the fabp6 mice   used in this study were produced in - house and have been backcrossed to the c57bl/6 background for 8 generations . the mice were housed in a controlled environment with a 12 h light / dark cycle and were given free access to water and standard diet ( 2020x teklad global rodent diet ) . immature ( 2325 days old ) female mice were superovulated by administration of equine chorionic gonadotropin ( ecg ; 5 iu , i.p . ) followed 48 h later by human chorionic gonadotropin ( hcg ; 5 iu , i.p . ) . mice were euthanized at defined time - points during the gonadotropin - stimulated follicular and luteal development . briefly , ovaries were placed in phosphate - buffered saline and large antral follicles were punctured using sterile 27.5 g needle . ovaries were gently squeezed with a spatula to flush out remaining granulosa cells from punctured follicles . the cell suspension was passed through a cell strainer ( 40 m , bd falcon ) to remove cumulus - oocyte complexes . granulosa cells from both ovaries of each 
 mouse were pooled for quantitative real - time pcr ( qpcr ) and immunoblot analyses . to determine ovulation rate , ovulated oocytes were collected from both oviducts and counted as a measure of number of ovulations for each mouse . total rna was purified from ovaries using the trizol reagent ( life technologies , burlington , on ) and then converted to cdna for qpcr analysis ( bio - rad cfx384 system , bio - rad , mississauga , on ) . the pre - validated primers used were : fabp6 ; aromatase ( cyp19a1 ) , 5-tggagaacaattcgccctttc-3 and 5-ccgaggtgtcggtgacttc-3 ; prostaglandin synthase 2 ( ptgs2 ) , 5- tgagcaactattccaaaccagc-3 and 5-gcacgtagtcttcgatcactatc-3 ; succinate dehydrogenase complex a ( sdha ) , 5-ggaacactccaaaaacagacct-3 and 5-ccaccactgggtattgagtagaa-3 ; -2-microglobulin ( b2 m ) , 5-ttctggtgcttggtctcactga-3 and 5-cagtatgttcggcttcccattc-3 ; glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , 5-aggtcggtgtgaacggatttg-3 and 5-tgtagaccatgtagttgaggtca-3 ; and ribosomal protein l19 ( rpl19 ) , 5-atgagtatgctcaggctacaga-3 and 5-gcattggcgatttcattggtc-3. the average combined 
 abundance of sdha , b2 m , gapdh and rpl19 mrnas was used to normalize the mrna abundance of other genes . the thermal cycler profile used in the analyses is as follows : initial denaturation step of 5 min at 95 c , followed by 35 cycles of 15 sec at 95 c denaturation step and 30 sec at 58 c annealing / extension step . for immunoblot analyses , protein extracts were resolved by sds - polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes . after blocking with 5% milk in tbs - t ( 50 mm tris - hcl , ph 7.5 ; 150 mm nacl ; 0.05% tween 20 ) , the membranes were incubated overnight at 4 c with primary antibodies followed by washing with tbs - t ( 3 times , 10 min each ) and incubation with secondary antibody ( 1:10000 ) for 1 h at room temperature . the immunoblotted proteins were detected using immun - star kit and chemidoc analyzer ( bio - rad ) . the mouse fabp6 antiserum ( used at 1:40,000 dilution ) was generated in rabbits using recombinant mouse fabp6 as antigen . other antibodies ( diluted 1:1000 as recommended by the suppliers ) were purchased from commercial sources : anti - star ( sc-25806 , santa cruz biotechnology , dallas , tx ) , anti - actb ( ab8227 , abcam , cambridge , ma ) . the secondary antibody , 
 goat anti - rabbit igg ( hrp - conjugated ) ( ab6721 , abcam ) , was used at 1:10,000 dilution . ovaries collected from superovulated mice were fixed in 10% neutral buffered formalin , embedded in paraffin and cut into 4 m sections ( leica rm2125rt ) . following initial steps of deparaffinization , dehydration , antigen retrieval and blocking ( blocking solution consisted of 3% bovine serum albumin , 0.4% triton x-100 and 10% normal goat serum ) , sections were incubated with or without murine fabp6 anti - serum ( 1:500 )   for 1 h. the slides were then washed with pbs and incubated with anti - rabbit secondary antibody ( 1:200 ) dilutions for 1 h. immunoreactivity to fabp6 antiserum ( brown staining ) was visualized using the substrate dab chromogen system ( k3467 , dako , burlington , on ) . differences between mrna abundance at different time points were evaluated by one - way analysis of variance followed by tukey s multiple comparisons post - hoc test . the fabp6 mice   used in this study were produced in - house and have been backcrossed to the c57bl/6 background for 8 generations . the mice were housed in a controlled environment with a 12 h light / dark cycle and were given free access to water and standard diet ( 2020x teklad global rodent diet ) . immature ( 2325 days old ) female mice were superovulated by administration of equine chorionic gonadotropin ( ecg ; 5 iu , i.p . ) followed 48 h later by human chorionic gonadotropin ( hcg ; 5 iu , i.p . ) . mice were euthanized at defined time - points during the gonadotropin - stimulated follicular and luteal development . briefly , ovaries were placed in phosphate - buffered saline and large antral follicles were punctured using sterile 27.5 g needle . ovaries were gently squeezed with a spatula to flush out remaining granulosa cells from punctured follicles . the cell suspension was passed through a cell strainer ( 40 m , bd falcon ) to remove cumulus - oocyte complexes . granulosa cells from both ovaries of each 
 mouse were pooled for quantitative real - time pcr ( qpcr ) and immunoblot analyses . to determine ovulation rate , ovulated oocytes were collected from both oviducts and counted as a measure of number of ovulations for each mouse . total rna was purified from ovaries using the trizol reagent ( life technologies , burlington , on ) and then converted to cdna for qpcr analysis ( bio - rad cfx384 system , bio - rad , mississauga , on ) . the pre - validated primers used were : fabp6 ; aromatase ( cyp19a1 ) , 5-tggagaacaattcgccctttc-3 and 5-ccgaggtgtcggtgacttc-3 ; prostaglandin synthase 2 ( ptgs2 ) , 5- tgagcaactattccaaaccagc-3 and 5-gcacgtagtcttcgatcactatc-3 ; succinate dehydrogenase complex a ( sdha ) , 5-ggaacactccaaaaacagacct-3 and 5-ccaccactgggtattgagtagaa-3 ; -2-microglobulin ( b2 m ) , 5-ttctggtgcttggtctcactga-3 and 5-cagtatgttcggcttcccattc-3 ; glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , 5-aggtcggtgtgaacggatttg-3 and 5-tgtagaccatgtagttgaggtca-3 ; and ribosomal protein l19 ( rpl19 ) , 5-atgagtatgctcaggctacaga-3 and 5-gcattggcgatttcattggtc-3. the average combined 
 abundance of sdha , b2 m , gapdh and rpl19 mrnas was used to normalize the mrna abundance of other genes . the thermal cycler profile used in the analyses is as follows : initial denaturation step of 5 min at 95 c , followed by 35 cycles of 15 sec at 95 c denaturation step and 30 sec at 58 c annealing / extension step . for immunoblot analyses , protein extracts were resolved by sds - polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes . after blocking with 5% milk in tbs - t ( 50 mm tris - hcl , ph 7.5 ; 150 mm nacl ; 0.05% tween 20 ) , the membranes were incubated overnight at 4 c with primary antibodies followed by washing with tbs - t ( 3 times , 10 min each ) and incubation with secondary antibody ( 1:10000 ) for 1 h at room temperature . the immunoblotted proteins were detected using immun - star kit and chemidoc analyzer ( bio - rad ) . the mouse fabp6 antiserum ( used at 1:40,000 dilution ) was generated in rabbits using recombinant mouse fabp6 as antigen . other antibodies ( diluted 1:1000 as recommended by the suppliers ) were purchased from commercial sources : anti - star ( sc-25806 , santa cruz biotechnology , dallas , tx ) , anti - actb ( ab8227 , abcam , cambridge , ma ) . the secondary antibody , 
 goat anti - rabbit igg ( hrp - conjugated ) ( ab6721 , abcam ) , was used at 1:10,000 dilution . ovaries collected from superovulated mice were fixed in 10% neutral buffered formalin , embedded in paraffin and cut into 4 m sections ( leica rm2125rt ) . following initial steps of deparaffinization , dehydration , antigen retrieval and blocking ( blocking solution consisted of 3% bovine serum albumin , 0.4% triton x-100 and 10% normal goat serum ) , sections were incubated with or without murine fabp6 anti - serum ( 1:500 )   for 1 h. the slides were then washed with pbs and incubated with anti - rabbit secondary antibody ( 1:200 ) dilutions for 1 h. immunoreactivity to fabp6 antiserum ( brown staining ) was visualized using the substrate dab chromogen system ( k3467 , dako , burlington , on ) . differences between mrna abundance at different time points were evaluated by one - way analysis of variance followed by tukey s multiple comparisons post - hoc test . detection and semi - quantitative estimation by conventional pcr using primers specific for the entire open reading frame of the murine fabp6 mrna sequence   demonstrated its presence in ovary of wild - type ( c57bl/6 ) mouse , but at a lower abundance compared to ileum ( fig . 1 . detection of fabp6 mrna in the wild - type ( c57bl/6 ) murine ovary . ( a ) the fabp6 open reading frame was amplified by conventional pcr to detect the fabp6 mrna in murine ovary and ileum . primers specific for the murine 18s ribosomal rna sequence were used to indicate the presence of templates in the reaction . ( b ) relative mrna abundance ( mean  sem ; n = 4 ) of fabp6 , cyp19a1 and ptgs2 in pure populations of granulosa and luteal cells isolated from ovaries at specific time points during superovulation protocol as described in the materials and methods section . relative mrna abundance was determined by normalization to the average abundance of sdha , b2 m , gapdh and rpl19 mrnas . therefore , we used total rna from granulosa and luteal cells collected at specific time - points during gonadotropin - induced follicular development and ovulation . analysis of cyp19a1 and ptgs2 mrna abundance by qpcr , in ecg and hcg dependent manner , confirmed that our cell populations represented precise stages of granulosa cell differentiation through follicular and luteal development . ( a ) the fabp6 open reading frame was amplified by conventional pcr to detect the fabp6 mrna in murine ovary and ileum . primers specific for the murine 18s ribosomal rna sequence were used to indicate the presence of templates in the reaction . ( b ) relative mrna abundance ( mean  sem ; n = 4 ) of fabp6 , cyp19a1 and ptgs2 in pure populations of granulosa and luteal cells isolated from ovaries at specific time points during superovulation protocol as described in the materials and methods section . relative mrna abundance was determined by normalization to the average abundance of sdha , b2 m , gapdh and rpl19 mrnas . the pattern of fabp6 gene expression in response to gonadotropin ( ecg and hcg ) treatment indicated that maximal transcript abundance ( p < 0.01 ) was at ecg-48h and hcg-1h ( fig . a previous report showed gonadotropin treatment of immature rat ovaries also increased fabp6 expression , albeit this study measured fabp6 mrna abundance in the whole ovary at 48 h after hcg treatment . our data further show that gonadotropin dependent induction of fabp6 in granulosa cells followed the expression pattern of cyp19a1 , suggesting that its expression in follicular granulosa cells is estrogen dependent . the fabp6 mrna was detected in rat luteal cells in previous studies [ 3 , 13 , 14 ] . likewise , it was detectable in murine luteal cells . also , park et al .   demonstrated that the transcription factor runx2 regulates fapb6 expression in luteinizing granulosa cells . immunoblot analysis confirmed that the fabp6 protein of the expected size is present in ovarian homogenates and granulosa cell lysates of c57bl/6 mice , but at a significantly lower level than in ileal homogenates ( fig . ( a ) homogenates of c57bl/6 mouse ( + /+ ) ileum ( 8 g ) , fabp6 mouse ovary ( 100 g ; collected at 24 h after hcg ) and purified c57bl/6 granulosa cells ( collected at 4 h after hcg ) were analyzed by immunoblotting using antiserum against recombinant murine fabp6 . antibodies against -actin ( actb ) and steroidogenic acute regulatory protein ( star ) were used as controls . ( b ) immunohistochemical detection of fabp6 in paraffin sections of the ovaries collected at specific time points of superovulation treatment from immature c57bl/6 mice . negative control tissue sections ( left ) collected 48 h after ecg treatment and 24 h after hcg treatment were not incubated with anti - fabp6 . g , granulosa cells ; t , theca cells ; o , oocyte ; cl , corpus luteum . our findings are consistent with the results of a previous report showing lower fabp6 abundance in human ovary and placenta compared to the ileum . we recently reported the creation of a mouse strain that lacks fabp6 , and the fabp6 protein was not detectable in ovarian homogenates of these mice ( fig . ( a ) homogenates of c57bl/6 mouse ( + /+ ) ileum ( 8 g ) , fabp6 mouse ovary ( 100 g ; collected at 24 h after hcg ) and purified c57bl/6 granulosa cells ( collected at 4 h after hcg ) were analyzed by immunoblotting using antiserum against recombinant murine fabp6 . antibodies against -actin ( actb ) and steroidogenic acute regulatory protein ( star ) were used as controls . ( b ) immunohistochemical detection of fabp6 in paraffin sections of the ovaries collected at specific time points of superovulation treatment from immature c57bl/6 mice . negative control tissue sections ( left ) collected 48 h after ecg treatment and 24 h after hcg treatment were not incubated with anti - fabp6 . g , granulosa cells ; t , theca cells ; o , oocyte ; cl , corpus luteum . ovary , we analyzed thin sections of ovaries collected at specific time - points of follicle and luteal development by immunohistochemistry using our validated antiserum to murine fabp6   ( fig . immnuoreactivity was observed only in tissue sections incubated with anti - fabp6 , but not in negative control tissue sections . granulosa cells of follicles at all stages and luteal cells of corpora lutea showed immunoreactivity to fabp6 antibody . fabp6 was abundant in the cytoplasm of the granulosa , cumulus and luteal cells compared to the cytoplasm of theca cells , interstitial cells and oocytes which showed very little or no immunoreactivity . granulosa cells close to the antrum showed more immunoreactivity compared to those near the basement membrane . our data partially agree with the previous report , which 
 showed fabp6 in corpora lutea of unstimulated rats of unknown estrous cycle stage . it is also worth mentioning that we used a validated fabp6 antibody and pure cell populations to determine the presence of fabp6 protein in specific cell - types . overall , these results provide in vivo evidence for the expression of fabp6 specifically in granulosa and luteal cells . to determine if fabp6 plays a role in ovarian function , we compared the ovulatory response of c57bl/6 mice and fapb6 mice to superovulatory treatment with ecg and hcg . mean body weight of mice of both genotypes was similar ( p > 0.05 ; fig . ( a ) mean body weight of mice on the first day of superovulation treatment protocol . ( b ) mean number of ovulations in response to superovulation treatment in c57bl/6 ( n = 4 ) and fabp6 ( n = 7 ) mice as described in the materials and methods section . the number of oocytes in oviducts of each mouse was determined at 1820 h after hcg stimulation . values shown are mean  sem . * p < 0.03 . ) on the day of ecg treatment suggesting that overall growth and development of fabp6 mice was normal . the number of oocytes ovulated in fabp6 mice was significantly lower ( p < 0.03 ) than c57bl/6 mice ( fig . 3b ) . nonetheless , the ovulated cumulus - oocyte - complexes of both genotypes appeared normal with expanded cumulus cell layers and mature oocytes . these data suggest that fabp6 plays a significant role in granulosa cell differentiation required for follicular rupture , but is dispensable for meiotic maturation of the oocyte . ( a ) mean body weight of mice on the first day of superovulation treatment protocol . ( b ) mean number of ovulations in response to superovulation treatment in c57bl/6 ( n = 4 ) and fabp6 ( n = 7 ) mice as described in the materials and methods section . the number of oocytes in oviducts of each mouse was determined at 1820 h after hcg stimulation . it is generally accepted that fabp6 ( also known as ileal lipid binding protein ; ileal bile acid binding protein ) is involved in intracellular transport of bile acids in enterocytes [ 10 , 16 ] . however , the fabp6 mrna and protein have also been detected in the ovaries of several species [ 3 , 14 , 15 , 17,18,19 ] , albeit the precise tissue localization and expression pattern in dynamic structures of the ovary were not established . further , the importance of this protein in ovarian function remains unknown since the ovary is not a major site for bile acid metabolism . in the present study , we surveyed murine ovaries for fabp6 distribution and explored its potential 
 importance in ovarian function . fabp6 is known to preferentially bind bile acids but the ovaries are not regarded as an active site for bile acid metabolism , although a recent study reported that some genes involved in bile acid synthesis are expressed in human granulosa cells . we reported recently that bile acids are found in the follicular fluid of the dominant follicle of lactating cows and that granulosa cells of the dominant follicles express slc10a2 and gpbar1 ( bile acid transporter and receptor , respectively ) , suggesting the novel idea that bile acid signaling might be occurring in ovarian cells . on the other hand , fabp6 has been shown to be capable of binding other ligands , including fatty acids [ 8 , 9 ] and progesterone , and therefore may be involved in metabolic pathways beyond bile acid metabolism . it is well established that the progesterone receptor is induced in granulosa cells of ovulating follicles by hcg and mice lacking this receptor do not ovulate . along with these observations , our data showing high mrna abundance of fabp6 at ecg-48h and hcg-1h are indicative of its role in progesterone signaling , which is needed for efficient ovulation . interestingly , despite the attenuated ovulation response displayed by fabp6 mice to superovulatory stimulation , they do not exhibit overt defects in fertility or fecundity . the loss of fabp6 function may not seriously impact the overall fecundity of polytocous animals ( such as mice ) , but may be important for species that release only one egg per cycle ( such as humans ) . detailed understanding of mechanisms by which fapb6 influences the ovulatory process requires further research . in summary , we demonstrated here that fabp6 is present in distinct cell populations within the murine ovaries and loss of fabp6 results in marked reduction in ovulatory response to superstimulation . these findings suggest that fabp6 may have important roles in ovarian physiology that are distinct from its function as an intracellular bile acid transporter in the ileum .