Guang-Peng Li

Review of Enucleation Methods and Procedures Used in Animal Cloning: State of the Art

Enucleation of a recipient oocyte is a crucially important process for nuclear transfer efficiency. Several procedures have been developed and used in the production of nuclear transfer embryos. Although the use of excitable fluorochromes and ultraviolet (UV) light are commonly used for complete enucleation, they also pose the risk of damaging the maternal cytoplast. Telophase and chemically assisted enucleation have also been used for cloning, but the quality and quantity of the recipient cytoplasm varies with the procedure used. This paper reviews various methods used for enucleation, and discusses their benefits and limitations with respect to cloning efficiency.

Enucleation of Demecolcine-Treated Bovine Oocytes in Cytochalasin-Free Medium: Mechanism Investigation and Practical Improvement

Demecolcine-assisted/induced enucleation has been used in nuclear transfer cloning procedures for many species, yet its mechanism of action remains unclear. Primarily because oocytoplasm protrusion induced by demecolcine is inhibited by the presence of cytochalasin, its use has had limited application. In this experiment, we investigated the microtubule and microfilament alterations in bovine oocytes after demecolcine and/or cytochalasin B (CB) treatments by immunocytochemical staining. We also examined mechanical enucleation of demecolcine-treated oocytes in cytochalasin-free medium. The results showed that demecolcine-treatment disrupts the balance between microtubule/microfilament interactions primarily by deleting microtubules and with little effect on the microfilaments that we believe accounts for the membrane protrusion. The CB treatment reduced the amount of microfilaments but had little effect on the microtubules. Most demecolcine-induced membrane protrusions disappeared when exposed to CB. Western blotting showed that CB treatment increases G-actin, which indicates a decrease in the microfilaments. High oocyte enucleation, survival, and developmental rates occurred when demecolcine-assisted enucleation was carried out in a CB-free solution. Higher blastocyst development rates and blastocyst cell numbers were achieved compared to control, indicating that CB is not necessary in the enucleation procedure of bovine oocytes. This study provides a clearer understanding of the mechanism for the demecolcine-induced oocyte membrane protrusion, and substantiates the practical use of demecolcine-assisted enucleation in a CB-free environment.

In Vitro Development and Chromosomal Configuration of Bovine Somatic Cloned Embryos with Nonenucleated Metaphase II Oocytes

This study was designed to examine the effects of the presence of oocyte nuclei on the donor cell nuclear remodeling, including premature chromosome condensation (PCC) and DNA configuration, and subsequent embryo development. The results showed that: (1) the presence of oocyte MII spindles was more likely to induce donor cell PCC. (2) The positional relationship between the fused donor cell and the oocyte metaphase spindle had an effect on oocyte PB2 extrusion. When the fused donor cell was widely separated from the MII spindle, 94.4% of the reconstructed oocytes expelled a PB2. When the donor cell was fused adjacently to the MII spindle, almost all of the reconstructed oocytes did not expel the PB2; the majority (67.9%) formed a very large M-phase spindle in which the oocyte and the donor cell chromosomes merged. (3) After activation, the oocyte and donor nuclei exhibited a variety of pronuclear patterns and asynchronous development. (4) The embryos reconstituted with nonenucleated oocytes resulted in a similar cleavage rate as observed in the control embryos reconstituted with enucleated oocytes. Blastocyst developmental rates were no different between nonenucleated and enucleated cloned embryos; however, the development rates from early to hatching blastocysts significantly decreased in the nonenucleation group compared to enucleation controls (0 vs. 23.1%; 27.5 vs. 67.8%), regardless with either cumulus cells or fibroblasts as donor cells. (5) All nonenucleated oocyte-derived blastocysts contained mixed polyploidy with a variety of compositions that included 2n/4n, 2n/6n, 2n/8n, and 2n/4n/8n. (6) Nuclear transfer preceding the oocyte enucleation experiment indicated that prolonged presence of oocyte nuclei induced abnormal DNA configuration and reduced in vitro development of transferred somatic nuclei, but short time presence of oocyte nuclei did not affect the in vitro development of cloned embryos. We conclude that oocyte MII spindles induce donor cell PCC, the developmental capacity of cloned embryos reconstituted with nonenucleated oocytes is inferior to those with enucleated oocytes, and that all such derived blastocysts are polyploidy.

Why clone horses and mules

The ability to produce offspring from animals otherwise incapable of reproduction has important implications in the equine. Routinely, male equines are electively castrated to produce animals (geldings) that are more easily managed and handled. These animals are then much more conveniently and safety used in various competitions such as cutting, reining, polo, roping, and racing. The ability to recover the genetics of animals that either prematurely die or die prior to sufficient impact of valuable genomics is an important consideration in the horse.

Nicotine combined with okadaic acid or taxol adversely affects bovine oocyte maturation and subsequent embryo development

OBJECTIVE To investigate the effects of nicotine in combination with okadaic acid (OA) or taxol on bovine oocyte maturation and subsequent embryonic development. DESIGN Prospective randomized study. SETTING University research laboratory. PATIENT(S) Bovine ovaries, oocytes, and embryos. INTERVENTION(S) Oocyte maturation and subsequent embryo development in the presence of nicotine in combination with okadaic acid and taxol. MAIN OUTCOME MEASURE(S) Haploid composition, cleavage rate, IVF and parthenogenetic embryo development, blastocyst cell number. RESULT(S) The results showed that nicotine and OA or nicotine and taxol significantly decreased oocyte maturation rates. Combinations of nicotine (10 or 50 micromol/L) and OA (0.01 or 0.05 micromol/L) did not affect oocyte haploid composition; however, taxol alone or combined with nicotine caused a decrease in haploid composition compared with control. Parthenogenetic activation of oocytes that were matured in nicotine, OA, or taxol resulted in blastocyst development rates of 12%-17%, which were not different from the control. Various combinations of nicotine and OA or nicotine and taxol significantly lowered (3%-8%) blastocyst development compared with control. The average cell number of blastocysts derived from nicotine + OA- and nicotine + taxol-treated oocytes was lower than all other treatment groups and control. For oocytes fertilized in vitro, oocytes matured in OA, taxol, or combinations of nicotine and OA or taxol-containing media resulted in significantly lower cleavage rates and blastocyst development compared with the control group and the 10 micromol/L nicotine treatment group. The IVF embryos cultured in nicotine + OA-containing medium had a significantly lower blastocyst development rate. CONCLUSION(S) Combinations of nicotine with OA or taxol adversely affect oocyte maturation and subsequently result in poor blastocyst development.

DNMT 1 maintains hypermethylation of CAG promoter specific region and prevents expression of exogenous gene in fat-1 transgenic sheep

Methylation is an important issue in gene expression regulation and also in the fields of genetics and reproduction. In this study, we created fat-1 transgenic sheep, investigated the fine-mapping and the modulatory mechanisms of promoter methylation. Sheep fetal fibroblasts were transfected by pCAG-fat1-IRES-EGFP. Monoclonal cell line was screened as nuclear donor and carried out nuclear transfer (441 transgenic cloned embryos, 52 synchronism recipient sheep). Six offsprings were obtained. Expressions of exogenous genes fat-1 and EGFP were detectable in 10 examined tissues and upregulated omega-3 fatty acid content. Interestingly, more or less EGFP negative cells were detectable in the positive transgenic fetal skin cells. EGFP negative and positive cells were sorted by flow cytometry, and their methylation status in the whole promoter region (1701 nt) were investigated by bisulphate sequencing. The fine-mapping of methylation in CAG promoter were proposed. The results suggested that exogenous gene expression was determined by the methylation status from 721–1346 nt and modulated by methylation levels at 101, 108 and 115 nt sites in CAG promoter. To clarify the regulatory mechanism of methylation, examination of four DNA methyltransferases (DNMTs) demonstrated that hypermethylation of CAG promoter is mainly maintained by DNMT 1 in EGFP negative cells. Furthermore, investigation of the cell surface antigen CD34, CD45 and CD166 indicated that EGFP positive and negative cells belong to different types. The present study systematically clarified methylation status of CAG promoter in transgenic sheep and regulatory mechanism, which will provide research strategies for gene expression regulation in transgenic animals.

SGO1 Maintains Bovine Meiotic and Mitotic Centromeric Cohesions of Sister Chromatids and Directly Affects Embryo Development

Shugoshin (SGO) is a critical factor that enforces cohesion from segregation of paired sister chromatids during mitosis and meiosis. It has been studied mainly in invertebrates. Knowledge of SGO(s) in a mammalian system has only been reported in the mouse and Hela cells. In this study, the functions of SGO1 in bovine oocytes during meiotic maturation, early embryonic development and somatic cell mitosis were investigated. The results showed that SGO1 was expressed from germinal vesicle (GV) to the metaphase II stage. SGO1 accumulated on condensed and scattered chromosomes from pre-metaphase I to metaphase II. The over-expression of SGO1 did not interfere with the process of homologous chromosome separation, although once separated they were unable to move to the opposing spindle poles. This often resulted in the formation of oocytes with 60 replicated chromosomes. Depletion of SGO1 in GV oocytes affected chromosomal separation resulting in abnormal chromosome alignment at a significantly higher proportion than in control oocytes. Knockdown of SGO1 expression significantly decreased the embryonic developmental rate and quality. To further confirm the function(s) of SGO1 during mitosis, bovine embryonic fibroblast cells were transfected with SGO1 siRNAs. SGO1 depletion induced the premature dissociation of chromosomal cohesion at the centromere and along the chromosome arm giving rise to abnormal appearing mitotic patterns. The results of this study infer that SGO1 is involved in the centromeric cohesion of sister chromatids and chromosomal movement towards the spindle poles. Depletion of SGO1 causes arrestment of cell division in meiosis and mitosis.