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a Laboratory of Animal Genetics and Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
b The Institute of Cetacean Research, Tokyo 104-0055, Japan
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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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The presence of CC surrounding oocytes is an important factor affecting in vitro maturation (IVM) of immature oocytes. It has been reported that oocytes with CC show a significantly higher maturation rate than denuded oocytes [2,16]. Moreover, the developmental capacity of oocytes after IVM and in vitro fertilization (IVF) was affected by the sexual maturity of donor animals in both cattle [17] and sheep [18].
When Fukui et al. [19] cultured minke whale follicular oocytes for 3.5–5 days, they found that the maturation rate peaked at approximately 30%. However, to date, no information has been available regarding cryopreservation of immature oocytes harvested from cetacean species such as minke whales or dolphins. If the cryopreservation of minke whale oocytes is established, IVM and IVF (including intracytoplasmic sperm injection) could be performed to produce minke whale embryos in a laboratory rather than on board the vessel [20]. Therefore, the goal of this study was to establish a technique to produce minke whale embryos in vitro as for other domestic animals including cattle.
In this study, the effect of the presence or absence of CC at the time of freezing and that of the sexual maturity of minke whales on postthaw viability of follicular oocytes were examined. In addition, the cryopreserved immature oocytes were processed to examine the ultrastructure of frozen-thawed oocytes by transmission electron microscopy (TEM).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was undertaken as a part of the Japanese Whale Research Programme with Special Permit in the Antarctic and was organized by the Institute of Cetacean Research in Tokyo, Japan, between 7 December 1997 and 13 March 1998, in the area of lat 60–68°S and long 70–130°E.
A total of 13 female southern minke whales (immature: n = 6, 7.7 ± 0.4 meters and 5.5 ± 0.8 tons; mature: n = 7, 9.0 ± 0.1 meters and 8.5 ± 0.3 tons for body length and body weight, respectively) were shot with grenaded harpoons, and ovaries were collected within 3 h after death according to the method reported by Fujise et al. [21].
Sexual maturity of each whale was determined by the presence or absence of a corpus luteum and/or corpora albicans on either ovary. Animals with neither corpus luteum or corpora albicans were considered immature. All mature whales were found to be pregnant.
Oocyte Collection
Aspiration of follicular oocytes was carried out by using an 18-gauge needle connected with a 5-ml syringe containing modified PBS (Embryo Tech; Nihon-Zenyaku Industrial Co., Kohriyama, Japan) supplemented with 0.38% (v:v) sodium citrate solution (Chitoral; Yamanouchi Pharmaceutical Co., Tokyo, Japan). Oocytes recovered from the follicles (2–10 mm in diameter) were assigned into 4 groups according to the maturity of the animals (immature and mature whales) and the presence or absence of the CC before freezing. Because of insufficiency of facilities for IVM culture on the vessel, all oocytes were frozen in order to investigate maturation ability.
Freezing and Thawing
Dulbecco's PBS (D-PBS; Nissui Pharmaceutical Co., Tokyo, Japan) containing 1.5 M ethylene glycol (EG; Wako Co., Osaka, Japan), 0.1 M sucrose, and 10% heat-treated fetal calf serum (FCS) was used as a cryoprotective medium.
Aspirated oocytes were transferred to the cryoprotective medium and equilibrated for 10 min at room temperature (20°C). After equilibration, a group of 10 oocytes were loaded into a 0.25-ml straw (Fujihira Industry Co., Tokyo, Japan), powder sealed, and transferred into a precooled methanol bath (0°C) within a programmable freezer (ET-1; Fujihira). The straws were cooled from 0°C to -6.5°C at 0.5°C/min and were seeded at -6.5°C for 10 min. After seeding, the straws were further cooled to -35°C at the rate of 0.3°C/min before being plunged into liquid nitrogen (LN2). Oocytes were kept in LN2 for 3–5 mo prior to thawing and subsequent experimentation. The straws were warmed rapidly (10 sec at 25°C in air, followed by 20 sec at 39°C in a water bath), and cryoprotectants were removed by transferring the oocytes directly into D-PBS supplemented with 10% FCS.
IVM
The medium used for IVM was Tissue Culture Medium 199 (TCM199; Dainippon Pharmaceutical Co., Osaka, Japan) supplemented with 10% (v:v) FCS, 10% (v:v) bovine follicular fluid, 25 mM NaHCO3, 0.3 mM sodium pyruvate (Wako), 1 mM glutamine (Wako), 2 mM taurine (Sigma Chemical Co., St. Louis, MO), 10 µg/ml insulin (Sigma), 1 µg/ml estradiol-17ß (Sigma), 0.02 AU/ml porcine pituitary FSH (Antrin; Denka Pharmaceutical Co., Kanagawa, Japan), and 5 x 105 bovine granulosa cells per milliliter. Bovine granulosa cells were collected from follicles (8–12 mm in diameter) obtained from Holstein cows at a local abattoir, according to the method of Moor and Trounson [22], and washed (500 x g for 10 min) twice with TCM199 containing 10 mM Hepes, 2 mM NaHCO3, and 0.3% (w:v) BSA (fatty acid free, fraction V; Sigma). Bovine follicular fluid was aspirated from follicles (5–8 mm in diameter); the supernatant was collected after centrifugation at 1000 x g for 20 min and then added to IVM medium.
Oocytes were divided into 4 groups (immature and mature whales, and cumulus-enclosed and cumulus-free oocytes: 2 x 2) and cultured in drops of IVM medium (100 µl) covered with mineral oil (Squibb & Sons, Princeton, NJ) for 132 h at 39°C in 5% CO2 in air and 
95% humidity. The IVM duration (132 h) was determined according to our previous studies [19,20]. Every 48 h in culture, 50 µl of the IVM medium was replaced with fresh medium.
Preparation of Oocytes for TEM
Postthawed oocytes (20 oocytes per each group) were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at 4°C, followed by 1% osmium tetraoxide in 0.1 M phosphate buffer for 2 h at 4°C according to the procedures described previously for bovine oocytes [23,24]. Briefly, after routine dehydration with a graded series of ethanol, the oocytes were infiltrated successively with 3:1, 1:1, and 1:3 mixtures of absolute propylene-oxide and Spurr's epoxy resin, then with absolute Spurr's epoxy resin. Oocytes were embedded in the beam capsules with fresh Spurr's epoxy resin. Ultrathin sections were cut out with a diamond knife and double-stained with uranyl acetate and lead citrate; they were then examined under an H-7500 transmission electron microscope (Hitachi, Tokyo, Japan).
Morphological Evaluation and Nuclear Maturation of Oocytes
All frozen-thawed oocytes were examined morphologically under a light microscope. Oocytes without damage, such those that had a homogeneous ooplasm, were considered normal (Fig. 1).
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The in vitro-matured oocytes were fixed in acetic acid:ethanol (1:3) in D-PBS (50%) followed by nondiluted acetic acid:ethanol for at least 48 h. They were subsequently stained with aceto-orcein (2% orcein in 45% acetic acid solution) and assessed for the nuclear stage of meiosis under a phase-contrast microscope. The nuclear stages were classified as previously described [19]: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I/anaphase I/telophase I (MI/AI/TI), metaphase II (M-II), or degenerated. Oocytes were considered to have matured when they reached the M-II stage.
Statistical Analysis
Six replicates were conducted for each experiment. The proportions of normal oocytes after cryopreservation and the nuclear maturation of oocytes after IVM were compared by ANOVA using the General Linear Models (GLM) procedure of the Statistical Analysis System (Cary, NC), combined with Duncan's Multiple Range test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Light Microscope Morphology of Thawed Oocytes
The morphology of minke whale oocytes as assessed by light microscopy is shown in Figure 1. The proportions of normal oocytes after thawing in the 4 groups are shown in Table 1. The total proportion of normal oocytes was 39.7% (83 of 209). There was no significant difference in the proportions of normal oocytes between immature and mature whales (42.0% and 35.9%, respectively) or between oocytes with and without CC (39.6% and 39.8%, respectively).
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Nuclear Maturation of Cultured Oocytes After Thawing
The nuclear status of IVM oocytes recovered from immature and mature whales, and of those with or without CC, is shown in Table 2, with a representative sample of these oocytes, after IVM, shown in Figure 2. Eighty-two oocytes dissolved during fixation and staining procedures, and the nuclear status of some oocytes could not be determined. No GV oocytes without CC were observed in mature whales. The proportion of oocytes at the GV stage tended to be lower in mature whales than in immature whales (8.6% vs. 19.8%). The rate of MI/AI/TI stages was significantly (P < 0.05) higher in oocytes with CC (29.1%) than in those without CC (13.5%). This difference was largely due to a significant difference that was seen in immature but not in mature whales. In contrast, few oocytes were found in the M-II stage (0–7.5%); overall, M-II oocytes constituted 3.8% of the cultured oocytes. Neither the maturity of the whales nor the presence or absence of CC appeared to influence this proportion (Table 2).
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Ultrastructure of Cryopreserved Minke Whale Oocytes
Electron microscopy revealed widespread damage in the cryopreserved oocytes, such as rupture of the ooplasm membrane, vacuolation of microvilli (Mv), migration of cortical granules (CG), and the presence of vacuolated mitochondria (Figs. 3–7). Organelles tended to be dispersed in the immature whale oocytes, but those in mature whales clustered to the periphery (Fig. 3b). Mv were flattened on the plasma membrane in immature whales (Fig. 3c), but Mv of mature whales were extended and attached to the zona pellucida and entangled with CC projections (Fig. 4c). The cytoplasm of oocytes from immature whales appeared more vacuolated than that of mature whales (Figs. 3 and 4). Mitochondria in immature whales were spherical, while those of mature whales were elongated (Fig. 5). In addition, the mitochondrial matrix varied between oocytes, with some vacuolated (Fig. 5a) or electron-dense (Fig. 5b) mitochondria. The CG were spherical in structure and comprised a highly electron-dense material (Fig. 6). They were peripherally located beneath the ooplasm membrane. The nuclei, endoplasmic reticulum, and a few vesicles were observed in CC attached to the zona pellucida (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In general, cells are sensitive to cryopreservation. During freezing and thawing, mammalian cells are at risk of damage by various factors, including toxicity of cryoprotectants, chilling injury, osmotic swelling, and shrinkage [9,25]. The present study demonstrated three major points: 1) more than one third of immature minke whale oocytes retained a "normal" appearance after freezing and thawing; 2) after IVM, about 20% of frozen-thawed immature oocytes resumed meiosis beyond MI stage; 3) electron microscopy of frozen-thawed minke whale oocytes revealed various aspects of damage to cytoplasmic organelles, normally associated with freezing and thawing.
The survival rates of cryopreserved immature oocytes have been reported in several mammalian species, such as mice [2], rats [3], cattle [26], and horses [6], with rates of survival ranging from 50% to 70%. The survival rate (40%) of postthawed minke whale oocytes was lower than reported in other studies [2,3,6,26]. The low survival rate indicates that the present procedures of freezing and thawing may not be appropriate for minke whale oocytes. There was no significant difference in the survival of the oocytes with and without CC. This result is similar to that reported by Whittingham [1] in the mouse. The presence of CC has been considered to be among the important factors influencing IVM [27] and IVF [28,29] results. Reasons for the negative effect of the presence of CC surrounding minke whale oocytes are unknown. In the present study, IVM was performed with a coculture system using bovine granulosa cells in the medium containing FCS, bovine follicular fluid, and additives. Therefore, the coculture IVM system may be not appropriate for investigating the differential maturation or survival ability of CC surrounding minke whale oocytes.
The maturation rate (0–7.5%) of the oocytes after thawing was extremely low compared with those in domestic animals and with the rate obtained in a previous study (27%) [19] using fresh minke whale oocytes. However, about 30% of the frozen-thawed oocytes surrounded with CC resumed maturation to MI/AI/TI stages with the present IVM culture. Therefore, it is possible that the IVM rate could be improved if the culture period was extended to more than 5.5 days. However, a consideration is that a portion of the oocytes may have been already meiotically activated before IVM culture, as shown by our previous study using fresh oocytes [19]. According to studies on cryopreservation of bovine and equine oocytes, resumption of nuclear maturation is disturbed by the damage in oocytes following freezing and thawing procedures and the toxicity of cryoprotectants [7]. However, it been has demonstrated that the physical damage is less in maturing and matured oocytes than in immature oocytes [6,30].
CC expansion during IVM has been considered an indicator of the maturity of oocytes in many species. CC expansion of minke whale oocytes cultured in this study was also observed within 24 h in culture. Chian et al. [31] and Shamsuddin et al. [32] reported that the presence of CC for at least 12 h from the beginning of IVM culture was necessary for maturation of bovine oocytes in vitro. The lower maturation rate of denuded oocytes could be associated with loss of direct communication with CC [2,5]. CC are metabolically coupled to the oocyte and are thought to be important for the competence of oocyte maturation in cattle [16,27].
O'Brien et al. [18,33] demonstrated the effect of sexual maturity of the donor animals on IVM rates of ovine oocytes. They reported that oocyte maturation was not different between oocytes obtained from prepubertal and adult ewes [33]. Sexual maturity of the minke whale was also not related to the viability of oocytes after thawing. It is likely that freeze-thaw damage to the meiotic spindle occurs regardless of the sexual maturity of donors. Such damage to the spindle induced by the low temperature and cryoprotectants is well described for immature bovine oocytes [9,34]. Yang et al. [26] also suggest that the developmental capacity of cryopreserved immature bovine oocytes is impaired because of severe ultrastructure damage. Damage to cytoplasmic organelles in oocytes of other mammals has also been observed using TEM [10,13,14,35]. Observations made with cryopreserved bovine [13,35] and mouse [14] oocytes showed that the ooplasm was often occupied by confluence vesicles and that the ooplasm was then ruptured. The confluence vesicles and ruptured plasma membrane of bovine oocytes [36] were recognized during oocyte shrinkage at the time of freezing and increased the vesicles' confluence resulting from shrinking and changes in cellular volume. The confluence vesicle and the destruction of plasma membrane in immature whale oocytes were observed in our study (Fig. 3). In cryopreserved horse oocytes [15], the freezing method itself induced some ultrastructural changes, such as vacuolation of mitochondria and destruction of peripheral ooplasm. Mitochondrial matrix in the ooplasm of the immature minke whale oocytes was also vacuolated, whereas mitochondrial matrix in the ooplasm of mature whale oocytes was quite dense (Fig. 5). On the basis of these observations it appears likely that vacuolated mitochondrial matrix was affected by the present cryopreservation method, while the granule structures in Figure 6 appear to be CG, judging from observations in murine [14], bovine [13], and human [14] oocytes. Migration of CG from clusters to solitary positions, and the formation of a line along the ooplasm membrane, were observed in matured bovine oocytes [13]; and CG release occurs in mature oocytes to prevent polyspermic fertilization [37,38]. In the present study, the appearance of CG might have been induced by the low temperature and cryoprotectant during freezing and thawing as reported for cryopreserved bovine oocytes [10,30]. The filament structures shown in Figure 6 could not be identified. The CC surrounding the oocytes (Fig. 7) was morphologically similar to bovine CC as reported by Zhao et al. [23]. As a whole, ultrastructural damage to the cryopreserved minke whale oocytes was similar to that seen in other mammalian species, and it appears that these observations were caused by the present freezing and thawing procedures. Further studies are needed to improve freezing and thawing methods for higher rates of survival and IVM of these oocytes.
In conclusion, the present study showed that cryopreserved minke whale follicular oocytes could resume meiosis to M-II stage, but their postthaw survival rate was lower than observed for other mammalian oocytes. The presence of CC and sexual maturity of minke whales did not affect the IVM rate, and 20–30% of the oocytes cultured resumed meiosis with 3.8% of oocytes reaching the M-II stage. Cytoplasmic organelles of the cryopreserved oocytes were impaired during the freezing and thawing procedures.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence. FAX: 81 155 49 5462; fukui{at}obihiro.ac.jp 
Accepted: September 9, 1999.
Received: April 26, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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