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a Chiba Prefectural Mineoka Dairy Experimental Station, Chiba 299-2507, Japan
b Nasu Institute, Nisshin Flour Milling Co. Ltd., Nasu, Tochigi 329-2763, Japan
c Department of Obstetrics and Gynaecology, The University of Adelaide, Adelaide, South Australia 5005, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nagashima et al. [5] recently reported that the extreme sensitivity of pre-morula-stage porcine embryos to low temperature relates to a high lipid content in their cytoplasm, and that removal of lipid droplets allows them to tolerate cryopreservation. Similarly, pre-morula-stage bovine embryos are also known to have large quantities of cytoplasmic lipid droplets and to show high sensitivity to freezing [3], while it is also known that the lipid droplet content declines after the morula stage, coinciding with the embryos' loss in sensitivity to cryopreservation [6]. Since these characteristics resemble those of pigs, we surmised that the sensitivity of pre-morula-stage bovine embryos to freezing could be altered by the removal of cytoplasmic lipids, with this interesting possibility leading to the present study designed to investigate whether tolerance to cryopreservation of pre-morula-stage embryos produced by IVM/IVF can be improved by removal of cytoplasmic lipid droplets.
Another purpose of the present study is to determine whether or not cryopreserved delipated IVM/IVF bovine embryos can be used as karyoplasts for nuclear transfer. Westhusin et al. [7] and Bondioli et al. [8] carried out nuclear transfer of bovine embryos using cryopreserved, in vivo-derived morulae as nucleus donors, yet the corresponding use of in vitro-derived pre-morula-stage embryos has never been reported. The ability to use cryopreserved IVM/IVF embryos as a source of the donor nucleus holds much promise, because in vitro production of embryos obtained from valuable breeding stock before their slaughter has become a practical technology.
In the study reported here, we studied in vitro development of delipated IVM/IVF bovine embryos at the pre-morula stage after cryopreservation, and then used blastomeres of these embryos as the donor nucleus for nuclear transfer. Parameters determining the efficacy of nuclear transfer, i.e., isolation and fusion rates of karyoplasts, and the developmental ability of reconstructed embryos were also investigated to evaluate the feasibility of using cryopreserved, delipated IVM/IVF bovine embryos for nuclear transfer.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used the following chemicals: BSA (fraction V), heparin, caffeine, cytochalasin B (CB), hyaluronidase, and cycloheximide (Sigma Chemical Co., St. Louis, MO); TCM-199 with Earle's salts, fetal calf serum (FCS), Giemsa stain, and colcemid (Gibco BRL, Grand Island, NY); streptomycin and penicillin-G (Meiji Chemical Co., Tokyo, Japan).
In Vitro Production of Bovine Embryos
Ovaries of Holstein cattle obtained from a slaughterhouse were placed in vacuum bottles and transported to our laboratory within 3 h in 0.9% saline while being kept at 30–35°C. To collect cumulus-oocyte complexes (COCs), follicles from 3 to 5 mm in diameter were aspirated through an 18-gauge needle into a disposable 10-ml syringe. Chosen for the experiment were oocytes with a compact layer of unexpanded cumulus oophorus and evenly granulated cytoplasm [9]. Collected COCs were washed four times with 25 mM Hepes-buffered TCM-199 supplemented with 5% (v:v) FCS, 50 µg/ml streptomycin, and 100 U/ml penicillin-G. In a single drop of the same medium, 50 COCs were cultured at 38.5°C in an atmosphere of 3% CO2 in air for 22 h [10]. Matured oocytes were subsequently inseminated with 5 x 106 sperm during a 5-h incubation period in a 500-µl droplet of BO medium [11] supplemented with 10 mg/ml BSA, 10 µg/ml heparin, and 5 mM caffeine [9]. After insemination, the oocytes were cultured at 38.5°C in a 500-µl droplet of the maturation medium with cumulus cells (in vitro culture [IVC] medium) in an atmosphere of 3% CO2 in air [9]. One-cell-stage embryos grown 24 h after insemination were used for the experiments. In our earlier work, the maturation rate, fertilization rate, and developmental rate of oocytes to blastocysts using this IVM/IVF/IVC system were respectively 90%, 90%, and 20%.
Removal of Cytoplasmic Lipid Droplets from IVM/IVF Embryos and Development of Delipated Embryos
We applied a previously described method used for porcine embryos to polarize cytoplasmic lipid droplets by centrifugation [5], after modification for IVM/IVF bovine embryos. Briefly, one-cell-stage embryos were preincubated in modified PBS (PB1) [12] + 20% FCS containing 7.5 µg/ml CB for 10 min and then were centrifuged (12 000 x g) in the same medium for 15 min.
Micromanipulation to remove polarized cytoplasmic lipid droplets from embryos, referred to as delipated embryos, was performed using the method of Nagashima et al. [5]. That is, the polarized lipid layer was aspirated by a beveled suction pipette (diam. 35 µm) attached to a Narishige micromanipulator (MO-202; Tokyo, Japan) while being viewed under a Nikon inverted microscope at x200 magnification (TMD model; Nikon Inc., Tokyo, Japan; Fig. 1). Also prepared were intact control embryos and sham-operated, undelipated embryos (sham-operated embryos) treated with CB and centrifuged (12 000 x g) to polarize cytoplasmic lipid droplets. The development to the blastocyst stage of all embryos was examined [9, 10] after culturing in IVC medium for 7 days as described above.
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Blastocysts were fixed by Tarkowski's air-dry method [13] with slight modification and stained with 5% Giemsa stain to allow counting of cells. Briefly, embryos were treated with 0.9% sodium citrate solution for 20 min at room temperature (r.t.); fixed onto a spot plate (Corning Labware and Equipment, Corning, NY) with ice-cooled fixative consisting of methanol, acetic acid, and distilled water (3:2:1) for 1 min; mounted on a glass slide using a small amount of the fixative; air-dried for 1 h at r.t.; and, finally, stained with Giemsa stain for 10 min. Total nuclei numbers of the blastocysts were counted under a microscope at x400 magnification.
Cryopreservation of Embryos
Embryos were equilibrated at r.t. with 1.5 M 1,2-propanediol (PROH) for 15 min and loaded into a 0.25-ml plastic straw with 1.5 M PROH + 0.1 M sucrose [14, 15]. Straws were cooled directly from r.t. to -6.5°C, seeded, cooled at a rate of 0.3°C/min to -30°C using a programmable freezer (ET-1; FHK, Tokyo, Japan), and finally plunged into liquid nitrogen. Straws were thawed in air for 10 sec and then in 35°C water for 10 sec, after which the embryos were expelled from the straw. Cryoprotectant was diluted at r.t. by step-wise exposure to 1 M PROH + 0.2 M sucrose for 10 min, 0.5 M PROH + 0.2 M sucrose for 5 min, 0.2 M sucrose for 5 min, and PB1 + 20% FCS for 10 min. We used PB1 + 20% FCS to dilute media for equilibration, freezing, and thawing.
Post-thaw survival of embryos was assessed by their in vitro development after culture for 6 days in IVC medium. Cell numbers of the developing blastocysts were counted as described above.
Nucleus Transfer of Frozen-Thawed Delipated Embryos
Recipient cytoplasts for nucleus transfer were obtained by IVM of oocytes using the same methods described above. After 20 h of maturation culture, cumulus cells of oocytes were removed by repeated pipetting through a fine-bore pipette in BSA-free PB1 containing 1 mg/ml hyaluronidase. Oocytes with the first polar body (metaphase II oocytes) were selected as recipient oocytes.
Enucleation of oocytes was carried out as previously described [16]. Briefly, they were enucleated by aspirating the first polar body and adjacent ooplasm (about 1/3 of an ooplasm) using a micropipette (diam. 35 µm) in PB1 + 20% FCS containing 5 µg/ml CB and 0.1 µg/ml colcemid. To confirm removal of the M II chromosomes, fragments of removed ooplasm were stained with 5 µg/ml acridine orange for 5 min and observed using a fluorescent microscope (Nikon TMD). After 22 h of maturation culture, enucleated oocytes were activated by exposure to 7% ethanol in maturation medium for 7 min [16] and further cultured in maturation medium containing 5 µg/ml cycloheximide for 6 h [17].
Donor blastomeres at the 8- to 16-cell stage were isolated from cryopreserved delipated embryos cultured for 24 h after thawing. Blastomeres at the same stage were similarly isolated from unfrozen undelipated control embryos. For isolation, zona pellucidae of the donor embryos were removed mechanically using a microneedle and holding pipette [16], with blastomeres then being disaggregated by repeated gentle pipetting through a fine-bore pipette (130 µm diameter) in PB1 + 20% FCS. Each blastomere was inserted into the perivitelline space of an enucleated recipient oocyte through a slit on the zona opened as a result of micromanipulation for enucleation.
Oocyte-blastomere complexes were placed between two platinum wire electrodes mounted 1 mm apart on a glass microscope slide (BTX Inc., San Diego, CA) and overlaid with fusion medium consisting of 0.3 M sucrose, 0.1 mM MgSO4, and 0.05 mM CaCl2. After an alternating current field (1 MHz, 11 V/mm) was applied for 20 sec, membrane fusion between a blastomere and recipient oocyte was induced by applying two direct-current pulses (75 V/mm for 50 µsec). Subsequently, oocyte-blastomere complexes were kept in PB1 + 20% FCS at 38.5°C for 1 h, after which they were examined under a stereoscopic microscope (SMZ model; Nikon) at x80 magnification to check for the occurrence of fusion, with those showing no sign of fusion [16] being discarded (11 of 285, 3.9%). In our previous study, we confirmed swelling of the donor nucleus after fusion [16]. The development of nucleus transfer embryos was examined over an 8-day culture period in IVC medium.
Statistics
Developmental and survival rates of the embryos, as well as the fusion rate of blastomeres, were analyzed by the chi-square test with Yates' correction for independence and by Fisher's exact probability test to determine the statistical difference between groups. Student's t-test was used to compare mean cell number of blastocysts and numbers of isolated donor blastomeres. A probability of less than 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Table 1 presents results summarizing in vitro development of the intact control, sham-operated, and delipated embryo groups. Regarding developmental timing, after insemination, intact control embryos developed to the 2- and 8- to 16-cell stages after 30 and 48 h, respectively. The development of these embryos tended to arrest at the 8- to 16-cell stage for 24–48 h, subsequently progressing to the morula and blastocyst stages after culturing in IVC medium for 7 days. Delipated and sham-operated embryos developed almost synchronously with the intact control embryos (Fig. 2). It should be noted that the developmental rate of delipated embryos to the blastocyst stage was not significantly different from that of intact control or sham-operated embryo groups, and the blastocysts derived from delipated embryos had cell counts comparable to those derived from intact control embryos, i.e., 90 ± 3.4 vs. 105 ± 5.7.
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Survival of Delipated IVM/IVF Embryosafter Cryopreservation
Table 2 presents the in vitro survival results of intact control, sham-operated, and delipated embryo groups after cryopreservation, showing that significantly more delipated embryos were recovered with no visible sign of cell damage in comparison with embryos in the sham-operated or intact control embryo groups (40% vs. 14% and 14%; p < 0.01). In addition, a higher percentage of delipated embryos developed to blastocysts than those of the other two groups (23% vs. 5% and 9%; p < 0.05).
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Nucleus Transfer of Frozen-Thawed Delipated Embryos
As shown in Table 3, although the efficiency of isolating blastomeres from frozen-thawed delipated embryos and the fusion rate of these blastomeres to recipient oocytes were significantly lower (p < 0.01) than those for the unfrozen, undelipated, control embryos, the developmental rate of the nucleus transfer embryos reconstituted with frozen, delipated blastomeres (16%) was comparable to that of the nucleus transfer embryos with nuclei from control embryos (25%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tolerance to freezing of bovine embryos produced by IVM/IVF/IVC is known to be lower than that of embryos collected in vivo [4, 19, 20]. Leibo and Loskutoff [19] found that IVM/IVF bovine embryos had darker cytoplasm and lower specific gravity than in vivo-derived embryos, suggesting that the cytoplasm of IVM/IVF embryos contained a higher content of lipid droplets, or the composition of component lipids of IVM/IVF embryos differed from that of their in vivo-derived counterparts. We surmised that changes in the quantity and quality of component lipids account for the low tolerance to cryopreservation of IVM/IVF bovine embryos.
Damage to membrane lipids can be caused by free oxygen radical formation due to suboptimal conditions in a long-term culture. This is known to change membrane fluidity and permeability such that the resistance of embryos to osmotic stress decreases [21]. Since embryos are exposed to osmotic stress during the course of freezing and thawing [22], this decrease in resistance could presumably have a major effect on the tolerance of embryos to freezing.
Since the damage to lipids due to in vitro culture may occur in cytoplasmic lipid droplets as well as membrane lipids, the removal of these droplets from IVM/IVF embryos could in turn remove the target of free oxygen radicals in the cytoplasm. Accordingly, such removal of targets could very well decrease the ensuing chain reaction of peroxidative damage to embryos, a phenomenon that ultimately improves the embryo's tolerance to freezing. In contrast, survival rate and the increase of cell numbers in the sham-operated embryos after freeze-thawing was lower, indicating that many of the blastomeres were damaged. A part of the polarized lipid droplets left unremoved in the perivitelline space of the sham-operated embryos was observed to redistribute into the blastomeres, particularly those located in the hemisphere contiguous to the polarized lipids. This implies that the embryos were incompletely delipated and may not have been rendered fully cryotolerant.
Several researchers have reported that the developmental ability of IVM/IVF bovine embryos is affected by culture conditions, i.e., low oxygen tension [23] and the addition of thiol compounds [24] or antioxidants [25–27]. These modifications, which may prevent an autocatalytic peroxidative chain reaction among cellular lipids [21], have also been shown to contribute to increasing the cryotolerance of in vitro-produced bovine embryos [28, 29]. Therefore, the use of delipated embryos for cryopreservation may provide valuable insights into the relation between lipid peroxidation and cryoinjury of embryonic cells. Such knowledge is expected to provide clues for developing a noninvasive method to produce IVM/IVF bovine embryos with improved freezability.
Results also indicate that delipated IVM/IVF bovine embryos develop into blastocysts in vitro at a developmental rate and culture period that is not significantly different from those of the controls. Total cell number of Day 7 blastocysts from delipated embryos was similar to cell numbers of blastocysts produced IVM/IVF/IVC in previous reports [30, 31]. Diez et al. [18] have obtained pregnancies by transferring blastocysts developed from delipated bovine embryos, while Nagashima et al. [32] showed that delipated porcine embryos developed into blastocysts in vitro and into normal offspring after transfer into recipients. While it is known that cytoplasmic lipid droplets in embryos are a source of energy and contain metabolic products and structural elements (for review see [33]), results of these two experiments indicate that cytoplasmic lipid droplets are not essential for the development of either bovine or porcine embryos in the early cleavage stage, which raises new questions about the significance of the presence of cytoplasmic lipid droplets in many mammalian embryos.
It has recently been demonstrated that nuclear transfer using cultured somatic cells can lead to normal offspring in sheep [34] and cattle [35], achievements implying that a potentially unlimited source of donor nuclei can be cryopreserved for cloning animals [36]. The efficiency of nuclear transfer with somatic cell nuclei, however, is still very limited [34, 37], and therefore the use of cryopreserved embryos as a source of donor nuclei is practically important.
Our results indicate that blastomeres of delipated, cryopreserved IVM/IVF bovine embryos can be successfully used as karyoplasts for nucleus transfer. To our knowledge, this is the first report of the use of cryopreserved 8- to 16-cell-stage bovine IVM/IVF embryos as nucleus donors for nuclear transfer. In contrast, when nucleus transfer was performed using intact control or sham-operated embryos after freeze-thawing, many blastomeres ruptured during micromanipulation or electric fusion, and therefore they were judged to be unsuitable nucleus donors for nucleus transfer (data not shown).
In the present study, the efficiency of isolating blastomeres from cryopreserved delipated embryos and the fusion rate of isolated blastomeres were found to be inferior to those of the unfrozen, undelipated blastomeres used as controls. Ushijima and Eto [38] have shown that the rate of rupture of undelipated blastomeres derived from in vitro-produced embryos by isolation and electric fusion is higher than that for those derived in vivo. Nagashima et al. [39] demonstrated that isolation of blastomeres from cryopreserved, delipated, 8- to 16-cell-stage porcine embryos derived in vivo and electric fusion of such isolated blastomeres can be performed with an efficiency equivalent to that of using unfrozen, undelipated embryos as controls. Taken together, these observations suggest that the susceptibility to rupture of blastomeres is greatly influenced by the use of IVM/IVF to derive the embryos.
In summary, our results demonstrate that the removal of cytoplasmic lipid droplets from bovine pre-morula-stage IVM/IVF embryos, which are in general highly sensitive to low temperatures, improved their tolerance to cryopreservation. They also show that the blastomeres isolated from cryopreserved, delipated IVM/IVF embryos can be used as nucleus donors for nucleus transfer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: Biomedical Research Center, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan.
Accepted: October 1, 1998.
Received: March 18, 1997.
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REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
