the domestic cat ( felis catus ) is seasonally polyestrous , with ovulation usually provoked by coitus [ 1 , 2 ] ; however in as much as 3050% of the cycles , ovulation is spontaneous . during the breeding season , which extends from february - march until late summer in moderate latitudes , the cat exhibits several estrous cycles .
if ovulation is not followed by pregnancy , the queen enters pseudopregnancy ( a nonpregnant luteal phase ) that lasts only about one - half of a normal gestation .
the length of the luteal phase in pregnant queens , accompanied by elevated progesterone ( p4 ) levels , is assumed to be partially driven by luteotrophic and luteolytic factors of placental origin . in the dog , the uteroplacental unit seems to serve as the major source for the prepartum release of the luteolytic pgf2 [ 79 ] . even though the luteal phase in pseudopregnant cats is much shorter than in the dog
, one may speculate that the feline cl possesses an intraluteal mechanism that triggers its demise at the appropriate time .
the life - span of the cl in the pregnant cat is longer than in pseudopregnant queens ; however , it needs to be precisely regulated to allow the onset of parturition .
although pgf2 seems to be a very potent luteolysin , its role in regulating the cl of pregnancy in cats is still uncertain .
shille and stabenfeldt   showed that high doses and repeated treatments with pgf2 on days 1115 after mating had almost negligible effects on progesterone levels in cats , which initially diminished but shortly afterwards recovered completely following treatment .
similarly , pgf2 given on days 4 and 5 or 12 and 13 had no effect nor on circulating p4 neither on cl size as monitored laparoscopically . in female cats on days 2125 after mating
, luteal function was transiently depressed after treatment , but eventually the treatment did not alter the length of the luteal phase compared with untreated cats . however , exogenously administered pgf2 or its analogue has a luteolytic effect on fully developed cls after day 33   or day 40 of gestation .
recently , the pgf2 inactive metabolite 13,14-dihydro-15-keto prostaglandin f2 ( pgfm ) , measured in the feces , was shown to be a precise pregnancy indicator in several felid species , including the domestic cat , during the last trimester of gestation .
we hypothesize that the placenta of the domestic cat synthesizes pgf2 in a time - dependent manner . to address this hypothesis
, we examined in the feline placenta ( 1 ) the cellular localization of pgfs ( akr1c3 ) , ( 2 ) the pgfs protein and mrna content , ( 3 ) the cellular localization of ptgs2 and its mrna expression , and ( 4 ) the tissue level of pgf2 at different gestational stages . moreover , in maternal blood plasma the pgf2 metabolite pgfm was measured throughout gestation .
all procedures were approved by the local animal care and use committee in olsztyn , poland ( 61/2010/dtn ) .
thirty domestic , healthy cats , aged 16 years were brought into a local veterinary clinic for neutering .
four groups were created with queens ovariohysterectomized according to the following schedule.postimplantation , early gestation , days 1821 ( 2.53 weeks ) , n = 8.mid gestation , days 2835 ( 4 - 5 weeks ) , n = 8.late gestation , days 4956 ( 7 - 8 weeks ) , n = 7.before parturition , days 6064 ( 8.59 weeks ) , n = 6 .
postimplantation , early gestation , days 1821 ( 2.53 weeks ) , n = 8 .
mid gestation , days 2835 ( 4 - 5 weeks ) , n = 8 .
late gestation , days 4956 ( 7 - 8 weeks ) , n = 7 . before parturition ,
additionally , samples were collected from one cat during hysterectomy around day 14 of gestation ( peri - implantation period ) .
the day of mating , known in 18 females , was recorded as day 0 .
confirmation of gestational age , whenever the day of mating was unknown , was done according to the measurements of the crown - rump length in fetuses and uterine ampullae diameters or lengths [ 15 , 16 ] .
in addition , measurements of the progesterone ( p4 ) values reported in the literature   and confirmed in our last report   were performed .
blood samples were collected from the cephalic vein into edta - containing tubes ( tyco healthcare group lp , mansfield , usa ) and transported to the laboratory at 4c .
plasma obtained after blood centrifugation ( 3500  g , 10  min ) was frozen at 20c until p4 and pgfm measurements .
tissues were washed immediately after surgery with sterile saline to remove blood contamination then were placed into fresh sterile saline at 4c and transported to the laboratory within 1  h. uterine horns were slit longitudinally and pieces of uterus with attached placenta were prepared , washed in fresh saline to remove blood , preserved overnight at 4c with rnalater ( ambion biotechnologie gmbh , wiesbaden , germany ) , and then stored at 80c until total - rna extraction .
another fragment of the same placenta was fixed in buffered 4% formaldehyde for 24  h , dehydrated , and wax - embedded .
sections ( 2 - 3  m thick ) were used to determine pgfs and ptgs2 protein localization by immunohistochemistry .
transcripts of pgfs and ptgs2 were determined using real - time pcr , while the content of pgfs protein was determined by western blot analysis .
total rna was isolated from feline placenta using trizol - reagent according to the manufacturer 's instructions ( sigma aldrich , warszawa , poland ) .
the rna content was measured with a nanodrop 1000 spectrophotometer ( thermo , usa ) . prior to reverse transcription , genomic dna contamination was removed by treatment with dnase ( sigma aldrich , warszawa , poland ) .
reverse transcription was performed using the improm - ii reverse transcription system ( promega , warszawa , poland ) according to the manufacturer 's protocol . in brief ,
an experimental reaction contained 1  g of rna in 12  l of reaction mix , 4  l of reaction buffer , 2.5  l of mgcl2 ( final concentration 3  mm ) , 1  l of dntp mix ( final concentration 0.5  mm each dntp ) , and 1  l of improm - ii reverse transcriptase .
the tubes were placed in a thermocycler ( bio - rad laboratories , hercules , ca , usa ) equilibrated at 25c and incubated for 5  min . for the next step
, tubes were incubated in a thermocycler for 1  h at 42c . for reverse transcriptase inactivation ,
the levels of mrna expression of target genes were examined by real - time pcr using specific primers for pgfs , ptgs2 , and cyclophilin a ( cyc ) . among several housekeeping genes that have been already examined for their stability and efficiency [ jursza , siemieniuch , 2013 , unpublished ] ,
all primers were purchased from sigma aldrich ( warszawa , poland ) and were tested in our previous report [ 18 , 19 ] .
the sequences were as follows : pgfs forward : 5-tcaaccagagcaaactgctg-3 , pgfs reverse : 5-cattccttccctgagttgga-3 ( genbank accession number hm490147 ) ; ptgs2 forward : 5-aacaggagcatccagaatgg-3 ; ptgs2 reverse : 5-gcagctctgggtcaaacttc-3 ( genbank accession number ef036473 ) ; cyc forward : 5-ccttctgtagctcgggtgag-3 ; cyc reverse : 5-cttggaggggaggtaaggag-3 ( genbank accession number ay029366 ) .
the real - time pcr reactions were carried out in an automated fluorometer abi prism 7300 sequence detection system ( applied biosystems , darmstadt , germany ) using sybr green master mix ( applied biosystems , applera , warsaw , poland ) .
the total reaction volume was 20  l containing 1  l cdna ( 200  ng ) , 500  nm each of forward and reverse primers , and 10  l sybr green pcr master mix .
real - time pcr was carried out as follows : initial denaturation ( 10  min at 95c ) , followed by 40 cycles of denaturation ( 15  s at 95c ) , and annealing ( 1  min at 60c ) .
after each pcr reaction , melting curves were obtained by stepwise increases in temperature from 60 to 95c to ensure single product amplification .
relative quantification was performed by normalizing the signals of target genes with the cyc signal using the miner method for quantifying qrt - pcr results , using calculations based on the kinetics of individual pcr reactions .
tissue homogenates were prepared with net-2 lysis buffer containing 50  mm tris - hcl , ph = 7.4 , 300  mm nacl , 0.05% np-40 , and protease inhibitor cocktail ( 10  l / ml ) .
tissues were centrifuged at 10,000  g for 10  min at 4c ; then proteins in the supernatant were disrupted using a sonicator ( 75  w for 15  s ) .
the concentrations of protein were determined by the bradford assay with a spectrophotometer ( smartspec plus , bio - rad ) .
proteins were solubilized in sample buffer ( 25  mmol / l tris - cl , ph = 6.8 , 1% sds , 5% -mercaptoethanol , 10% glycerol , and 0.01% bromophenol blue ) , placed onto 12% sds - polyacrylamide gel , and separated at 120  v. afterwards , proteins were transferred onto methanol - activated pvdf membranes for 1  h at 100  v. to block nonspecific binding , membranes were incubated for 1  h in nonfat dry milk , 5% in pbs with 0.25% tween 20 .
membranes were incubated overnight at 4c with canine - specific guinea pig polyclonal affinity purified anti - pgfs ( akr1c3 ) custom - made antibody ( eurogentec s.a .
, seraing , belgium ) , at a dilution of 1  :  400 , as described by gram et al . .
after this , membranes were washed three times for 10  min in pbs with tween-20 at 20c .
next , membranes were incubated with horseradish peroxidase ( hrp)-conjugated secondary antibody ( sigma , dilution 1  :  15.000 ) for 1  h at room temperature ( rt ) . as a loading control
, pvdf were reblotted with anti--actin mouse monoclonal antibody ( sc-69879 , santa cruz biotechnology , rockford , il , usa , dilution 1  :  1000 ) .
results were visualized using the supersignal west femto maximum sensitivity substrate according to the manufacturer 's protocol ( thermo scientific , usa ) and the chemidoc xrs+ system and image lab ( biorad ) .
formalin - fixed , paraffin - embedded tissues were cut with a microtome ( 2 - 3  m ) and mounted on superfrost plus microscope slides ( menzel - glser , braunschweig , germany ) .
briefly , slides were deparaffinized and rehydrated in a graded ethanol series and then incubated in citrate buffer ( 10  nm , ph 6.0 ) for 15  min under microwave irradiation at 560  w for antigen retrieval .
then , sections were incubated in 0.3% h2o2 in methanol for 30  min to quench endogenous peroxidase and then washed in ihc - buffer/0.3% triton x ph 7.27.4 ( 0.8  mm na2hpo4 , 1.47  mm kh2po4 , 2.68  mm kcl , and 1.37  mm nacl ) .
the following primary antibodies were used : canine - specific guinea pig polyclonal affinity purified anti - pgfs ( akr1c3 ) antibody ( dilution 1  :  400 ) ( eurogentec s.a . , seraing , belgium ) , the same as for western blot and as recently applied for canine uteroplacental tissue , and affinity purified goat anti - rat polyclonal antibody raised against the n - terminus of cox2 ( santa cruz biotechnology , inc . , ca , usa ) .
the negative controls were as follows : slides without the primary antibody and slides incubated with nonimmunized guinea pig or goat serum , respectively , at the same dilution and protein concentration as primary antibodies .
, slides were incubated for 30  min at rt with either biotinylated goat igg ( secondary antibody , dilution 1  :  100 ) against guinea pig immunoglobulin ( vector laboratories , burlingame , us ) or biotinylated rabbit igg antibody ( dilution 1  :  100 ) against goat immunoglobulin ( vectastain abc kit , vector laboratories ) . for enhancing signals
, sections were incubated with the avidin - biotin - peroxidase complex ( vectastain abc kit , vector laboratories ) for 30  min at rt . after washing with ihc - buffer ,
sections were allowed to react with the substrate diamino - benzidine ( dab ; dakocytomation , glostrup , denmark ) according to the manufacturer 's instructions .
slides were counterstained with hematoxylin , rinsed under running tap water for 5  min , dehydrated in a graded ethanol series , and mounted in mounting medium dpx ( panreac quimica sau , barcelona , spain ) . for pgf2 extraction , the protocol elaborated by tsang et al .   and tested for feline placental progesterone extraction by siemieniuch et al .   was applied , with some minor modifications . briefly , placental fragments weighing 200220  mg were stored at 80c . after thawing , each tissue sample was homogenized in a glass vial using a tissue disruptor with 400  l of a tris buffered saline containing proteins and sodium azide as preservative and acidified by addition of 45  l 1  n hcl . after adding 3  ml of ethyl ether to the samples
, they were vortexed for 10  min and incubated at 20c for 4  h. afterwards , the supernatant was collected and evaporated to dryness under a stream of nitrogen at 40c .
finally , 400  l of the tris buffered saline was added , mixed , and allowed to sit for 15  min at 20c .
the samples were stored at 20c until the immunoassay was run . for pgf2 and pgfm measurements , the commercial pgf2 high sensitivity eia kit ( enzo life sciences inc . ,
farmingdale , ny , usa ) and 13,14-dihydro-15-keto prostaglandin f2 eia kit ( cayman chemical company , ann arbor , mi , usa ) were used , respectively , and run according to the manufacturers ' instructions .
this assay is based on the competition between free pgf2 and a pgf2 tracer for a limited number of pgf2-specific sheep antiserum binding sites .
the cross - reactivity for various prostaglandins and their metabolites was as follows : pgf2 100% , pgf1 11.82% , pgd2 3.62% , 6-keto - pgf1 1.38% , pgi2 1.25% , and pge2 0.77% .
this assay is based on the competition between free 13,14-dihydro-15-keto pgf2 and a 13,14-dihydro-15-keto prostaglandin f2 tracer for a limited number of 13,14-dihydro-15-keto pgf2-specific rabbit antiserum binding sites .
the cross - reactivity for various prostaglandins and their metabolites was as follows : 13,14-dihydro-15-keto pgf2 100% , 13,14-dihydro-15-keto pge2 2.7% , and 15-keto pgf2 1.8% .
for p4 measurement , a commercial progesterone elisa kit ( enzo life science ) was used and run in accordance with the manufacturer 's instructions .
this kit uses a polyclonal antibody to p4 to bind , in a competitive manner , p4 in a sample or p4 with a covalently attached alkaline phosphatase molecule .
the cross - reactivity for a number of related steroids was as follows : p4 100% , 5-pregnane-3,20-dione 100% , 10-oh - progesterone 3.46% , corticosterone 0.77% , and deoxycorticosterone 0.28% .
the inter- and intraassay precision variation was 8.1% and 9.5% , respectively . to test the effect of different stages of pregnancy on mrna levels and on pgfm or pgf2 concentrations , the kruskal - wallis test ( a nonparametric anova )
was used followed by the newman - keuls multiple comparison test using the statistical software program graphpad6 ( graphpad prism v 6.0 ; graphpad software inc . , san diego , ca , usa ) .
pgfm concentrations in plasma or pgf2 concentrations in the placenta are shown as a mean  standard deviation .
in the uterus at the peri - implantation period ( 2nd week of pregnancy ) , no or very weak signals were observed in the glandular epithelium ( figure 1(a ) ) . after implantation ( 3rd week of pregnancy ) , and in fully developed placenta at the 7th week of gestation , strong placental signals were localized in fetal trophoblast cells ( figures 1(b ) and 1(c ) , resp . ) . towards the end of pregnancy ( 9th week )
signals in trophoblast cells were much weaker , and were localized in the maternal decidual cells ( figures 1(d ) and 1(e ) ) .
negative controls showed no staining ( figure 1(f ) ) . in the western blot analysis ,
the pgfs was detected as a protein with a molecular size of approximately 3436  kda .
the expression of pgfs protein was strongly affected by gestational age ( figure 2 ) .
the pgfs protein was upregulated , particularly in the first period studied , at 2.53 weeks of pregnancy , compared to samples collected at 7 - 8 gestational weeks ( p < 0.05 ) or 8.59 weeks ( p < 0.001 ) .
the expression of transcripts for placental pgfs as determined by real - time pcr was strongly time - dependent ( figure 3(a ) ) .
the pgfs mrna was significantly upregulated at the 3rd week of pregnancy and then gradually declined towards the end of gestation ( p < 0.001 ) .
strong staining was observed in the invading fetal trophoblast cells surrounding maternal blood vessels ( figure 4(a ) ) .
similarly , strongly positive signals were observed in the maternal decidual cells in the 9th week of pregnancy ( figure 4(b ) ) .
no signals were noted in the negative controls ( figure 4(c ) ) . only weak signals were observed for placental ptgs2 protein expression during earlier stages of pregnancy .
the expression of transcripts for placental ptgs2 , as determined by real - time pcr , was time - dependent ( figure 3(b ) ) .
ptgs2 mrna expression was basal during early and midgestation , started to increase during late gestation , and was significantly upregulated during the prepartal phase ( p < 0.001 ) .
the levels of pgf2 extracted from placentas mirrored the pgfm levels measured in peripheral blood plasma of pregnant females throughout gestation , as shown in figures 5(a ) and 5(b ) , respectively .
both placental pgf2 and plasma pgfm were elevated towards the end of pregnancy compared to the values obtained at the 3rd week of pregnancy ( p < 0.001 ) .
to examine the role of prostaglandins in the feline placenta near the end of pregnancy , the transcripts for ptgs2 and its cellular localization were analyzed .
ptgs2 , the crucial enzyme in generating prostaglandin , catalyzes the conversion of arachidonic acid into endoperoxide pgh2 , which is a precursor of all prostaglandins [ 22 , 23 ] . in the present study , transcripts for ptgs2 were upregulated in placentas solely from queens in the last week before termination of the pregnancy .
these findings are in agreement with a previous report of an acute increase of ptgs2 transcripts in the placenta in the dog during prepartum luteolysis . in the present study ,
the protein localization of ptgs2 was found in both fetal trophoblast and maternal decidual cells .
in contrast , both protein and transcripts for pgfs ( akr1c3 ) were strongly upregulated soon after implantation , which takes place on days 1214 after ovulation in the cat .
transcripts for pgfs and pgfs protein were rather low in the placenta near the end of pregnancy .
these findings are in agreement with a report of decreased pgfs transcripts in preparturient dogs , which led to the conclusion that pgfs ( akr1c3 ) might be not responsible for the sharp pgf2 elevation just before term in the bitch .
one of the questions addressed in this study was whether pgfs ( akr1c3 ) , that was shown to promote the direct conversion of pgh2 to pgf2 , is responsible for placental elevation of pgf2. in the dog , pgfs ( akr1c3 ) staining was predominantly localized in the fetal trophoblast cells in the fetomaternal contact zone , justifying the assumption that pgfs may play a role in decidualization and placentation ; moreover , pgfs expression was low prior to implantation but distinctly increased thereafter .
the cellular localization of pgfs in the feline placenta from 3 weeks of gestation is similar to that observed in the dog .
therefore , pgfs is implicated in the processes of fetal membrane development and placentation . the discrepancy observed between elevated pgf2 concentration in the placenta and downregulation of pgfs ( akr1c3 ) protein and mrna levels
may be explained by the number of enzymes involved in generation of the wide variety of prostaglandins .
it can be directly synthesized from pgh2 by pgf2 synthase ( pgfs ) , like akr1c3 presented herein .
however , other pathways of pgf2 synthesis , including enzymes belonging to the aldo - ketoreductases family , for example , akr1b1 or akr1b5 , are possible .
the canine pgfs cdna sequence has been cloned   and classified as an akr1c3 and is the only canine - specific pgfs isoform known so far .
the feline pgfs cdna sequencing was based on a dog cdna sequence and showed 87% homology with the canine pgfs .
furthermore , pgf2 may originate from conversion of several other prostaglandins , including catalysis of pge2 to pgf2 by 9-keto - reductase ( 9kpgr ) , or perhaps even at relatively low expression levels akr1c3 is still not rate - limiting for pgf2 synthesis .
recently , elevated concentrations of a stable pgf2 metabolite ( pgfm ) were found in the feces of several felid species in the last trimester of pregnancy . in the present study ,
the pgfm level , measured in feline plasma blood collected just before ovariohysterectomy , was low during the first and second trimesters of pregnancy and then started to increase for the last 3 weeks of gestation .
nevertheless , to examine whether the placenta might support synthesis of pgf2 , placental tissues from different gestational weeks were extracted to obtain pgf2. the pgfm blood plasma level was mirrored by the placental pgf2 concentration .
the lowest amount of pgf2 was observed in tissue collected soon after implantation , indicating there is no likely biological role at this time .
it explains why in the earlier studies by wildt and coworkers   or shille and stabenfeldt   pgf2 failed to influence p4 production or reduce cl size .
the first rise in concentration was seen at the beginning of the second trimester , but the most distinct increase coincided with the last week of gestation .
the very similar pattern of placental pgf2 and its stable metabolite in feline blood plasma may suggest the placental origin of pgfm .
however , this finding does not rule out an intraluteal and/or uterine source of pgf2 during pregnancy , especially since components of the underlying synthetic pathways were already found in the domestic cat cl ( zschockelt and siemieniuch 2013 , unpublished ) .
this is in contrast with the canine cl , in which intraluteal prostaglandin synthesis is associated with luteal formation but not with luteal regression or luteolysis [ 8 , 26 , 27 ] .
the strong prepartum increase in circulating pgf2 observed in the dog seems to be originated from the uteroplacental compartment , where strongly upregulated expression of the prostaglandin system was reported   and was evidenced by the increased output of pgf2 from the canine placenta at the time of parturition .
furthermore , the prepartum increase in peripheral pgf2 in the dog is accompanied by a steep p4 decline , implicating its role during luteolysis and/or fetal expulsion [ 28 , 29 ]
. these results partially agree with our present observations concerning elevated pgfm levels in maternal blood in the domestic cat and are consistent with the previous report from our laboratory presenting a considerable decline of p4 in both maternal plasma and placental tissue in the last week of feline pregnancy .
the latter observation , however , concerning the p4 levels does not relate to the immediate prepartum time period but describes the strongly diminished p4 levels during the last gestational week .
at least , the luteolytic role of pgf2 in the second half of feline gestation is generally accepted , since pgf2 or its analogues given to females with fully developed cls induces luteolysis and consequent abortion after day 33   or day 40 of gestation . in conclusion , the data presented here confirms that the placenta of the domestic cat is capable of synthesizing pgf2 in a time - dependent manner .
besides the fact that akr1c3 is not a rate limiting enzyme even at relatively low expression levels , it may still be determinant for pgf2 synthesis in the feline placenta .
the peak of both placental pgf2 and maternal plasma pgfm coincides with the beginning of the last week of pregnancy in cats , compared to dogs , in which peak plasma pgfm is observed 2448  h before term .
other pathways involved in pgf2 synthesis in the cat placenta are possible and need to be elucidated .
 in the present study , the question was addressed whether the feline placenta can synthesize prostaglandin f2 ( pgf2 ) . 
 the pgfs protein was elevated , particularly at 2.53 weeks of pregnancy compared to 7 - 8 ( p < 0.05 ) and 8.59 weeks ( p < 0.001 ) . transcripts for pgfs were significantly upregulated at 2.53 weeks of pregnancy and then gradually declined towards the end of gestation ( p < 0.001 ) . 
 transcripts for ptgs2 were only upregulated in placentas from queens close to term ( p < 0.001 ) compared with earlier phases . 
 staining of ptgs2 showed distinct positive signals in placentas obtained during the last week before labor , particularly in the strongly invading trophoblast surrounding blood vessels , and also in decidual cells . shortly after implantation 
 , signals for pgfs were localized in the trophoblast cells . near term 
 , pgfs staining was seen mainly in decidual cells . both placental pgf2 and plasma pgfm 
 were elevated towards the end of pregnancy ( p < 0.001 ) compared with earlier weeks of pregnancy . 
 the content of pgf2 in extracted placenta mirrored the pgfm level in plasma of pregnant females . during late gestation 
 there is a significant increase in pgfm levels in maternal blood and of pgf2 levels in placental tissue concomitant with an upregulation of placental ptgs2 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion