Recipient Oocytes Research Articles

DNA Methylation, Histone Modifications and Behaviour of AKAP95 during Mouse Oocyte Growth and Upon Nuclear Transfer of Foreign Chromatin into Fully Grown Prophase Oocytes

The poor efficiency of mammalian cloning is due to inappropriate/incomplete epigenetic reprogramming of the donor chromatin. As the success in reprogramming of the donor nucleus may require activity of similar mechanisms which reprogram the chromatin in the course of gametogenesis, we decided to follow the status of some epigenetic markers in the late phase of oogenesis in mice, i.e. in prophase oocytes during their growth and after completion of the growth phase. Our analysis reveals an increase in the level of global DNA methylation starting in oocytes with diameters around 60 microm which was further elevated until completion of oocyte growth. A similar increase was observed in respect to the acetylation of histone H4. On the other hand, the methylation of histone H4 Arg3 was constantly high until the end of oocyte growth, although it differed between fully grown oocytes depending on the type of spatial chromatin organization. We have also studied the AKAP95 protein which was abundant at earlier stages but decreased in fully grown oocytes according to changes in their chromatin organization. The nuclear transfer of different types of donor nuclei with hypomethylated DNA into fully grown prophase oocytes did not increase the global level of methylation of transferred foreign chromatin, regardless if the recipient oocyte was devoid of its own nucleus or its nucleus was left intact. This suggests a major problem in the ability of recipient oocytes to modify donor DNA methylation.

Segregation of donor cell mitochondrial DNA in gaur–bovine interspecies somatic cell nuclear transfer embryos, fetuses and an offspring

The fate of foreign mitochondrial DNA (mtDNA) following somatic cell nuclear transfer (SCNT) is still controversial. In this study, we examined the transmission of the heteroplasmic mtDNA of gaur donor cells and recipient bovine oocytes to an offspring and aborted and mummified fetuses at various levels during the development of gaur–bovine interspecies SCNT (iSCNT) embryos. High levels of the donor cell mtDNA were found in various tissue samples but they did not have any beneficial effect to the survival of iSCNT offspring. However, the factors on mtDNA inheritance are unique for each iSCNT experiment and depend on the recipient oocyte and donor cell used, which might play an important role in the efficiency of iSCNT.

Improved Cloning Efficiency and Developmental Potential in Bovine Somatic Cell Nuclear Transfer with the Oosight Imaging System

In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.

Exposure of in vitro-matured porcine oocytes to SYBR-14 and fluorescence impairs their developmental capacity

Staining with Hoechst 33342 followed by ultraviolet irradiation is frequently used to aid or confirm the enucleation of recipient oocytes in porcine somatic cell nuclear transfer programs. However, the procedure has a clearly deleterious effect on the developmental ability of oocytes. This study evaluated the effectiveness of a longer-wavelength fluorochrome (SYBR-14) for visualizing maternal chromosomes in in vitro-matured porcine oocytes and the effects of this dye in combination with fluorescence excitation on the subsequent in vitro fertilization and embryo development of the oocytes. In the first experiment, the oocytes were exposed to different concentrations (1, 3, 5 and 7μg/mL) of SYBR-14 at different incubation times (5, 10 and 30min) in a 4×3 factorial design. The optimal condition for proper metaphase-II plate and first polar body visualization was a 10-min incubation with 5μg/mL of SYBR-14. In the second experiment, the degeneration rate of the oocytes 18h after exposure to SYBR-14 (5μg/mL for 10min) and fluorescence excitation for 5 or 30s was significantly higher (p<0.002) than that obtained for non-exposed oocytes. The fertilization parameters were not influenced by the treatments. The cleavage and blastocyst rates during culture were lower (p<0.001) for the oocytes exposed to SYBR-14 and fluorescence than for those in the non-exposed group. These results indicate that the exposure of mature oocytes to SYBR-14 and fluorescence for periods as short as 5s increased the rate of oocyte degeneration and limited their subsequent developmental competence.

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

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

Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin

The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT.

L-carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression

The objective of this study was to examine the effect of L-carnitine treatment during in vitro maturation (IVM) of immature pig (Sus scrofa) oocytes. Specifically, the effects of L-carnitine treatment on nuclear maturation and oocyte intracellular glutathione (GSH) levels, embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT pig embryos were determined. During IVM culture, immature oocytes were either treated or not treated with 10 mM L-carnitine. L-carnitine treatment did not improve the nuclear maturation of oocytes but significantly increased intracellular GSH levels, which led to a reduction of reactive oxygen species (ROS) levels in IVM oocytes. Oocytes treated with L-carnitine showed higher (P < 0.05) rates of blastocyst formation after PA (39.4% vs. 27.1%) and SCNT (23.2% vs. 14.9%) compared with untreated oocytes. SCNT embryos that were derived from L-carnitine-treated oocytes showed increased (P < 0.05) expression levels of DNMT1, PCNA, FGFR2, and POU5F1 mRNA compared with control embryos. Treatment of recipient oocytes with L-carnitine increased (P < 0.05) the expression of both BAX and p-Bcl-xl mRNA in SCNT blastocysts. However, the increase was more prominent in BAX than in p-Bcl-xl mRNA. Our results demonstrate that L-carnitine treatment during IVM improves the developmental competence of SCNT embryos. This effect is probably due to increased intracellular GSH synthesis in recipient ooplasts, which reduces ROS levels, and the stimulation of nuclear reprogramming via increased expression of POU5F1 and transcription factors.

Chromosome transfer in mature oocytes

To demonstrate step-by-step micromanipulation procedures required for transfer of spindle-chromosomal complexes between mature oocytes. Video presentation of reproductive biology study. In vitro fertilization and embryo manipulation laboratory. Rhesus (Macaca mulatta) primates. Transplantation of the genetic material between mammalian oocytes offers many opportunities to study various aspects of nuclear-cytoplasmic interactions during oogenesis, fertilization and embryo development. We demonstrate the feasibility of isolation and transfer of chromosomes between mature metaphase II (MII) primate oocyte. After fertilization, manipulated oocytes were capable of producing healthy offspring or embryonic stem cells. In this video, we show micromanipulation procedures required for isolation and transfer of spindle-chromosomal complexes between rhesus MII oocytes. In brief, the spindle is visualized using a polarized microscope and extracted into a membrane enclosed karyoplast. Karyoplasts are then reintroduced into an enucleated recipient oocyte (cytoplast, derived from an another female) by karyoplast-cytoplast membrane fusion. Newly reconstructed oocytes consist of nuclear genetic material from one female and cytoplasmic components, including mitochondria and mitochondrial DNA from another. This video demonstrates the protocol developed for primate oocytes that successfully allowed of isolation and transfer of chromosomes between mature metaphase II (MII) oocytes. Potential clinical applications include mitochondrial gene replacement therapy to prevent transmission of mtDNA mutations and treatment of infertility caused by cytoplasmic defects in oocytes. Video is available at http://fertstertforum.com/2012974tachibana/.

Follicular Growth-Stimulated Cows Provide Favorable Oocytes for Producing Cloned Embryos

We examined the influence of recipient oocytes on in vitro development, oxygen consumption, and gene expression in the resulting cloned bovine embryos. Oocytes derived from slaughterhouse ovaries and ovum pickup (OPU)-derived oocytes were used as recipient cytoplasts for the production of cloned embryos. A series of OPU sessions was conducted on Holstein cows without follicular growth treatment (FGT). In the same cows, we then performed dominant follicle ablation and subsequently administered follicle-stimulating hormone and prostaglandin F(2α) with controlled internal drug release device before a second series of OPU. Cumulus cells collected from single Holstein cows were used as donor cells. After measurement of oxygen consumption at the blastocyst stage with modified scanning electrochemical microscopy, analysis of 10 genes (CDX2, IFN-tau, PLAC8, OCT4, SOX2, NANOG, ATP5A1, GLUT1, AKR1B1, and IGF2R) was performed with real-time RT-PCR. Rates of fusion, cleavage, and blastocyst formation were not different among the treatment groups. Levels of oxygen consumption in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT were significantly lower than in blastocysts derived from artificial insemination (AI). However, oxygen consumption was increased in cloned blastocysts derived from OPU with FGT, depending on the individual oocyte donor. Furthermore, gene expression of IFN-tau and OCT4 in cloned blastocysts derived from OPU with FGT was similar to that in AI-derived blastocysts, whereas expression of those genes in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT was significantly different from that in AI-derived blastocysts. Thus, recipient oocytes collected by OPU in combination with manipulation of follicular growth in donor cows are suitable for producing cloned embryos.

Comparative Proteomic Analysis of Liver Mitochondrial Proteins Derived from Cloned Adult Pigs Reconstructed with Meishan Pig Fibroblast Cells and European Pig Enucleated Oocytes

Somatic cell nuclear transfer (SCNT) has been exploited in efforts to clone and propagate valuable animal lineages. However, in many instances, recipient oocytes are obtained from sources independent of donor cell populations. As such, influences of potential nuclear-cytoplasmic incompatibility, post SCNT, are largely unknown. In the present study, alterations in mitochondrial protein levels were investigated in adult SCNT pigs produced by microinjection of Meishan pig fetus fibroblast cells into enucleated matured oocytes (maternal Landrace genetic background). Mitochondrial fractions were prepared from liver samples by mechanical homogenization and differential centrifugation. Liver mitochondria were then subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Protein expression changes were confirmed with a volume ratio greater than 2 fold (P<0.05). 2-D DIGE analysis further revealed differential expression of three proteins between the Meishan (n=3) and Landrace (n=3) breeds. Differential expression patterns of 16 proteins were detected in SCNT pig liver tissue (n=3) when compared with Meishan control samples. However, none of the 16 proteins correlated with the three differentially expressed Meishan and Landrace liver mitochondrial proteins. In summary, alteration of mitochondrial protein expression levels was observed in adult SCNT pigs that did not reflect the breed difference of the recipient oocytes. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.

15 EFFECTS OF DEMECOLCINE ON THE MEIOTIC CELL-CYCLE AND MICROTUBULAR KINETICS OF ACTIVATED BOVINE OOCYTES SUBMITTED TO CHEMICAL ENUCLEATION

Enucleation in traditional nuclear transfer (NT) is an invasive procedure for which alternative protocols have been sought. The present study was designed to explore the time course effects of demecolcine, a microtubule-depolymerizing agent, in bovine-activated oocytes submitted to induced chemical enucleation. For that purpose, after 26 h of in vitro maturation, the oocytes were parthenogenetically activated (5 μM ionomycin for 5 min and 10 μg mL–1 cycloheximide for 4 h) and treated with demecolcine (0.05 μg mL–1 for 2 h) 2 h after activation. Three groups were established: control (untreated oocytes), activated (oocytes exposed to activation) and deme (oocytes activated and treated with demecolcine). Then the nuclear and microtubular dynamics of the oocytes were evaluated by immunofluorescence microscopy of tubulin and chromatin (Liu et al. 1998 Biol. Reprod. 59, 537–545). In each one of 3 replicates, 15 to 30 oocytes were evaluated per group. Oocytes were classified according to microtubule (MT) patterns as follows: present (evident MT), reduced (MT with reduced density), or absent. The results in percentage were submitted to ANOVA and means were compared by Tukey test, with a significance level of 5%. Effects of activation were observed after 2 h, when higher rates of oocytes presenting second polar body (2nd PB) extrusion were observed in the groups activated and deme (49.3% in both groups) compared with control (1.7%). At the end of activation treatment (4 h), the activated group presented 81.8% of oocytes with 2nd PB extrusion, whereas it was observed only in 37.8% of oocytes in the deme group. Effects of demecolcine on the microtubules initiated after only 0.5 h of treatment, when an increase (P &lt; 0.05) of oocytes with reduced MT was observed in the deme group (26%; control – 3%; activated – 0%). After 6 h of culture in demecolcine-free medium, the deme group displayed ∼50% of oocytes with reduced MT (control – 0%; activated – 39%) and absence of MT in 23% of oocytes, which was superior to other groups (control – 0%; activated – 2%). Therefore, we detected a reduction of MT density after exposition of activated oocytes to demecolcine. However, MT were not completely absent in most of the evaluated oocytes, as previously described for bovine oocytes submitted to chemically assisted enucleation (Saraiva et al. 2009 Cloning Stem Cells 11, 141–152; Meng et al. 2011 Cell. Reprogram., in press). Apparently, there was no immediate repolymerization of MT after culture in demecolcine-free medium and this could result in negative consequences for subsequent embryo development. Moreover, demecolcine impaired the second PB extrusion during the activation process, probably due to inhibition of spindle rotation caused by the MT-disrupting drug. Nonetheless, considering the higher cytoplasmatic volume obtained with chemical enucleation and the lesser extent of injuries suffered by recipient oocytes, further studies focusing on the potential of embryo development and the quality of embryos are advisable. Financial support: FAPESP.

19 CHANGES IN PLASMA STEROID CONCENTRATIONS DURING GESTATION IN COWS WITH SPONTANEOUS ABORTION OF SOMATIC CELL CLONED FETUSES

Cloning of mammals by nuclear transfer frequently results in gestational failure with a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Monitoring the placental function during gestation is important to clarify the cause of abnormalities in cloned animals. Sex steroids are produced in the bovine placenta and their levels in maternal peripheral blood are a useful measure of placentation. The objective of this study was to investigate changes in plasma concentrations of sex steroids during gestation in cows aborting cloned fetuses. Donor cells for nuclear transfer were obtained from subculture of cumulus cells retrieved from ovarian follicles of a Japanese Black cow. Recipient oocytes were derived from ovaries obtained at an abattoir and matured in vitro. Metaphase II oocytes were enucleated and each fused with a donor cell by DC pulses. Nuclear-transferred oocytes were activated and cultured for 7 days. Embryos developed to the blastocyst stage were each transferred into the uterine horn ipsilateral to the ovary bearing the CL of 39 multiparous Japanese Black and Holstein crossbred cows at 7 to 8 days after the day of standing oestrus (day 0). Fourteen recipient cows were diagnosed pregnant on Day 40 by ultrasonography and 7 cows delivered at full term. The other seven miscarried on Day 66, 81, 85, 89, 97, 104 and 211. Blood was collected from these cows at least once a week following the pregnancy diagnosis. Progesterone, estrone, oestradiol-17β and estrone sulfate in the blood plasma were measured by RIA and were compared with those in pregnant AI cows. Statistical differences at stages of gestation were analysed with repeated-measures ANOVA. In all miscarried cows, progesterone concentrations were similar to those in AI cows until several days before abortion and then rapidly decreased to the basal level. Concentrations of all estrogens stayed low until abortion in six cows aborting by day 104, whereas estrone and oestradiol-17β started to increase around Day 80 and estrone sulfate gradually increased from around Day 50 and started to increase drastically around Day 80 in AI cows. In another cow aborting on Day 211, profiles of estrone and oestradiol-17β were similar to those in AI cows until around Day 150. Thereafter, concentrations of these estrogens gradually decreased to the basal levels by Day 160 and stayed low until abortion. In this cow, gradual increase in estrone sulfate during Day 50 to 80 was not observed, but the difference in the concentration was not statistically significant from AI cows. The following profile of estrone sulfate was similar to those in active estrogens. The fetus was still alive on day 160 and fetal death was confirmed on day 180 by ultrasonography. These results suggest the possibility that developmental or functional failure of placenta associated with steroid synthesis may be a cause of mid-term miscarriage of a cloned fetus.

Human oocytes: still in the game

Retaining the recipient oocyte genome after human somatic cell nuclear transfer permits development to the blastocyst stage and derivation of triploid human embryonic stem cell lines.

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation.

Development of Embryos Reconstructed from Different Age of Recipient Oocytes and Activation Time after Fusion of Fetal Fibroblasts in Buffalo (Bubalus bubalis) Nuclear Transfer.

Development of Embryos Reconstructed from Different Age of Recipient Oocytes and Activation Time after Fusion of Fetal Fibroblasts in Buffalo (Bubalus bubalis) Nuclear Transfer.

Widespread Transcription in an Amphibian Oocyte Relates to Its Reprogramming Activity on Transplanted Somatic Nuclei

Amphibian oocytes have the special ability to directly induce the transcription of pluripotency and other genes in transplanted somatic nuclei. To this extent, oocytes induce a stem cell-like pattern of transcription in somatic cell nuclei. We ask whether the induced transcription in transplanted nuclei reflects the normal transcriptional activity of oocyte genes. We describe here the transcript content of a wide range of genes in Xenopus tropicalis oocytes. Using accurate quantitation, we find that each mature oocyte has accumulated several hundred transcripts of cell-type specific genes. This value is several orders of magnitude greater than the "leakage" level found in most somatic cells and about the same level found in somatic cells where these genes are fully expressed. Illumina sequencing confirms the high transcript content of a mature Xenopus oocyte. Most of the transcripts from these highly expressed genes in oocytes are correctly and efficiently spliced. Our results contribute a more quantitative view of certain amphibian oocyte transcripts than previously available. Our results also show that transplanted somatic nuclei conform, with respect to the genes analyzed, to the transcriptional characteristics of the recipient oocytes.

Interspecies Somatic Cell Nuclear Transfer Is Dependent on Compatible Mitochondrial DNA and Reprogramming Factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 µM 2′,3′-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.

Caffeine Can Be Used for Oocyte Enucleation

The removal of chromosomes from recipient oocytes is one of the key steps in nuclear transfer cloning. Although microtubule interrupters have been successfully used for oocyte enucleation, their potential side effect on oocyte developmental potential should be considered, and less harmful drugs should be explored for chemical-assisted enucleation. Based on our previous findings that any maturation promoting factor-activating agent induces ooplasmic protrusion without disrupting microtubules, we have studied the feasibility to use caffeine or MG132 for chemical-assisted enucleation. Experiments using goat oocytes showed that treatments for 30 min with 1-mM caffeine or 5-μM MG132-induced ooplasmic protrusions in about 85% of the oocytes, a percentage similar to that achieved with optimal demecolcine treatment. Rates of enucleation, cell fusion and in vitro blastulation were similar among caffeine, MG132, and demecolcine enucleation but significantly higher than blind aspiration. Furthermore, neither rates of pregnancy on days 90 and 120 nor the general rate of live births/embryos transferred differed significantly (p > 0.05) between caffeine and demecolcine enucleation. Although oocytes treated with caffeine did not retract protrusions until 2 h, many oocytes treated with MG132 withdrew protrusions as early as 0.5 h after treatment. The optimal treatment to induce ooplasmic protrusion in 75% pig oocytes was 8-mM caffeine for 60 min. Mouse oocytes responded poorly to demecolcine or caffeine with less than 40% forming inconspicuous protrusions following optimal treatments. It is concluded that caffeine can be used for enucleation of goat and pig oocytes with similar results as demecolcine, and live kids were born after caffeine-assisted enucleation.

In Vitro Developmental Potential of Nuclear Transfer Embryos Cloned with Enucleation Methods using Pre-denuded Bovine Oocytes

Enucleation of a recipient oocyte is an important essential process in the procedure of somatic cell nuclear transfer (SCNT). The present study investigated a method for the improvement of enucleation efficiency. Oocytes were denuded of cumulus cells before the completion of nuclear maturation (pre-denuded) after 12 h of culture at MI stage and subsequently cultured for additional 6 h until the completion of nuclear maturation and extrusion of the first polar body (PB1). The extrusion rate of PB1 was not significantly different in the pre-denuded oocyte group, compared with control oocyte group matured for 18 h. However, the number of oocytes showing the metaphase II (MII) located just underneath the PB1 was significantly higher (p<0.05) in the pre-denuded oocyte group than those in control oocyte group. To test the effect of pre-denuding on the enucleation rate and developmental potential of embryos to blastocyst stage, subsequent somatic cell nuclear transfer comparisons were made with three different methods of enucleation at MII stage using vital dyes (demicoline and Hoescht) or the PB1 (blind enucleation) to localize the chromosome plate. Enucleation rate of the oocytes with demicoline, Hoechst and pre-denuding enucleation groups were significantly higher (p<0.05) than those of blind enucleation groups. However, cleavage rate to two-cell stage and, developmental rate to blastocyst and hatched blastocyst stage, the mean numbers of total and ICM cells in the SCNT embryos with Hoechst enucleation groups were significantly decreased (p<0.05), compared to those of blind, demicoline and pre-denuding enucleation groups. Moreover, the level of telomerase activity was also significantly (p<0.05) decreased in SCNT blastocysts of Hoechst enucleation group, compared to those of blind, demicoline and pre-denuding enucleation groups. Taken together, pre-denuding enucleation group using pre-denuded oocytes was a useful and simple enucleation method for bovine SCNT embryos.

Interspecies Somatic Cell Nuclear Transfer Technique for Researching Dog Cloning and Embryonic Stem Cells

Large quantities of high-quality recipient oocytes with uniform cytoplasm are needed for research in the promising field of somatic cell nuclear transfer (SCNT) and embryonic stem cell research. In canines, however, it is difficult to obtain large quantities of oocytes because each donor produces a limited number of mature oocytes in vivo. Although in vitro maturation (IVM) is considered an alternative approach to oocyte production, this technique is still too rudimentary to be used for the production of highquality, uniform oocytes in large quantities. One technique for overcoming this difficulty is to use oocytes obtained from different species. This technique is known as interspecies SCNT (iSCNT). This review provides an overview of recent advances in canine - porcine interspecies SCNT.