A micro-fabricated device (microICSI) improves porcine blastocyst development and procedural efficiency for both porcine intracytoplasmic sperm injection and human microinjection.

PURPOSE: Intracytoplasmic sperm injection (ICSI) imparts physical stress on the oolemma of the oocyte and remains among the most technically demanding skills to master, with success rates related to experience and expertise. ICSI is also time-consuming and requires workflow management in the laboratory. This study presents a device designed to reduce the pressure on the oocyte during injection and investigates if this improves embryo development in a porcine model. The impact of this device on laboratory workflow was also assessed. METHODS: Porcine oocytes were matured in vitro and injected with porcine sperm by conventional ICSI (C-ICSI) or with microICSI, an ICSI dish that supports up to 20 oocytes housed individually in microwells created through microfabrication. Data collected included set-up time, time to align the polar body, time to perform the injection, the number of hand adjustments between controllers, and degree of invagination at injection. Developmental parameters measured included cleavage and day 6 blastocyst rates. Blastocysts were differentially stained to assess cell numbers of the inner cell mass and trophectoderm. A pilot study with human donated MII oocytes injected with beads was also performed. RESULTS: A significant increase in porcine blastocyst rate for microICSI compared to C-ICSI was observed, while cleavage rates and blastocyst cell numbers were comparable between treatments. Procedural efficiency of microinjection was significantly improved with microICSI compared to C-ICSI in both species. CONCLUSION: The microICSI device demonstrated significant developmental and procedural benefits for porcine ICSI. A pilot study suggests human ICSI should benefit equally.

Characterisation of transgenic pigs expressing a human T cell-depleting anti-CD2 monoclonal antibody.

BACKGROUND: Pig islet xenotransplantation is a potential treatment for type 1 diabetes. We have shown that maintenance immunosuppression is required to protect genetically modified (GM) porcine islet xenografts from T cell-mediated rejection in baboons. Local expression of a depleting anti-CD2 monoclonal antibody (mAb) by the xenograft may provide an alternative solution. We have previously reported the generation of GGTA1 knock-in transgenic pigs expressing the chimeric anti-CD2 mAb diliximab under an MHC class I promoter (MHCIP). In this study, we generated GGTA1 knock-in pigs in which MHCIP was replaced by the ß-cell-specific porcine insulin promoter (PIP), and compared the pattern of diliximab expression in the two lines. METHODS: A PIP-diliximab knock-in construct was prepared and validated by transfection of NIT-1 mouse insulinoma cells. The construct was knocked into GGTA1 in wild type (WT) porcine fetal fibroblasts using CRISPR, and knock-in cells were used to generate pigs by somatic cell nuclear transfer (SCNT). Expression of the transgene in MHCIP-diliximab and PIP-diliximab knock-in pigs was characterised at the mRNA and protein levels using RT-qPCR, flow cytometry, ELISA and immunohistochemistry. Islets from MHCIP-diliximab and control GGTA1 KO neonatal pigs were transplanted under the kidney capsule of streptozotocin-diabetic SCID mice. RESULTS: NIT-1 cells stably transfected with the PIP-diliximab knock-in construct secreted diliximab into the culture supernatant, confirming correct expression and processing of the mAb in ß cells. PIP-diliximab knock-in pigs showed a precise integration of the transgene within GGTA1. Diliximab mRNA was detected in all tissues tested (spleen, kidney, heart, liver, lung, pancreas) in MHCIP-diliximab pigs, but was not detectable in PIP-diliximab pigs. Likewise, diliximab was present in the serum of MHCIP-diliximab pigs, at a mean concentration of 1.8 µg/mL, but was not detected in PIP-diliximab pig serum. An immunohistochemical survey revealed staining for diliximab in all organs of MHCIP-diliximab pigs but not of PIP-diliximab pigs. Whole genome sequencing (WGS) of a PIP-diliximab pig identified a missense mutation in the coding region for the dixilimab light chain. This mutation was also found to be present in the fibroblast knock-in clone used to generate the PIP-diliximab pigs. Islet xenografts from neonatal MHCIP-diliximab pigs restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated expression of diliximab in situ. CONCLUSION: Diliximab was widely expressed in MHCIP-diliximab pigs, including in islets, consistent with the endogenous expression pattern of MHC class I. Further investigation is required to determine whether the level of expression in islets from the MHCIP-diliximab pigs is sufficient to prevent T cell-mediated islet xenograft rejection. The unexpected absence of diliximab expression in the islets of PIP-diliximab pigs was probably due to a mutation in the transgene arising during the generation of the knock-in cells used for SCNT.

Mitochondrial DNA Deficiency and Supplementation in Sus scrofa Oocytes Influence Transcriptome Profiles in Oocytes and Blastocysts.

Mitochondrial DNA (mtDNA) deficiency correlates with poor oocyte quality and fertilisation failure. However, the supplementation of mtDNA deficient oocytes with extra copies of mtDNA improves fertilisation rates and embryo development. The molecular mechanisms associated with oocyte developmental incompetence, and the effects of mtDNA supplementation on embryo development are largely unknown. We investigated the association between the developmental competence of Sus scrofa oocytes, assessed with Brilliant Cresyl Blue, and transcriptome profiles. We also analysed the effects of mtDNA supplementation on the developmental transition from the oocyte to the blastocyst by longitudinal transcriptome analysis. mtDNA deficient oocytes revealed downregulation of genes associated with RNA metabolism and oxidative phosphorylation, including 56 small nucleolar RNA genes and 13 mtDNA protein coding genes. We also identified the downregulation of a large subset of genes for meiotic and mitotic cell cycle process, suggesting that developmental competence affects the completion of meiosis II and first embryonic cell division. The supplementation of oocytes with mtDNA in combination with fertilisation improves the maintenance of the expression of several key developmental genes and the patterns of parental allele-specific imprinting gene expression in blastocysts. These results suggest associations between mtDNA deficiency and meiotic cell cycle and the developmental effects of mtDNA supplementation on Sus scrofa blastocysts.

Does supplementation of oocytes with additional mtDNA influence developmental outcome?

Introducing extra mitochondrial DNA (mtDNA) into oocytes at fertilization can rescue poor quality oocytes. However, supplementation alters DNA methylation and gene expression profiles of preimplantation embryos. To determine if these alterations impacted offspring, we introduced mtDNA from failed-to-mature sister (autologous) or third party (heterologous) oocytes into mature oocytes and transferred zygotes into surrogates. Founders exhibited significantly greater daily weight gain (heterologous) and growth rates (heterologous and autologous) to controls. In weaners, cholesterol, bilirubin (heterologous and autologous), anion gap, and lymphocyte count (autologous) were elevated. In mature pigs, potassium (heterologous) and bicarbonate (autologous) were altered. mtDNA and imprinted gene analyses did not reveal aberrant profiles. Neither group exhibited gross anatomical, morphological, or histopathological differences that would lead to clinically significant lesions. Female founders were fertile and their offspring exhibited modified weight and height gain, biochemical, and hematological profiles. mtDNA supplementation induced minor differences that did not affect health and well-being.

Mitochondrial supplementation of Sus scrofa metaphase II oocytes alters DNA methylation and gene expression profiles of blastocysts.

BACKGROUND: Mitochondrial DNA (mtDNA) copy number in oocytes correlates with oocyte quality and fertilisation outcome. The introduction of additional copies of mtDNA through mitochondrial supplementation of mtDNA-deficient Sus scrofa oocytes resulted in: (1) improved rates of fertilisation; (2) increased mtDNA copy number in the 2-cell stage embryo; and (3) improved development of the embryo to the blastocyst stage. Furthermore, a subset of genes showed changes in gene expression. However, it is still unknown if mitochondrial supplementation alters global and local DNA methylation patterns during early development. RESULTS: We generated a series of embryos in a model animal, Sus scrofa, by intracytoplasmic sperm injection (ICSI) and mitochondrial supplementation in combination with ICSI (mICSI). The DNA methylation status of ICSI- and mICSI-derived blastocysts was analysed by whole genome bisulfite sequencing. At a global level, the additional copies of mtDNA did not affect nuclear DNA methylation profiles of blastocysts, though over 2000 local genomic regions exhibited differential levels of DNA methylation. In terms of the imprinted genes, DNA methylation patterns were conserved in putative imprint control regions; and the gene expression profile of these genes and genes involved in embryonic genome activation were not affected by mitochondrial supplementation. However, 52 genes showed significant differences in expression as demonstrated by RNAseq analysis. The affected gene networks involved haematological system development and function, tissue morphology and cell cycle. Furthermore, seven mtDNA-encoded t-RNAs were downregulated in mICSI-derived blastocysts suggesting that extra copies of mtDNA affected tRNA processing and/or turnover, hence protein synthesis in blastocysts. We also showed a potential association between differentially methylated regions and changes in expression for 55 genes due to mitochondrial supplementation. CONCLUSIONS: The addition of just an extra ~ 800 copies of mtDNA into oocytes can have a significant impact on both gene expression and DNA methylation profiles in Sus scrofa blastocysts by altering the epigenetic programming established during oogenesis. Some of these changes may affect specific tissue-types later in life. Consequently, it is important to determine the longitudinal effect of these molecular changes on growth and development before considering human clinical practice.

Low guanine content and biased nucleotide distribution in vertebrate mtDNA can cause overestimation of non-CpG methylation.

Mitochondrial DNA (mtDNA) methylation in vertebrates has been hotly debated for over 40 years. Most contrasting results have been reported following bisulfite sequencing (BS-seq) analyses. We addressed whether BS-seq experimental and analysis conditions influenced the estimation of the levels of methylation in specific mtDNA sequences. We found false positive non-CpG methylation in the CHH context (fpCHH) using unmethylated Sus scrofa mtDNA BS-seq data. fpCHH methylation was detected on the top/plus strand of mtDNA within low guanine content regions. These top/plus strand sequences of fpCHH regions would become extremely AT-rich sequences after BS-conversion, whilst bottom/minus strand sequences remained almost unchanged. These unique sequences caused BS-seq aligners to falsely assign the origin of each strand in fpCHH regions, resulting in false methylation calls. fpCHH methylation detection was enhanced by short sequence reads, short library inserts, skewed top/bottom read ratios and non-directional read mapping modes. We confirmed no detectable CHH methylation in fpCHH regions by BS-amplicon sequencing. The fpCHH peaks were located in the D-loop, ATP6, ND2, ND4L, ND5 and ND6 regions and identified in our S. scrofa ovary and oocyte data and human BS-seq data sets. We conclude that non-CpG methylation could potentially be overestimated in specific sequence regions by BS-seq analysis.

Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9.

Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.

Energy balance influences number of ovulations rather than embryo quality in the pig.

The present study was undertaken to examine the effect of feed restriction on ovulation rate and in vivo blastocyst development in gilts and sows. In the first experiment, gilts were feed restricted (1 vs. 2.5 times maintenance requirement) during the luteal and follicular phases before ovulation. In the second experiment, primiparous sows were feed restricted (ad lib vs. 60% thereof) during the last week of lactation before weaning. Gilts and sows were slaughtered at 5 days after ovulation to determine ovulation rate and blastocyst development. Blastocysts were also differentially stained to determine the effect of feed restriction on total, trophectoderm, and inner cell mass cell numbers. In both experiments, feed restriction delayed ovulation and reduced the number of ovulations in gilts (14.8 ± 1.3 vs. 12.0 ± 0.2; P < 0.05) and in sows (19.9 ± 1.0 vs. 18.4 ± 0.7). The number of blastocysts recovered on Day 5 was similarly reduced in gilts (12.0 ± 1.7 vs. 9.1 ± 1.1; P < 0.10) and in sows (15.9 ± 1.5 vs. 14.7 ± 1.0). However, feed restriction did not affect total, trophectoderm, or inner cell mass cell numbers in gilts or sows. In conclusion, the present study reported that energy balance influences ovulation rate and blastocyst number rather than blastocyst viability as measured by cell number.

Isolation and in vitro characterization of putative porcine embryonic stem cells from cloned embryos treated with trichostatin A.

We report here the establishment and characterization of putative porcine embryonic stem cell (ESC) lines derived from somatic cell nuclear transfer embryos (NT-ESCs). These cells had a similar morphology to that described previously by us for ESCs derived from in vitro produced embryos, namely, a polygonal shape, a relatively small (10-15 µm) diameter, a small cytoplasmic/nuclear ratio, a single nucleus with multiple nucleoli and multiple lipid inclusions in the cytoplasm. NT-ESCs could be passaged at least 15 times and vitrified repeatedly without changes in their morphology, karyotype, or Oct-4 and Nanog expression. These cells formed embryoid bodies and could be directed to differentiate in vitro to cell types representative of all three germ layers. Following their injection into blastocysts, these cells preferentially localized in the inner cell mass. In conclusion, we have isolated putative porcine ESCs from cloned embryos that have the potential to be used for a variety of applications including as a model for human therapeutic cloning.

In vitro and in vivo characterization of putative porcine embryonic stem cells.

We have developed a new method for the isolation of porcine embryonic stem cells (ESCs) from in vivo-derived and in vitro-produced embryos. Here we describe the isolation and characterization of several ESC lines established using this method. Cells from these lines were passaged up to 14 times, during which they were repeatedly cryopreserved. During this time, ESCs maintained their morphology and continued to express Oct 4, Nanog, and SSEA1. These cells formed embryoid bodies in suspension culture, and could be directed to differentiate into various lineages representative of all three germ layers in vitro. When injected into blastocysts these cells localized in the inner cell mass of blastocysts. To examine their pluripotency further, cells were injected into host blastocysts and transferred to recipient animals. Of the six transfers undertaken, one recipient became pregnant and gave birth to a litter of one male and three female piglets. Microsatellite analysis of DNA extracted from the tail tissue of these piglets indicated that two female piglets were chimaeric.

Development of culture conditions for the isolation of pluripotent porcine embryonal outgrowths from in vitro produced and in vivo derived embryos.

In the present study we examined the effect of culture media and protein source on the formation of pluripotent primary outgrowths from in vitro produced and in vivo derived porcine embryos as the first step towards the isolation of embryonic stem cells (ESCs). To do this we compared high glucose Dulbeccos Modified Eagles Medium (DMEM) with Minimal Essential Alpha Medium (αMEM) both supplemented with fetal bovine serum (FBS) or serum replacement (SR) in a 2 × 2 factorial design. Culture in DMEM or αMEM supplemented with 10% SR resulted in the establishment of homogenous populations of cells which expressed Oct 4 and Nanog. In contrast culture in either media with FBS resulted in the formation of embryonal outgrowths composed entirely of differentiated cells or a mixture of differentiated cells and putative ESCs which grew poorly and could not be passaged. Using αMEM medium containing 10% SR and culturing in 5% oxygen, putative ESC lines were isolated from in vitro and in vivo derived embryos at efficiencies of 2 and 10% respectively.

Cytochalasin B and trichostatin a treatment postactivation improves in vitro development of porcine somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) is a useful technique for the production of transgenic pigs that can be used for biomedical research. However, the efficiency of SCNT in pigs is low. In this study, we examined the effect of two postactivation treatments, cytochalasin B (CB) and trichostatin A (TSA), on the in vitro development of porcine SCNT embryos. Treating porcine parthenotes with 7.5 microg/mL CB for 3 h after electrical activation was effective in preventing the extrusion of the second polar body in 65% of the oocytes compared to 17% in the control group. Treating SCNT embryos with CB for 3 h after electrical activation significantly increased the average blastocyst cell number compared to the control group (CB treatment 51, Control 34, p < 0.05). Treatment of porcine SCNT embryos with CB for 3 h and 50 nM TSA for 24 h after electrical activation resulted in a threefold increase in blastocyst rate (CB + TSA 64%, CB 20%, p < 0.05) and an increase in the average blastocyst cell number (CB + TSA 63, CB 46, p < 0.05), compared to CB treatment alone. These results show that treatment with TSA and CB significantly improves the in vitro morphological development and quality of porcine SCNT embryos.

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

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

Production of homozygous alpha-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer.

We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White x Landrace female heterozygous Gal KO founders produced using SCNT were mated with Hampshire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half-sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half-sib and subsequently a full-sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts.

Use of adult mesenchymal stem cells isolated from bone marrow and blood for somatic cell nuclear transfer in pigs.

Mesenchymal stem cells (MSCs) isolated from bone marrow were used to examine the hypothesis that a less differentiated cell type could increase adult somatic cell nuclear transfer (SCNT) efficiencies in the pig. SCNT embryos were produced using a fusion before activation protocol described previously and the rate at which these developed to the blastocyst stage compared with that using fibroblasts obtained from ear tissue from the same animal. The use of bone marrow MSCs did not increase cleavage rates compared with adult fibroblasts. However, the percentage of embryos that developed to the blastocyst stage was almost doubled, providing support for the hypothesis that a less differentiated cell can increase cloning efficiencies. As MSCs are relatively difficult to isolate from the bone marrow of live animals, a second experiment was undertaken to determine whether MSCs could be isolated from the peripheral circulation and used for SCNT. Blood MSCs were successfully isolated from four of the five pigs sampled. These cells had a similar differentiation capacity and marker profile to those isolated from bone marrow but did not result in increased rates of development. This is the first study to our knowledge, to report that MSCs can be derived from peripheral blood and used for SCNT for any species. These cells can be readily obtained under relatively sterile conditions compared with adult fibroblasts and as such, may provide an alternative cell type for cloning live animals.

An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs.

We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.

Relationship between donor animal age, follicular fluid steroid content and oocyte developmental competence in the pig.

The developmental competence of oocytes recovered from the ovaries of slaughtered prepubertal and adult pigs was evaluated after in vitro maturation, parthenogenetic activation and culture in vitro. In addition, the effect of prepubertal and adult follicular fluid (FF) on the developmental competence of prepubertal and adult oocytes was investigated. When matured in adult FF, the rates of cleavage (92 v. 73% P < 0.01) and blastocyst formation (57 v. 38%; P < 0.05) were greater for adult oocytes than for prepubertal oocytes. Blastocysts derived from adult oocytes had more trophectoderm cells (43 v. 30; P < 0.05) and total cells (51 v. 36; P < 0.05) than blastocysts derived from prepubertal oocytes. The developmental competence of prepubertal oocytes was not affected by the FF donor age, whereas the developmental competence of adult oocytes was. Blastocysts derived from adult oocytes matured in adult FF had more trophectoderm cells (38 v. 24; P < 0.005), inner cell mass cells (7 v. 3; P < 0.01) and total cells (45 v. 27; P < 0.001) than blastocysts derived from adult oocytes matured in prepubertal FF. Characterization of the steroid content of the FF used to supplement the maturation medium revealed that adult FF contained more progesterone (42 v. 23 ng mL(-1); P < 0.005) and androstenedione (70 v. 16 ng mL(-1); P < 0.05) than prepubertal FF. In addition, the molar ratios of progesterone to androstenedione, androstenedione to 17beta-oestradiol and androstenedione to testosterone differed (P < 0.05) between prepubertal and adult FF. The results support the hypothesis that a greater proportion of adult oocytes than of prepubertal oocytes has completed 'oocyte capacitation'. The differences in FF steroid content are indicative of the different follicular environments from which the prepubertal and adult oocytes were isolated, and may be attributed to the observed effects on oocyte developmental competence.

Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes.

The effect of the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the maturation promoting factor (MPF) activity, pronuclear formation, and parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes was investigated. Oocytes were activated by exposure to two DC pulses, each of 1.5 kV/cm field strength and 60 microsec duration, applied 1 sec apart. In the first experiment, subsequent incubation with 2 or 5 mM 6-DMAP for 3 hr increased the incidence of blastocyst formation compared with no treatment, whereas incubation with 2 or 5 mM 6-DMAP for 5 hr did not. In the proceeding experiments, oocytes exposed to 6-DMAP were incubated with 2 mM of the reagent for 3 hr. Assaying histone H1 kinase activity in the second experiment revealed that the levels of active MPF in electrically activated oocytes treated with 6-DMAP were depleted more rapidly and remained depleted for longer compared with electrical activation alone. The kinetics of MPF activity following 6-DMAP treatment were similar to that found in inseminated oocytes in the third experiment. The effect of 6-DMAP was correlated with an increased incidence of parthenogenetic blastocyst formation. A fourth experiment was undertaken to examine the diploidizing effect of 6-DMAP. Electrically activated oocytes treated with 6-DMAP and cytochalasin B, either alone or in combination, displayed a higher incidence of second polar body retention compared with those that were untreated or treated with cycloheximide alone. After 6 days of culture in vitro, parthenotes exposed to 6-DMAP, either alone or in combination with cytochalasin B, formed blastocysts at a greater rate compared with those exposed to cytochalasin B alone, cycloheximide alone or no treatment. The combined 6-DMAP and cytochalasin B treatment induced the highest rate of blastocyst formation (47%), but the numbers of trophectoderm and total cells in these blastocysts were lower compared with those obtained following exposure to 6-DMAP alone. These results suggest that the increased developmental potential of 6-DMAP-treated parthenotes may be attributable to the MPF-inactivating effect of 6-DMAP, rather than the diploidizing effect of 6-DMAP.

Production of cloned pigs from cultured fetal fibroblast cells.

Somatic cell nuclear transfer was used to produce live piglets from cultured fetal fibroblast cells. This was achieved by exposing donor cell nuclei to oocyte cytoplasm for approximately 3 h before activation by chemical means. Initially, an experiment was performed to optimize a cell fusion system that prevented concurrent activation in the majority of recipient cytoplasts. Cultured fibroblast cells were fused in medium with or without calcium into enucleated oocytes flushed from superovulated gilts. Cybrids fused in the presence of calcium cleaved at a significantly (P < 0.05) greater rate (69%, 37 out of 54) after 2 days of culture compared with those fused without calcium (10%, 7 out of 73), suggesting that calcium-free conditions are needed to avoid activation in the majority of recipient cytoplasts during fusion. In the second experiment, cybrids fused in calcium-free medium were activated approximately 3 h later with ionomycin, followed by incubation in 6-dimethylaminopurine to determine development in vitro. Following 2 days of culture, cleavage rates of chemically activated and unactivated cybrids (fusion without activation control) were 93% (100 out of 108) and 7% (2 out of 27), respectively. After an additional 5 days of culture, activated cloned embryos formed blastocysts at a rate of 23% (25 out of 108) with an average inner cell mass and trophectoderm cell number of 10 (range, 3 to 38) and 31 (range, 16 to 58), respectively. In the third experiment, activated nuclear transfer embryos were transferred to the uteri of synchronized recipients after 3 days of culture to assess their development in vivo. Of 10 recipients receiving an average of 80 cleaved embryos (range, 40 to 107), 5 became pregnant (50%) as determined by ultrasound between Day 25 and Day 35 of gestation. Of the five pregnant recipients, two subsequently farrowed one piglet per litter originating from two different cell culture lines. In this study, efficient reprogramming of porcine donor nuclei by fusing cells in the absence of calcium followed by chemical activation of recipient cytoplasts was reflected in high rates of development to blastocyst and pregnancy initiation leading to full term development.