Cryopreservation of pig spermatozoa using carboxylated poly-L-lysine as cryoprotectant.

In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro. However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.

The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones.

Recent advances in genome editing systems such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) have facilitated genomic modification in mammalian cells. However, most systems employ transient treatment with selective drugs such as puromycin to obtain the desired genome-edited cells, which often allows some untransfected cells to survive and decreases the efficiency of generating genome-edited cells. Here, we developed a novel targeted toxin-based drug-free selection system for the enrichment of genome-edited cells. Cells were transfected with three expression vectors, each of which carries a guide RNA (gRNA), humanized Cas9 (hCas9) gene, or Clostridium perfringens-derived endo-β-galactosidase C (EndoGalC) gene. Once EndoGalC is expressed in a cell, it digests the cell-surface α-Gal epitope, which is specifically recognized by BS-I-B4 lectin (IB4). Three days after transfection, these cells were treated with cytotoxin saporin-conjugated IB4 (IB4SAP) for 30 min at 37 °C prior to cultivation in a normal medium. Untransfected cells and those weakly expressing EndoGalC will die due to the internalization of saporin. Cells transiently expressing EndoGalC strongly survive, and some of these surviving clones are expected to be genome-edited bi-allelic knockout (KO) clones due to their strong co-expression of gRNA and hCas9. When porcine α-1,3-galactosyltransferase gene, which can synthesize the α-Gal epitope, was attempted to be knocked out, 16.7% and 36.7% of the surviving clones were bi-allelic and mono-allelic knockout (KO) cells, respectively, which was in contrast to the isolation of clones in the absence of IB4SAP treatment. Namely, 0% and 13.3% of the resulting clones were bi-allelic and mono-allelic KO cells, respectively. A similar tendency was seen when other target genes such as DiGeorge syndrome critical region gene 2 and transforming growth factor-β receptor type 1 gene were targeted to be knocked out. Our results indicate that a combination of the CRISPR/Cas9 system and targeted toxin technology using IB4SAP allows efficient enrichment of genome-edited clones, particularly bi-allelic KO clones.

Selection of human blastocysts with a high implantation potential based on timely compaction.

PURPOSE: Recently, we established a noninvasive system for selecting human blastocysts with a high pre-transfer implantation potential based on first and second division patterns. The present study was carried out to improve the selection system. METHODS: Embryos that completed first and second divisions within 25.90 and 37.88 h after culture, respectively, were selected using a time-lapse incubator. We examined the effects of compaction and blastocyst formation times on pregnancy rates after transferring these embryos at the blastocyst stage. RESULTS: The completion of compaction and blastocyst formation times (79.93 and 97.47 h after culture, respectively) of embryos resulting in pregnancies after transfer were significantly (P < 0.01) shorter than those (86.46 and 100.34 h after culture, respectively) of embryos that failed to induce pregnancies. Embryo selection based on completion of compaction time improved pregnancy rates (40.9 vs. 74.6%, P < 0.01). CONCLUSIONS: Of the embryos that formed two cells during the first division within 25.90 h after culture and four cells during the second division within 37.88 h after culture, those that completed compaction within 79.93 h after culture before reaching the blastocyst stage had a high implantation potential.

Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System.

The recent advancement in genome editing such a CRISPR/Cas9 system has enabled isolation of cells with knocked multiple alleles through a one-step transfection. Somatic cell nuclear transfer (SCNT) has been frequently employed as one of the efficient tools for the production of genetically modified (GM) animals. To use GM cells as SCNT donor, efficient isolation of transfectants with mutations at multiple target loci is often required. The methods for the isolation of such GM cells largely rely on the use of drug selection-based approach using selectable genes; however, it is often difficult to isolate cells with mutations at multiple target loci. In this study, we used a novel approach for the efficient isolation of porcine cells with at least two target loci mutations by one-step introduction of CRISPR/Cas9-related components. A single guide (sg) RNA targeted to GGTA1 gene, involved in the synthesis of cell-surface α-Gal epitope (known as xenogenic antigen), is always a prerequisite. When the transfected cells were reacted with toxin-labeled BS-I-B4 isolectin for 2 h at 37 C to eliminate α-Gal epitope-expressing cells, the surviving clones lacked α-Gal epitope expression and were highly expected to exhibit induced mutations at another target loci. Analysis of these α-Gal epitope-negative surviving cells demonstrated a 100% occurrence of genome editing at target loci. SCNT using these cells as donors resulted in the production of cloned blastocysts with the genotype similar to that of the donor cells used. Thus, this novel system will be useful for SCNT-mediated acquisition of GM cloned piglets, in which multiple target loci may be mutated.

Developmental ability of embryos produced from oocytes with fragile oolemma by intracytoplasmic sperm injection.

PURPOSE: In intracytoplasmic sperm injection (ICSI) of oocytes with a fragile oolemma (fragile oocytes), breakage can occur at injection. In this study, we produced embryos from oocytes with a fragile and normal oolemma (normal oocytes) by ICSI and compared their ability to be fertilized and develop in vitro. We also investigated whether fragile oocyte-derived embryos could implant after blastocyst transfer to determine whether fragile oocytes should be used for assisted reproductive technology treatment. METHODS: Oocytes were divided into three groups-normal oocytes from cycles containing no fragile oocytes (group A), normal oocytes from cycles containing at least one fragile oocyte (group B), and fragile oocytes (group C), and their fertilization abilities after ICSI and the developmental abilities of resultant embryos were compared. RESULTS: The fertilization rate in group C (65.3 %) was significantly (P < 0.01) lower than those in groups A (84.6 %) and B (86.9 %), and the degeneration rate in group C (24.2 %) was significantly (P < 0.01) higher than those in groups A (0.71 %) and B (0.28 %). However, there were no significant differences in the blastocyst formation rates (59.7-67.5 %) of embryos among the different groups. In addition, the pregnancy rate after transfer of blastocysts in group C (50.0 %) was not significantly different from those in groups A (35.6 %) and B (45.8 %). CONCLUSIONS: The fertilization ability after ICSI of fragile oocytes is lower than that of normal oocytes but the resultant embryos have the same developmental ability as those of normal oocyte-derived embryos.

The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.

The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes.

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations.

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.

The combinational use of CRISPR/Cas9-based gene editing and targeted toxin technology enables efficient biallelic knockout of the α-1,3-galactosyltransferase gene in porcine embryonic fibroblasts.

BACKGROUND: The recent development of the type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled genome editing of mammalian genomes including those of mice and human; however, its applicability and efficiency in the pig have not been studied in depth. Here, using the CRISPR/Cas9 system, we aimed to destroy the function of the porcine α-1,3-galactosyltransferase (α-GalT) gene (GGTA1) whose product is responsible for the synthesis of the α-Gal epitope, a causative agent for hyperacute rejection upon pig-to-human xenotransplantation. METHODS: Porcine embryonic fibroblasts were transfected with a Cas9 expression vector and guide RNA specifically designed to target GGTA1. At 4 days after transfection, the cells were incubated with IB4 conjugated with saporin (IB4SAP), which eliminates α-Gal epitope-expressing cells. Therefore, the cells surviving after IB4SAP treatment would be those negative for α-Gal epitope expression, which in turn indicates the generation of GGTA1 biallelic knockout (KO) cells. RESULTS: Of the 1.0 × 10(6) cells transfected, 10-33 colonies survived after IB4SAP treatment, and almost all colonies (approximately 90%) were negative for staining with red fluorescence-labeled IB4. Sequencing of the mutated portion of GGTA1 revealed a frameshift of the α-GalT protein. Porcine blastocysts derived from the somatic cell nuclear transfer of these α-Gal epitope-negative cells also lacked the α-Gal epitope on their surface. CONCLUSIONS: These results demonstrated that the CRISPR/Cas9 system can efficiently induce the biallelic conversion of GGTA1 in the resulting somatic cells and is thus a promising tool for the creation of KO cloned piglets.

Osmolarity- and stage-dependent effects of glycine on parthenogenetic development of pig oocytes.

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.

Improved fertility of frozen-thawed porcine spermatozoa with 3,3-dimethylglutaric anhydride poly-L-lysine as a novel cryoprotectant.

In this study, we determined the efficacy of 3,3-dimethylglutaric anhydride poly-L-lysine (DMGA-PLL) as a cryoprotectant for porcine spermatozoa. Porcine spermatozoa were cryopreserved in a freezing extender containing 3% (v/v) glycerol and various concentrations of DMGA-PLL. At 12 h after thawing, the motility index of spermatozoa cryopreserved with 0.25% (v/v) DMGA-PLL (25.9) was significantly (P < 0.01) higher than that of spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA-PLL (10.0-16.3). In addition, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% DMGA-PLL (22.8%) was significantly (P < 0.01) higher than that of embryos derived from spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA-PLL (7.9%-10.9%). The mean number of total piglets born to sows inseminated with spermatozoa cryopreserved without DMGA-PLL (9.0) was significantly (P < 0.05) lower than that of total piglets born to sows inseminated with spermatozoa stored at 17°C (13.8). However, when spermatozoa cryopreserved with 0.25% DMGA-PLL were used for artificial insemination, the mean number of total piglets (11.7) was not significantly different from that obtained following artificial insemination using spermatozoa stored at 17°C. The results showed the usefulness of DMGA-PLL as a cryoprotectant in the cryopreservation of porcine spermatozoa.

Gene expression profile differences in embryos derived from prepubertal and adult Japanese Black cattle during in vitro development.

The present study was carried out to compare the gene expression profiles of in vitro-generated embryos derived from adult and prepubertal Japanese Black cattle oocytes using GeneChip Bovine Genome Array (containing 24072 probe sets representing over 23000 transcripts). Microarray experiments were performed on populations of 8- to 16-cell stage embryos and blastocysts derived from adult (24-35 months old) versus prepubertal (9-10 months old) Japanese Black cattle oocytes matured and fertilised in vitro. In total, 591 (2.4%) and 490 (2.0%) genes were differentially expressed in prepubertal and adult bovine in 8- to 16-cell and blastocyst stage embryos, respectively. Out of these, 218 and 248 genes were upregulated, while 373 and 242 were downregulated in prepubertal and adult 8- to 16-cell and blastocysts stage embryos, respectively. Gene ontology classification regarding biological process, molecular functions and cellular component revealed diversity in transcript abundances between prepubertal and adult groups in both the distinct developmental stages. Quantitative reverse transcription-PCR validated the expression differences of some selected transcripts as identified by microarray analysis. To our knowledge, this is the first report indicating the significant number of genes differentially expression (>2-fold, P<0.01) in preimplantition embryos between adult and prepubertal Japanese Black cattle during in vitro development.

Successful suppression of endogenous α-1,3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro.

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation.

Effect of postactivation treatment with latrunculin A on in vitro and in vivo development of cloned embryos derived from kidney fibroblasts of an aged Clawn miniature boar.

The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 µM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 µM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 µM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term.

Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate.

BACKGROUND: The recent availability of alpha-1,3-galatosyltransferase knockout pigs has eliminated anti-Gal antibodies to the galalpha1-3gal (alphagal epitope) as the major barrier to xenotransplantation. These alphagal epitope-negative animals can also be produced by somatic cell nuclear transfer of cells overexpressing endo-beta-galactosidase (EndoGalC), an enzyme capable of digesting the alphagal epitope. For this, selection of cells with highly reduced synthesis of alphagal epitope is a prerequisite. In this study, we developed a novel method of selection using isolectin BS-I-B(4)-conjugated saporin (IB4-SAP), a targeted cytotoxin, that is specific for the terminal alphagal epitope. METHODS: A mixture of alphagal epitope-expressing and non-expressing pig cells was obtained by transfection with an EndoGalC expression vector. These cells were incubated with a solution containing IB4-SAP for 2 h at 37 degrees C, and subsequently cultivated for over 2 months under general conditions. RESULTS: Almost all (98%) of surviving cells were completely negative for expression of alphagal epitope, as confirmed by cytochemical staining using fluorescence-labeled IB4. FACS analysis also confirmed that the IB4-SAP-treated cells exhibited a staining pattern similar to that of the IB4-negative human cells. Extended cultivation (more than 6 months) of these IB4-SAP-treated cells did not alter the above staining pattern. RT-PCR analysis revealed the presence of EndoGalC mRNA in these cells. CONCLUSIONS: This IB4-SAP-mediated method of selection of alphagal epitope-negative cells will provide an alternative to the present method of cytotoxicity-based selection using specific antibody and complement.

Production of genetically modified porcine blastocysts by somatic cell nuclear transfer: preliminary results toward production of xenograft-competent miniature pigs.

Galα1-3Gal (α-Gal epitope) is the major xenoantigenic epitope responsible for hyperacute rejection upon pig-to-human xenotransplantation. Endo-ß-galactosidase C (EndoGalC) from Clostridium perfringens can digest the α-Gal epitope. In this study, gene-engineered primary cultured porcine embryonic fibroblasts (PEF) expressing EndoGalC were obtained and subjected to somatic cell nuclear transfer (SCNT) to test whether xenograft-competent pigs can be created. The EndoGalC-expressing PEF clones exhibited highly reduced expression of α-Gal epitope, as revealed by cytochemical staining with BS-I-B(4) isolectin, a lectin that specifically binds to α-Gal epitope, and FACS analysis. The pattern of low level of α-Gal epitope expression continued for at least 6 months (more than 10 generations) after isolation. SCNT of nuclei from these cells resulted in the generation of blastocysts that displayed nearly complete loss of α-Gal epitope from their cell surface. This is the first study to demonstrate that SCNT using EndoGalC-expressing PEFs as donors would be useful for production of genetically modified cloned pigs suitable for xenotransplantation.

Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes.

Recently, successful one-step genome editing by microinjection of CRISPR/Cas9-related mRNA components into the porcine zygote has been described. Given the relatively long gestational period and the high cost of housing swine, the establishment of an effective microinjection-based porcine genome editing method is urgently required. Previously, we have attempted to disrupt a gene encoding α-1,3-galactosyltransferase (GGTA1), which synthesizes the α-Gal epitope, by microinjecting CRISPR/Cas9-related nucleic acids and enhanced green fluorescent protein (EGFP) mRNA into porcine oocytes immediately after electrical activation. We found that genome editing was indeed induced, although the resulting blastocysts were mosaic and the frequency of modified cells appeared to be low (50%). To improve genome editing efficiency in porcine oocytes, cytoplasmic injection was performed 6 h after electrical activation, a stage wherein the pronucleus is formed. The developing blastocysts exhibited higher levels of EGFP. Furthermore, the T7 endonuclease 1 assay and subsequent sequencing demonstrated that these embryos exhibited increased genome editing efficiencies (69%), although a high degree of mosaicism for the induced mutation was still observed. Single blastocyst-based cytochemical staining with fluorescently labeled isolectin BS-I-B4 also confirmed this mosaicism. Thus, the development of a technique that avoids or reduces such mosaicism would be a key factor for efficient knock out piglet production via microinjection.

In vitro development of cloned embryos derived from miniature pig somatic cells after activation by ultrasound stimulation.

The present study was carried out to examine the activation and development of cloned embryos produced by transferring miniature pig somatic cells into enucleated farm pig oocytes after exposing to ultrasound. The rates of the pronucleus-like structure formation and polar body-like structure extrusion in embryos exposed to ultrasound did not differ from those applied electric pulses. Although there was no significant difference in the blastocyst formation rates between different activation methods, the mean number of cells in the blastocysts developed from embryos activated by exposing to ultrasound was significantly (p < 0.05) higher than that obtained by applying electric pulses. The results of the present study showed that ultrasound stimulation can induce the activation and in vitro development of cloned embryos derived from miniature pig somatic cells.

Production of cloned calves using roscovitine-treated adult somatic cells as donors.

The stage of the donor cell cycle is a major factor in the success of cloning. Quiescent cells arrested in the G0/G1 phases of the cell cycle by either serum starvation or growth arrest when cultured cells reach confluence have been used as donors to produce cloned animals. Recently, we have developed a novel and effective method using roscovitine to synchronize adult bovine granulosa cells in the G0/G1 cell cycle stage. The resulting fetal and calf survival after transfer of cloned embryos was enhanced in the roscovitine-treated group compared with serum-starved controls. The methods described in this chapter outline (1) the preparation of donor cells, (2) the preparation of recipient oocytes, and (3) the production of cloned embryos. The first section involves methods for the preparation of donor cell stocks from isolated granulosa cells and the roscovitine treatment of the cells before nuclear transfer. The second section explains procedures of in vitro maturation of recipient oocytes. The last section involves methods for the production of cell-oocyte complexes, the fusion of the complexes, and the activation, in vitro culture, and transfer into recipient females of cloned embryos.

Birth of Cloned Microminipigs Derived from Somatic Cell Nuclear Transfer Embryos That Have Been Transiently Treated with Valproic Acid.

In our previous study, we found that treatment of miniature pig somatic cell nuclear transfer (SCNT) embryos with 4 mM valproic acid (VPA), a histone deacetylase inhibitor, for 48 hours after activation enhanced blastocyst formation rate and octamer-binding transcription factor-3/4 (Oct-3/4) gene expression at the late blastocyst stage; however, the production of viable cloned pups failed, when those VPA-treated SCNT embryos were transferred to recipients. This failure suggests that the present VPA treatment is suboptimal. In the present study, we explored the optimal conditions for VPA to have beneficial effects on the development of SCNT embryos. When miniature pig SCNT embryos were treated with 8 mM VPA for 24 hours after activation, both the rates of blastocyst formation and blastocysts expressing the Oct-3/4 gene were significantly (p < 0.05) improved. A similar increase in blastocyst formation was also observed when microminipig-derived cells were used as SCNT donors. Five cloned piglets were obtained after the transfer of 152 microminipig SCNT embryos that had been treated with 8 mM VPA for 24 hours. The results indicated that a short duration of treatment with VPA improves the development of both miniature pig and microminipig SCNT embryos, possibly via an enhanced reprogramming mechanism.

Effects of early cleavage patterns of human embryos on subsequent in vitro development and implantation.

OBJECTIVE: To establish a noninvasive selection system of human embryos with a high implantation potential before transferring. DESIGN: Cohort study. SETTING: Independent IVF clinics. PATIENT(S): One hundred sixty-four patients, with 791 embryos fertilized, checked, and recorded. INTERVENTION(S): Embryos were divided into nine groups according to the number of cells they contained and the extent of their fragmentation during the first and second divisions. We examined the effects of the cell division pattern and division time of embryos on subsequent development in vitro and in vivo using a time-lapse incubator. MAIN OUTCOME MEASURE(S): Rates of blastocyst formation, high-quality blastocyst formation, and pregnancy. RESULT(S): The rates of embryos developed into blastocysts and blastocysts in which both the inner cell mass and trophectoderm grades were scored B or higher (good-quality blastocysts) were high in groups that formed two cells during the first division and four cells during the second division, regardless of the presence or absence of fragmentation. In these groups the first and second division times (25.90 and 37.88 hours after culture, respectively) of embryos developed into good-quality blastocysts were significantly shorter than those of other embryos. Although embryo selection based on the first and second division times did not affect the pregnancy rates after transfer at day 2 or 3 of culture, it improved the pregnancy rates after blastocyst transfer at day 5. CONCLUSION(S): Transfer of blastocysts derived from embryos that completed first and second divisions within 25.90 and 37.88 hours after culture, respectively, brings about high pregnancy rates.