Min-Kang Wang

Viable rabbits derived from reconstructed oocytes by germinal vesicle transfer after intracytoplasmic sperm injection (ICSI).

Abnormal oocyte spindle due to the improper function of ooplasm is associated with female infertility of advanced maternal age. A possible way to overcome this problem is to transfer an oocyte germinal vesicle (GV) which contains genetic materials of a patient with a history of poor embryo development to the cytoplast from a donor oocyte. Here we demonstrate that GV transfer is feasible using a rabbit model. When the GVs were transferred to auto‐ or hetero‐cytoplasts of GV stage oocytes, around 80% of the reconstructed oocytes could mature in vitro and 7.1–9.4% of the oocytes developed to blastocyst stage after intracytoplasmic sperm injection (ICSI). Transfer of 93 fertilized eggs reconstructed via GV transfer into six recipients resulted in two live offspring. Results of this experiment indicate that GV transfer can potentially become a new approach in treatment of infertility because of advanced maternal age. Mol. Reprod. Dev. 58:180–185, 2001.

The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg

The giant panda skeletal muscle cells, uterus epithelial cells and mammary gland cells from an adult individual were cultured and used as nucleus donor for the construction of intenpecies embryos by transferring them into enucleated rabbit eggs. All the three kinds of somatic cells were able to reprogram in rabbit ooplasm and support early embryo development, of which mammary gland cells were proven to be the best, followed by uterus epithelial cells and skeletal muscle cells. The experiments showed that direct injection of mammary gland cell into enucleated rabbit ooplasm, combined within vim development in ligated rabbit oviduct, achieved higher blastocyst development thanin vitro culture after the somatic cell was injected into the perivitelline space and fused with the enucleated egg by electrical stimulation. The chromosome analysis demonstrated that the genetic materials in reconstructed blastocyst cells were the same as that in panda somatic cells. In addition, giant panda mitochondrial DNA (mtDNA) was shown to exist in the intenpecies reconstructed blastocyst. The data suggest that (i) the ability of ooplasm to dedifferentiate somatic cells is not speciesspecific; (ii) there is compatibility between intenpecies somatic nucleus and ooplasm during early development of the reconstructed egg.

In vitro fertilisation of mouse oocytes reconstructed by transfer of metaphase II chromosomes results in live births.

The interaction between nucleus and cytoplasm can be explored through nuclear transfer. We describe here another tool to investigate this interaction: MII meiotic apparatus transfer (MAT) between mouse oocytes. In this study, the MII oocyte meiotic apparatus or spindle from C57BL/6 mice, a black strain, was transferred into an enucleated metaphase oocyte from Kunming mouse, a white strain. The results showed that the enucleation rate by treating oocytes with 3% sucrose was 100%, but the electrofusion efficiency was very low, with only 17.6% of reconstructed karyoplast-recipient cytoplasm pairs fused. When the fused oocytes were exposed to spermatozoa from C57BL/6 mice, 9 of 11 (82%) were fertilised. Eight reconstructed embryos at 1- to 4-cell stages were transferred into the oviducts of two synchronously pregnant Kunming strain fosters and one delivered two normal C57BL/6 offspring. This study indicates that MII meiotic apparatus or spindle sustains normal structure and function after micromanipulation and electrofusion. MAT provides a model for further research on the application of this technique to assisted human reproduction.

Effect of telophase enucleation on bovine somatic nuclear transfer.

Telophase enucleation has been proven to be an efficient method for preparing recipient cytoplasts in bovine embryonic nuclear transfer (2, 11). This research was designed to study in vitro development of bovine oocytes containing transferred somatic cell nuclei, reconstructed by using enucleated in vitro-matured oocytes 32 h of age at telophase II stage as recipient cytoplasts, compared with those 24 h of age at metaphase II stage. Two protocols for donor cell injection were adopted, i.e., subzonal injection (SUZI) and intracytoplasmic injection (ICI). Bovine oviduct epithelial cells (BOECs) and bovine cumulus cells (BCCs) from an adult cow were used as nuclear donors for these experiments. In SUZI groups, the fusion rate of donor cells, both BOECs and BCCs, with MII enucleated oocytes were higher than those with TII enucleated oocytes (54% vs. 41% and 53% vs. 39%, respectively; P<0.05), but the development rates to morula plus blastocyst stage in MII groups were lower than those in TII groups (22% vs. 39% and 21% vs. 41%, respectively; P<0.05). In ICI groups, about 26% of enucleated MII oocytes injected with BOECs or BCCs cleaved and only small parts of them developed to blastocyst stage (4% and 3%, respectively; P>0.05). When BOECs or BCCs were intracytoplasmically injected into oocytes enucleated at TII stage, no blastocyst was formed in either donor cell group and no cleavage occurred in BOEC group. Our data demonstrated that telophase enucleation is beneficial to early embryo development when bovine somatic nuclei are transferred by subzonal injection. However, it is harmful when donor cells are directly injected into the cytoplast of the enucleated oocytes.

Sucrose pretreatment for enucleation: an efficient and non-damage method for removing the spindle of the mouse MII oocyte.

Oocytes enucleated at metaphase II stage can support reprogramming of transferred nucleus and further developing to term. However, the first polar body in mice sometimes migrates away from the original place of expulsion, so the chromosomes of the oocyte will displace from the first polar body. Thus, it is not always possible to successfully enucleate according to the position of the first polar body. Here we use sucrose treatment to visualize metaphase spindle fibers and chromosomes with standard light microscopy. In the manipulation medium containing 3% sucrose, oocytes of poor quality become shrunken, deformed or fragmented, while oocytes of good quality in the same medium would show a swelling around the metaphase chromosomes and a transparent spindle area, shaped like “∞” and “0”. So it is easy to remove the well‐distinguished spindle and chromosomes in oocytes of good quality. Re‐examined by Hoechst 33342 stain under the UV light, the enucleation rate was 100%. There was no significant difference in IVF and cleavage rates between the sucrose treatment and the control group. In conclusion, this study demonstrated that 3% sucrose pretreatment can give a method for evaluating embryo quality and more importantly, it can, under a common microscope, allow the visualization of the spindle and chromosomes in oocytes of good quality and hence efficiently improve enucleation rate without any harm. Mol. Reprod. Dev. 58:432–436, 2001.

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals.

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.

Maturation of the reconstructed oocytes by germinal vesicle transfer in rabbits and mice.

The present study was designed to evaluate the feasibility of germinal vesicle (GV) transfer in rabbits and mice. The GV oocytes were collected from ovaries and cultured in 20 microg/mL 3-isobutyl-1-methylxanthin (IBMX) in TCM199 medium, which caused oocytes to shrink, enlarging the perivitelline space to facilitate the GV removal and transfer. Pairs of GV-cytoplast complexes were fused with electric pulses, and the fused, reconstructed oocytes were cultured in TCM199 for 24 h. Results are as follows: 1) The exposure time of rabbit GV oocytes to IBMX medium affected the success of GV removal. For oocytes cultured for 2 and 3 h in IBMX medium, removed rates were 56% and 44, respectively, significantly higher (P < 0.05) than removal rates of GV oocytes cultured for 1 and 4 h (27% and 27%, respectively); 2) There was no significant difference (P > 0.1) in fusion and maturation rates of rabbit reconstructed oocytes collected at 72 and 84 h after initiation of FSH injection to donors; 3) eCG in the maturation media improved development of rabbit-to-rabbit GV transferred oocytes but had no positive effect on mouse-to-rabbit GV transferred oocytes; 4) When mouse GV-karyoplasts were injected into enucleated rabbit oocytes, fusion rates of GV-karyoplasts measuring 40- to 50-microm and 80- to 90-microm in diameters obtained were 84% and 93%, respectively. The rates were significantly higher (P < 0.05) than fusion rates after transferring GV-karyoplasts measuring 30- to 35-microm in diameter (63%). The maturation rate (89%) of reconstructed oocytes composed of 80- to 90-microm mouse GV-karyoplasts and rabbit GV-enucleated cytoplasts was higher than that seen for oocytes composed of 40- to 50-microm (77%, P<0.05) or 30- to 35-microm (59%, P<0.01) mouse karyoplasts. Thirty-five of the 63 (56%) mature mouse-to-rabbit reconstructed oocytes had the normal complement of 20 chromosomes.

NUCLEAR TRANSFER USING NONQUIESCENT ADULT FIBROBLASTS FROM A BOVINE EAR

The natural reproduction of mammal is sexual reproduction, which needs fertilization involving sperm and oocyte. Nuclear transfer provided an asexual reproduction method for mammal. Donor cells used in previous experiments of nuclear transfer were mostly from undifferentiated or non-terminally differentiated cells, such as embryonic or fetal cells. However, since Wilmutet al. obtained a viable lamb by transfer of an adult sheep somatic cell into an enucleated oocyte, nuclear transfer using adult somatic cell has been successful in several species. Wilmutet al. suggested that it was a key factor for the success of somatic nuclear transfer to induce the donor cells into GO phase (“GO-phase hypothesis”). In order to verify the Gophase hypothesis, nonquiescent adult fibroblasts from a bovine ear were transferred into enucleated bovine oocytes. The experiments showed that the rate of electrofusion after micromanipulation was above 50%, the cleaving rate was 54.5% and 9.1% of those reconstructed embryos developed to 32-cell stage. These results indicate that for cattle, nuclei from nonquiescent adult somatic cells introduced into enucleated oocytes are at least capable of supporting early development.

Delivery and storage of single embryos, sperm, or cells in microglass capillaries.

Dear Editor: In general, a large number of cells can be placed in glass or plastic tubes for delivery or storage (or both). In these cases, the loss of some cells does not affect further experimental procedures or analyses. However, in some cases, only a few cells, such as gametes and embryos, can be obtained from humans or rare animals for further analysis. For example, it has been reported that single cells can be analyzed by polymerase chain reaction (Li et al., 1988; Monk and Holding, 1990; Zhang et al., 1992; Dietmaier et al., 1999). In human-assis ted reproduction, offspring can be produced by intracytoplasmic sperm injection (ICSI), and normal fetuses may be obtained by mieroinsemination with frozen-thawed round spermatids collected from obstructive azoospennic males (Abuzeid et al., 1997). Offspring of mice may be obtained by microinsemination with frozen-thawed spermatids of azoospermic males and spermatozoa stored in alcohol (Tanemura et al., 1997; Tateno et al., 1998). Nomml offspring may also be obtained from mnuse oocytes injected with spermatozoa after cryopreserving with or without cryoprotectants or by using freeze-dried spermatozoa (Wakayama et al., 1998; Wakayama and Yanagimachi, 1998). Indeed, for further analysis or research, reliable and safe delivery of one cell or embryo from one location to another is often necessary. In this study the two separated halves of zona pellucida, the embryos reconstructed by nuclear transfer, and the sperm and somatic cells at different densities were stored in glass capillaries and delivered more than 1000 kin by mail within 1 wk. Glass capillaries with 1.0or 1.3-mm outer diameter (O.D.) and 0.9-mm inner diameter (I.D.) were employed. Using an alcohol burnei, a tip of about 30 mm in length and having a 100-1xm O.D. is pulled on to one end of the glass capillary; the tip and the opposite end (base) of the glass pipette are then fire-polished. A silicone tube with 1.0-ram I.D. is connected to the base of the pulled capillary, which in turn is connected to another silicone tube with 2.0-mm I.D. to form a mouth-controlled pipette (Wang et al., 1999). Using a stereomicroscope or inverted microscope, mineral oil and an air bubble are aspirated into the tip of the capillary. Then, a column of medium containing the cell, the embryo, or the cellular structure to be stored or delivered is aspirated into the capillary, followed by mineral oil. The last oil droplet is at least 5 mm from the tip of the capillary tube. The tip of the capillary is checked using an inverted microscope to ensure that the cell or the embryo is contained within the medium. The arrangement in the filled capillary is shown in Fig. 1. The two ends of the capillary are then flame-sealed. Coded marks are then made on the neck or the midregion (or on both) of the capillary for identification. In total, six marks can be made with a marking pen along the surface of the capilla17, as shown in Fig. 1, and can reliably identify up to 20 individual capillaries when employed in the manner depicted. For delivery to other laboratories, the capillaries are placed into a hardwall container, such as a plastic or metal tube or box whose space is filled with cotton or soft material to fix the position of the capillaries.

Microsatellite DNA analysis proves nucleus of interspecies reconstructed blastocyst coming from that of donor giant panda

A method for DNA isolation from early development of blastocyst and further analysis of nuclear and mitochondrial DNA was developed in present study. Total DNA was prepared from interspecies reconstructed blastocyst and a giant panda specific microsatellite locus g010 was successfully amplified. DNA sequencing of the PCR product showed that two sequences of reconstructed blastocysts are the same as that of positive control giant panda. Our results prove that the nucleus of interspecies reconstructed blastocyst comes from somatic nucleus of donor giant panda.