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Term placenta shows methylation independent down regulation of Cyp19 gene in animals with retained fetal membranes
Research in Veterinary Science 92 (2012) 53–59
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Term placenta shows methylation independent down regulation of Cyp19 gene in 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, India Livestock Production and Management Division, National Dairy Research Institute, Karnal-132001, Haryana, India c Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India b
a r t i c l e
i n f o
Article history: Received 6 March 2010 Accepted 14 October 2010
Keywords: Retention of fetal membranes Cyp19 gene Promoter methylation Estrogen Cotyledons
a b s t r a c t Retention of fetal membranes (RFM) is the major post-partum disorder in dairy cattle. Cyp19 gene encodes 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 comparative analysis of Cyp19 gene expression and its epigenetic regulation in placental cotyledons of animals with and without RFM. Significantly lower expression of Cyp19 gene was found in placental samples of RFM affected animals in comparison to normal animals. Methylation analysis of 5 CpG dinucleotides of placenta specific Cyp19 gene promoter I.1 and proximal promoter, PII showed hypo-methylation of both PI.1 and PII in term placenta of normal and diseased animals. In conclusion, a mechanism other than promoter methylation is responsible for decreased aromatase expression in placental cotyledons of animals suffering from RFM. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Post parturition reproductive diseases are a serious problem in dairy cattle. These diseases not only lower productivity and fertility but they also bring economic loss. It is therefore, important to detect 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 is retention of fetal membranes (RFM) (Stephen, 2008; El-Wishy, 2007). In cattle, fetal membranes are expelled within 6–12 h after calving. The failure to push out all or a part of the placenta from the uterus within 12 h of calving leads to RFM (Drillich et al., 2006). In dairy cattle, 4–11% of spontaneous calvings have been reported to result in RFM (Hashem and Hussein, 2009) which is comparatively higher in buffalo (Arthur et al., 1989; Laven and Peters, 1996; Ahmed et al., 2009). A wide variation (2.89–12.23%) has been reported in buffaloes with a maximum at the fifth parity (30%) and associated with malnutrition (Choudhury et al., 1993; Gupta et al., 1999). Surprisingly, 54% of Iraqi buffaloes studied developed RFM (Azawi et al., 2008) while the incidence of RFM in buffalo in India is 21% (Satya pal, 2003). A failure in the separation of cotyledonary villi from the crypts of the maternal caruncles results in RFM (Weithril, 1965). The etiology of this disorder however, is yet to be completely understood.
⇑ Corresponding author. Tel.: +91 184 2259135; fax: +91 184 2250042. E-mail addresses: [email protected], [email protected] (D. Singh). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.10.008
Complex metabolic disturbances during the pre-partum period have been proposed to be the probable reason of RFM (Michal et al., 2006). It has been suggested that a surge in estrogen concentration 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 is important in normal parturition and placental expulsion. It is generally recognized that estrogens play an important role in the maturation process of the placentomes (Grunert et al., 1989). Cows and buffaloes with RFM have been observed to possess higher levels of progesterone and lower levels of estradiol 17-b compared to normal animals (Matton et al., 1987; Thomas et al., 1992; Kankofer et al., 1996; Hashem and Hussein, 2009; Ali et al., 2009). Moreover, it has been shown that in cattle and goats, low levels of estrogens and the reduction or absence of the estrogen peak near delivery are associated with abortion, dystocia and placental retention (Engeland et al., 1999; Zhang et al., 1999a,b). Therefore, downregulation of estradiol is supposed to be an important factor responsible for RFM. The Cytochrome P450 aromatase enzyme encoded by the Cyp19 gene has been found to play an important role in converting progesterone to estrogen in the placenta (Flint et al., 1975; Mason et al., 1989; Nelson et al., 1996). A change in the estradiol levels in animals affected with RFM thus might be due to the change in expression levels of Cyp19 gene. However, there is no report of the expression status of Cyp19 gene in RFM- affected farm animals (cattle and buffalo). Cyp19 gene has been found to be down regulated by the promoter switching phenomenon during
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folliculogenesis in cattle and buffalo (Vanselow et al., 2005; Sharma et al., 2009). Methylation of the proximal promoter region is partly responsible for this promoter switching in ovary (Vanselow et al., 2005; Fürbass et al., 2008) and differential promoter usage in placenta (Vanselow et al., 2008). Recently, we found that DNA methylation is also involved in stage specific Cyp19 gene expression in buffalo placenta (Ghai et al., 2010). DNA methylation patterns are also found to change in response to environmental stimuli such as diet or toxins due to which the epigenome seems to be most susceptible during early in utero development. Aberrant DNA methylation changes have been detected in several diseases, particularly cancer where genome-wide hypo-methylation coincides with gene-specific hypermethylation (Tost, 2009). Therefore, the role of promoter methylation linked down regulation of the Cyp19 gene in animals with RFM cannot be ignored. The decrease in estradiol levels in placental cotyledons of animals affected with RFM may probably be due to the down regulation of Cyp19 gene which in turn might be regulated by promoter methylation. In view of the above points, the present study was focused on determining the Cyp19 gene expression in normal and RFM affected buffalo which is major milk yielding animal in India and contributes more than 60% of country’s milk production. Further studies were done to understand the relationship between Cyp19 gene expression and promoter methylation levels in buffaloes affected with RFM. 2. Materials and methods 2.1. Collection of placental tissues Placenta of the animals which expelled their fetal membranes naturally within 12 h of parturition was considered as normal placenta. The animals which did not expel their fetal membranes naturally but the placenta that was ejected manually after oxytocin injection was considered as retained placenta. Tissue samples (n = 3) were collected from NDRI cattle yard immediately after expulsion. The samples were washed with chilled normal saline containing antibiotics to remove contamination and blood and then brought to the laboratory as early as possible (10–15 min). The samples to be used for RNA isolation were immediately kept in RNAlaterÒ (Qiagen, GmbH, Germany) till further processing.
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 final volume of 20 ll. The contents were incubated at 25 °C for 10 min, 42 °C for 30 min and 95 °C for 3 min. The cDNA was amplified with gene specific primers (Table 1) in a 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 with nuclease free water. The amplification was done in a thermocycler under two different cycle conditions. For Cyp19 gene expression, two-step PCR was performed as nested PCR. For the first amplification, 2 ll of cDNA was used as template, for the second amplification, 5 ll of PCR product from the first reaction was used. PCR reactions were performed by incubating the contents at 94 °C for 2 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 GAPDH 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 for Cyp19 gene in normal and retained placental tissues. The reaction mixture containing 2 SYBRÒ Universal PCR Master Mix (Fermentas), 200 nM gene specific primers (CYPF and CYPR), Table 1 and 2 ll of cDNA to a final volume of 10 ll was incubated in the M J MiniCycler (BioRad). The reaction conditions used were 94 °C for 2 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. GAPDH was used as internal control. Cyp19 and GAPDH expressions were determined by measuring PCR product fluorescence compared to cycle number to determine CT values. Relative Cyp19 expression for each tissue sample was calculated using the formula:
DCT ¼ CTCyp19 CT GAPDH The fold change in Cyp19 gene expression (DCyp19) between normal and RFM animals was calculated with the formula
ðDCyp19Þ ¼ 2ðDCTNormal DCTRFM Þ
2.2. Separation of placental cotyledons and isolation of DNA and RNA
2.5. Bisulfite modification of DNA
The buffalo’s placental cotyledons were excised and removed with the help of sterilized scissors and forceps. The appropriate amount of tissue (100 mg) required for DNA and RNA isolation was chopped into pieces and homogenized into DNAzolÒ and TRIzolÒ (Life Technologies (India) Pvt. Ltd.), respectively. Total RNA was isolated by modified TRIzolÒ method (Chomczynski and Sacchi, 1987) and DNA was isolated using DNAzolÒ reagent following manufacturer’s instructions. The DNA and RNA were quantified spectrophotometrically and the respective integrities were evaluated by normal (0.7%) and denaturing (1.5%) agarose gel electrophoresis.
Genomic DNA (1 lg) isolated from placental cotyledons of animals with normal and retained fetal membrane was bisulfite-treated using the EZ DNA Methylation Gold Kit (Zymo Research, Orange, CA). The procedure is based on the method developed by Frommer et al. (1992). It is based on the principle that treatment of single stranded DNA with sodium bisulfite under acidic conditions changes all the cytosines in the DNA to uracil while 50 -methyl cytosines resist this change. Amplification of the bisulfite-treated DNA by PCR followed by sequencing reveals the positions of 5methylcytosine in the gene. Bisulfite-treated DNA was eluted in 10 ll volumes of elution buffer with 4 ll used for each PCR. The frequency of methylation at 5 individual CpG sites within the 363-bp and 340-bp region of respective PI.1, the distal promoter responsible for placenta specific Cyp19 gene expression and PII, the proximal promoter responsible for ovary specific Cyp19 gene expression was assessed using bisulfite-specific sequencing. Bisulfite sequencing PCR (BSP) primer pairs specific for modified DNAs were designed to contain no CpG sites. The Methyl Primer Express softwareÒ v1.0 (Applied Biosystems) was used to design primers specific for BSP (Table 2) and positions of the CpG residues to be analyzed were also located using the same.
2.3. Reverse transcription-polymerase chain reaction (RT-PCR) The cDNA was synthesized using a Fermentas First strand cDNA synthesis kit (Fermentas, Germany). The reaction mixture contained 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 incubated at 65 °C for 10 min followed by 2 min incubation at room temperature. The reagents added subsequently were: 4 ll of 5 reaction buffer (250 mM Tris–HCl, pH 8.3; 250 mM KCl, 20 mM MgCl2,
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S. Ghai et al. / Research in Veterinary Science 92 (2012) 53–59 Table 1 Primers used for gene specific RT-PCR and real-time PCR. Primer name P6F P7R P5F P8R CYPF CYPR G3F G3R
Primer sequence 0
0
5 -CATGGCAAGCTCTCCTTCTC-3 50 -GCAGGGACTGACCAAACTTC-30 50 -ACTGGAGGGGTGAAGAAACC-30 50 -TTGGCATGGATAGCACACAG-30 50 -CCTGTGCGGGAAAGTACATCAC-30 50 -TCTTCTCAACGCACCGACCTT-30 50 -AAACCCATCACCATCTTCCAG-30 50 -AGGGGCCATCCACAGTCTTCT-30
Accession no. (NCBI)
Size of amplification product
Z32741 Z32741
866 bp 302 bp
NM_174305.1
105 bp
M17701
361 bp
Table 2 Primers used for amplification of promoter regions in bisulfite treated genomic DNA. Primer name B1.1 F B1.1 R BI.1NF BI.1NR BS2F BS2R
Primer sequence 0
0
5 -GTGTAAAAGAGTTAAGAAAGGTGA-3 50 -AATCCTTTAAAAAACAATAACAAAA-30 50 -TCCTCACTATTCACTCCTCTCTAAA-30 50 -TAGTTAGTGGTTTTTTTATTAGAGATTG-30 50 -TGTTGATGAAGTTATAGAATGA-30 50 -CCCCAAAATATACATTCAAAA-30
For amplification of region I.1 and II in bisulfite treated genomic DNA, 4 ll of treated DNA was amplified in a reaction mixture containing 0.4 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], 0.4 mM dNTP mix, 1 U Taq polymerase (1 U/ll) made to 25 ll with nuclease free water. The amplification was done in a thermocycler under two different cycle conditions. For amplification of region I.1, two-step PCR was performed as nested PCR. For the first amplification, 4 ll of treated DNA was used as template, for the second amplification, 5 ll of PCR product from the first reaction was used. PCR conditions used were 94 °C for 5 min; 40 cycles at 94 °C for 30 s, 54.0 °C for 30 s, and 70 °C for 2 min; and a final extension at 70 °C for 5 min. In a second PCR, nested primers were used with 5 ll of the outer PCR product as 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; and a final extension at 70 °C for 5 min. The PCR conditions used for amplification of PII in bisulfite treated genomic DNA were 94 °C for 5 min; 40 cycles at 94 °C for 30 s, 58 °C for 30 s, and 70 °C for 2 min; and a final extension at 70 °C for 5 min. The PCR products were separated by electrophoresis through a 1.5% agarose gel containing ethidium bromide, extracted from the gel using WizardÒ SV Gel and PCR Clean-Up System, (Promega, USA) as per manufacturer’s instructions and cloned into pGEMT-Easy vector. At least four clones of each sample were custom sequenced by an ABI model 3730 sequencer using M13F and M13R primers (Bangalore Genei, India).
Region amplified
Size of amplification product
Region I.1 (outer)
628 bp 363 bp
Region I.1 (Inner) Region II
340 bp
3. Results 3.1. Expression of cytochrome P450 aromatase (Cyp19) mRNA in buffalo placental cotyledons using RT-PCR and real-time PCR The relative expression of aromatase mRNA in cotyledons of normal and retained term placenta is shown in Fig. 1 The expression was exhibited by both types of placenta (Fig. 1A). The nested PCR experiment was performed to confirm the aromatase mRNA expression in cotyledons of normal and retained term placenta. The results showed Cyp19 gene expression in cotyledons of both normal and retained term placenta (Fig. 1A). However, the expression was detected in both the tissues only after second amplification (Fig. 2A). The comparative expression of aromatase mRNA (quantified by Image J software) is shown in Figs. 1C and 2C, where
2.6. Statistical analysis The relationship between degree of methylation and level of expression was evaluated using a linear correlation model. The sequencing data were analyzed and methylation density of each tissue sample was calculated using BDPC (Bisulfite Sequencing Data Presentation and Compilation) online software (available at: http://biochem.jacobs-university.de/BDPC/). The mRNA levels of Cyp19 gene in each tissue was measured by measuring the intensity of the band using Image J software (available at: http://rsbweb.nih.gov/ij/). The statistical analysis of the mRNA levels of two tissues was done using one way ANOVA.
Fig. 1. Expression of Cyp19 (aromatase) gene mRNA in buffalo placental cotyledons of normal and RFM affected animals (N = 3) by semi-quantitative RT-PCR. PCR reaction 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 Cyp19 gene mRNA in both the tissues. The data represent the mean value of three different experiments. Error Bars represent means ± SEM. NP, term placental cotyledons of normal animals; RP, term placental cotyledons of animals affected with RFM.
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samples. All together, five non-converted C were found in six different 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 very efficient and should not produce considerable artifacts due to incomplete conversion. 3.4. Methylation profiles of individual CpG dinucleotides in buffalo placenta
Fig. 2. Expression of Cyp19 (aromatase) gene mRNA in buffalo placental cotyledons by 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, first amplification; lanes 3, 5, second amplification in retained placenta and normal placenta, respectively. (B) GAPDH mRNA expression in respective tissues. (C) Quantitative analysis of Cyp19 gene mRNA in different tissues. The data represent the mean value of three different experiments. Error Bars represent means ± SEM. NP, term placental cotyledons of normal animals; RP, term placental cotyledons of animals affected with RFM.
placental cotyledons of buffalo with RFM were shown to exhibit significantly lower Cyp19 gene expression than those of normal animals when compared taking GAPDH as an internal control (Figs. 1B and 2B). Quantitative real-time PCR analysis further confirmed the change in the expression profiles of Cyp19 gene in animals with normal and retained term placenta. A 2.5-fold change in the expression levels of the Cyp19 gene was observed in the two tissues studied with retained placenta possessing 2.5-fold lesser expression than the normal term placental tissues (Fig. 3). 3.2. Distribution of CpG dinucleotides within regions I.1 and II of Cyp19 gene For DNA methylation analysis two different regions of Cyp19 gene were included, regions I.1 including exon I.1 and PI.1, the main promoter in the placenta and region II including exon II and PII, the most proximal and major granulosa cells promoter (Sharma et al., 2009). Both the regions lack a CpG island but instead contain 5 CpG dinucleotides each. The positions of CpG relative to the major start sites of transcription are shown in Fig. 4. Of the 5 CpG sites analyzed, four and three CpG residues of PI.1 (Fig. 4A) and PII (Fig. 4B) respectively, were upstream of the transcription start site (TSS) and a single and two CpG residues of the respective promoters were downstream. The positions of the CpG dinucleotides were located on the basis of translation initiation site (TIS) to rule out any anomaly due to the presence of multiple transcription start sites in region II. 3.3. Efficiency of bisulfite treatment The accuracy of quantitative DNA methylation analysis depends on the efficiency of the conversion of unmethylated C into U. In order to evaluate the efficiency of the bisulfite modification step, the extent of conversion of C that is not in the context of a CpG dinucleotide was examined. For this, 363 and 340 bp of each sequence that included 79 and 71 C, respectively, were evaluated in all the
In placental cotyledons of normal and retained term placenta, both regions I.1 and II were almost unmethylated, as calculated by the percentage methylation (10.73% and 8.35%, respectively, calculated from all five CpG dinucleotides of both the regions). Placental cotyledons of normal animals showed methylation of region I.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 animals with 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 therefore hypomethylated 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 not significantly methylated in both the tissues. Further, no correlation between the methylation levels and expression status was observed in any of the tissues (Fig. 5C). 4. Discussion The present study demonstrates significantly lower levels of Cyp19 gene expression (P < 0.05) in the placental cotyledons of animals affected with RFM in comparison to normal animals. As the Cyp19 gene encodes cytochrome P450 aromatase enzyme, catalyzing the rate limiting step of estrogen biosynthesis, the decreased expression of Cyp19 gene from normal to retained placenta indicates low estradiol-17b synthesis in placental cotyledons of RFM animals. These results are in agreement with the findings that showed comparatively lower levels of 17b-estradiol in animals with RFM (Hashem and Hussein, 2009; Kornmatitsuk et al., 2000). It has been reported earlier that estrogens play an important role in the maturation process of the placentomes (Grunert et al., 1989). In line with the above reports it is thus hypothesized that down regulation of Cyp19 gene results in insufficient estradiol17b production required for placental maturation leading to RFM. To the best of our knowledge, this is the first report of the expression levels of Cyp19 gene in animals suffering from RFM. After the finding that animals with RFM possess comparatively lower levels of Cyp19 mRNA, the probable mechanism behind the down-regulation of the Cyp19 gene was of interest to us. Methylation of the promoter region is one of the important mechanisms for the down regulation of genes at the level of transcription. In our previous studies, we found that Cyp19 gene regulation is under the control of methylation in buffalo placenta (Ghai et al., 2010). Besides, several earlier reports of the importance of DNA methylation for development and differentiation of placental cell types have been published (Serman et al., 2007, Vlahovic et al., 1999). Methylation analysis of individual CpG dinucleotides was therefore done using the bisulfite direct sequencing method. This method enables the strand specific methylation analysis of all CpG dinucleotides within a given genomic region. Two strategies are usually applied to analyze modified bisulfite-treated and PCR amplified regions of interest viz., direct sequencing of the PCR products and sequencing of the PCR products after cloning (Archey et al., 2002; Paul and Clark, 1996). Firstly, we went for direct sequencing of
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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). PCR reaction 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 the tissues 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 two tissues. The data represent the mean value of three different experiments. Error Bars represent means ± SEM. NP, term placental cotyledons of normal animals; RP, term placental cotyledons of animals affected with RFM.
PCR products however, after facing continuous failures in direct sequencing, due to the presence of a number of heterozygous peaks, we switched to the cloning of PCR products which is, although a tedious process, a more accurate approach for providing methylation data. In buffalo placenta, the major transcript of Cyp19 gene comprises the 50 UTR of exon I.1 and thus was suggested as the major placental promoter (Sharma et al., 2009). Ovary specific promoter, promoter II is the most proximal promoter and is considered as the strongest of all the Cyp19 gene promoters. We therefore, focused on the analysis of the aforesaid two promoters only. The two promoters of Cyp19 gene analyzed do not possess any CpG island and are thus recognized as non-CpG island promoters (Brena et al., 2006). Because of the differences in methylation levels in different tissues, the analyzed regions are defined as tissue-specific differentially methylated regions (T-DMRs) in cattle and sheep (Vanselow et al., 2008). Generally, it is believed that T-DMRs are involved in tissue-specific regulation of transcription. It has been shown in bovine granulosa cells and luteal cells also that Cyp19 includes TDMRs, which might play a role during development, differentiation, and luteinization of follicles (Vanselow et al., 2005). Considering the number of biological replicates of the two tissues studied, the methylation status of 120 individual CpG of each Cyp19 gene promoter in buffalo placental cotyledons was determined. No correlation between methylation levels of the promoters and expression status of the gene was observed during this
study. Within promoter I.1, all the CpG dinucleotides in both the normal term placenta as well as retained placenta were found to be unmethylated despite of the variation in the Cyp19 mRNA levels. The methylation levels of region I.1 were similar in both the tissues and were much below the expected threshold value of 25% (Vanselow et al., 2008). It is thus inferred that methylation of PI.1 is not responsible for the down regulation of Cyp19 gene in animals suffering from RFM. In case of the proximal promoter, promoter II, the methylation data showed contrary results. It has been observed that the placenta of animals affected with RFM, possessing low concentrations of Cyp19 mRNA, has the hypomethylated form of the PII (6.68%). Term placenta of normal animals possessing comparatively higher levels of Cyp19 mRNA has however; comparatively more methylated (11.44%) PII. The percentage of methylation was below the threshold value of 25% and thus might not be able to show any significant effect on the Cyp19 gene expression in placental cotyledons of both the tissues. Promoter methylation which has been found to be responsible for decreased expression of Cyp19 gene in luteal cells (Vanselow et al., 2005) and differential promoter usage in sheep and bovine placenta (Vanselow et al., 2008) thus does not seem to have any role in the decline in Cyp19 mRNA levels in RFM affected animals. A mechanism other than promoter methylation is therefore involved in the decreased expression of Cyp19 gene in animals suffering from RFM. An alternative mechanism for the regulation of gene at the level of transcription is through various regulatory molecules. IL-6 is one
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concentrations of IL-6 are higher in post-partum normal animals than in those with RFM, which have been found to possess comparatively lower pre-partum levels of IL-6 (Ishikawa et al., 2004). IL-6 has been reported to be up regulated by PGE-2a leading to the induction of the Cyp19 gene expression (Zhang et al., 1988; Hinson et al., 1996). PGE-2a has been found to activate the Cyp19 gene expression by stimulating binding of GATA-4 to its proximal promoter (Cai et al., 2007). Recently we have found by in silico analysis the presence of a GATA-4 binding site in Buffalo Cyp19 proximal promoter (Sharma et al., 2009). The proximal promoter has been found to play an important role in the expression of Cyp19 gene during pregnancy (Ghai et al., 2010), so, it is quite possible that the lower levels of IL-6 in RFM affected animals may lead to the down regulation of Cyp19 gene expression either directly or by reducing the GATA-4 binding. This results in insufficient estrogen production leading to the formation of immature placentomes which are not expelled out naturally causing RFM. The present data gives an insight into the mechanism behind the RFM in dairy cattle but further studies are still to be done to elucidate the exact molecular mechanism.
5. Conclusion
Fig. 4. DNA sequences of the buffalo Cyp19 (A) region 1.1, including P1.1 and exon 1.1, and (B) region II, including PII, and exon II. Promoter sequences are separated from the exon sequences on the basis of identified transcription start site (TSS). The CpG dinucleotides studied are indicated in grey together with their respective positions in both the regions. The numbering of the CG dinucleotides is done on the basis of their respective positions from the translation initiation site (TIS). SS indicates the splice site present in both the regions.
such factor that might be involved in the down regulation of Cyp19 gene in RFM affected animals. Toda et al. (1995) found that binding of nuclear factor interleukin-6 to a distal element increases promoter activity of Cyp19 gene. It has been reported that pre-partum
In conclusion, the data of the present study suggests that the buffalo Cyp19 gene possess significant variation in expression llevels with lower expression in RFM affected tissue and higher expression in normal. In addition, methylation status of the promoters indicates that both the major promoters are hypomethylated in the two tissues and a mechanism other than differential methylation is involved in the change in Cyp19 gene expression. This is the first report of Cyp19 gene expression and methylation status in animals with RFM and may prove useful for elucidating the molecular mechanisms behind the cause of RFM in dairy animals.
Fig. 5. Average methylation levels of individual CpG dinucleotides within (A) region II (PII and exonII) and (B) region I.1 (PI.1 and exon I.1) of Cyp19 gene in buffalo placental cotyledons of two tissues. (C) The total percentage of methylation (sum of percentage methylation of region I.1 and region II) were compared with the overall expression of Cyp19 gene in two tissues of placenta. NP, term placental cotyledons of normal animals; RP, term placental cotyledons of animals affected with RFM.
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Acknowledgements The authors are grateful to the Director, National Dairy Research Institute, Karnal for providing necessary facilities for this work. A special thanks to Dr. Jens Vanselow, FBN Dummerstorf, Germany for helpful discussion in calculating percentage of methylation. This work was supported by Department of Biotechnology (DBT) project grant (to DS). References Ahmed, W.M., Hameed, A.R.A.E., Khadrawy, H.H.E., Hanafi, E.M., 2009. investigations of retained placenta in Egyptian buffaloes. Global Veterinaria 3, 120–124. Ali, M.A., Lodhi, L.A., Ahmad, I., Younas, M., 2009. Serum progesterone and estradiol17b profiles in nili ravi buffaloes (bubalus bubalis) with and without retention of fetal membranes. Pakistan Veterinary Journal 29, 64–66. Archey, W.B., McEachern, K.A., Robson, M., Offit, K., Vaziri, S.A., Casey, G., Borg, A., Arrick, B.A., 2002. Increased CpG methylation of the estrogen receptor gene in BRCA1-linked estrogen receptornegative breast cancers. Oncogene 21, 7034– 7041. Arthur, G.H., Noakes, D.E., Pearson, H., 1989. Veterinary Reproduction and Obstetrics, Sixth ed. Bailliere Tindall, London, UK. pp. 591–599. Azawi, O.I., Rahawy, M.A., Hadad, J.J., 2008. Bacterial isolates associated with dystocia and retained placenta in Iraqi buffaloes. Reproduction of Domestic Animals 43, 286–292. Brena, R.M., Huang, T.H., Plass, C., 2006. Toward a human epigenome. Nature Genetics 38, 1359–1360. Cai, Z., Kwintkiewicz, J., Young, M.E., Stocco, C., 2007. Prostaglandin E2 increases cyp19 expression in rat granulosa cells: implication of GATA-4. Molecular and Cellular Endocrinology 263, 181–189. Chomczynski, P., Sacchi, N., 1987. Single method of RNA isolation by acid guanidinium-thiocyanate–phenol–chloroform extraction. Analytical Biochemistry 162, 156–160. Choudhury, M.N., Bhattacharyya, B., Ahmed, S., 1993. Incidence, biochemical and histopathological profiles of retained placenta in cattle and buffalo. Environmental Ecology 11, 34–37. Drillich, M., Mahlstedt, M., Reichert, U., Tenhagen, B.A., Heuwieser, W., 2006. Strategies to improve the therapy of retained fetal membranes in dairy cows. Journal of Dairy Science 89, 627–635. El-Wishy, A.B., 2007. The postpartum buffalo: I. Endocrinological changes and uterine involution. Animal Reproduction Science 97, 201–215. Engeland, I.V., Ropstad, E., Kindahl, H., Andresen, O., Waldeland, H., Tverdal, A., 1999. Foetal loss in dairy goats: function of the adrenal glands, corpus luteum and the foetal-placental unit. Animal Reproduction Science 55, 205–222. Flint, A.P.F., Ricketts, A.P., Craig, V.A., 1975. The control of placental steroid synthesis at parturition in domestic animals. Animal Reproduction Science 2, 239–251. Frommer, M., MacDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L., Paul, C.L., 1992. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proceedings of the National academy of Sciences of the United States of America 89, 1827– 1831. Fürbass, R., Selimyan, R., Vanselow, J., 2008. DNA methylation and chromatin accessibility of the proximal Cyp19 promoter region 1.5/2 correlate with expression levels in sheep placentomes. Molecular Reproduction and Development 75, 1–7. Ghai, S., Monga R, Mohanty T.K., Chuahan M.S. and Singh D., 2010. Tissue-specific promoter methylation coincides with Cyp19 gene expression in buffalo (Bubalus bubalis) placenta of different stages of gestation. General and Comparative Endocrinology doi:10.1016/j.ygcen.2010.07.012. Grunert, E., Ahlers, D., Heuwieser, W., 1989. The role of endogenous estrogens in the maturation process of the bovine placenta. Theriogenology 31, 1081–1091. Gupta, A., Pandit, R.K., Jogi, S., Agarwal, R.G., 1999. Retention of placenta in relation to parity, season and sex of calf in Murrah buffaloes. Buffalo Bulletin 18, 5–7. Hashem, M.A., Hussein, A.A., 2009. Hormonal and biochemical anomalies in dairy cows affected by retained fetal membranes. Veterinary On-Line 2009. Hinson, R.M., Williams, J.A., Shacter, E., 1996. Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: possible role of cyclooxygenase 2. Proceedings of the National Academy of Sciences of the United States of America 93, 4885–4890. Hoffmann, B., Wagner, W.C., Gimenez, 1976. Free and conjugated steroids in maternal and fetal plasma in the cow near term. Biology of Reproduction 15, 126–133. Hunter, T., Fairclough, R.J., Peterson, A.J., Welch, R.A.S., 1977. Foetal and maternal hormonal changes preceding normal bovine parturition. Acta Endocrinologica 84, 653–662. Inaba, T., Oka, A., Koketsu, Y., Nakama, S., Imori, 1983. Progesterone and estrogen synthesis by the bovine placenta. Japanese Journal of Animal Reproduction 29, 88–93. Ishikawa, Y., Nakada, K., Hagiwara, K., Kirisawa, R., Iwai, H., Moriyoshi, M., Sawamukai, Y., 2004. Changes in interleukin-6 concentration in peripheral
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Ovine placental steroid 17a-hydroxylase/C-17, 20-lyase, aromatase and sulphatase in dexamethasone-induced and natural parturition. Journal of Endocrinology 122, 351–359. Matton, P., Adelakoun, V., Dufour, J., 1987. Corpus luteum activity and prostaglandin levels after parturition in cows with retained fetal membranes. Canadian Journal of Animal Science 67, 21–26. Michal, K., Edward, M., Hanna, M., 2006. Some hormonal and biochemical blood indices in cows with retained placenta and puerperal metritis. Bulletin Veterinary Institute of Pulawy 50, 89–92. Nelson, D.R., Koymans, L., Kamataki, T., Stegeman, J.J., Feyereisen, R., et al., 1996. P450 superfamily: update on new sequences, genemapping, accession numbers and nomenclature. Pharmacogenetics 6, 1–42. Paul, C.L., Clark, S.J., 1996. Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis. BioTechniques 21, 126–133. Peterson, A.J., Hunter, J.T., Welch, R.A.S., Fairclough, R.J., 1975. 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Vanselow, J., Pohland, R., Fürbass, R., 2005. Promoter 2 derived Cyp19 expression in bovine granulosa cells coincides with gene-specific DNA hypomethylation. Molecular and Cellular Endocrinology 233, 57–64. Vanselow, J., Selimyan, R., Fürbass, R., 2008. DNA methylation of placenta-specific Cyp19 promoters of cattle and sheep. Experimental and Clinical Endocrinology & Diabetes 116, 437–442. Veenhuizen, E.L., Erb, R.E., Gorski, J., 1960. Quantitative determination of free estrone, estradiol 17-Ect, and estradiol 17-ß in bovine fetal cotyledons. Journal of Dairy Science 43, 270–277. Vlahovic, M., Bulic-Jakus, F., Juric-Lekic, G., Fucic, A., Maric, S., Serman, D., 1999. Changes in the placenta and in the rat embryo caused by the demethylating agent 5-azacytidine. International Journal of Developmental Biology 43, 843– 846. Weithril, G.D., 1965. Retained placenta in the bovine – a brief review. Canadian Veterinary Journal 6, 290–294. 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Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Term placenta shows methylation independent down regulation of Cyp19 gene in 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, India Livestock Production and Management Division, National Dairy Research Institute, Karnal-132001, Haryana, India c Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India b
a r t i c l e
i n f o
Article history: Received 6 March 2010 Accepted 14 October 2010
Keywords: Retention of fetal membranes Cyp19 gene Promoter methylation Estrogen Cotyledons
a b s t r a c t Retention of fetal membranes (RFM) is the major post-partum disorder in dairy cattle. Cyp19 gene encodes 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 comparative analysis of Cyp19 gene expression and its epigenetic regulation in placental cotyledons of animals with and without RFM. Significantly lower expression of Cyp19 gene was found in placental samples of RFM affected animals in comparison to normal animals. Methylation analysis of 5 CpG dinucleotides of placenta specific Cyp19 gene promoter I.1 and proximal promoter, PII showed hypo-methylation of both PI.1 and PII in term placenta of normal and diseased animals. In conclusion, a mechanism other than promoter methylation is responsible for decreased aromatase expression in placental cotyledons of animals suffering from RFM. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Post parturition reproductive diseases are a serious problem in dairy cattle. These diseases not only lower productivity and fertility but they also bring economic loss. It is therefore, important to detect 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 is retention of fetal membranes (RFM) (Stephen, 2008; El-Wishy, 2007). In cattle, fetal membranes are expelled within 6–12 h after calving. The failure to push out all or a part of the placenta from the uterus within 12 h of calving leads to RFM (Drillich et al., 2006). In dairy cattle, 4–11% of spontaneous calvings have been reported to result in RFM (Hashem and Hussein, 2009) which is comparatively higher in buffalo (Arthur et al., 1989; Laven and Peters, 1996; Ahmed et al., 2009). A wide variation (2.89–12.23%) has been reported in buffaloes with a maximum at the fifth parity (30%) and associated with malnutrition (Choudhury et al., 1993; Gupta et al., 1999). Surprisingly, 54% of Iraqi buffaloes studied developed RFM (Azawi et al., 2008) while the incidence of RFM in buffalo in India is 21% (Satya pal, 2003). A failure in the separation of cotyledonary villi from the crypts of the maternal caruncles results in RFM (Weithril, 1965). The etiology of this disorder however, is yet to be completely understood.
⇑ Corresponding author. Tel.: +91 184 2259135; fax: +91 184 2250042. E-mail addresses: [email protected], [email protected] (D. Singh). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.10.008
Complex metabolic disturbances during the pre-partum period have been proposed to be the probable reason of RFM (Michal et al., 2006). It has been suggested that a surge in estrogen concentration 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 is important in normal parturition and placental expulsion. It is generally recognized that estrogens play an important role in the maturation process of the placentomes (Grunert et al., 1989). Cows and buffaloes with RFM have been observed to possess higher levels of progesterone and lower levels of estradiol 17-b compared to normal animals (Matton et al., 1987; Thomas et al., 1992; Kankofer et al., 1996; Hashem and Hussein, 2009; Ali et al., 2009). Moreover, it has been shown that in cattle and goats, low levels of estrogens and the reduction or absence of the estrogen peak near delivery are associated with abortion, dystocia and placental retention (Engeland et al., 1999; Zhang et al., 1999a,b). Therefore, downregulation of estradiol is supposed to be an important factor responsible for RFM. The Cytochrome P450 aromatase enzyme encoded by the Cyp19 gene has been found to play an important role in converting progesterone to estrogen in the placenta (Flint et al., 1975; Mason et al., 1989; Nelson et al., 1996). A change in the estradiol levels in animals affected with RFM thus might be due to the change in expression levels of Cyp19 gene. However, there is no report of the expression status of Cyp19 gene in RFM- affected farm animals (cattle and buffalo). Cyp19 gene has been found to be down regulated by the promoter switching phenomenon during
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folliculogenesis in cattle and buffalo (Vanselow et al., 2005; Sharma et al., 2009). Methylation of the proximal promoter region is partly responsible for this promoter switching in ovary (Vanselow et al., 2005; Fürbass et al., 2008) and differential promoter usage in placenta (Vanselow et al., 2008). Recently, we found that DNA methylation is also involved in stage specific Cyp19 gene expression in buffalo placenta (Ghai et al., 2010). DNA methylation patterns are also found to change in response to environmental stimuli such as diet or toxins due to which the epigenome seems to be most susceptible during early in utero development. Aberrant DNA methylation changes have been detected in several diseases, particularly cancer where genome-wide hypo-methylation coincides with gene-specific hypermethylation (Tost, 2009). Therefore, the role of promoter methylation linked down regulation of the Cyp19 gene in animals with RFM cannot be ignored. The decrease in estradiol levels in placental cotyledons of animals affected with RFM may probably be due to the down regulation of Cyp19 gene which in turn might be regulated by promoter methylation. In view of the above points, the present study was focused on determining the Cyp19 gene expression in normal and RFM affected buffalo which is major milk yielding animal in India and contributes more than 60% of country’s milk production. Further studies were done to understand the relationship between Cyp19 gene expression and promoter methylation levels in buffaloes affected with RFM. 2. Materials and methods 2.1. Collection of placental tissues Placenta of the animals which expelled their fetal membranes naturally within 12 h of parturition was considered as normal placenta. The animals which did not expel their fetal membranes naturally but the placenta that was ejected manually after oxytocin injection was considered as retained placenta. Tissue samples (n = 3) were collected from NDRI cattle yard immediately after expulsion. The samples were washed with chilled normal saline containing antibiotics to remove contamination and blood and then brought to the laboratory as early as possible (10–15 min). The samples to be used for RNA isolation were immediately kept in RNAlaterÒ (Qiagen, GmbH, Germany) till further processing.
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 final volume of 20 ll. The contents were incubated at 25 °C for 10 min, 42 °C for 30 min and 95 °C for 3 min. The cDNA was amplified with gene specific primers (Table 1) in a 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 with nuclease free water. The amplification was done in a thermocycler under two different cycle conditions. For Cyp19 gene expression, two-step PCR was performed as nested PCR. For the first amplification, 2 ll of cDNA was used as template, for the second amplification, 5 ll of PCR product from the first reaction was used. PCR reactions were performed by incubating the contents at 94 °C for 2 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 GAPDH 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 for Cyp19 gene in normal and retained placental tissues. The reaction mixture containing 2 SYBRÒ Universal PCR Master Mix (Fermentas), 200 nM gene specific primers (CYPF and CYPR), Table 1 and 2 ll of cDNA to a final volume of 10 ll was incubated in the M J MiniCycler (BioRad). The reaction conditions used were 94 °C for 2 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. GAPDH was used as internal control. Cyp19 and GAPDH expressions were determined by measuring PCR product fluorescence compared to cycle number to determine CT values. Relative Cyp19 expression for each tissue sample was calculated using the formula:
DCT ¼ CTCyp19 CT GAPDH The fold change in Cyp19 gene expression (DCyp19) between normal and RFM animals was calculated with the formula
ðDCyp19Þ ¼ 2ðDCTNormal DCTRFM Þ
2.2. Separation of placental cotyledons and isolation of DNA and RNA
2.5. Bisulfite modification of DNA
The buffalo’s placental cotyledons were excised and removed with the help of sterilized scissors and forceps. The appropriate amount of tissue (100 mg) required for DNA and RNA isolation was chopped into pieces and homogenized into DNAzolÒ and TRIzolÒ (Life Technologies (India) Pvt. Ltd.), respectively. Total RNA was isolated by modified TRIzolÒ method (Chomczynski and Sacchi, 1987) and DNA was isolated using DNAzolÒ reagent following manufacturer’s instructions. The DNA and RNA were quantified spectrophotometrically and the respective integrities were evaluated by normal (0.7%) and denaturing (1.5%) agarose gel electrophoresis.
Genomic DNA (1 lg) isolated from placental cotyledons of animals with normal and retained fetal membrane was bisulfite-treated using the EZ DNA Methylation Gold Kit (Zymo Research, Orange, CA). The procedure is based on the method developed by Frommer et al. (1992). It is based on the principle that treatment of single stranded DNA with sodium bisulfite under acidic conditions changes all the cytosines in the DNA to uracil while 50 -methyl cytosines resist this change. Amplification of the bisulfite-treated DNA by PCR followed by sequencing reveals the positions of 5methylcytosine in the gene. Bisulfite-treated DNA was eluted in 10 ll volumes of elution buffer with 4 ll used for each PCR. The frequency of methylation at 5 individual CpG sites within the 363-bp and 340-bp region of respective PI.1, the distal promoter responsible for placenta specific Cyp19 gene expression and PII, the proximal promoter responsible for ovary specific Cyp19 gene expression was assessed using bisulfite-specific sequencing. Bisulfite sequencing PCR (BSP) primer pairs specific for modified DNAs were designed to contain no CpG sites. The Methyl Primer Express softwareÒ v1.0 (Applied Biosystems) was used to design primers specific for BSP (Table 2) and positions of the CpG residues to be analyzed were also located using the same.
2.3. Reverse transcription-polymerase chain reaction (RT-PCR) The cDNA was synthesized using a Fermentas First strand cDNA synthesis kit (Fermentas, Germany). The reaction mixture contained 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 incubated at 65 °C for 10 min followed by 2 min incubation at room temperature. The reagents added subsequently were: 4 ll of 5 reaction buffer (250 mM Tris–HCl, pH 8.3; 250 mM KCl, 20 mM MgCl2,
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S. Ghai et al. / Research in Veterinary Science 92 (2012) 53–59 Table 1 Primers used for gene specific RT-PCR and real-time PCR. Primer name P6F P7R P5F P8R CYPF CYPR G3F G3R
Primer sequence 0
0
5 -CATGGCAAGCTCTCCTTCTC-3 50 -GCAGGGACTGACCAAACTTC-30 50 -ACTGGAGGGGTGAAGAAACC-30 50 -TTGGCATGGATAGCACACAG-30 50 -CCTGTGCGGGAAAGTACATCAC-30 50 -TCTTCTCAACGCACCGACCTT-30 50 -AAACCCATCACCATCTTCCAG-30 50 -AGGGGCCATCCACAGTCTTCT-30
Accession no. (NCBI)
Size of amplification product
Z32741 Z32741
866 bp 302 bp
NM_174305.1
105 bp
M17701
361 bp
Table 2 Primers used for amplification of promoter regions in bisulfite treated genomic DNA. Primer name B1.1 F B1.1 R BI.1NF BI.1NR BS2F BS2R
Primer sequence 0
0
5 -GTGTAAAAGAGTTAAGAAAGGTGA-3 50 -AATCCTTTAAAAAACAATAACAAAA-30 50 -TCCTCACTATTCACTCCTCTCTAAA-30 50 -TAGTTAGTGGTTTTTTTATTAGAGATTG-30 50 -TGTTGATGAAGTTATAGAATGA-30 50 -CCCCAAAATATACATTCAAAA-30
For amplification of region I.1 and II in bisulfite treated genomic DNA, 4 ll of treated DNA was amplified in a reaction mixture containing 0.4 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], 0.4 mM dNTP mix, 1 U Taq polymerase (1 U/ll) made to 25 ll with nuclease free water. The amplification was done in a thermocycler under two different cycle conditions. For amplification of region I.1, two-step PCR was performed as nested PCR. For the first amplification, 4 ll of treated DNA was used as template, for the second amplification, 5 ll of PCR product from the first reaction was used. PCR conditions used were 94 °C for 5 min; 40 cycles at 94 °C for 30 s, 54.0 °C for 30 s, and 70 °C for 2 min; and a final extension at 70 °C for 5 min. In a second PCR, nested primers were used with 5 ll of the outer PCR product as 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; and a final extension at 70 °C for 5 min. The PCR conditions used for amplification of PII in bisulfite treated genomic DNA were 94 °C for 5 min; 40 cycles at 94 °C for 30 s, 58 °C for 30 s, and 70 °C for 2 min; and a final extension at 70 °C for 5 min. The PCR products were separated by electrophoresis through a 1.5% agarose gel containing ethidium bromide, extracted from the gel using WizardÒ SV Gel and PCR Clean-Up System, (Promega, USA) as per manufacturer’s instructions and cloned into pGEMT-Easy vector. At least four clones of each sample were custom sequenced by an ABI model 3730 sequencer using M13F and M13R primers (Bangalore Genei, India).
Region amplified
Size of amplification product
Region I.1 (outer)
628 bp 363 bp
Region I.1 (Inner) Region II
340 bp
3. Results 3.1. Expression of cytochrome P450 aromatase (Cyp19) mRNA in buffalo placental cotyledons using RT-PCR and real-time PCR The relative expression of aromatase mRNA in cotyledons of normal and retained term placenta is shown in Fig. 1 The expression was exhibited by both types of placenta (Fig. 1A). The nested PCR experiment was performed to confirm the aromatase mRNA expression in cotyledons of normal and retained term placenta. The results showed Cyp19 gene expression in cotyledons of both normal and retained term placenta (Fig. 1A). However, the expression was detected in both the tissues only after second amplification (Fig. 2A). The comparative expression of aromatase mRNA (quantified by Image J software) is shown in Figs. 1C and 2C, where
2.6. Statistical analysis The relationship between degree of methylation and level of expression was evaluated using a linear correlation model. The sequencing data were analyzed and methylation density of each tissue sample was calculated using BDPC (Bisulfite Sequencing Data Presentation and Compilation) online software (available at: http://biochem.jacobs-university.de/BDPC/). The mRNA levels of Cyp19 gene in each tissue was measured by measuring the intensity of the band using Image J software (available at: http://rsbweb.nih.gov/ij/). The statistical analysis of the mRNA levels of two tissues was done using one way ANOVA.
Fig. 1. Expression of Cyp19 (aromatase) gene mRNA in buffalo placental cotyledons of normal and RFM affected animals (N = 3) by semi-quantitative RT-PCR. PCR reaction 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 Cyp19 gene mRNA in both the tissues. The data represent the mean value of three different experiments. Error Bars represent means ± SEM. NP, term placental cotyledons of normal animals; RP, term placental cotyledons of animals affected with RFM.
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samples. All together, five non-converted C were found in six different 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 very efficient and should not produce considerable artifacts due to incomplete conversion. 3.4. Methylation profiles of individual CpG dinucleotides in buffalo placenta
Fig. 2. Expression of Cyp19 (aromatase) gene mRNA in buffalo placental cotyledons by 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, first amplification; lanes 3, 5, second amplification in retained placenta and normal placenta, respectively. (B) GAPDH mRNA expression in respective tissues. (C) Quantitative analysis of Cyp19 gene mRNA in different tissues. The data represent the mean value of three different experiments. Error Bars represent means ± SEM. NP, term placental cotyledons of normal animals; RP, term placental cotyledons of animals affected with RFM.
placental cotyledons of buffalo with RFM were shown to exhibit significantly lower Cyp19 gene expression than those of normal animals when compared taking GAPDH as an internal control (Figs. 1B and 2B). Quantitative real-time PCR analysis further confirmed the change in the expression profiles of Cyp19 gene in animals with normal and retained term placenta. A 2.5-fold change in the expression levels of the Cyp19 gene was observed in the two tissues studied with retained placenta possessing 2.5-fold lesser expression than the normal term placental tissues (Fig. 3). 3.2. Distribution of CpG dinucleotides within regions I.1 and II of Cyp19 gene For DNA methylation analysis two different regions of Cyp19 gene were included, regions I.1 including exon I.1 and PI.1, the main promoter in the placenta and region II including exon II and PII, the most proximal and major granulosa cells promoter (Sharma et al., 2009). Both the regions lack a CpG island but instead contain 5 CpG dinucleotides each. The positions of CpG relative to the major start sites of transcription are shown in Fig. 4. Of the 5 CpG sites analyzed, four and three CpG residues of PI.1 (Fig. 4A) and PII (Fig. 4B) respectively, were upstream of the transcription start site (TSS) and a single and two CpG residues of the respective promoters were downstream. The positions of the CpG dinucleotides were located on the basis of translation initiation site (TIS) to rule out any anomaly due to the presence of multiple transcription start sites in region II. 3.3. Efficiency of bisulfite treatment The accuracy of quantitative DNA methylation analysis depends on the efficiency of the conversion of unmethylated C into U. In order to evaluate the efficiency of the bisulfite modification step, the extent of conversion of C that is not in the context of a CpG dinucleotide was examined. For this, 363 and 340 bp of each sequence that included 79 and 71 C, respectively, were evaluated in all the
In placental cotyledons of normal and retained term placenta, both regions I.1 and II were almost unmethylated, as calculated by the percentage methylation (10.73% and 8.35%, respectively, calculated from all five CpG dinucleotides of both the regions). Placental cotyledons of normal animals showed methylation of region I.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 animals with 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 therefore hypomethylated 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 not significantly methylated in both the tissues. Further, no correlation between the methylation levels and expression status was observed in any of the tissues (Fig. 5C). 4. Discussion The present study demonstrates significantly lower levels of Cyp19 gene expression (P < 0.05) in the placental cotyledons of animals affected with RFM in comparison to normal animals. As the Cyp19 gene encodes cytochrome P450 aromatase enzyme, catalyzing the rate limiting step of estrogen biosynthesis, the decreased expression of Cyp19 gene from normal to retained placenta indicates low estradiol-17b synthesis in placental cotyledons of RFM animals. These results are in agreement with the findings that showed comparatively lower levels of 17b-estradiol in animals with RFM (Hashem and Hussein, 2009; Kornmatitsuk et al., 2000). It has been reported earlier that estrogens play an important role in the maturation process of the placentomes (Grunert et al., 1989). In line with the above reports it is thus hypothesized that down regulation of Cyp19 gene results in insufficient estradiol17b production required for placental maturation leading to RFM. To the best of our knowledge, this is the first report of the expression levels of Cyp19 gene in animals suffering from RFM. After the finding that animals with RFM possess comparatively lower levels of Cyp19 mRNA, the probable mechanism behind the down-regulation of the Cyp19 gene was of interest to us. Methylation of the promoter region is one of the important mechanisms for the down regulation of genes at the level of transcription. In our previous studies, we found that Cyp19 gene regulation is under the control of methylation in buffalo placenta (Ghai et al., 2010). Besides, several earlier reports of the importance of DNA methylation for development and differentiation of placental cell types have been published (Serman et al., 2007, Vlahovic et al., 1999). Methylation analysis of individual CpG dinucleotides was therefore done using the bisulfite direct sequencing method. This method enables the strand specific methylation analysis of all CpG dinucleotides within a given genomic region. Two strategies are usually applied to analyze modified bisulfite-treated and PCR amplified regions of interest viz., direct sequencing of the PCR products and sequencing of the PCR products after cloning (Archey et al., 2002; Paul and Clark, 1996). Firstly, we went for direct sequencing of
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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). PCR reaction 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 the tissues 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 two tissues. The data represent the mean value of three different experiments. Error Bars represent means ± SEM. NP, term placental cotyledons of normal animals; RP, term placental cotyledons of animals affected with RFM.
PCR products however, after facing continuous failures in direct sequencing, due to the presence of a number of heterozygous peaks, we switched to the cloning of PCR products which is, although a tedious process, a more accurate approach for providing methylation data. In buffalo placenta, the major transcript of Cyp19 gene comprises the 50 UTR of exon I.1 and thus was suggested as the major placental promoter (Sharma et al., 2009). Ovary specific promoter, promoter II is the most proximal promoter and is considered as the strongest of all the Cyp19 gene promoters. We therefore, focused on the analysis of the aforesaid two promoters only. The two promoters of Cyp19 gene analyzed do not possess any CpG island and are thus recognized as non-CpG island promoters (Brena et al., 2006). Because of the differences in methylation levels in different tissues, the analyzed regions are defined as tissue-specific differentially methylated regions (T-DMRs) in cattle and sheep (Vanselow et al., 2008). Generally, it is believed that T-DMRs are involved in tissue-specific regulation of transcription. It has been shown in bovine granulosa cells and luteal cells also that Cyp19 includes TDMRs, which might play a role during development, differentiation, and luteinization of follicles (Vanselow et al., 2005). Considering the number of biological replicates of the two tissues studied, the methylation status of 120 individual CpG of each Cyp19 gene promoter in buffalo placental cotyledons was determined. No correlation between methylation levels of the promoters and expression status of the gene was observed during this
study. Within promoter I.1, all the CpG dinucleotides in both the normal term placenta as well as retained placenta were found to be unmethylated despite of the variation in the Cyp19 mRNA levels. The methylation levels of region I.1 were similar in both the tissues and were much below the expected threshold value of 25% (Vanselow et al., 2008). It is thus inferred that methylation of PI.1 is not responsible for the down regulation of Cyp19 gene in animals suffering from RFM. In case of the proximal promoter, promoter II, the methylation data showed contrary results. It has been observed that the placenta of animals affected with RFM, possessing low concentrations of Cyp19 mRNA, has the hypomethylated form of the PII (6.68%). Term placenta of normal animals possessing comparatively higher levels of Cyp19 mRNA has however; comparatively more methylated (11.44%) PII. The percentage of methylation was below the threshold value of 25% and thus might not be able to show any significant effect on the Cyp19 gene expression in placental cotyledons of both the tissues. Promoter methylation which has been found to be responsible for decreased expression of Cyp19 gene in luteal cells (Vanselow et al., 2005) and differential promoter usage in sheep and bovine placenta (Vanselow et al., 2008) thus does not seem to have any role in the decline in Cyp19 mRNA levels in RFM affected animals. A mechanism other than promoter methylation is therefore involved in the decreased expression of Cyp19 gene in animals suffering from RFM. An alternative mechanism for the regulation of gene at the level of transcription is through various regulatory molecules. IL-6 is one
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concentrations of IL-6 are higher in post-partum normal animals than in those with RFM, which have been found to possess comparatively lower pre-partum levels of IL-6 (Ishikawa et al., 2004). IL-6 has been reported to be up regulated by PGE-2a leading to the induction of the Cyp19 gene expression (Zhang et al., 1988; Hinson et al., 1996). PGE-2a has been found to activate the Cyp19 gene expression by stimulating binding of GATA-4 to its proximal promoter (Cai et al., 2007). Recently we have found by in silico analysis the presence of a GATA-4 binding site in Buffalo Cyp19 proximal promoter (Sharma et al., 2009). The proximal promoter has been found to play an important role in the expression of Cyp19 gene during pregnancy (Ghai et al., 2010), so, it is quite possible that the lower levels of IL-6 in RFM affected animals may lead to the down regulation of Cyp19 gene expression either directly or by reducing the GATA-4 binding. This results in insufficient estrogen production leading to the formation of immature placentomes which are not expelled out naturally causing RFM. The present data gives an insight into the mechanism behind the RFM in dairy cattle but further studies are still to be done to elucidate the exact molecular mechanism.
5. Conclusion
Fig. 4. DNA sequences of the buffalo Cyp19 (A) region 1.1, including P1.1 and exon 1.1, and (B) region II, including PII, and exon II. Promoter sequences are separated from the exon sequences on the basis of identified transcription start site (TSS). The CpG dinucleotides studied are indicated in grey together with their respective positions in both the regions. The numbering of the CG dinucleotides is done on the basis of their respective positions from the translation initiation site (TIS). SS indicates the splice site present in both the regions.
such factor that might be involved in the down regulation of Cyp19 gene in RFM affected animals. Toda et al. (1995) found that binding of nuclear factor interleukin-6 to a distal element increases promoter activity of Cyp19 gene. It has been reported that pre-partum
In conclusion, the data of the present study suggests that the buffalo Cyp19 gene possess significant variation in expression llevels with lower expression in RFM affected tissue and higher expression in normal. In addition, methylation status of the promoters indicates that both the major promoters are hypomethylated in the two tissues and a mechanism other than differential methylation is involved in the change in Cyp19 gene expression. This is the first report of Cyp19 gene expression and methylation status in animals with RFM and may prove useful for elucidating the molecular mechanisms behind the cause of RFM in dairy animals.
Fig. 5. Average methylation levels of individual CpG dinucleotides within (A) region II (PII and exonII) and (B) region I.1 (PI.1 and exon I.1) of Cyp19 gene in buffalo placental cotyledons of two tissues. (C) The total percentage of methylation (sum of percentage methylation of region I.1 and region II) were compared with the overall expression of Cyp19 gene in two tissues of placenta. NP, term placental cotyledons of normal animals; RP, term placental cotyledons of animals affected with RFM.
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