptosis ( di - poi et al ., 2002 ; liou et al ., 2006 ). however , the mechanism by which pparδ promotes embryo cell proliferation is unknown . it was reported that over - expression of pparδ in the vascular smooth muscle cells increases post - confluent cell proliferation by increasing cyclin a and cdk2 as well as decreasing p57kip2 ( zhang et al ., 2002 ). it is possible that pparδ - mediated 14 - 3 - 3 upregulation is involved in cell proliferation because 14 - 3 - 3 proteins are considered to be scaffolds for a large number of proteins , including signaling molecules and receptors ( fu et al ., 2000 ; tzivion and avruch , 2002 ). work is in progress to address this possibility . pparδ may also play an important role in protecting embryo cells from apoptosis via 14 - 3 - 3ε ( liou et al ., 2006 ) and pdk - 1 ( di - poi et al ., 2002 ). it was reported that the inner cell mass ( icm ) of blastocytes in culture are vulnerable to oxidative apoptosis ( brison and schultz , 1997 ; schratt et al ., 2004 ). pgi2 - activated pparδ may protect icm from apoptosis by upregulating 14 - 3 - 3ε , which sequesters bad phosphorylated via the pdk - 1 and akt pathway ( datta et al ., 1997 ; zha et al ., 1996 ). the anti - apoptotic function of pparδ may contribute to increased cell numbers in blastocysts and enhanced hatching . it has been reported that cox - 2 derived pgi2 in the uterus is involved in embryo implantation , and pparδ has been implicated for the action of pgi2 ( lim et al ., 1999 ). the role of the embryo pparδ in implantation has not been reported . results from the present study provide direct evidence for a crucial role of embryo pparδ in implantation . to mimic ivf procedures , we cultured wt and pparδ −/− two - cell embryos in vitro for 48 h . embryos at various stages of development were counted and transferred to receptive gestational carriers . gestation sacs in the uterus were counted 72 h later . the number of gestation sacs from pparδ −/− embryos was significantly less than that from wt embryos . reduced implantation of pparδ −/− embryos is correlated with the retarded embryo development and blastocyst formation of pparδ −/− embryos . these results underscore the importance of pparδ - mediated , enhanced cell proliferation in promoting the growth , maturation ( hatching ) and implantation of preimplantation embryos . a major goal of our studies is to improve ivf . we have previously reported that iloprost , a stable pgi2 analog , enhances embryo implantation and the potential of live birth ( huang et al ., 2004b ). in the present study , we confirmed the previous data and shed light on the mechanism by which exogenous iloprost enhances embryo hatching and implantation . our results show that pparδ is the target of pgi2 in the preimplantation embryos . enhanced blastocyst hatching by iloprost was completely abrogated in pparδ −/− embryos . a synthetic pparδ ligand , l - 165041 , exerted an effect similar to that of iloprost , but a combination of iloprost with l - 165041 did not have additional effects . these results further suggest that endogenous pgi2 production in cultured embryos is limited and that exogenous pgi2 analog or synthetic pparδ ligand can further activate pparδ . pparδ activation by synthetic ligand or pgi2 analog ( iloprost ) promotes further cell proliferation resulting in a higher hatching rate . it is worth noting that the effect of l - 165041 is not limited to two - cell - staged embryos . l - 165041 was quite effective in enhancing embryo hatching even in morula - staged embryos . however , l - 165041 did not appear to be effective in blastocyst - staged embryos . the afore - mentioned result is probably because some blastocysts had advanced development and were ready to hatch . this observation is of practical importance with respect to the timing of supplementing media with iloprost or pparδ ligands . naturally , the application of pparδ ligands in human ivf requires a thorough investigation of its safety profiles . l - 165041 appears to enhance embryo implantation by a mechanism independent of its effect on blastocyst formation , because the developmental stages of control and experimental embryos at the time of embryos transfer were similar , yet transferred experimental embryos resulted in more gestation sacs . the molecular basis for the delayed action is unclear . we speculate that l - 165041 induces a sustained signaling activation via pparδ to enhance implantation . there probably is a crosstalk between the transcriptional signalings of uterine and embryo pparδ . the coordination between embryo development and endometrial decidualization ensures a successful implantation . elucidation of the molecular mechanism ( s ) will have major impacts on our understanding of embryo implantation and the potential to improve ivf success . in summary , preimplantation embryos express pparδ , which plays an essential role in embryo development , blastocyst formation , embryo hatching and implantation . activation of embryo pparδ is a novel therapeutic strategy to improve the outcome of ivf § & lt ; morula stage refers to two - cell , four - cell , and eight - cell staged embryos adderley , s . r ., and fitzgerald , d . j . 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( 2000 ) human oviductal cells reduce the incidence of apoptosis in cocultured mouse embryos . fertil steril , 74 , 1215 - 1219 without further elaboration , it is believed that one skilled in the art can , using the description herein , utilize the present invention to its fullest extent . the foregoing embodiments are to be construed as illustrative , and not as constraining the remainder of the disclosure in any way whatsoever . while the preferred embodiments of the invention have been shown and described , modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention . the embodiments described herein are exemplary only , and are not intended to be limiting . many variations and modifications of the invention disclosed herein are possible and are within the scope of the invention . the disclosures of all patents , patent applications and publications cited herein are hereby incorporated herein by reference , to the extent that they provide procedural or other details supplementary to those set forth herein .