Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

Effect of pronase treatment, microdissection, and zona pellucida removal on the development of porcine embryos and blastomeres in vitro

The in vitro development of porcine blastomeres and the effects of pronase treatment, microdissection, and zona pellucida removal used in the isolation procedure were investigated. Seven hundred and forty-nine two to eight-cell embryos were collected from 11 sows and 74 gilts. Zona-free porcine blastomeres (ISOL BL) were obtained by treating embryos with 2.5 or 5.0% pronase for 3.0 min and microdissecting with finely drawn siliconized glass pipettes. The effect of the pronase treatment on subsequent in vitro development was evaluated by treating two to eight-cell embryos with 5.0% pronase for 3.0 min (PTD EMB). The effect of pronase treatment and microdissection on in vitro development was evaluated by microdissecting PTD EMB, leaving one blastomere bounded by the zona pellucida (BL ZP). Untreated two to eight-cell embryos were cultured as controls (CONTROLS). Embryos and blastomeres were cultured individually in microdrops of Whitten's medium with 15 mg/ml bovine serum albumin (WM + BSA) under paraffin oil in a humidified atmosphere of 5% CO2 in air at 37 degrees C. Observations were conducted at 24-h intervals and at the cessation of division embryos were fixed, stained, nuclei enumerated, and cleavage indices assigned. Blastocysts and vesiculated embryos which developed were measured using an ocular micrometer. The incidence of blastocyst formation was greater (P less than 0.05) for ISOL BL from four-cell than from two or eight-cell embryos. The presence of the zona pellucida did not significantly affect the incidence of blastocyst formation by single blastomeres. Although ISOL BL did not develop as well as CONTROLS or PTD EMB (P less than 0.05), development of BL ZP was not significantly different from the respective PTD EMB. Blastocysts developing from blastomeres had fewer cells and were smaller than CONTROLS or PTD EMB (P less than 0.05). Although development of ISOL BL may have been impaired by the isolation procedures employed, BL ZP are capable of in vitro development comparable to their respective PTD EMB.     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

Cleavage in vivo and in culture in the Dasyurid marsupial Antechinus stuartii (macleay)

Cleavage in the brown marsupial mouse, Antechinus stuartii, from the zygote to the unilaminar blastocyst, was observed in vivo and in culture and in sections of embryos. The first three divisions were meridional and passed from the yolk pole to the opposite pole. Deutoplasmolysis, resulting in a distinct yolk mass, occurred during the first two divisions. Prior to the third and fourth divisions, the blastomeres elongated and flattened against the zona pellucida. The fourth division was latitudinal and resulted in two histologically distinct rings of eight blastomeres which were at first rounded and then became flattened against the zona. Further divisions and flattening of the blastomeres resulted in a complete unilaminar blastocyst by the time the blastocyst numbered 22 to 30 cells. Some expansion, causing compression of the zona and mucoid layers, occurred before completion of the blastocyst, but most expansion occurred once the blastocyst was complete. No histological differences could be detected between the blastomeres at this stage. Embryos were successfully cultured from the zygote to the rounded four-cell stage and from the flattened four-cell stage to the completed unilaminar blastocyst of around 32 cells. Total estimated cleavage times were slower in culture than in vivo, but the relative lengths of time for each division were approximately the same.     

Cell surface interaction induces polarization of mouse 8-cell blastomeres at compaction

The development of the polarized surface binding of the fluoresceinated ligand concanavalin A (FITC-Con A) was studied in blastomeres of the early mouse embryo. Single 8-cell blastomeres, natural 8-cell couplets derived from the in vitro division of individual 4-cell blastomeres, and reaggregated couplets made from dissociated 8-cells were cultured for varying periods of time and on a variety of substrata. The development of surface polarity was found to be highly dependent upon cell contact. Over 50% of the cells in couplets were polarized after 4–5 hr in culture, with the smaller cell in the couplet usually more advanced in its polarization than the larger cell. The orientation of the poles of FITC-Con A binding was opposite the point of contact between cells in the couplets regardless of their previous orientation within the embryo or the plane of cleavage.     

Determination of protein concentration in cleaving mammalian ova by the capillary spectrophotometry method]

A micromodification of the Lowry's method is described which allows to measure reliably the protein content in 5-20 embryos of white rats and CBA mice. Differences in the protein content in rat embryos at the stages of 2 blastomeres and blastocyst were shown to be statistically unreliable (20.2 +/- 1.4 and 18.9 +/- 1.3 ng, resp.). The protein content in the mouse embryos at the same two stages differs reliably (19.1 +/- 1.1 and 22.0 +/- +/- 1.7 ng, resp.). The protein content in zona pellucida does not differ reliably from those in rat embryos both at the stages of 2 blastomeres (4.5 ng) and blastocyst (2.3 ng). The protein content in embryos devoid of zona pellucida decreased after 3 hours incubation in the medium 199 at 37 degrees at the stages of morula and blastocyst by 17-20% in rats and by 50% in mice. Addition of 1% serum albumin to the incubation medium did not prevent the partial "loss" of protein by the embryos. The protein content in the rat and mouse embryos at the stage of 2 blastomeres suffered no changes under long-term incubation in the medium 199.     

激光辅助制作小鼠嵌合体胚胎的研究

目的：探索激光辅助体外制作小鼠嵌合体胚胎的方法。方法：激光辅助去除不同发育阶段体外受精胚胎的透明带,随机组合卵裂球体外培养,观察胚胎融合情况。结果：没有透明带的卵裂球体外能够“自发”融合,并且融合胚胎在体外培养环境中,能够发育至囊胚期,显微镜观察其形态基本与二倍体胚胎无差别。结论：激光辅助方法获得裸露的卵裂球能够在体外培养环境制作嵌合体胚胎。     

Inhibition of zona shedding of rabbit blastocysts in culture by the presence of a mucin coat.

Rabbit ova, with and without mucin coats, were cultured to the expanding blastocyst stage. The presence of a mucin coat appeared to inhibit shedding of the zona pellucida by the expanding blastocysts.     

Developmental rate and allocation of transgenic cells in rabbit chimeric embryos

The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR< >N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage. All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR< >N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175+/-13.10, of which 58+/-2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24+/-5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage. Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.     

Cell surface carbohydrates of preimplantation embryos as assessed by lectin binding

Preimplantation embryos were obtained from the uteri and oviducts of 2 strains of mice, Swiss CD-1 and B₆ CBA. After removal of the zona pellucida by treatment with pronase, FITC-lectins were bound to the embryonic cell surfaces at either 4°C or 37°C. Both morula and blastocyst stage embryos bound the following lectins, FITC-ConA, FITC-WGA, FITC-RCA_II and FITC-RCA_I. No difference in binding was observed between the morula stage and the blastocyst stage within each mouse strain for each specific lectin. However B₆ CBA embryos bound less FITC-ConA and FITC-WGA than the corresponding Swiss CD-1 embryos. The topographical arrangement of the lectin receptors was observed to differ between 4°C and 37°C for FITC-Con A, FITC-RCA_II, and FITC-RCA_I. While lectins bound at 4°C showed a pattern of continuous labeling, the same lectin at 37°C showed aggregation of lectin receptors into patches indicating lateral mobility of these receptors within the embryonic cell membranes. In contrast FITC-WGA bound at 4°C and 37°C demonstrated continuous labeling of embryos at both temperatures. FITC-fucose binding protein did not bind to Swiss CD-1 embryos. The invasiveness of trophoblastic cells of mouse blastocysts was studied by culturing isolated embryos without prior enzyme treatment on reconstituted collagen gels. After 4 days in BME containing only glutamine and bovine serum albumin as supplements, the embryos shed their zona pellucida and implanted into the collagen gel as indicated by zones of lysis in proximity to the embryonic cells when analyzed by scanning electron microscopy.     

Effects of transferring in vitro-cultured rabbit embryos to recipient oviducts on mucin coat deposition, implantation and development

A mucin coat is deposited on rabbit embryos during passage through the oviduct; rabbit blastocysts cultured from the 1-cell stage in vitro have no mucin coat. When cultured blastocysts are transferred to recipients, the lack of mucin coat might account in part for subsequent failure of pregnancy. We have investigated the possibility that mucin coat deposition is induced following transfer of in vitro 72 h-cultured blastocysts to oviducts of asynchronous or synchronous recipients. One-cell embryos were collected by flushing oviducts 19-20 h post-coitus and were cultured in vitro for 72 h until they reached the blastocyst stage. The blastocysts were transferred to the oviducts of recipients that were synchronized either with the donors (synchronous) or 1 day later than the donors (asynchronous). They were recovered after 24-48 h and the mucin coat thickness and embryo degeneration rate were measured. The degeneration rate of blastocysts recovered from uteri of synchronous recipients was higher than that from asynchronous recipients (72.2% vs 40.0%). The mucin coats around embryos recovered from oviducts of asynchronous recipients after 48 h were thicker than those from synchronous recipients. More asynchronous recipients were pregnant and gave birth to more pups than synchronous recipients. These results indicate that the oviducts of asynchronous recipients secreted more mucin around the transferred embryos, causing higher rates of implantation of the in vitro-cultured blastocysts.     

High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method.

Rabbit morulae were exposed to a vitrification solution-modified PBS [PB1] medium containing 40% ethylene glycol + 18% Ficoll + 0.3 M sucrose (EFS) for 2, 5, or 10 min at 20 degrees C and were vitrified in liquid nitrogen. When morulae were rapidly warmed, 96% had an intact zona pellucida. When embryos were cultured after removal of the mucin coat, high proportions of them formed blastocoel (79-100%), but the percentage of embryos developed to fully expanded blastocysts decreased with increased exposure time 87%, 40%, and 17%). The survival rate of morulae vitrified after removal of the mucin coat was lower than that of mucin-intact embryos. To assess the development potential in vivo, 131 embryos were vitrified after 2 min of exposure to EFS solution; all the embryos were recovered and 120 were transferred to recipients without removal of the mucin coat, resulting in 78 (65%) full-term fetuses or young. This simple method, which yields high survival both in vitro and in vivo, will be of practical use for vitrifying rabbit embryos.     

Production of mouse chimeras by injection of embryonic stem cells into the perivitelline space of one-cell stage embryos

Generation of mouse chimeras is useful for the elucidation of gene function. In the present report, we describe a new technique for the production of chimeras by injection of R1 embryonic stem (ES) cells into the perivitelline space of one-cell stage mouse embryos. One-cell embryos are injected with 2–6 ES cells into the perivitelline space under the zona pellucida without laser-assistance. Our embryo culture experiments reveal that ES cells injected at the one-cell stage embryo start to be incorporated into the blastomeres beginning at the 8-cell stage and form a chimeric blastocyst after 4 days. We have used this approach to successfully produce a high rate of mouse chimeras in two different mouse genetic backgrounds permitting the establishment of germ line transmitters. This method allows for the earlier introduction of ES cells into mouse embryos, and should free up the possibility of using frozen one-cell embryos for this purpose.     

Fucosylated glycoconjugates in mouse preimplantation embryos

Preimplantation mouse embryos were metabolically labelled with 3H or 14C-fucose to investigate the synthesis of fucosylated macromolecules. Scintillation counting revealed that there was a progressive increase in both total fucose taken up by the embryo and incorporation of fucose into TCA-precipitable material as embryos developed from the 4-cell to the blastocyst stage. This was reflected in the increasing intensity of bands on autoradiographs of radioactive fucose labelled proteins separated on 10% SDS-PAGs between the 4-cell embryo (at which stage bands were first detectable) and the blastocyst. Minor qualitative changes in fucoproteins were detected at the time of compaction and additional bands appeared at the blastocyst stage. Preliminary analysis of fucolipids in 6- to 8-cell embryos indicated that an approximately equal amount of fucose was incorporated into lipid and protein. Autoradiographs of semi-thin sections of 3H-fucose-labelled embryos showed substantial amounts of radioactive material in the vicinity of the plasma membrane both adjacent to other cells and facing the zona pellucida. These data would support a predominant role for fucoconjugates in cell surface events in the preimplantation embryo from the 8-cell stage.     

Use of zona drilling for safe and effective biopsy of murine oocytes and embryos

With the mouse as a model, we have used zona drilling to devise procedures for safe removal of the first polar body or one or more blastomeres from cleaving embryos. These methods require minimal disruption of the zona pellucida and little or no direct contact between microtools and the materials to be biopsied. Of 175 eggs subjected to the polar body biopsy procedure, 1 was killed, and 165/174 survivors were fertilized (94.8%). For blastomere biopsy, embryos from the 2- to 16-cell stage were incubated in a chelating medium containing 100 mM sucrose for at least 30 min. The zonae were then drilled, and one or more blastomeres were "pushed" out through the hole by pressure exerted against the zona at some distance from the drilling site. In all 85 embryos biopsied, one or more additional intact blastomeres were successfully removed. Moreover, 83/84 biposied embryos that were subsequently cultured developed into blastocysts (98.8%). Although acid Tyrode's solution was used in this study, mechanical methods of zona opening were also effective. The data indicate that oocyte and embryo biopsy assisted by zona drilling is safe and does not appear to affect fertilization or development, and as such, it is applicable to genetic diagnostic procedures.     

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.     

Electrofusion of mouse blastomeres

Fusion of blastomeres of 2-cell mouse embryos with an intact zona pellucida can be induced with electric pulses. Fusion was most frequent with the field strength of 1 kV/cm and direct current pulses of 100-250 microsec duration. An electrolyte solution (PBS) can be used instead of a non-electrolyte solution (0.3 M mannitol). The viability of blastomeres fused in these two types of solution is similar. Fused 2-cell blastomeres develop into tetraploid blastocysts but die after implantation. Embryos in which blastomeres failed to fuse despite the treatment (diploid controls) can develop till term. The technique can also be applied to 3- and 4-cell embryos and to zona-free oocytes and blastomeres.