Postimplantation development of tetraploid mouse embryos produced by electrofusion

Despite the fact that a variety of experimental techniques have been devised over the years to induce tetraploid mammalian embryonic development, success rates to date have been limited. Apart from the early study by Snow, who obtained development to term of a limited number of cytochalasin B-induced tetraploid mouse embryos, no other researchers have achieved development of tetraploid embryos beyond the early postimplantation period. We now report advanced postimplantation development of tetraploid mouse embryos following electrofusion of blastomeres at the 2-cell stage, and subsequent transfer of these 1-cell 'fused' embryos to appropriate recipients. Cytogenetic analysis of the extraembryonic membranes of all of the postimplantation embryos encountered in the present study has provided an unequivocal means of confirming their tetraploid chromosome constitution. A preliminary morphological and histological analysis of the tetraploid embryos obtained by this technique has revealed that characteristic craniofacial abnormalities particularly involving the forebrain and eyes were consistently observed, and these features were often associated with abnormalities of the vertebral axis and heart. The most advanced viable embryo in this series was recovered on the 15th day of gestation, and its morphological features suggest that it was developmentally equivalent to a normal embryo of about 13.5-14 days p.c.     

Development of reconstituted mouse embryos produced from bisected and electrofused pronuclear-stage embryos

The present study was designed to investigate the in vitro and in vivo development potential of reconstituted mouse embryos produced by bisection and electrofusion of pronuclear stage embryos (PN-E). Pronuclear-stage ICR and F1 (C57BL x CBA) strain mouse embryos were bisected manually with a fine glass needle under the dissecting microscope to produce karyoplasts (KP) and cytoplasts (CP). The KP of ICR PN-E and CP of F1 PN-E (KP: ICR + CP:F1) or the KP of F1 PN-E and CP of F1 PN-E (KP:F1 + CP:ICR) were attached using phytohemagglutinin-P (PHA-P) and then electrofused. High fusion rates of the KP and CP of PN-E were obtained (93.5%). The fused embryos were encapsulated in alginate gel and cultured for 72 or 96 hours. The cleavage rates of reconstituted embryos were also high (98.8%). Developmental rates to the blastocyst stage in vitro for the 96-hour culture of reconstituted embryos were 68.9% (KP:ICR + CP:F1) and 78.4% (KP:F1 + CP:ICR). Furthermore, the developmental ability of reconstituted embryos in vivo was investigated, and some live young were obtained (KP:ICR + CP:F1, 7.5% and KP:F1 + CP:ICR, 10.8%). In this study, it was confirmed that reconstituted embryos produced by bisection and electrofusion of pronuclear stage embryos were able to develop into blastocysts in vitro and into live young in vivo.     

Chromosome aberrations in mouse embryos and fetuses produced by assisted reproductive technology

The occurrence of structural chromosome aberrations in mouse one-cell embryos produced by intracytoplasmic sperm injection (ICSI) with mature spermatozoa was dependent on the type of sperm incubation medium and sperm incubation time. When cauda epididymal spermatozoa were used following incubation in bicarbonate-buffered TYH medium for 0h (no incubation) and 0.5h, the chromosome aberration rates (6.9% and 7.4%, respectively) in the resultant embryos were significantly higher than that (2.3%) in the IVF embryos. However, when the spermatozoa were incubated for 2-2.5h and 6h in the same medium, the chromosome aberration rates were reduced to the IVF embryo level (3.8% and 4.3%, respectively). When spermatozoa incubated in Hepes-buffered H-mCZB and phosphate-buffered PB1 media were used for ICSI, chromosome aberration rates in embryos were significantly high (8.6-28.1%) and increased in a time-dependent manner. On the other hand, when immature testicular spermatozoa were incubated in those three media for 0.5h and 6h, the incidences of resultant embryos with structural chromosome aberrations ranged between 7.4% and 11.7%, and there was no medium- and time-dependent change in these aberration rates. To evaluate transmissible risk of chromosome aberrations to offspring, two- or four-cell embryos derived from cauda epididymal spermatozoa were transferred into the oviducts of pseudopregnant females and chromosomes of live fetuses were examined on gestational day 16. One (2.0%) mosaic fetus was found when spermatozoa were incubated in TYH for 2-2.5h, and there were four (6.7%) fetuses displaying a structurally abnormal karyotype when spermatozoa were incubated in H-mCZB for 2-2.5h, indicating that structural chromosome aberrations generated in ICSI one-cell embryos are transmissible to offspring. The causal mechanism of structural chromosome aberrations in ICSI one-cell embryos is discussed in relation to the acrosomal plasma membrane cholesterol and the acrosome.     

Development of androgenetic mouse embryos produced by in vitro fertilization of enucleated oocytes

Enucleated mouse oocytes were successfully fertilized in vitro, and the resultant androgenetic eggs developed to the blastocyst stage. The proportion of enucleated oocytes fertilized in vitro was high (87–99%) at sperm concentrations ranging from 10–100 × 10⁴/ml. At high sperm concentrations (100–1,000 × 10⁴), 35–45% of the fertilized eggs resulted in heterozygous bispermic androgenones. The proportion of hemizygous haploid and heterozygous diploid androgenones developing to blastocysts was 11% and 43%, respectively. Hemizygous diploidization, however, showed no positive effect on development. These results clearly show that the procedure reported here is efficient and reliable for the production of androgenetic eggs. © 1993 Wiley-Liss, Inc.     

Electron microscopic studies of chimeric blastocysts experimentally produced by aggregating blastomeres of rat and mouse embryos

Xenogeneic chimeras between rat and mouse were produced by aggregating embryos at the 8- to 12-cell stages. Of 114 combinations we have made so far, 26 chimeric embryos developed into blastocysts. The origin of each cell in the composite embryos can be identified unambiguously by the ultrastructural appearance of the cytoplasmic inclusions. Both the rat- and the mouse-derived cells differentiated equally well into either ICM or trophoblast cells. However, the mouse-derived cells gave rise to ICM cells more frequently than the rat-derived cells. Furthermore, when the ratderived cells formed trophoblast cells, they were predominantly mural trophoblast cells, while the mouse-derived cells differentiated predominantly into the polar trophoblast cells. Cells of the same species tend to remain as a group in the chimeric blastocysts.     

Genetically identical parthenogenetic mouse embryos produced by inhibition of the first meiotic cleavage with cytochalasin D

The microfilament inhibitor cytochalasin D inhibits extrusion of the first polar body when present during the first meiotic division of mouse oocytes; however, it does not interfere with anaphase movement of chromosomes, and thus induces the formation of tetraploid oocytes. After the separation of chromosomes in anaphase, two spindles start to assemble. However, they merge rapidly and a single meiotic spindle forms. During the transition between metaphase I and metaphase II, in the presence of cytochalasin D, a drop in histone kinase activity takes place demonstrating a transitional decrease in the activity of the maturation promoting factor. These oocytes can be activated parthenogenetically a few hours after washing out the inhibitor. After completion of the second meiotic division and extrusion of a polar body, they contain a diploid number of chromosomes. They are genetically identical to each other and to their mother. Such eggs develop to the blastocyst stage and can implant in the uteri of foster mothers. Most of these fetuses die before the 9th day of gestation, as do diploid control fetuses treated with cytochalasin D during the second meiotic division. The heterozygous state of the experimental embryos obtained after activation of eggs recovered from heterozygous females and treated with cytochalasin D during the first meiotic division was confirmed using a glucose-phosphate isomerase assay. This technique allows the production of genetic clones of parthenogenetic embryos by simple means.     

Development of mouse embryos cryopreserved by vitrification

Eight-cell mouse embryos were cryopreserved by vitrification in a concentrated solution of dimethylsulphoxide, acetamide, propylene glycol and polyethylene glycol. This solution (designated VS1) does not crystallize when cooled to subzero temperatures but instead forms a glassy transparent solid. Embryos were exposed in three steps to a stock VS1 solution or a saline solution containing 90% of the cryoprotectants in the stock VS1 (90% VS1) and then the suspensions were vitrified by rapid cooling in liquid nitrogen. Of 568 embryos vitrified in 90% VS1, 80% developed in vitro and 98 normal fetuses or young (17% of the total) were produced after transfer to pseudopregnant recipients. By contrast, 22% of 153 embryos vitrified in the stock VS1 developed in vitro, but only one normal fetus was obtained after transfer. These results demonstrate that normal fetuses and young can be produced from embryos cryopreserved by the simple and rapid method of vitrification.     

绵羊胞内单精子注射技术

In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.     

Development of reconstituted mouse embryos produced from the cytoplast of bisected oocytes or pronuclear-stage embryos and single blastomeres of 2-cell stage embryos

The present study investigated the in vitro developmental potential of reconstituted mouse embryos produced from the cytoplast of pronuclear-stage embryos or oocytes and single blastomeres of 2-cell stage embryos by electrofusion. The cytoplast of pronuclear-stage embryos and oocytes were obtained by manual bisection with a fine glass needle under a dissecting microscope. The fusion rates of the reconstituted embryos produced from the cytoplast of oocytes and single blastomeres of 2-cell stage embryos (O-SB2: 38.1 and 41.5%) were significantly lower than those produced from the cytoplast of pronuclear-stage embryos and single blastomeres of 2-cell stage embryos (P-SB2: 91.2 and 97.6%; P<0.001). Reconstituted embryos were encapsulated in alginate gel and were cultured for 96 hours. Similarly, the cleavage and development rates to the blastocyst stage of O-SB2 (56.3, 61.2 and 2.0, 3.1%, respectively) were significantly lower than those of the P-SB2 (91.0, 91.2 and 18.6, 20.7%; respectively, P<0.05). The cleavage and development rates to the blastocyst stage (61.2 and 2.0%) of reconstituted embryos produced from single blastomeres of late 2-cell stage embryos and oocyte cytoplast improved after activation by ethanol treatment (76.1 and 21.7%). However, the use of single blastomeres of early 2-cell stage embryos as nuclear donors did not enhance the cleavage and development rates of the reconstituted embryos to the blastocyst stage.     

Handling of actinomycin D by preimplantation mouse embryos

A protein isolated from serum is required in the cell suspension for the chemotactic response of normal mouse peritoneal macrophages to complement-derived C5a. Macrophage stimulating protein (MSP) has a molecular weight of 100000 and an isoelectric point of 7.0. It is resistant to changes in pH over a range of 1.3–11, is heat labile especially after partial purification and does not survive proteolytic enzyme attack. Binding to ConA Sepharose suggests that it contains a carbohydrate moiety. Its concentration in normal serum is very low and it is detectable only by virtue of a sensitive bioassay. An upper limit has been estimated at 75 ng/ml; the actual concentration may be considerably lower.     

Protein synthesis in mouse embryos with experimentally produced asynchrony between chromosome replication and cell division

Summary The cleavage of fertilized mouse eggs was prevented during cytochalasin B incubation and consequently these eggs became tetraploid the following day during in vitro culture. When the eggs were cultured further in normal medium, they cleaved and gave rise to tetraploid blastocysts. Protein synthesis was analysed in these embryos at different developmental stages using two-dimensional polyacrylamide gel electrophoresis. The protein synthesis pattern of one-cell tetraploid eggs was intermediate between those of normal one- and two-cell embryos. Tetraploid two-cell embryos expressed protein sets equivalent to those of untreated four-cell embryos, and tetraploid four-cell embryos synthesized proteins similar to those of four- to eight-cell controls. At subsequent pre-implantation stages the asynchrony was no longer detectable. When fertilized eggs were cultured continuously in the presence of cytochalasin B, they became tetraploid, octoploid and more and more polyploid without cleavage occurring. The protein synthesis patterns expressed by these one-cell polyploid eggs did not resemble that of normal fertilized eggs, but were similar to those of cleaving control embryos and blastocysts of equivalent age and nuclear division. These results strongly suggest that in early mouse embryos stage-specific translation is temporally correlated with chromosome replication (karyokinesis) and independent of cell division (cytokinesis) or cell interaction.Some of these results were presented at the IX Congress of the International Society of Developmental Biologists in Basle, Switzerland, August 28–September 1, 1981     

Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells

Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.     

小鼠电激活孤雌胚胎早期体内外发育能力的比较

目的探讨小鼠电激活孤雌胚胎的早期体内、外发育能力。方法 利用不同电脉冲参数和激活液对小鼠卵母细胞进行活化,观察激活后的小鼠孤雌胚体外发育状况和移植后的发育能力。结果非电解质激活液优于电解质液,脉冲强度、脉冲宽度和脉冲次数3个参数各自处于某一范围内时,他们之间存在某种相关性,降低其中1个参数可通过升高另外2个参数得到补偿,经筛选较适宜的电脉冲参数为：1.0 kV/cm、40μs、2 p,或者1.5kV/cm、30/μs、2 p,分别为74.65%和71.19%,体外囊胚发育率分别为43.40%和47.62%。电激活孤雌胚体外发育时序比正常胚胎慢,但囊胚细胞数与对照组差异不显著。它们经胚胎移植后,其中的一部分能够着床,但着床率仅为3.6%,极显著低于对照组（67%,P〈0.01）。结论电刺激能够较好地模拟正常受精过程激活小鼠卵母细胞,但激活后的多数小鼠孤雌胚胎着床能力较低,不能够顺利着床。     

Suppressor factor produced by neonatal mouse spleen cells

Spleen cells from 5- to 6-day- old BDF₁ mice produce a soluble suppressor factor (SF) when cultured for 2–4 days in the presence of 10% fetal calf serum. This suppressor factor inhibits the mixed lymphocyte reactivity of adult mouse spleen cells as well as the in vitro generation of cytotoxic cells. The SF which is not H₂-restricted or antigen specific is most effective when added in the early phase of the culture period. The SF is resistant to heat and uv treatment and appears to consist of a large and small component. It is resistant to treatment with pronase or trypsin. The SF appears to be produced by neonatal spleen cells which are not adherent to plastic or Sephadex G-10 and are insensitive to treatment with anti-Thy 1.2 and complement. Incubation of SF with peritoneal exudate cells reduces suppressor activity.