Survivin protein expression in bovine follicular oocytes and their in vitro developmental competence

Animal Reproduction Science 108 (2008) 319–333

Survivin protein expression in bovine follicular oocytes and their in vitro developmental competence Kilsoo Jeon a,b,1 , Eun Young Kim a,1 , Jin Cheol Tae a,c , Chang Hyun Lee a,b , Keum Sil Lee a,b , Yeon Ok Kim a , Dong Kee Jeong d , Somi K. Cho d , Jae Hoon Kim d , Hyo Yeon Lee d , Key Zung Riu d , Ssang Goo Cho a,b,∗∗ , Se Pill Park a,d,∗ a

c

Mirae Biotech./Cheju National University Stem Cell Research Center, Seoul 143-854, South Korea b Department of Animal Biotechnology, Konkuk University, Seoul 143-702, South Korea Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk 361-763, South Korea d Faculty of Biotechnology, College of Applied Life Sciences, Cheju National University, 66 Jejudaehakno, Jeju 690-756, South Korea Received 18 April 2007; received in revised form 24 July 2007; accepted 1 September 2007 Available online 19 September 2007

Abstract This study examined the relationship between survivin expression and the stage of development of in vitro cultured bovine oocytes and embryos; and whether survivin expression is affected by the quality of cumulus–oocyte complexes (COCS) or the quality of pre-implantation embryos. A polyclonal antibody was prepared using recombinant bovine survivin protein. Expression of survivin mRNA and protein was analyzed by real-time quantitative RT-PCR and immunocytochemistry. In the first experiment, survivin mRNA expression was examined at developmental stages from germinal vesicle (GV) oocyte to blastocyst, it was significantly decreased after fertilization of matured oocytes (P < 0.05), then increased slightly to the 8-cell stage followed by rapid increases at the morula and blastocyst stages (P < 0.05). In the second experiment, the effect of oocyte quality on survivin protein, pro-apoptotic (bax, caspase-3) and anti-apoptotic (survivin, bax inhibitor) mRNA expression was examined. Survivin protein was more strongly expressed in good quality COCS than in poor quality COCS. The expression of the anti-apoptotic genes, survivin and bax inhibitor, was significantly higher (P < 0.05) and that of the pro-apoptotic genes, bax and caspase-3, was significantly lower (P < 0.05) in good compared to poor quality COCS. The developmental competence of good quality COCS (30.4% blastocysts) was significantly better than that of poor quality COCS. In the last

∗ ∗∗ 1

Corresponding author. Tel.: +82 2 457 8758; fax: +82 2 457 8753. Corresponding author. Tel.: +82 2 450 4207; fax: +82 2 457 8753. E-mail addresses: [email protected] (S.G. Cho), [email protected] (S.P. Park). These authors contributed equally to this work.

0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.09.003

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experiment also, we confirmed that significantly higher expression of survivin and bax inhibitor genes and significantly lower expression of bax and caspase-3 genes was resulted in good quality blastocysts than in poor quality blastocysts (P < 0.05). It was concluded that the expression of survivin was related to the quality of COCS, their developmental competence and the quality of in vitro produced blastocysts. Consequently, survivin may be a good candidate marker for embryo quality. © 2007 Elsevier B.V. All rights reserved. Keywords: Bovine; In vitro; Embryo; Survivin; Apoptotic genes

1. Introduction In vitro production of bovine embryos has been a useful tool for basic studies as well as for application. Compared to their in vivo counterparts, in vitro produced bovine embryos displayed a number of marked differences in gene expression for developmental capacity (Niemann and Wrenzycki, 2000; de Oliveira et al., 2005). However, only competent oocytes are able to undergo complete maturation and normal embryonic development. Therefore, the identification of genes that are differentially expressed in competent oocytes would contribute to our understanding of the factors controlling competency (Dode et al., 2006). Especially, in bovine germinal vesicle (GV) oocytes cumulus cell configuration is a good marker to assess their developmental capacity (Sirard et al., 1988; Park et al., 1990). In developing bovine embryos, cell viability is determined by alterations in the expression of members of the anti-apoptotic and pro-apoptotic gene families such as survivin, Bcl 2, Bcl-XL and bax (Augustin et al., 2003; Yang and Rajamahendran, 2002; Kolle et al., 2002; Lonergan et al., 2003a,b). Among them, focusing gene in this report, survivin is a 16.5-kDa, an inhibitor of apoptotic protein [IAP containing a single BIR (baculoviral IAP repeat)] domain (Ambrosini et al., 1997). IAP has been reported to be an essential anti-apopotic gene expressed in all preimplantation mouse embryonic stages. It is thought to protect the embryos from apoptosis by inhibiting an apoptotic pathway involving caspase activity (Kawamura et al., 2003). Survivin has also been shown to regulate cellular division as a chromosomal passenger protein (Skoufias et al., 2000; Uren et al., 2000; Wheatley et al., 2001). To investigate the survivin expression is intimately related to the embryo developmental capacity, we developed an anti-bovine survivin antibody. We analyzed the expression levels of survivin mRNA and protein in all development stages of bovine embryos using real-time RT-PCR and immunocytochemistry, respectively. Also we examined the survivin expression levels in the COCS and in vitro produced blastocysts classified into good and poor by their morphological quality. Our aim was to determine if survivin levels could be used as a marker of ovum viability. 2. Materials and methods 2.1. Production of bovine pre-implantation embryos The culture procedures used to produce pre-implantation embryos from follicular oocytes are described by Park et al. (1998). Briefly, bovine ovaries were obtained from a slaughterhouse, and cumulus–oocyte complexes (COCS) were aspirated from visible follicles. The COCS were then washed with HEPES-buffered Tyrode’s medium and cultured in maturation medium composed of TCM199 with 10% fetal bovine serum (FBS), 0.2 mM sodium-pyruvate, 1 ␮g/ml follicle-

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stimulating hormone, 1 ␮g/ml estradiol −17␤, and 25 ␮g/ml gentamycin sulfate at 39◦ C in a 5% CO2 incubator. After incubation for 22–24 h in the in vitro maturation (IVM) medium, the COCS underwent in vitro fertilization (IVF) in TL-STOCK medium using highly motile sperm recovered from frozen bull semen, separated on a discontinuous Percoll column. Fertilization was assessed by the cleavage rate (≥2-cell) after 44 ± 2 h of incubation. Cleaved embryos were cultured (in vitro culture; IVC) in CR1aa medium supplemented with fatty acid-free BSA (3 mg/ml). The embryos were then transferred to CR1aa medium containing 10% FBS for 4 days after IVF. In total experimental design, oocytes were matured in groups of 10, fertilized in groups of 10 and presumptive zygotes were cultured in groups of 30–40 in each 50 ␮l drop, respectively. To investigate survivin expression levels at different developmental stage, 15 oocytes or cleaved embryos were recovered from each distributed drops at each different development time; GV, IVM 0 h: MII, 22–24 h after IVM: 1-cell, 18–20 h post insemination (hpi): 2-cell, 30–32 hpi: 4cell, 46–48 hpi: 8-cell, 70–72 hpi: 16-cell, 94–96 hpi: 32-cell, 110–112 hpi: morula, 130–132 hpi: blastocyst, 180–182 hpi. 2.2. mRNA extraction Massenger RNA for real-time RT-PCR was prepared from GV oocytes to blastocysts using magnetic beads (Dynabeads mRNA purification kit; Dynal, Oslo, Norway), following the manufacturer’s instructions. Briefly, 15 oocytes or embryos were resuspended in 100 ␮l of lysis/binding buffer, 100 mM Tris–HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA pH 8.0, 1% LiDS and 5 mM DTT, and vortexed at room temperature for 5 min to lyse tissue. A 50 ␮l aliquot of an oligo(dT)25 magnetic-bead suspension was added to the samples, and the samples were incubated at room temperature for 5 min. The hybridized mRNA and oligo(dT) beads were washed twice with washing buffer A, containing 10 mM Tris–HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA and 1% LiD, and then washed once with washing buffer B, containing 10 mM Tris–HCl, pH 7.5, 0.15 M LiCl and 1 mM EDTA. mRNA samples were eluted from beads in 15 ␮l of double-distilled DEPC-treated water. 2.3. Real-time RT-PCR quantification Massenger RNA was extracted as described above and standard cDNA was synthesized using an oligo(dT)12–18 primer and superscript reverse transcriptase (Invitrogen). Real-time RT-PCR (Bio-Rad, Chromo4) was performed using the primer sets as shown in Table 1. In all experiments, histone H2a mRNA served as an internal standard. The threshold cycle (Ct ) value represents the cycle number at which the sample fluorescence rises statistically above background noise. To monitor the reactions, we followed the protocol described by the DyNAmo SYBR Green qPCR kit, which contains a modified Tbr DNA polymerase, SYBR Green, optimized PCR buffer, 5 mM MgCl2 and a dNTP mix that includes dUTP (Finnzyme Oy, Espoo, Finland). The PCR protocol used a denaturation step at 95 ◦ C for 15 min, followed by amplification and quantification cycles which were repeated 40 times at 94 ◦ C for 30 s, 50 or 56 ◦ C for 1 min and 72 ◦ C for 1 min using a single fluorescence measurement, and a melting curve program of 65–95 ◦ C, with a heating rate of 0.2 ◦ C/s and continuous fluorescence measurement. The samples were then cooled to 12 ◦ C. Fluorescence of SYBR Green was measured after the extension step during the PCR reactions. PCR products were then analyzed by generating a melting curve. Since the melting curve of a product is sequence-specific, it can be used to distinguish non-specific from specific PCR products. To do this it is necessary to determine the crossing points (CP) for each transcript. The CP is defined

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Table 1 Primers used for real-time RT-PCR assay of various pro-apoptotic and anti-apoptotic bovine genes Genes

Primer sequence

Annealing temperature (◦ C)

H2A

5 -GTCTTGGAGTACCTGACCGC 5 -ACAACGAGGGCTTCTTCTGA

56

Survivin

5 -CCTGGCAGCTCTACCTCAAG 5 -TAAGTAGGCCAACACGAAAG

56

Bax inhibitor

5 -GCTCTGGACTTGTGCATT 5 -GCCAAGATCATCATGAGC

56

Bax

5 -GCTCTGAGCAGATCAAG 5 -AGCCGCTCTCGAAGGAAGTC

56

Caspase-3

5 -CGATCTGGTACAGACGTG 5 -GCCATGTCATCCTCA

50

Product size (bp) 201 233 374 400 359

as the point at which fluorescence rises appreciably above background noise. Gene expression was quantified by the 2-ddCt method (Livak and Schmittgen, 2001). 2.4. Construction of bovine (b) survivin expression vector The survivin cDNA gene from bovine embryos was cloned into a pGEM-T easy vector (Promega) using RT-PCR. Primer sequences were: forward, 5 -CCTGGCAGCTCTACCTCAAG3 and reverse, 5 -GAAAGCACAACCGGATGAAT-3 . The cloned DNA was digested with EcoRI. After digestion, gene products were purified by agarose gel and ligated into a pET-32A vector (Novagen) cut with the same enzyme. The resultant expression plasmid, pETbsurvivin, was sequenced using the ABI prismTM terminator cycle sequencing Ready reaction kit V. 3.1 (Applied Biosystems). 2.5. Expression of recombinant (rec)-bsurvivin in E. coli and extraction of rec-bsurvivin E. coli (BL21 DE3) was transformed into pETbsurvivin according to the manufacturer’s instructions (Novagen). Single colonies were inoculated into 3 Ml Luria Broth (LB) medium containing 50 ␮g/Ml ampicillin and incubated overnight at 37 ◦ C. They were then inoculated into 300 Ml of LB medium, which contained ampicillin. Incubation was continued at 37 ◦ C for 2 h to reach an optical density (OD) at 600 of 0.35–0.4. We then added 0.1 mM of isopropyl-␤d-thio-galactopyranoside (IPTG) to the culture. Cells were incubated for 3 h and harvested by centrifugation at 5000 rpm for 20 min. The pellet was resuspended in 20 ml of PBS, centrifuged at 5000 rpm for 5 min at 4 ◦ C, resuspended again and lysed for 20 min on ice in 400 ␮l of PROPREPTM solution (Intron Biotechnology, Inc.). Lysates were centrifuged at 13,000 rpm for 5 min at 4 ◦ C, and the supernatant was assayed for protein concentration using the BCA assay. 2.6. Western blot analysis of rec-bsurvivin protein Recombinant proteins were analyzed using 12% SDS-PAGE gels (Laemmli, 1970). The samples were boiled for 5 min and run on the gels at 40 mA/gel. Gels were then stained with Coomassie blue (0.1%, w/v) in methanol/acetic acid/H2 O and transferred to nitrocellulose membranes accord-

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ing to the manufacturer’s instructions using a TE22 Mini Tank Transfer Unit (Hofer). Membranes were probed for bovine survivin proteins with alkaline phosphatase-conjugated goat anti-rabbit antibody (Sigma), using a NBT/BCIP Western blotting detection system (Promega). 2.7. Immunization To generate antibodies, two New Zealand white rabbits were injected intramuscularly with 300 ␮g each of rec-bsurvivin in a complete Freund’s adjuvant, and boosted every 2 weeks for 8 weeks with antigen in incomplete Freund’s adjuvant. Antibodies were purified from the rabbit serum with a Protein-A antibody purification kit (Pro-Chem, Inc.) 2.8. Purification of recombinant-bsurvivin and antibody production To produce recombinant proteins in E. coli (BL21 DE3), we isolated survivin cDNA from a bovine embryo cDNA library and cloned it into the expression vector pET-32A (Fig. 1). The survivin protein was purified in E. coli (BL21 DE3) through target protein elution after SDS-PAGE. Purified protein (MW, 28 kDa) was identified as rec-bsurvivin through s-tag Western blotting using an s-protein conjugate. We next generated polyclonal antibodies from survivin using rabbits that were injected intramuscularly with the purified rec-bsurvivin protein. Antibody production and

Fig. 1. Plasmid construct of pET-A/bsurvivin. The plasmid containing cDNA corresponding to the amino acid residues 78–143 of mature bovine survivin protein. The cDNA was placed under the control of a plasmid T7 lac promoter in pET-32A vector.

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Fig. 2. SDS-PAGE and Western blot analysis of recombinant bovine survivin protein. After incubation with IPTG for 3 h, E. coli transformations with pET-32A and pETbsurvivin were lysed with SDS-PAGE sample buffer. Extracted recbsurvivin was solubilized in the same buffer. The samples were subjected to SDS-PAGE (lanes 1–3) and Western blotting (lanes 4–6). Lanes 1 and 6 represent pET-32A transformed E. coli. Lanes 2 and 5 show the overexpression of pETbsurvivin transformed E. coli. Lanes 3 and 4 are purified rec-bsurvivin. Proteins were stained with CBB (lanes 1–3) and probed with homebrew anti-survivin antibody (lanes 4–6). The molecular mass standards are indicated on the left.

titration were analyzed by both dot and Western blotting. We found that survivin antibody titer was sufficient to isolate the antibodies after 8 weeks. The specificity of the antibody was reconfirmed by Western blotting after antibody purification using Protein-A antibody purification kit (Fig. 2). 2.9. Immunofluorescence of bovine oocytes and embryos Oocytes or embryos were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min. After washing with PBS, they were incubated for 1 h in 10% normal goat serum to block any non-specific binding. They were then incubated with our inhouse polyclonal anti-bovine survivin antibodies for 1 h. After washing the oocytes or embryos with PBS, they were incubated with an FITC-labeled secondary antibody (1:200, Jackson Immuno Research Lab), using 40 ␮g/ml PI (Sigma) to stain the nuclei. All treated samples were loaded onto slide glass and mounted with a coverslip. Slides were examined for the combined excitation of fluorescence for proteins and PI for DNA using laser scanning confocal microscopy performed with a Leica DM IRB which was equipped with a krypton–argon ion laser. The COCS staining to examine the survivin protein expression in cumulus cells was also carried out by the same method. 2.10. Experimental design This study consists of the following three experimental designs. 2.10.1. Experiment 1: Expression of survivin mRNA and protein during in vitro development of bovine follicular oocytes Survivin protein expression was examined by immunofluorescence at eight stages of development from germinal vesicle oocyte to blastocyst in three replicates using 15 oocytes/embryos

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Table 2 In vitro development of bovine follicular oocytes classified by cumulus cell mass configuration Cumulus cell mass configuration of COCS*

Compacted (good COCS) Dispersed (poor COCS)

138 125

No. of oocytes

No. (%) of embryos developed to†

≥2-cell on day 2

≥16-cell on day 4

Blastocyst on day 8

114 (82.6)b 73 (58.4)c

67 (48.6)b 15 (12.0)c

42 (30.4)b 7 (5.6)c

* Oocytes were classified into compacted and dispersed group according to their cumulus cell pattern of oocyte cumulus complexes (COCS). † Different superscripts (a and b) within columns denote significant differences (P < 0.05).

per development stage/replicate. Response was evaluated by a qualitative scoring system. The survivin mRNA expression of a 233 bp band (a survivin amplification product) was examined by real-time RT-PCR in a similarly designed and replicated experiment at seven of these stages of development from GV to blastocyst. 2.10.2. Experiment 2: Comparison of survivin protein, apoptotic genes expression and in vitro development between good quality and poor quality bovine COCS Using 2 (good versus poor quality) × 3 replicate designs with 15 oocytes per treatment/replicate, the effect of oocyte quality on survivin protein, pro-apoptotic (bax; caspase-3) and anti-apoptotic (survivin, bax inhibitor) mRNA expression was analyzed. Also, to examine the effect of oocyte quality on developmental competence, three replicates (40–50 COCS/replicate) of IVM/IVF/IVC were carried out. GV oocytes were classified into good quality or poor quality COCS; we assessed by cumulus cell attachment and cytoplasm color level. Cumulus cellcompacted and bright gray-colored COCS were regarded as good quality COCS and cumulus cell-dispersed and brown-colored COCS were poor quality. 2.10.3. Experiment 3: Comparison of survivin protein and apoptotic genes expression between in vitro produced good quality and poor quality bovine blastocysts The same 2 (good versus poor quality) × 3 replicate design with groups of 15 embryos per treatment/replicate was used to assess the effect of blastocyst quality on the expression of survivin protein and, pro-apoptotic and anti-apoptotic mRNA by immunocytochemistry and real-time RT-PCR, respectively. Blastocyst quality was classified by their development potential and morphological healthy level at day 8 IVC (180–182 hpi); good quality embryos were expanding, expanded or hatching developed and simultaneously healthy formed blastocysts, and poor quality embryos were included inner cell mass and trophectoderm cell poor expanding blastocyst, delayed early blastocyst or degenerating blastocysts. 2.11. Statistical analysis In survivin mRNA expression (Figs. 3I, 4G and 5C ), the relative abundance of gene expression was evaluated by analyses of variance using the general linear model (PROC-GLM) (SAS Software) (Anon, 1992). Differences in the developmental rates between good quality and poor quality bovine follicular oocytes in Table 2 were assessed using the SAS Software. Differences of P < 0.05 were considered statistically significant.

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Fig. 3. Expression of bovine survivin protein and mRNA during in vitro development of bovine follicular oocytes. Laser scanning confocal microscopic images of survivin protein in bovine pre-implantation embryos (A–I, 600×). Stages shown are (A) GV, germinal vesicle oocyte (strong positive, ++); (B) MII, metaphase II oocyte (strong positive, ++); (C) 2-cell (positive, +); (D) 4-cell (positive, +); (E) 8-cell (positive, +); (F) 16-cell (strong positive, ++); (G) 32-cell (very strong positive, +++) and (H) blastocyst (very strong positive, +++). Panel I shows a negative control in which non-immunized

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3. Results 3.1. Expression of survivin protein and mRNA during in vitro development of bovine follicular oocytes Survivin protein expression at representative eight developmental stages (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell, 32-cell and blastocyst) of the bovine follicular oocytes during in vitro development was examined (Fig. 3A–I). The immunoreactivity was predominantly localized to the cytoplasm and their expression was variable to their progressive developmental stages; GV, germinal vesicle oocyte (strong positive, ++); MII, metaphase II oocyte (strong positive, ++); 2-cell (positive, +); 4-cell (positive, +); 8-cell (positive, +); 16-cell (strong positive, ++); 32-cell (very strong positive, +++) and blastocyst (very strong positive, +++). The intensity assessment standard was the lowest positive intensity, 2-cell embryos. Negative control shows no expression (Fig. 3I). This result indicated that survivin expression of follicular oocytes during in vitro development was decreased after fertilization, and it was gradually increased after genomic activation stage (>8-cell). Real-time RT-PCR was performed to detect mRNAs encoding survivin in bovine 15 oocytes and in 15 embryos at seven different development stages (GV, MII, 1-cell, 2-cell, 8-cell, morula and blastocyst) (Fig. 3J). DNA sequencing confirmed that the band was a 233 bp survivin amplification product (data not shown). Survivin mRNA was detected at all representative seven developmental stages of bovine pre-implantation embryo. Similar to protein expression results, survivin mRNA expression was significantly decreased after fertilization of matured oocytes (P < 0.05), then increased slightly to the 8-cell stage followed by rapid increases at the morula and blastocyst stages (P < 0.05).

3.2. Comparison of survivin protein and apoptotic gene expression and in vitro development between good quality and poor quality bovine COCS Survivin protein expression between good and poor quality COCS was examined by immunofluorescence. As shown in Fig. 4, the good quality COCS (Fig. 4A) demonstrated strong immunostaining (Fig. 4C), while the poor quality COCS (Fig. 4B) did not (Fig. 4D). Also, mRNA expression of survivin and other anti-apoptotic gene (bax inhibitor), or proapoptotic genes (bax and caspase-3) between two different COCS group was examined, survivin and bax inhibitor were significantly higher (P < 0.05) and the expression of bax and caspase-3, was significantly lower (P < 0.05) in good compared to poor quality COCS (Fig. 4G). When good and poor COCS groups were compared for their in vitro developmental capacity, as shown in Table 2, the developmental competence of good quality COCS (30.4% blastocysts) was significantly better than that of poor quality COCS (5.6% blastocysts; P < 0.05).

rabbit IgG was used in place of the in-house polyclonal surviving antibody at the same concentration. Green, survivin protein; red, chromatin. (J) A 233 bp band corresponding to survivin was detected at all stages of bovine oocyte and embryos using real-time RT-PCR; GV, MII, 1-cell, 2-cell, 8-cell, morula and blastocyst. In real-time RT-PCR, the average of three independent experiments was represented as a bar graph. Values are mean ± S.E.M. Error bar (*) indicates statistically significant differences (P < 0.05).

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Fig. 4. Survivin protein and apoptotic gene expression in morphologically good and poor bovine cumulus–oocyte complexes (COCS). Fluorescent microscopic images showing survivin protein expression in bovine good (A, C and E) and poor (B, D and F) quality COCS. The compacted and healthy formed cumulus cell mass configuration, good quality COCS demonstrated (A) strong expression of the survivin protein compared to the poor quality COCS; (B) dispersed and degenerating cumulus cell mass configuration; (C and D) green, survivin protein and (E and F) red, chromatin. Bars, 150 ␮m. Figure (G) shows survivin and bax inhibitor (anti-apoptotic), bax, caspase-3 (pro-apoptotic) mRNA levels in cumulus cells of good (black bar) and poor (white bar) COCS using real-time RT-PCR. In real-time RT-PCR, the average of three independent experiments is represented as a bar graph. Values are mean ± S.E.M. Error bar (*) indicates statistically significant differences between two cumulus cell groups (P < 0.05). Original magnification, 200×.

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Fig. 5. Apoptotic gene expression in good and poor bovine blastocysts produced in vitro. In vitro produced day 8 blastocyst quality was classified by their development stage and morphological healthy rate; (A) good embryos were included expanding, expanded and hatching development and simultaneously healthy formed blastocysts and (B) poor embryos were delayed developed early blastocyst and simultaneously degenerating or unhealthy formed blastocysts. Figure (C) shows survivin and bax inhibitor (anti-apoptotic), bax and caspase-3 (pro-apoptotic) mRNA levels in good (black bar) and poor (white bar) blastocysts using real-time RT-PCR. In real-time RT-PCR, the average of three independent experiments is represented as a bar graph. Values are mean ± S.E.M. Error bar (*) indicates a statistically significant difference between the two groups (P < 0.05).

3.3. Comparison of survivin protein and apoptotic genes expression between in vitro produced good quality and poor quality bovine blastocysts We performed real-time RT-PCR to compare the mRNA expression level of survivin and other anti-apoptotic gene (bax inhibitor), and pro-apoptotic genes (bax and caspase-3) between good (Fig. 5A) and poor (Fig. 5B) quality blastocysts. As shown in Fig. 5C, there was a significantly higher expression of survivin and bax inhibitor genes and significantly lower expression of bax and caspase-3 genes in good quality blastocysts than in poor quality blastocysts (P < 0.05). Also, the immunostaining results showed that poor quality blastocysts (Fig. 6D and F) presented decreased survivin protein expression around some blastomere nucleus compared to an even strong expression in all blastomere of good quality blastocysts (Fig. 6C and E). 4. Discussion In this study we demonstrate that survivin mRNA and protein are expressed in bovine COCS and in all developmental stages of the bovine pre-implantation embryos. Also, we confirmed that survivin mRNA expression in bovine COCS or blastocysts was significantly different according

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Fig. 6. Expression of survivin protein in good and poor bovine blastocysts produced in vitro. Laser scanning confocal microscopic images show the expression of survivin protein in good quality (A, C and E) or poor quality (B, D and F) bovine blastocysts. Good quality embryo (A) has many inner cell mass (ICM) cells and trophectoderm (TE) cells, (C) each blastomere expressed equally strong survivin protein, (B) while poor quality embryo has not many ICM and TE cells and some blastomere did not express survivin protein as dot-circle (D and F). Survivin protein, green (A and B); chromatin, red, (C and D); merge (E and F) (600×).

to their quality (P < 0.05). And good quality COCS were indicated significantly higher embryo development than poor quality COCS (P < 0.05). Therefore, survivin expression may be a marker to expect in vitro developmental capacity of bovine follicular oocytes. However, we previously reported that both DNA fragmentation and apoptosis-related gene expression were increased in freeze-thawed bovine blastocysts. The expression of survivin, Fas, caspase-3 and Hsp 70 was higher in the freeze-thawed blastocysts than in the non-frozen control group; this may be the cause of reduced developmental capacity of blastocysts subjected to freezing and thawing conditions (Park et al., 2006). The reason why survivin level was higher in frozen-thawed blastocysts than control has not been found, but after thawing embryos might have more anti-apoptotic characteristics for survival than control, even though they were exposed in pro-apoptotic environment.

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Survivin is known to have two functions, that is, inhibiting apoptosis and regulating cellular division. In a recent study using a murine model, it was reported that survivin is an essential antiapopotic gene and is expressed in pre-implantation mouse embryos in all stages of development. The authors concluded that survivin could protect the embryos from apoptosis by inhibiting an apoptotic pathway involving caspases (Kawamura et al., 2003). In this study, we focused that survivin is related to regulating cellular division (especially in quality and developmental capacity) of bovine pre-implantation embryos including GV oocytes. Although follicular oocytes were cultured under normal conditions in vitro, the bovine survivin expression was similar to other important genes essential for embryonic development such as glucose transporter 5, mitochondrial Mn-superoxide dismutase, connexin 43, interferon tau, insulin-like growth factor II and insulinlike growth factor-I receptor (Lonergan et al., 2003a,b). In this study, we were able to detect the expression of survivin mRNA at all embryonic developmental stages. Survivin mRNA was highly expressed in the GV oocyte, significantly decreased after fertilization and then gradually increased starting at the 8-cell stage (P < 0.05). There is no data about survivin protein expression of bovine GV and MII oocyte yet. However, survivin is abundantly expressed after embryonic day 11.5 but is not expressed in most terminally differentiated adult tissues in mouse (Adida et al., 1998; Kobayashi et al., 1999). Also, targeted deletion of mouse results in early embryonic both survivin alleles in lethality with polyploidy (Uren et al., 2000). In this study, using quantitative real-time RT-PCR and immunocytochemistry with an antibody that we generated here, we present evidence that survivin mRNA and protein are expressed well in all stages of pre-implantation embryos and even in GV or MII oocytes. The expression of survivin mRNA was significantly increased by about six times in the blastocyst stage embryos than in the oocytes (P < 0.05). When comparing the expression levels of survivin in COCS and blatsocysts, significant differences were found between good and poor quality ovum (P < 0.05). The good quality somatic cells also demonstrated strong immunofluorescence (P < 0.05). Also, we searched other apoptotic related gene expression against COCS and in vitro produced day 8 blastocyst. In both samples similar results were found; good groups presented significantly increased mRNA expression of anti-apoptotic gene and significant decrease of pro-apoptotic gene expression than those of poor groups (P < 0.05). These results demonstrate that analysis of the levels of survivin expression may assist in determining embryo quality. In this study, we demonstrate that our developed rabbit anti-bovine survivin polyclonal antibodies are very sensitive for detecting survivin expression in both somatic and germ cells. Recent studies have focused on the expression of survivin in humans and mice. To explore the expression of survivin in bovine, we used recombinant methods to generate bovine recombinant survivin and produced bovine anti-survivin polyclonal antibodies. Recombinant proteins from bovine survivin were produced in E. coli (BL21 DE3) by use of an expression vector, pET-32a. Survivin antibody titers were sufficient to retrieve antibodies from the rabbit sera and the antibodies proved to be specific for the protein, using both dot and Western blotting. Through immunocytochemical analysis we demonstrated evidence that survivin is expressed in all developmental stages in the cytoplasm and around the nucleus of pre-implantation embryos and oocytes. Similarly, several studies have reported that survivin is found in the cytosol of tumor-derived cell lines and non-neoplastic cells (Ambrosini et al., 1997; Grossman et al., 2001). It has been reported that in mouse pre-implantation embryos, survivin is found predominantly in the cytoplasm, and the intensity of survivin immunostaining is reportedly similar in ICM and TE cells in the mouse blastocyst (Kawamura et al., 2003). Survivin has also been found in microtubules and/or kinetochores (Fortugno et al., 2002; Skoufias et al., 2000; Uren et al., 2000) during mitosis.

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