Recipient Cytoplasts Research Articles

Stain-free enucleation of mouse and human oocytes with a 1033nm femtosecond laser.

Preparation of a recipient cytoplast by oocyte enucleation is an essential task for animal cloning and assisted reproductive technologies in humans. The femtosecond laser is a precise and low-invasive tool for oocyte enucleation, and it should be an appropriate alternative to traditional enucleation by a microneedle aspiration. However, until recently, the laser enucleation was performed only with applying a fluorescent dye. This work is aimed to (1)achieve femtosecond laser oocyte enucleation without applying a fluorescent dye and (2)to study the effect of laser destruction of chromosomes on the structure and dynamics of the spindle. We applied polarized light microscopy for spindle visualization and performed stain-free mouse and human oocyte enucleation with a 1033nm femtosecond laser. Also, we studied transformation of a spindle after metaphase plate elimination by a confocal microscopy. We demonstrated a fundamental possibility of inactivating the metaphase plate in mouse and human oocytes by 1033nm femtosecond laser radiation without applying a fluorescent dye. Irradiation of the spindle area, visualized by polarized light microscopy, resulted in partly or complete metaphase plate destruction but avoided the microtubules impairment. After the metaphase plate elimination, the spindle reorganized, however, it was not a complete depolymerization. This method of recipient cytoplast preparation is expected to be useful for animal cloning and assisted reproductive technologies.

Femtosecond laser oocyte enucleation as a low-invasive and effective method of recipient cytoplast preparation.

Recipient cytoplast preparation, commonly performed by DNA aspiration with a needle, inevitably leads to the loss of reprogramming factors. As an alternative to the traditional enucleation technique, femtosecond laser enucleation can eliminate DNA effectively without loss of reprogramming factors and without oocyte puncturing. In this work we have performed oocyte enucleation by destructing the metaphase plate using a 795 nm femtosecond laser. The disability of the enucleated oocytes to develop after the parthenogenetic activation, as well as the lack of DNA staining luminescence, strongly confirms the efficiency of the femtosecond laser enucleation. The parthenogenetic development of oocytes after the cytoplasm treatment suggests a low-invasive effect of the laser enucleation technique.

Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, and In Vivo Survival of Cloned Sheep Embryos.

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

Embryo aggregation allows the production of kodkod (Leopardus guigna) blastocysts after interspecific SCNT

The kodkod (Leopardus guigna) is a small felid endemic of Chile and is considered a vulnerable species. Domestic cat oocytes have been successfully used as recipient cytoplast to reprogram somatic cells from different felids by interspecific somatic cell nuclear transfer (iSCNT). The developmental competence of felid embryos generated by iSCNT can be improved by the aggregation method using a zona-free culture system. The objective of this research was to evaluate the developmental competence of kodkod embryos generated by iSCNT using domestic cat oocytes and the aggregation method. For this purpose, five experimental group were done: (1) cat embryos generated by IVF, (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by iSCNT (K1x) and (5) aggregated kodkod embryos generated by iSCNT (K2x). Cleavage, morulae and blastocyst rates were estimated. The blastocyst diameter was evaluated. The gene expression level of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6) was analyzed in blastocysts. Morulae rate was higher in the IVF group and when cloned embryos were cultured in aggregates (IVF: 68.2%, Ca2x: 58.0% and K2x: 62.4%) compared to individually cultured kodkod embryos (K1x: 37.0%) (P < 0.05). Embryo aggregation increased blastocysts formation in the Ca2x group (30.9%) to a similar rate compared to the IVF group (44.5%) (P > 0.05). No blastocysts were generated in the K1x group, whereas blastocysts formation was obtained in K2x group (5.9%). The diameter of blastocysts from the K2x group (172.8 μm) was significantly lower than blastocysts from the Ca2x group (P < 0.05). The relative expression of OCT4 was lower in blastocysts from Ca1x than in blastocysts from IVF (P < 0.05). Furthermore, CDX2 expression was lower in blastocysts from Ca2x than in blastocysts from Ca1x and IVF groups (P < 0.05). In kodkod embryos, only one blastocyst from the K2x group expressed OCT4. No expression of SOX2, NANOG, CDX2 and GATA6 was detected in kodkod blastocysts. In conclusion, after iSCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. The aggregation method increases the morulae rate of kodkod cloned embryos and allows blastocysts formation. However, kodkod blastocysts have a poor morphological quality and a lacking expression of pluripotency and differentiation markers, probably caused by an incomplete nuclear reprogramming.

Do the Cytoplast and Nuclear Material of Germinal Vesicle Oocyte Support Developmental Competence Upon Reconstruction with Embryonic/Somatic Nucleus.

Maturation conditions and oocytes quality have substantial roles on developmental competence of unreconstructed or reconstructed oocytes. Cloning has been reported successfully with low efficiency through embryonic or somatic nuclear transfer into enucleated metaphase II oocytes. It has been suggested that introducing embryonic or somatic nucleus to cytoplast at earlier stage might improve reprogramming of the introduced nucleus. In addition, the synchronization between the donor nucleus and recipient cytoplasts might effect on reprogramming and further embryonic development. Therefore, the question arises; does the cytoplast of germinal vesicle (GV) oocyte containing nuclear sap improve developmental competence upon reconstruction with embryonic/somatic nucleus compared with MII cytoplast. It has been indicated that GV material is essential for remodeling of sperm or embryonic or somatic nucleus in GV oocyte cytoplast and their further embryonic developmental competence. GV cytoplasts could be obtained through micromanipulation. Different micromanipulation techniques of immature oocytes at different stages were adapted in addition to introducing donor nuclei at G0/G1, S and G2/M phase, and enucleolation technique as well. Upon micromanipulation, it could obtain GV cytoplasts; cumulus-free and without GV material, cumulus-complexes and without GV material, cumulus-free and with GV material, and cumulus-complexes and with GV material in addition to enucleolated GV oocytes. Therefore, this short review will address briefly the importance of maturation conditions, cumulus cells, oocyte quality, the techniques of enucleation GV oocyte, the cell cycle stage of the introduced donor cell, or nucleus for oocyte maturation and embryo development.

34 Recent Advances in Cloning by Somatic Cell Nuclear Transfer in Camelids

In addition to its application for production of elite males, racing champions, animals with the highest potential for milk production, and the prized beauty camels, somatic cell nuclear transfer (SCNT) can be utilised for the conservation of endangered wild Bactrian camels and vicunas by using the technique of interspecies SCNT (iSCNT). In the present study, embryos were reconstructed by using skin fibroblast cells from a Bactrian camel (Camelus bactrianus), a llama (Llama glama), and a dromedary camel (Camelus dromedarius) as donor karyoplasts and dromedary oocytes as recipient cytoplasts to evaluate in vitro and in vivo developmental potential of these embryos. Mature oocytes were collected from super-stimulated dromedary camels by ultrasound guided transvaginal ovum pick-up. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated oocytes. The fibroblast cell and recipient cytoplasm were fused by 2 DC pulses of 100V for 15 µs each. Reconstructs were activated 1 to 1.5 h post-fusion with 5 µM ionomycin, followed by exposure to 6-DMAP for 4 h. The activated oocytes were then cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture. A lower blastocyst production rate was observed when llama fibroblast cells were used as karyoplast (13.3 ± 3.3) compared with those obtained from the embryos reconstructed with Bactrian (34.4 ± 3.9) and dromedary (32.2 ± 6.2) fibroblast cells; however, no difference was observed in their cell numbers. Out of 26 and 20 blastocysts from reconstructs with Bactrian and dromedary fibroblast cells, transferred to 23 and 12 synchronized dromedary recipients, 3 and 2 pregnancies were achieved at Day 60, respectively. One pregnancy from each group reached term and both (a Bactrian and a dromedary) calves were delivered normally after completing the gestation period. We demonstrated birth of a Bactrian calf conceived from the reconstructed embryo by iSCNT using the somatic cell from a Bactrian camel and enucleated dromedary oocyte. The present study also demonstrated that dromedary camel can be used as a surrogate to carry these pregnancies to term (Wani et al. 2017 PLoS One 12, e0177800; https://doi.org/10.1371/journal.pone.0177800). This study has opened doors for enhanced multiplication and preservation of the wild Bactrian camels, which are threatened with extinction, being the eighth most endangered large mammal on the planet.

Source, treatment and type of nuclear donor cells influences in vitro and in vivo development of embryos cloned by somatic cell nuclear transfer in camel (Camelus dromedarius)

Experiments were conducted to evaluate the effect of source, treatment and type of nuclear donor cells on embryonic and fetal development of somatic cell nuclear-transfer reconstructs in dromedary camel. In experiment 1, actively growing, serum starved or confluent skin fibroblast cells were used as nuclear donors. In experiment 2, skin fibroblasts from 4 different animals while in experiment 3, skin fibroblasts and cumulus cells from the same animal were used as nuclear donors. In all the three experiments, mature oocytes collected by transvaginal ovum pick up were used as recipient cytoplasts. All the blastocysts obtained were transferred to synchronized recipients on Day 5–6 after ovulation. In experiment 1, pregnancies were achieved from the embryos reconstructed with all the groups of cells, however, only 1 full term calf was delivered from the embryos reconstructed with serum-starved cells. In experiment 2, significant differences were observed in embryo development and establishment of pregnancies among the donor cell lines from different animals. Five cloned calves were delivered from the embryos reconstructed with skin fibroblast cells of 3 animals, while the sole pregnancy from fourth animal aborted on Day 224 of gestation. Three full term calves were delivered from pregnancies achieved by the embryos reconstructed with cumulus cells in experiment 3, while a single pregnancy achieved from skin fibroblast cells was lost on Day 296 of gestation. In conclusion, we observed that cell donor, cell type and their treatment affect the outcome of cloning by somatic cell nuclear transfer in camels.

CLONAGEM POR TRANSFERÊNCIA NUCLEAR EM BUBALINOS

The success of core transfer (CT) depends on the origin of the donor cell, on the stage of development of the recipient cytoplast and on the synchronization between the cell cycle of the donor and recipient cells. The somatic nucleus must be reprogrammed after CT, thus restoring the totipotent state, and then resuming cellular development. However, it is noted that the efficiency of CT is still low, especially with a deficiency of the overall gene expression of the cloned embryo. However, the number of species of cloned mammals has been increasing in the last years, being this technique an important tool that does not aid in the effectiveness of buffalo reproduction. Thus, this review focuses on the description of the main processes pertinent to this process, as well as to analyze as future implications, as well as some factors that affect the success of nuclear transfer.

First cloned Bactrian camel (Camelus bactrianus) calf produced by interspecies somatic cell nuclear transfer: A step towards preserving the critically endangered wild Bactrian camels.

Studies were conducted to explore the possibility of employing dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for the development of interspecies somatic cell nuclear transfer (iSCNT) embryos using skin fibroblast cells of an adult Bactrian camel (Camelus bactrianus) and Llama (Llama glama) as donor nuclei. Also, the embryos reconstructed with Bactrian cells were transferred into the uterus of synchronized dromedary camel recipients to explore the possibility of using them as surrogate mothers. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated mature oocytes, collected from super-stimulated dromedary camels, and fused using an Eppendorf electroporator. After activation with 5μM ionomycin and 6-dimethylaminopurine, they were cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. In experiment 1, Day 7 blastocysts were stained with Hoechst to count their cell numbers, while in experiment 2, they were transferred to synchronized dromedary recipients. A lower number (P < 0.05) of blastocysts were obtained from reconstructs utilizing fibroblast cells from Llama when compared with those reconstructed with dromedary and Bactrian fibroblast cells. However, no difference was observed in their cell numbers. In experiment 2, a higher (P < 0.05) proportion of blastocysts were obtained from the cleaved embryos reconstructed with Bactrian fibroblast cells when compared to those reconstructed with dromedary cells. Twenty-six Day 7 blastocysts reconstructed with Bactrian cells were transferred to 23 synchronized dromedary recipients with 5 pregnancies established on Day 30, however, only one of the pregnancies developed to term and a healthy calf weighing 33 kgs was born after completing 392 days of gestation. Unfortunately, the calf died on day 7 due to acute septicemia. In conclusion, the present study reports, for the first time, birth of a cloned Bactrian calf by iSCNT using dromedary camel as a source for oocytes as well as a surrogate for carrying the pregnancy to term.

Combined positive effect of oocyte extracts and brilliant cresyl blue stained recipient cytoplasts on epigenetic reprogramming and gene expression in buffalo nuclear transfer embryos.

This study examined the effects of buffalo oocyte extracts (BOE) on donor cells reprogramming and molecular characterisation of oocytes screened via brilliant cresyl blue (BCB) staining and comparison of gene expression profiles of developmentally important genes in blastocysts from IVF and cloned derived from BOE treated donor cells with BCB selected recipient cytoplasts. Relative abundance (RA) of OCT4 and NANOG was increased (P<0.05) and HDAC-1, DNMT-1, and DNMT-3A decreased (P<0.05) in extract treated cells (ETCs). This ETCs dedifferentiated into neuron-like lineage under appropriate induction condition. The RA of NASP, EEF1A1, DNMT1, ODC1 and RPS27A was increased (P<0.05) in BCB+ oocytes, whereas ATP5A1 and S100A10 increased (P<0.05) in BCB- oocytes. Total cell number and RA of OCT4, NANOG, SOX2, DNMT1, IGF2, IGF2R, MNSOD, GLUT1, BAX and BCL2 in cloned blastocysts derived from BCB+ oocytes with ETC more closely followed that of IVF counterparts compared to BCB+ oocytes with extract untreated cell and BCB- oocytes with ETC derived blastocysts. In conclusion, BOE influenced epigenetic reprogramming of buffalo fibroblasts making them suitable donors for nuclear transfer (NT). BCB staining can be effectively used for selection of developmentally competent oocytes for NT. The combined effects of epigenetic reprogramming of donor nuclei by BOE and higher nuclear reprogramming capacity of BCB+ oocytes improve developmentally important gene expression in cloned blastocysts. Whether these improvements have long-term effects on buffalo calves born following embryo transfer remains unknown.

Resilience of oocyte germinal vesicles to microwave-assisted drying in the domestic cat model.

The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing. The objective of the present study was to understand the impact of water loss and storage time on GV DNA integrity. Immature cat oocytes were exposed to 1.5 M trehalose for 10 min before microwave-assisted dehydration for 0, 5, 10, 15, 20, 25, 30, or 40 min. Oocytes then were rehydrated to assess chromatin configuration and the incidence of DNA fragmentation (TUNEL assay). The moisture content progressively decreased (p<0.05) from 1.7 to 0.1 gH2O/gDW over the first 30 min, but did not decrease further (p>0.05) after 40 min. Chromatin configuration was unaffected (p>0.05) over time. The percentage of GVs with DNA fragmentation was unaltered (p>0.05) from 0 to 30 min of treatment (range, 6.1%-12%), but increased (p<0.05) to 32.5% after 40 min. Next, the influence of storage at two different supra-zero temperatures after 30 min of drying was investigated. Oocyte-loaded, microwave-treated filters were individually sealed in Dri-Shield moisture barrier bags and stored at 4°C or ambient temperature for 0 to 8 weeks. Moisture contents gradually decreased (p<0.05) from 0.12 to 0.10 gH2O/gDW after 8 weeks of storage at 4°C or ambient temperature. The percentage of GVs with DNA fragmentation more than doubled (p<0.05) from 0 (14.3%) to 2 days (30.0%-33.0%), but remained stable (p>0.05) thereafter (1 through 4 weeks, 25.0%-35.0%). Collective results demonstrate the feasibility of using microwave processing to dehydrate the mammalian GV to a moisture content that is nonlethal and enables nonfrozen storage, an alternative approach for preserving the maternal genome at cool or ambient temperature.

Expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocytes extract and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized embryos.

To compare the expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocyte extracts and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized (IVF) embryos. Hand-made cloned embryos were produced using oocyte extracts treated donor cells and brilliant cresyl blue (BCB) stained recipient cytoplasts. IVF embryos were produced by culturing 15-20 COCs in BO capacitated sperms from frozen thawed buffalo semen and the mRNA expression patterns of genes implicated in metabolism (GLUT1), pluripotency (OCT4), DNA methylation (DNMT1), pro- apoptosis (BAX) and anti-apoptosis (BCL2) were evaluated at 8- to16- cell stage embryos. A significantly (P < 0.05) higher number of 8- to16- cell and blastocyst stages (73.9 %, 32.8 %, respectively) were reported in hand-made cloning (HMC) as compared to in vitro fertilization (49.2 %, 24.2 %, respectively). The amount of RNA recovered from 8- to 16- cell embryos of HMC and in vitro fertilization did not appear to be influenced by the method of embryo generation (3.76 ± 0.61 and 3.82 ± 0.62 ng/μl for HMC and in vitro fertilization embryos, respectively). There were no differences in the expression of the mRNA transcripts of genes (GLUT1, OCT4, DNMT1, BAX and BCL2) were analysed by real-time PCR between hand-made cloned and IVF embryos. Pre-treatment of donor cells with oocyte extracts and selection of developmentally competent oocytes through BCB staining for recipient cytoplast preparations may enhance expression of developmentally important genes GLUT1, OCT4, DNMT1, BAX, and BCL2 in hand-made cloned embryos at levels similar to IVF counterparts. These results also support the notion that if developmental differences observed in HMC and in vitro fertilization produced foetuses and neonates are the results of aberrant gene expression during the pre-implantation stage, those differences in expression are subtle or appear after the maternal to zygotic transition stage of development.

Identification and Characterization of an Oocyte Factor Required for Porcine Nuclear Reprogramming

Nuclear reprogramming of somatic cells can be induced by oocyte factors. Despite numerous attempts, the factors responsible for successful nuclear reprogramming remain elusive. In the present study, we found that porcine oocytes with the first polar body collected at 42 h of in vitro maturation had a stronger ability to support early development of cloned embryos than porcine oocytes with the first polar body collected at 33 h of in vitro maturation. To explore the key reprogramming factors responsible for the difference, we compared proteome signatures of the two groups of oocytes. 18 differentially expressed proteins between these two groups of oocytes were discovered by mass spectrometry (MS). Among these proteins, we especially focused on vimentin (VIM). A certain amount of VIM protein was stored in oocytes and accumulated during oocyte maturation, and maternal VIM was specifically incorporated into transferred somatic nuclei during nuclear reprogramming. When maternal VIM function was inhibited by anti-VIM antibody, the rate of cloned embryos developing to blastocysts was significantly lower than that of IgG antibody-injected embryos and non-injected embryos (12.24 versus 22.57 and 21.10%; p < 0.05), but the development of in vitro fertilization and parthenogenetic activation embryos was not affected. Furthermore, we found that DNA double strand breaks dramatically increased and that the p53 pathway was activated in cloned embryos when VIM function was inhibited. This study demonstrates that maternal VIM, as a genomic protector, is crucial for nuclear reprogramming in porcine cloned embryos.

Selection and Synchronization of Recipient Cytopalsts for Improving the Efficiency of Porcine Somatic Cell Nuclear Transfer

The development of embryos reconstructed by nuclear transfer (NT) is dependent upon numerous factors. Central to development is the quality and development of the recipient cytoplast. Typically oocytes Corresponding author: Joon-Hee Lee Tel: +82-55-772-1886 Fax: +82-55-772-1889 E-mail: sbxjh@gnu.ac.kr 168 ... Journal of Agriculture & Life Science 47(6) at either AI/TI or MII are potential candidates used as recipient cytoplasts for NT, because they contain active MPF, which causes NEBD and PCC in the transferred nucleus and may be essential for nuclear reprogramming. In vitro maturation of porcine oocytes generally spend longer times (44-48h), progress asynchronously from immature stage and also present insufficient MPF for NEBD and PCC. Here, we have developed three stages establishment of in vitro porcine oocyte maturation to select good quality of oocytes used as recipient cytoplasts for NT. Firstly, oocytes were assessed by the activity of glucose-6-phosphate dehydrogenase (G6PD) before in vitro oocyte maturation. Positively BCB stained oocytes reached to MII (67.0% vs. 58.4%) and produced more parthenogenetic blastocysts (50.2% vs. 29.6%) as compared with negatively BCB stained oocytes (P < 0.05). Secondly, positively BCB stained oocytes were treated with 5 μg/ml CHXM to block meiotic progression for synchronizing the cell cycle of oocytes for 16 hr. All of oocytes (130/130) were efficiently synchronized at GV stage by treatment with CHXM, whereas most of oocytes (w/o CHXM) were passed through GV stage (80/176). In addition, retrieval from treatment of CHXM could reversibly induce meiotic resumption and progresses synchronously in porcine oocytes. However, there was no difference between CHXM treated and non-treated group (21.9% vs. 22.3%) in the developmental rate to the blastocyst stage of parthenogenetic embryos. Following treatment of 5 mM caffeine (a phosphatase inhibitor) for 12 hr, all of oocytes were significantly increased the activity of MPF (P < 0.05). Finally, a piglet was produced from NT embryos reconstructed by using non-caffeine treated recipients.

Transgenic Chicken, Mice, Cattle, and Pig Embryos by Somatic Cell Nuclear Transfer into Pig Oocytes

This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p<0.05) SCNT embryos than in cattle (6.3±2.5%), mice (4.2±1.4%), or chicken (5.1±2.4%) iSCNT embryos. The iSCNT embryos also contained a significantly less number of cells per blastocyst than those of SCNT pig embryos (p<0.05). All (100%) iSCNT embryos expressed the EGFP gene, as evidenced by the green florescence under ultraviolet (UV) illumination. Microinjection of purified mitochondria from cattle somatic cells into pig oocytes did not have any adverse effect on their postfertilization in vitro development and embryo quality (p>0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle-pig iSCNT embryos.

Effects of Caffeine on Maturation-Promoting Factor (MPF) Activity in Bovine Oocytes and on the Development of Somatic Cell Nuclear Transfer Embryos in White-Hanwoo

The technique of SCNT is now well established but still remains inefficient. The in vitro development of SCNT embryos is dependent upon numerous factors including the recipient cytoplast and karyoplast. Above all, the metaphase of the second meiotic division (MII) oocytes have typically become the recipient of choice. Generally high level of MPF present in MII oocytes induces the transferred nucleus to enter mitotic division precociously and causes NEBD and PCC, which may be the critical role for nuclear reprogramming. In the present study we investigated the in vitro development and pregnancy of White-Hanwoo SCNT embryos treated with caffeine (a protein kinase phosphatase inhibitor). As results, the treatment of 10 mM caffeine for 6 h significantly increased MPF activity in bovine oocytes but does not affect the developmental competence to the blastocyst stage in bovine SCNT embryos. However, a significant increase in the mean cell number of blastocysts and the frequency of pregnant on 150 days of White-Hanwoo SCNT embryos produced using caffeine treated cytoplasts was observed. These results indicated that the recipient cytoplast treated with caffeine for a short period prior to reconstruction of SCNT embryos is able to increase the frequency of pregnancy in cow.

Developmental potential of vitrified goat oocytes following somatic cell nuclear transfer and parthenogenetic activation

Oocyte cryopreservation is an alternative tool in assisted reproductive technology. The objective of this study was to determine the developmental potential of vitrified goat oocytes after somatic cell nuclear transfer (SCNT). In vitro matured goat oocytes were vitrified by Cryotop method. The survival rate of vitrified oocytes at 1h after thawing determined by fluorescein diacetate staining was 92.1% which was significantly lower than that of fresh oocytes (99.3%). Live oocytes from both vitrified and fresh groups were used as recipient cytoplasts for SCNT. No significant difference in fusion rate was found between vitrified (97.6%) and fresh (93.2%) oocytes. The cleavage rates of vitrified oocytes in the SCNT and parthenogenetic activation (PA) embryos (29.6% and 27.9%) were significantly lower than those from fresh SCNT and PA oocytes (80.9% and 91.1%). The developmental rates to 8-cell stage of SCNT and PA embryos from vitrified oocytes were also lower than that of fresh oocytes. There was no significant difference among the groups in the development to morula stage (11–27%). However, the blastocyst rate of PA embryos derived from fresh oocytes (12.5%) was significantly higher than the other groups (1.2–3.3%). Although high survival rate of vitrified oocytes was obtained, the cleavage, 8-cell, and blastocyst formation rates of SCNT and PA embryos from vitrified oocytes were still lower than those of fresh oocytes.

Production of myostatin-targeted goat by nuclear transfer from cultured adult somatic cells

Myostatin, a member of the transforming growth factor-β family, acts as a negative regulator of skeletal muscle mass. In this study, myostatin-targeted caprine fibroblasts were obtained and subjected to SCNT to determine whether myostatin-knockout goats could be created. Fibroblasts from a 2-mo-old goat were transfected with a myostatin-targeted vector to prepare transgenic donor cells for nuclear transfer. After serum-starvation (for synchronization of the cell cycle), the percentage of transgenic fibroblasts in the G0/G1 phase increased (66.2% vs. 82.9%; P < 0.05) compared with that in the control group, whereas the apoptosis rate and mitochondrial membrane potential were unaffected (P > 0.05). There were no significant differences between in vivo- and in vitro-matured oocytes as recipient cytoplasts for rates of fusion (86.5% vs. 78.4%), pregnancy (21.6% vs. 16.7%), or kidding (2.7% vs. 0%). One female kid from an in vivo-matured oocyte was born, but died a few hours later. Microsatellite analysis and polymerase chain reaction identification confirmed that this kid was genetically identical to the donor cells. Based on Western blot analysis, myostatin of the cloned kid was not expressed compared with that of nontransgenic kids. In conclusion, SCNT using myostatin-targeted 2-mo-old goat fibroblasts as donors has potential as a method for producing myostatin-targeted goats.

38. Impact of microwave processing on dehydration and structural integrity of germinal vesicles from domestic cat oocytes

The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing and could enable drying and storage of GV at supra-zero temperatures. Previous studies demonstrated that (1) 60% of GVs can survive simple air-drying in the presence of trehalose followed by 4 weeks of storage at 4 °C and (2) 10% of reconstructed oocytes with rehydrated GV can achieve nuclear maturation (Graves-Herring et al., 2011). However, air-drying is an uncontrolled process that often results in non-uniform distributions of water within and between samples. Compared to existing techniques, microwave assisted dehydration appears to be a simple and reliable approach to dry biological samples at ambient temperatures (Chakraborty et al., 2008). The objective of the study therefore was to (1) explore the feasibility of drying GV exposed to trehalose using microwave processing and (2) assess the impact on GV structural integrity (chromatin configuration and incidence of DNA damage). Immature cat oocytes from adult ovaries were denuded from the cumulus cells and permeabilized with hemolysin. Groups of five denuded oocytes were exposed to Tris–HCL EDTA (pH 8.0) buffer containing 200 mM trehalose for 10 min and then transferred into 10-μl droplets placed on small pieces of Millipore Isopore Membrane filters (n = 182 total oocytes in 5 replicates). A maximum of eight droplets were arranged on the turntable of a CEM SAM 255 microwave system. Drying was conducted at 20% power for 5, 8, 20, 25 or 30 min. After each time point, samples were weighed immediately to establish the extent of water loss and then were rehydrated before fixation in 4% paraformaldehyde followed by GV chromatin configuration assessment (Hoechst staining) and DNA fragmentation assay (TUNEL). Fresh denuded oocytes (n = 60 total oocytes) were fixed as controls. The average ambient humidity during all measurements was 43.2 ± 1.1%. From time 0 through 30 min of microwave treatment, moisture contents (g water/g dry weight) progressively decreased (0 min: 12.3 ± 0.5; 5 min: 8.3 ± 1.2; 8 min: 8.9 ± 1.2; 20 min: 5.0 ± 0.5; 25 min: 1.9 ± 0.3; 30 min: 1.3 ± 0.4). GV chromatin configuration was not affected by the microwave treatment. The proportion of degenerated oocytes (absence of GV) ranged from 10% to 15% after the different time points and was not different (P > 0.05; Chi-square test) from the fresh controls. Interestingly, pooled proportions of GV with DNA damage following 5–8 min of microwave dehydration were not different (P > 0.05) from fresh controls (14.3% and 10.9%, respectively). However, the incidence of DNA fragmentation was higher (43.5%; P

Improvement of the nuclear transfer efficiency by using the same genetic background of recipient oocytes as the somatic donor cells in goats

We have compared the effect of the genetic background of recipient oocytes on the in vitro and in vivo development of nuclear transfer reconstructed embryos in goats. Adult fibroblast cells from Boer goats were used as donor cells, and recipient oocytes were obtained from Boer goats and Boer cross-breeds (Boer♂×Huanghuai♀). Nuclear transfer reconstructed embryos were cultured in vitro, or transferred into recipient goats. The mitochondrial origin of 2 cloned Boer goats was investigated by analysing the D-loop region based on polymorphisms via DNA sequencing. There was no significant difference in the fusion rate and cleavage rate of reconstructed embryos (P>0.05), when using Boer and cross-breeding goat oocytes as recipient cytoplast respectively. However, in vitro morula development of reconstructed embryos from Boer oocytes was significantly higher than that of cross-breeding embryos (34.1% versus 19.1%, P<0.05). There was no significant difference in the rate of pregnancy and foetus loss between the 2 breeds. However, the live-birth rate was significantly higher with Boer goat oocyte recipients than the cross-breeds (3.1% versus 0.8%, P<0.05). Mitochondrial analysis showed that the 2 cloned goats were similar to their respective oocyte donor goats, and significantly different from the nucleus donor. In conclusion, genetic background of recipient oocytes affected in vitro and in vivo development of reconstructed embryos, with the homologous background of cytoplast and nuclear donor benefiting development of reconstructed embryos. The mitochondrial origin of the 2 cloned Boer goats came from recipient oocytes, not donors.