Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, gamma-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. gamma-tubulin translocated into the two poles of the transient spindles, while no accumulated gamma-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, gamma-tubulin was translocated to the spindle poles. The distribution of gamma-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. gamma-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as pericentriolar material surrounding a pair of centrioles, is degraded in the 1-cell reconstituted embryos after activation; (2) components of donor cell centrosomes contribute to the formation of the transient spindle and normal functional mitotic spindle, although the contribution of centrosomal material stored in the recipient ooplasm is not excluded; and (3) components of donor cell centrosomes involved in spindle assembly may not be species-specific.     

Current status and applications of somatic cell nuclear transfer in dogs

Although somatic cell nuclear transfer (SCNT) technology and applications are well developed in most domesticated and laboratory animals, their use in dogs has advanced only slowly. Many technical difficulties had to be overcome before preliminary experiments could be conducted. First, due to the very low efficiency of dog oocyte maturation in vitro, in vivo matured oocytes were generally used. The nucleus of an in vivo matured oocyte was removed and a donor cell (from fetal or adult fibroblasts) was injected into the oocyte. Secondly, fusion of the reconstructed oocytes was problematic, and it was found that a higher electrical voltage was necessary, in comparison to other mammalian species. By transferring the resulting fused oocytes into surrogate females, several cloned offspring were born. SCNT was also used for producing cloned wolves, validating reproductive technologies for aiding conservation of endangered or extinct breeds. Although examples of transgenesis in canine species are very sparse, SCNT studies are increasing, and together with the new field of gene targeting technology, they have been applied in many fields of veterinary or bio-medical science. This review summarizes the current status of SCNT in dogs and evaluates its potential future applications.     

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.     

Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.     

Gene profiling of cattle blastocysts derived from nuclear transfer, in vitro fertilization and in vivo development based on cDNA library

Gene expression analysis of cloned embryos would enable us to better understand the early biological events during preimplantation after NT (nuclear transfer). Routine RT-PCR and Northern-blot were limited because it could not analyze tens of thousands of genes at one time and were impeded by minimum material. Based on the developed RT-PCR methodology, we previously constructed cDNA libraries with equivalent to single embryo from the pooled AI-blastocysts (artificial insemination and in vivo developed blastocysts) of cattle. To identify gene expression profiles in NT- and IVF (in vitro fertilized)-blastocysts, and search for new candidate genes involved during this period, here we created cDNA sources from three types of blastocysts (AI-, IVF- and NT-blastocysts). The expressions of 60 genes previously identified from cDNA library were compared in three types of blastocyst. Results showed that the gene expression profile of NT-blastocysts was more similar to that of AI-blastocysts than that of created from IVF-blastocysts. Several important genes, such as Oct-4 and IFN-ι, only detected in the early embryonic development, were highly expressed in three types of blastocysts and showed no significant difference, it indicated that the donor nuclear undergone efficient reprogramming by the blastocyst stage and gained totipotential after nuclear transfer. The gene expression profiles in three types of blastocysts suggested that nuclear transfer and in vitro culture environments impaired the viability of embryos in different ways.     

Optimization of embryo culture conditions in the production of cloned goat embryos, following somatic cell nuclear transfer

This study was initiated to investigate the effect of culture conditions on the in vitro development of cloned goat embryos, as well as the pregnancy and delivery success after embryo transfer. The fused embryos were randomly distributed to four culture groups: (i) 72 h in G1 medium then 96 h in G2 medium (group G1/G2); (ii) 168 h in mSOFBSA (group mSOFBSA); (iii) 72 h in mSOFBSA then 96 h in mSOFBSA supplemented with 10% FBS (group mSOFBSA-FBS); (iv) 168 h in mSOFBSA supplemented with 10% FBS (group mSOFFBS). A higher proportion of reconstructed embryos developed to the hatching blastocyst stage in the mSOFBSA group, compared to the G1/G2 group (30.8% vs. 11.4%; P < 0.05). Furthermore, supplementation of 10% FBS to mSOFBSA at the beginning of culture, or 72 h later both significantly improved the hatching rates, compared to the control group (48.9%, 51.2% vs. 28.2%, respectively; P < 0.05). The pregnancy rate following the transfer of one-cell stage embryos was higher than that of in vitro cultured blastocysts (32.0% vs. 6.9%; P < 0.05). However, no significant differences were observed regarding NT efficiency, based on the number of embryos transferred between the two groups (2.0% and 1.3%, respectively). Results suggest that cloned goat embryos cultured to the blastocyst stage in vitro in mSOFFBS may develop to term.     

Birth of calves expressing the enhanced green fluorescent protein after transfer of fresh or vitrified/thawed blastocysts produced by somatic cell nuclear transfer

The present study examined effects of genetic manipulation and serum starvation on in vitro developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos and vitrification on in vivo developmental competence of transgenic SCNT blastocysts. Fetal oviduct epithelial cells (FOECs) were isolated from the oviduct of a Day 147 bovine fetus and transfected with a plasmid (pCE-EGFP-IRES-NEO) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes. There were no significant differences (P > 0.05) in cleavage rates or development rates to the blastocyst stage for SCNT embryos derived from FOECs (72.5 and 47.8%, respectively) or transfected FOECs (TFOECs, 73.8 and 47.7%, respectively); nor from serum-fed (73.6 and 47.2%, respectively) or serum-starved (72.7 and 48.3%, respectively) cells. Seventeen of Day 7 GFP-embryos (eight fresh blastocysts and nine vitrified/thawed blastocysts ) were transferred to recipients with one embryo per recipient. Two (25%) recipients were confirmed pregnant at Day 60 in fresh blastocysts group, and three recipients (33%) were confirmed pregnant at Day 60 in vitrified/thawed blastocysts group. Two healthy calves (25%) were obtained from fresh blastocysts and one (11%) from vitrified/thawed blastocysts. Microsatellite analysis confirmed that the three clones were genetically identical to the donor cells. Moreover, PCR and Southern blot demonstrated integration of transgene in genomic DNA of all three cloned calves. Expression of GFP in skin biopsies isolated from transgenic cloned calves and fibroblasts derived from the skin biopsies revealed the activity of EGFP gene, and G418 resistance in vitro of these fibroblasts confirmed the activity of Neor gene. Our results show that genetic manipulation and serum starvation of donor cells (FOECs) do not affect in vitro developmental competence of bovine SCNT embryos, and vitrified transgenic SCNT blastocysts can develop to term successfully.     

Effects of exogenous hexoses on bovine in vitro fertilized and cloned embryo development: Improved blastocyst formation after glucose replacement with fructose in a serum-free culture medium

To evaluate the embryotrophic role of three hexoses (glucose, fructose, and galactose), bovine embryos derived from somatic cell nuclear transfer (SCNT) or in vitro-fertilization (IVF) were cultured in a modified synthetic oviductal fluid (mSOF), which contained either glucose (1.5 or 5.6 mM), fructose (1.5 or 5.6 mM), or galactose (1.5 or 5.6 mM). Compared to 1.5 mM glucose, use of 1.5 mM fructose significantly enhanced blastocyst formation in both SCNT (23 vs. 33%) and IVF embryos (26 vs. 34%), while 5.6 mM fructose did not improve blastocyst formation. Using 1.5 mM galactose did not improve blastocyst formation in SCNT embryos (22 vs. 23%), whereas it significantly inhibited blastocyst formation in IVF embryos (26 vs. 0%). In both SCNT and IVF embryos, 5.6 mM glucose or galactose significantly inhibited embryo development. In a second experiment, in glucose-free mSOF, fructose at concentrations of 0.75, 1.5, 3.0, or 5.6 mM was able to support to morula (32-42 vs. 12%) and blastocyst formation (30-38 vs. 12%) compared to 0 mM fructose. In Experiment 3, addition of fructose (1.5, 3.0, or 5.6 mM) to mSOF containing 1.5 mM glucose did not further promote blastocyst formation in SCNT embryos compared with replacement with 1.5 mM fructose only. Replacement of glucose with 1.5 mM fructose significantly increased total blastomeres (143 vs. 123 cells) and trophectodermal (TE) cells (116 vs. 94 cells) and decreased inner cell mass (ICM) to TE cell ratio (0.24 vs. 0.31) in blastocysts, compared to 1.5 mM glucose. The combined addition of 1.5 mM fructose and glucose significantly increased ICM cell number (36.7 cells) and ICM/TE ratio (0.46). In conclusion, fructose might be a more efficient energy substrate than glucose for producing large number of transferable blastocysts derived from SCNT.     

父系遗传背景对小鼠体细胞核移植效率的影响

为了评价父系遗传背景对小鼠体细胞核移植效率的影响,本试验用129/Sv小鼠、C3H小鼠和ICR的雄鼠分别与昆明雌鼠(KM)杂交的F1代为研究对象,以KM自交鼠F1代为对照,比较卵母细胞的可操作性以及重构胚的激活率、卵裂率和囊胚发育率。结果显示:129/Sv×KM、C3H×KM和KM×KM的去核效率显著高于ICR×KM(78.0%、82.9%、81.0%vs63.9%;P<0.05);129/Sv×KM的注核成功率显著高于C3H×KM、ICR×KM和KM×KM(83.0%vs59.6%、55.5%、71.4%;P<0.05);129/Sv×KM的重构胚激活率显著高于C3H×KM、ICR×KM和KM×KM(97.3%vs85.2%、81.7%、78.3%;P<0.05);C3H×KM的卵裂率和囊胚率显著高于ICR×KM和KM×KM(84.5%、28.2%vs63.2%、11.4%,64.5%、16.5%;P<0.05)。研究表明129/Sv、C3H和ICR3个品系父系遗传背景影响小鼠体细胞核移植效率,其中C3H父系遗传背景的卵母细胞可提高体细胞核移植效率。     

Quantifying difference in gene expression profile between bovine blastocysts derived by in vitro fertilization and somatic cell nuclear transfer

Epigenetic reprogramming intensely occurs in somatic-cell nuclear transfer (SCNT) embryos, which highlights the importance of proper expressions of reprogramming-related genes in SCNT embryos. We here assessed gene expression profiles (GEPs) difference between bovine blastocyst groups derived by in-vitro fertilization (IVF) or SCNT; in SCNT, cumulus cells and ear skin fibroblasts were used for cSCNT and fSCNT blastocysts, respectively. We obtained GEPs of 15 reprogramming-related genes in single blastocysts using multiplex PCR and found a broad range of variations in their GEPs. Weighted root-mean-square deviation (wRMSD) analysis, which calculates the deviation of SCNT blastocysts' GEPs from IVF blastocysts' mean GEP, found a significant difference between IVF and fSCNT and between cSCNT and fSCNT blastocysts (p < 0.001) but not between IVF and cSCNT. Since the fibroblasts' GEP was more distant from the IVF blastocysts' than the cumulus cells', it might partly explain the less similarity of fSCNT blastocysts' GEPs to the IVF's mean GEP. Our wRMSD method succeeds in expressing in figures how different two comparable embryo groups of different derivations are in GEP, which would be useful to select a better embryo derivation protocol among the candidates prior to field applications.     

Ovum pick up,intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle,buffalo and horses: from the research laboratory to clinical practice

Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production to advance the implementation of embryo-based biotechnologies in that industry, although it is still mainly in the developmental phase. In the horse, OPU is now an established procedure for breeding from infertile and sporting mares throughout the year. It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is very efficient and the only option because conventional IVF does not work. Somatic cell nuclear transfer is destined to fill a very small niche for generating animals of extremely high commercial value. The efficiency is low, but because normal animals can be generated it is likely that advancing our knowledge in that field might improve the technology and reduce its cost.     

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.     

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.     

A diet of somatic cell nuclear transfer cloned-cattle meat produced no toxic effects on behavioral or reproductive characteristics of F1 rats derived from dams fed on cloned-cattle meat

Background: The composition and nutritional value of meat and milk derived from cloned animals and their progeny has not been demonstrated to be different from normal animals, but possible food consumption risks that might arise from unidentified hazards remain. In this study, we investigated the effects of somatic cell nuclear transfer cloned‐cattle meat diet on the behavioral and reproductive characteristics of F1 rats derived from dams that were also fed on cloned‐cattle meat. Methods and results: F1 rats were divided into five diet groups with their dams: commercial pellets (control), pellets containing 5% (N‐5) and 10% (N‐10) of normal‐cattle meat, and diets containing 5% (C‐5) and 10% (C‐10) of cloned‐cattle meat. In most cases, the cloned‐cattle meat diet did not affect body weight and food consumption in both male and female F1 rats during 11 weeks, except for significantly higher body weight in both N‐5 and N‐10 (3–5 weeks, p<0.05 or p<0.01) and significantly higher food consumption in the both normal‐ and cloned‐cattle meat groups (7–9 weeks, p<0.05 or p<0.01), as compared with the controls, respectively. We detected no signs of test substance‐related toxicities on organ weights and behavioral characteristics (sensory reflex, motor function, and spatial learning and memory tests). Reproductive functions did not significantly differ among all examined rats (mating, fertility, and implantation). Conclusions: These behavioral and reproductive toxicity results suggest that there are no obvious food safety concerns related to cloned‐cattle meat in these parameters. Birth Defects Res (Part B) 92:224–230, 2011. © 2011 Wiley‐Liss, Inc.     

Effect of addition of hyaluronan to embryo culture medium on survival of bovine embryos in vitro following vitrification and establishment of pregnancy after transfer to recipients

Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1 mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1 mg/mL, hyaluronan increased (P < 0.05) the percentage of oocytes that were blastocysts and re-expansion rate at 24 h after warming. At 0.5 mg/mL, hyaluronan tended (P < 0.10) to increase re-expansion rate at 48 h after warming and increased (P < 0.05) embryo hatching rate at 24 and 72 h. Treatment with BSA caused a slight reduction in cleavage rate (P < 0.05), but only for cultures containing hyaluronan (BSA × hyaluronan, P = 0.10), an increase in the percentage of oocytes that became blastocysts (P < 0.001), and a reduction in re-expansion rates (P < 0.001) and hatching rates (P < 0.05 or P < 0.01) at all times examined. In Experiment 2, embryos were produced in vitro and cultured in modified SOF containing 4 mg/mL BSA, with or without 1 mg/mL hyaluronan. At 159-162 h after insemination, grade 1 morula, blastocysts and expanded blastocysts were harvested for embryo transfer. Harvested embryos were transferred individually to lactating Holstein recipients with a palpable corpus luteum on Day 7 after presumptive ovulation. There was an interaction (P < 0.05) between hyaluronan and embryo stage on pregnancy rate. Recipients that received morula and blastocyst stage embryos treated with hyaluronan had a higher pregnancy rate than recipients that received control embryos of the same stage. There was no effect of hyaluronan on pregnancy rates of recipients that received expanded blastocysts. In conclusion, addition of hyaluronan to embryo culture enhanced blastocyst yield, improved survival following vitrification, and enhanced the post-transfer survival of fresh morula and blastocyst stage embryos.     

Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction

Therapeutic cloning or nuclear transfer for stem cells (NTSC) seeks to overcome immune rejection through the development of embryonic stem cells (ES cells) derived from cloned blastocysts. The successful derivation of a human embryonic stem cell (hESC) line from blastocysts generated by somatic cell nuclear transfer (SCNT) provides proof-of-principle for "therapeutic cloning," though immune matching of the differentiated NT-hES remains to be established. Here, in nonhuman primates (NHPs; rhesus and cynomologus macaques), the strategies used with human SCNT improve NHP-SCNT development significantly. Protocol improvements include the following: enucleation just prior to metaphase-II arrest; extrusion rather than extraction of the meiotic spindle-chromosome complex (SCC); nuclear transfer by electrofusion with simultaneous cytoplast activation; and sequential media. Embryo transfers (ET) of 135 SCNT-NHP into 25 staged surrogates did not result in convincing evidence of pregnancies after 30 days post-ET. These results demonstrate that (i) protocols optimized in humans generate preimplantation embryos in nonhuman primates; (ii) some, though perhaps not yet all, hurdles in deriving NT-nhpES cells from cloned macaque embryos (therapeutic cloning) have been overcome; (iii) reproductive cloning with SCNT-NHP embryos appears significantly less efficient than with fertilized embryos; (iv) therapeutic cloning with matured metaphase-II oocytes, aged oocytes, or "fertilization failures" might remain difficult since enucleation is optimally performed prior to metaphase-II arrest; and (v) challenges remain for producing reproductive successes since NT embryos appear inferior to fertilized ones due to spindle defects resulting from centrosome and motor deficiencies that produce aneuploid preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming.     

Effect of follicle-stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer

The effects of luteinizing hormone (LH) (0, 100, 10,000 lU/ml) and follicle-stimulating hormone (FSH) (20 μg/ml) supplementation during in vitro maturation of slaughterhouse-derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Go-nadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 lU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05). © 1993 Wiley-Liss, Inc.