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Phytohemagglutinin improves efficiency of electrofusing mammary gland epithelial cells into oocytes in goats
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Theriogenology 69 (2008) 1165–1171 www.theriojournal.com
Technical note
Phytohemagglutinin improves efficiency of electrofusing mammary gland epithelial cells into oocytes in goats Y.L. Zhang a, F.J. Liu b, D.Q. Sun a, X.Q. Chen a, Y. Zhang a,*, Y.M. Zheng a, M.T. Zhao a, G.H. Wang a a
b
Institute of Biotechnology, Northwest Sci-Tech University of Agriculture & Forestry, Yangling, Shaanxi 712100, China College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, Henan 471003, China Received 10 August 2007; received in revised form 28 October 2007
Abstract The objective was to investigate the effect of phytohemagglutinin (PHA) on the fusion of mammary gland epithelial (MGE) cells into enucleated oocytes in goats. The toxicity of PHA was evaluated by testing its effect on the development of parthenogenetic caprine oocytes. The effective dose and duration of PHA treatment (100 mg/mL, 20 min incubation) was selected and used to compare fusion efficiency and embryo development following nuclear transfer. Two electrofusion protocols, chamber fusion (CF) and pressurized microelectrode fusion (pMEF), were also compared, when couplets were treated with and without PHA (100 mg/ mL, 20 min). Fusion rate of couplets increased from 52.8 to 74.0% for the CF protocol (P < 0.05), but was not significantly different for the pMEF protocol (72.7% vs. 78.1%) after PHA treatment. There were no significant differences between treated group and control in rates of subsequent cleavage or blastocyst development. Following transfer of the cloned blastocysts derived from the PHA-treated group and the control group into synchronized recipients, pregnancy rates (Day 30) were not significantly different between treated group and control (28.6% vs. 25.0%). However, all recipients aborted within 120 d, microsatellite DNA analyses confirmed that the aborted fetuses were genetically identical to the donor goat. In conclusion, the fusion rate of caprine MGE cell couplets was improved by pre-incubating couplets in medium containing 100 mg/mL PHA prior to electrical pulsing, and embryos derived from PHA treatment established early pregnancies. # 2008 Published by Elsevier Inc. Keywords: Electrofusion; Mammary gland epithelial cells; Nuclear transfer; Phytohemagglutinin; Goat
1. Introduction Somatic cell nuclear transfer (SCNT) is a useful technique for producing large scale numbers of genetically identical animals [1,2]. Various somatic cell types have been used as donor cells in SCNT research [1,3]. However, the technique is still inefficient for some cell types, such as mammary gland epithelial (MGE) cells. Kishi et al. reported an overall efficiency of <1% with * Corresponding author. Tel.: +86 29 87080085; fax: +86 29 87080085. E-mail address: [email protected] (Y. Zhang). 0093-691X/$ – see front matter # 2008 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2007.10.028
MGE cells [4], partly due to the low efficiency of cell fusion. Therefore, achieving a higher rate of fusion has been one of the challenges of MGE cell nuclear transfer (NT). To date, few studies have been focused on caprine NT using MGE cells as donor cells, which may be due to the difficulty in MGE cells fusing to enucleated oocytes. In that regard, Kishi et al. [4] suggested that the contact between MGE cells and the enucleated oocytes might be the cause of this problem. Phytohemagglutinin (PHA) has the potential to induce closer contacts between adjacent cell membranes; it is an N-acetylgalactosamine/galactose sugarspecific lectin with a wide variety of biological activities
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[5], widely used for enhancing cell agglutination and fusion in various mammalian cells. Moreover, PHA has been successfully used for membrane-induced fusion in human oocytes [6], bovine oocytes [7], and caprine oocytes [8]. Thus, we hypothesized that PHA would promote contact between caprine MGE cells and oocyte cytoplast cells, and lead to higher fusion efficiency. The purpose of this study was to improve the fusion efficiency of caprine MGE cells into oocyte cytoplast cells using PHA. The MGE cell–oocyte cytoplast cell couplets were treated by PHA before electrofusion, with fusion rates and subsequent developmental potential of reconstructed embryos being determined. In addition, this study also compared two fusion methods, chamber fusion and pressurized microelectrode fusion (pMEF), for fusing caprine MGE cells into oocyte cytoplast cells. 2. Materials and methods 2.1. Chemicals Unless otherwise stated, all media and components were purchased from Sigma–Aldrich Corp. (St. Louis, MO, USA). 2.2. Oocyte recovery and in vitro maturation Caprine cumulus–oocyte complexes were recovered from abattoir-derived ovaries and cultured in TCM199 (31100-027; Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), 0.075 U/mL human menopausal gonadotropin (Livzon, China), 1 mg/mL estradiol-17b, 10 ng/mL EGF, 1% (v/v) insulin-transferrin-seuim (ITS), and 0.2 mM sodium pyruvate for IVM at 38.5 8C in 5% CO2 in air. After 20–24 h of IVM, the cumulus cells were removed from the oocytes by repeated pipetting in DPBS containing 0.1% hyaluronidase. The oocytes with the first polar body were selected for either parthenogenetic activation or enucleation for NT. 2.3. Preparation of mammary gland epithelial cells for nuclear transfer Mammary gland epithelial cells were derived from colostrum of a Saanen dairy goat by the modified method as described by Kishi et al. [4]. Cells were cultured in DMEM/F12 (11300-037; Gibco) supplemented with 10% FBS, 1% ITS, 100 IU/mL penicillin, and 0.1 mg/ mL streptomycin at 38.5 8C in 5% CO2 in air. After 3–5 passages, the confluent cells were starved in 0.5% FBS for 3–6 d before SCNT, and then trypsinized using 0.25%
trypsin–EDTA. The dissociated cells were washed three or four times by centrifugation (300 g for 2 min), and 100–200 cells were inserted into the injection droplets. 2.4. Nuclear transfer and cell fusion Before enucleation, denuded MII oocytes were incubated in M199-Hepes (12350-039; Gibco) containing 5 mg/mL Hoechst 33342, 7.5 mg/mL cytochalasin B, and 5% FBS for 10–15 min (to visualize the metaphase plate). Groups of 30 oocytes were then transferred to a micromanipulation droplet (30 mL) overlaid with warmed mineral oil. Using the holding pipette to stabilize the oocyte, the metaphase plate was rotated into focus with the enucleation pipette while under a brief exposure (<10 s) to UV-light (Nikon Inc., Tokyo, Japan). A slight negative pressure was applied to the holding pipette, and the enucleation pipette was pushed through the zona pellucida until it was adjacent to the metaphase chromosomes. While exposed to UV-light (<5 s), the chromosomes and the first polar body were withdrawn from the oocyte by gentle suction applied to the enucleation pipette. Enucleated oocytes were washed and transferred into 30 mL droplets of M199 and held for at least 30 min (38.5 8C and 5% CO2) to await reconstruction. A single round donor cell with smooth membrane was then injected into the perivitelline space of each enucleated oocyte in M199-Hepes containing 5% FBS with the same enucleation pipette. Karyoplast–cytoplast couplets were transferred into Zimmermann’s fusion media (0.3 M mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium chloride, 0.05% BSA) to equilibrate for 5 min. Before fusion, for pMEF protocol, a couplet was aligned and gently pressurized by a pair of platinum electrodes (outside diameter, 15 mm) connected to the micromanipulator in the fusion medium droplet (30 mL), or for CF protocol, 5–10 couplets in each group were manually aligned with a fine glass pipette in a chamber with two stainless steel electrodes (0.5 mm apart), overlaid with fusion medium. Electric fusion was accomplished by a double electrical pulse (2.2 kV/cm for 10 ms). Following electric pulsing, couplets were incubated for 4–5 h in 10% FBS M199 at 38.5 8C in 5% CO2 in air, following being subjected to further activation procedures. Fusion rates were determined 60 min after the electrical pulse. 2.5. Activation, in vitro culture of embryos and cell number counting of blastocysts Caprine MII oocytes and NT embryos were activated by 5 min exposure to 5 mM ionomycin, and then
Y.L. Zhang et al. / Theriogenology 69 (2008) 1165–1171
incubated for 4 h in 2 mM 6-dimethylaminopurine, after being washed extensively in synthetic oviductal fluid (SOF) [9] modified by addition of 2% (v/v) Eagle basal medium amino acids solution, 1% (v/v) Eagle medium non-essential amino acids solution, 1 mM glutamine, and 8 mg/mL fatty acid free-BSA (mSOFaa). After activation, the embryos were washed again and co-cultured with granulosa cells in a 30 mm U shape glass dish with 500 mL mSOFaa medium covered with mineral oil for 7 d at 38.5 8C in 5% CO2 in air. The culture medium was replaced by the same medium but with 10% FBS at 72 h. The development of blastocysts was examined at Day 7 of culture. The cell number of some NT blastocysts was determined by counting stained nuclei. In brief, cloned blastocysts were dyed in 5 mg/mL Hoechst 33342 for 5 min, and then the blastocysts were crushed on a carrier slip. Stained nuclei were counted under ultraviolet light, each was counted three times, and average values were recorded. 2.6. Embryo transfer Recipients (Saanen dairy goats) were synchronized by inserting intravaginal sponges (IBT, Yangling, Shaanxi, China) containing 60 mg of medroxyprogesterone acetate into the vagina of recipient goats. Sponges were removed on Day 10, and an injection of 0.1 mg of cloprostenol (IOZCAS, Beijing, China) was given. Estrus was observed 24–48 h after sponge removal. All blastocysts were transferred surgically to recipient goats 5–7 d after estrus (2 or 3 embryos/ recipient). Pregnancy diagnosis (ultrasonography) was done at 30, 60, 90, and 120 d of gestation.
MII oocytes were first stained with TB (0.05%, 2 min). Unstained oocytes were classified as viable, stained oocytes as dead, and oocytes with intermediate degrees of staining were called ‘damaged’. Unstained oocytes were washed three times with PBS and randomly allocated to five treatments with PHA in 10% FBS M199 medium (dose of PHA was 0, 50, 100, 150, 300, or 600 mg/mL), and for 0, 10, 20, 30, or 60 min (orthogonal experimental design). Thereafter, oocytes treated by PHA were restained with TB to evaluate the survival rate. Unstained oocytes were washed, activated and cultured in mSOFaa/ co-culture system as described above to determine their developmental potential. 2.8.2. Experiment 2: Fusion rate and subsequent development of cloned embryos incubated with PHA After determining the optimized PHA parameters (100 mg/mL for 20 min; Experiment 1), couplets were randomly allocated to four groups and were fused respectively with the pMEF protocol (Fig. 1C) or CF protocol, with PHA treatment for 20 min at a concentration of either 0 or 100 mg/mL in 10% FBS M199. The NT embryos were subsequently allocated to test in vitro developmental potential. 2.9. Statistical analysis Five replicates for each experiment were conducted. All percentage data were pooled and then tested by Chisquare analysis using the Data Processing System software (version 6.01; Refine Information Tech. Co., Ltd., China). Differences were considered significant at P < 0.05.
2.7. Microsatellite analysis
3. Results
Genomic DNAs were extracted from aborted fetal tissues and the white blood cells collected from the donor goat and recipient goats, and typed for microsatellite markers [2].
3.1. Experiment 1
2.8. Experimental design 2.8.1. Experiment 1: Determining the toxic effect of PHA on the development of parthenogenetic embryos Parthenogenetic activation was used to test the toxicity of PHA on embryo development in vitro according to the report of Du et al. [10] with some modifications. The viability of the oocytes was evaluated by trypan blue (TB) staining. To eliminate any negative effects caused by original unviable oocytes, after 24 h of IVM, denuded
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The survival rate of oocytes was not different when treated with PHA at a dose from 0 to 100 mg/mL for 20 min (Table 1). Likewise, the potential of blastocyst development was similar among groups exposed to 0, 50, or 100 mg/mL (55.9, 46.3, and 46.2%, respectively; P > 0.05). In contrast, groups exposed to >100 mg/mL had reduced oocyte development. For oocytes treated with PHA (100 mg/mL) for 0–20 min (Table 2), rates of survival (91.6–94.3%), cleavage (75.1–82.6%), and blastocyst development (44.8– 55.3%) were not significantly affected. However, when duration of incubation exceeded 30 min, oocyte development was reduced, compared with shorter duration treatments (P < 0.05).
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Fig. 1. Mammary gland epithelial cells nuclear transfer using phytohemagglutinin as an agglutination agent and pressurized microelectrode fusion protocol in goat (150). The donor cell often stuck to the oocyte’s zona pellucida without PHA treatment (A). The donor cell and recipient oocyte adhered together after PHA treatment (B). A karyoplast–cytoplast couplet was subjected to cell fusion induced by direct electrical pulses using the pressurized microelectrode fusion protocol (C). A donor MGE cell fusing into a cytoplast following an electric pulse (D). Bar = 50 mm.
3.2. Experiment 2 In Experiment 2 (Table 3), the fusion rate of MGE cells into oocytes increased from 52.8 to 74.0% for the CF
protocol (P < 0.05), but was not significantly different for the pMEF protocol after PHA treatment. There was not significant difference between groups in subsequent cleavage rates and blastocysts development. Further-
Table 1 Effect of phytohemagglutinin concentration on parthenogenetic development of activated caprine oocytes PHA* (mg/mL) 0 50 100 150 300 600 a–d
No. of oocytes examined 246 258 297 239 221 265
No. of oocytes survived (%) ** 236 244 279 203 165 136
a
(95.9) (94.6)a (93.9)a (84.9)b (74.7)c (51.3)d
No. of cleaved (%)*** 195 190 210 141 102 81
Within a column, values without a common superscript differ (P < 0.05). Phytohemagglutinin. ** Survival rate = no. of viable oocytes after try pan blue staining/no. of examined oocytes. *** Percentage of the number of survived embryos. **** Percentage of the number of cleaved embryos. *
a
(82.6) (77.9)ab (75.3)ab (69.5)bc (61.8)bc (59.6)c
No. of blastocysts (%) **** 109(55.9)a 88 (46.3)a 97 (46.2)a 44 (31.2)c 29 (28.4)cd 14 (17.3)d
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Table 2 Effect of duration of phytohemagglutinin (100 mg/mL) treatment on parthenogenetic development of activated caprine oocytes Duration (min)
No. of oocytes examined
No. of oocytes survived (%) *
0 10 20 30 60
244 256 263 239 272
230 232 241 213 240
No. of cleaved (%) **
(94.3)a (90.6)ab (91.6)ab (89.1)ab (88.2)b
190 175 181 127 129
(82.6)a (75.4)a (75.1)a (59.6)b (53.8)b
No. of blastocysts (%)*** (55.3)a (44.3)ab (44.8)ab (32.3)bc (20.9)c
105 74 81 41 27
a–c
Within a column, values without a common superscript differ (P < 0.05). Survival rate = no. of viable oocytes after trypan blue staining/no. of examined oocytes. ** Percentage of the number of survived embryos. *** Percentage of the number of cleaved embryos. *
Table 3 Effects of phytohemagglutinin treatment and fusion protocols on the fusion efficiency and development of reconstructed caprine embryos Fusion protocols
PHA* (mg/mL)
No. of couplets examined
No. of fused couplets (%) **
No. of cleaved (%) ***
No. of blastocysts (%) ****
Total efficiency (%) **
Chamber fusion
0 100
195 204
103 (52.8) a 151 (74.0) b
80 (77.7) 118 (78.1)
13 (16.3) 21 (17.8)
13 (6.7)a 21 (10.3)ab
Pressurized microelectrode fusion
0 100
205 215
149 (72.7) b 168 (78.1) b
113 (75.8) 133 (79.2)
21 (18.6) 27 (20.3)
21 (10.2)ab 27 (12.6)b
a,b
Within a column, values without a common superscript differ (P < 0.05). Phytohemagglutinin. ** Percentage of the number of examined couplets. *** Percentage of the number of fused couplets. **** Percentage of the number of cleaved embryos. *
more, the efficiency of NT blastocyst development was markedly improved from 6.7 to 12.6% when PHA and pMEF were combined. The quality of NT blastocysts, estimated by their mean cell numbers, was not different between the PHA-treated group (115, n = 34) and the control group (109, n = 38). 3.3. Development in vivo of cloned embryos following PHA treatment To evaluate the complete developmental potential of cloned embryos derived from the couplets treated by PHA, 32 embryos derived from the treated group (PHA+) and 28 embryos from the control group (PHA ) were surgically transferred to 26 recipients
(12 for PHA+ and 14 for PHA ). At 30 d, there were three pregnancies in the PHA+ group and four in the PHA group (Table 4). However, all recipients aborted within 120 d, there was no difference in the pregnancy rate between PHA+ and PHA group (25.0% vs. 28.6%). Microsatellite DNA analyses confirmed that the aborted fetuses were genetically identical to the donor goat and different from the recipient goats (data not shown). 4. Discussion It has been proposed that cell fusion occurs by a mechanism involving three stages [11]. In brief, (1) cell fusogens induced close contact between adjacent cell
Table 4 Development in vivo of cloned caprine embryos derived from phytohemagglutinin treatment PHA* (mg/mL)
0 100
No. of blastocysts transferred
No. of recipients transferred
No. (%) of day of pregnant recipients on 30
60
90
120
32 28
14 12
4 (28.6) 3 (25.0)
2 (14.3) 2 (16.7)
1 (7.1) 1 (8.3)
0 0
No significant difference between groups for any end point. * Phytohemagglutinin.
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membranes, (2) membrane fusion occurred at small localized sites of cell contact, and (3) expansion of sites of membrane fusion to form spherical fused cells by a process of cell swelling. It was thought that PHA facilitated close cell contact by binding to the N-linked carbohydrate core structure (beta 1–6 branching) of glycoproteins on the cell membrane [12,13]. Since the cell membranes of caprine oocytes and MGE donor cells contained a variety of glycoproteins containing beta 1–6 branching, we concluded that PHA recognized and mediated adhesion/fusion. Based on Experiment 1, PHA treatment (appropriate dose and duration of incubation) did not affect the development of parthenogenetic embryos. In previous reports, the dose of PHA used in SCNT was 10 mg/mL [14], 20 mg/mL [15], 50 mg/mL [16], 100 mg/mL [4], 300 mg/mL [17], even up to 500 mg/mL [18,19]. In this research, PHA reduced parthenogenic development of oocytes at concentrations >100 mg/mL and exposure of >30 min duration. The difference between the abovementioned reports and the present research was that the treatment duration of PHA was longer (20 min) in our experiments than that of other studies (3 s) [18,19]. In addition, they treated either cytoplasm (zona-free) or donor cells with PHA, so cell sticking may have occurred very quickly. In the present study, couplets were enveloped by a zona pellucida; therefore, PHA had to penetrate the zona pellucida to enhance sticking, which may have accounted for the longer duration required. Similar results were reported by Du et al. [10]. In this study, the fusion rate was improved by preincubating couplets in medium containing 100 mg/mL PHA before electrical pulsing and embryos derived from the PHA treatment established early pregnancies. The efficiency of fusion was markedly increased (from 52.8 to 74.0%) using PHA for the CF protocol, presumably due to its agglutinating factors that assisted in the adhesion between a MGE cell and a recipient cytoplast. Similar results were reported for NT studies using other donor cell types and species. Du et al. [10] treated cattle couplets (derived from fibroblasts and cumulus cells) with 150 mg/mL PHA for 20 min; consequently, electrofusion rate and cloning efficiency were significantly improved compared with the control group (without PHA treatment). Similarly, Begin et al. [8] increased the fusion rate of couplets reconstructed with caprine cumulus–granulosa cells and oocytes with 150 mg/mL PHA. However, in the present study, the efficiency of fusion was not markedly improved following PHA treatment for pMEF protocol (72.7% vs. 78.1%), which was probably attributed to the donor cell-to-cytoplast membrane contact enhanced by micro-
electrode pressurization. Perhaps pressurization had a similar function of PHA to improve tightness of membrane contact. Unfortunately, the cloned embryos derived from MGE cells did not develop into normal offspring, which probably was due to few recipient numbers, donor cell type, or potential cell toxicity of PHA. Begin et al. [8] established pregnancies resulting from caprine NT embryos produced by fusing couplets reconstructed with cumulus–granulosa cells and the enucleated oocytes in the presence of lectin (100 mg/mL). However, they also failed to get viable offspring. Therefore, pregnancy failure might be related to the potential cell toxicity of PHA. Whether PHA negatively effects full term development of caprine cloned embryos needs further study. The loss of all pregnancies might be attributed to uncompleted epigenetic reprogramming of MGE cells induced by the limitations of the present NT technique. There is no report regarding birth of offspring cloned with caprine MGE cells, the present NT protocol of caprine MGE cells need to be further optimized. In conclusion, both PHA and pressurization had a positive effect on the fusion of MGE cell–cytoplast couplets. Furthermore, the fusion efficiency of NT was improved without decreasing the early development potential of the reconstructed embryos. Although the embryos derived from the PHA treatment established early pregnancies, they were not maintained until term. Acknowledgment This work was supported by the National High Technique Research and Development Plan (863) Item (Project No. 2004AA213072). References [1] Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385:810–3. [2] Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, et al. Eight calves cloned from somatic cells of a single adult. Science 1998;282:2095–8. [3] Cho JK, Lee BC, Park JI, Lim JM, Shin SJ, Kim KY, et al. Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation. Theriogenology 2002;57:1819–28. [4] Kishi M, Itagaki Y, Takakura R, Imamura M, Sudo T, Yoshinari M, et al. Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells. Theriogenology 2000;54:675–84. [5] Gupta MK, Uhm SJ, Han DW, Lee HT. Embryo quality and production efficiency of porcine parthenotes is improved by phytohemagglutinin. Mol Reprod Dev 2007;74: 435–444.
Y.L. Zhang et al. / Theriogenology 69 (2008) 1165–1171 [6] Tesarik J, Nagy ZP, Mendoza C, Greco E. Chemically and mechanically induced membrane fusion: nonactivating methods for nuclear transfer in mature human oocytes. Hum Reprod 2000;15:1149–54. [7] Hong SB, Uhm SJ, Lee HY, Park CY, Gupta MK, Chung BH, et al. Developmental ability of bovine embryos nuclear transferred with frozen–thawed or cooled donor cells. Asian Aust J Anim Sci 2005;18:1242–8. [8] Begin I, Bhatia B, Rao K, Keyston R, Pierson JT, Neveu N, et al. Pregnancies resulted from goat NT embryos produced by fusing couplets in the presence of lectin. Reprod Fertil Dev 2004;16: 136. [9] Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972;30: 493–7. [10] Du F, Shen PC, Xu J, Sung LY, Jeong BS, Nedambale TL, et al. The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus). Theriogenology 2006;6:642–57. [11] Knutton S. Studies of membrane fusion. III. Fusion of erythrocytes with polyethylene glycol. J Cell Sci 1979;36: 61–72. [12] Hamelryck TW, Dao-Thi MH, Poortmans F, Chrispeels MJ, Wyns L, Loris R. The crystallographic structure of phytohemagglutinin-L. J Biol Chem 1996;271:20479–85.
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[13] Sharma V, Surolia A. Analyses of carbohydrate recognition by legume lectins: size of the combining site loops and their primary specificity. J Mol Biol 1997;267:433–45. [14] Lavoir MC, Rumph N, Moens A, King WA, Plante Y, Johnson WH, et al. Development of bovine nuclear transfer embryos made with oogonia. Biol Reprod 1997;56:194–9. [15] Keefer CL, Stice SL, Matthews DL. Bovine inner cell mass cells as donor nuclei in the production of nuclear transfer embryos and calves. Biol Reprod 1994;50:935–9. [16] Yang BC, Im GS, Kim DH, Yang BS, Oh HJ, Park HS, et al. Development of vitrified-thawed bovine oocytes after in vitro fertilization and somatic cell nuclear transfer. Anim Reprod Sci 2008;103:25–37. [17] Booth PJ, Viuff D, Tan S, Holm P, Greve T, Callesen H. Numerical chromosome errors in Day 7 somatic nuclear transfer bovine blasto. Biol Reprod 2003;68:922–8. [18] Vajta G, Bartels P, Joubert J, de la Rey M, Treadwell R, Callesen H. Production of a healthy calf by somatic cell nuclear transfer without micromanipulators and carbon dioxide incubators using the handmade cloning (HMC) and the submarine incubation system (SIS). Theriogenology 2004;62:1465–72. [19] Bhojwani S, Alm H, Torner H, Kanitz W, Poehland R. Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer. Theriogenology 2007;67:341–5.
Theriogenology 69 (2008) 1165–1171 www.theriojournal.com
Technical note
Phytohemagglutinin improves efficiency of electrofusing mammary gland epithelial cells into oocytes in goats Y.L. Zhang a, F.J. Liu b, D.Q. Sun a, X.Q. Chen a, Y. Zhang a,*, Y.M. Zheng a, M.T. Zhao a, G.H. Wang a a
b
Institute of Biotechnology, Northwest Sci-Tech University of Agriculture & Forestry, Yangling, Shaanxi 712100, China College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, Henan 471003, China Received 10 August 2007; received in revised form 28 October 2007
Abstract The objective was to investigate the effect of phytohemagglutinin (PHA) on the fusion of mammary gland epithelial (MGE) cells into enucleated oocytes in goats. The toxicity of PHA was evaluated by testing its effect on the development of parthenogenetic caprine oocytes. The effective dose and duration of PHA treatment (100 mg/mL, 20 min incubation) was selected and used to compare fusion efficiency and embryo development following nuclear transfer. Two electrofusion protocols, chamber fusion (CF) and pressurized microelectrode fusion (pMEF), were also compared, when couplets were treated with and without PHA (100 mg/ mL, 20 min). Fusion rate of couplets increased from 52.8 to 74.0% for the CF protocol (P < 0.05), but was not significantly different for the pMEF protocol (72.7% vs. 78.1%) after PHA treatment. There were no significant differences between treated group and control in rates of subsequent cleavage or blastocyst development. Following transfer of the cloned blastocysts derived from the PHA-treated group and the control group into synchronized recipients, pregnancy rates (Day 30) were not significantly different between treated group and control (28.6% vs. 25.0%). However, all recipients aborted within 120 d, microsatellite DNA analyses confirmed that the aborted fetuses were genetically identical to the donor goat. In conclusion, the fusion rate of caprine MGE cell couplets was improved by pre-incubating couplets in medium containing 100 mg/mL PHA prior to electrical pulsing, and embryos derived from PHA treatment established early pregnancies. # 2008 Published by Elsevier Inc. Keywords: Electrofusion; Mammary gland epithelial cells; Nuclear transfer; Phytohemagglutinin; Goat
1. Introduction Somatic cell nuclear transfer (SCNT) is a useful technique for producing large scale numbers of genetically identical animals [1,2]. Various somatic cell types have been used as donor cells in SCNT research [1,3]. However, the technique is still inefficient for some cell types, such as mammary gland epithelial (MGE) cells. Kishi et al. reported an overall efficiency of <1% with * Corresponding author. Tel.: +86 29 87080085; fax: +86 29 87080085. E-mail address: [email protected] (Y. Zhang). 0093-691X/$ – see front matter # 2008 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2007.10.028
MGE cells [4], partly due to the low efficiency of cell fusion. Therefore, achieving a higher rate of fusion has been one of the challenges of MGE cell nuclear transfer (NT). To date, few studies have been focused on caprine NT using MGE cells as donor cells, which may be due to the difficulty in MGE cells fusing to enucleated oocytes. In that regard, Kishi et al. [4] suggested that the contact between MGE cells and the enucleated oocytes might be the cause of this problem. Phytohemagglutinin (PHA) has the potential to induce closer contacts between adjacent cell membranes; it is an N-acetylgalactosamine/galactose sugarspecific lectin with a wide variety of biological activities
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[5], widely used for enhancing cell agglutination and fusion in various mammalian cells. Moreover, PHA has been successfully used for membrane-induced fusion in human oocytes [6], bovine oocytes [7], and caprine oocytes [8]. Thus, we hypothesized that PHA would promote contact between caprine MGE cells and oocyte cytoplast cells, and lead to higher fusion efficiency. The purpose of this study was to improve the fusion efficiency of caprine MGE cells into oocyte cytoplast cells using PHA. The MGE cell–oocyte cytoplast cell couplets were treated by PHA before electrofusion, with fusion rates and subsequent developmental potential of reconstructed embryos being determined. In addition, this study also compared two fusion methods, chamber fusion and pressurized microelectrode fusion (pMEF), for fusing caprine MGE cells into oocyte cytoplast cells. 2. Materials and methods 2.1. Chemicals Unless otherwise stated, all media and components were purchased from Sigma–Aldrich Corp. (St. Louis, MO, USA). 2.2. Oocyte recovery and in vitro maturation Caprine cumulus–oocyte complexes were recovered from abattoir-derived ovaries and cultured in TCM199 (31100-027; Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), 0.075 U/mL human menopausal gonadotropin (Livzon, China), 1 mg/mL estradiol-17b, 10 ng/mL EGF, 1% (v/v) insulin-transferrin-seuim (ITS), and 0.2 mM sodium pyruvate for IVM at 38.5 8C in 5% CO2 in air. After 20–24 h of IVM, the cumulus cells were removed from the oocytes by repeated pipetting in DPBS containing 0.1% hyaluronidase. The oocytes with the first polar body were selected for either parthenogenetic activation or enucleation for NT. 2.3. Preparation of mammary gland epithelial cells for nuclear transfer Mammary gland epithelial cells were derived from colostrum of a Saanen dairy goat by the modified method as described by Kishi et al. [4]. Cells were cultured in DMEM/F12 (11300-037; Gibco) supplemented with 10% FBS, 1% ITS, 100 IU/mL penicillin, and 0.1 mg/ mL streptomycin at 38.5 8C in 5% CO2 in air. After 3–5 passages, the confluent cells were starved in 0.5% FBS for 3–6 d before SCNT, and then trypsinized using 0.25%
trypsin–EDTA. The dissociated cells were washed three or four times by centrifugation (300 g for 2 min), and 100–200 cells were inserted into the injection droplets. 2.4. Nuclear transfer and cell fusion Before enucleation, denuded MII oocytes were incubated in M199-Hepes (12350-039; Gibco) containing 5 mg/mL Hoechst 33342, 7.5 mg/mL cytochalasin B, and 5% FBS for 10–15 min (to visualize the metaphase plate). Groups of 30 oocytes were then transferred to a micromanipulation droplet (30 mL) overlaid with warmed mineral oil. Using the holding pipette to stabilize the oocyte, the metaphase plate was rotated into focus with the enucleation pipette while under a brief exposure (<10 s) to UV-light (Nikon Inc., Tokyo, Japan). A slight negative pressure was applied to the holding pipette, and the enucleation pipette was pushed through the zona pellucida until it was adjacent to the metaphase chromosomes. While exposed to UV-light (<5 s), the chromosomes and the first polar body were withdrawn from the oocyte by gentle suction applied to the enucleation pipette. Enucleated oocytes were washed and transferred into 30 mL droplets of M199 and held for at least 30 min (38.5 8C and 5% CO2) to await reconstruction. A single round donor cell with smooth membrane was then injected into the perivitelline space of each enucleated oocyte in M199-Hepes containing 5% FBS with the same enucleation pipette. Karyoplast–cytoplast couplets were transferred into Zimmermann’s fusion media (0.3 M mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium chloride, 0.05% BSA) to equilibrate for 5 min. Before fusion, for pMEF protocol, a couplet was aligned and gently pressurized by a pair of platinum electrodes (outside diameter, 15 mm) connected to the micromanipulator in the fusion medium droplet (30 mL), or for CF protocol, 5–10 couplets in each group were manually aligned with a fine glass pipette in a chamber with two stainless steel electrodes (0.5 mm apart), overlaid with fusion medium. Electric fusion was accomplished by a double electrical pulse (2.2 kV/cm for 10 ms). Following electric pulsing, couplets were incubated for 4–5 h in 10% FBS M199 at 38.5 8C in 5% CO2 in air, following being subjected to further activation procedures. Fusion rates were determined 60 min after the electrical pulse. 2.5. Activation, in vitro culture of embryos and cell number counting of blastocysts Caprine MII oocytes and NT embryos were activated by 5 min exposure to 5 mM ionomycin, and then
Y.L. Zhang et al. / Theriogenology 69 (2008) 1165–1171
incubated for 4 h in 2 mM 6-dimethylaminopurine, after being washed extensively in synthetic oviductal fluid (SOF) [9] modified by addition of 2% (v/v) Eagle basal medium amino acids solution, 1% (v/v) Eagle medium non-essential amino acids solution, 1 mM glutamine, and 8 mg/mL fatty acid free-BSA (mSOFaa). After activation, the embryos were washed again and co-cultured with granulosa cells in a 30 mm U shape glass dish with 500 mL mSOFaa medium covered with mineral oil for 7 d at 38.5 8C in 5% CO2 in air. The culture medium was replaced by the same medium but with 10% FBS at 72 h. The development of blastocysts was examined at Day 7 of culture. The cell number of some NT blastocysts was determined by counting stained nuclei. In brief, cloned blastocysts were dyed in 5 mg/mL Hoechst 33342 for 5 min, and then the blastocysts were crushed on a carrier slip. Stained nuclei were counted under ultraviolet light, each was counted three times, and average values were recorded. 2.6. Embryo transfer Recipients (Saanen dairy goats) were synchronized by inserting intravaginal sponges (IBT, Yangling, Shaanxi, China) containing 60 mg of medroxyprogesterone acetate into the vagina of recipient goats. Sponges were removed on Day 10, and an injection of 0.1 mg of cloprostenol (IOZCAS, Beijing, China) was given. Estrus was observed 24–48 h after sponge removal. All blastocysts were transferred surgically to recipient goats 5–7 d after estrus (2 or 3 embryos/ recipient). Pregnancy diagnosis (ultrasonography) was done at 30, 60, 90, and 120 d of gestation.
MII oocytes were first stained with TB (0.05%, 2 min). Unstained oocytes were classified as viable, stained oocytes as dead, and oocytes with intermediate degrees of staining were called ‘damaged’. Unstained oocytes were washed three times with PBS and randomly allocated to five treatments with PHA in 10% FBS M199 medium (dose of PHA was 0, 50, 100, 150, 300, or 600 mg/mL), and for 0, 10, 20, 30, or 60 min (orthogonal experimental design). Thereafter, oocytes treated by PHA were restained with TB to evaluate the survival rate. Unstained oocytes were washed, activated and cultured in mSOFaa/ co-culture system as described above to determine their developmental potential. 2.8.2. Experiment 2: Fusion rate and subsequent development of cloned embryos incubated with PHA After determining the optimized PHA parameters (100 mg/mL for 20 min; Experiment 1), couplets were randomly allocated to four groups and were fused respectively with the pMEF protocol (Fig. 1C) or CF protocol, with PHA treatment for 20 min at a concentration of either 0 or 100 mg/mL in 10% FBS M199. The NT embryos were subsequently allocated to test in vitro developmental potential. 2.9. Statistical analysis Five replicates for each experiment were conducted. All percentage data were pooled and then tested by Chisquare analysis using the Data Processing System software (version 6.01; Refine Information Tech. Co., Ltd., China). Differences were considered significant at P < 0.05.
2.7. Microsatellite analysis
3. Results
Genomic DNAs were extracted from aborted fetal tissues and the white blood cells collected from the donor goat and recipient goats, and typed for microsatellite markers [2].
3.1. Experiment 1
2.8. Experimental design 2.8.1. Experiment 1: Determining the toxic effect of PHA on the development of parthenogenetic embryos Parthenogenetic activation was used to test the toxicity of PHA on embryo development in vitro according to the report of Du et al. [10] with some modifications. The viability of the oocytes was evaluated by trypan blue (TB) staining. To eliminate any negative effects caused by original unviable oocytes, after 24 h of IVM, denuded
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The survival rate of oocytes was not different when treated with PHA at a dose from 0 to 100 mg/mL for 20 min (Table 1). Likewise, the potential of blastocyst development was similar among groups exposed to 0, 50, or 100 mg/mL (55.9, 46.3, and 46.2%, respectively; P > 0.05). In contrast, groups exposed to >100 mg/mL had reduced oocyte development. For oocytes treated with PHA (100 mg/mL) for 0–20 min (Table 2), rates of survival (91.6–94.3%), cleavage (75.1–82.6%), and blastocyst development (44.8– 55.3%) were not significantly affected. However, when duration of incubation exceeded 30 min, oocyte development was reduced, compared with shorter duration treatments (P < 0.05).
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Fig. 1. Mammary gland epithelial cells nuclear transfer using phytohemagglutinin as an agglutination agent and pressurized microelectrode fusion protocol in goat (150). The donor cell often stuck to the oocyte’s zona pellucida without PHA treatment (A). The donor cell and recipient oocyte adhered together after PHA treatment (B). A karyoplast–cytoplast couplet was subjected to cell fusion induced by direct electrical pulses using the pressurized microelectrode fusion protocol (C). A donor MGE cell fusing into a cytoplast following an electric pulse (D). Bar = 50 mm.
3.2. Experiment 2 In Experiment 2 (Table 3), the fusion rate of MGE cells into oocytes increased from 52.8 to 74.0% for the CF
protocol (P < 0.05), but was not significantly different for the pMEF protocol after PHA treatment. There was not significant difference between groups in subsequent cleavage rates and blastocysts development. Further-
Table 1 Effect of phytohemagglutinin concentration on parthenogenetic development of activated caprine oocytes PHA* (mg/mL) 0 50 100 150 300 600 a–d
No. of oocytes examined 246 258 297 239 221 265
No. of oocytes survived (%) ** 236 244 279 203 165 136
a
(95.9) (94.6)a (93.9)a (84.9)b (74.7)c (51.3)d
No. of cleaved (%)*** 195 190 210 141 102 81
Within a column, values without a common superscript differ (P < 0.05). Phytohemagglutinin. ** Survival rate = no. of viable oocytes after try pan blue staining/no. of examined oocytes. *** Percentage of the number of survived embryos. **** Percentage of the number of cleaved embryos. *
a
(82.6) (77.9)ab (75.3)ab (69.5)bc (61.8)bc (59.6)c
No. of blastocysts (%) **** 109(55.9)a 88 (46.3)a 97 (46.2)a 44 (31.2)c 29 (28.4)cd 14 (17.3)d
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Table 2 Effect of duration of phytohemagglutinin (100 mg/mL) treatment on parthenogenetic development of activated caprine oocytes Duration (min)
No. of oocytes examined
No. of oocytes survived (%) *
0 10 20 30 60
244 256 263 239 272
230 232 241 213 240
No. of cleaved (%) **
(94.3)a (90.6)ab (91.6)ab (89.1)ab (88.2)b
190 175 181 127 129
(82.6)a (75.4)a (75.1)a (59.6)b (53.8)b
No. of blastocysts (%)*** (55.3)a (44.3)ab (44.8)ab (32.3)bc (20.9)c
105 74 81 41 27
a–c
Within a column, values without a common superscript differ (P < 0.05). Survival rate = no. of viable oocytes after trypan blue staining/no. of examined oocytes. ** Percentage of the number of survived embryos. *** Percentage of the number of cleaved embryos. *
Table 3 Effects of phytohemagglutinin treatment and fusion protocols on the fusion efficiency and development of reconstructed caprine embryos Fusion protocols
PHA* (mg/mL)
No. of couplets examined
No. of fused couplets (%) **
No. of cleaved (%) ***
No. of blastocysts (%) ****
Total efficiency (%) **
Chamber fusion
0 100
195 204
103 (52.8) a 151 (74.0) b
80 (77.7) 118 (78.1)
13 (16.3) 21 (17.8)
13 (6.7)a 21 (10.3)ab
Pressurized microelectrode fusion
0 100
205 215
149 (72.7) b 168 (78.1) b
113 (75.8) 133 (79.2)
21 (18.6) 27 (20.3)
21 (10.2)ab 27 (12.6)b
a,b
Within a column, values without a common superscript differ (P < 0.05). Phytohemagglutinin. ** Percentage of the number of examined couplets. *** Percentage of the number of fused couplets. **** Percentage of the number of cleaved embryos. *
more, the efficiency of NT blastocyst development was markedly improved from 6.7 to 12.6% when PHA and pMEF were combined. The quality of NT blastocysts, estimated by their mean cell numbers, was not different between the PHA-treated group (115, n = 34) and the control group (109, n = 38). 3.3. Development in vivo of cloned embryos following PHA treatment To evaluate the complete developmental potential of cloned embryos derived from the couplets treated by PHA, 32 embryos derived from the treated group (PHA+) and 28 embryos from the control group (PHA ) were surgically transferred to 26 recipients
(12 for PHA+ and 14 for PHA ). At 30 d, there were three pregnancies in the PHA+ group and four in the PHA group (Table 4). However, all recipients aborted within 120 d, there was no difference in the pregnancy rate between PHA+ and PHA group (25.0% vs. 28.6%). Microsatellite DNA analyses confirmed that the aborted fetuses were genetically identical to the donor goat and different from the recipient goats (data not shown). 4. Discussion It has been proposed that cell fusion occurs by a mechanism involving three stages [11]. In brief, (1) cell fusogens induced close contact between adjacent cell
Table 4 Development in vivo of cloned caprine embryos derived from phytohemagglutinin treatment PHA* (mg/mL)
0 100
No. of blastocysts transferred
No. of recipients transferred
No. (%) of day of pregnant recipients on 30
60
90
120
32 28
14 12
4 (28.6) 3 (25.0)
2 (14.3) 2 (16.7)
1 (7.1) 1 (8.3)
0 0
No significant difference between groups for any end point. * Phytohemagglutinin.
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membranes, (2) membrane fusion occurred at small localized sites of cell contact, and (3) expansion of sites of membrane fusion to form spherical fused cells by a process of cell swelling. It was thought that PHA facilitated close cell contact by binding to the N-linked carbohydrate core structure (beta 1–6 branching) of glycoproteins on the cell membrane [12,13]. Since the cell membranes of caprine oocytes and MGE donor cells contained a variety of glycoproteins containing beta 1–6 branching, we concluded that PHA recognized and mediated adhesion/fusion. Based on Experiment 1, PHA treatment (appropriate dose and duration of incubation) did not affect the development of parthenogenetic embryos. In previous reports, the dose of PHA used in SCNT was 10 mg/mL [14], 20 mg/mL [15], 50 mg/mL [16], 100 mg/mL [4], 300 mg/mL [17], even up to 500 mg/mL [18,19]. In this research, PHA reduced parthenogenic development of oocytes at concentrations >100 mg/mL and exposure of >30 min duration. The difference between the abovementioned reports and the present research was that the treatment duration of PHA was longer (20 min) in our experiments than that of other studies (3 s) [18,19]. In addition, they treated either cytoplasm (zona-free) or donor cells with PHA, so cell sticking may have occurred very quickly. In the present study, couplets were enveloped by a zona pellucida; therefore, PHA had to penetrate the zona pellucida to enhance sticking, which may have accounted for the longer duration required. Similar results were reported by Du et al. [10]. In this study, the fusion rate was improved by preincubating couplets in medium containing 100 mg/mL PHA before electrical pulsing and embryos derived from the PHA treatment established early pregnancies. The efficiency of fusion was markedly increased (from 52.8 to 74.0%) using PHA for the CF protocol, presumably due to its agglutinating factors that assisted in the adhesion between a MGE cell and a recipient cytoplast. Similar results were reported for NT studies using other donor cell types and species. Du et al. [10] treated cattle couplets (derived from fibroblasts and cumulus cells) with 150 mg/mL PHA for 20 min; consequently, electrofusion rate and cloning efficiency were significantly improved compared with the control group (without PHA treatment). Similarly, Begin et al. [8] increased the fusion rate of couplets reconstructed with caprine cumulus–granulosa cells and oocytes with 150 mg/mL PHA. However, in the present study, the efficiency of fusion was not markedly improved following PHA treatment for pMEF protocol (72.7% vs. 78.1%), which was probably attributed to the donor cell-to-cytoplast membrane contact enhanced by micro-
electrode pressurization. Perhaps pressurization had a similar function of PHA to improve tightness of membrane contact. Unfortunately, the cloned embryos derived from MGE cells did not develop into normal offspring, which probably was due to few recipient numbers, donor cell type, or potential cell toxicity of PHA. Begin et al. [8] established pregnancies resulting from caprine NT embryos produced by fusing couplets reconstructed with cumulus–granulosa cells and the enucleated oocytes in the presence of lectin (100 mg/mL). However, they also failed to get viable offspring. Therefore, pregnancy failure might be related to the potential cell toxicity of PHA. Whether PHA negatively effects full term development of caprine cloned embryos needs further study. The loss of all pregnancies might be attributed to uncompleted epigenetic reprogramming of MGE cells induced by the limitations of the present NT technique. There is no report regarding birth of offspring cloned with caprine MGE cells, the present NT protocol of caprine MGE cells need to be further optimized. In conclusion, both PHA and pressurization had a positive effect on the fusion of MGE cell–cytoplast couplets. Furthermore, the fusion efficiency of NT was improved without decreasing the early development potential of the reconstructed embryos. Although the embryos derived from the PHA treatment established early pregnancies, they were not maintained until term. Acknowledgment This work was supported by the National High Technique Research and Development Plan (863) Item (Project No. 2004AA213072). References [1] Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385:810–3. [2] Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, et al. Eight calves cloned from somatic cells of a single adult. Science 1998;282:2095–8. [3] Cho JK, Lee BC, Park JI, Lim JM, Shin SJ, Kim KY, et al. Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation. Theriogenology 2002;57:1819–28. [4] Kishi M, Itagaki Y, Takakura R, Imamura M, Sudo T, Yoshinari M, et al. Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells. Theriogenology 2000;54:675–84. [5] Gupta MK, Uhm SJ, Han DW, Lee HT. Embryo quality and production efficiency of porcine parthenotes is improved by phytohemagglutinin. Mol Reprod Dev 2007;74: 435–444.
Y.L. Zhang et al. / Theriogenology 69 (2008) 1165–1171 [6] Tesarik J, Nagy ZP, Mendoza C, Greco E. Chemically and mechanically induced membrane fusion: nonactivating methods for nuclear transfer in mature human oocytes. Hum Reprod 2000;15:1149–54. [7] Hong SB, Uhm SJ, Lee HY, Park CY, Gupta MK, Chung BH, et al. Developmental ability of bovine embryos nuclear transferred with frozen–thawed or cooled donor cells. Asian Aust J Anim Sci 2005;18:1242–8. [8] Begin I, Bhatia B, Rao K, Keyston R, Pierson JT, Neveu N, et al. Pregnancies resulted from goat NT embryos produced by fusing couplets in the presence of lectin. Reprod Fertil Dev 2004;16: 136. [9] Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972;30: 493–7. [10] Du F, Shen PC, Xu J, Sung LY, Jeong BS, Nedambale TL, et al. The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus). Theriogenology 2006;6:642–57. [11] Knutton S. Studies of membrane fusion. III. Fusion of erythrocytes with polyethylene glycol. J Cell Sci 1979;36: 61–72. [12] Hamelryck TW, Dao-Thi MH, Poortmans F, Chrispeels MJ, Wyns L, Loris R. The crystallographic structure of phytohemagglutinin-L. J Biol Chem 1996;271:20479–85.
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[13] Sharma V, Surolia A. Analyses of carbohydrate recognition by legume lectins: size of the combining site loops and their primary specificity. J Mol Biol 1997;267:433–45. [14] Lavoir MC, Rumph N, Moens A, King WA, Plante Y, Johnson WH, et al. Development of bovine nuclear transfer embryos made with oogonia. Biol Reprod 1997;56:194–9. [15] Keefer CL, Stice SL, Matthews DL. Bovine inner cell mass cells as donor nuclei in the production of nuclear transfer embryos and calves. Biol Reprod 1994;50:935–9. [16] Yang BC, Im GS, Kim DH, Yang BS, Oh HJ, Park HS, et al. Development of vitrified-thawed bovine oocytes after in vitro fertilization and somatic cell nuclear transfer. Anim Reprod Sci 2008;103:25–37. [17] Booth PJ, Viuff D, Tan S, Holm P, Greve T, Callesen H. Numerical chromosome errors in Day 7 somatic nuclear transfer bovine blasto. Biol Reprod 2003;68:922–8. [18] Vajta G, Bartels P, Joubert J, de la Rey M, Treadwell R, Callesen H. Production of a healthy calf by somatic cell nuclear transfer without micromanipulators and carbon dioxide incubators using the handmade cloning (HMC) and the submarine incubation system (SIS). Theriogenology 2004;62:1465–72. [19] Bhojwani S, Alm H, Torner H, Kanitz W, Poehland R. Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer. Theriogenology 2007;67:341–5.