Development and expression of the green fluorescent protein in porcine embryos derived from nuclear transfer of transgenic granulosa-derived cells.

Nuclear transfer (NT) techniques have advanced in the last few years, and cloned animals have been produced from somatic cells in several species including pig. In this study we examined the feasibility of using granulosa-derived cells (GCs) as donor cells combined with a microinjection procedure to transfer those nuclei. In vitro matured oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm. Mural GCs infected with an enhanced green fluorescence protein (EGFP) gene were serum-starved (0.5% serum, 7 days), injected directly into cytoplasm of enucleated oocytes and the oocytes were electrically activated. The reconstructed embryos were cultured for 7 days and stained with Hoechst 33342 to determine the number of nuclei. Non-manipulated oocytes were electrically activated and cultured as controls. At 9 h post-activation, the pronuclear formation rates were 78.7+/-3.7% in NT and 97.4+/-4.4% in controls at 9 h post-activation. After 7 days culture, the cleavage rates were 24.5+/-7.2% in NT and 79.3+/-5.6% in controls. The blastocysts formation rates were 4.9+/-2.4% in NT and 26.8+/-3.8% in controls. To examine the effect of activation time on development of NT embryos, oocytes were activated at 0-0.5, 1-2, or 3-4 h post-injection. At 18 h post-activation the pronuclear formation rates were higher (62.5+/-7.3%) in the 3-4 h group as compared to the 0-0.5 h (22.0+/-12.5%) or 1-2h (44.5+/-6.3%) groups (P<0.05). However, the cleavage rates (9.6+/-4.6 to 10.7+/-4.2%) and the blastocysts formation rates (1.2+/-2.4 to 4.9+/-3.7%) were not different among treatments (P>0.05). The mean cell number of blastocysts was 15.7+/-5.7 in NT and 25.3+/-24.7 in controls. Green fluorescence was observed in roughly half of the embryos from the one-cell to the blastocyst stage. These results indicate that granulosa-derived cell nuclei can be remodeled in the cytoplasm of porcine oocytes, and that the reconstructed embryos can develop to the blastocyst stage. In addition, EGFP can be used as a marker for gene expression of donor nuclei.

Feasibility of producing porcine nuclear transfer embryos by using G2/M-stage fetal fibroblasts as donors.

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide useful information to answer this question, G2/M- and G0/G1-stage fetal fibroblasts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both groups, nuclear envelope breakdown and premature chromosome condensation were completed within 1-2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and formed a spindle within 1-1.5 h after activation. Decondensation of chromosomes could be seen within 2-4 h after activation. Reformation of the new nuclear envelope occurred 4-6 h after activation and was followed by nuclear swelling and formation of a pronucleus-like structure (PN) 8-12 h after activation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells extruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 donors. A variety of PN and PB combinations were observed in reconstructed oocytes derived from G2/M-stage donors, including 1PN+0PB, 1PN+1PB, 1PN+2PB, 2PN+0PB, 2PN+1PB, 2PN+2PB, and 3PN+1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blastocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage can be morphologically remodeled by cytoplasm of MII oocytes in pigs. To maintain normal ploidy, the extra chromosomes derived from G2/M-stage cells could be expelled by oocytes as a second polar body. G2/M-stage fibroblast nuclei could direct reconstructed embryos to develop to the blastocyst stage.

Fertilization and embryo recovery rates in superovulated chios ewes after laparoscopic intrauterine insemination.

Forty superovulated dairy ewes of the Greek Chios breed were used in an experiment to evaluate the efficiency of laparoscopic intrauterine insemination on fertilization and embryo recovery rates as well as embryo quality. Estrus was synchronized by intravaginal progestagen impregnated sponges and superovulation was induced by administration of 8.8 mg o-FSH i.m. following a standard 8 dose protocol. A small volume (0.3 mL) of diluted fresh ram semen was deposited in each uterine horn 24 to 28 h after onset of the estrus by a laparoscopic technique. The animals were allocated randomly into two groups (Group A and B) of 20 animals each. In Group A, embryos were recovered 18 to 24 h after the intrauterine insemination and in Group B on Day 6. The average number of corpora lutea was 12.8 +/- 1.2 and 11.5 +/- 1.1 (+/- SEM); the overall embryo recovery was 66.4% and 57% and the percentage of recovered fertilized ova was 81% and 82.8% in Groups A and B, respectively. More fertilized ova were collected per ewe from Group A (P < or = 0.1). Results indicated that in Chios breed, superovulation using homologous FSH combined with laparoscopic AI leads to good ovarian response with satisfactory results in fertilization, embryo recovery and quality of embryos. This could lead to improved and more efficient methods for obtaining large numbers of high quality oocytes and embryos for embryo transfer programs which could contribute to genetic improvement and increase of the population size.

Synchronization of porcine fetal fibroblast cells with topoisomerase-inhibitor hoechst 33342.

Proper synchronization of donor nuclei has been shown to have a major influence on the developmental potential of nuclear transfer embryos. In the present study, a protocol was established to synchronize porcine fetal fibroblasts in the G2 stage of the cell cycle. Cell cycle analyses were performed by flow cytometry. Cells were pre-synchronized by serum deprivation or aphidicoline-treatment; then incubated in medium containing 0.1 microg/ml Hoechst 33342 (H342). The fluorochrome H342 has been shown to be a topoisomerase-inhibitor that can inhibit progression through the cell cycle. There was no significant difference in the percentage of fibroblasts in G2/M whether cells were pre-synchronized in medium supplemented with 0.1% serum for 48h or 0.5% serum for 6 days. Compared with controls, pre-synchronization in early S-phase before incubation in H342 increased the proportion of G2/M fibroblasts; also an increase from 0 and 6 versus 12h culture in complete medium before incubation in H342 resulted in an increased percentage of cells in G2/M at the end of the synchronization period (9.3 and 13.1% versus 33.7%; P<0.001). Neither an increase in the concentration of H342 (0.1-1.0 microg/ml) nor a longer exposure time (12h versus 18h versus 24h) increased the proportion of G2/M fibroblasts. The protocol established in this study arrested porcine fibroblasts reversibly in the G2/M-stage and is therefore suitable to provide synchronized cells for nuclear transfer experiments.

Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs.

Different factors are believed to influence the outcome of nuclear transfer (NT) experiments. Besides the cell cycle stage of both recipient cytoplast and donor karyoplast, the origin of the donor cells (embryonic, fetal, and adult) is of interest. We compared in vitro development of NT embryos derived from small serum-starved (G0) or small cycling (G1) porcine fetal fibroblast cells. Serum starvation did not have a positive effect on cleavage rate or the percentage of embryos that developed to the morula and blastocyst stages. Next, we investigated the development of porcine NT embryos derived from different transgenic clonal cell lines that had originated from the same fetus. When different clonal lines of fetal fibroblasts were fused to enucleated metaphase II oocytes, differences in fusion rates as well as in development to the morula and blastocyst stages were observed (P < 0.05). When oocytes derived from sow ovaries were used as recipient cytoplasts, significantly better cleavage (P = 0.03) and blastocyst formation (P < 0.014) was obtained when compared with oocytes derived from gilts. Our data indicate that not only different cell lines, but also different clones derived from one primary cell line, result in different development when used for NT. In addition, the use of sow oocytes as a cytoplast source also improves the efficiency of NT experiments.

Dynamic events are differently mediated by microfilaments, microtubules, and mitogen-activated protein kinase during porcine oocyte maturation and fertilization in vitro.

The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.

Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein.

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of "large offspring syndrome" were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.

Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.

Here we report the production of transgenic pigs that express enhanced green fluorescent protein (eGFP). Porcine oocytes were matured in vitro in a serum-free, chemically defined maturation medium, subsequently infected with a replication deficient pseudotyped retrovirus, fertilized and cultured in vitro before being transferred to a recipient female. Two litters were born from these embryo transfers; one pig from each litter was identified as transgenic and both expressed eGFP. From a tool in basic research to direct applications in production agriculture, domestic livestock capable of expressing foreign genes have many scientific applications.

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

Porcine oocyte activation induced by a cytosolic sperm factor.

It is not known how the fertilizing sperm elicits the release of Ca(2+) from the oocyte's intracellular stores. We investigated whether a crude extract isolated from boar sperm could induce the Ca(2+) release and trigger subsequent early and late activation events upon injection into matured porcine oocytes. The sperm extract induced an immediate rise in the intracellular free Ca(2+) concentration in all oocytes tested, which was followed by repetitive Ca(2+) transients in 11 out of 14 oocytes. Heat or trypsin treatment of the extract totally abolished the Ca(2+) releasing activity of the sperm factor. The injected oocytes showed cortical granule exocytosis, they resumed meiosis and entered first interphase: pronuclei were formed in 89.2% (132/148) of the cases. Pronuclear formation was accompanied by the appearance of a new 22 kDa protein as normally seen at fertilization. Of the successfully injected oocytes 51.7% (105/203) cleaved and 2.0% (4/203) developed to the blastocyst stage after being cultured for 7 days in NCSU 23 medium. Injection of the carrier medium could not trigger these changes. The results indicate that the sperm might activate porcine oocytes by introducing a soluble factor into the oocyte's cytoplasm after gamete fusion.

Advances in livestock nuclear transfer.

Cloning and transgenic animal production have been greatly enhanced by the development of nuclear transfer technology. In the past, genetic modification in domestic animals was not tightly controlled. With the nuclear transfer technology one can now create some domestic animals with specific genetic modifications. An ever-expanding variety of cell types have been successfully used as donors to create the clones. Both cell fusion and microinjection are successfully being used to create these animals. However, it is still not clear which stage(s) of the cell cycle for donor and recipient cells yield the greatest degree of development. While for the most part gene expression is reprogrammed in nuclear transfer embryos, all structural changes may not be corrected as evidenced by the length of the telomeres in sheep resulting from nuclear transfer. Even after these animals are created the question of "are they really clones?" arises due to mitochondrial inheritance from the donor cell versus the recipient oocyte. This review discusses these issues as they relate to livestock.

Long-term culture and characterization of goat primordial germ cells.

While the culture and identification of primordial germ cells (PGCs) in mice is established, only limited investigations on PGCs in livestock have been reported. This study was performed to characterize goat PGCs after culture and cryopreservation. Goat PGCs were isolated from Day 32 fetuses and cultured on a continuous cell line of murine embryonal fibroblasts (STO) as feeder-cells in the presence of leukemia inhibitory factor (LIF). The PGCs proliferated slowly and showed colony formation in early passages. Frozen-thawed PGCs continued to proliferate when stem cell factor (SCF) was added to the culture medium. However, differentiation into epithelial-like polygonal cells or neuronal cells was observed after 1 or 2 passages. The PGCs of 1 female and 1 male cell line were characterized by immunocytochemistry. The PGCs showed positive staining for anti stage-specific embryonic antigen-1 (SSEA-1) and FMA-1 (monoclonal antibody produced against a glycoprotein cell surface antigen of the embryonal carcinoma Nulli SCC1), whereas the reactivity to alkaline phosphatase (AP), an established marker for PGCs in mice, was inconsistent. After differentiation, PGCs lost their positive reaction to SSEA-1, EMA-1 and AP. In conclusion, SSEA-1 and EMA-1 can be used as reliable markers for identifying goat PGCs in addition to morphological criteria. The results indicate that goat PGCs can be kept in long-term culture without losing their morphological characteristics and their positive reaction to SSEA-1 and EMA-1, thus providing a promising source of donor-karyoplasts for nuclear transfer procedures.

Production of transgenic porcine blastocysts by nuclear transfer.

In this study the in vitro development of porcine nuclear transfer (NT) embryos was investigated. Transgenic fetal fibroblast cells that were frozen after 5 days of serum starvation were injected immediately after thawing into enucleated metaphase II (MII) oocytes. Reconstructed embryos were activated by incubation in 200 microM thimerosal followed by a 30-min treatment of 8 mM DTT. The embryos were subsequently cultured in NCSU23, supplemented with 4 mg/ml BSA for 7 days. The actual cleavage rate (embryos showing > or =2 nuclei) in 6 replicates was 33% (ranging from 15% to 50%). Three blastocysts with cell numbers of 14, 15, and 18 were obtained. The blastocyst rate was significantly lower for NT embryos as opposed to parthenogenetically activated embryos (1% vs. 5%; P<0.05). The neomycin-resistance gene was amplified by PCR in all three NT embryos, indicating their origin from the injected transgenic fibroblasts. Efforts are now being directed in improvements in the nuclear transfer technology, whereby viable fetuses or offspring can be produced from these NT-embryos.

Changes in the structure of nuclei after transfer to oocytes.

Nuclear transfer and the potential for cloning animals have refocused attention on the oocyte. This focus is not limited to the use of the oocyte as a recipient in nuclear transfer procedures, but more broadly in terms of what factors are present in the oocyte that are responsible for establishing the developmental pattern of RNA synthesis and subsequent protein production. Deviations in the pattern of RNA synthesis can result in abortions, as well as abnormalities at birth. This paper will focus on the changes to nuclear structure that result from transfer to the cytoplasm of an oocyte, as well as some of the changes in the patterns of RNA synthesis that have been described.

In vivo development of microinjected embryos from superovulated prepuberal slaughter lambs.

This study was performed to investigate the developmental potential of microinjected embryos recovered from superovulated prepuberal lambs. Fifty-nine mixed-bred lambs (about 3 mo old) were superovulated either with 18 mg FSH-P with (Group FSH/+S) or without (Group FSH/-S) progestagen treatment, or with 10 mL Ovagene following progestagen treatment (Group OVA/+S). All animals received hCG to induce ovulation. Ovulation rates and the number of ova recovered per animal for the different groups were 8.7 and 4.7 (55%, FSH/+S); 7.3 and 3.2 (42%, FSH/-S); and 6.4 and 4.0 (65%, OVA/+S), respectively. No significant differences were seen in the ovulation and the recovery rates, but animals without progestagen treatment showed a significantly lower fertilization rate (44%) when compared with progestagen-treated groups (87%; P<0.001). Foreign DNA was microinjected into the pronuclei of fertilized ova (n = 155), which were transferred (n = 98) into synchronized recipient ewes (n = 21). Two animals were detected pregnant and both gave birth to a single lamb. Results of superovulation and embryo recovery from prepuberal lambs were promising, but the low rate of development to term indicates that ova recovered from prepuberal lambs have reduced developmental competence in vivo. Although 2 lambs were born, it seems that this is not a successful method for use in future gene transfer programs.

Repeated endoscopic ovum pick-up in sheep.

Endoscopy is an effective and minimally invasive technique which offers the possibility of repeated ovum pick-up (OPU). In this study 4 different treatment programs (Groups A, B, C and D) for repeated endoscopic OPU in sheep were investigated. The number of follicles and oocytes, quality of cumulus-oocyte-complexes (COCs), and detectable effects on fertility of the donor ewes were compared. Each group consisted of 5 East Friesian Milksheep. In Group A, follicles were punctured twice a week, in Group B once a week, and in Group C once a week followed by administration of 1500 IU PMSG 48 h prior to OPU. In Group D follicles were punctured and the sheep stimulated with 1500 IU PMSG 48 h prior to OPU once every 2 weeks. The PMSG-stimulated sheep received anti-PMSG immediately after OPU. Over a period of 10 weeks 216 OPU-sessions were performed. A total of 1978 follicles was punctured, and 1098 oocytes were recovered, for a collection rate of 55.5%. In the Groups A, B, C and D an average of 6.8, 8.6, 12.2 and 14.9 follicles per animal and session was aspirated, and an average of 3.8, 4.9, 7.0 and 7.6 COCs per animal per session was recovered, respectively. No significant differences between groups were observed in the collection rates (51.1 to 57.1%) or in the quality of the COCs, and 65 to 70% of the COCs were suitable for in vitro production of ovine embryos. Seven sheep developed small adhesions between the ovary and infundibulum. After the study 15 ewes became pregnant following natural mating with the same fertile ram (5 from Group A, 1 from Group B, 4 from Group C and 5 from Group D). In conclusion, OPU once a week in PMSG/anti-PMSG treated ewes was found to be the most effective treatment program for oocyte collection.

Laparoscopic recovery of pronuclear-stage goat embryos.

The oviducts of 16 Saanen does, superovulated with follicle-stimulating hormone (FSH) and synchronised with prostaglandin F2 alpha were flushed 75 to 86 hours after the injection of prostaglandin. The mean (sd) ovulation rate was 13.7 (3.9). The flushings were directed orthograde through a flexible intravenous catheter, which was introduced into the oviduct via the infundibulum. The flushing medium was recovered by a balloon-catheter, which was placed in the uterine lumen near the uterotubal junction. Five does were flushed unilaterally either because they had one blocked oviduct or because they had ovulated on only one ovary. The overall embryo recovery rate was 72 per cent. Nine weeks later 11 of the donor ewes were examined laparoscopically and no adhesions of the reproductive organs were observed. Eight of these does were synchronised with progestagen-vaginal sponges, superovulated with FSH and their oviducts were flushed again. Their mean ovulation rate was 16.0 (4.3) and 86 per cent of the embryos were recovered. The optimal time to obtain pronuclear stage embryos was 75 to 78 hours after the injection of prostaglandin. All the embryos recovered within this period were at the pronuclear stage whereas 28 per cent of those recovered one to six hours later were at the two- or four-cell stage.

Repeated endoscopic ovum pick-up in hormonally untreated ewes: a new technique.

Multiple ovulation and embryo transfer (MOET) are cumbersome and may not be used in all animals. An alternative method for obtaining embryos is to harvest ova and subject them to in vitro maturation/fertilization (IVM/IVF). We tested a modified endoscopic technique designed to allow repeated recovery of oocytes from donor ewes without any hormonal treatment. Seventeen randomly chosen Merino donors were used for ovum pick-up 5 times (OPU1 to OPU5) at 1-wk intervals. The OPUs were performed by ventral laparoscopy. The follicular fluid was aspirated through a needle (0.9 x 75 mm) connected to a 5 ml-syringe. A total of 385 oocytes was collected from 567 aspirated follicles for a collection rate of 67.9%. The number of follicles and oocytes per ewe and the collection rate did not differ significantly (P > 0.05) between collection periods; however, a large variability in the number of follicles and oocytes was observed among individual animals. The collection rate tended to be higher in donors which showed a higher number of follicles. Possible reasons for this are discussed. Oocytes were divided into 4 classes based upon morphological criteria. The portion of oocytes suitable for in vitro production (Classes I to III) were similar among collection periods (83% in OPU1 vs 84% in OPU5). The technique described in this study is useful for obtaining large numbers of oocytes from individual animals during a defined period. This method combined with established in vitro production programs provides a way to increase the number of offspring from genetically valuable animals even when they are affected by infertility due to adhesions or deformations of the oviduct and/or uterus.