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Parthenogenetic development of in vitro matured porcine oocytes treated with chemical agents
Animal Reproduction Science 105 (2008) 226–233
Parthenogenetic development of in vitro matured porcine oocytes treated with chemical agents Erika Varga a,∗ , Ren´ata Pataki a , Zsuzsanna L˝orincz b , ´ Bali Papp a Judit Koltai b , Agnes a b
Institute of Animal Breeding, University of West Hungary, Faculty of Agricultural and Food Sciences, H-9200 Mosonmagyar´ov´ar, V´ar 4, Hungary Institute of Economic Sciences, University of West Hungary, Faculty of Agricultural and Food Sciences, H-9200 Mosonmagyar´ov´ar, V´ar 2, Hungary Received 24 May 2006; received in revised form 26 February 2007; accepted 2 March 2007 Available online 6 March 2007
Abstract Parthenogenetic activation is a possible way to produce homogeneous embryos with the same ploidy. These embryos could develop to the blastocyst stage during the cultivation. Probably such embryos could be used in other areas of biotechnology. The objectives of the present study were first to assess the ability of strontium-chloride to induce activation and parthenogenetic development in porcine oocytes in comparison with cycloheximide and 6dimethylaminopurine; second to verify whether the combination of the two treatments improved activation and parthenogenetic development rates. At first, the effects of cycloheximide, 6-dimethylaminopurine and strontium-chloride on oocyte activation and embryonic development were compared. Oocytes from slaughterhouse ovaries were matured for 42 h in tissue culture medium (TCM) 199 at 38.5 ◦ C, 5% CO2 in air. Matured oocytes were activated with 10 mM strontium-chloride (S), 0.04 mM cycloheximide (CX), 2 mM 6-dimethylaminopurine (D) for 5 h. The activation rate was judged by pronuclear formation of oocytes. Following the activation, oocytes were incubated in NCSU 37 medium for 6 days and in all groups more than 45% of oocytes activated. The activation rate for CX treatment was significantly higher (P < 0.05) than for D (57.37 ± 4.21% and 48.09 ± 3.43%, respectively). In a second experiment in vitro matured porcine oocytes were activated using a combined treatment of strontium-chloride with cycloheximide (SCX) and strontium-chloride combined with 6dimethylaminopurine (SD). In S and SCX groups more than 50% of oocytes were activated (53.29 ± 5.39% and 54.3 ± 7.29%, respectively). However a large portion of embryos stopped their development at the twoor four-cell stage. Significantly higher numbers of embryos could reach the eight-cell stage in SD and SCX
∗
Corresponding author. Tel.: +36 96 566 613; fax: +36 96 566 695. E-mail address: [email protected] (E. Varga).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.03.004
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than for S (7.8 ± 1.0%, 7.2 ± 4.0% and 3.9 ± 3.1%, respectively). Blastocyst formation was only observed in S, CX and SCX. These results show that porcine in vitro matured oocytes can be artificially activated by cycloheximide, 6-dimethylaminopurine and strontium-chloride. © 2007 Elsevier B.V. All rights reserved. Keywords: Activation; Strontium; Cycloheximide; 6-Dimethylaminopurine; Porcine oocytes; Embryo development
1. Introduction Oocyte activation is important to nuclear transfer and could be used for cytogenetic studies of embryos because in the resultant parthenotes maternal chromosomes can be analyzed independent of paternal chromosomes. There are much published protocols for producing parthenogenetic embryos by artificial activation of in vitro matured mammalian oocytes. Oocytes could be electrically activated (Kure-bayashi et al., 2000; Ozil and Huneau, 2001). A variety of agents activate mammalian oocytes in vitro, including ethanol (Zernicka-Goetz, 1991; Meo et al., 2004), ionophore A23187 (Funahashi et al., 1994; Wang et al., 1998), cycloheximide (Nussbaum and Prather, 1995; Cha et al., 1997), strontium (Fraser, 1987; Kline and Kline, 1992; Okada et al., 2003), 6-dimethylaminopurine (Grupen et al., 2002) and calcium chloride (Machaty et al., 1996). In this study, the effects of strontium-chloride, cycloheximide and 6-dimethylaminopurine on oocyte activation were examined. The objectives of the present study were first to assess the ability of strontium-chloride to induce activation and parthenogenetic development in porcine oocytes in comparison with cycloheximide and 6-dimethylaminopurine; second, to verify whether the combination of two treatments improves activation and parthenogenetic development rates. 2. Materials and methods All chemical reagents used for oocyte maturation, activation and embryo culture were purchased from Sigma–Aldrich Chemical Co. (Budapest, Hungary) unless otherwise noted. 2.1. Recovery and in vitro maturation (IVM) of oocytes Ovaries of Hungarian Large White gilts were collected from a local abbatoir and stored at 38 ◦ C during transportation. At the laboratory, they were washed three times with warmed hexadecyl trimethyl-ammonium bromide (CETAB) solution (H-5882) and saline solution, and then stored in a water bath at 38 ◦ C before use. The cumulus-oocyte complexes (COCs) were collected by aspiration of 3–6 mm non-atretic follicles using an 18-gauge needle attached to a 5 ml disposable syringe. The medium for oocyte collection was Hepes (H-3375) medium. The COCs were selected under a stereomicroscope and washed three times in tissue culture medium (TCM) 199 (M-7528). Oocytes surrounded by three or more compact layers of cumulus cells and with an evenly granulated ooplasm were used for in vitro maturation. Oocytes were matured in groups of 50/500 l of maturation medium for 42 h at 38 ◦ C, 5% CO2 in air. The basic medium used for oocyte maturation was TCM199 supplemented with 10% pig follicular fluid, 1.25 mM l-glutamine (G-3126), 0.9 mM Na-pyruvate (S-3362), 100 M cysteamine (M-9768), 0.1 mg/ml streptomycin sulphate, 10 IU/ml PMSG (Werftt-Chemie GmbH) and 10 IU/ml hCG (Werftt-Chemie GmbH).
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2.2. Oocyte activation After 42 h of maturation oocytes were denuded by pipetting on a warm stage at 38 ◦ C. Denuded oocytes were washed three times in Ca2+ -free North Carolina State University (NCSU) 37 medium (Kikuchi, 2002). The basic medium for oocyte activation was NCSU 37 without Ca2+ . For activation oocytes were incubated in activation medium containing 10 mM SrCl2 (S-group), 0.04 mM cycloheximide (CX-group) and 2 mM 6-dimethylaminopurine (D-group) for 5 h at 38 ◦ C in a CO2 incubator. For combined chemical treatments stocks of strontium-chloride and cycloheximide (SCXgroup: S, 15.85 mg/ml; CX, 1 mg/ml) and strontium-chloride and 6-dimethylaminopurine (SDgroup: S, 15.85 mg/ml; D, 32.36 mg/ml) were prepared in NCSU 37 medium without Ca2+ . The SCX and SD stock solutions were diluted in NCSU 37 medium without Ca2+ to 10 g/ml and use for 5 h oocyte activation. 2.3. Embryo culture The basic medium for embryo cultivation was NCSU 37. After 5 h of activation embryos were removed, and washed three times in preincubated NCSU 37 medium and then cultured in groups of 30/500 l of the culture medium for 6 days at 38 ◦ C in an atmosphere of 5% CO2 in air. 2.4. Experimental design 2.4.1. Experiment 1: single stimulus by each chemical agent In these experiment oocytes were activated by single stimulus of each chemical. Cumulus free IVM oocytes were exposed to 10 mM strontium-chloride in S-group (number of oocytes (n) were 319) treated with 2 M 6-dimethylaminopurine in D-group (n = 319) and activate with 0.04 mM cycloheximide in CX-group (n = 366). Following treatments with chemical agents oocytes were washed three times in NCSU 37 medium and transferred to a droplet (0.5 ml) NCSU 37 medium. Thereafter the embryos were cultured in incubator. Oocytes in control group were not treated with chemical agents, only matured for 42 h and then were cultured for 6 days in incubator. 2.4.2. Experiment 2: combined chemical stimuli The effects of strontium-chloride combined with cycloheximide and strontium-chloride combined with 6-dimethylaminopurine were examined. The treated oocytes were rinsed three times in NCSU 37 medium and transferred to a droplet of NCSU 37 medium for further culture. 2.5. Evaluation of oocytes and embryos • Evaluation of activation status The rate of activation was judged by morphological appearance of oocytes examined 7 h after incubation. Those oocytes that formed visible pronuclei were recorded as activated. • Evaluation of embryo development At 48 h and 6 days after activation, oocytes, zygotes were mounted on glass slides with coverslips and were fixed in acetic alcohol (1/3 w/v) for at least 3 days. Fixed oocytes were then stained with 0.1% (w/v) orcein in 45% (v/v) acetic acid and evaluated under phase contrast microscopy.
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2.6. Statistical analysis Each experiment was repeated four times. All data were analyzed by ANOVA, followed Duncan’s multiple range test (P < 0.05). 3. Results 3.1. Single stimulus by each chemical agent At first, we compared the effects of cycloheximide, 6-dimethylaminopurine and strontiumchloride on oocyte activation and embryonic development. In all groups more than 45% of oocyte activation was observed. The activation rate for CX was significantly higher (P < 0.05) than for D (57.37 ± 4.21% and 48.09 ± 3.43%, respectively), see Table 1. 3.2. Combined chemical stimuli In a second experiment in vitro matured porcine oocytes were activated using a combined treatment of strontium-chloride with cycloheximide (SCX) and strontium-chloride combined with 6-dimethylaminopurine (SD). In S and SCX groups more than 50% of oocytes activated (53.29 ± 5.39% and 54.3 ± 7.29%, respectively). However a large portion of embryos stopped their development in two- or four-cell stage. A significantly higher number of embryos could reach the eight-cell stage in SD and SCX than for S (7.8 ± 1.0%, 7.2 ± 4.0% and 3.9 ± 3.1%, respectively). Blastocyst formation was only observed in S, CX and SCX, see Table 2. 4. Discussion Several chemical agents, including ethanol, inhibitors of protein synthesis (like cycloheximide), strontium-chloride, 6-dimethylaminopurine, ionophore A 23187 could stimulate oocytes in vitro. CX and ionophore A 2187 increase intracellular Ca2+ and strontium-chloride could induce Ca2+ oscillation, which occur during normal fertilization process. Increased Ca2+ destroys the existing Ca-sensitive cytostatic factor (CSF), resulting in the reduction of maturation promoting factor (MPF) activity (Swann and Ozil, 1994). In mammalian oocytes, activity of MPF, which consist of cdc2 kinase and cyclin B, is essential for meiotic arrest at metaphase II (Nurse, 1990). Porcine oocytes are arrested at the metaphase II stage of meiosis and the activation is the release from this arrested stage and progression to first interphase. Cycloheximide is believed to inhibit the synthesis of calcium-sensitive cytostatic factor in M II oocytes (Siracusa et al., 1978; Clarke and Masui, 1983). The increased intracellular Ca2+ and the decreased CSF cause the inactivation of MPF, resulting parthenogenetic activation. In our experiments 57.37% of oocytes could be activated with cycloheximide. A lower fragmentation rate was observed in CX group (21.05%). In CX group the blastocyst rate was 3.16%. Martinaz Diaz et al. (2003) demonstrated that protein synthesis inhibition treatment required for the induction of oocyte activation promotes the development of nuclear transfer embryos. Strontium is a very effective activation agent for parthenogenetic activation of mouse, rat and bovine oocytes (Roh and Hwang, 2002; Meo et al., 2004). Strontium induces the Ca2+ oscillation in mouse (Kline and Kline, 1992). Chinese hamster oocytes could not be activated with strontium (Tateno and Kamiguchi, 1997). Krivokharchenko et al. (2003) successful stimulate rat oocytes with
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Treatments
S D CX Control
Number of oocytes
319 366 319 180
Number (%)a activated
170 (53.29) 176 (48.09) a 183 (57.37) a 3 (1.67)
Parthenogenetic development day 6 (%)b Non cleaved
Fragmented
Two-cell
Four-cell
Eight-cell
Morula
Blastocyst
25 (14.71) 16 (9.09) 21 (11.48) 0
53 (31.17) 60 (34.09) 38 (20.77) 0
32 (18.82) 20 (11.36) 23 (12.57) 3 (100)
15 (8.84) 31 (17.61) 37 (20.22) 0
20 (11.76) 33 (18.75) 35 (19.13) 0
20 (11.76) 16 (9.09) 23 (12.57) 0
5 (2.94) 0 6 (3.28) 0
Values with different letter in the same column differ significantly. Each treatment was repeated three times. Abbreviations—S: oocytes were activated with 10 mM strontiumchloride; D: oocytes were activated with 2 mM 6-dimethylaminopurine; CX: oocytes were activated with 0.04 mM cycloheximide; control: oocytes were only matured and cultured for 6 days. a Percentages mean of the original number of oocytes. b Percentages mean the number of oocytes activated.
E. Varga et al. / Animal Reproduction Science 105 (2008) 226–233
Table 1 Parthenogenetic development of porcine oocytes activated by strontium-chloride, cycloheximide and 6-dimethylaminopurine
Treatments
S SD SCX
Number of oocytes
319 346 302
Number (%)a activated
170 (53.29) 170 (49.13) 164 (54.30)
Parthenogenetic development day 6 (%)b Non cleaved
Fragmented
Two-cell
Four-cell
Eight-cell
Morula
25 (14.71) 36 (21.18) a 16 (9.75) a
53 (31.17) 53 (31.18) a 40 (24.39) a
32 (18.82) 40 (23.53) 21 (12.80)
15 (8.84) 15 (8.82) 21 (12.80)
20 (11.76) a 20 (11.76) 15 (8.82) b 11 (6.47) 32 (19.51) ab 21 (12.80)
Blastocyst 5 (2.94) 0 14 (8.54)
Values with different letters in the same column differ significantly. Each treatment was repeated three times. Abbreviations—S: oocytes were activated with 10 mM strontiumchloride; SD: oocytes were activated with strontium-chloride combined with dimethylaminopurine; SCX: oocytes were activated with strontium-chloride combined with cycloheximide. a Percentages mean of the original number of oocytes. b Percentages mean the number of oocytes activated.
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Table 2 Parthenogenetic development of porcine oocytes activated by combined chemical stimuli
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2 mM strontium. Lei et al. (2002) demonstrated that mouse oocytes could be activated with 10 mM strontium. Meo et al. (2004) produced bovine blastocysts (6.3%) from oocytes activated with strontium. Okada et al. (2003) activated pig oocytes by intracytoplasmic injection of strontium. The blastocyst rate was 29%. It has been shown that some combinations of chemical stimuli improve parthenogenetic activation in mouse (Hagemann et al., 1995), bovine (Presicce and Yang, 1994) and Chinese hamster oocytes (Tateno and Kamiguchi, 1997). In our experiments oocytes were activated by 10 mM strontium-chloride and strontium-chloride combined with cycloheximide and with 6-dimethylaminopurine. For the 6 day after activation the blastocyst rate was 2.94% in S-group. Until recently, activation of in vitro matured oocytes has been associated with low blastocyst development, slow embryo growth, and low cell numbers in blastocysts. Lee et al. (2004) noticed faster embryo development and observed expanded blastocysts 5 days after electrical activation. The greatest number of blastocyst (8.64%) was in SCX group, in which the oocytes were activated by strontium combined with cycloheximide. The lower fragmentation rate (21.05%) and the greatest number of blastocyst (8.64%) were in SX group. However most of the embryos stopped there development at the two- or four-cell stage. This could be explained by the low sensitivity of the 4-cell porcine embryos. Acknowledgements Financial support was provided by the grants of the Hungarian Scientific Research Fund-T 43131. The authors are grateful to Dr. Zolt´an Machaty for critical reading of the manuscript. References Cha, S.K., Kim, N.H., Lee, S.M., Baik, C.S., Lee, H.T., Chung, K.S., 1997. Effect of cytochalsin B and cycloheximide on the activation rate, chromosome constituent and in vitro development of porcine oocytes following parthenogenetic stimulation. Reprod. Fertil. Dev. 9, 441–446. Clarke, H., Masui, J., 1983. The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes. Dev. Biol. 97, 291–301. Fraser, L.R., 1987. Strontium supports capacitacion and the acrosome reaction in mouse sperm and rapidly activates mouse eggs. Gamete Res. 18, 363–374. Funahashi, H., Cantley, T.C., Stumpf, T.T., Terlow, S.L., Day, B.N., 1994. In vitro development of in vitro matured porcine oocytes following chemical activation or in vitro fertilization. Biol. Reprod. 50, 1072–1077. Grupen, C.G., Mau, J.C., McLlfitrick, S.M., Maddocks, S., Nottle, M.B., 2002. Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes. Mol. Reprod. Dev. 62, 387–396. Hagemann, L.J., Hillery-Weinhold, F.L., Rutledge, M.L., First, N.L., 1995. Activation of murine oocytes with Ca2+ ionophore and cycloheximide. J. Exp. Zool. 271, 57–61. Kikuchi, K., 2002. Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biol. Reprod. 66, 1033–1041. Kline, D., Kline, J., 1992. Repetitive calcium transients and the role of calcium in exocitosis and cell cycle activation in the mouse egg. Dev. Biol. 149, 80–89. Krivokharchenko, A., Popova, E., Zaitseva, I., Vilianovich, L., Ganten, D., Bader, M., 2003. Development of parthenogenetic rat embryos. Biol. Reprod. 68, 829–836. Kure-bayashi, S., Miyake, M., Okada, K., Kato, S., 2000. Succesful implantation of in vitro matured, electro-activated oocytes in the pig. Theriogenology 53, 1105–1119. Lee, J.W., Tian, X.C., Yang, X., 2004. Optimization of parthenogenetic activation protocol in porcine. Mol. Reprod. Dev. 68, 51–57. Lei, L., Liu, Z.H., Kou, Z.H., Chen da, Y., 2002. Effects of different treatments on oocyte activation and parthenogenesis in mouse. Shi Yan Sheng Wu Xue Bao 35, 236–238.
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Machaty, Z., Funahasi, H., Mayes, M.A., Day, B.N., 1996. Effects of injecting calcium chloride into in vitro matured porcine oocytes. Biol. Reprod. 54, 316–322. Martinaz Diaz, M.A., Suzuki, M., Kogawa, M., Ikeda, K., Takahashi, Y., 2003. Effects of cycloheximide treatment on in vitro development of porcine parthenotes and somatic cell nuclear transfer embryos. Jpn. J. Vet. Res. 50, 147–155. Meo, S.C., Leal, C.L., Garcia, J.M., 2004. Activation and early parthenogenesis of bovine oocytes treated with ethanol and strontium. Anim. Reprod. Sci. 81, 35–46. Nurse, P., 1990. Universal control mechanism regulating onset of M-phase. Nature 334, 503–508. Nussbaum, D.J., Prather, R.S., 1995. Differential effects of protein synthesis inhibitors on porcine oocyte activation. Mol. Reprod. Dev. 41, 70–75. Okada, K., Miyano, T., Miyake, M., 2003. Activation of pig oocytes by intracytoplasmic injection of strontium and barium. Zygote 11, 159–165. Ozil, J.P., Huneau, D., 2001. Activation of rabbit oocytes: the important of the Ca2+ signal regime on development. Development 128, 917–928. Presicce, G., Yang, X., 1994. Parthenogenetic development of bovine oocytes matured in vitro for 24 h and activated by ethanol and cycloheximide. Mol. Reprod. Dev. 38, 380–385. Roh, S., Hwang, W.S., 2002. In vitro development of porcine parthenogenetic and cloned embryos: comparison of oocyte activating techniques, various culture systems and nuclear transfer methods. Reprod. Fertil. Dev. 14, 93–99. Siracusa, G., Whittingham, D.G., Molinaro, M., Vivarelli, E., 1978. Parthenogenetic activation of mouse oocytes induced by inhibitors of protein synthesis. J. Embryol. Exp. Morphol. 43, 157–166. Swann, K., Ozil, J.P., 1994. Dynamics of the calcium signal that triggers mammalian egg activation. Int. Rev. Cytol. 1152, 183–222. Tateno, H., Kamiguchi, Y., 1997. Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation. Mol. Reprod. Dev. 47, 72–78. Wang, W.H., Machaty, Z., Abeydera, L.R., Prather, R.S., Day, B.N., 1998. Parthenogenetic activation of pig oocytes with Ca-ionophore and the block to sperm penetration after activation. Biol. Reprod. 58, 1357–1366. Zernicka-Goetz, M., 1991. Spontaneous and induced activation of rat oocytes. Mol. Reprod. Dev. 28, 169–176.
Parthenogenetic development of in vitro matured porcine oocytes treated with chemical agents Erika Varga a,∗ , Ren´ata Pataki a , Zsuzsanna L˝orincz b , ´ Bali Papp a Judit Koltai b , Agnes a b
Institute of Animal Breeding, University of West Hungary, Faculty of Agricultural and Food Sciences, H-9200 Mosonmagyar´ov´ar, V´ar 4, Hungary Institute of Economic Sciences, University of West Hungary, Faculty of Agricultural and Food Sciences, H-9200 Mosonmagyar´ov´ar, V´ar 2, Hungary Received 24 May 2006; received in revised form 26 February 2007; accepted 2 March 2007 Available online 6 March 2007
Abstract Parthenogenetic activation is a possible way to produce homogeneous embryos with the same ploidy. These embryos could develop to the blastocyst stage during the cultivation. Probably such embryos could be used in other areas of biotechnology. The objectives of the present study were first to assess the ability of strontium-chloride to induce activation and parthenogenetic development in porcine oocytes in comparison with cycloheximide and 6dimethylaminopurine; second to verify whether the combination of the two treatments improved activation and parthenogenetic development rates. At first, the effects of cycloheximide, 6-dimethylaminopurine and strontium-chloride on oocyte activation and embryonic development were compared. Oocytes from slaughterhouse ovaries were matured for 42 h in tissue culture medium (TCM) 199 at 38.5 ◦ C, 5% CO2 in air. Matured oocytes were activated with 10 mM strontium-chloride (S), 0.04 mM cycloheximide (CX), 2 mM 6-dimethylaminopurine (D) for 5 h. The activation rate was judged by pronuclear formation of oocytes. Following the activation, oocytes were incubated in NCSU 37 medium for 6 days and in all groups more than 45% of oocytes activated. The activation rate for CX treatment was significantly higher (P < 0.05) than for D (57.37 ± 4.21% and 48.09 ± 3.43%, respectively). In a second experiment in vitro matured porcine oocytes were activated using a combined treatment of strontium-chloride with cycloheximide (SCX) and strontium-chloride combined with 6dimethylaminopurine (SD). In S and SCX groups more than 50% of oocytes were activated (53.29 ± 5.39% and 54.3 ± 7.29%, respectively). However a large portion of embryos stopped their development at the twoor four-cell stage. Significantly higher numbers of embryos could reach the eight-cell stage in SD and SCX
∗
Corresponding author. Tel.: +36 96 566 613; fax: +36 96 566 695. E-mail address: [email protected] (E. Varga).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.03.004
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than for S (7.8 ± 1.0%, 7.2 ± 4.0% and 3.9 ± 3.1%, respectively). Blastocyst formation was only observed in S, CX and SCX. These results show that porcine in vitro matured oocytes can be artificially activated by cycloheximide, 6-dimethylaminopurine and strontium-chloride. © 2007 Elsevier B.V. All rights reserved. Keywords: Activation; Strontium; Cycloheximide; 6-Dimethylaminopurine; Porcine oocytes; Embryo development
1. Introduction Oocyte activation is important to nuclear transfer and could be used for cytogenetic studies of embryos because in the resultant parthenotes maternal chromosomes can be analyzed independent of paternal chromosomes. There are much published protocols for producing parthenogenetic embryos by artificial activation of in vitro matured mammalian oocytes. Oocytes could be electrically activated (Kure-bayashi et al., 2000; Ozil and Huneau, 2001). A variety of agents activate mammalian oocytes in vitro, including ethanol (Zernicka-Goetz, 1991; Meo et al., 2004), ionophore A23187 (Funahashi et al., 1994; Wang et al., 1998), cycloheximide (Nussbaum and Prather, 1995; Cha et al., 1997), strontium (Fraser, 1987; Kline and Kline, 1992; Okada et al., 2003), 6-dimethylaminopurine (Grupen et al., 2002) and calcium chloride (Machaty et al., 1996). In this study, the effects of strontium-chloride, cycloheximide and 6-dimethylaminopurine on oocyte activation were examined. The objectives of the present study were first to assess the ability of strontium-chloride to induce activation and parthenogenetic development in porcine oocytes in comparison with cycloheximide and 6-dimethylaminopurine; second, to verify whether the combination of two treatments improves activation and parthenogenetic development rates. 2. Materials and methods All chemical reagents used for oocyte maturation, activation and embryo culture were purchased from Sigma–Aldrich Chemical Co. (Budapest, Hungary) unless otherwise noted. 2.1. Recovery and in vitro maturation (IVM) of oocytes Ovaries of Hungarian Large White gilts were collected from a local abbatoir and stored at 38 ◦ C during transportation. At the laboratory, they were washed three times with warmed hexadecyl trimethyl-ammonium bromide (CETAB) solution (H-5882) and saline solution, and then stored in a water bath at 38 ◦ C before use. The cumulus-oocyte complexes (COCs) were collected by aspiration of 3–6 mm non-atretic follicles using an 18-gauge needle attached to a 5 ml disposable syringe. The medium for oocyte collection was Hepes (H-3375) medium. The COCs were selected under a stereomicroscope and washed three times in tissue culture medium (TCM) 199 (M-7528). Oocytes surrounded by three or more compact layers of cumulus cells and with an evenly granulated ooplasm were used for in vitro maturation. Oocytes were matured in groups of 50/500 l of maturation medium for 42 h at 38 ◦ C, 5% CO2 in air. The basic medium used for oocyte maturation was TCM199 supplemented with 10% pig follicular fluid, 1.25 mM l-glutamine (G-3126), 0.9 mM Na-pyruvate (S-3362), 100 M cysteamine (M-9768), 0.1 mg/ml streptomycin sulphate, 10 IU/ml PMSG (Werftt-Chemie GmbH) and 10 IU/ml hCG (Werftt-Chemie GmbH).
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2.2. Oocyte activation After 42 h of maturation oocytes were denuded by pipetting on a warm stage at 38 ◦ C. Denuded oocytes were washed three times in Ca2+ -free North Carolina State University (NCSU) 37 medium (Kikuchi, 2002). The basic medium for oocyte activation was NCSU 37 without Ca2+ . For activation oocytes were incubated in activation medium containing 10 mM SrCl2 (S-group), 0.04 mM cycloheximide (CX-group) and 2 mM 6-dimethylaminopurine (D-group) for 5 h at 38 ◦ C in a CO2 incubator. For combined chemical treatments stocks of strontium-chloride and cycloheximide (SCXgroup: S, 15.85 mg/ml; CX, 1 mg/ml) and strontium-chloride and 6-dimethylaminopurine (SDgroup: S, 15.85 mg/ml; D, 32.36 mg/ml) were prepared in NCSU 37 medium without Ca2+ . The SCX and SD stock solutions were diluted in NCSU 37 medium without Ca2+ to 10 g/ml and use for 5 h oocyte activation. 2.3. Embryo culture The basic medium for embryo cultivation was NCSU 37. After 5 h of activation embryos were removed, and washed three times in preincubated NCSU 37 medium and then cultured in groups of 30/500 l of the culture medium for 6 days at 38 ◦ C in an atmosphere of 5% CO2 in air. 2.4. Experimental design 2.4.1. Experiment 1: single stimulus by each chemical agent In these experiment oocytes were activated by single stimulus of each chemical. Cumulus free IVM oocytes were exposed to 10 mM strontium-chloride in S-group (number of oocytes (n) were 319) treated with 2 M 6-dimethylaminopurine in D-group (n = 319) and activate with 0.04 mM cycloheximide in CX-group (n = 366). Following treatments with chemical agents oocytes were washed three times in NCSU 37 medium and transferred to a droplet (0.5 ml) NCSU 37 medium. Thereafter the embryos were cultured in incubator. Oocytes in control group were not treated with chemical agents, only matured for 42 h and then were cultured for 6 days in incubator. 2.4.2. Experiment 2: combined chemical stimuli The effects of strontium-chloride combined with cycloheximide and strontium-chloride combined with 6-dimethylaminopurine were examined. The treated oocytes were rinsed three times in NCSU 37 medium and transferred to a droplet of NCSU 37 medium for further culture. 2.5. Evaluation of oocytes and embryos • Evaluation of activation status The rate of activation was judged by morphological appearance of oocytes examined 7 h after incubation. Those oocytes that formed visible pronuclei were recorded as activated. • Evaluation of embryo development At 48 h and 6 days after activation, oocytes, zygotes were mounted on glass slides with coverslips and were fixed in acetic alcohol (1/3 w/v) for at least 3 days. Fixed oocytes were then stained with 0.1% (w/v) orcein in 45% (v/v) acetic acid and evaluated under phase contrast microscopy.
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2.6. Statistical analysis Each experiment was repeated four times. All data were analyzed by ANOVA, followed Duncan’s multiple range test (P < 0.05). 3. Results 3.1. Single stimulus by each chemical agent At first, we compared the effects of cycloheximide, 6-dimethylaminopurine and strontiumchloride on oocyte activation and embryonic development. In all groups more than 45% of oocyte activation was observed. The activation rate for CX was significantly higher (P < 0.05) than for D (57.37 ± 4.21% and 48.09 ± 3.43%, respectively), see Table 1. 3.2. Combined chemical stimuli In a second experiment in vitro matured porcine oocytes were activated using a combined treatment of strontium-chloride with cycloheximide (SCX) and strontium-chloride combined with 6-dimethylaminopurine (SD). In S and SCX groups more than 50% of oocytes activated (53.29 ± 5.39% and 54.3 ± 7.29%, respectively). However a large portion of embryos stopped their development in two- or four-cell stage. A significantly higher number of embryos could reach the eight-cell stage in SD and SCX than for S (7.8 ± 1.0%, 7.2 ± 4.0% and 3.9 ± 3.1%, respectively). Blastocyst formation was only observed in S, CX and SCX, see Table 2. 4. Discussion Several chemical agents, including ethanol, inhibitors of protein synthesis (like cycloheximide), strontium-chloride, 6-dimethylaminopurine, ionophore A 23187 could stimulate oocytes in vitro. CX and ionophore A 2187 increase intracellular Ca2+ and strontium-chloride could induce Ca2+ oscillation, which occur during normal fertilization process. Increased Ca2+ destroys the existing Ca-sensitive cytostatic factor (CSF), resulting in the reduction of maturation promoting factor (MPF) activity (Swann and Ozil, 1994). In mammalian oocytes, activity of MPF, which consist of cdc2 kinase and cyclin B, is essential for meiotic arrest at metaphase II (Nurse, 1990). Porcine oocytes are arrested at the metaphase II stage of meiosis and the activation is the release from this arrested stage and progression to first interphase. Cycloheximide is believed to inhibit the synthesis of calcium-sensitive cytostatic factor in M II oocytes (Siracusa et al., 1978; Clarke and Masui, 1983). The increased intracellular Ca2+ and the decreased CSF cause the inactivation of MPF, resulting parthenogenetic activation. In our experiments 57.37% of oocytes could be activated with cycloheximide. A lower fragmentation rate was observed in CX group (21.05%). In CX group the blastocyst rate was 3.16%. Martinaz Diaz et al. (2003) demonstrated that protein synthesis inhibition treatment required for the induction of oocyte activation promotes the development of nuclear transfer embryos. Strontium is a very effective activation agent for parthenogenetic activation of mouse, rat and bovine oocytes (Roh and Hwang, 2002; Meo et al., 2004). Strontium induces the Ca2+ oscillation in mouse (Kline and Kline, 1992). Chinese hamster oocytes could not be activated with strontium (Tateno and Kamiguchi, 1997). Krivokharchenko et al. (2003) successful stimulate rat oocytes with
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Treatments
S D CX Control
Number of oocytes
319 366 319 180
Number (%)a activated
170 (53.29) 176 (48.09) a 183 (57.37) a 3 (1.67)
Parthenogenetic development day 6 (%)b Non cleaved
Fragmented
Two-cell
Four-cell
Eight-cell
Morula
Blastocyst
25 (14.71) 16 (9.09) 21 (11.48) 0
53 (31.17) 60 (34.09) 38 (20.77) 0
32 (18.82) 20 (11.36) 23 (12.57) 3 (100)
15 (8.84) 31 (17.61) 37 (20.22) 0
20 (11.76) 33 (18.75) 35 (19.13) 0
20 (11.76) 16 (9.09) 23 (12.57) 0
5 (2.94) 0 6 (3.28) 0
Values with different letter in the same column differ significantly. Each treatment was repeated three times. Abbreviations—S: oocytes were activated with 10 mM strontiumchloride; D: oocytes were activated with 2 mM 6-dimethylaminopurine; CX: oocytes were activated with 0.04 mM cycloheximide; control: oocytes were only matured and cultured for 6 days. a Percentages mean of the original number of oocytes. b Percentages mean the number of oocytes activated.
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Table 1 Parthenogenetic development of porcine oocytes activated by strontium-chloride, cycloheximide and 6-dimethylaminopurine
Treatments
S SD SCX
Number of oocytes
319 346 302
Number (%)a activated
170 (53.29) 170 (49.13) 164 (54.30)
Parthenogenetic development day 6 (%)b Non cleaved
Fragmented
Two-cell
Four-cell
Eight-cell
Morula
25 (14.71) 36 (21.18) a 16 (9.75) a
53 (31.17) 53 (31.18) a 40 (24.39) a
32 (18.82) 40 (23.53) 21 (12.80)
15 (8.84) 15 (8.82) 21 (12.80)
20 (11.76) a 20 (11.76) 15 (8.82) b 11 (6.47) 32 (19.51) ab 21 (12.80)
Blastocyst 5 (2.94) 0 14 (8.54)
Values with different letters in the same column differ significantly. Each treatment was repeated three times. Abbreviations—S: oocytes were activated with 10 mM strontiumchloride; SD: oocytes were activated with strontium-chloride combined with dimethylaminopurine; SCX: oocytes were activated with strontium-chloride combined with cycloheximide. a Percentages mean of the original number of oocytes. b Percentages mean the number of oocytes activated.
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Table 2 Parthenogenetic development of porcine oocytes activated by combined chemical stimuli
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2 mM strontium. Lei et al. (2002) demonstrated that mouse oocytes could be activated with 10 mM strontium. Meo et al. (2004) produced bovine blastocysts (6.3%) from oocytes activated with strontium. Okada et al. (2003) activated pig oocytes by intracytoplasmic injection of strontium. The blastocyst rate was 29%. It has been shown that some combinations of chemical stimuli improve parthenogenetic activation in mouse (Hagemann et al., 1995), bovine (Presicce and Yang, 1994) and Chinese hamster oocytes (Tateno and Kamiguchi, 1997). In our experiments oocytes were activated by 10 mM strontium-chloride and strontium-chloride combined with cycloheximide and with 6-dimethylaminopurine. For the 6 day after activation the blastocyst rate was 2.94% in S-group. Until recently, activation of in vitro matured oocytes has been associated with low blastocyst development, slow embryo growth, and low cell numbers in blastocysts. Lee et al. (2004) noticed faster embryo development and observed expanded blastocysts 5 days after electrical activation. The greatest number of blastocyst (8.64%) was in SCX group, in which the oocytes were activated by strontium combined with cycloheximide. The lower fragmentation rate (21.05%) and the greatest number of blastocyst (8.64%) were in SX group. However most of the embryos stopped there development at the two- or four-cell stage. This could be explained by the low sensitivity of the 4-cell porcine embryos. Acknowledgements Financial support was provided by the grants of the Hungarian Scientific Research Fund-T 43131. The authors are grateful to Dr. Zolt´an Machaty for critical reading of the manuscript. References Cha, S.K., Kim, N.H., Lee, S.M., Baik, C.S., Lee, H.T., Chung, K.S., 1997. Effect of cytochalsin B and cycloheximide on the activation rate, chromosome constituent and in vitro development of porcine oocytes following parthenogenetic stimulation. Reprod. Fertil. Dev. 9, 441–446. Clarke, H., Masui, J., 1983. The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes. Dev. Biol. 97, 291–301. Fraser, L.R., 1987. Strontium supports capacitacion and the acrosome reaction in mouse sperm and rapidly activates mouse eggs. Gamete Res. 18, 363–374. Funahashi, H., Cantley, T.C., Stumpf, T.T., Terlow, S.L., Day, B.N., 1994. In vitro development of in vitro matured porcine oocytes following chemical activation or in vitro fertilization. Biol. Reprod. 50, 1072–1077. Grupen, C.G., Mau, J.C., McLlfitrick, S.M., Maddocks, S., Nottle, M.B., 2002. Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes. Mol. Reprod. Dev. 62, 387–396. Hagemann, L.J., Hillery-Weinhold, F.L., Rutledge, M.L., First, N.L., 1995. Activation of murine oocytes with Ca2+ ionophore and cycloheximide. J. Exp. Zool. 271, 57–61. Kikuchi, K., 2002. Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biol. Reprod. 66, 1033–1041. Kline, D., Kline, J., 1992. Repetitive calcium transients and the role of calcium in exocitosis and cell cycle activation in the mouse egg. Dev. Biol. 149, 80–89. Krivokharchenko, A., Popova, E., Zaitseva, I., Vilianovich, L., Ganten, D., Bader, M., 2003. Development of parthenogenetic rat embryos. Biol. Reprod. 68, 829–836. Kure-bayashi, S., Miyake, M., Okada, K., Kato, S., 2000. Succesful implantation of in vitro matured, electro-activated oocytes in the pig. Theriogenology 53, 1105–1119. Lee, J.W., Tian, X.C., Yang, X., 2004. Optimization of parthenogenetic activation protocol in porcine. Mol. Reprod. Dev. 68, 51–57. Lei, L., Liu, Z.H., Kou, Z.H., Chen da, Y., 2002. Effects of different treatments on oocyte activation and parthenogenesis in mouse. Shi Yan Sheng Wu Xue Bao 35, 236–238.
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