Research in Veterinary Science 92 (2012) 53–59

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Research in Veterinary Science

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Term placenta shows methylation independent down regulation of Cyp19 genein animals with retained fetal membranes

Sandeep Ghai a, Rachna Monga a, T.K. Mohanty b, M.S. Chauhan c, Dheer Singh a,⇑a Molecular Endocrinology Laboratory, Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, Indiab Livestock Production and Management Division, National Dairy Research Institute, Karnal-132001, Haryana, Indiac Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 March 2010Accepted 14 October 2010

Keywords:Retention of fetal membranesCyp19 genePromoter methylationEstrogenCotyledons

0034-5288/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.rvsc.2010.10.008

⇑ Corresponding author. Tel.: +91 184 2259135; faxE-mail addresses: dheer@ndri.res.in, drdheer.singh

Retention of fetal membranes (RFM) is the major post-partum disorder in dairy cattle. Cyp19 geneencodes the aromatase enzyme responsible for catalyzing the rate limiting step in estrogen biosynthesis,an important hormone for placental maturation and expulsion. The present study was aimed for compar-ative analysis of Cyp19 gene expression and its epigenetic regulation in placental cotyledons of animalswith and without RFM. Significantly lower expression of Cyp19 gene was found in placental samples ofRFM affected animals in comparison to normal animals. Methylation analysis of 5 CpG dinucleotides ofplacenta specific Cyp19 gene promoter I.1 and proximal promoter, PII showed hypo-methylation of bothPI.1 and PII in term placenta of normal and diseased animals. In conclusion, a mechanism other than pro-moter methylation is responsible for decreased aromatase expression in placental cotyledons of animalssuffering from RFM.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Post parturition reproductive diseases are a serious problem indairy cattle. These diseases not only lower productivity and fertil-ity but they also bring economic loss. It is therefore, important todetect post-partum reproductive disorders as early as possible,and to develop prophylactic measures to prevent these disorders.One of the most important post-partum diseases in dairy cattle isretention of fetal membranes (RFM) (Stephen, 2008; El-Wishy,2007). In cattle, fetal membranes are expelled within 6–12 h aftercalving. The failure to push out all or a part of the placenta from theuterus within 12 h of calving leads to RFM (Drillich et al., 2006). Indairy cattle, 4–11% of spontaneous calvings have been reported toresult in RFM (Hashem and Hussein, 2009) which is comparativelyhigher in buffalo (Arthur et al., 1989; Laven and Peters, 1996;Ahmed et al., 2009). A wide variation (2.89–12.23%) has been re-ported in buffaloes with a maximum at the fifth parity (30%) andassociated with malnutrition (Choudhury et al., 1993; Guptaet al., 1999). Surprisingly, 54% of Iraqi buffaloes studied developedRFM (Azawi et al., 2008) while the incidence of RFM in buffalo inIndia is 21% (Satya pal, 2003).

A failure in the separation of cotyledonary villi from the cryptsof the maternal caruncles results in RFM (Weithril, 1965). The eti-ology of this disorder however, is yet to be completely understood.

ll rights reserved.

: +91 184 2250042.@gmail.com (D. Singh).

Complex metabolic disturbances during the pre-partum periodhave been proposed to be the probable reason of RFM (Michalet al., 2006). It has been suggested that a surge in estrogen concen-tration in bovine maternal blood (Robertson, 1974; Peterson et al.,1975; Hoffmann et al., 1976; Hunter et al., 1977) and placenta(Veenhuizen et al., 1960; Inaba et al., 1983) before parturition isimportant in normal parturition and placental expulsion. It is gen-erally recognized that estrogens play an important role in the mat-uration process of the placentomes (Grunert et al., 1989). Cows andbuffaloes with RFM have been observed to possess higher levels ofprogesterone and lower levels of estradiol 17-b compared to nor-mal animals (Matton et al., 1987; Thomas et al., 1992; Kankoferet al., 1996; Hashem and Hussein, 2009; Ali et al., 2009). Moreover,it has been shown that in cattle and goats, low levels of estrogensand the reduction or absence of the estrogen peak near deliveryare associated with abortion, dystocia and placental retention(Engeland et al., 1999; Zhang et al., 1999a,b). Therefore, down-regulation of estradiol is supposed to be an important factorresponsible for RFM.

The Cytochrome P450 aromatase enzyme encoded by the Cyp19gene has been found to play an important role in converting pro-gesterone to estrogen in the placenta (Flint et al., 1975; Masonet al., 1989; Nelson et al., 1996). A change in the estradiol levelsin animals affected with RFM thus might be due to the change inexpression levels of Cyp19 gene. However, there is no report ofthe expression status of Cyp19 gene in RFM- affected farm animals(cattle and buffalo). Cyp19 gene has been found to be downregulated by the promoter switching phenomenon during

54 S. Ghai et al. / Research in Veterinary Science 92 (2012) 53–59

folliculogenesis in cattle and buffalo (Vanselow et al., 2005; Shar-ma et al., 2009). Methylation of the proximal promoter region ispartly responsible for this promoter switching in ovary (Vanselowet al., 2005; Fürbass et al., 2008) and differential promoter usage inplacenta (Vanselow et al., 2008). Recently, we found that DNAmethylation is also involved in stage specific Cyp19 gene expres-sion in buffalo placenta (Ghai et al., 2010). DNA methylation pat-terns are also found to change in response to environmentalstimuli such as diet or toxins due to which the epigenome seemsto be most susceptible during early in utero development. AberrantDNA methylation changes have been detected in several diseases,particularly cancer where genome-wide hypo-methylation coin-cides with gene-specific hypermethylation (Tost, 2009). Therefore,the role of promoter methylation linked down regulation of theCyp19 gene in animals with RFM cannot be ignored.

The decrease in estradiol levels in placental cotyledons of ani-mals affected with RFM may probably be due to the down regula-tion of Cyp19 gene which in turn might be regulated by promotermethylation. In view of the above points, the present study was fo-cused on determining the Cyp19 gene expression in normal andRFM affected buffalo which is major milk yielding animal in Indiaand contributes more than 60% of country’s milk production. Fur-ther studies were done to understand the relationship betweenCyp19 gene expression and promoter methylation levels in buffa-loes affected with RFM.

2. Materials and methods

2.1. Collection of placental tissues

Placenta of the animals which expelled their fetal membranesnaturally within 12 h of parturition was considered as normal pla-centa. The animals which did not expel their fetal membranes nat-urally but the placenta that was ejected manually after oxytocininjection was considered as retained placenta. Tissue samples(n = 3) were collected from NDRI cattle yard immediately afterexpulsion. The samples were washed with chilled normal salinecontaining antibiotics to remove contamination and blood andthen brought to the laboratory as early as possible (10–15 min).The samples to be used for RNA isolation were immediately keptin RNAlater� (Qiagen, GmbH, Germany) till further processing.

2.2. Separation of placental cotyledons and isolation of DNA and RNA

The buffalo’s placental cotyledons were excised and removedwith the help of sterilized scissors and forceps. The appropriateamount of tissue (100 mg) required for DNA and RNA isolationwas chopped into pieces and homogenized into DNAzol� and TRI-zol� (Life Technologies (India) Pvt. Ltd.), respectively. Total RNAwas isolated by modified TRIzol� method (Chomczynski and Sac-chi, 1987) and DNA was isolated using DNAzol� reagent followingmanufacturer’s instructions. The DNA and RNA were quantifiedspectrophotometrically and the respective integrities were evalu-ated by normal (0.7%) and denaturing (1.5%) agarose gelelectrophoresis.

2.3. Reverse transcription-polymerase chain reaction (RT-PCR)

The cDNA was synthesized using a Fermentas First strand cDNAsynthesis kit (Fermentas, Germany). The reaction mixture con-tained 2 lg of total RNA, 1 ll of random hexamers (0.2 lg/ll)and DEPC treated water up to 11 ll. The contents were incubatedat 65 �C for 10 min followed by 2 min incubation at room temper-ature. The reagents added subsequently were: 4 ll of 5� reactionbuffer (250 mM Tris–HCl, pH 8.3; 250 mM KCl, 20 mM MgCl2,

50 mM DTT), 1 ll of RNase inhibitor (20 IU), 2 ll of dNTP mix(10 mM), 2 ll of M-MuLV reverse transcriptase (200 IU) to a finalvolume of 20 ll. The contents were incubated at 25 �C for10 min, 42 �C for 30 min and 95 �C for 3 min.

The cDNA was amplified with gene specific primers (Table 1) ina reaction mixture containing 2 ll of RT product, 0.2 lM primers(gene-specific forward and reverse primers), 1� PCR buffer[10 mM Tris HCl (pH 9), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin],0.2 mM dNTP mix, 1 U Taq polymerase (1 U/ll) made to 50 ll withnuclease free water. The amplification was done in a thermocyclerunder two different cycle conditions. For Cyp19 gene expression,two-step PCR was performed as nested PCR. For the first amplifica-tion, 2 ll of cDNA was used as template, for the second amplifica-tion, 5 ll of PCR product from the first reaction was used. PCRreactions were performed by incubating the contents at 94 �C for2 min, followed by 32 cycles of 94 �C for 1 min, 60 �C for 1 min,72 �C for 1 min and the final extension of 4 min at 72 �C. The GAP-DH was used as a housekeeping gene.

2.4. Real-time PCR

For mRNA expression analysis by quantitative real-time PCR,SYBR green dye-based expression assays were performed forCyp19 gene in normal and retained placental tissues. The reactionmixture containing 2� SYBR� Universal PCR Master Mix (Fermen-tas), 200 nM gene specific primers (CYPF and CYPR), Table 1 and2 ll of cDNA to a final volume of 10 ll was incubated in the M JMiniCycler (BioRad). The reaction conditions used were 94 �C for2 min, followed by 32 cycles of 94 �C for 20 s, 57 �C for 15 s,72 �C for 15 s. and the final extension of 4 min at 72 �C. GAPDHwas used as internal control. Cyp19 and GAPDH expressions weredetermined by measuring PCR product fluorescence compared tocycle number to determine CT values. Relative Cyp19 expressionfor each tissue sample was calculated using the formula:

DCT ¼ CTCyp19 � CTGAPDH

The fold change in Cyp19 gene expression (DCyp19) between nor-mal and RFM animals was calculated with the formula

ðDCyp19Þ ¼ 2� DCTNormalDCTRFMð Þ

2.5. Bisulfite modification of DNA

Genomic DNA (1 lg) isolated from placental cotyledons of ani-mals with normal and retained fetal membrane was bisulfite-trea-ted using the EZ DNA Methylation Gold Kit (Zymo Research,Orange, CA). The procedure is based on the method developed byFrommer et al. (1992). It is based on the principle that treatmentof single stranded DNA with sodium bisulfite under acidic condi-tions changes all the cytosines in the DNA to uracil while 50-methylcytosines resist this change. Amplification of the bisulfite-treatedDNA by PCR followed by sequencing reveals the positions of 5-methylcytosine in the gene. Bisulfite-treated DNA was eluted in10 ll volumes of elution buffer with 4 ll used for each PCR. Thefrequency of methylation at 5 individual CpG sites within the363-bp and 340-bp region of respective PI.1, the distal promoterresponsible for placenta specific Cyp19 gene expression and PII,the proximal promoter responsible for ovary specific Cyp19 geneexpression was assessed using bisulfite-specific sequencing. Bisul-fite sequencing PCR (BSP) primer pairs specific for modified DNAswere designed to contain no CpG sites. The Methyl Primer Expresssoftware� v1.0 (Applied Biosystems) was used to design primersspecific for BSP (Table 2) and positions of the CpG residues to beanalyzed were also located using the same.

Table 1Primers used for gene specific RT-PCR and real-time PCR.

Primer name Primer sequence Accession no. (NCBI) Size of amplification product

P6F 50-CATGGCAAGCTCTCCTTCTC-30 Z32741 866 bpP7R 50-GCAGGGACTGACCAAACTTC-30 Z32741 302 bpP5F 50-ACTGGAGGGGTGAAGAAACC-30

P8R 50-TTGGCATGGATAGCACACAG-30 NM_174305.1 105 bpCYPF 50-CCTGTGCGGGAAAGTACATCAC-30

CYPR 50-TCTTCTCAACGCACCGACCTT-30

G3F 50-AAACCCATCACCATCTTCCAG-30 M17701 361 bpG3R 50-AGGGGCCATCCACAGTCTTCT-30

Table 2Primers used for amplification of promoter regions in bisulfite treated genomic DNA.

Primer name Primer sequence Region amplified Size of amplification product

B1.1 F 50-GTGTAAAAGAGTTAAGAAAGGTGA-30 Region I.1 (outer) 628 bpB1.1 R 50-AATCCTTTAAAAAACAATAACAAAA-30 363 bpBI.1NF 50-TCCTCACTATTCACTCCTCTCTAAA-30 Region I.1 (Inner)BI.1NR 50-TAGTTAGTGGTTTTTTTATTAGAGATTG-30 Region II 340 bpBS2F 50-TGTTGATGAAGTTATAGAATGA-30

BS2R 50-CCCCAAAATATACATTCAAAA-30

S. Ghai et al. / Research in Veterinary Science 92 (2012) 53–59 55

For amplification of region I.1 and II in bisulfite treated genomicDNA, 4 ll of treated DNA was amplified in a reaction mixture con-taining 0.4 lM primers (gene-specific forward and reverse prim-ers), 1� PCR buffer [10 mM Tris HCl (pH 9), 50 mM KCl, 1.5 mMMgCl2, 0.01% gelatin], 0.4 mM dNTP mix, 1 U Taq polymerase(1 U/ll) made to 25 ll with nuclease free water. The amplificationwas done in a thermocycler under two different cycle conditions.For amplification of region I.1, two-step PCR was performed asnested PCR. For the first amplification, 4 ll of treated DNA wasused as template, for the second amplification, 5 ll of PCR productfrom the first reaction was used. PCR conditions used were 94 �Cfor 5 min; 40 cycles at 94 �C for 30 s, 54.0 �C for 30 s, and 70 �Cfor 2 min; and a final extension at 70 �C for 5 min. In a secondPCR, nested primers were used with 5 ll of the outer PCR productas template. PCR conditions for second PCR were 94 �C for 5 min;39 cycles at 94 �C for 30 s, 55 �C for 30 s, and 70 �C for 90 s; anda final extension at 70 �C for 5 min.

The PCR conditions used for amplification of PII in bisulfitetreated genomic DNA were 94 �C for 5 min; 40 cycles at 94 �Cfor 30 s, 58 �C for 30 s, and 70 �C for 2 min; and a final extensionat 70 �C for 5 min. The PCR products were separated byelectrophoresis through a 1.5% agarose gel containing ethidiumbromide, extracted from the gel using Wizard� SV Gel and PCRClean-Up System, (Promega, USA) as per manufacturer’s instruc-tions and cloned into pGEMT-Easy vector. At least four clones ofeach sample were custom sequenced by an ABI model 3730sequencer using M13F and M13R primers (Bangalore Genei,India).

Fig. 1. Expression of Cyp19 (aromatase) gene mRNA in buffalo placental cotyledonsof normal and RFM affected animals (N = 3) by semi-quantitative RT-PCR. PCRreaction was carried out in 1.5 mM MgCl2 at 60 �C (annealing) for 32 cycles. (A)Lane 1, 100 bp DNA ladder; lane 2, normal placenta; lane 3, retained placenta. (B)GAPDH mRNA expression in respective tissues (C) Quantitative analysis of Cyp19gene mRNA in both the tissues. The data represent the mean value of three differentexperiments. Error Bars represent means ± SEM. NP, term placental cotyledons ofnormal animals; RP, term placental cotyledons of animals affected with RFM.

2.6. Statistical analysis

The relationship between degree of methylation and level ofexpression was evaluated using a linear correlation model. Thesequencing data were analyzed and methylation density ofeach tissue sample was calculated using BDPC (BisulfiteSequencing Data Presentation and Compilation) online software(available at: http://biochem.jacobs-university.de/BDPC/). ThemRNA levels of Cyp19 gene in each tissue was measured bymeasuring the intensity of the band using Image J software(available at: http://rsbweb.nih.gov/ij/). The statistical analysisof the mRNA levels of two tissues was done using one wayANOVA.

3. Results

3.1. Expression of cytochrome P450 aromatase (Cyp19) mRNA inbuffalo placental cotyledons using RT-PCR and real-time PCR

The relative expression of aromatase mRNA in cotyledons ofnormal and retained term placenta is shown in Fig. 1 The expres-sion was exhibited by both types of placenta (Fig. 1A). The nestedPCR experiment was performed to confirm the aromatase mRNAexpression in cotyledons of normal and retained term placenta.The results showed Cyp19 gene expression in cotyledons of bothnormal and retained term placenta (Fig. 1A). However, the expres-sion was detected in both the tissues only after second amplifica-tion (Fig. 2A). The comparative expression of aromatase mRNA(quantified by Image J software) is shown in Figs. 1C and 2C, where

Fig. 2. Expression of Cyp19 (aromatase) gene mRNA in buffalo placental cotyledonsby nested PCR (N = 3). PCR reaction was carried out in 1.5 mM MgCl2 at 60 �C(annealing) for 32 cycles. Lane 1, 100 bp DNA ladder. (A) lanes 2, 4, firstamplification; lanes 3, 5, second amplification in retained placenta and normalplacenta, respectively. (B) GAPDH mRNA expression in respective tissues. (C)Quantitative analysis of Cyp19 gene mRNA in different tissues. The data representthe mean value of three different experiments. Error Bars represent means ± SEM.NP, term placental cotyledons of normal animals; RP, term placental cotyledons ofanimals affected with RFM.

56 S. Ghai et al. / Research in Veterinary Science 92 (2012) 53–59

placental cotyledons of buffalo with RFM were shown to exhibitsignificantly lower Cyp19 gene expression than those of normalanimals when compared taking GAPDH as an internal control (Figs.1B and 2B).

Quantitative real-time PCR analysis further confirmed thechange in the expression profiles of Cyp19 gene in animals withnormal and retained term placenta. A 2.5-fold change in theexpression levels of the Cyp19 gene was observed in the two tis-sues studied with retained placenta possessing 2.5-fold lesserexpression than the normal term placental tissues (Fig. 3).

3.2. Distribution of CpG dinucleotides within regions I.1 and II of Cyp19gene

For DNA methylation analysis two different regions of Cyp19gene were included, regions I.1 including exon I.1 and PI.1, themain promoter in the placenta and region II including exon IIand PII, the most proximal and major granulosa cells promoter(Sharma et al., 2009). Both the regions lack a CpG island but insteadcontain 5 CpG dinucleotides each. The positions of CpG relative tothe major start sites of transcription are shown in Fig. 4. Of the 5CpG sites analyzed, four and three CpG residues of PI.1 (Fig. 4A)and PII (Fig. 4B) respectively, were upstream of the transcriptionstart site (TSS) and a single and two CpG residues of the respectivepromoters were downstream. The positions of the CpG dinucleo-tides were located on the basis of translation initiation site (TIS)to rule out any anomaly due to the presence of multiple transcrip-tion start sites in region II.

3.3. Efficiency of bisulfite treatment

The accuracy of quantitative DNA methylation analysis dependson the efficiency of the conversion of unmethylated C into U. In or-der to evaluate the efficiency of the bisulfite modification step, theextent of conversion of C that is not in the context of a CpG dinu-cleotide was examined. For this, 363 and 340 bp of each sequencethat included 79 and 71 C, respectively, were evaluated in all the

samples. All together, five non-converted C were found in six dif-ferent samples of each promoter, which means that almost 99.4%of all C had been chemically converted to U by bisulfite treatment.This demonstrates that the DNA modification procedure was veryefficient and should not produce considerable artifacts due toincomplete conversion.

3.4. Methylation profiles of individual CpG dinucleotides in buffaloplacenta

In placental cotyledons of normal and retained term placenta,both regions I.1 and II were almost unmethylated, as calculatedby the percentage methylation (10.73% and 8.35%, respectively,calculated from all five CpG dinucleotides of both the regions). Pla-cental cotyledons of normal animals showed methylation of regionI.1 (starting from 50 to 30 direction) viz., 0.00%, 0.00% 16.7%, 16.7%and 16.7%, whereas the corresponding mean values in animalswith RFM were 16.7%, 0.00%, 16.7%, 0.00% and 16.7%, respectively(Fig. 5A). Almost all the CpG residues of region I.1 were thereforehypomethylated in normal and retained term placental cotyledons,with no significant difference in methylation levels.

The corresponding values of region II were 0.00%, 0.00%, 28.6%,28.6% and 0.00% for normal term placenta and 0.00%, 16.7%, 0.00%,16.7%, 0.00% for retained placenta (Fig. 5B). PII, therefore was notsignificantly methylated in both the tissues. Further, no correlationbetween the methylation levels and expression status wasobserved in any of the tissues (Fig. 5C).

4. Discussion

The present study demonstrates significantly lower levels ofCyp19 gene expression (P < 0.05) in the placental cotyledons of ani-mals affected with RFM in comparison to normal animals. As theCyp19 gene encodes cytochrome P450 aromatase enzyme, catalyz-ing the rate limiting step of estrogen biosynthesis, the decreasedexpression of Cyp19 gene from normal to retained placenta indi-cates low estradiol-17b synthesis in placental cotyledons of RFManimals. These results are in agreement with the findings thatshowed comparatively lower levels of 17b-estradiol in animalswith RFM (Hashem and Hussein, 2009; Kornmatitsuk et al.,2000). It has been reported earlier that estrogens play an importantrole in the maturation process of the placentomes (Grunert et al.,1989). In line with the above reports it is thus hypothesized thatdown regulation of Cyp19 gene results in insufficient estradiol-17b production required for placental maturation leading to RFM.To the best of our knowledge, this is the first report of the expres-sion levels of Cyp19 gene in animals suffering from RFM.

After the finding that animals with RFM possess comparativelylower levels of Cyp19 mRNA, the probable mechanism behind thedown-regulation of the Cyp19 gene was of interest to us. Methyl-ation of the promoter region is one of the important mechanismsfor the down regulation of genes at the level of transcription. Inour previous studies, we found that Cyp19 gene regulation is underthe control of methylation in buffalo placenta (Ghai et al., 2010).Besides, several earlier reports of the importance of DNA methyla-tion for development and differentiation of placental cell typeshave been published (Serman et al., 2007, Vlahovic et al., 1999).Methylation analysis of individual CpG dinucleotides was thereforedone using the bisulfite direct sequencing method. This methodenables the strand specific methylation analysis of all CpG dinucle-otides within a given genomic region. Two strategies are usuallyapplied to analyze modified bisulfite-treated and PCR amplified re-gions of interest viz., direct sequencing of the PCR products andsequencing of the PCR products after cloning (Archey et al., 2002;Paul and Clark, 1996). Firstly, we went for direct sequencing of

Fig. 3. Relative quantification of Cyp19 (aromatase) gene mRNA in buffalo placental cotyledons of normal and RFM affected animals by quantitative real time-PCR (N = 3). PCRreaction was carried out in 1.5 mM MgCl2 at 57 �C (annealing) for 32 cycles. (A) Graphical representation of the amplification of Cyp19 gene mRNA and GAPDH mRNA. (B)Melt curve analysis of amplified product to verify the absence of non-specific amplification in both the tissues (C) Quantitative analysis of Cyp19 gene mRNA in both thetissues represented by fold change. The amplification is compared by assuming the amplification in NP as 1. A 2.5-fold change in expression was observed between the twotissues. The data represent the mean value of three different experiments. Error Bars represent means ± SEM. NP, term placental cotyledons of normal animals; RP, termplacental cotyledons of animals affected with RFM.

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PCR products however, after facing continuous failures in directsequencing, due to the presence of a number of heterozygouspeaks, we switched to the cloning of PCR products which is,although a tedious process, a more accurate approach for providingmethylation data.

In buffalo placenta, the major transcript of Cyp19 gene com-prises the 50 UTR of exon I.1 and thus was suggested as the majorplacental promoter (Sharma et al., 2009). Ovary specific promoter,promoter II is the most proximal promoter and is considered as thestrongest of all the Cyp19 gene promoters. We therefore, focusedon the analysis of the aforesaid two promoters only. The two pro-moters of Cyp19 gene analyzed do not possess any CpG island andare thus recognized as non-CpG island promoters (Brena et al.,2006). Because of the differences in methylation levels in differenttissues, the analyzed regions are defined as tissue-specific differen-tially methylated regions (T-DMRs) in cattle and sheep (Vanselowet al., 2008). Generally, it is believed that T-DMRs are involved intissue-specific regulation of transcription. It has been shown in bo-vine granulosa cells and luteal cells also that Cyp19 includes T-DMRs, which might play a role during development, differentia-tion, and luteinization of follicles (Vanselow et al., 2005).

Considering the number of biological replicates of the two tis-sues studied, the methylation status of 120 individual CpG of eachCyp19 gene promoter in buffalo placental cotyledons was deter-mined. No correlation between methylation levels of the promot-ers and expression status of the gene was observed during this

study. Within promoter I.1, all the CpG dinucleotides in both thenormal term placenta as well as retained placenta were found tobe unmethylated despite of the variation in the Cyp19 mRNA levels.The methylation levels of region I.1 were similar in both the tissuesand were much below the expected threshold value of 25% (Vanse-low et al., 2008). It is thus inferred that methylation of PI.1 is notresponsible for the down regulation of Cyp19 gene in animals suf-fering from RFM. In case of the proximal promoter, promoter II, themethylation data showed contrary results. It has been observedthat the placenta of animals affected with RFM, possessing lowconcentrations of Cyp19 mRNA, has the hypomethylated form ofthe PII (6.68%). Term placenta of normal animals possessing com-paratively higher levels of Cyp19 mRNA has however; compara-tively more methylated (11.44%) PII. The percentage ofmethylation was below the threshold value of 25% and thus mightnot be able to show any significant effect on the Cyp19 geneexpression in placental cotyledons of both the tissues. Promotermethylation which has been found to be responsible for decreasedexpression of Cyp19 gene in luteal cells (Vanselow et al., 2005) anddifferential promoter usage in sheep and bovine placenta (Vanse-low et al., 2008) thus does not seem to have any role in the declinein Cyp19 mRNA levels in RFM affected animals. A mechanism otherthan promoter methylation is therefore involved in the decreasedexpression of Cyp19 gene in animals suffering from RFM. An alter-native mechanism for the regulation of gene at the level of tran-scription is through various regulatory molecules. IL-6 is one

Fig. 4. DNA sequences of the buffalo Cyp19 (A) region 1.1, including P1.1 and exon1.1, and (B) region II, including PII, and exon II. Promoter sequences are separatedfrom the exon sequences on the basis of identified transcription start site (TSS). TheCpG dinucleotides studied are indicated in grey together with their respectivepositions in both the regions. The numbering of the CG dinucleotides is done on thebasis of their respective positions from the translation initiation site (TIS). SSindicates the splice site present in both the regions.

58 S. Ghai et al. / Research in Veterinary Science 92 (2012) 53–59

such factor that might be involved in the down regulation of Cyp19gene in RFM affected animals. Toda et al. (1995) found that bindingof nuclear factor interleukin-6 to a distal element increases pro-moter activity of Cyp19 gene. It has been reported that pre-partum

Fig. 5. Average methylation levels of individual CpG dinucleotides within (A) region II (Pcotyledons of two tissues. (C) The total percentage of methylation (sum of percentage mCyp19 gene in two tissues of placenta. NP, term placental cotyledons of normal animals

concentrations of IL-6 are higher in post-partum normal animalsthan in those with RFM, which have been found to possess compar-atively lower pre-partum levels of IL-6 (Ishikawa et al., 2004). IL-6has been reported to be up regulated by PGE-2a leading to theinduction of the Cyp19 gene expression (Zhang et al., 1988; Hinsonet al., 1996). PGE-2a has been found to activate the Cyp19 geneexpression by stimulating binding of GATA-4 to its proximal pro-moter (Cai et al., 2007). Recently we have found by in silico analysisthe presence of a GATA-4 binding site in Buffalo Cyp19 proximalpromoter (Sharma et al., 2009). The proximal promoter has beenfound to play an important role in the expression of Cyp19 geneduring pregnancy (Ghai et al., 2010), so, it is quite possible thatthe lower levels of IL-6 in RFM affected animals may lead to thedown regulation of Cyp19 gene expression either directly or byreducing the GATA-4 binding. This results in insufficient estrogenproduction leading to the formation of immature placentomeswhich are not expelled out naturally causing RFM. The present datagives an insight into the mechanism behind the RFM in dairy cattlebut further studies are still to be done to elucidate the exact molec-ular mechanism.

5. Conclusion

In conclusion, the data of the present study suggests that thebuffalo Cyp19 gene possess significant variation in expression llev-els with lower expression in RFM affected tissue and higherexpression in normal. In addition, methylation status of the pro-moters indicates that both the major promoters are hypomethylat-ed in the two tissues and a mechanism other than differentialmethylation is involved in the change in Cyp19 gene expression.This is the first report of Cyp19 gene expression and methylationstatus in animals with RFM and may prove useful for elucidatingthe molecular mechanisms behind the cause of RFM in dairyanimals.

II and exonII) and (B) region I.1 (PI.1 and exon I.1) of Cyp19 gene in buffalo placentalethylation of region I.1 and region II) were compared with the overall expression of; RP, term placental cotyledons of animals affected with RFM.

S. Ghai et al. / Research in Veterinary Science 92 (2012) 53–59 59

Acknowledgements

The authors are grateful to the Director, National DairyResearch Institute, Karnal for providing necessary facilities for thiswork. A special thanks to Dr. Jens Vanselow, FBN Dummerstorf,Germany for helpful discussion in calculating percentage of meth-ylation. This work was supported by Department of Biotechnology(DBT) project grant (to DS).

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