Effects of a Sonic Hedgehog agonist on ovine oocyte maturation, epigenetic changes and development of parthenogenetic embryos

Small Ruminant Research 141 (2016) 84–90

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Effects of a Sonic Hedgehog agonist on ovine oocyte maturation, epigenetic changes and development of parthenogenetic embryos Parisa Nadri a , Saeid Ansari-Mahyari a,∗ , Azadeh Zahmatkesh b , Ahmad Riasi a , Samira Zarvandi c , Mohammad Salehi d,∗ a

Department of Animal Science, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran c Medical Biotechnology and Nanotechnology Department, School of Medicine, Zanjan University of Medical Science, Zanjan, Iran d Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 2 January 2016 Received in revised form 30 April 2016 Accepted 4 July 2016 Available online 5 July 2016 Keywords: Nuclear maturation Cytoplasmic maturation Purmorphamine Parthenogenesis Ovine oocyte Epigenetic

a b s t r a c t Purmorphamine is a Sonic Hedgehog agonist that plays an important role in activity regulation of receptors and transcription factors. This study was carried out to assess the effects of Purmorphamine on nuclear and cytoplasmic maturation, epigenetic changes of in vitro matured oocytes and development of parthenogenetic ovine embryos. Ovine ovaries were randomly collected from industrial slaughterhouse, and cumulus-oocyte complexes were cultured in media containing 250 or 500 ng/ml Purmorphamine or without Purmorphamine as a control group. Then, after in vitro maturation (IVM), Hoechst stain, cell tracker blue and histone H4K12 antibody were used, respectively to assess the nuclear and cytoplasmic maturation, and histone acetylation rate of matured oocytes. Matured oocytes were activated and cultured to the blastocysts stage in order to determine the parthenogenetic embryo cleavage rate. Expression of Histone Deacetylase 1, 2 and 3 (Hdac1, Hdac2 and Hdac3) genes were evaluated in matured oocytes by quantitative Real Time PCR. Results showed that although the concentration of 500 ng/ml Purmorphamine had no significant influence on the oocyte nuclear maturation, it led to an increase in oocyte cytoplasmic maturation. Also, no significant difference in the rates of cleavage and blastocysts per cleavage was observed (P > 0.05) in the groups treated with Purmorphamine compared to the control. Quantitative PCR analysis indicated that in 500 ng/ml Purmorphamine treatment, Hdac 2 and 3 transcripts decreased and the H4K12 acetylation increased significantly (P < 0.05). Consequently, this study showed that 500 ng/ml Purmorphamine can induce the cytoplasmic maturation of oocytes, and may possibly be a good addative for improvement of ovine oocyte cytoplasmic maturation. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Oocyte maturation can be divided into nuclear and cytoplasmic steps. Nuclear maturation causes the formation of the first polar body and encompasses the process driving the progression of meiosis to metaphase II. Cytoplasmic maturation determines the quantity and quality of blastocysts (Pavlok et al., 1992; Sirard, 2001). Cytoplasmic glutathione (l-␥- glutamyl-l-cysteinyl-glycine; GSH) is the major non-protein sulphydryl compound in mammalian gametes (Pastore et al., 2003), which has been introduced

∗ Corresponding authors. E-mail addresses: [email protected] (S. Ansari-Mahyari), [email protected] (M. Salehi). http://dx.doi.org/10.1016/j.smallrumres.2016.07.002 0921-4488/© 2016 Elsevier B.V. All rights reserved.

as an important indicator of cytoplasmic maturation (Kim et al., 2007). There have been wide-spread studies focusing on in vitro culture (IVC) systems of the oocytes and embryos which have improved in vitro applications of oocytes (Van Langendonckt et al. 1997; Lonergan et al. 2003; Rizos et al. 2003; Wrenzycki et al., 2004). Altering the culture conditions of oocyte maturation and embryo development will help manipulate gene expression patterns in order to simulate in vivo conditions and enhance embryo quality (Nguyen et al., 2011). Maturation of oocytes is influenced by different factors such as steroids and growth factors (Driancourt and Thuel, 1998). Therefore, researchers have been trying to enhance the environmental conditions by introducing these factors, which may improve the process of oocyte maturation and subsequent development of in vitro embryos (Ikeda et al., 2000). Sonic Hedge-

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hog (SHH) is a factor that seems to have an effect on oocyte maturation, proliferation and embryonic development in human and animal species (Nguyen et al., 2011). Sonic Hedgehog signaling pathway is mediated through a cell surface receptor system consisting of two proteins: the receptor Patched (Ptc) and its co-receptor Smoothened (Smo) (Hooper and Scott, 2005). In the absence of SHH, SMO is inhibited by PTC receptors resulting in inactivation of Glioma-Associated Oncogene (GLI) transcription factor. However, in the presence of SHH, SMO is released causing translocation of GLI protein to the nucleus, which could increase transcription of Histone Deacetylase (Hdac) genes in the nucleus (Ruiz i Altaba et al., 2002; Gueripel et al., 2006; Canettieri et al., 2010). Histone Deacetylase 1, 2 and 3 enzymes belong to the Hdac family and are encoded by Hdac 1, 2 and 3 genes in human, respectively. These proteins have notable roles in epigenetic programming, regulation of gene expression and transcription, cell cycle progression and development of embryos (Mehnert and Kelly, 2007). Several porcine studies have demonstrated that inclusion of exogenous SHH in the IVM or IVC media enhances oocyte maturation and development of parthenogenetic embryos (Nguyen et al., 2009b, 2010). Hedgehog (Hh) molecular signaling pathway has been shown to be affected by many growth factors and small molecules such as Purmorphamine. Purmorphamine is a SHH signaling activating agonist that has been developed by Wu and his colleagues (Wu et al., 2002). This small molecule plays a crucial role in regulating the activity of SMO and PTCH receptors and GLI transcription factors (Nguyen et al., 2011). According to the positive effect of SHH on porcine oocyte maturation and embryo development (Nguyen et al., 2011), this idea has emerged that Purmorphamine may also affect ovine oocyte maturation process in the same way. Therefore, the aims of this study were to investigate the effects of this factor on ovine oocyte nuclear and cytoplasmic maturation, histone H4K12 acetylation, expression of Hdac1, 2 and 3 genes in ovine in vitro matured oocytes and also on development of parthenogenetic embryos. The findings of this study may be helpful in giving an insight for possible application of Purmorphamine in IVM media in order to enhance the quality of resulting ovine embryos. 2. Material and methods All chemicals and media were obtained from Sigma-Aldrich Company (St Louis, MO, USA), unless otherwise specified. Experimental procedures for collecting the samples of the animals were approved by the review committee of the Stem Cell Technology Research Center (Tehran, Iran), and animal experimentations including ovine slaughter and ovarian sample collections were in agreement with the ethical commission. 2.1. Oocytes collection and in vitro maturation (IVM) Ovaries from adult sheep (an Iranian breed called Shal) were randomly collected from an industrial abattoir and maintained at 35 ◦ C in physiological saline, and transported to the laboratory within 2 h. Cumulus oocyte-complexes (COCs) were aspirated from medium-sized follicles (3–7 mm in diameter) with an 18 gauge needle. Then COCs were washed in Hepes-TCM 199 supplemented with 10% FBS (Gibco, Grand Island, NY, USA). Only COCs with at least two layers of cumulus cells and homogeneous ooplasm were collected under a microscope and washed three times in maturation medium. Ten to 12 COCs were randomly allocated to each 50 ␮l droplet of TCM 199 maturation medium supplemented with 10% FBS, 5 ␮g/ml LH, 5 ␮g/ml FSH, 1 ␮g/ml estradiol and different concentrations of Purmorphamine (250 ng/ml and 500 ng/ml Purmorphamine; (Santa Cruz Inc., California, USA). For control group, concentration of Purmorphamine was zero. Then COCs were cul-

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tured at 39 ◦ C, in a 5% CO2 incubator for 22 h (Thompson et al., 1995). The selected Purmorphamine concentrations were according to a previous study (Nguyen et al., 2009b, 2010). 2.2. Staining of oocytes nuclei Immediately after IVM, oocytes were denuded using Hyaluronidase enzyme. In order to stain the nuclei and determine nuclear maturation rate, the oocytes were washed with 100 ml phosphate buffer saline, containing 1 mg PVA and then fixed in 4% paraformaldehyde for 30 min. Then, oocytes were stained in 10 ␮g/ml Hoechst solution for 5 min. Oocytes were inserted in 10 ␮l glycerol droplets, squashed on a glass slide and then observed under an epifluorescence microscope (Nikon, Tokyo, Japan) (Mohammadi-Sangcheshmeh et al., 2011). Based on maturation stages, oocytes were classified as being in germinal vesicle (GV), germinal vesicle break- down (GVBD), metaphase-I (MI), and metaphase-II (MII) according to Mohammadi-Sangcheshmeh et al. (2011). 2.3. Glutathione assessment in matured oocytes Glutathione content was measured according to a previously method described by You and Park, (2010). Briefly, immediately after IVM, oocytes were denuded and incubated in a medium containing 100 ml phosphate buffer saline (PBS), 1 mg PVA (Polyvinyl Alcohol) and 10 ␮g/ml Cell Tracker Blue (Invitrogen, USA) for 30 min. The oocytes were subsequently washed in 100 ml PBS medium, containing 1 mg PVA and were placed into 10 ␮l glycerol droplets, observed under an epifluorescence microscope (Nikon, Tokyo, Japan) with UV filters and then all fluorescent images were saved as graphic files. The assessment of blue pixels was analyzed by ImageJ software (Version 1.45 s, National Institutes of Health, USA), and compared with control oocytes. 2.4. RNA extraction and cDNA synthesis In order to analyze the effect of Purmorphamine on the expression of Hdac1, 2 and 3 genes in in vitro-matured oocytes, three biological replicates for each sample, each containing 10 denuded matured oocytes, were used for RNA extraction. The oocytes were washed in PBS droplets and transferred into Eppendorf tubes containing 1.5 ml cellular lysis buffer (Zuccotti et al., 2002). The RNA from oocytes was extracted using Qiazol reagent (Qiagen, Hilden, Germany). Complementary DNA was synthesized using First Strand cDNA Synthesis Kit (Fermentas, Germany) according to the manufacture’s instruction. Briefly, 3 ␮g/ml random hexamer and 5 ␮l nuclease-free water were added to RNA, and tubes were placed in a thermal cycler (Bio-Rad, Hercules, CA, USA) for 5 min at 75 ◦ C. Then tubes were placed on ice and 5 ␮l RT buffer 5X, 1 ␮l RT enzyme (200 u/␮l), 3 ␮l dNTP (10 mM) and 0.25 ␮l RNA inhibitor (20 u/␮l) were added in a 10 ␮l-total reaction volume. The reverse transcription program was as follows: 25 ◦ C for 10 min, 37 ◦ C for 15 min, 42 ◦ C for 45 min and 72 ◦ C for 10 min. 2.5. Real time PCR Quantitative Real-time PCR was performed to assess the expression of Hdac1, 2 and 3 genes in three biological replicates (each cDNA in duplicates) for each sample using Rotor Gene Q instrument (Qiagen, Hilden, Germany). Reactions were in a total volume of 13 ␮l including 6.5 ␮l SYBR Green PCR master mix (Takara, Japan), 4.5 ␮l distilled water, 1 ␮l of forward and reverse primers (10 pmol/␮l) and 1 ␮l cDNA. Tubes were transferred to the RotorGene cycler. The amplification program was as follows: 3 min at 95◦ C for enzyme activation and 40 cycles of 5 s denaturation at

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Table 1 Primer sequences, product size and accession numbers. Gene name

Primer sequences

Product size (bp)

Accession numbers

Sh-HDAC1 Sh.HDAC2 Sh.HDAC3 Sh.GAPDH

F: GAC AAA CGC ATC TC AT R:TTG GCT TTC TTG AAG TT F:ATG GAA AAG ATA AAA CAGR:CTA TTC GTT TGT GTG ATG CT F:ACA AAT ACG GAA ACT T R:GAA AAG GTGCTT GTA ACT C F:AGT GTC GTT GAA GTC R:GAA ACC TGC CAA GTA TGAT

119 169 131 121

XM XM XM XM

004005023.1 004011189.1 00400891.1 001190390.1

Fig. 1. Hoechst-stained oocytes in different maturation stages. (a) No change is observed in the nucleus after culture (GV), (b) The oocyte nuclear membrane is broken but it has not reached the final maturation (GVBD), (c) Maturation has occurred but polar body is not observed yet (MI), (d) Final maturation has occurred and the polar body can be observed (MII), (100× magnification).

Fig. 2. Oocytes staining by Cell Tracker Blue for assessment of glutathione level. (a) Control group (without Purmorphamine), (b) 250 ng/ml Purmorphamine, and (c) 500 ng/ml Purmorphamine, (100× magnification). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

95◦ C, 15 s annealing at 60◦ C and 10 s extension at 72 ◦ C. Finally, a melting step was included to confirm the specificity of amplification. Ovine GAPDH primers were utilized as internal control (Livak and Schmittgen, 2001), and the mRNA levels of the samples were normalized to GAPDH. Gene expression data were analyzed using Rest-2009 software based on Pfaffl mathematical methods (Pfaffl et al., 2002). Sequences of the primers are shown in Table 1.

2.6. Immunocytochemistry assey for measurement of acetylation (H4K12) After IVM, denuded matured oocytes were fixed in 0.4% paraformaldehyde for 30 min, and then incubated in 0.2% triton for 30 min. Oocytes were incubated in PBS + 0.5% BSA for 24 h in fridge, then were incubated in droplets containing H4K12 antibody

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(Stemgen, Milano, Italy) at 37 ◦ C for 1 h. Oocytes were washed in PBS + 0.1% BSA and incubated in PBS + 0.1% BSA droplets containing anti Rabbit IgG secondary antibody (Stemgen, Milan, Italy) for 1 h. Subsequently after washing, oocytes were incubated in Hoechst solution with PBS + 0.1% BSA for 5 min. Finally, they were washed in PBS + 0.1% BSA and evaluated by fluorescence microscopy (Endo et al., 2005). The amount of acetylation was measured after photography using ImageJ software (Version 1.45 s; National Institutes of Health, USA), and acetylation rates were compared in matured oocytes of different treatments (0 ng/ml, 250 ng/ml and 500 ng/ml Purmorphamine). 2.7. Oocytes activation (parthenogenesis) and in vitro culture After IVM, COCs were vortexed in HTCM medium supplemented with 10% FBS and 100 ␮l Hyaluronidase for 3 min in 1.5 ml centrifuge tubes to disperse the cumulus cells. For chemical activation, the denuded oocytes were exposed to 5 mg/ml ionomycin and 995 ␮g/ml HTCM for 5 min, followed by 5 min incubation in HTCM supplemented with 30 mg/ml bovine serum albumin (BSA). Oocytes were incubated in 50 ␮l droplets of embryo culture medium (CR1aa) containing 2 mM 6-dimethylaminopurine for 2 h and then, incubated in CR1aa medium at 39 ◦ C, 5% CO2 and 5% O2 for 18 h. After activation, 6–8 oocytes were cultured in 20 ␮l CR1aa droplets for 8 days. The rates of cleavage and blastocysts per cleavage were assessed on days 3 and 7, respectively (Nguyen et al., 2010). 2.8. Statistical analysis The effect of Purmorphamine on oocyte nuclear maturation, cytoplasmic maturation (glutathione level in each treatment), H4K12 acetylation and Hdac genes expression in different treatments was evaluated by one-way ANOVA procedure, SAS software, version 9.1 (2003). The comparison of treatment means was carried out by Tukey test at 5% probability level. Furthermore to evaluate the effects of Purmorphamine on parthenogenetic embryo development, according to the relativity of numerical data (relative to the total number), GENMOD procedure was used. In this procedure, binomial distribution and logit link function with Wald’s statistics for type 3 contrasts were used with treatments as fixed effects. 3. Results In this study approximately 100 oocytes were used for IVM in each of the 0 ng/ml, 250 ng/ml and 500 ng/ml Purmorphamine treatments to assess the effects of Purmorphamine on ovine oocyte nuclear maturation process. In vitro maturation results are presented in Table 2 and Hoechst staining is shown in Fig. 1. In 250 ng/ml Purmorphamine, 81% of oocytes were in meiosis II. This was not significantly different from the control group in which, 75% of oocytes were in meiosis II. Among oocytes treated with 500 ng/ml Purmorphamine, 72% proceeded to meiosis II, which did not show any significant difference in nuclear maturation with the control group. However, the percentage of GVBD increased significantly in 500 ng/ml Purmorphamine compared to the other groups. No significant difference in oocyte nuclear maturation between the 250 ng/ml and 500 ng/ml Purmorphamine groups was observed. In order to find the effect of Purmorphamine on ovine oocyte cytoplasmic maturation, Glutathione level (emitted fluorescent from oocytes, based on blue pixels) was measured in 0 ng/ml, 250 ng/ml and 500 ng/ml Purmorphamine groups after IVM. The results did not show any significant difference between 250 ng/ml Purmorphamine (232.3 ± 3.3) and the control groups (236.4 ± 2.1). On the contrary, rising the concentration of Purmorphamine to

Fig. 3. The effect of Purmorphamine on gene expression (Hdac1, Hdac2 and Hdac3) in ovine matured oocytes. Different superscripts within the columns indicate significant difference (P < 0.05).

500 ng/ml led to an increase (241.3 ± 1.3) in cytoplasmic maturation compared to the control group. Fig. 2 indicates the stained oocytes for assessment of glutathione level. In this study, the expression levels of Hdac1, 2 and 3 genes in mature oocyts in different treatment groups of 0 ng/ml, 250 ng/ml and 500 ng/ml Purmorphamine were analyzed by Real Time PCR and the results are shown in Fig. 3. According to the results, there was no significant difference in gene expression of Hdac1 between treatments of 250 ng/ml and 500 ng/ml Purmorphamine compared to the control (P > 0.05). Besides, Hdac2 transcripts increased significantly (P < 0.05) in 250 ng/ml Purmorphamine compared with the control group, but reduced by increasing the concentration to 500 ng/ml Purmorphamine. Treatment with 250 ng/ml and 500 ng/ml Purmorphamine was significantly associated with reduction in Hdac3 transcripts (P < 0.05). After IVM, acetylation rate was measured following photography of stained oocytes with H4K12 antibody by ImageJ software, which is shown in Fig. 4. The difference in acetylation rate between the 250 ng/ml Purmorphamine level and the control group was not significant. Increasing the concentration of Purmorphamine to 500 ng/ml led to a significant increase in acetylation rate (P < 0.05). Furthermore, a significant difference was observed between the 250 ng/ml and 500 ng/ml Purmorphamine treatments (P < 0.05) (Fig. 5). After IVM, 60, 64 and 62 oocytes were activated in the 250 ng/ml, 500 ng/ml Purmorphamine treatments and the control groups, respectively. In terms of the rate of cleavage, there was no significant difference in 250 ng/ml (1.0169 ± 0.2830) and 500 ng/ml (1.4663 ± 0.3203) Purmorphamine treatments compared to the control group (1.4069 ± 0.3221), and also between the two treatments. No significant difference was observed in rate of blastocysts per cleavage in 250 ng/ml (-1.3083 ± 0.3564) and 500 ng/ml (-1.7047 ± 0.3844) treatments compared to the control (−2.4204 ± 0.5217) and also between the two treatments. Fig. 6 indicates the blastocyst stage embryos of parthenogenetic process.

4. Discussion The quality of oocytes is crucial in oocyte maturation process and in vitro fertilization, therefore, extensive research has been carried out in order to improve these outcomes (Bavister, 2002). Undoubtedly, the basic conditions for fertilization process and development of preimplantation embryo are the quality of immature oocytes and differentiated cumulus cell. Therefore, to produce

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Table 2 Effect of Purmorphamine on different stages of oocyte nuclear maturation (LSM ±SE). Group

control 250 ng/ml Purmorphamine 500 ng/ml Purmorphamine

Nuclear status No. oocytes

GV%

GVBD%

MI%

MII%

100 100 100

4.47 ± 1.68 4.21 ± 2.70 2.92 ± 2.33

4.47 ± 1.67 4.83 ± 1.29 14.42 ± 5.80

7.37 ± 3.52 9.73 ± 4.6 9.99 ± 2.46

75.68 ± 4.3 81.22 ± 3.72 72.66 ± 3.33

Fig. 4. Immunocytochemistry assey for H4K12 acetylation. Right: Hoechst, Left: H4K12 antibody. Acetylation rate is indicated in meiosis II, (100× magnification).

in vitro embryos with high success rate, high quality matured oocytes are required (Anderiesz et al., 2000). Several researchers have studied the effect of adding growth factors (Kim et al., 2006), cytokines (Uhm et al., 2007), vitamins and amino acids (Gupta et al., 2007) to in vitro maturation media that have led to improvement in the quality of in vitro embryos in different species. Many studies have looked at the effect of different factors on increasing the quality of in vitro matured oocytes. Mo et al. (2014) has reported that adding 25 ng leukemia inhibitory factor to IVM medium increases bovine oocyte nuclear maturation and decreases cytoplasmic maturation in vitro. The same study on porcine showed that leukemia inhibitory factor increased nuclear maturation while there was no effect on cytoplasmic maturation (Dang-Nguyen et al., 2014). In further related studies on the effect of Brain-Derived Nerve Growth Factor on bovine oocyte maturation, it has been observed that this factor enhances cytoplasmic maturation without any effect on the nuclear maturation (Martins da Silva et al., 2005). Another study demonstrated that supplementing porcine oocyte maturation medium with SHH could lead to an increase in either nuclear or cytoplasmic maturation, and also the following parthenogenetic embryo development (Nguyen et al., 2009a,b). Due to the positive effect of SHH factor on oocyte maturation and development of porcine embryos, the effect of its agonist on ovine oocyte nuclear and cytoplasmic maturation and development of parthenogenetic embryos was evaluated in the present study. In this study, treating the oocyte maturation medium with 500 ng/ml Purmorphamine, had no effect on oocyte nuclear maturation, but increased oocyte cytoplasmic maturation. We further observed that treating oocytes with 250 ng/ml and 500 ng/ml Purmorphamine showed no significant difference in the rates of cleavage and blastocysts per cleavage, compared to the control group. Oocyte cytoplasmic maturation determines the quantity and quality of obtained blastocysts from parthenogenetic embryos and

in vitro fertilization (Pavlok et al., 1992; Sirard, 2001). Intracellular GSH is a molecular marker in mature oocytes that has been reported to predict cytoplasmic maturation in porcine oocytes (Wang et al., 1997) and is involved in various cellular processes, including DNA and protein synthesis, metabolism of chemicals, cellular protection, and amino acid transport (Meister et al., 1983). It also plays an important role protecting cells from oxidative damage (Meister, 1983) and regulating intracellular redox metabolism (Luberda, 2005). Glutathione concentration increases during oocyte maturation, and inhibition of its synthesis suppresses the formation of male pronucleus (Perreault et al., 1988; Yoshida, 1993). Hence, glutathione production is found to play an essential role in cytoplasmic maturation (Eppig, 1996).Our results demonstrated that treating oocytes with 500 ng/ml Purmorphamine led to a significant increase in glutathione level in ovine, which implicates its possible role in inducing oocyte maturation in different species. Gene activating process should be reversible; otherwise, activated genes will be active constantly. Histone acetylation and deacetylation are catalyzed by multiple subsets and perform important roles in regulation of eukaryotic gene expression. Histone deacetylases are kinds of enzymes that cause removal of acetyl group (O C CH3) from N-acetyl lysine residue of histones which results in tight turns of DNA around the histones. Therefore, condensation of DNA is vitally important because expression of DNA is regulated by deacetylation (Stebbins-Boaz et al., 1996). There has been some research on gene expression of histone deacetylases in matured oocyte and different tissues in different animal species. A bovine study showed that the expression of Hdac1 gene is higher than Hdac2 and Hdac3 in matured oocyte (Segev et al., 2001). In another study, adding Smoothened Agonist (SAG), a SHH agonist, to the medium of cerebellar granule precursor (CGP) cells derived from medulloblastoma, were accompanied by an increase in expression and function of Hdac1, 2 and 3 genes (Lee et al., 2013).

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Fig. 5. The effect of Purmorphamine on H4K12 acetylation in ovine mature oocytes. Different letters (a and b) indicate significant difference (P < 0.05).

Real Time PCR results demonstrated that Hdac2 transcripts increased significantly in 250 ng/ml Purmorphamine, and increasing the concentration to 500 ng/ml was followed by a notable reduction inHdac2 and Hdac3 transcripts, when compared to the control. It has been known that fully grown oocytes undergo transcriptional quiescence prior to meiotic resumption. Transcription is suppressed during oocyte maturation and fertilization, until zygotic genome activation (ZGA) (Schultz, 1993). Hence the changes in amounts of Hdac transcripts in this study would probably be a result of changes in utilization of these transcripts during or after the maturation process. In other words, Purmorphamine had an influence on the need of oocytes to use Hdac transcripts which led to a different reduction/increase in different transcripts. Despite the fact that Hdac1, 2 and 3 are homologous genes, they showed various patterns of use in response to Purmorphamine during the maturation, which indicates a possible difference in their utility pathway, and probably their next function. Histone H4K12 acetylation rate has been shown to decrease during oocytes maturation in mouse (Kim et al., 1996) and bovine (Maalouf et al., 2008). It has been further reported that by adding a histone acetylase inhibitor (TSA) to oocyte maturation medium, the acetylation rate increased in matured oocyte (Maalouf et al., 2008). There is no report on the effect of Purmorphamine on histone acetylation in other species. In the present study, H4K12 acetylation in ovine matured oocytes showed a significant increase only in 500 ng/ml Purmorphamine level. It is known that oocyte maturation helps carry out a proper epigenetic remodeling of parental chromatin, reflected by pronuclear methylation and acetylation (Gioia et al., 2005). Researchers have noted that oocyte maturation is accompanied by changes in histone modification, and the maturation-associated reduction in acetylation is due to the histone deacetylase activation and a lack of histone acetyltransferase activity in the oocytes (Brunmeir et al., 2009). These reports have shown that epigenetic modifications in the genome during oocyte maturation exert a carryover effect on

Fig. 6. Parthenogenetic blastocysts, (100× magnification).

cell proliferation and differentiation of preimplantation embryos. In general, the concentration of 500 ng/ml Purmorphamine has led to an increase in oocyte cytoplasmic maturation, acetylation in matured oocytes, as well as a reduction in Hdac2 and 3transcripts. Hence, one may speculate that there might be a relation between the cytoplasmic maturation process and histone acetylation and deacetylation, which should be further investigated. As a conclusion, although no effect of 500 ng/ml Purmorphamine on the cleavage and blastocyst rates of parthenogenetic embryos was detected, the quality of parthenogenetic embryos such as blastocyst cell number or developmental competence of IVF embryos should also be studied in order to further demonstrate the effectiveness of 500 ng/ml Purmorphamine as an appropriate concentration for improving oocyte cytoplasmic maturation and further embryonic development. In order to improve cytoplasmic maturation as an important determinant of successful in vitro embryonic devel-

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