Introduction
Growth differentiation factor 9 (GDF9) is an oocyte-specific paracrine factor which is expressed throughout most stages of folliculogenesis and even persists after fertilization (Pennetier et al., Reference Pennetier, Uzbekova, Perreau, Papollier, Mermillod and Dalbies-Tran2004; Knight & Glister, Reference Knight and Glister2006; Kristensen et al., Reference Kristensen, Andersen, Clement, Franks, Hardy and Andersen2014). It plays an important role in the communication process between oocytes and its surrounding cumulus cells which is important for acquisition of oocyte’s development competence and subsequent embryogenesis (Matzuk et al., Reference Matzuk, Burns, Vivieros and Eppig2002; Qinglei et al., Reference Qinglei, McKenzie and Matzuk2008; Gupta et al., Reference Gupta, Pandey, Parmar, Somal, Paul, Panda, Bhat, Baiju, Bharti, Saikumar, Sarkar, Chandra and Sharma2017). It also plays key regulatory roles in cumulus cell functions such as differentiation, metabolism, expansion and gene expression, which in turn are necessary for oocyte survival and successful ovulation (Eppig, Reference Eppig2001; Gilchrist et al., Reference Gilchrist, Ritter and Armstrong2004; Bhardwaj et al., Reference Bhardwaj, Ansari, Pandey, Parmar, Chandra, Kumar and Sharma2016). It has been indicated that oocytes matured in vitro have compromised development competence possibly due to disrupted oocyte–cumulus communication resulting from inappropriate expression levels of oocyte-secreted factors such as GDF9 (Yeo et al., Reference Yeo, Gilchrist, Thompson and Lane2008). In an in vitro fertilization (IVF) procedure, in vitro maturation (IVM) of oocytes represents the most critical step because events during IVM have been demonstrated to affect not only the process of fertilization but also the subsequent stages of early cleavage divisions, blastocyst formation and successful implantation which are highly dependent on the fine tuning of genes expressed during IVM (De Sousa et al., Reference De sousa, Westhusin and Watson1998; Watson et al., Reference Watson, De Sousa, Caveney, Barcroft, Natale, Urquhart and Westhusin2000; Knijn et al., Reference Knijin, Wrenzycki, Hendriksen, Vos, Hermann, Vander, Niemann and Dielemen2002; Jones et al., Reference Jones, Cram, Song, Magli, Gianaroli, Kaplan, Findlay, Jenkin and Trounson2008). It is also reported that persistent alterations of the normal gene expression patterns take place under a given in vitro condition which may be responsible for poor development fate of IVF-produced embryos (Knijin et al., Reference Knijin, Wrenzycki, Hendriksen, Vos, Hermann, Vander, Niemann and Dielemen2002; Warzych et al., Reference Warzych, Wrenzycki, Peippo and Leichniak2007; Jones et al., Reference Jones, Cram, Song, Magli, Gianaroli, Kaplan, Findlay, Jenkin and Trounson2008). Thus, a better understanding of the molecular mechanisms regulating oocyte maturation is necessary.
In spite of the important role of GDF9 in oocyte maturation events, however, only a few sporadic information is available on its exact transcription control mechanism (Lan et al., Reference Lan, Gu, Xu, Jackson, DeMayo, O’Malley and Cooney2003; Rajkovic et al., Reference Rajkovic, Pangas, Ballow, Suzumori and Matzuk2004; Choi & Rajkovic, Reference Choi and Rajkovic2006; Yan et al., Reference Yan, Elvin, Lin, Hadsell, Wang, DeMayo and Matzuk2006; Choi et al., Reference Choi, Ballow, Xin and Rajkovic2008; Roy et al., Reference Roy, Rajput, Raghav, Kumar, Verma, Kumar, De, Goswami and Datta2012, Reference Roy, Rajput, Raghav, Kumar, Verma, Jain, Jain, Singh, De, Goswami and Datta2013). Earlier work from our laboratory reported the predicted transcription factor binding sites in putative buffalo GDF9 promoter (Roy et al., Reference Roy, Rajput, Raghav, Kumar, Verma, Jain, Jain, Singh, De, Goswami and Datta2013). In silico analysis of buffalo, GDF9 promoter region revealed presence of oestrogen response elements, DR0 element along with consensus E boxes. Based on the previous information generated in our lab and literature available, we hypothesized that nuclear receptor (NR) superfamily transcription factors (TFs) i.e., oestrogen receptor 2 (ESR2) and germ cell nuclear factor (GCNF), and basic helix-loop-helix (bHLH) TFs viz. factor in germ line alpha (FIGLA), upstream stimulatory factor 1 and 2 (USF1 and USF2) and twist-related protein 1 (TWIST1) might be associated with GDF9 gene regulation in oocytes. In the present study, we intended to generate some hint on possible relationship between the expression pattern of GDF9 in buffalo oocytes vis-à-vis the selected NR and bHLH transcription factors during in vitro maturation of oocytes. Thus, the present work was intended to generate clues that can help in more physiological manipulation of buffalo oocytes towards better IVF/ assisted reproductive technology success.
Materials and methods
All media and chemicals were obtained from Sigma Aldrich Chemical Co. Ltd, St. Louis, MO, USA unless otherwise indicated. Disposable plastic wares used were from Falcon NJ, USA and Nunc, Denmark. Foetal bovine serum was from Hyclone, Canada.
Oocyte maturation and production of buffalo embryos
Since ovaries were collected from buffaloes that were routinely slaughtered, no ethical approval was required. Buffalo ovaries were collected at an abattoir, regardless of the oestrous cycle and transported within 3–4 h to the laboratory in phosphate buffer saline (PBS) containing strepto-penicillin (0.05 mg/ml). Cumulus–oocyte complexes (COCs) were aspirated from visible ovarian follicles (3–8 mm in size) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered hamster embryo culture (HH) medium (Keefer et al., Reference Keefer, Stice and Matihews1994). COCs were picked up under stereo zoom microscope and washed three times in HH medium. Only excellent-grade oocytes with more than five compact layers of cumulus cells and homogenous cytoplasm were used for IVM and IVF (Jain et al., Reference Jain, Jain, Kumar, Goswami, De, Singh and Datta2012). A cluster of 25 COCs was put for in vitro maturation (IVM) in 4 different supplementation medium groups viz. 1) All supplementation group (Control): TCM-199 with 0.005% streptomycin, 0.01% sodium pyruvate and 0.005% glutamine, 64 µg/ml cysteamine and 50 µl ITS as base medium and supplemented with 10% foetal bovine serum (FBS), 5.0 µg/ml porcine follicle stimulating hormone (pFSH), 10 µg/ml luteinizing hormone (LH), 1µg/ml oestradiol 17-β (E2) and 50 ng/ml epidermal growth factor; 2) FSH group: TCM-199 base medium containing PVA (1 mg/ml) and 5.0 µg/ml pFSH; 3) IGF1 group: same as media 2 where FSH was replaced with 100 ng/ml of IGF1 and 4) oestradiol (E2) group: same as media 2 where FSH was replaced with 1µg/ml of E2. For IVM, oocytes were placed in drops of 100 µl maturation medium and overlaid with mineral oil and cultured for 24 h at 38.5°C in an atmosphere of 5% CO2 in air.
In-vitro fertilization (IVF) was done in 100 μl droplets of Brackett and Oliphant (BO) medium (Brackett and Oliphant, Reference Brackett and Oliphant1975) supplemented with 1% bovine serum albumin (BSA; fatty acid free), 1.9 mg/ml caffeine sodium benzoate, 0.14 mg/ml sodium pyruvate and 0.01 mg/ml heparin. Matured COCs were washed thrice in BO medium and then transferred into fertilization drops. The frozen-thawed buffalo semen was processed for in vitro capacitation as described earlier (Chauhan et al., Reference Chauhan, Singla, Palta, Manik and Madan1998). Briefly, two frozen semen straws were thawed and washed twice with BO medium (without 1% fatty acid-free BSA). The sperm pellet was re-suspended in BO medium and 50 μl of the sperm suspension (at final concentration of 1X106 /ml) was added to each fertilization drops having 15 -20 COCs and incubated at 38.5°C with 5% CO2 for 14 h. Sperm counting was done using haemocytometer. Presumptive zygotes were removed from the fertilization drops after 14 h of insemination (HPI), and adhered cumulus cells were mechanically removed by vortexing and washed five times in mCR2aa medium (Kumar et al., Reference Kumar, Palta, Manik, Singla and Chauhan2007). After washing, 15–20 presumptive zygotes were co-cultured with monolayers of granulosa cells in 100 µl drops of IVC-I medium (mCR2aa supplemented with 0.8% BSA, 1 mM glucose, 0.33 mM Na pyruvate, 1 mM glutamine, 1x MEM essential amino acid, 1x non-essential amino acid and 50 µg/ml gentamycin. After 48 h of insemination (HPI) zygotes were evaluated for evidence of cleavage. At 72 HPI all cleaved embryos were transferred to IVC-II medium (same as IVC-I except BSA replaced with 10% FBS) and maintained for 8 days at 5% CO2 and 38.5ºC with replacement of medium after every 48 h. Representative batches of oocyte samples were collected at 8 h of IVM for gene expression studies.
Observations made on developing oocytes and embryos
All IVM and IVF experiments were repeated at least four times. The nuclear maturation status was determined after 24 h of IVM by denuding oocytes and staining with Hoechst 33342 using protocol described before (Smith, Reference Smith1993) with slight modification. Briefly, denuded oocytes from all experimental groups were fixed in 4% paraformaldehyde solution (in PBS, pH 7.4) for 1 h at room temperature. After fixing and washing, groups of 50 oocytes were transferred to 200 µl drop of 10 µg/ml Hoechst 33342 dye solution for 20 min under dark condition. Stained oocytes were washed three times in PBS-PVP solution and placed on glass slides and mounted with Pro-Long mounting medium (Invitrogen, USA) and observed under the fluorescent microscope with UV filter (Olympus, Japan), and counted manually. Oocytes nuclei revealing two blue dots were considered as matured (M-II) oocytes. M-II % was calculated for each IVM groups for at least 250 oocytes. Cleavage and blastocyst stages of the embryos were recorded on day 2 and day 7 post insemination, respectively, in all four experimental groups. Cleavage and blastocyst rates were counted from the number of immature oocytes used for IVM.
RNA Isolation and cDNA preparation
Total RNA was extracted using the RNAqueous Micro Kit (Ambion) from a fixed number of 10 oocytes as per manufacturer’s instructions. Briefly, ten COCs were taken out from maturation drops after 8 h of culture and washed with chilled 1 × PBS. Washed COCs were then denuded and taken in RNase-free Eppendorf tube with 50 μl of lysis buffer (RNAqueous, Ambion). Total RNA was eluted in 12 µl of elution buffer and treated with RNase-free DNase I (Ambion). Quality of the total RNA was checked by Microvolume spectrophotometer (NanoDrop). Ratio of A260/A280 in an individual RNA sample above 1.9 was considered good quality and was taken for cDNA synthesis. Total RNA from each sample was reverse-transcribed using Reverse Transcription (RT) System Revert-Aid (Fermentas, USA) following the manufacturer’s instructions. Briefly, the cDNA was synthesized using oligo-dT primers and H Minus M-MuLV reverse transcriptase in a final volume of 20 µl. After termination of cDNA synthesis, each RT reactions were diluted with nuclease-free water (Ambion, USA) to a final volume of 80 µl and resultant cDNA was stored at −80oC till further use.
Quantitative real-time RT-PCR
Quantification of GDF9, GCNF, ESR2, FIGLA, TWIST1, USF1 and USF2 genes transcript was done by quantitative real-time PCR (qPCR) using Maxima SYBR Green qPCR Master Mix (Fermentas, USA). Primers used for amplification were designed using the Beacon Designer 7.0 (Premier Biosoft, International; Table 1). RSP18 was used as an internal calibrator. A working primer concentration of 10 pmol was used to set a primer matrix experiment to optimize the primer concentrations for valid transcript quantification (Bettegowda et al., Reference Bettegowda, Patel, Lee, Park, Salem, Yao, Ireland and Smith2008). qPCR reaction mixtures were consisted of 10 µl of SYBR Green qPCR mix, 2 µl of cDNA template, optimized primer quantities and nuclease-free water to make the total reaction volume of 20 µl. Reactions were performed in duplicate for each sample using Mx3005P Real Time PCR System (Stratagene). PCR conditions used were 95ºC for 10 min, then 40 cycles consisting of denaturation at 95ºC for 30 s, annealing at 57ºC for 20 s and extension at 72ºC for 30 s. No template control reactions were carried out to negate PCR contamination and dissociation curve analysis was performed to confirm authenticity of amplified products. Mean sample threshold cycle values (CT) for genes under study were calculated for duplicate samples and relative transcript abundance for target gene expression was calculated using the formula 2-(ΔΔC T ) as described by Livak and Schmittgen (Reference Livak and Schmittgen2001). Relative transcript abundance values for genes under study were interpreted considering the corresponding CT value of all supplement medium group as calibrator.
Table 1. Primers used for real-time PCR amplification of genes under study
Statistical analysis
Data for relative abundance values for genes under study were analyzed using SYSTAT version 12 software package. Differences of means were analyzed using one-way ANOVA followed by Duncan’s multiple range test. Significance of differences between means was calculated at 5% level of significance (P < 0.05). Correlation values were calculated using Pearson product-moment correlation analysis. Significance of correlation values was calculated at 5% (P < 0.05) and 1% levels (P < 0.01).
Result
Development rate of buffalo oocytes under different IVM media supplements
Development rate of oocytes under different IVM media supplements was assessed in terms of maturation (M-II) %, cleavage rate and blastocyst development rates. Results are described in Figure 1. A significantly higher number of oocytes attained M-II in all supplement and FSH IVM groups as compared to the IGF1 group with least percentage in E2 group. For cleavage and blastocyst rates, the trend was similar to that of M-II. Representative figures of matured oocytes and respective cleavage stage embryos are depicted in Figure 2.
Figure 1. Effect of different in vitro maturation groups on (A) Maturation, (B) Cleavage and (C) Blastocyst rates. Maturation rate was assessed after Hoechst stain. Cleavage rate was counted after 48 hr post insemination (pi). Blastocyst rate was counted on day 7 of pi.
Figure 2. Oocytes and different cell stage embryos. A (A- grade COCs), B (Expanded oocytes), C (2-cell embryos), D (4-cell embryos), E (8-cell embryos) and F (Expanded blastocysts).
Effect of different IVM groups on GDF9 and transcription factor expression in oocyte
In the present study, we decided to assay the gene expression status of GDF9 and associated NR and bHLH transcription factors at 8 h of IVM with consideration that transcriptional activity in germinal vesicle intact oocytes remains very active which subsequently diminishes on progression towards M-II stage and our earlier study established that the GDF9 mRNA abundance values are most predictive of their development competence at 8 h. Results are described in Figure 3. The trend of GDF9 expression in different media groups was found to be in the same order as that of their development rate data with the highest level found in all supplement group followed by the FSH, IGF1 and the least in the E2 group. Expression of two NR TFs viz. ESR2 and GCNF were similar to the extent that in both cases the expression was significantly higher in E2 group as compared to the control (all supplement) group which yielded maximum GDF9 expression as well as the highest blastocyst rate (Figure 3). Similar trend of their expression was also observed in 2 of the 4 bHLH TFs (viz. USF2 and TWIST1) studied in the present work. For either of these 2 genes, the expression was highest in E2 group which eventually yielded lowest GDF9 expression as well as least blastocyst rate. On the other hand, the expression of the USF1 was very similar to that of the GDF9.
Figure 3. Expression pattern of A-GDF9, B- GCNF, C- ESR2, D- TWIST1, E- FIGLA, F- USF1, G-USF2. in buffalo oocytes harvested at 8 h of maturation in different IVM groups. Relative mRNA abundance values are indicated as ± SEM. Bars with different letters indicate significant differences between different in vitro maturation groups (P < 0.05).
Correlation of transcription factors with GDF9 expression
Correlation analysis of the expression levels of respective genes under study across different IVM media groups with GDF9 revealed the significant influence of USF1 as positive regulator and GCNF, TWIST1, and ESR2 as potential negative regulators of GDF9 expression (Figure 4). Further, the TFs revealing significant association with GDF9 expression were studied for correlation amongst themselves (Table 2). Factors, TWIST1 & ESR2 revealing negative correlation with GDF9 expression were found to be positively correlated amongst themselves also. Factors, GCNF & USF1 revealing highly significant correlation with GDF9 expression in opposite manner were found to be negatively correlated.
Figure 4. Pearson correlation coefficients of transcription factor expression data with growth differentiation factor 9 expression at 8 h of maturation. Single star (*) represents p < 0.05 and double star (**) represents p < 0.01.
Table 2. Correlation matrix for transcription factors revealing significant correlation with GDF9 expression. Single star (*) represents p < 0.05 and double star (**) represents p < 0.01
Discussion
Considering the fact that the expression of GDF9 exerts a significant influence with future development ability of oocytes (Hussein et al., Reference Hussein, Thompson and Gilchrist2006; Yeo et al., Reference Yeo, Gilchrist, Thompson and Lane2008; Gode et al., Reference Gode, Gulekli, Dogan, Korhan, Dogan, Bige, Cimrin and Atabey2011; Gomez et al., Reference Gomez, Kang, Koo, Kim, Kwon, Park, Atikuzzaman, Hong, Jang and Lee2012; Jain et al., Reference Jain, Jain, Kumar, Goswami, De, Singh and Datta2012; Pandey et al., Reference Pandey, Somal, Parmar, Gupta, Bharti, Bhat, Indu, Chandra, Kumar and Sharma2018) and also the fact that the buffalo GDF9 5’ upstream sequence typically differs from many other species (Roy et al., Reference Roy, Rajput, Raghav, Kumar, Verma, Jain, Jain, Singh, De, Goswami and Datta2013), in the present study, we intended to generate some clue on the relationship between GDF9 expression in buffalo oocytes during IVM with predicted NR and bHLH TFs known to influence many oocyte-specific gene’s expression including GDF9. We evaluated the effect of different IVM medium supplements on the expression modulation of GDF9 and other TF genes under study at the peak of oocyte’s transcription activity (8 h of IVM) and tried to look at their possible association.
The two NR superfamily TFs considered under the study were GCNF and ESR2. Both GCNF (P < .01) and ESR2 in the present study were found to be negatively correlated with GDF9 expression across the media supplement groups. Role of GCNF as repressor of GDF9 by binding with corresponding response element (DR0) has been studied in mice oocytes (Lan et al., Reference Lan, Gu, Xu, Jackson, DeMayo, O’Malley and Cooney2003). Roy et al. (Reference Roy, Rajput, Raghav, Kumar, Verma, Jain, Jain, Singh, De, Goswami and Datta2013) also predicted the GCNF binding site (DR0 element) in buffalo GDF9 promoter. Lan et al. (Reference Lan, Gu, Xu, Jackson, DeMayo, O’Malley and Cooney2003) reported direct upregulation of BMP15 and GDF9 by inactivating GCNF gene in oocytes at the diestrus stage of the oestrous cycle and further demonstrated direct repression of BMP 15 and GDF9 by GCNF interaction with multiple DR0 elements in the promoters of both the genes.
The E2-supplemented group in the present set of experiment was found to yield the least blastocyst rate as well as the GDF9 expression. Strikingly the ESR2 expression was found very significantly higher in E2 group as compared to others. A previous study reported that E2 can potentially autoregulate ESR2 gene expression (Tessier et al., Reference Tessier, Deb, Prigent-Tessier, Ferguson-Gottschall, Gibori, Shiu and Gibori2000; Kowalski et al., Reference Kowalski, Graddy, Vale-Cruz, Choi, Katzenellenbogen, Simmen and Simmen2002). Li et al. (Reference Li, Yeh, Nojima and Dahiya2000) also reported the presence of oestrogen response elements (EREs) in the human ESR2 gene promoter having regulatory role on its transcription. Our finding that lower GDF9 expression in E2 group was consistent with the reported up regulation of GDF9 mRNA in E2 deficient aromatase knockout (ArKO) ovaries and the effect of E2 to decrease GDF9 mRNA levels in ArKO ovaries (Britt et al., Reference Britt, Saunders, McPherson, Misso, Simpson and Findlay2004). The ESR is the NR TF that binds to EREs with high affinity to influence transcription activity in response to E2 (Ramsey and Klinge, Reference Ramsey and Klinge2001). Multi protein complexes containing coregulators assemble in response to E2 and activate ESR-mediated transcription (McKenna et al., Reference McKenna, Lanz and O’Malley1999). Nevertheless, Hall and McDonnell (Reference Hall and McDonnell2005) reported about the diverse functions of E2 depending on the differential recruitment of either activators or repressors to the E2-ER complex. Thus the results from the present study prompts us to speculate about the possible involvement of ESR2 in GDF9 repression either directly by recruiting corepressors through oestrogen response elements predicted in 5’ upstream region of GDF9 (Roy et al., Reference Roy, Rajput, Raghav, Kumar, Verma, Jain, Jain, Singh, De, Goswami and Datta2013) or indirectly through transactivating other TFs having repressor activity for GDF9 expression viz. TWIST1. This TWIST1 was detected to have very high correlation (P < 0.01) with ESR2 in the present study (Table 2).
We studied the expression pattern of four bHLH TFs (viz. FIGLA, TWIST1, USF1 and USF2) for their possible association with GDF9 expression. Interaction of bHLH TFs with E-box elements in controlling oocyte-expressed genes has been described before (Ebara et al., Reference Ebara, Kawasaki, Nakamura, Tsutsumimoto, Nakayama, Nikaido and Takaoka1997; Liang et al., Reference Liang, Soyal and Dean1997; Kawasaki et al., Reference Kawasaki, Ebara, Nakayama and Takaoka1999). Interestingly, multiple E-box elements along with several TWIST subfamily TF binding sites have been identified on 5’ upstream sequence of buffalo GDF9 also (Roy et al., Reference Roy, Rajput, Raghav, Kumar, Verma, Jain, Jain, Singh, De, Goswami and Datta2013). We observed both TWIST1 (p < 0.05) and USF1 (p < 0.01) to be highly significantly correlated (p < 0.05) with GDF9 expression although in opposite relationship. Involvement of the E-box elements in oocyte-specific expression of GDF9 in mice has been studied before (Yan et al., Reference Yan, Elvin, Lin, Hadsell, Wang, DeMayo and Matzuk2006). In general, the bHLH TFs binds with E-box element as homo or hetero dimer to regulate target gene expression (Sirito et al., Reference Sirito, Lin, Maity and Sawadogo1994; Liang et al., Reference Liang, Soyal and Dean1997; Sosic et al., Reference Sosic, Brand-Saberi, Schmidt, Christ and Olson1997; Bayne et al., Reference Bayne, Martins da Silva and Anderson2004). Notably, we observed significant positive correlation (p < 0.01) among FIGLA, TWIST1 USF1 and USF2. Based on these observations, it will be tempting to interpret that these TFs possibly play synergistic role in influencing GDF9 expression through E-box elements as heterodimerization partners. Some preliminary works to support our speculation are already available (Liang et al., Reference Liang, Soyal and Dean1997; Sosic et al., Reference Sosic, Richardson, Yu, Ornitz and Olson2003; Bayne et al., Reference Bayne, Martins da Silva and Anderson2004). FIGLA heterodimerizes with a ubiquitous bHLH E12 protein and regulates the zona pellucida (ZP) genes by binding to an E-box (Liang et al., Reference Liang, Soyal and Dean1997; Bayne et al., Reference Bayne, Martins da Silva and Anderson2004).
Significant negative correlation between GCNF and USF1 suggest their synergistic role in GDF9 regulation via DR0 and E box element, respectively. Role of DR0 element and E box element in regulation of GDF9 expression has been demonstrated in mice (Lan et al., Reference Lan, Gu, Xu, Jackson, DeMayo, O’Malley and Cooney2003; Yan et al., Reference Yan, Elvin, Lin, Hadsell, Wang, DeMayo and Matzuk2006). Further Lan et al. (Reference Lan, Gu, Xu, Jackson, DeMayo, O’Malley and Cooney2003) also reported repression of GDF9 expression by direct binding of GCNF to DR0 element in promoter region. Yan et al. (Reference Yan, Elvin, Lin, Hadsell, Wang, DeMayo and Matzuk2006) speculated FIGLA as a likely candidate TF for germ cell-specific regulation of GDF9 and the expression window of FIGLA preceded and coincided with the GDF9 expression during folliculogenesis (Rajkovic et al., Reference Rajkovic, Pangas, Ballow, Suzumori and Matzuk2004; Pangas and Rajkovic, Reference Pangas and Rajkovic2006). FIGLA is reported to be expressed at high levels in primordial oocytes and persists in growing oocytes (Huntriss et al., Reference Huntriss, Gosden, Hinkins, Oliver, Miller, Rutherford and Picton2002; Bayne et al., Reference Bayne, Martins da Silva and Anderson2004). Joshi et al. (Reference Joshi, Davies, Sims, Levy and Dean2007) reported that although FIGLA affects the expression of target genes by directly regulating its downstream target genes, it may have indirect effects through the activation (or suppression) of other TFs. The possibility of FIGLA heterodimers increasing the potential E-boxes that can be bound, or may sequester these proteins in inactive complexes has also been cited (Firulli et al., Reference Firulli, Hadzie, McDaid and Firulli2000). No previous work described the role of USFs and TWIST1 in GDF9 expression regulation. TWIST1 and ubiquitously expressed USF-1 and USF-2 proteins have been reported to interact with high affinity to cognate E-box regulatory elements (CANNTG) which were also found conserved in buffalo GDF9 (Corre and Galibert, Reference Corre and Galibert2006; Asaka et al.,Reference Asaka, Terada, Ogasawara, Katsura and Inui2007; Roy et al., Reference Roy, Rajput, Raghav, Kumar, Verma, Jain, Jain, Singh, De, Goswami and Datta2013). TWIST has been reported to work as a repressor or an activator depending on its dimerization partner (Sosic et al., Reference Sosic, Richardson, Yu, Ornitz and Olson2003). It utilizes several mechanisms to inhibit transcriptional activation of targets, one of which is direct inhibition of number of transactivators including the bHLH factors (Spicer et al., Reference Spicer, Augustine and McDonald1996; Hebrok, et al., Reference Hebrok, Fuchtbauer and Fuchtbauer1997; Hamamori et al., Reference Hamamori, Wu, Sartorelli and Kedes1997, Reference Hamamori, Sartorelli, Ogryzko, Puri, Wu, Wang, Nakatani and Kedes1999). We observed strong association among the expression of TWIST1, USF1, USF2 and FIGLA (Table 2) which raises possibility of their heterodimerization partner in repressing GDF9 expression.
Conclusion
Based on the results we conclude that the expression of GDF9 in buffalo oocytes remains under control through the involvement of NR and bHLH TFs. GCNF and USF1 appeared as the most potent factors along with other TFs (ESR2 & TWIST1) studied. Their exact mechanism of action through the involvement of E Box, DR0, ER elements and TWIST subfamily binding sites would be worth exploring. More direct evidences in this direction will help in improve the efficiency of IVF system of buffalo in particular and enhance the already realized reproduction constraints of this species in broader perspective.
Acknowledgements and funding
The authors are thankful for support of fund under NAIP/C4/C1056 to the corresponding author, CSIR Jr. Research Fellowship to Tripti Jain and to Mr. Gian Singh, Computer Centre, NDRI, Karnal for analysis of data.
Competing interests
The authors declare nothing to disclose as conflict of interest in preparation of this manuscript.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
