Osmotic stress induced by sodium chloride, sucrose or trehalose improves cryotolerance and developmental competence of porcine oocytes

2009 ◽  
Vol 21 (2) ◽  
pp. 338 ◽  
Author(s):  
Lin Lin ◽  
Peter M. Kragh ◽  
Stig Purup ◽  
Masashige Kuwayama ◽  
Yutao Du ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Control Groups ◽  
Concentrated Solutions ◽  
Parthenogenetic Activation ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Osmotic Agents

Exposure of porcine oocytes to increased concentrations of NaCl prior to manipulation has been reported not only to increase cryotolerance after vitrification, but also to improve developmental competence after somatic cell nuclear transfer (SCNT). In the present study we compared the effects of NaCl with those of concentrated solutions of two non-permeable osmotic agents, namely sucrose and trehalose, on the cryotolerance and developmental competence of porcine oocytes. In Experiment 1, porcine in vitro-matured cumulus–oocyte complexes (COCs; n = 1200) were exposed to 588 mOsmol NaCl, sucrose or trehalose solutions for 1 h, allowed to recover for a further 1 h, vitrified, warmed and subjected to parthenogenetic activation. Both Day 2 (where Day 0 is the day of activation) cleavage and Day 7 blastocyst rates were significantly increased after NaCl, sucrose and trehalose osmotic treatments compared with untreated controls (cleavage: 46 ± 5%, 44 ± 7%, 45 ± 4% and 26 ± 6%, respectively; expanded blastocyst rate: 6 ± 1%, 6 ± 2%, 7 ± 2% and 1 ± 1%, respectively). In Experiment 2, COCs (n = 2000) were treated with 588 mOsmol NaCl, sucrose or trehalose, then used as recipients for SCNT (Day 0). Cleavage rates on Day 1 did not differ between the NaCl-, sucrose-, trehalose-treated and the untreated control groups (92 ± 3%, 95 ± 3%, 92 ± 2% and 94 ± 2%, respectively), but blastocyst rates on Day 6 were higher in all treated groups compared with control (64 ± 2%, 69 ± 5%, 65 ± 3% and 47 ± 4%, respectively). Cell numbers of Day 6 blastocysts were higher in the control and NaCl-treated groups compared with the sucrose- and trehalose-treated groups. In conclusion, treatment of porcine oocytes with osmotic stress improved developmental competence after vitrification combined with parthenogenetic activation, as well as after SCNT.

18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES

2014 ◽  
Vol 26 (1) ◽  
pp. 123
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
P. Hassel ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Handmade Cloning

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

79 DEVELOPMENTAL COMPETENCE OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE: IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 145
Author(s):  
A. R. Moawad ◽  
I. Choi ◽  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Control Groups ◽  
High Frequencies ◽  
Parthenogenetic Activation ◽  
And Control

Oocyte cryopreservation represents an important development in the field of assisted reproductive technologies. This study investigated the effects of vitrification on spindle morphology following subsequent in vitro maturation (IVM), cleavage, and development following IVF and parthenogenetic activation. The developmental competence of ovine oocytes vitrified at the germinal vesicle (GV) stage, matured, and used as cytoplast recipients for somatic cell nuclear transfer (SCNT) was also determined. Cumulus–oocyte complexes obtained at slaughter were divided into 3 groups: 1) untreated (control), 2) toxicity (exposed to vitrification solutions without freezing), and 3) vitrified (2008 Reprod. Fertil. Dev. 20, 122). At 24 hpm (hours post onset of maturation), oocytes were subjected to 1) immunostaining, 2) IVF, or 3) activation by 2 different protocols [calcium ionophore, cycloheximide, and cytochalasin B (CA+CHX/CB), or strontium and CB (Sr/CB)]. The SCNT was performed as previously described (2010 Reprod. Fertil. Dev. 22, 1000–1014). Presumptive zygotes were cultured in vitro for 7 days. No significant differences (P > 0.05; chi-square) were observed in the frequencies of oocytes with normal spindle configuration between vitrified, toxicity, and control groups (50.0, 54.9, and 70.4%, respectively). Cleavage 24, 48 hpi, and morula development (5 days pi) were significantly decreased (P < 0.01) in the vitrified group (17.3, 42.9, and 36.4%) compared with toxicity (47.0, 85.3, and 60.7%) and control (68.9, 89.7, and 62.6%) groups. Blastocyst development significantly decreased (P < 0.01) in the vitrified group (12.3%) compared with toxicity (42.7%) and control (40.4%) groups. Based on cleaved embryos, no significant difference was observed between vitrified and control groups (29.4 v. 45.1%). Post-activation, cleavage 24 hpa (hours post-activation, 6.2 v. 3.8%) and 48 hpa (28.4 v. 27.5%) was significantly lower (P < 0.05) in vitrified oocytes activated by (CA+CHX/CB and Sr/CB) than other groups. No blastocyst developed from vitrified oocytes activated by CA+CHX/CB; however, 3.8% developed from Sr/CB oocytes. This was significantly (P < 0.05) lower than toxicity and control (20.0 and 27.3%) groups. Following SCNT, high frequencies of enucleation (99%) and fusion (98%) were achieved in vitrified and control groups. Cleavage 24 and 48 hpa significantly decreased (P < 0.05) in the vitrified group (31.0 and 48.0%) compared with the control (55.1 and 85.0%). No significant differences were observed in morula (38.0 v. 46.7%) and blastocyst (13.0 v. 23.4%) development. The proportion of cleaved embryos that developed to blastocyst stages was similar in both groups (27.0%). No significant differences (t-test) were observed in total cell numbers, apoptotic nuclei, and proportion of diploid embryos. In conclusion, ovine oocytes vitrified at GV stage can be matured, fertilized, and develop in vitro with high developmental potential. Strontium can be used effectively for activation of vitrified/thawed ovine oocytes. Vitrified/thawed ovine oocytes were used successfully for the first time as recipient cytoplasts for SCNT and produced high frequencies of good-quality blastocyst stage embryos.

41 STUDY ON THE ESTABLISHMENT OF SOMATIC CELL NUCLEAR TRANSFER USING IN VIVO-MATURED OOCYTES IN DOGS

2006 ◽  
Vol 18 (2) ◽  
pp. 129 ◽  
Author(s):  
G. Jang ◽  
M. Kim ◽  
H. J. Oh ◽  
F. Y. Heru ◽  
M. S. Hossein ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Chemical Activation ◽  
Developmental Competence ◽  
Perivitelline Space ◽  
Parthenogenetic Activation ◽  
Vaginal Cytology

The present study was performed to collect in vivo matured canine oocytes for somatic cell nuclear transfer (SCNT) and to investigate the developmental competence of canine parthenogenetic and SCNT embryos as the preliminary research for producing cloned dog. The day of ovulation as described by Hase et al. (2000 J. Vet. Med. Sci. 62, 243-248) was determined by serum progesterone levels and at that time vaginal cytology was performed to assess the cornified index. In vivo-matured oocytes were recovered by retrograde flushing of the oviducts at around 48 h (n = 20) or 72 h (n = 25) after the estimated time of ovulation. Overall size of each oocyte, as well as ooplasmic diameter, zona pellucida thickness, and perivitelline space width, was determined after removing the cumulus cells by pipetting (Exp. 1). To determine activation protocols, two treatments, (1) chemical activation (10 �M Ca ionophore for 4 min, followed by incubation for 4 h with 1.9 mM 6-dimethylaminopurine) and (2) electrical stimulation (3.1?3.4 kV/cm in 0.25M mannitol solution), were evaluated to induce parthenogenetic activation of oocytes (Exp. 2). Donor cells were obtained from the primary cell culture of a canine ear skin biopsy, and SCNT was performed according to our laboratory procedures (Jang et al. 2004 Theriogenology 62, 512-521). Three voltages (1.7?2.0 kV/cm, 2.1-2.4 kV/cm, and 3.1-3.4 kV/cm) were tested for fusion. The fused couplets were subjected to chemical or electrical stimulation as in parthenogenetic activation and in vitro developmental competence was monitored (Exp. 3). As a result, more in vivo-matured canine oocytes were obtained at 72 h (92%) than at 48 h (15%) after ovulation; the 72-h occytes had progesterone concentrations of 4-8 ng/mL and a cornified index (vaginal cytology) of 83.34. The average number of oocytes recovered was 12 and sizes of ooplasmic diameter, cytoplasm, zona pellucida, and perivitelline space in in vivo canine-matured oocytes (n = 120) were 178.8 � 9.3 �m, 125.0 � 8.2 �m, 21.7 � 3.7 �m, and 12.7 � 3.5 �m, respectively. Parthenogenetically activated oocytes developed to the 16-cell and morula stages, but failed to develop to the blastocyst stage. Among the three voltages, in the highest voltage (75.2%) the number of fused couplets was increased compared to either of the other voltages (33.3% and 44.0%). Cleavage rates (60.9% vs. 58.0%) of cloned embryos were not significantly affected by method of activation. In terms of in vitro developmental competence, cloned embryos developed to the 16-cell or morula stage in vitro after electrical or chemical activation, respectively. In conclusion, in the present study we demonstrated that measurement of progesterone levels, in combination with evaluation of vaginal cytology, can be used to determine the estimated time of ovulation in bitches. In addition, we determined fusion/activation protocols that resulted in in vitro development of a portion of parthenogenetically activated and cloned embryos to the 16-cell and morula stages. This study was supported by grants from the Biogreen 21-1000520030100000.

43 COMPARISON OF THE EFFECTS OF PRE-TREATMENT WITH SODIUM CHLORIDE, SUCROSE AND TREHALOSE ON DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 121
Author(s):  
L. Lin ◽  
P. Kragh ◽  
S. Purup ◽  
Y. Du ◽  
X. Zhang ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Nuclear Transfer ◽  
Technical Assistance ◽  
Recovery Period ◽  
Cell Number ◽  
Developmental Competence ◽  
Fetal Fibroblasts ◽  
Pre Treatment ◽  
Maximum Humidity

Modified environmental stress was reported to improve the developmental competence and cryotolerance of porcine oocytes, such as high hydrostatic pressure (HHP; Du et al. 2008 Cloning Stem Cells, Epub ahead of print) and osmotic stress (Lin et al. 2008 Reprod. Biomed. Online, in press). HHP also improved the cryotolerance of bovine and murine blastocysts (Pribenszky et al. 2005a Reprod. Dom. Anim. 40, 338–344; Pribenszky et al. 2005b Anim. Reprod. Sci. 87, 143–150). In the present study we compared the effects of NaCl with that of concentrated solutions of two non-permeable osmotic agents, sucrose and trehalose on in vitro maturated oocytes. A total of 2050 slaughterhouse-derived porcine cumulus–oocyte complexes (COCs) were matured for 41–42 h, and then put into 800 μL T2 (HEPES-buffered TCM-199 [Earle’s salts] with 2% cattle serum) supplemented with additional NaCl, sucrose or trehalose with the same osmotic level (588 mOsmol) in 4-well dishes and incubated for 1 h at 38.5°C in air. COCs incubated in T2 under the same conditions without supplementation were used as controls. Subsequently COCs were incubated in IVM medium for 1 h at 38.5°C in 5% CO2 with maximum humidity. After this recovery period cumulus cells were removed with 1 mg mL–1 hyaluronidase and pipetting, and oocytes were used as recipients for somatic nuclear transfer with handmade cloning (HMC) method. Porcine fetal fibroblasts were used as nuclear donor cells. Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) in 5% CO2, 5% O2 and 90% N2 and maximum humidity. Cleavage and blastocyst rates were checked on Day 1 and Day 6, respectively. Cell numbers were counted after fixation in glycerol containing 20 μg mL–1 Hoechst 33342 fluorochrome on Day 6. t-test was performed for statistical calculations with SPSS 11.0 program (SPSS, Chicago, IL, USA). Results are shown in Table 1. Osmotic stress with both permeable and non-permeable agents increased developmental competence of porcine IVM oocytes. NaCl seems to be more appropriate for the purpose, as the other two components resulted in decreased cell number in blastocysts after somatic cell nuclear transfer (SCNT). In conclusion, a simple NaCl pre-treatment of oocytes has improved the in vitro efficiency of porcine SCNT. Table 1.Developmental competence of porcine HMC embryos derived from oocytes treated with different agents The authors thank Ruth Kristensen, Anette Pedersen, Janne Adamsen and Klaus Villemoes for their help and excellent technical assistance.

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer

2015 ◽  
Vol 27 (3) ◽  
pp. 544 ◽  
Author(s):  
H. S. Pedersen ◽  
Y. Liu ◽  
R. Li ◽  
S. Purup ◽  
P. Løvendahl ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Oocyte Growth ◽  
Parthenogenetic Activation ◽  
Selection Of

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter (‘small’ ≤110 µm; ‘medium’ >110 µm; ‘large’ ≥120 µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

134 EFFECTS OF VITAMIN E AND VITAMIN C ON THE DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION, IN VITRO FERTILIZATION, AND NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 171
Author(s):  
F. Lu ◽  
Z. Zhang ◽  
S. Zhang ◽  
N. Li ◽  
J. Jiang ◽  
...  
Keyword(s):  
Vitamin E ◽  
Nuclear Transfer ◽  
Bubalus Bubalis ◽  
Development Rate ◽  
The Other ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation

The purpose of this study was to explore the effects of vitamin E (VE) and vitamin C (VC) on the in vitro development of embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (NT) in buffalo (Bubalus bubalis). Buffalo oocytes obtained from ovaries at slaughter were matured in vitro for 22 to 24 h. After maturation, oocytes were separated to 3 groups: one group of oocytes was fertilized in vitro with buffalo sperm; one group of oocytes was parthenogenetically activated by exposing them to 5 μM ionomycin for 5 min and then cultured in 2 mM 6-DMAP for 3 h; the other group of oocytes was enucleated, and fibroblasts in DMEM + 10% FBS for 4 to 5 days were transferred into enucleated oocytes by electronic fusion (100 v mm–1, 15 μs, and 3 pulses). After fusion, the activation of reconstructed embryos was induced by exposure to 5 μM ionomycin for 5 min and then cultured in 2 mM 6-DMAP for 3 h. The embryos of PA, IVF, and NT were respectively cultured in the culture medium (CM) containing different concentrations of VE, VC, or VE + VC for 7 to 9 days to evaluate embryonic development. As a result, when the embryos were cultured in the CM with different concentrations of VE (0, 50, 100, 150, and 200 μM), the blastocyst development rate of the embryos derived from PA, IVF, and NT gradually rose with increasing concentrations of VE and reached the highest amount [PA: 32.9% (81/246); IVF: 21.4% (45/210); and NT: 21.1% (47/223)] in the group containing 150 μM of VE; it was significantly higher than that of other groups (P < 0.05). When the different concentrations of VC (0, 50, 100, 150, and 200 μM) were added to the CM, the blastocyst development rate of the embryos derived from PA, IVF, and NT also enhanced according to the increasing concentration of VC, and more embryos developed to blastocysts in the group containing 150 μM of VC [PA: 31.2% (72/231); IVF: 20.2% (43/213); NT: 19.8% (48/243)] than in the other groups (P < 0.05). Compared with the control group (0 μM), the blastocyst rate of PA and IVF, as well as NT embryos, cultured in the CM with 150 μM VE + 150 μM VC groups was significantly higher (P < 0.05), but there were no significant differences in the percentage of blastocysts among groups of the 150 μM VE, 150 μM VC, and 150 μM VE + 150 μM VC (P > 0.05). These results indicated that adding VE (150 μM), VC (150 μM), or VE (150 μM) + VC (150 μM) in the CM could efficiently enhance the developmental competence of buffalo embryos during in vitro culture. This work was funded by China High Technology Development Program (2007AA100505), Guangxi Science Foundation (0718005-3A), Fok Ying Tung Education Foundation (111034).

28 HIGH HYDROSTATIC PRESSURE IMPROVED DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES FOR HANDMADE CLONING

2008 ◽  
Vol 20 (1) ◽  
pp. 94 ◽  
Author(s):  
Y. Du ◽  
L. Lin ◽  
C. Pribenszky ◽  
M. Molnár ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Nuclear Transfer ◽  
Technical Assistance ◽  
Developmental Competence ◽  
Control Groups ◽  
Donor Cells ◽  
And Control ◽  
Handmade Cloning

High hydrostatic pressure (HHP) has been introduced into the field of embryology recently, with the possible mechanism that a sublethal HHP could induce the synthesis of molecular chaperons to protect the embryos from further stresses. Improved cryotolerance has been achieved successfully in HHP-treated mouse (Pribenszky 2005 Anim. Reprod. Sci. 87, 143–150) and bovine (Pribenszky 2005 Reprod. Domest. Anim. 40, 338) embryos, and the semen of bull (Pribenszky 2007 Reprod. Fertil. Dev. 19, 181–182) and boar (Pribenszky 2005 Reprod. Fertil. Dev. 18, 162–163). The objective of the present study was to apply this new technique to in vitro-matured (IVM) porcine oocytes and further investigate its effect in the procedure of handmade cloning (HMC). After 40 h IVM, cumulus–oocyte complexes (COCs) were loaded in 0.5-mL straws by a 2-mL syringe, with HEPES-buffered TCM199 as the loading medium. COCs were then treated with 20 MPa (200 times greater than atmospheric pressure) for 60 min by a pressurizing device (Cryo-Innovation Inc., Budapest, Hungary), with an interval of 120 min between HHP treatment and subsequent HMC. Two different cell lines (from Day 40 fetuses of Yucatan and Danish Landrace breeds (LW1-2)) were used as donor cells for nuclear transfer. A total of 592 reconstructed embryos were produced from both HHP-treated and control groups and were in vitro cultured for 6 days to evaluate the developmental competence through to blastocyst formation. The effect of donor cells on blastocyst development was also investigated. SPSS 11.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis; values with P < 0.05 were regarded as significant. Blastocyst rates of the different groups are shown in Table 1. Our results indicated that COCs treated with HHP had a much higher blastocyst rate than those untreated (P < 0.01) and this improvement was not affected by using different donor cells for nuclear transfer. In conclusion, the sublethal HHP treatment could improve the in vitro developmental competence of porcine IVM oocytes when they are used for HMC. Further in vivo experiments are required to investigate the long-term effect of HHP on embryo development. Table 1. Day 6 blastocyst rates of HHP-treated and control groups with different donor cells for nuclear transfer The authors thank Ruth Kristensen and Janne Adamsen for their help and excellent technical assistance.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment

Zygote ◽  
2017 ◽  
Vol 25 (4) ◽  
pp. 453-461 ◽  
Author(s):  
Xiao-Chen Li ◽  
Qing Guo ◽  
Hai-Ying Zhu ◽  
Long Jin ◽  
Yu-Chen Zhang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Formation Rate ◽  
Developmental Competence ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation

SummaryWe examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P < 0.05). The blastocyst formation rate was higher in the group treated with AZD5438 for 4 h than in the groups treated with AZD5438 for 2 or 6 h (42.8% vs. 38.6% and 37.2%, respectively; P > 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

Caffeine and dithiothreitol delay ovine oocyte ageing

2010 ◽  
Vol 22 (8) ◽  
pp. 1254 ◽  
Author(s):  
Xiao-Fang Ye ◽  
Shi-Bin Chen ◽  
Li-Qin Wang ◽  
Yun-Cheng Zhao ◽  
Xue-Feng Lv ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Limited Extent ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation ◽  
Cell Numbers ◽  
Reconstructed Embryos ◽  
Intracellular Glutathione ◽  
Post Onset

The intracellular glutathione levels and developmental competence of aged oocytes after parthenogenetic activation, somatic cell nuclear transfer and intracytoplasmic sperm injection in the presence or absence of caffeine or dithiothreitol (DTT) were examined. The following results were found: (1) ovine oocytes were fully aged 30 h post-onset of maturation culture; (2) the appropriate concentrations of caffeine and DTT for oocyte culture were 5 mM and 1 mM, respectively; (3) when nuclear transfer-reconstructed embryos were treated with caffeine or DTT following fusion, no increase in the frequency of development to blastocyst was observed (P > 0.05), but the cell numbers of blastocysts increased (P < 0.05); (4) both caffeine and DTT increased the blastocyst formation rates of intracytoplasmic sperm-injected embryos (P < 0.05); (5) caffeine increased the glutathione content of aged oocytes (P < 0.05). The glutathione content of DTT-treated aged oocytes was higher than that of oocytes matured for 36 h (P < 0.05). In conclusion, caffeine and dithiothreitol delay oocyte ageing but only to a limited extent.

36 DEVELOPMENTAL COMPETENCE OF HUMAN AGED OOCYTES AS HOST CELLS FOR NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 126
Author(s):  
V. Hall ◽  
D. Compton ◽  
P. Stojkovic ◽  
M. Nesbitt ◽  
M. Herbert ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Calcium Ionophore ◽  
Developmental Competence ◽  
Host Cells ◽  
Cell Stage ◽  
Double Labeling ◽  
Immunocytochemical Analysis ◽  
Parthenogenetic Activation

The use of aged metaphase II oocytes (cultured in vitro for more than 14 h) for somatic cell nuclear transfer (SCNT) in varying species has resulted in lower developmental outcomes compared with non-aged in vitro- or in vivo-matured oocytes. However, due to limited resources of fresh oocytes for the derivation of nuclear transfer stem cell lines, further investigation in using spare oocytes is required. Aged spare oocytes (48 h post oocyte retrieval) were consigned for research (under HFEA and local ethics approval) by couples undergoing either in vitro fertilization (failed IVF oocytes, f-IVF) or intracytoplasmic sperm injection (failed-ICSI oocytes, f-ICSI) treatments. Aged oocytes were randomly assigned for double-labeling immunocytochemical analysis (f-IVF, n = 10; f-ICSI, n = 7) for the microtubule markers, NuMA and �-tubulin, or parthenogenetic activation. Immunocytochemical analysis was performed as previously described (Chatzimeletiou et al. 2005 Hum. Reprod. 20, 672-682) using primary anti-rabbit NuMA (gift from D. Compton, Dartmouth Medical School, Hanover, NH, USA) and anti-mouse DM1-�. Secondary antibodies were donkey anti-rabbit and anti-mouse immunoglobulins. Oocytes were counterstained with Hoechst 33342. Negative controls were performed as above with blocking solution substituting for primary antibodies. Parthenogenetic activation was performed for 4 h using 10 �M calcium ionophore (5 min) and 2 mM 6-dimethylaminopurine (Ca-I/DMAP) for f-IVF (n = 10) and f-ICSI oocytes (n = 11) or 10 �g/mL puromycin (Ca-I/Pur) for f-IVF (n = 12) and f-ICSI oocytes (n = 10) (4 h). Activated oocytes were cultured in a biphasic system, G1.3" and G2.3" (Vitrolife UK, Ltd., Ediburgh, Lothian, UK) for 5 days at 37 �C in 5% CO2 in humidified air. NuMA was localized to the metaphase spindle in 6/10 (60%) and 7/7 (100%) oocytes for f-IVF and f-ICSI, respectively, and/or in cytoplasmic cytasters. One f-IVF oocyte and four f-ICSI oocytes had visible tetrapolar spindles. Unusual patterns of diffuse NuMA staining containing dense foci within these regions, but not associated with the cytasters or metaphase spindle, were also observed in two f-IVF oocytes. The majority of oocytes displayed ring-like staining of DM1-�, which was aberrant in two f-ICSI oocytes. Parthenogenetic development was poor for both treatments. Cleavage rates were 17% and 20% for f-IVF using Ca-I/PUR and Ca-I/DMAP, respectively, and 40% and 45% for f-ICSI using Ca-I/PUR and Ca-I/DMAP, respectively. Fragmentation rates were high across all treatments. No parthenogenetic embryos developed beyond the 6-cell stage. Thus, the use of aged human oocytes for SCNT may be difficult due to their incapacity to artificially activate using current activation protocols and, in addition, due to the microtubule abnormalities observed in many of these aged oocytes.

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