Analysis of actinomycin D treated cattle oocytes and their use for somatic cell nuclear transfer

Animal Reproduction Science 109 (2008) 40–49

Analysis of actinomycin D treated cattle oocytes and their use for somatic cell nuclear transfer Marcelo Tigre Moura a,b , Regivaldo Vieira de Sousa a,b , Ligiane de Oliveira Leme a,b , Rodolfo Rumpf a,b,∗ a

Laborat´orio de Reprodu¸ca˜ o Animal, Embrapa Recursos Gen´eticos e Biotecnologia, C.P. 02372 Bras´ılia, DF, CEP 70770-900, Brazil b Departamento de Agronomia e Medicina Veterin´ aria, Universidade de Bras´ılia, CEP 70910-900 Bras´ılia, DF, Brazil Received 17 May 2007; accepted 19 October 2007 Available online 26 December 2007

Abstract The present work aimed to evaluate the transcription and replication inhibitor, actinomycin D, for oocyte chemical enucleation. Cattle oocytes matured in vitro were treated with actinomycin D according to the following treatments: T1, control; T2 = 1.0 ␮g/ml for 16 h; T3 = 1.0 ␮g/ml for 14 h; T4 = 2.5 ␮g/ml for 14 h; T5 = 5.0 ␮g/ml for 14 h. The oocytes were denuded and activated during 24–26 h of maturation. Oocytes were fixed to determine the maturation status and for chromosome morphology evaluation. Furthermore, oocytes treated with actinomycin D were used for somatic cell nuclear transfer (SCNT). Parthenogenetic and SCNT embryos were fixed to evaluate the percentage of apoptotic nuclei by the TUNEL assay. The maturation (T1 = 90.4%; T2 = 82.3%; T3 = 79.1%; T4 = 83.4%; T5 = 74.7%), cleavage (T1 = 68.9%; T2 = 46.0%; T3 = 49.7%; T4 = 33.4%; T5 = 29.3%) and blastocyst rate at D8 (T1 = 41.1%; T2 = 1.8%; T3 = 1.3%; T4 = 0.9%; T5 = 0.0%) after actinomycin D treatment were significantly different. There was a significant chromosome uncoiling when treated with greater concentrations (2.5 and 5.0 ␮g/ml). After SCNT, the cleavage rate (61.3%) was similar to the actinomycin D-treated control group (61.3%) and less than the non-treated control (70.2%), although the blastocyst rate was greater in the SCNT group (11.8%) comparing with the treated control (3.6%) and less than the untreated control (38.0%). Treated parthenogenetic embryos had more apoptotic cells than the parthenogenetic controls (24.2% compared with 4.8%). However, the SCNT group using treated cytoplasts was similar from the SCNT control (9.3 compared ∗ Corresponding author at: Embrapa Genetics Resources and Biotechnology, W5 Norte Final, 70770-900, Bras´ılia, Distrito Federal, Brazil. Tel.: +55 6134484693; fax: +55 6133403658. E-mail address: [email protected] (R. Rumpf).

0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.10.013

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with 13.0%). Actinomycin D treatment was efficient in blocking embryonic development. Moreover, it was possible to obtain reconstructed embryos that possess an apoptotic cell index indistinguishable from controls. © 2007 Elsevier B.V. All rights reserved. Keywords: Oocyte; Enucleation; Actinomycin D; Nuclear Transfer

1. Introduction Cloning by somatic cell nuclear transfer (SCNT) is a process that can reverse a committed cell phenotype into a totipotent one (Hochedlinger and Jaenisch, 2006). Although the technology is a powerful tool for basic research (Eggan et al., 2004), animal production (Faber et al., 2004) and holds great promise to the development of genetically matched pluripotent cell lines for therapy (Solter, 2006), it remains inefficient and labor intensive (Jaenisch et al., 2002). Any progress on these limitations will permit a more use of the technology. The removal of the maternal chromosomes is an essential step during the production of cloned embryos, to maintain the correct ploidy (Li et al., 2004). Tetraploid cells are normally directed to placenta development and alone will not attain full term development (Eakin et al., 2005; Nagy et al., 1993). However, the process damages the oocyte and reduces its developmental potential. Particularly in non-human primates, it has been demonstrated that enucleation also removes important proteins involved in spindle assembly, resulting in aneuploidy (Simerly et al., 2003). With the objective to reduce the effect of enucleation and to facilitate the production of cytoplasts, many alternative strategies were developed: centrifugation (Tatham et al., 1995), demecolcine (Baguisi and Overstrom, 2000), etoposide with cyclohexamide (Fulka and Moor, 1993), X-ray (Kim et al., 2004), ultraviolet (UV) light and Hoechst 33342 staining (Wagoner et al., 1996). However, these methods do not allow for enucleation with great efficiency while maintaining oocyte viability. More recently, the use of demecolcine, nocodazole or sucrose have proven to be useful to assist the mechanical removal of the metaphase plate, avoiding UV exposure (Wang et al., 2001; Kawakami et al., 2003; Liu et al., 2002). To our knowledge, there is no report using transcription and/or replication inhibitor to enucleate oocytes. Actinomycin D is an irreversible transcription and replication inhibitor that binds to guanines and interferes with polimerase mediated elongation (Sobell, 1985). It has been successfully used as a chemical enucleation method for somatic cells (Hikawa and Takenaka, 1996; Bayona-Bafaluy et al., 2003). The objective of the present study was to evaluate the effectiveness of actinomycin D in blocking embryonic development by treating oocytes during in vitro maturation (IVM) and to use them as recipient cytoplast for SCNT. 2. Material and methods 2.1. Chemicals Chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. 2.2. In vitro oocyte maturation Oocyte maturation was as described earlier (Pereira et al., 2005). Briefly, cattle ovaries were obtained from local slaughterhouses (Distrito Federal, Brazil) and follicles between 2 and 8 mm

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were aspirated using an 18 gauge needle with a vacuum system. Cumulus oocytes complexes (COC) recovered with at least three cumulus layers and a homogeneous granulated cytoplasm were selected. The maturation medium consisted of TCM-199 medium (Invitrogen, CA, USA) supplemented with 10% (V/V) FBS (Invitrogen, CA, USA), 24 IU/ml of LH, 0.01 IU/ml of FSH and antibiotics (100 IU/ml of penicillin and 50 ␮g/ml of streptomycin), with 5% CO2 in air at 39 ◦ C for 20 h or as described later. 2.3. Oocyte treatment with actinomycin D Different treatments of actinomycin D (Fluka, Switzerland) were tested to block further embryonic transcription and replication. Oocytes were randomly allocated to five treatments: 0 ␮g/ml, control; 1.0 ␮g/ml for 14 h; 1.0 ␮g/ml for 16 h; 2.5 ␮g/ml for 14 h; and 5.0 ␮g/ml for 14 h. All groups were initially cultured in maturation medium without actinomycin D. The group treated for 16 h was washed after 4 h of maturation and cultured in actinomycin D containing medium during a 20 h maturation period. The groups treated for 14 h were washed after 6 h in maturation medium and subsequently cultured in actinomycin D containing medium. 2.4. Maturation evaluation Oocytes matured for 20 h were washed from actinomycin D containing medium, denuded by 0.2% hialuronidase treatment with gentle pipetting and cultured in maturation medium for 4 h. After 24 h of maturation, the number of oocytes with a polar body (PB) was recorded. Subsequently, oocytes were fixed or submitted to parthenogenetic activation. 2.5. Cytogenetics Oocytes considered immature as a result of the absence of a PB were washed in PBS and fixed in a 2:1 ethanol/acetic acid solution for at least 24 h. After fixation, the oocytes were placed on a slide and stained with a 2% lacmoyd solution. Later, the oocytes were washed with fixation solution and analyzed for meiotic phases under immersion oil with a light microscope under a 1000× magnification. Oocytes with a PB were removed from culture and washed with PBS. Groups of five to nine oocytes were kept in a 0.9% sodium citrate solution for five minutes and transferred to a slide with the least volume as possible. One drop of 2:1 methanol/acetic acid was immediately deposited over the oocytes. After removal of excess and initial cell swelling, another drop was deposited. The last procedure was repeated, but using a 3:1 solution. The slides were air dried before being immersed in the 3:1 solution for 24 h. The slides were dried and stained with a 1% orcein solution for 2 min. The chromosome morphology was recorded under a light microscope with a 1000× magnification. 2.6. Somatic cell nuclear transfer SCNT was performed essentially as previously described (Iguma et al., 2005). Briefly, oocytes matured for 20 h were denuded as described above. Oocytes with homogeneous cytoplasm and a visible PB were selected and incubated in 7.5 ␮g/ml citochalasin D and 10 ␮g/ml Hoechst 33342 containing maturation medium for 15 min. Micromanipulation was conducted with a Nikon TDM inverted microscope (Nikon, Japan) with Narishige 188 micromanipulators (Narishige, Japan) at

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room temperature. After being oriented to the position analogous to five or four o’clock on a clock face, the PB with 10–30% of the adjacent cytoplasm was removed with a beveled injection pipette. The biopsy was exposed to UV light to confirm enucleation success. A fibroblast cell obtained from an adult cow was inserted into the perivitelline space through the same slit. After reconstruction, the couplets were immediately fused by applying two 30 ␮s pluses of 1.9 Kva/cm delivered by an electro cell manipulator (BTX 200, San Diego, CA, USA). Fusion results were evaluated under a stereomicroscope 30 min after fusion. Non-fused couplets were submitted to a second fusion. Fused units were cultured in SOFaaci medium (Holm et al., 1999) for 3 h before parthenogenetic activation, as described below. When oocytes treated with actinomycin D were used as recipient cytoplasts, these were positioned with the PB at the analogous three or four o’clock positions on a clock face for it’s careful removal and donor cell introduction at 6 or 12 o’clock position. Fusion and activation was the same as for the control SCNT. 2.7. Parthenogenetic activation, embryo culture and evaluation Oocytes and fused couplets were activated using 5 ␮M of Ionomycin for 5 min and 2 ␮M of 6-dimethylaminopurine (6-DMAP) for 4–5 h. After activation, the presumptive zygotes were washed and cultured in 200 ␮l drops of SOFaaci medium supplemented with 2.77 mM of myoinositol and 5% FCS covered with mineral oil with 39 ◦ C with 5% of CO2 in air. The embryos were co-cultured on a cumulus monolayer for 8 days. Embryo development was recorded 48, 168 and 192 h post-activation. 2.8. Tunel staining Embryos 64 h post-activation (D3) were washed in PBS and fixed with 4% paraformaldehyde for 1 h. Embryos were permeabilized with 0.5% triton X-100 solution for 1 h and submitted to the TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling) assay (Roche, Germany). The analysis was conducted as described earlier (Gjorret et al., 2003, 2005). All DNA stained blue and TUNEL positive cells stained green. Only the nuclei that had apoptotic morphology accompanied by TUNEL staining were recorded as apoptotic (M + T). The apoptotic index was obtained by the percentage of affected blastomeres from the total number of blastomeres. TUNEL staining was analyzed and recorded at the 40× magnification using an Axiophot fluorescence microscope (Zeiss, Germany). 2.9. Statistical analysis The maturation, cleavage and blastocyst rates were analyzed by the chi-square test. For normal distribution analysis, the Shapiro–Wilk/Stephens’ test was used. The blastomere mean number was analyzed by the Kruskal-Wallis and the percentage of apoptotic nuclei was evaluated by the Mann–Whitney non-parametric test. Differences with probability of p < 0.05 were considered significant. 3. Results Initially, the inhibitory effect of actinomycin D on maturation and embryo development was investigated. Oocytes exposed to actinomycin D during IVM were less efficient to reach Metaphase II (MII) than controls as evaluated by the presence of the first PB (Table 1). After parthenogenetic

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Table 1 Maturation and embryonic development following activation of oocytes with different actinomycin D treatments Concentration (h)

No. of oocytes

MII (%)

Cleavage (%)

Blastocyst D7 (%)

Blastocyst D8 (%)

0.0 (control) 1.0 (16) 1.0 (14) 2.5 (14) 5.0 (14)

386 356 379 206 194

349 (90.4)a 293 (82.3)b 300 (79.1)b 172 (83.4)b 145 (74.7)b

266 (68.9)a 164 (46.0)b 181 (49.7)b 69 (33.4)c 57 (29.3)c

144 (37.3)a 4 (1.4)b 5 (1.3)b 1 (0.4)b 0 (0.0)b

159 (41.1)a 5 (1.8)b 5 (1.3)b 2 (0.9)b 0 (0.0)b

Within a column, percentages with different superscripts (a, b, c) differ significantly, p < 0.05. Concentrations of actinomycin D in ␮g/ml; MII: metaphase II. Table 2 Maturation cell cycle distribution of oocytes without a polar body after 24 h of in vitro maturation with different actinomycin D treatments Concentration (h)

GV

PI

MI

AI

TI

MII

0.0 (control) 1.0 (16) 1.0 (14) 2.5 (14) 5.0 (14)

– – – – 1

– – 1 2 1

1 – 8 3 2

– 4 1 2 2

– 1 3 2 2

5 22 9 14 12

Total

1

4

14

9

8

62

GV, germinal vesicle; PI, prophase I; MI, metaphase I; AI, anaphase I; TI, telophase I; MII, metaphase II. Concentrations of actinomycin D in ␮g/ml.

activation, the cleavage rate was also affected in actinomycin D treated oocytes, irrespectively of the treatment used. Blastocyst development was severely reduced, but only 5.0 ␮g/ml for 14 h completely abolished development to the blastocyst stage (Table 1). Cytogenetic analysis was also conducted to assess if the oocytes without a PB were indeed immature (Table 2). These data indicated an increased total maturation rate, and there remained a significant difference in maturation efficiency compared with controls (Table 3). Oocytes matured were fixed to observe the chromosome morphology after treatment (n = 16). Control oocytes and treatments with a concentration of 1.0 ␮g/ml of actinomycin D had no visible chromosome morphology alteration (Fig. 1A and B). However, oocytes treated with 2.5 or 5.0 ␮g/ml for 14 h had a very loose chromosomal structure, making it difficult to distinguish individual chromosomes (Fig. 1C and D). The remaining oocytes were activated and used as controls for their replicates, with similar results to Table 1 (data not shown). Table 3 Effect of actinomycin D treatment on oocyte in vitro maturation assessed by polar body identification and oocyte fixation Concentration (h)

No. of oocytes

MII with PB (%)

No. of oocytes

MII with PB and Fixation (%)

0.0 (control) 1.0 (16) 1.0 (14) 2.5 (14) 5.0 (14)

140 146 151 150 128

122 (87.1)a 110 (75.3)b 128 (84.7)a 115 (76.6)b 98 (76.5)b

128 137 150 138 118

127 (99.2)a 132 (96.3)a 137 (91.3)b 129 (93.4)b 110 (93.2)b

Within a column, percentages with different superscripts (a, b) differ significantly, p < 0.05. MII, metaphase II; PB, polar body. Concentrations of actinomycin D in ␮g/ml; only oocytes without a polar body were fixed.

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Fig. 1. Chromosome morphology of oocytes treated with actinomycin D during oocyte in vitro maturation. (A) Control oocyte; (B) oocyte treated with 1.0 ␮g/ml for 16 h; (C) oocyte treated with 2.5 ␮g/ml for 14 h; (D) oocyte exposed to 5.0 ␮g/ml for 14 h. Orcein staining with 1000× magnification.

To functionally evaluate cytoplasm viability of treated oocytes, these oocytes were used as recipient cytoplasts. The treatment with 1 ␮g/ml for 14 h was chosen based on the previously detailed results. Due to the difference on enucleation methodology, a small modification to the SCNT protocol was established. In actinomycin D-treated oocytes, after the mechanical removal of the PB, the somatic cell was immediately inserted into the perivitelline space through the same slit on the zona pellucida. Further steps were identical to conventional SCNT. The reconstructed embryo cleavage rate was similar to the treated control and lower than non-treated controls (Table 4). Table 4 Development of reconstructed embryos following oocyte chemical enucleation by actinomycin D Group P 0.0 P 1.0 SCNT 1.0

No. of oocytes 84 220 101

Cleavage (%) (70.2)a

59 135 (61.3)b 62 (61.3)b

Blastocyst D7 (%)

Blastocyst D8 (%)

32 (38.0)a

32 (38.0)a 8 (3.6)c 12 (11.8)b

8 (3.6)c 12 (11.8)b

Within a column, percentages with different superscripts (a, b, c) differ significantly, p < 0.05. P 0.0, parthenogenetic control group; P 1.0, parthenogenetic group treated with 1.0 ␮g/ml of actinomycin D for 14 h; SCNT 1.0, somatic cell nuclear transfer group using oocytes treated with 1.0 ␮g/ml of actinomycin D for 14 h.

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Table 5 Apoptotic index of parthenogenetic and reconstructed embryos by SCNT produced using oocytes submitted to chemical enucleation by actinomycin D Group P 0.0 P 1.0 SCNT 0.0 SCNT 1.0

No. of embryos

Mean no. of blastomeres

Mean no. of apoptotic cells (%)

36 30 16 20

6.91a

4.80a 24.2b 13.00y 9.37y

5.96a 6.37a 6.15a

Within a column, percentages with different superscripts (a, b, y) differ significantly, p < 0.05. P 0.0, parthenogenetic control group; P 1.0, parthenogenetic group treated with 1.0 ␮g/ml of actinomycin D for 14 h; SCNT 0.0, somatic cell nuclear transfer group using non-treated oocytes; SCNT 1.0, somatic cell nuclear transfer group using oocytes treated with 1.0 ␮g/ml of actinomycin D for 14 h.

However, the blastocyst rate from SCNT embryos was greater than the treated parthenogenetic control, but again less than the non-treated control (Table 4). With the objective to evaluate embryo quality, SCNT and parthenogenetic embryos were fixed to analyze the percentage of apoptotic cells by the TUNEL assay (Table 5). Cloned embryos produced using non-treated oocytes were used as controls for SCNT. The remaining oocytes were activated and also used as controls (data not shown). There was no difference on blastomere mean number between groups (Table 5). Parthenogenetic embryos derived from treated oocytes had a greater percentage of apoptotic cells than the non-treated controls (P = 0.0016). However, there was no difference between the cloned embryo groups (P = 0.814). 4. Discussion Although many different methods have been developed, there are not efficient alternative strategies for oocyte enucleation available (Galli et al., 2003). .Actinomycin D, a transcription and replication inhibitor, has been successfully used to functionally enucleate somatic cells (Hikawa and Takenaka, 1996; Bayona-Bafaluy et al., 2003). Based on these previous reports, the inhibitor was tested as a chemical enucleation method for cattle oocytes. In the present work, there was a reduction on maturation rates after treatment with actinomycin D, although oocytes are transcriptionally inactive (Andreu-Vieyra et al., 2006). There is considerable evidence that indicates the cumulus cells are important during the initial hours of oocyte in vitro maturation (Kastrop et al., 1991; Tatemoto and Terada, 1995; Li et al., 2006). To avoid the negative effect of early inhibition of cumulus transcription, the actinomycin D treatment in the present work started at 4 or 6 h after the beginning of maturation. However, the exposure to actinomycin D reduced maturation efficiency. After or during the incubation with the inhibitor, it is possible that the cells are secreting a factor harmful to the oocyte, affecting nuclear maturation (Mattioli et al., 1991). In agreement, the percentage of mouse denuded oocytes with a PB was not affected by actinomycin D treatment at a 50 ␮g/ml concentration for up to 24 h (Grondahl et al., 2000). Moreover, the effect of ␣-amanitin on maturation is dependent on the presence of cumulus cells (Mattioli et al., 1991; Tatemoto and Terada, 1995). These results do not seem to be dependent on the inhibition method, because different inhibitors (␣-amanitin and 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole) with different modes of action generate similar results with zygotes from cattle (Chandolia et al., 1999). Furthermore, oocytes that lacked a PB after 24 h of maturation were fixed. Including these data, however, indicated there was still a negative effect of actinomycin D treatment on oocyte maturation.

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Actinomycin D is a molecule that intercalates with DNA (Sobell, 1985), resulting indirectly in chromosome morphology alterations and breaks in the chromosome structure (Jagiello, 1969; Trask and Muller, 1988). To analyze the oocytes in the present research, oocytes were fixed for classical cytogenetic analysis. The chromosome morphology of oocytes treated with the concentration of 2.5 and 5.0 ␮g/ml for 14 h was severely affected, with obvious uncoiling of the structure. The alterations observed in the present work were more severe than observed before (Jagiello, 1969), probably due to differences in the concentration and/or time of treatments. However, these alterations do not suggest cytoplasmatic inviability, because cells treated with ␣-amanitin, a reversible inhibitor, also have significant alterations in the nucleolus (Goldberg et al., 1962). The actinomycin D treatment during oocyte maturation significantly reduced the cleavage rate, as previously reported on treated zygotes (Chandolia et al., 1999). The treatments were efficient in reducing blastocyst formation. When exposed to the 1.0 and 2.5 ␮g/ml concentrations, only a few embryos reached the blastocyst stage. However, the 5 ␮g/ml concentration was capable of totally blocking blastocyst formation. To functionally test the viability of the cytoplasts, treated oocytes were used for SCNT. With four replicates, it was possible to demonstrate that some were capable of further development following SCNT. The introduction of a somatic cell nucleus “rescued” treated oocytes, producing significantly more blastocysts than the control counterparts. Although promising, a direct comparison with the mechanical method of enucleation is necessary. A negative aspect of actinomycin D is the capacity to induce apoptosis (Hietanen et al., 2000; Riley et al., 2004; Fabian et al., 2003). Due to the effect of actinomycin D and to estimate embryo quality, the TUNEL assay was used to assess if treatment would induce apoptosis in embryos derived from treated oocytes. Based on previous work, we used a more reproducible methodology to assess apoptotic death on preimplantation embryos (Gjorret et al., 2003, 2005). Moreover, we used early developing embryos (D3) that have minor incidence of apoptosis and could be efficiently recovered from all groups. In terms of blastomere mean number, there was no statistical difference between all groups. While comparing parthenogenetic embryos from treated oocytes to respective controls, the former group had a significantly greater percentage of apoptotic cells, validating the analysis. When comparing embryos produced by SCNT, the groups were similar. These data suggest that the inhibition during oocyte maturation in vitro does not induce apoptosis in blastomeres that originated from the somatic nucleus. 5. Conclusion Based on the results described above, actinomycin D treatment during bovine oocyte IVM can efficiently block further embryonic development. However, following SCNT, reconstructed embryos derived from treated oocytes can develop up to the blastocyst stage, with an apoptotic index indistinguishable from the control. Acknowledgements To Maur´ıcio Machaim Franco, Carlos Frederico Martins, Eduardo Oliveira Melo for helpful discussions, Cezar Martins de S´a for reagents and to Romero Moura for critically reviewing the manuscript. The authors are also grateful to CAPES and Embrapa Genetics Resources and Biotechnology for the financial support of this study.

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