A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72-78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.     

Efficient production of nuclear transferred rat embryos by modified methods of reconstruction

In this study we investigated spontaneous oocyte activation and developmental ability of rat embryos of the SD-OFA substrain. We also tried to improve the somatic cell nuclear transfer (SCNT) technique in the rat by optimizing methods for the production of reconstructed embryos. About 20% of oocytes extruded the second polar body after culture for 3 hr in vitro and 84% of oocytes were at the MII stage. MG132 blocked spontaneous activation but decreased efficiency of parthenogenetic activation. Pronuclear formation was more efficient in strontium-activated oocytes (66.1-80.9%) compared to roscovitine activation (24.1-54.5%). Survival rate after enucleation was significantly higher (89.4%) after slitting the zona pellucida and then pressing the oocyte with a holding pipette in medium without cytochalasin B (CB) compared to the conventional protocol using aspiration of the chromosomes after CB treatment (67.7%). Exposure of rat ova to UV light for 30 sec did not decrease their in vitro developmental capacity. Intracytoplasmic cumulus cell injection dramatically decreased survival rate of oocytes (42%). In contrast, 75.9% of oocytes could be successfully electrofused. Development to the 2-cell stage was reduced after SCNT (24.6% compared 94.6% in controls) and none from 244 reconstructed embryos developed in vitro beyond this stage. After overnight in vitro culture, 74.4% of the SCNT embryos survived and 56.1% formed pronuclei. The pregnancy rate of 33 recipients after the transfer of 695 of these cloned embryos was, however, very low (18.2%) and only six implantation sites could be detected (0.9%) without any live fetuses and offspring.     

Demecolcine化学辅助去核技术在牛体细胞核移植中的初步研究

本实验目的是研究demecolcine辅助去核的卵母细胞能否支持牛的核移植胚胎的发育。通过化学药物demecolcine处理牛MII期卵母细胞来辅助去除牛卵母细胞核,并用去核的卵母细胞做受体,进行核移植的研究。实验结果显示,demecolcine辅助去核后的卵母细胞质膜有明显一个或二个突起,并且突起内都含有卵母细胞染色体组,显示去核效果较好（57.89%～73.3%）。药物处理一小时为最适时间,去核率可达73.3%。对demecolcine辅助去核的卵母细胞的核移植胚胎发育情况显示囊胚率较盲吸法核移植胚胎较好（12.5%VS10.2%）,但二者差异不显著(p>0.05)。Demecolcine药物处理后的卵母细胞能够支持核移植胚胎的发育。Demecolcine辅助去核可以在牛体细胞核移植中的到应用。     

Demecolcine- and nocodazole-induced enucleation in mouse and goat oocytes for the preparation of recipient cytoplasts in somatic cell nuclear transfer procedures

Treatment of pre-activated oocytes with demecolcine (DEM) has been shown to induce the extrusion of all oocyte chromosomes within the second polar body (PB2). However, induced enucleation (IE) rates are generally low and the competence of these cytoplasts to support embryonic development following somatic cell nuclear transfer (SCNT) is impaired. Here, we explored whether short treatments with DEM or another antimitotic, nocodazole (NOC), improve IE efficiency, and determined the most appropriate timing for nuclear transfer in the cytoplasts produced. We show, for the first time, that IE can be accomplished in mouse and goat oocytes using NOC and that short treatments with DEM or NOC result in similar IE rates, which proved to be strain- and species-specific. Because enucleation induced by both antimitotic drugs is reversible, the IE protocol was combined with the mechanical aspiration of PB2s to increase permanent enucleation rates in mouse oocytes. None of the cloned mouse embryos produced from the resultant cytoplasts developed to the blastocyst stage. However, when they were reconstructed prior to the activation and antimitotic treatment, their in vitro embryonic development was similar to that of cloned embryos produced from mechanically-enucleated oocytes.     

Birth of Beagle dogs by somatic cell nuclear transfer

The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 μs electrical stimulation, chemically activated after 1 h of fusion using 10 μM calcium ionophore for 4 min and cultured 4 h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4–10, 11–25 and 26–40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P > 0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4–10 embryos (n = 7) and 26–40 embryos (n = 12) while pregnancy was detected in 23.53% recipients received a group of 11–25 embryos (n = 17). Among them, five (1.76%) live puppies were born (P < 0.05). These data show an increase in the overall efficiency of SCNT in canine species.     

Chromosomes in the Porcine First Polar Body Possess Competence of Second Meiotic Division within Enucleated MII Stage Oocytes

To determine whether chromosomes in the porcine first polar body (PB1) can complete the second meiotic division and subsequently undergo normal pre-implantation embryonic development, we examined the developmental competence of PB1 chromosomes injected into enucleated MII stage oocytes by nuclear transfer method (chromosome replacement group, CR group). After parthenogenetic activation (PA) or in vitro fertilization (IVF), the cleavage rate of reconstructed oocytes in the IVF group (CR-IVF group, 36.4 ± 3.2%) and PA group (CR-PA group, 50.8 ± 4.2%) were significantly lower than that of control groups in which normal MII oocytes were subjected to IVF (MII-IVF group, 75.8 ± 1.5%) and PA (MII-PA group, 86.9 ± 3.7%). Unfertilized rates was significantly higher in the CR-IVF group (48.6 ± 3.3%) than in the MII-IVF group (13.1 ± 3.4%). The blastocyst formation rate was 8.3 ± 1.9% in the CR-PA group, whereas no blastocyst formation was observed in the CR-IVF group. To produce tetraploid parthenogenetic embryos, intact MII stage oocytes injected with PB1chromosomes were electrically stimulated, treated with 7.5 μg/mL cytochalasin B for 3 h (MII oocyte + PB1 + CB group), and then cultured without cytochalasin B. The average cleavage rate of reconstructed oocytes was 72.5% (48 of 66), and the blastocyst formation rate was 18.7% (9 of 48). Chromosome analysis showed similar proportions of haploid and diploid cells in the control (normal MII oocytes) and CR groups after PA; overall, 23.6% of blastocysts were tetraploid in the MII oocyte + PB1 + CB group. These results demonstrate that chromosomes in PB1 can participate in normal pre-implantation embryonic development when injected into enucleated MII stage oocytes, and that tetraploid PA blastocysts are produced (although at a low proportion) when PB1 chromosomes are injected into intact MII stage oocytes.     

Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein

A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1). received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2). were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3). were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P < 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%-69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P < 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P < 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE-6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%-69%) were higher than those with gilt oocytes (23%-27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFF-SCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.     

Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries

For production of viable somatic cell nuclear transferred (SCNT) miniature pig embryos, in vitro condition for controlling the quality of recipient oocytes derived from domestic pig ovaries should be evaluated. In the present study, to get information on optimal in vitro maturation (IVM) condition of oocytes, we investigated the effect of IVM duration of recipient oocytes on subsequent development of SCNT miniature pig embryos, the maturation-promoting factor (MPF) activity in recipient oocytes before and after SCNT, and the occurrence of premature chromosome condensation (PCC) and spindle morphologies of donor nuclei following SCNT. The optimal window of the IVM period in terms of in vitro developmental ability of SCNT embryos was determined to be 36-40 h after the start of IVM. The use of recipient oocytes matured for 36 and 40 h resulted in a high level of MPF activity before and after SCNT, and increased the occurrence of PCC in transferred nuclei compared to the use of oocytes matured for 44 and 52 h. The proportion of abnormal spindle-like structures increased as the IVM period was prolonged. In addition, SCNT embryos constructed from recipient cytoplasts obtained after 40 h of maturation by using fetal fibroblasts of miniature pigs were transferred to surrogate miniature pigs, and developed to full term. These results suggest that recipient oocytes matured for 36 h and 40 h effectively induce PCC with a normal cytoskeletal structure because of a high level of MPF activity; furthermore, the 40-h IVM period improves in vitro development of SCNT embryos to the blastocyst stage, resulting in the production of viable cloned miniature pigs.     

Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer

Successful cryopreservation of porcine embryos offers a promising perspective in the fields of agriculture, animal science, and human medical research. The objective of the present work was to establish a system facilitating the cryopreservation of porcine embryos produced by somatic cell nuclear transfer (SCNT). Several key techniques including micromanipulator-based enucleation, noninvasive delipation, zona-free fusion, and activation were combined with high efficiency. After a partial zona digestion and high-speed centrifugation, 89.8+/-2.1% (mean+/-SEM) of enucleated oocytes were successfully delipated. Delipated cytoplasts were incubated for an additional 0.5 or 2 h before fusion with somatic cells. After activation and 6 days of in vitro culture, no significant difference in the rate of blastocysts per reconstructed embryo was observed between the two groups (33.1+/-1.8% and 26.0+/-4.3% for 0.5 and 2 h recovery time, respectively). Cryopreservation of the blastocysts was performed with a Cryotop device and factory-prepared vitrification and warming solutions. One hundred fifty-five vitrified SCNT embryos were transferred surgically into two recipient sows to test their developmental capacity in vivo. One recipient became pregnant and delivered six piglets. In conclusion, our simplified delipation and SCNT procedure resulted in viable piglets after vitrification and embryo transfer at the blastocyst stage.     

A comparison of two in vitro maturation media for use with adult porcine oocytes for adult somatic cell nuclear transfer

Two media used to mature adult porcine oocytes for somatic cell nuclear transfer were compared. In the first experiment, parthenogenetic embryos were produced using a maturation medium used by us previously to clone pigs (OMM199) and that described by Kühholzer et al. (2001) to transport oocytes overnight (BOMED). There was no difference in maturation rates between the two different media. However, BOMED medium increased the percentage of parthenogenetic embryos that developed to the blastocyst stage compared with OMM199 (49% vs. 29%, respectively). In a second experiment, BOMED medium increased the percentage of SCNT embryos that developed to the blastocyst stage compared with OMM199 (22% vs. 8%, respectively). The efficiency of our cloning protocol using adult oocytes matured in BOMED medium was then determined by transferring SCNT embryos reconstructed using adult fibroblasts to synchronized recipients. Primary cultures of adult fibroblasts were obtained from two adult male pigs and used for SCNT (passages 2-4). Between 82 and 146 fused couplets were transferred to seven recipients synchronized 1 day behind the embryos. Five recipients (71% pregnancy rate) subsequently farrowed a total of 23 piglets (4.4 average litter size). Overall efficiencies (liveborn/embryos transferred) were 3.2% for all transfers and 4.3% for animals that gave birth.     

Cloning of non-human primates: the road "less traveled by"

Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model.     

Maturity at collection and the developmental potential of rhesus monkey oocytes

The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear and cytoskeletal dynamics during oocyte maturation and development of somatic cell cloned pig embryos injected with membrane disintegrated donor cells

The objectives of this study were to characterize the nuclear and cytoskeletal changes of pig oocytes during in vitro maturation (IVM) and the development of the reconstructed embryos after injection with membrane intact or disintegrated donor cells. Cumulus-oocyte complexes (COCs) were collected from abattoir ovaries by follicle (2-8mm) aspiration. In Experiment 1, COCs were cultured in NCSU-23 medium for 0, 11, 22, 33, and 44 h. Oocytes were fixed at different time points for nuclear and cytoskeletal labeling. Forty-three percent and 75% oocytes progressed to MII stage at 33 and 44 h after IVM culture, respectively. Dynamic shift of spindle and cytoplasmic microtubules was evident. In Experiment 2, matured oocytes were injected with either the whole cumulus cell with or without intact cell membranes after enucleation. The reconstructed oocytes were fixed at 0, 2, or 4 h after cell injection for nuclear and cytoskeletal evaluation. When an intact cumulus cell was injected, the injected cell remained intact within 4h after injection. When a cell with disintegrated membrane was injected, 59-63% (n=146) of the injected cell underwent premature chromosome condensation (PCC). In Experiment 3, the reconstructed pig oocytes received membrane-disintegrated cumulus cells or fetal fibroblasts were cultured in PZM medium. The blastocyst rate of the fibroblast-injected embryos was 10%, which was lower than the non-cloned parthenotes (33%, P<0.05) but higher than the cumulus cell-injected embryos (2.7%). These results suggest that pig oocytes are subjected to nuclear and cytoskeletal reorganization during maturation. Pig oocytes injected with membrane-disintegrated fibroblast cells support better blastocyst development of the cloned embryos.     

In vivo aging of oocytes influences the behavior of nuclei transferred to enucleated rabbit oocytes

The present study examined nuclear remodeling in rabbit nuclear transfer (NT) embryos formed from metaphase II (MII) oocytes aged in vivo until 19 hr postcoitum (hpc), enucleated, and fused at 22–26 hpc with 32-cell morula blastomeres by means of electric fields, which also induced recipient oocyte activation. Post-activation events observed during the first hour following the fusion/activation pulse were studied in terms of chromatin, lamins, and micro-tubules, and revealed that transferred nuclei underwent premature chromosomes condensation (PCC) in only one-third of NT embryos and remained in interphase in others. Recipient oocytes were mostly not activated by manipulations performed before the fusion/activation pulse. The persistance of transferred nuclei in interphase resulted from the rapid progression of recipient oocytes to interphase after activation, suggesting that the cytoplasmic state of MII oocytes aged in vivo was poised for the approach to interphase. Studying micro-tubular organization in MII oocytes before nuclear transfer manipulations, we found that 19 hpc MII oocytes aged in vivo differed from 14 hpc MII oocytes (freshly ovulated) and from 19-hpc MII oocytes aged in vitro (collected at 14 hpc and cultured for 5 hr), notably by the presence of microtubule asters and tubulin foci or only tubulin foci dispersed throughout the cytoplasm. When PCC was avoided, remodeling of the transferred nucleus was well advanced 1 hr after nuclear transfer, and NT embryos developed better to the blastocyst stage. Mol. Reprod. Dev. 46:325–336, 1997. © 1997 Wiley-Liss, Inc.     

Piglets produced from cloned blastocysts cultured in vitro with GM‐CSF

In general, pig embryos established by somatic cell nuclear transfer (SCNT) are transferred at the one‐cell stage because of suboptimal embryo culture conditions. Improvements in embryo culture can increase the practical application of late embryo transfer. The goal of this study was to evaluate embryos cultured with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in vitro, and to track the in vivo developmental competency of SCNT‐derived blastocysts from these GM‐CSF embryos. The receptor for GM‐CSF was up‐regulated in in vitro‐produced embryos when compared to in vivo‐produced cohorts, but the level decreased when GM‐CSF was present. In vitro fertilized (IVF) embryos, supplemented with GM‐CSF (2 or 10 ng/ml), showed a higher frequency of development to the blastocyst stage compared to controls. The total cell numbers of the blastocysts also increased with supplementation of GM‐CSF. Molecular analysis demonstrates that IVF‐derived blastocysts cultured with GM‐CSF exhibit less apoptotic activity. Similarly, an increase in development to the blastocyst stage and an increase in the average total‐cell number in the blastocysts were observed when SCNT‐derived embryos were cultured with either concentration of GM‐CSF (2 or 10 ng/ml). When SCNT‐derived embryos, cultured with 10 ng/ml GM‐CSF, were transferred into six surrogates at Day 6, five of the surrogates became pregnant and delivered healthy piglets. Our findings suggest that supplementation of GM‐CSF can provide better culture conditions for IVF‐ and SCNT‐derived embryos, and pig SCNT‐derived embryos cultured with GM‐CSF in vitro can successfully produce piglets when transferred into surrogates at the blastocyst stage. Thus, it may be practical to begin performing SCNT‐derived embryo transfer at the blastocyst stage. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.     

山羊M II期卵母细胞胞质支持异种间克隆胚的着床前发育

研究去核山羊(Capra hircus)体内成熟的M II期卵母细胞与异种成年的哺乳动物(包括山羊、波尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力。结果显示这些异种体细胞核移植重构胚可以完成着床前发育, 并形成囊胚。种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469); 亚种间或种间体细胞核移植胚的融合率和囊胚发育率分别为: 波尔山羊78.18%(541/692)、33.90%(40/118), 牛70.53%(146/207)、22.52%(25/111), 塔尔羊53.51%(61/114)、5.26%(3/570), 熊猫79.82%(1159/1452)、8.35%(75/898), 人68.76%(317/461)、5.41%(16/296)。由此结果得出以下结论: (1)山羊M II期卵母细胞胞质与供核细胞之间的亲缘性不影响两者的融合率; (2)山羊M II期卵母细胞的胞质能支持异种间体细胞核移植胚的着床前发育; (3)亲缘关系近的种间核移植胚的囊胚发育率高于亲缘关系远的种间核移植胚的。     

Effects of enucleation and caffeine on maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities in ovine oocytes used as recipient cytoplasts for nuclear transfer

In general, oocytes arrested at metaphase of the second meiotic division (MII) are used as recipient cytoplasts for nuclear transfer (NT) procedures. MII oocytes contain high levels of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), which cause nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the transferred nucleus and have been implicated in nuclear reprogramming. However, the occurrence of NEBD and the extent of PCC are variable between individual oocytes and species and are dependent on donor cell type and cell cycle stage. Enucleation, which removes oocyte cytoplasm, may reduce MPF and MAPK activities and reduce reprogramming; conversely, increasing kinase activities may increase reprogramming. We compared the effects of enucleation of ovine oocytes at anaphase/telophase of the first meiotic division (AI-TI) and at MII. MPF and MAPK activities were maximal at MII; blind enucleation at AI-TI was more efficient than at MII and removed a smaller volume of cytoplasm. Neither protocol significantly affected the activity of either kinase and the fate of the donor nucleus; however, enucleation per se significantly reduced the occurrence of NEBD in NT embryos. Treatment with 10 mM caffeine significantly increased the activities of both kinases and the occurrence of NEBD but did not affect the frequency of development to the blastocyst stage; however, a significant increase in total cell numbers was observed. The results show that caffeine can increase MPF and MAPK activities in ovine oocytes and that this may contribute to an increased reprogramming in NT embryos.     

Developmental competence of somatic cell nuclear transfer embryos reconstructed from oocytes matured in vitro with follicle shells in miniature pig

The developmental competence of domestic pig oocytes that were transferred to somatic cell nuclei of miniature pig was examined. A co-culture system of oocytes with follicle shells was used for the maturation of domestic pig oocytes in vitro. Co-cultured oocytes progressed to the metaphase II stage of meiosis more quickly and more synchronously than non co-cultured oocytes. Oocytes were enucleated and fused with fibroblast cells of Potbelly miniature pig at 48 h of maturation. The blastocyst formation rate of nuclear transfer (NT) embryos using cocultured oocytes (24%) was significantly higher (p < 0.05) than that of non-co-cultured oocytes (13%). Cleaved embryos at 48 h after nuclear transfer using co-cultured oocytes were transferred to the oviducts of 14 G?ttingen miniature pigs and four Meishan pigs. Estrus of all G?ttingens returned at around 20-31 days of pregnancy. Two of the four Meishans became pregnant. Three and two cloned piglets were born after modest number of embryo transfer (15 and 29 embryos transferred), respectively. These results indicated that oocytes co-cultured with follicle shells have a high developmental competence after nuclear transfer and result in full-term development after embryo transfer.     

Sucrose pretreatment for enucleation: an efficient and non-damage method for removing the spindle of the mouse MII oocyte

Oocytes enucleated at metaphase II stage can support reprogramming of transferred nucleus and further developing to term. However, the first polar body in mice sometimes migrates away from the original place of expulsion, so the chromosomes of the oocyte will displace from the first polar body. Thus, it is not always possible to successfully enucleate according to the position of the first polar body. Here we use sucrose treatment to visualize metaphase spindle fibers and chromosomes with standard light microscopy. In the manipulation medium containing 3% sucrose, oocytes of poor quality become shrunken, deformed or fragmented, while oocytes of good quality in the same medium would show a swelling around the metaphase chromosomes and a transparent spindle area, shaped like "infinity" and "0". So it is easy to remove the well-distinguished spindle and chromosomes in oocytes of good quality. Re-examined by Hoechst 33342 stain under the UV light, the enucleation rate was 100%. There was no significant difference in IVF and cleavage rates between the sucrose treatment and the control group. In conclusion, this study demonstrated that 3% sucrose pretreatment can give a method for evaluating embryo quality and more importantly, it can, under a common microscope, allow the visualization of the spindle and chromosomes in oocytes of good quality and hence efficiently improve enucleation rate without any harm.     

山羊M II期卵母细胞胞质支持异种间克隆胚的着床前发育

研究去核山羊(Capra hircus)体内成熟的M II期卵母细胞与异种成年的哺乳动物(包括山羊、波尔山羊、牛、塔尔羊、熊猫)及人的成纤维细胞融合形成的体细胞核移植胚胎着床前的发育能力。结果显示这些异种体细胞核移植重构胚可以完成着床前发育, 并形成囊胚。种内体细胞核移植胚的融合率和囊胚发育率分别为78.67%(557/708)和56.29%(264/469); 亚种间或种间体细胞核移植胚的融合率和囊胚发育率分别为: 波尔山羊78.18%(541/692)、33.90%(40/118), 牛70.53%(146/207)、22.52%(25/111), 塔尔羊53.51%(61/114)、5.26%(3/570), 熊猫79.82%(1159/1452)、8.35%(75/898), 人68.76%(317/461)、5.41%(16/296)。由此结果得出以下结论: (1)山羊M II期卵母细胞胞质与供核细胞之间的亲缘性不影响两者的融合率; (2)山羊M II期卵母细胞的胞质能支持异种间体细胞核移植胚的着床前发育; (3)亲缘关系近的种间核移植胚的囊胚发育率高于亲缘关系远的种间核移植胚的。