animals and collection of endometrial tissue : reproductive tracts of 
 holstein cows used in the present study were obtained from a local abattoir . evidently 
 healthy uteri without a conceptus were transported to the laboratory within 30 min . days of 
 the estrous cycle were assessed by morphological observations of follicles and cls in 
 ovaries according to a previous report . for 
 measurement of the p4 concentration and mrna expression , endometrial tissue was collected 
 from cows at three different stages of the luteal phase . based on ovarian morphology , the 
 stages of the estrous cycle were estimated as follows : elp ( days 5 and 6 , day 0=estrus ) , mlp 
 ( days 812 ) and late luteal phase ( llp , days 1517 ) . endometrial tissue was collected from the cranial and middle parts of uterine horns both 
 ipsilateral and contralateral to the ovary containing a cl ( the ipsi - horn and contra - horn , 
 respectively ; elp , n=6 ; mlp , n=6 ; and llp , n=5 ) . cranial endometrial tissue samples were 
 collected at approximately 2 cm from the uterotubal junction . middle endometrial tissue 
 samples were collected from the uterus just above the external bifurcation . endometrial 
 tissue samples were cut into small pieces , weighed and rinsed in 1 ml of 
 physiological saline and then placed on absorbent paper to dry . approximately 50 mg of each 
 uterine endometrial tissue was frozen in liquid nitrogen and stored at 80c until processed 
 for p4 measurement and rna isolation . the experimental procedures complied with the guide for care and use of agricultural 
 animals of obihiro university . endometrial p4 concentration : the concentrations of p4 in the endometrial 
 tissue were determined in duplicate using a second - antibody enzyme immunoassay ( eia ) . endometrial tissue samples were placed into a glass tube with the assay buffer for the 
 steroid eia ( 0.1 g tissue per 1 ml buffer solution ) in cold water and 
 homogenized using a multipro ( dremel germany ) at 15,000 rpm for 2 min . the homogenate was 
 centrifuged at 1,450 g for 15 min , and the supernatant was collected . the standard curve ranged from 0.05 ng / ml to 
 50 ng / ml for p4 , the median effective dose ( ed50 ) of the 
 assay was 2.4 ng / ml for p4 , and the intra- and interassay 
 cvs were 8.2% and 10.5% at 1.5 ng / ml , respectively . rna isolation and cdna synthesis : frozen uterine endometrial tissue 
 samples were individually ground using a mortar and pestle and then transferred immediately 
 to total rna extraction solution ( isogen , nippon gene , tokyo , japan ) . the yield of total rna was quantified by 
 measuring the absorbance at 260 nm using a nanodrop spectrophotometer ( nanodrop 2000c , 
 thermo fisher scientific , waltham , ma , u.s.a . ) . total rna ( 1 g ) was 
 treated with dnase using a commercial kit ( rq1 dnase , promega , madison , wi , u.s.a . ) . single - stranded cdna was reverse transcribed from total rna using a first - strand cdna 
 synthesis kit ( superscript vilo reaction mix , invitrogen , carlsbad , ca , u.s.a . ) . the reverse 
 transcription conditions consisted of 10 min of annealing at 25c , 60 min of cdna synthesis 
 at 42c and 5 min of inactivation at 85c . real - time polymerase chain reaction : gene expression was determined by 
 real - time polymerase chain reaction ( pcr ) using a rotor - gene q ( qiagen , tokyo , japan ) and 
 sybr premix ex taq ii ( takara , tokyo , japan ) . the primers for real - time pcr 
 were designed using primer3 based on the bovine sequences and are shown in table 1table 1.primers used in real - time pcrgenesequence of nucleotide ( 53)size ( bp)annealingtemperature ( c)accession no.pgrftaatctgtggggatgaagca18158nm 001205356rcagcactttctaaggcgacapgrmc1faggagtgaggtcggaaaggt16559 nm 001075133ratcaatggcaaggtgttcgpgrmc2fccagaggactggcaacattt16756 nm 001099060racggttcttcccctggtttgapdhfccacttgatgttggcaggat6659xm 001252511rgaagctcgtcatcaatggaaaa ) forward , b ) reverse .. the amplification program consisted of 30 sec activation at 95c , followed by 
 40 cycles of pcr steps ( 15 sec denaturation at 94c , 30 sec annealing and 20 sec extension 
 at 72c ) . the values were normalized using glyceraldehyde 3-phosphate 
 dehydrogenase ( gapdh ) as the housekeeping gene . the relative quantification of mrna 
 expression of all the factors was performed according to the method of pfaffl . a ) forward , b ) reverse . statistical analysis : statistical analyses were performed using statview 
 version 5.0 ( sas institute , cary , nc , u.s.a . ) . for endometrial tissue p4 concentrations and 
 pgr , pgrmc1 and pgrmc2 mrna expression levels , a two - way factorial anova was used to 
 determine the main effects of location ( ipsi - horn vs contra - horn ) and estrous stage ( elp vs 
 mlp vs llp ) and their interaction , respectively . when a significant interaction was 
 detected , scheffe s f - test was used as a multiple comparison test to detect 
 significant differences between locations , within stages and among stages within locations . correlations between endometrial tissue p4 and pgr , pgrmc1 and pgrmc2 mrna expressions were 
 assessed using the nonparametric spearman rank correlation test . endometrial tissue p4 concentration : the endometrial tissue p4 
 concentrations in the cranial and middle parts of the uterine horn are presented in tables 2table 2.concentration of endometrial tissue p4 in the cranial part of the ipsilateral and 
 contralateral hornsstages ( n)endometrial tissue p4 ( ng / g)ipsi - horncontra - hornoverall meanselp ( 6)32.6  3.715.9  2.024.3  3.2mlp ( 6)25.8  1.414.4  2.720.1  2.2llp ( 5)18.8  3.712.5 2.713.7  2.4overall means26.1  2.114.4  1.4a , b ) significant differences at p<0.01 between the horn 
 ipsilateral to a cl ( ipsi - horn ) and horn contralateral to the cl ( contra - horn ) . the 
 luteal phase was divided into three stages ( elp , early luteal phase , n=6 ; mlp , 
 mid - luteal phase , n=6 ; and llp , late luteal phase , n=5 ) . 3table 3.concentration of endometrial tissue p4 in the middle part of the ipsilateral and 
 contralateral hornsstages ( n)endometrial tissue p4 ( ng / g)ipsi - horncontra - hornoverall meanselp ( 6)16.4  4.214.4  2.715.4  2.4mlp ( 6)11.9  2.69.2  2.310.6  1.7llp ( 5)15.6  4.78.9  3.012.3  2.9overall means14.6  2.110.9  1.6the luteal phase was divided into three stages ( elp , early luteal phase , n=6 ; mlp , 
 mid - luteal phase , n=6 ; and llp , late luteal phase , n=5 ) . , respectively . in the cranial part , the endometrial tissue p4 concentrations 
 were higher in the ipsi - horn than in the contra - horn during all stages of the luteal phase 
 ( p<0.01 ) . in both the ipsi- and contra - horns , the endometrial tissue 
 p4 concentrations tended to be high at the elp and then gradually decreased at the mlp and 
 llp . however , there were no statistical differences in endometrial tissue p4 concentrations 
 among the three stages of the luteal phase . in the middle part , the endometrial tissue p4 concentrations in 
 both the ipsi- and contra- horns tended to be high at the elp and then decreased from the 
 elp to mlp . the location and stages had no effect on the endometrial tissue p4 
 concentrations . a , b ) significant differences at p<0.01 between the horn 
 ipsilateral to a cl ( ipsi - horn ) and horn contralateral to the cl ( contra - horn ) . the 
 luteal phase was divided into three stages ( elp , early luteal phase , n=6 ; mlp , 
 mid - luteal phase , n=6 ; and llp , late luteal phase , n=5 ) . the luteal phase was divided into three stages ( elp , early luteal phase , n=6 ; mlp , 
 mid - luteal phase , n=6 ; and llp , late luteal phase , n=5 ) . expression of p4 receptor mrna in endometrial tissue : the pgr and pgrmc1 
 mrna expressions are shown in figs . 1 and 2fig . 1.comparative changes of the relative amounts of pgr mrna expression during the luteal 
 phase ( elp , n=12 ; mlp , n=12 ; llp , n=10 ) in the cranial part of the uterus . since the 
 effect of location was not observed , the data for the ipsilateral ( elp , n=6 ; mlp , n=6 ; 
 llp , n=5 ) and contralateral horns ( elp , n=6 ; mlp , n=6 ; llp , n=5 ) were combined . significant differences 
 at p<0.05 within the respective stages are indicated by letters 
 ( a , b ) . the luteal phase was divided into 
 three stages ( elp , early luteal phase ; mlp , mid - luteal phase ; and llp , late luteal 
 phase ) . p4 receptor mrna expression levels were normalized using the 
 housekeeping gene gapdh . in the cranial part , the effects of stages on the pgr mrna 
 expression were significant ; however , location effects were not observed . the pgr mrna 
 expression was higher at the elp than at the mlp and llp ( fig . both main effects ( location and stages ) and the location by stages 
 interaction on the pgrmc1 mrna expression were significant in the cranial part . at the elp , the pgrmc1 mrna expression in the ipsi - horn was higher compared with the contra - horn 
 ( p<0.05 , fig . 2.comparative changes of the relative amounts of the pgrmc1 mrna expression during the 
 luteal phase ( elp , n=6 ; mlp , n=6 ; and llp , n=5 ) in the cranial part of the uterus . in 
 the cranial part , both main effects ( location and stages ) and the location by stages 
 interaction were significant . significant differences at p<0.05 within 
 respective stages between the horns ipsilateral ( ipsi - horn ) and contralateral 
 ( contra - horn ) to a cl are indicated by the letters a and b , whereas differences within 
 the ipsi - horn among the three stages are indicated by the letters x and y. results are 
 presented as the mean  sem . the luteal phase was divided into three stages ( elp , 
 early luteal phase ; mlp , mid - luteal phase ; and llp , late luteal phase ) . ) . however , the pgrmc2 mrna expression was not affected by location and stages in the 
 cranial part of both horns . in the middle part , nevertheless , the location and stages had no 
 effect on the pgr , pgrmc1 and pgrmc2 mrna expressions . comparative changes of the relative amounts of pgr mrna expression during the luteal 
 phase ( elp , n=12 ; mlp , n=12 ; llp , n=10 ) in the cranial part of the uterus . since the 
 effect of location was not observed , the data for the ipsilateral ( elp , n=6 ; mlp , n=6 ; 
 llp , n=5 ) and contralateral horns ( elp , n=6 ; mlp , n=6 ; llp , n=5 ) were combined . significant differences 
 at p<0.05 within the respective stages are indicated by letters 
 ( a , b ) . the luteal phase was divided into 
 three stages ( elp , early luteal phase ; mlp , mid - luteal phase ; and llp , late luteal 
 phase ) . comparative changes of the relative amounts of the pgrmc1 mrna expression during the 
 luteal phase ( elp , n=6 ; mlp , n=6 ; and llp , n=5 ) in the cranial part of the uterus . in 
 the cranial part , both main effects ( location and stages ) and the location by stages 
 interaction were significant . significant differences at p<0.05 within 
 respective stages between the horns ipsilateral ( ipsi - horn ) and contralateral 
 ( contra - horn ) to a cl are indicated by the letters a and b , whereas differences within 
 the ipsi - horn among the three stages are indicated by the letters x and y. results are 
 presented as the mean  sem . the luteal phase was divided into three stages ( elp , 
 early luteal phase ; mlp , mid - luteal phase ; and llp , late luteal phase ) . the relationship between p4 concentrations and p4 receptors expression in 
 endometrial tissue : in the cranial part of the ipsi - horn at the elp , pgr mrna 
 expression had a negative correlation ( r=0.871 , 
 p<0.05 ) with endometrial tissue p4 . in addition , there was a significant 
 positive correlation ( r=0.913 , p<0.05 ) between pgrmc1 
 mrna expression and the endometrial tissue p4 concentrations . however , there was no 
 correlation of pgrmc2 mrna expression with endometrial tissue p4 . in the cranial part of the 
 ipsi - horn at the mlp and llp , the p4 concentration showed no correlation with pgr , pgrmc1 
 and pgrmc2 mrna expression in the endometrial tissue . in the cranial part of the contra - horn 
 and the middle part of both the ipsi- and contra - horns , p4 receptors mrna expression showed 
 no correlations with endometrial tissue p4 concentrations regardless of the stage of the 
 luteal phase . the results of the present study show the differences in p4 concentrations and pgr and 
 pgrmc1 mrna expressions in the bovine endometrial tissue between the ipsilateral and 
 contralateral horns through each stage of the luteal phase . the endometrial tissue p4 
 concentrations were higher in the cranial part of the ipsilateral horn than in the 
 contralateral horn , consistent with the results of previous reports . the present study showed that the endometrial tissue p4 
 concentrations tended to be higher at the elp in the cranial part of ipsilateral horn 
 compared with the mlp and llp . however , during the luteal phase , the endometrial tissue p4 
 concentration did not change significantly . on the other hand , a previous study reported 
 that endometrial tissue collected during the early stage of the luteal phase ( from day 1 to 
 day 5 ) showed the highest concentration of p4 as compared with the mid and late stages in 
 the ipsilateral horn . in the present study , the differences in the timing of tissue 
 collection may have resulted in the differences in profile of p4 concentrations of 
 endometrial tissue between the two studies . differences in endometrial tissue p4 between the ipsilateral horn and the contralateral 
 horn during the luteal phase in the present study indicate the locational effects of the cl 
 on the p4 concentrations in the endometrium of uterine horns in cattle . the p4 concentration 
 in the uterine artery in the ipsilateral horn was previous reported to be higher than that 
 in the contralateral horn . however , the reasons 
 for high levels of p4 in the uterine artery , which provides p4 to the endometrial tissue in 
 the ipsilateral horn in cattle , are as yet unclear . there is a possibility of a counter 
 current transfer system among the ovarian vein , ovarian artery and uterine artery . in ewes , 
 p4 has been shown to transfer from the utero - ovarian vein into the uterine artery . in cattle , estradiol-17 transfers from the ovarian 
 vein into the ovarian artery . the ovarian artery 
 and a branch of the uterine artery are interconnected by a prominent anastomotic vessel in 
 cattle . these aforementioned studies show that 
 steroid hormones in the ovarian vein may be transported into the ovarian artery at the 
 arterial anastomosis , which connects to the ovarian artery and the neighboring branch of the 
 uterine artery supplying the oviduct and uterine horn . therefore , p4 released from the cl 
 may migrate into the ovarian vein , ovarian artery and uterine artery and then reach the 
 uterine endometrium in cattle . finally , the p4 supply to the uterus by the counter current 
 transfer system may contribute to high concentrations of p4 in the ipsilateral horn in 
 cattle . interestingly , there were no differences in the endometrial tissue p4 concentrations among 
 the elp , mlp and llp . generally , bovine serum p4 concentrations gradually increase during 
 the elp and rise to the maximum level during the mlp and llp [ 8 , 15 ] . the results of the present study 
 indicate that the dynamics of the endometrial tissue p4 concentrations may be different from 
 those of the serum p4 concentrations during the luteal phase , both in the ipsilateral and 
 contralateral horn . it is unclear why the dynamics of the endometrial tissue p4 
 concentrations were different from those of the serum p4 concentrations . in the bovine , the 
 blood flow toward the uterus during the periestrous period this may efficiently enhance the supply of p4 from 
 the developing cl to the uterine horn . it is additionally speculated that higher p4 receptor 
 mrna expression at the elp in the endometrial tissue allows the sequestration of a large 
 amount of circulating p4 . consequently , the blood flow toward the uterus and/or expression 
 of the p4 receptors in the bovine endometrium may create differences in concentration 
 between tissue p4 and serum p4 . however , further investigation of p4 concentrations in the 
 uterine artery during the estrous cycle and p4 binding ability in the endometrial tissue is 
 required . p4 action provides an intrauterine environment that is suitable for pregnancy succession in 
 cattle . in cattle , the decrease of the pgr mrna expression in the 
 endometrium at the mlp and llp is consistent with previous reports that demonstrate a 
 decrease in pgr mrna expression at 810 days after estrus . reported that disappearance of the pgr mrna expression allows for increased secretion of 
 uterine gland products , which is required to support development of the conceptus in the 
 ipsilateral horn . a previous report demonstrated that pgrmc1 and pgrmc2 gene expressions in the bovine 
 endometrium did not differ among the stages of the estrous cycle .in both the ipsi- and contra - horns , there was no change in the pgrmc2 
 mrna expression during the luteal phase in the present study . on the 
 other hand , pgrmc1 mrna expression in the cranial part of the ipsi - horn was higher at the 
 elp . the reason why the profiles of pgrmc1 mrna expression during the luteal phase are 
 different between the previous study and the present study is not clear . in the present 
 study , the tissues from both the cranial and middle parts of the uterus were collected and 
 analyzed separately . however , the location of the endometrial tissue collected in the 
 previous study was not mentioned clearly . therefore , the differences in results between the 
 previous study and the present study might be caused by differences in the sampling location 
 within the uterine horn . the pgr mrna expression was not different between the ipsilateral horn and the 
 contralateral horn . it is well known that pgr expression is downregulated by treatment with 
 p4   in the bovine endometrium . in the present 
 study , there were no differences in pgr mrna expression between the ipsilateral and 
 contralateral horns , although the endometrial tissue p4 concentration was higher in the 
 ipsilateral horn than in the contralateral horn . in fact , a negative correlation between p4 
 concentration and pgr mrna expression was observed in endometrial tissue at the elp only in 
 the ipsilateral horn . from these results , the pgr mrna expression in pregnant cattle was 
 lower than that in nonpregnant cattle [ 2 , 25 ] . moreover , forde et al .   reported that low p4 concentrations delay the 
 downregulation of pgr mrna expression . in view of this fact , downregulation of pgr mrna 
 expression was attributed to high levels of endometrial tissue p4 in the ipsilateral horn , 
 which may create the intrauterine environment for pregnancy succession in the ipsilateral 
 horn . in the present study , there was a positive correlation between the p4 concentration and 
 pgrmc1 mrna expression in the cranial part of the uterine horn at the elp . however , the 
 factor regulating pgrmc1 expression has not yet been investigated in cattle . in 
 ovariectomized mice , pgrmc1 mrna expression was found to be upregulated in the uterus by p4 
 treatment . the present study indicates the 
 possibility that pgrmc1 mrna expression in the cranial part of the ipsilateral horn may be 
 upregulated locally by a higher p4 concentration in the endometrial tissue . in the cranial part , the pgr and pgrmc1 mrna expression and p4 concentrations in the 
 ipsilateral horn at the elp were different from those of the contralateral horn and other 
 stages . the role of changes in pgr and membrane progesterone receptor expressions and 
 endometrial tissue p4 concentration in pregnancy succession is not well known . okumu 
 et al .   reported high p4 
 causes early downregulation of the pgr expression in the bovine endometrium , which is a 
 critical event for the expression of genes required for embryo development . because pgr and 
 pgrmc1 mrna expressions may be regulated differently between the ipsi- and contra - horns , the 
 action of p4 on the pgr and pgrmc1 may create differences in the uterine environment for 
 embryo development between the ipsi- and contra - horns . on the other hand , there was a possibility that some other route exists via mpr- , mpr- 
 and mpr- to provide p4 functions . in humans , 
 mpr- , mpr- and mpr- mrna expressions were confirmed in the endometrium . in particular , 
 the mpr- expression was higher at the early - mid secretory phase than the proliferative 
 phase . in the ovine endometrium , the mpr- mrna 
 expression increased 4-fold at day 4 of the estrous cycle compared with 3 hr post onset of 
 estrus . there were also no differences in mrna 
 expressions of mpr- , mpr- and mpr- in endometrial tissue at 15.5 days between pregnant 
 and nonpregnant cattle . however , the mpr- , mpr- 
 and - mrna expressions were not analyzed in the present study . to elucidate the mechanism 
 of the intrauterine environment for pregnancy succession , analysis for p4 receptor 
 expression in the bovine endometrium , not only pgr and pgrmcs but also mpr- , mpr- and 
 mpr- , during the early luteal phase is required . in conclusion , the present study demonstrates differences in dynamics of endometrial tissue 
 p4 concentrations and expression of the p4 receptors between the two horns ipsilateral and 
 contralateral to the ovary containing a cl . the pgr or pgrmc1 mrna expression showed a 
 negative or positive relationship , respectively , with endometrial tissue p4 concentrations 
 of the cranial part of the ipsilateral horn . differences in dynamics of endometrial tissue 
 p4 concentrations and expression of p4 receptors between the horns ipsilateral and 
 contralateral to the ovary containing a cl may cause differences in intrauterine environment 
 for both the ipsi- and contra - horns .
because the establishment of pregnancy begins at the uterine horn ipsilateral to the 
 corpus luteum ( ipsi - horn ) in cattle , levels of progesterone ( p4 ) and receptor expression 
 in the endometrial tissue , which regulate the intrauterine environment for embryo 
 development , may differ between the ipsi - horn and the uterine horn contralateral to corpus 
 luteum ( contra - horn ) . 
 the aim of the present study was to determine the endometrial tissue 
 p4 concentrations and nuclear progesterone receptor ( pgr ) , progesterone receptor membrane 
 component 1 ( pgrmc1 ) and pgrmc2 mrna expressions in the cranial and middle parts of the 
 uterine horns during the luteal phase . 
 the results showed higher endometrial tissue p4 
 concentrations in the cranial part of the ipsi - horn than in that of the contra - horn 
 ( p<0.01 ) ; however , no change in the endometrial tissue p4 
 concentrations was evident during the luteal phase . 
 the pgr mrna expression was higher 
 during the early luteal phase ( p<0.05 ) , but no differences between the 
 horns were evident . 
 however , pgrmc1 mrna expression during the early luteal phase was 
 higher in the cranial part of the ipsi - horn than in that of the contra - horn 
 ( p<0.05 ) . in the middle part 
 , there were no changes in the 
 endometrial tissue p4 concentrations and p4 receptor expressions during the luteal phase . 
 in conclusion , 
 the differences in dynamics of endometrial tissue p4 concentrations and p4 
 receptor expressions between the uterine horns ipsilateral and contralateral to the ovary 
 containing a corpus luteum may cause differences in the intrauterine environment for both 
 the ipsi- and contra - horns .