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 .
   the fatty acid binding protein 6 ( fabp6 ) is commonly regarded as a bile acid binding protein found in the distal portion of the small intestine and has been shown to be important in maintaining bile acid homeostasis . 
 previous studies have also reported the presence of fabp6 in human , rat and fish ovaries , but the significance of fabp6 in this organ is largely unknown . 
 therefore , we surveyed murine ovaries for fabp6 gene expression and evaluated its role in ovarian function using mice with whole body fabp6 deficiency . here 
 we show that the fabp6 gene is expressed in granulosa and luteal cells of the mouse ovary . 
 treatment with gonadotropins stimulated fabp6 gene expression in large antral follicles . 
 the ovulation rate in response to superovulatory treatment in fabp6-deficient mice was markedly decreased compared to wildtype ( c57bl/6 ) mice . 
 the results of this study suggest that expression of fabp6 gene in 
 granulosa cells serves an important and previously unrecognized function in fertility . 
Materials and Methods
Mice
Superovulation and sample collection
RNA and protein detection
Immunohistochemistry
Statistical analyses
Results
Discussion