Simplification of bovine somatic cell nuclear transfer by application of a zona-free manipulation technique

Contemporary nuclear transfer techniques often require the involvement of skilled personnel and extended periods of micromanipulation. Here, we present details of the development of a nuclear transfer technique for somatic cells that is both simpler and faster than traditional methods. The technique comprises the bisection of zona-free oocytes and the reconstruction of embryos comprising two half cytoplasts and a somatic cell by adherence using phytohaemagglutinin-P (PHA) followed by an electropulse and subsequent culture in microwells (termed WOWs--well of the well). The development of the system was based on results using parthenogenetic and in vitro fertilized zygotes in order to (a) select the optimal primary activation agent that induced the lowest lysis rate but highest parthenogenetic blastocyst yield, (b) evaluate the quantity and quality of zona-free blastocysts produced in WOWs, and (c) establish any potential embryotoxic effects of PHA-P. The initial data indicated that, of calcium ionophore A23187, ionomycin, and electropulse treatments as primary activation agents, the two former were equally efficient even with reduced exposure times. WOW-culture of zona-free versus zona-intact zygotes were not different in either blastocyst yield (44.6 +/- 2.4% versus 51.8 +/- 13.5% [mean +/- SEM]) or quality (126.3 +/- 48.4 versus 119.9 +/- 32.6 total cells), and exposure of zygotes to PHA-P did not reduce blastocyst yields compared to vehicle control (40.8 +/- 11.6% versus 47.1 +/- 20.8% of cultured oocytes). Subsequent application of the optimized technique for nuclear transfer using nine different granulosa cell primary cultures (cultured in 0.5% serum for 5-12 days) generated 37.6 +/- 3.9% (11 replicates; range, 16.4-58.1%) blastocysts per successfully fused and surviving reconstructed embryo (after activation), and 33.6 +/- 3.7% blastocysts per attempted reconstructed embryo. Mean day 7 total blastocyst cell numbers from 5 clone families was 128.1 +/- 15.3. The ongoing pregnancy rate of recipients each receiving two nuclear transfer blastocysts is 3/13 (23.1%) recipients pregnant at 5 months after transfer. These results suggest that the zona-free nuclear transfer technique generates blastocysts of equivalent quantity and quality compared to conventional micromanipulation methods, requires less technical expertise, is less time consuming and can double the daily output of reconstructed embryos (even after taking into consideration the rejection of the half oocytes containing the metaphase plate).     

Production of a cloned calf using zona-free serial nuclear transfer

The efficiency of generating cloned animals following somatic cell nuclear transfer appears to have reached a plateau, despite ongoing research to improve developmental outcomes. A major limitation appears in the restricted nature of the adult/donor cell to de-differentiate to form a totipotent nucleus. Serial nuclear transfer, a modified cloning technique, has increased the developmental competence of amphibian, murine and porcine cloned embryos. This procedure involves a second nuclear transfer step; pronuclear-like cloned nuclei are transferred into pronuclear stage zygotic cytoplasts. The present study reports on the development of a serial nuclear transfer technique in the bovine, based on a zona-free method (hand-made cloning), resulting in the birth of a cloned calf. Comparisons were made between embryos produced by hand-made cloning and serial nuclear transfer. There were no differences between in vitro development or differential cell counts in the blastocysts produced. Transfer of 16 serial hand-made cloned blastocysts resulted in the production of one healthy calf (6%), whereas hand-made cloning resulted in the birth of 1 calf from 23 transferred blastocysts (4%). One serial nuclear transfer pre-term fetus had renal and hepatic abnormalities (previously observed in clones from this cell line). Although it may not be as beneficial in the bovine as in other species, normal placentation (size, placentomes and umbilicus) was encouraging. Refinement of this technique may help to identify species-specific differences in zygotic competence that affect reprogramming of donor cell nuclei and that may improve efficiency.     

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.     

Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplasts

The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.     

Differential cytoplast requirement for embryonic and somatic cell nuclear transfer in cattle

Effective activation of a recipient oocyte and its compatibility with the nuclear donor are critical to the successful nuclear reprogramming during nuclear transfer. We designed a series of experiments using various activation methods to determine the optimum activation efficiency of bovine oocytes. We then performed nuclear transfer (NT) of embryonic and somatic cells into cytoplasts presumably at G1/S phase (with prior activation) or at metaphase II (MII, without prior activation). Oocytes at 24 hr of maturation in vitro were activated with various combinations of calcium ionophore A23187 (A187) (5 microM, 5 min), electric pulse (EP), ethanol (7%, 7 min), cycloheximide (CHX) (10 micro g/ml, 6 hr), and then cultured in cytochalasin D (CD) for a total of 18 hr. Through a series of experiments (Exp. 1-4), an improved activation protocol (A187/EP/CHX/CD) was identified and used for comparison of NT efficiency of embryonic versus somatic donor cells (Exp. 5). When embryonic cells from morula and blastocysts (BL) were used as nuclear donors, a significantly higher rate of blastocyst development from cloned embryos was obtained with G1/S phase cytoplasts than with MII-phase cytoplasts (36 vs. 11%, P < 0.05). In contrast, when skin fibroblasts were used as donor cells, the use of an MII cytoplast (vs. G1/S phase) was imperative for blastocyst development (30 vs. 6%, P < 0.05). Differential staining showed that parthenogenetic, embryonic, and somatic cloned BL contained 26, 29, and 33% presumptive inner cell mass (ICM) cells, respectively, which is similar to that of frozen-thawed in vivo embryos at a comparable developmental stage (23%). These data indicate that embryonic and somatic nuclei require different recipient cytoplast environment for remodeling/ reprogramming, and this is likely due to the different cell cycle stage and profiles of molecular differentiation of the transferred donor nuclei.     

Cloning of Asian yellow goat (C. hircus) by somatic cell nuclear transfer: telophase enucleation combined with whole cell intracytoplasmic injection

Our and other previous studies have shown that telophase enucleation is an efficient method for preparing recipient cytoplasts in nuclear transfer. Conventional methods of somatic cell nuclear transfer either by electro-fusion or direct nucleus injection have very low efficiency in animal somatic cell cloning. To simplify the manipulation procedure and increase the efficiency of somatic cell nuclear transfer, this study was designed to study in vitro and in vivo development of Asian yellow goat cloned embryos reconstructed by direct whole cell intracytoplasmic injection (WCICI) into in vitro matured oocytes enucleated at telophase II stage. Our results demonstrated that the rates of cleavage and blastocyst development of embryos reconstructed by WCICI were slightly higher than in conventional subzonal injection (SUZI) group, but no statistic difference (P > 0.05) existed between these two methods. However, the percentage of successful embryonic reconstruction in WCICI group was significantly higher than that in SUZI group (P < 0.05). After embryo transfer at 4-cell stage, the foster in both groups gave birth to offspring. Therefore, the present study suggests that the telophase ooplasm could properly reprogram the genome of somatic cells, produce Asian yellow goat cloned embryos and viable kids, and whole cell intracytoplasmic injection is an efficient protocol for goat somatic cell nuclear transfer.     

Cloned cattle derived from a novel zona-free embryo reconstruction system

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.     

Establishment of an efficient somatic cell nuclear transfer system for production of transgenic pigs

Handmade cloning (HMC) is now an established procedure used in several species for somatic cell nuclear transfer, but only applied in two related laboratories for pigs. The aim of this review is to facilitate widespread application by summarizing the process of establishment and explaining the background of the incorporated special approaches. Optimized steps of traditional cloning in pigs (in vitro maturation, activation, embryo culture) were merged with those of the micromanipulation-free HMC that has been modified according to the specific needs of sensitive porcine oocytes (partial zona digestion before enucleation, two-step zona-free fusion with the somatic cell; initiation of activation with the second fusion). The zona-free approach required embryo culture to the blastocyst stage before surgical transfer of embryos to the uterine horns of recipient sows in the proper phase of an unstimulated cycle. Eventually a competitive, inexpensive and reliable alternative to traditional porcine nuclear transfer cloning techniques evolved that is also suitable to produce transgenic offspring containing various genetic modifications to establish models for several human diseases with genetic background. Further improvements and involvement of additional techniques to increase the overall efficiency and facilitate practical applications are expected in the foreseeable future.     

Production of calves by transfer of nuclei from cultured somatic cells obtained from Japanese black bulls

We investigated the possibility of producing calves from transferable bovine embryos obtained by nuclear transfer using somatic cell-derived cell lines. Muscle cells obtained from 2 Japanese Black bulls were dispersed in Hank's solution supplemented with collagenase Type-I. The separated muscle cells were cultured in Dulbecco's Modified Eagle's medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) at 39 degrees C in an atmosphere of 5% CO2 in air. Cells were passaged at least 4 times, and for 5 d prior to nuclear transfer they (donor cells: karyoplasts) were cultured in D-MEM supplemented with 0.5% FBS (to induce quiescence) or 10% FBS. Recipient oocytes were produced by in vitro culture of bovine oocytes that were obtained at a slaughterhouse and then enucleated in modified phosphate buffered saline supplemented with cytochalasin B. Embryos were reconstructed by 3 protocols using karyoplasts cultured in the medium with 0.5% FBS. 1) Group A: recipient oocytes (cytoplasts; n = 157) were treated with Ca ionophore A 23187, ethanol and cycloheximide, and then a karyoplast was fused to an activated cytoplast. 2) Group B: karyoplasts were transferred to cytoplasts (n = 117), and the couplets were treated with electric stimulation and then Ca ionophore A 23187 and cycloheximide. 3) Group C: cytoplasts (n = 104) were cultured for a further 12 h before fusion, and then the couplets were treated with electric stimulation and cycloheximide. 4) Group D: in addition to the above 3 groups, karyoplasts cultured in the medium with 10% FBS were transferred to recipient cytoplasts (n = 137) and treated as in Protocol 2. Reconstructed embryos were cultured in modified CR1aa for 8 d, and the development of embryos was assessed. In total 73 blastocysts were obtained, and the frequency of development to the blastocyst stage in Group A (2.5%) was lower than that of Groups B, C and D (20.5, 18.3 and 19.0%, respectively; P < 0.01). Of these the sex of 21 blastocysts was determined by rapid Y-chromosome detection assay, and all were male, suggesting that nuclear replacement had been achieved successfully. When 26 blastocysts were transferred to 20 recipient cows, 8 of them became pregnant; 4 cows subsequently aborted about 60 d after embryo transfer while the remaining 4 cows calved. These results indicate that reconstructed embryos obtained by nuclear transfer of muscle cell-derived cell lines can develop to the blastocyst stage, and some are sufficiently competent to develop to term. Particularly important was the finding that special culture protocols for somatic cells prior to nuclear transfer were not necessary in our system.     

Development to the blastocyst stage of porcine somatic cell nuclear transfer embryos reconstructed by the fusion of cumulus cells and cytoplasts prepared by gradient centrifugation

The present study was designated to examine the possibility of producing somatic cell nuclear transfer (SCNT) embryos in pigs using oocyte cytoplasm fragments (OCFs), prepared by centrifugations, as recipient cytoplasts. In Experiment 1, in vitro matured oocytes were centrifuged at 13,000 x g for 3, 6, and 9 min to stratify the cytoplasm, and then the oocytes were freed from zona pellucida and recentrifuged at 5,000 x g for 4 sec in Percoll gradient solution to produce OCFs as the source of recipient cytoplasts. It was found that a long duration of the first centrifugation tends to produce large-sized OCFs after the second centrifugation. In Experiment 2, two or three cytoplasts without chromosomes were aggregated, and then they were fused with a cumulus cell to produce SCNT embryos. The results showed that 66.4 +/- 9.4% of the reconstructed embryos underwent premature chromosome condensation at 1 h after activation, and 85.2 +/- 7.1% and 61.6 +/- 7.0% of them had pseudopronuclei at 10 and 24 h after activation, respectively. In Experiment 3, when SCNT embryos reconstructed by the fusion of three cytoplasts and one cumulus cell, a significantly higher (p < 0.05) rate of reconstructed embryos developed to the blastocyst stage (10.6 +/- 1.8%) than that of reconstructed with two cytoplasts and one cumulus cell (5.2 +/- 1.5%). These results indicate that cytoplasts obtained by two centrifugations can support the remodeling of a transferred somatic nucleus, resulting in the development of the reconstructed porcine embryos to the blastocyst stage.     

Optimization strategies for production of mammalian embryos by nuclear transfer

In order to optimize each of the individual steps in the nuclear transfer procedure, we report alternative protocols useful for producing recipient cytoplasts and for improving the success rate of nuclear transfer embryos in cattle, rhesus monkey, and hamster. Vital labeling of maternal chromatin/spindle is accomplished by long wavelength fluorochromes Sybr14 and rhodamine labeled tubulin allowing constant monitoring and verification during enucleation. The use of Chinese hamster ovary (CHO) donor cells expressing the viral influenza hemagglutinin fusion protein (HA-300a+), to adhere and induce fusion between the donor cells and enucleated cow, rhesus and hamster oocytes was examined. Cell surface hemagglutinin was activated with trypsin prior to nuclear transfer and fusion was induced by a short incubation of a newly created nuclear transfer couplet at pH 5.2 at room temperature. Donor cell cytoplasm was dynamically labeled with CMFDA, or further transfected with the green fluorescence protein (GFP) gene, so that fusion could be directly monitored using live imaging. High rates of fusion were observed between CHO donor cells and hamster (100%), rhesus (100%), and cow recipient cytoplasts (81.6%). Live imaging during fusion revealed rapid intermixing of cytoplasmic components between a recipient and a donor cell. Prelabeled donor cytoplasmic components were uniformly distributed throughout the recipient cytoplast, within minutes of fusion, while the newly introduced nucleus remained at the periphery. The fusion process did not induce activation as evidenced by unchanged distribution and density of cortical granules in the recipient cytoplasts. After artificial activation, the nuclear transfer embryos created in this manner were capable of completing several embryonic cell divisions. These procedures hold promise for enhancing the efficiency of nuclear transfer in mammals of importance for biomedical research, agriculture, biotechnology, and preserving unique, rare, and endangered species.     

Heterogeneous nuclear-transferred-embryos reconstructed with camel (Camelus bactrianus) skin fibroblasts and enucleated ovine oocytes and their development H-M

This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and ovine oocytes as recipient cytoplasts for investigating the developmental potential of the reconstructed embryos. Serum-starved adult camel skin fibroblast cells were used as donor somatic cells. Ovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electrofusion. The fused oocytes were activated by 5mM/ml inomycin with 2mM/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168h. A total of 300 enucleated ovine oocytes were available for xenonuclear embryo reconstruction. The results showed that 71% of the nuclear transfer couplets were successfully fused, 55% of the fused oocytes cleaved within 48h after activation, 82% of the cleaved oocytes developed to 2-16-cell embryo stages and 18% of the cleaved nuclear transfer zygotes developed to the morula stage. This study demonstrated that the xenonuclear transfer camel embryos can undergo the first embryonic division and subsequent development to morula stage in vitro.     

Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro

Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.     

Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep

Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.     

Production of rabbit chimeric embryos by aggregation of zona-free nuclear transfer blastomeres

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p < 0.05), but significantly lower than in TR<>N chimeric blastocysts (p < 0.001). Similarly, a higher proportion (p < 0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.     

Telophase-stage host ooplasts support complete reprogramming of roscovitine-treated somatic cell nuclei in cattle

Nuclear-cytoplasmic incompatibilities are known to play a significant role in the developmental outcome of embryos produced by nuclear transfer, particularly when metaphase arrested oocytes are used as hosts for interphase donor nuclei. To further our understanding of how cell cycle coordination affects somatic cell cloning, somatic cells at different stages of the cell cycle were fused to host oocytes either before (metaphase II, M-II) or after (telophase II, T-II) activation. To obtain cells at different stages of the cell cycle, fetal fibroblast (FF) and granulosa cells (GC) were treated with roscovitine, an inhibitor of cyclin-dependent kinases (CDKs) resulting in a large percentage of cells in S/G(2)-phase. In contrast to the M-II group, which did better with confluent cells, embryos reconstructed with T-II cytoplasts resulted in higher rates of blastocyst formation when fused to cells recovered at 16-24 h after passage. Embryos reconstructed with FF treated with roscovitine and T-II cytoplasts (Rosc/T-II) resulted in similar blastocyst rate compared to those produced with confluent cells and M-II cytoplasts (Conf/M-II). Transfer of blastocysts to surrogate heifers resulted pregnancies and birth of healthy calves from Rosc/T-II and Conf/M-II reconstructed embryos. These results indicate that, when combined with nuclear donor cells at specific cell cycle stages, M-II and T-II bovine oocytes are similarly effective in supporting the reprogramming of somatic cell nuclei.     

Effect of post-fusion holding time, orientation and position of somatic cell-cytoplasts during electrofusion on the development of handmade cloned embryos in buffalo (Bubalus bubalis)

The present study was conducted primarily to optimize electrofusion conditions for efficient production of zona-free nuclear transfer embryos in buffalos (Bubalus bubalis). We found that 4V AC current for proper triplet alignment and single step fusion method, using a single DC pulse of 3.36 kV/cm for 4-μs duration, produced the most convincing results for efficient reconstitution of zona-free cloned embryos. Lysis rate was very high (84.28 ± 2.59%) when triplets were in physical contact with negative electrode after applying DC current, however, cleavage rate and blastocyst rate were found to be similar when the triplets were not in physical contact with either positive or negative electrodes or when they were in physical contact with the positive electrode. Significant improvement in blastocyst production was observed when the somatic cell faced the positive electrode than when it faced the negative electrode (39.17 ± 2.74% vs. 25.91 ± 2.00%, respectively) during electrofusion. Similarly, the blastocyst rate (52.0 ± 3.4%) was found to be significantly higher when reconstructed embryos were activated 6 h post electrofusion as compared to 0, 2, 4 and 8 h (16.04 ± 6.3%; 18.36 ± 1.4%; 22.44 ± 3.7% and 30.02 ± 4.6%, respectively). This study establishes the application of zona-free nuclear transfer procedures for the production of handmade cloned buffalo embryos through optimization of electrofusion parameters and post fusion holding time for enhancing their preimplantation development.     

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.     

Isolation of bovine cardiomyocytes for reprogramming studies based on nuclear transfer

The goal of this study was to establish and validate a protocol for preparing bovine cardiomyocytes from slaughterhouse material for nuclear transfer experiments. The cardiomyocyte was selected because it is a terminally differentiated cell and strongly expresses a unique subset of genes which can be monitored during the reprogramming period. A total of 39 trials were conducted, and an optimized protocol was developed yielding individual contractile cardiomyocytes from 3-5-month-old bovine fetuses The basic protocol involves stabilization of bovine heart tissue for transportation from the slaughterhouse to the laboratory by perfusion with Custodiol. This was followed by an enzymatic dissociation with collagenase in calcium-free medium and yielded individual contractile rod-shaped cardiomyocytes. Subsequent addition of Ca2+ caused the cardiomyocytes to round up which was an essential pre-condition for drawing them into glass transfer pipettes for delivery into the perivitelline space and for efficient electrofusion with cytoplasts derived from in vitro matured bovine oocytes. The use of cardiomyocytes maintained at 37 degrees C in nuclear transfer, resulted in a significantly reduced proportion of blastocysts compared to adult fibroblasts (14.0% versus 32.7%). Storage of cardiomyocytes at 4 degrees C prior to nuclear transfer was not compatible with blastocyst development. It is expected that this system will be valuable for investigating the reprogramming of gene expression which occurs after somatic cell nuclear transfer.     

Effect of telophase enucleation on bovine somatic nuclear transfer

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.