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Effect of chilling on the organization of tubulin and chromosomes in rhesus monkey oocytes
FERTILITY AND STERILITY威 VOL. 77, NO. 4, APRIL 2002 Copyright ©2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Effect of chilling on the organization of tubulin and chromosomes in rhesus monkey oocytes Nucharin Songsasen, D.V.M., Ph.D.,a Il-Jeoung Yu, D.V.M., Ph.D.,a Marion S. Ratterree, D.V.M.,b Catherine A. VandeVoort, Ph.D.,c and Stanley P. Leibo, Ph.D.a University of New Orleans and Audubon Center for Research of Endangered Species, New Orleans, Louisiana
Received May 22, 2001; revised and accepted October 4, 2001. Supported by National Institutes of Health grant no. 5R01-RR13439 (to C.A.V.), and by grant no. NIH-5P51 RR00164-39 to Tulane Regional Primate Research Center. Reprint requests: Stanley P. Leibo, Ph.D., Audubon Center for Research of Endangered Species, 14001 River Rd., New Orleans, Louisiana 70131 (FAX: 504-391-7707; E-mail: [email protected]). a Department of Biological Sciences. b Tulane Regional Primate Research Center, Covington, Louisiana. c California Regional Primate Research Center, Davis, California. 0015-0282/02/$22.00 PII S0015-0282(01)03240-X
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Objective: To determine the effects of chilling on the organization and distribution of tubulin and chromosomes in rhesus monkey oocytes. Design: Comparative laboratory study. Setting: Academic research laboratory. Animal(s): Eight adult female rhesus monkeys (Macaca mulatta) aged 6 –16 years. Intervention(s): A total of 171 oocytes retrieved from eight rhesus monkeys were separated into nine groups. One group of control oocytes was held at 37°C during the experiment. Four groups of oocytes were rapidly cooled to 0°C and held for 1, 5, 10, or 30 minutes and then fixed and stained. Four other groups of oocytes were cooled to 0°C, held for 1, 5, 10, or 30 minutes, warmed and incubated at 37°C for 60 minutes, and then fixed and stained. Main Outcome Measure(s): Organization of cytoskeleton and chromosomes. Result(s): Exposure of rhesus oocytes to 0°C for only 1 minute resulted in complete depolymerization of tubulin. Incubation of chilled oocytes at 37°C for 60 minutes caused partial restoration of tubulin, although most oocytes exhibited abnormal alignment of chromosomes and disorganized meiotic spindles. Conclusion(s): We conclude that rhesus monkey oocytes are extremely sensitive to chilling injury. Their successful cryopreservation may require rapid cooling to outpace this injury. (Fertil Steril威 2002;77:818 –25. ©2002 by American Society for Reproductive Medicine.) Key Words: Chilling injury, rhesus monkey, oocytes, cytoskeleton
The ability to effectively cryopreserve oocytes would yield many benefits for the assisted reproduction of many species. In the rhesus monkey, it would permit the collection of oocytes during the breeding season, yet allow their year-round use. This would remove the current barrier of reproductive seasonality in studies of rhesus monkey oocytes. Oocytes could be stockpiled for experiments requiring the simultaneous use of large numbers of oocytes. Furthermore, oocyte cryopreservation could be used to preserve genetic material from females of specific genotypes to increase their numbers in studies of genetically inherited diseases. Despite these potential benefits of oocyte cryopreservation, it is generally accepted that procedures developed for mammalian embryos are much less effective when used for oocytes (1,
2). Although live young have been produced from cryopreserved oocytes, success rates have been low and limited to only a few species, such as the mouse, rabbit, cattle, and human (3– 6). Schroeder et al. (3) cryopreserved 487 in vitro– matured mouse oocytes, of which 158 developed into embryos; these yielded 41 live young (26%) after ET. Vajta et al. (5) produced three bull calves after transferring 17 blastocyst-stage embryos produced from cryopreserved bovine oocytes. In humans, a small number of babies have been produced by conventional IVF of cryopreserved oocytes (1, 6). More recently, ICSI of cryopreserved oocytes has been widely used to produce human embryos, although the overall success rate in terms of the number of babies born as a percentage of cryopreserved
oocytes is still only about 1% (6). Very recently, vitrification of human oocytes has been attempted with encouraging results. Chung et al. (7) vitrified 89 oocytes, of which 33 developed to the 2 pronuclear stage after ICSI and 12 developed into euploid blastocysts. Kuleshova et al. (8) vitrified 17 oocytes and produced a single baby after transferring three embryos created by ICSI of cryopreserved oocytes. In many species, chilling injury may contribute to low survival and decreased developmental competence of cryopreserved oocytes (1, 2, 9, 10). Cooling metaphase II oocytes to nonphysiological temperatures results in depolymerization of meiotic spindles in mouse (11, 12), cattle (13), and human oocytes (14, 15), although the degree of injury varies among species. Disruption of the meiotic spindle of mouse oocytes can be reversed after rewarming and incubation at 37°C (11, 12), but irreversible damage is observed in bovine and human oocytes (13, 14). This damage is usually manifested as dispersal of chromosomes, resulting in chromosomal anomalies after fertilization, such as aneuploidy and polyploidy (13, 14). Moreover, the kinetics of chilling injury varies among species. Bovine oocytes appear to be very sensitive to chilling injury, since cooling them to 4°C for only 1 minute results in disassembly of the meiotic spindle (13); cooling them to 0°C for 30 seconds significantly reduces their cleavage after fertilization (10). Human oocytes seem to be less sensitive to chilling injury than bovine oocytes, since depolymerization of the spindle occurs only after they have been cooled to 0°C for 3– 4 minutes (15). Little is known about the response of rhesus monkey oocytes to cryopreservation procedures or even to cooling. Younis et al. (16) investigated the cryobiology of rhesus monkey oocytes by determining the effects of hypertonic stress and cooling to 0°C on the organization of F-actin within oocytes at various stages of maturation. Exposure of rhesus monkey oocytes to 1.0 or 2.0 M glycerol resulted in depolymerization of F-actin; however, the presence of glycerol appeared to protect microfilaments against the effects of cooling oocytes to 0°C. Microtubules and microfilaments are major cytoskeletal components that modulate the movement of chromosomes during meiosis and cell division after fertilization (17). Although the effect of cooling on microfilaments in rhesus monkey oocytes has been previously investigated, its effect on tubulin organization has not previously been examined, nor has the kinetics of chilling injury been described. The objective of this study was to determine the effects of cooling on the organization of tubulin and on the distribution of chromosomes in oocytes obtained from superovulated female monkeys. It has been shown in mouse and rabbit oocytes that cryoprotectants alter microtubule organization (9). Therefore, to identify just the effects of cooling alone on the cytoskeletal organization, we did not use cryoprotectants in FERTILITY & STERILITY威
this study. For this investigation, we collected oocytes from females that had received gonadotropins to induce resumption of meiosis, since such oocytes are the most developmentally competent that can be obtained from the rhesus monkey (18).
MATERIALS AND METHODS Animals, Superovulation Treatment, and Oocyte Retrieval Eight adult female rhesus monkeys (Macaca mulatta; ages 6 –16 years) used in the present study were housed at the Tulane Regional Primate Research Center (TRPRC), and were maintained according to the Guide for the Care and Use of Laboratory Animals (19). All procedures for handling and treatment of the animals were reviewed and approved in advance by the Institutional Animal Care and Use Committee of TRPRC and of the University of New Orleans. The monkeys were individually caged in rooms with a 06:00 to 18:00 light cycle and a temperature maintained at 25–27°C. Monkeys were fed a diet of monkey chow (Harlan TEKLAD NIB Primate diet; Madison, WI) twice daily and had free access to water. They were monitored every morning for menses. Beginning on day 1 or 2 of the menstrual cycle (day 1 ⫽ first day of menstruation), a menstruating animal received twice-daily i.m. injections of 37.5 IU human recombinant FSH (hrFSH; Serono Laboratory, Norwell, MA) for 7 or 8 days. The ovaries of sedated monkeys were examined on the fifth or sixth day of hrFSH injection by ultrasound to evaluate their follicular response to the gonadotropin. To induce oocyte maturation, a single i.m. injection of 1,000 IU recombinant hCG (hrCG; Serono) was given on day 8 or 9 to those monkeys that had responded to hrFSH. Oocyte collection was performed in an operating theater, and aseptic techniques were strictly practiced. Females were preanesthetized with acepromazine maleate (0.2 mg/kg body weight) and glycopyrrolate (0.01 mg/kg body weight), anesthetized with ketamine hydrochloride (10 mg/kg body weight), and intubated and maintained on isoflurane/oxygen (1.5%) during the procedure. Oocytes were aspirated laparoscopically 27–32 hours after the injection of rhCG from follicles ⬎3 mm in diameter, using a 20-gauge spinal needle, into Tyrode’s lactate-pyruvate-Hepes medium (TALPHEPES; (20) containing 10 IU/mL heparin at 37°C. After surgery, the monkeys were given injections of buprenorphine (0.1 mg/kg body weight) as an analgesic for 3 days. Chemicals used in this study were obtained from the Sigma Chemical Company (St. Louis, MO), unless otherwise stated. Cumulus-oocyte complexes (COCs) were recovered using a stereomicroscope and transported in TALP-HEPES medium at 37°C from the Primate Center in Covington to the laboratory in New Orleans (an approximately 1.5-hour drive) in a portable incubator (Minitube, Verona, WI). Care was taken to maintain the oocytes continuously at 37°C from the time of their collection until treatment. Upon arrival at the 819
laboratory, the COCs were placed into TALP-HEPES containing 1.5% (w/v) hyaluronidase (Type IV-S from bovine testes), and cumulus cells were removed using mechanical aspiration. Denuded oocytes were washed three times in TALP-HEPES and then subjected to various treatments as described below. Oocytes were handled in a room maintained at 32°C and on a stage warmer at 37°C.
Cooling of Rhesus Monkey Oocytes On four separate occasions, a total of 171 oocytes collected from eight monkeys were used in this study. Oocytes from each monkey were treated separately and were allocated without intentional selection or bias to five groups: control oocytes (group 1), oocytes cooled to 0°C and held for 1 minute (group 2), 5 minutes (group 3), 10 minutes (group 4), and 30 minutes (group 5). Control oocytes consisted of the following: [1] oocytes that were fixed within 1 hour after being collected while still at the Primate Center; [2] oocytes that were transported at 37°C in TALP-HEPES and fixed shortly after arrival at the laboratory (approximately 4 hours after collection); and [3] oocytes that were fixed at the end of the experiment on a given day (8 hours). The oocytes in groups 2–5 were rinsed in fresh TALPHEPES medium and loaded into 0.25 mL plastic straws. The straws were then placed directly into a well-stirred ethanol bath at 0°C in a controlled-rate freezer (Bio-Cool IV, FTS Systems, Stone Ridge, NY). At the end of each holding period, the chilled oocytes from each group were separated into two sets. The first set was transferred directly into a fixative solution (described below) without being warmed. The second set was warmed rapidly in a 37°C water bath, and the oocytes were expelled from the straws and cultured in CMRL 1066 medium (Connaught Medical Research Laboratories; Gibco BRL, Grand Island, NY) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 in air before being fixed.
Immunofluorescence
and stained with propidium iodide (0.1 mg/mL). Finally, the stained oocytes were washed briefly in PBS, mounted on slides in 90% glycerol in PBS containing 100 mg/mL triethylenediamine, and observed under a Nikon TE300 fluorescent microscope (Nikon Corporation, Tokyo, Japan) with excitation filters for rhodamine and fluorescein-labeled reagents.
Microscopic Evaluation Analysis of oocytes was performed using the four criteria described by Zenzes et al. (15): [1] meiotic stage, [2] the order in which the chromosomes were aligned on the spindle, [3] the amount of fluorescent microtubules, and [4] the number of oocytes at metaphase I, anaphase I, telophase I, and metaphase II in which there was a bipolar spindle. The meiotic stage was defined using the following criteria. The germinal vesicle (GV) stage showed fine filamentous chromatin and a nucleolus. The GV breakdown (GVBD) stage exhibited thickening of the chromatin filaments and disappearance of the nuclear membrane and nucleolus. The prometaphase I (pro-MI) stage exhibited chromosomes becoming aligned near each other, but with no clear evidence of a meiotic spindle. The metaphase I (MI) stage showed a meiotic spindle with chromosomes aligned between the two spindle poles. The anaphase I and telophase I (AI and TI) stages exhibited meiotic spindles with two sets of chromosomes each migrating toward opposite spindle poles. The metaphase II (MII) stage showed a meiotic spindle with chromosomes aligned along the midplane between the two spindle poles, and there was a distinct first polar body. To assess the amount of fluorescent microtubules, the oocytes were assigned to one of four categories: nil [0], weak [1], moderate [2], or strong [3]. In an attempt to provide a semiquantitative estimate of the amount of fluorescence for each group of oocytes, we assigned a fluorescence intensity index (FIS), using the same approach as did Zenzes et al. (15). This value was calculated using the following formula:
Immunostaining of oocytes was performed using the method described by Zenzes et al. (15). After being treated as described above, oocytes were fixed in microtubule stabilizing buffer containing 2.0% formaldehyde, 0.5% triton X-100, and 1 M taxol for 20 minutes. Subsequently, fixed oocytes were washed three times in a blocking solution of phosphate-buffered saline (PBS) containing 2% bovine serum albumin, 2% Carnation powdered skim milk (Nestle´ USA, Glendale, CA), 2% normal goat serum, 0.1 M glycine, and 0.01% Triton X-100. Oocytes were stored for 24 hours in this solution at 4°C.
FIS ⫽ [(number of oocytes) ⫻ (fluorescent intensity)]/
For staining, oocytes were treated with monoclonal mouse antibody to ␣-tubulin (Clone DM-IA; ICN Biomedicals, Costa Mesa, CA) for 1 hour at 37°C. Then, the oocytes were placed in blocking solution and incubated for 1 hour at room temperature followed by incubation in fluoresceinconjugated goat–anti-mouse Ig (IgG; ICN Biomedicals). Next the oocytes were washed in PBS-sodium azide solution
The oocytes were photographed using a digital camera (Coolpix 950; Nikon, Lewisville, TX) attached to an inverted microscope (TE300; Nikon) equipped with epifluorescent UV light and an excitation filter between 450 and 490 nm. Selected micrographs and figures were composed using Paint Shop Pro 5.03 (JASC Software, Eden Prairie, MN).
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(total number of oocytes in each group) We recognized that the assessment of fluorescent intensity was rather subjective. To reduce the degree of subjectivity, all oocytes were evaluated by the same investigator. However, the assessment was performed in a blinded fashion, in which the slides were coded by another person and the observer did not know the identity of each sample during the evaluation.
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Statistical Analysis Statistical analyses were performed using GraphPad Instat program (version 3.00 for Windows 95, GraphPad Software, San Diego, CA). Comparison of meiotic stages of control oocytes fixed at 1, 4, or 8 hours after oocyte retrieval was performed using a 2 test. Comparison of each meiotic stage between control oocytes fixed at 4 hours after oocyte collection and chilled oocytes fixed immediately after being cooled was performed using Fisher’s exact test. Comparison of each meiotic stage between control oocytes fixed at 8 hours after oocyte collection and chilled oocytes fixed after being incubated at 37°C was also performed using Fisher’s exact test. Comparison of fluorescent intensity indices among groups was performed using the Kruskal-Wallis test. Data for chromosome alignment and bipolar spindles were analyzed using the Fisher’s exact test. Differences were considered to be significant when P⬍.05.
FIGURE 1 Meiotic status of rhesus oocytes fixed and stained at 1 (n ⫽ 12), 4 (n ⫽ 12), or 8 hours (n ⫽ 22) after oocyte retrieval.
RESULTS Meiotic Status of Untreated Oocytes Since the interval between oocyte retrieval and the end of the experiment was approximately 8 hours, this time was sufficient for oocytes to have undergone meiotic maturation. Therefore, the meiotic status of control oocytes held at 37°C was examined at three intervals after oocyte retrieval. The first interval was within 1 hour after oocyte retrieval. This group of oocytes provided basic information as to the meiotic status of oocytes as they were collected from the gonadotropin-treated females. The second interval was 4 hours after oocyte retrieval when the oocytes had arrived at the laboratory. This was the starting point of the chilling experiment. Thus, this group of oocytes served as a control for oocytes that were fixed immediately after chilling but were not incubated. The third interval was 8 hours after oocyte retrieval, which was the end of experiment. Therefore, this group of oocytes served as a control for oocytes that were chilled and then fixed after being incubated at 37°C for 60 minutes. Figure 1 shows the distribution of the meiotic status of oocytes observed at 1, 4, or 8 hours after oocyte retrieval. At 1 hour after oocyte collection, the meiotic stages of the oocytes ranged from the GV to MII stages. However, at 4 and 8 hours after oocyte retrieval none of the oocytes were at the GV stage. Except for that difference, although the distribution of meiotic stages of the oocytes at a given interval varied somewhat, there were no significant differences among them.
Meiotic Status of Chilled Oocytes The meiotic configuration of chilled oocytes was observed either after they had been cooled and held for various times or after they had been cooled, warmed, and incubated at 37°C. The distribution by meiotic stage after chilling alone and after chilling and incubation at 37°C is shown in Figure 2. There were no differences in the meiotic status of oocytes FERTILITY & STERILITY威
Songsasen. Cooling sensitivity of rhesus monkey oocytes. Fertil Steril 2002.
cooled and held for various times before being fixed without warming, as the majority of oocytes exhibited pro-MI and MI configurations (Fig. 2A). Therefore, the data from different holding periods were pooled. The percentages of meiotic stages of chilled oocytes fixed without being warmed were not different from those of the control oocytes fixed at 4 hours after oocyte collection. As was true of those oocytes that were fixed without being warmed, the meiotic status of oocytes fixed after being warmed and incubated exhibited a similar pattern regardless of the holding period at 0°C. However, when chilled oocytes were incubated at 37°C for 60 minutes, their meiotic stages appeared to significantly progress compared with those fixed without being warmed (P⬍.01; Fig. 2B). That is, we compared the distribution in Figure 2A with that in Figure 2B and found a significant difference between them. Finally, the distribution of meiotic stages of chilled oocytes fixed after being warmed and incubated for 60 minutes was not significantly different from that of the control oocytes fixed at 8 hours after collection.
Configuration of Tubulin and Chromosome of Chilled Oocytes The FIS of control oocytes from each female ranged from 2 to 3. The FIS appeared to be dependent on the meiotic stage of the oocytes. The fluorescence of AI/TI and MII stage oocytes was the highest, whereas that of GVBD was the lowest (data are not shown). Cooling oocytes to 0°C and holding them at that temperature for only 1 minute resulted in depolymerization of 821
FIGURE 2 (A) Meiotic status of rhesus oocytes cooled to 0°C and held for 1 (n ⫽ 15), 5 (n ⫽ 16), 10 (n ⫽ 14), or 30 minutes (n ⫽ 10). (B) Meiotic status of rhesus oocytes cooled to 0°C, held for 1 (n ⫽ 17), 5 (n ⫽ 14), 10 (n ⫽ 20), or 30 minutes (n ⫽ 15) and then warmed to 37°C and incubated for 60 minutes.
Songsasen. Cooling sensitivity of rhesus monkey oocytes. Fertil Steril 2002.
tubulin. There were significant decreases in fluorescence intensity of tubulin in chilled oocytes (Table 1). However, oocytes from each monkey varied in their sensitivity to chilling. Complete tubulin depolymerization occurred in oocytes from seven of eight monkeys after the oocytes were cooled to 0°C for 1 minute, whereas those from the eighth monkey exhibited only slight depolymerization of tubulin after being cooled to 0°C for the same period. Repolymerization of tubulin occurred after chilled oo-
cytes were warmed and incubated for 60 minutes. The fluorescent intensity of tubulin increased after warming and incubation, but these increases were not significantly different from those in oocytes fixed without warming (Table 1). Moreover, the fluorescence indices of tubulin in chilled and warmed oocytes were significantly lower than those of the controls (Table 1). Figure 3 shows the fluorescent image of a control oocyte held at 37°C and of two chilled oocytes. Figure 3A shows a
TABLE 1 Effect of chilling on fluorescent intensity index, chromosome alignment, and organization of meiotic spindles in rhesus monkey oocytes. Time at 0°C (minutes) 0 1 5 10 30 1 5 10 30
Time at 37°C (minutes)
Oocytes (n)
FISa
Chromosomes aligned (%)
Bipolar spindle (%)b
120 0 0 0 0 60 60 60 60
22 17 16 15 10 17 14 20 16
2.2c 0.3d 0.4d 0.2d 0.3d 1.2d 1.3c,d 0.9d 0.9d
22 (100)c 10 (59)d,e 13 (81)c,d 7 (47)d,e 6 (60)d,e 6 (35)e 6 (43)d,e 9 (45)d,e 7 (44)d,e
17/17 (100)c 2/9 (22)d,e 2/13 (15)e 3/6 (50)d,e 1/9 (11)e 8/11 (73)c,d 4/12 (33)d,e 3/13 (23)e 5/13 (38)d,e
a
FIS is the fluorescence intensity index estimated by microscopic examination of each oocyte. The fraction shows the oocytes at MI, AI/TI, and MII oocytes in which there was a bipolar spindle. c,d,e Different letters within the same column indicate significant differences (P⬍.05). b
Songsasen. Cooling sensitivity of rhesus monkey oocytes. Fertil Steril 2002.
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Cooling sensitivity of rhesus monkey oocytes
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FIGURE 3 Fluorescent micrographs of rhesus oocytes: (A) metaphase I oocyte exhibiting chromosomes (yellow) aligned between two spindle poles (green); (B) chilled oocyte exhibiting multipolar spindles (green) and three sets of chromosomes (red) at each end; (C) chilled oocyte exhibiting irregularly shaped tubulin (green) and chromosomes (yellow) aligned underneath the tubulin. Bar ⫽ 10 m.
Songsasen. Cooling sensitivity of rhesus monkey oocytes. Fertil Steril 2002.
normal oocyte at MI (control) with the chromosomes aligned between two spindle poles. After oocytes had been chilled, they exhibited various abnormal configurations of tubulin and chromosomes. These abnormalities included multipolar spindles (Fig. 3B) and irregularly shaped tubulin with dispersed chromosomes (Fig. 3C), as well as complete and apparently irreversible depolymerization. Abnormal distribution of chromosomes in chilled oocytes was observed even when oocytes were cooled to 0°C for only 1 minute (Table 1). Longer exposure to 0°C increased the number of oocytes exhibiting abnormal distribution of chromosomes as well as the extent of damage to the meiotic FERTILITY & STERILITY威
spindles. Restoration of organized tubulin within the oocytes after incubation at 37°C also varied among females. For example, none of nine oocytes collected from female E540 exhibited a normal bipolar spindle after they were cooled to 0°C and then warmed and incubated at 37°C for 60 minutes. However, seven of nine oocytes obtained from female R037 regained their normal bipolar spindle after being chilled and incubated at 37°C for 60 minutes.
DISCUSSION In this study, we have demonstrated that rhesus monkey oocytes are similar to those of the human and of cattle in that 823
they are very sensitive to chilling injury. Cooling oocytes to 0°C and holding them at that temperature for only 1 minute resulted in complete depolymerization of tubulin in oocytes of seven of eight monkeys. Incubation of chilled oocytes at 37°C resulted in restoration of normal organization of tubulin and nuclear maturation in some oocytes. However, even after being incubated at 37°C, many of the chilled oocytes exhibited an abnormal organization of tubulin; this was accompanied by abnormal configurations of the chromosomes. We have also found that there were significant differences in chilling sensitivity of oocytes obtained from different females. We have no explanation for these differences among females. Many factors contribute to freezing sensitivity of mammalian oocytes (1, 2). Sensitivity of oocytes to cooling to low temperatures is believed to be a major factor contributing to the low survival of cryopreserved oocytes, since with conventional slow cooling methods, oocytes must be exposed to deleterious temperatures (ranging from 20°C to 0°C) for several minutes (1, 2, 9, 10). Exposure of MII oocytes to these temperatures affects the cytoskeletal organization and chromosome configuration (9 –15) and causes premature release of cortical granules that may compromise subsequent fertilization and embryonic development (10, 21). Evidence that rhesus monkey oocytes are sensitive to chilling injury was that even a 1-minute exposure of oocytes to 0°C caused the average fluorescent intensity index to decrease from 2.2 in control oocytes to only 0.2 to 0.4 in the chilled ones. This suggests that rhesus oocytes are more sensitive than human oocytes, since the fluorescent intensity index of human oocytes that were treated similarly declined only slightly after being cooled to 0°C for 1 minute (15). Repolymerization of tubulin and progression of nuclear maturation were observed in some rhesus oocytes after they were warmed and incubated at 37°C for 60 minutes. However, even after warming, a high proportion of oocytes exhibited abnormal organization of tubulin and irregular chromosomal configurations. Because of limitations of the epifluorescent microscope, we were only able to observe the organization of tubulin within oocytes in which a meiotic spindle was present (MI to MII stage). For those oocytes, we grouped abnormal tubulin organization into three categories: multipolar spindle, irregularly shaped tubulin, and complete tubulin depolymerization. Abnormal chromosome configurations were found in a high proportion of oocytes, especially after warming and incubation. This irreversible damage after brief chilling indicates that rhesus monkey oocytes are more like those of the human and bovine than like those of the mouse. In the mouse, chilled oocytes regained normal meiotic spindles after they were warmed to 37°C for 60 minutes (12). In contrast, fewer than half of human oocytes exposed to room temperature for 10 minutes returned to a normal 824
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appearance after 4 hours at 37°C (14). In the bovine, 90% of oocytes exhibited abnormal meiotic spindles after being cooled to 25°C for 60 minutes followed by incubation at 37°C for 30 minutes (13). In addition to centrosomes associated with the meiotic spindle, mouse oocytes contain microtubule organizing centers in the cortical cytoplasm (2, 9). Although a microtubule-enhancing drug, taxol, can induce formation of microtubule asters in the cytoplasm of human oocytes (22), cytoplasmic microtubules are not normally observed in the cortical cytoplasm of human and rhesus oocytes (14, 23). The absence of centrosomes and microtubules in the cortical cytoplasm (such as those that occur in mouse oocytes) is believed to be responsible for irreversible damage to the meiotic spindle after the cooling of human and bovine oocytes to low temperatures (13, 14). Perhaps this also may have contributed to chilling sensitivity of the meiotic spindle in rhesus monkey oocytes observed in the present study. In human and bovine oocytes, alterations of the meiotic spindle from cooling are influenced by the duration of exposure (13, 15). In the present study, holding oocytes at 0°C for longer periods did not substantially increase the amount of damage observed. This suggests that rhesus monkey oocytes are especially sensitive to cooling, since holding them for only 1 minute at 0°C was sufficient to cause irreversible damage in a majority of the oocytes. The developmental competency of in vitro–matured rhesus oocytes is rather low compared with their in vivo– matured counterparts (18). Therefore, we obtained oocytes for this study from rhCG-stimulated monkeys. The majority of the oocytes had already resumed meiosis with approximately 10% of the oocytes at the GVBD stage within 4 hours after oocyte retrieval. So far, little is known about the effects of cooling on the configuration of chromosomes in GVBD-stage oocytes. In the present study, six of 18 GVBD-stage oocytes exhibited an abnormal chromosomal configuration with chromosomal clumping. Perhaps the absence of the nuclear membrane in GVBD-stage oocytes might render them more sensitive to chilling injury. However, the small number of GVBD stages oocytes examined in the present study is insufficient to draw any firm conclusions regarding their sensitivity to chilling injury. We also found that there were differences in chilling sensitivity of oocytes obtained from different females. Among the eight females used in the present study, two females (N821 and R037) produced oocytes that appeared to be very resistant to chilling injury. Between 60% and 70% of the oocytes exhibited normal tubulin organization and chromosomal configuration after being chilled, warmed, and incubated at 37°C for 60 minutes. In contrast, oocytes from another female (E540) seemed to be extremely sensitive to cooling, as none of her oocytes at the MI or at MII stage exhibited normal spindle organization after being chilled and then incubated at 37°C.
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Characteristics of mammalian gametes are likely influenced by their genetic backgrounds. Using oocytes from outbred Institute of Cancer Research (ICR) mice, Benson and Critser (24) found that there were differences in water permeability and its activation energy of oocytes obtained from different females. In contrast, oocytes from different females of an inbred strain do not exhibit such differences (25). This difference between inbred and outbred mice is prima facie evidence that membrane differences among females have a genetic basis, although factors contributing to these differences among individuals have not been elucidated. It is possible that differences in membrane characteristics could play a role in differences among individuals in the freezing sensitivity of their gametes. However, formation of the meiotic spindle and alignment of chromosomes during meiosis in mammalian oocytes are regulated by intracellular factors, for example, the amount of free tubulin contained within the cytoplasm and its nucleating capacity (17). Therefore, it is possible that oocytes obtained from different females contain different amounts or types of these cytoplasmic factors that modulate spindle organization. In species whose oocytes are very sensitive to chilling injury, such as the bovine and human, ultrarapid cooling has proven to be the method of choice for oocyte cryopreservation. The rationale of this method is to cool oocytes at very high rates to enable them to pass through the damaging temperature zone in the fluid state fast enough to circumvent the chilling injury (5, 7, 8, 26). Martino et al. (26) demonstrated that significantly more bovine oocytes that were cryopreserved by ultrarapid cooling on electron microscope grids developed into blastocysts after IVF, compared with those cooled much less rapidly. Recently, Wu et al. (27) reported a human pregnancy after transferring embryos produced from human oocytes also cryopreserved by ultrarapid cooling on electron microscope grids. Vajta et al. (5) cryopreserved bovine oocytes in very fine plastic straws and produced three live calves. Using this vitrification technique described by Vajta et al. (5), Kuleshova et al. (8) reported the birth of a human baby after transferring embryos produced from cryopreserved in vitro–matured oocytes. In conclusion, the present study has shown that rhesus oocytes are at least as sensitive to chilling injury as those of humans and cattle. Therefore, we conclude that cryopreservation of rhesus monkey oocytes may require that they be cooled extremely rapidly to circumvent damage caused by chilling injury.
Acknowledgments: The authors thank R. Harrison, Ph.D., and SueAnn Schneider, B.A., of the TRPRC, as well as Melissa Johnston, B.S., of the Audubon Research Center, for their assistance throughout the study.
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References 1. Bernard A, Fuller BJ. Cryopreservation of human oocytes: a review of current problems and perspectives. Hum Reprod Update 1996;2:193– 207. 2. Parks JE, Ruffing NA. Factors affecting low temperature survival of mammalian oocytes. Theriogenology 1992;37:59 –73. 3. Schroeder AC, Champlin AK, Mobraaten LE, Eppig JJ. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J Reprod Fertil 1990;89:43–50. 4. Al-Hasani S, Kirsch J, Diedrich K, Blanke S, van der Ven H, Krebs D. Successful embryo transfer of cryopreserved and in-vitro fertilized rabbit oocytes. Hum Reprod 1989;4:77–9. 5. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51: 53– 8. 6. Ludwig M, Al-Hasani S, Felberbaum R, Diedrich K. New aspects of cryopreservation of oocytes and embryos in assisted reproduction and future perspectives. Hum Reprod 1999;14(Suppl 1):162– 85. 7. Chung HM, Hong SW, Lim JM, Lee SH, Cha WT, Ko JJ, et al. In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages. Fertil Steril 2000;73:545–51. 8. Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes. Hum Reprod 1999;14:3077–9. 9. Vincent C, Johnson MH. Cooling, cryoprotectants and the cytoskeleton of the mammalian oocyte. Oxford Rev Reprod Biol 1992;14: 73–100. 10. Martino A, Pollard JW, Leibo SP. Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev 1996;45:503– 12. 11. Magistrini M, Szo¨ llo¨ si D. Effects of cold and isopropyl-N-phenylcarbomate on the second meiotic spindle of mouse oocytes. J Cell Biol 1980;22:699 –707. 12. Pickering SJ, Johnson MH. The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 1987;2: 207–16. 13. Aman RR, Parks JE. Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro–matured bovine oocytes. Biol Reprod 1994;50:103–10. 14. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990;54:102– 8. 15. Zenzes MT, Bielecki R, Casper RF, Leibo SP. Effects of chilling to 0°C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril 2001;75:769 –77. 16. Younis AI, Toner M, Albertini DF, Biggers JD. Cryobiology of nonhuman primate oocytes. Hum Reprod 1996;11:156 – 65. 17. Simerly C, Navara C, Wu G-J, Schatten G. Cytoskeletal organization and dynamics in mammalian oocytes during maturation and fertilization. In: Grudzinskas JG, Yovich JL, eds. Gametes—the oocyte. Cambridge: Cambridge University Press, 1995:54 –94. 18. Lanzendorf SE, Zelinski-Wooten MB, Stouffer RL, Wolf DP. Maturity at collection and the developmental potential of rhesus monkey oocytes. Biol Reprod 1990;42:703–11. 19. Guide for the Care and Use of Laboratory Animals. DHHS publication no. NIH 85-23, revised, 1985. 20. Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988;38:1171– 80. 21. Zeron Y, Pearl M, Borochov A, Arav A. Kinetic and temporal factors influence chilling injury to germinal vesicle and mature bovine oocytes. Cryobiology 1999;38:35– 42. 22. Kim NH, Chung HM, Cha KY, Chung KS. Microtubule and microfilament organization in maturing human oocytes. Hum Reprod 1998;13: 2217–22. 23. Wu GJ, Simerly C, Zoran SS, Funte LR, Schatten G. Microtubule and chromatin dynamics during fertilization and development in rhesus monkeys, and regulation by intracellular calcium ions. Biol Reprod 1996;55:260 –70. 24. Benson CT, Critser JK. Variation of water permeability (Lp) and its activation energy (Ea) among unfertilized golden hamster and ICR murine oocytes. Cryobiology 1994;31:215–23. 25. Leibo SP. Water permeability and its activation energy of fertilized and unfertilized mouse ova. J Membr Biol 1980;53:179 – 88. 26. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996b;54:1059–69. 27. Wu J, Zhang L, Wang X. In vitro maturation, fertilization and embryo development after ultrarapid freezing of immature human oocytes. Reproduction 2001;121:389 –93.
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Effect of chilling on the organization of tubulin and chromosomes in rhesus monkey oocytes Nucharin Songsasen, D.V.M., Ph.D.,a Il-Jeoung Yu, D.V.M., Ph.D.,a Marion S. Ratterree, D.V.M.,b Catherine A. VandeVoort, Ph.D.,c and Stanley P. Leibo, Ph.D.a University of New Orleans and Audubon Center for Research of Endangered Species, New Orleans, Louisiana
Received May 22, 2001; revised and accepted October 4, 2001. Supported by National Institutes of Health grant no. 5R01-RR13439 (to C.A.V.), and by grant no. NIH-5P51 RR00164-39 to Tulane Regional Primate Research Center. Reprint requests: Stanley P. Leibo, Ph.D., Audubon Center for Research of Endangered Species, 14001 River Rd., New Orleans, Louisiana 70131 (FAX: 504-391-7707; E-mail: [email protected]). a Department of Biological Sciences. b Tulane Regional Primate Research Center, Covington, Louisiana. c California Regional Primate Research Center, Davis, California. 0015-0282/02/$22.00 PII S0015-0282(01)03240-X
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Objective: To determine the effects of chilling on the organization and distribution of tubulin and chromosomes in rhesus monkey oocytes. Design: Comparative laboratory study. Setting: Academic research laboratory. Animal(s): Eight adult female rhesus monkeys (Macaca mulatta) aged 6 –16 years. Intervention(s): A total of 171 oocytes retrieved from eight rhesus monkeys were separated into nine groups. One group of control oocytes was held at 37°C during the experiment. Four groups of oocytes were rapidly cooled to 0°C and held for 1, 5, 10, or 30 minutes and then fixed and stained. Four other groups of oocytes were cooled to 0°C, held for 1, 5, 10, or 30 minutes, warmed and incubated at 37°C for 60 minutes, and then fixed and stained. Main Outcome Measure(s): Organization of cytoskeleton and chromosomes. Result(s): Exposure of rhesus oocytes to 0°C for only 1 minute resulted in complete depolymerization of tubulin. Incubation of chilled oocytes at 37°C for 60 minutes caused partial restoration of tubulin, although most oocytes exhibited abnormal alignment of chromosomes and disorganized meiotic spindles. Conclusion(s): We conclude that rhesus monkey oocytes are extremely sensitive to chilling injury. Their successful cryopreservation may require rapid cooling to outpace this injury. (Fertil Steril威 2002;77:818 –25. ©2002 by American Society for Reproductive Medicine.) Key Words: Chilling injury, rhesus monkey, oocytes, cytoskeleton
The ability to effectively cryopreserve oocytes would yield many benefits for the assisted reproduction of many species. In the rhesus monkey, it would permit the collection of oocytes during the breeding season, yet allow their year-round use. This would remove the current barrier of reproductive seasonality in studies of rhesus monkey oocytes. Oocytes could be stockpiled for experiments requiring the simultaneous use of large numbers of oocytes. Furthermore, oocyte cryopreservation could be used to preserve genetic material from females of specific genotypes to increase their numbers in studies of genetically inherited diseases. Despite these potential benefits of oocyte cryopreservation, it is generally accepted that procedures developed for mammalian embryos are much less effective when used for oocytes (1,
2). Although live young have been produced from cryopreserved oocytes, success rates have been low and limited to only a few species, such as the mouse, rabbit, cattle, and human (3– 6). Schroeder et al. (3) cryopreserved 487 in vitro– matured mouse oocytes, of which 158 developed into embryos; these yielded 41 live young (26%) after ET. Vajta et al. (5) produced three bull calves after transferring 17 blastocyst-stage embryos produced from cryopreserved bovine oocytes. In humans, a small number of babies have been produced by conventional IVF of cryopreserved oocytes (1, 6). More recently, ICSI of cryopreserved oocytes has been widely used to produce human embryos, although the overall success rate in terms of the number of babies born as a percentage of cryopreserved
oocytes is still only about 1% (6). Very recently, vitrification of human oocytes has been attempted with encouraging results. Chung et al. (7) vitrified 89 oocytes, of which 33 developed to the 2 pronuclear stage after ICSI and 12 developed into euploid blastocysts. Kuleshova et al. (8) vitrified 17 oocytes and produced a single baby after transferring three embryos created by ICSI of cryopreserved oocytes. In many species, chilling injury may contribute to low survival and decreased developmental competence of cryopreserved oocytes (1, 2, 9, 10). Cooling metaphase II oocytes to nonphysiological temperatures results in depolymerization of meiotic spindles in mouse (11, 12), cattle (13), and human oocytes (14, 15), although the degree of injury varies among species. Disruption of the meiotic spindle of mouse oocytes can be reversed after rewarming and incubation at 37°C (11, 12), but irreversible damage is observed in bovine and human oocytes (13, 14). This damage is usually manifested as dispersal of chromosomes, resulting in chromosomal anomalies after fertilization, such as aneuploidy and polyploidy (13, 14). Moreover, the kinetics of chilling injury varies among species. Bovine oocytes appear to be very sensitive to chilling injury, since cooling them to 4°C for only 1 minute results in disassembly of the meiotic spindle (13); cooling them to 0°C for 30 seconds significantly reduces their cleavage after fertilization (10). Human oocytes seem to be less sensitive to chilling injury than bovine oocytes, since depolymerization of the spindle occurs only after they have been cooled to 0°C for 3– 4 minutes (15). Little is known about the response of rhesus monkey oocytes to cryopreservation procedures or even to cooling. Younis et al. (16) investigated the cryobiology of rhesus monkey oocytes by determining the effects of hypertonic stress and cooling to 0°C on the organization of F-actin within oocytes at various stages of maturation. Exposure of rhesus monkey oocytes to 1.0 or 2.0 M glycerol resulted in depolymerization of F-actin; however, the presence of glycerol appeared to protect microfilaments against the effects of cooling oocytes to 0°C. Microtubules and microfilaments are major cytoskeletal components that modulate the movement of chromosomes during meiosis and cell division after fertilization (17). Although the effect of cooling on microfilaments in rhesus monkey oocytes has been previously investigated, its effect on tubulin organization has not previously been examined, nor has the kinetics of chilling injury been described. The objective of this study was to determine the effects of cooling on the organization of tubulin and on the distribution of chromosomes in oocytes obtained from superovulated female monkeys. It has been shown in mouse and rabbit oocytes that cryoprotectants alter microtubule organization (9). Therefore, to identify just the effects of cooling alone on the cytoskeletal organization, we did not use cryoprotectants in FERTILITY & STERILITY威
this study. For this investigation, we collected oocytes from females that had received gonadotropins to induce resumption of meiosis, since such oocytes are the most developmentally competent that can be obtained from the rhesus monkey (18).
MATERIALS AND METHODS Animals, Superovulation Treatment, and Oocyte Retrieval Eight adult female rhesus monkeys (Macaca mulatta; ages 6 –16 years) used in the present study were housed at the Tulane Regional Primate Research Center (TRPRC), and were maintained according to the Guide for the Care and Use of Laboratory Animals (19). All procedures for handling and treatment of the animals were reviewed and approved in advance by the Institutional Animal Care and Use Committee of TRPRC and of the University of New Orleans. The monkeys were individually caged in rooms with a 06:00 to 18:00 light cycle and a temperature maintained at 25–27°C. Monkeys were fed a diet of monkey chow (Harlan TEKLAD NIB Primate diet; Madison, WI) twice daily and had free access to water. They were monitored every morning for menses. Beginning on day 1 or 2 of the menstrual cycle (day 1 ⫽ first day of menstruation), a menstruating animal received twice-daily i.m. injections of 37.5 IU human recombinant FSH (hrFSH; Serono Laboratory, Norwell, MA) for 7 or 8 days. The ovaries of sedated monkeys were examined on the fifth or sixth day of hrFSH injection by ultrasound to evaluate their follicular response to the gonadotropin. To induce oocyte maturation, a single i.m. injection of 1,000 IU recombinant hCG (hrCG; Serono) was given on day 8 or 9 to those monkeys that had responded to hrFSH. Oocyte collection was performed in an operating theater, and aseptic techniques were strictly practiced. Females were preanesthetized with acepromazine maleate (0.2 mg/kg body weight) and glycopyrrolate (0.01 mg/kg body weight), anesthetized with ketamine hydrochloride (10 mg/kg body weight), and intubated and maintained on isoflurane/oxygen (1.5%) during the procedure. Oocytes were aspirated laparoscopically 27–32 hours after the injection of rhCG from follicles ⬎3 mm in diameter, using a 20-gauge spinal needle, into Tyrode’s lactate-pyruvate-Hepes medium (TALPHEPES; (20) containing 10 IU/mL heparin at 37°C. After surgery, the monkeys were given injections of buprenorphine (0.1 mg/kg body weight) as an analgesic for 3 days. Chemicals used in this study were obtained from the Sigma Chemical Company (St. Louis, MO), unless otherwise stated. Cumulus-oocyte complexes (COCs) were recovered using a stereomicroscope and transported in TALP-HEPES medium at 37°C from the Primate Center in Covington to the laboratory in New Orleans (an approximately 1.5-hour drive) in a portable incubator (Minitube, Verona, WI). Care was taken to maintain the oocytes continuously at 37°C from the time of their collection until treatment. Upon arrival at the 819
laboratory, the COCs were placed into TALP-HEPES containing 1.5% (w/v) hyaluronidase (Type IV-S from bovine testes), and cumulus cells were removed using mechanical aspiration. Denuded oocytes were washed three times in TALP-HEPES and then subjected to various treatments as described below. Oocytes were handled in a room maintained at 32°C and on a stage warmer at 37°C.
Cooling of Rhesus Monkey Oocytes On four separate occasions, a total of 171 oocytes collected from eight monkeys were used in this study. Oocytes from each monkey were treated separately and were allocated without intentional selection or bias to five groups: control oocytes (group 1), oocytes cooled to 0°C and held for 1 minute (group 2), 5 minutes (group 3), 10 minutes (group 4), and 30 minutes (group 5). Control oocytes consisted of the following: [1] oocytes that were fixed within 1 hour after being collected while still at the Primate Center; [2] oocytes that were transported at 37°C in TALP-HEPES and fixed shortly after arrival at the laboratory (approximately 4 hours after collection); and [3] oocytes that were fixed at the end of the experiment on a given day (8 hours). The oocytes in groups 2–5 were rinsed in fresh TALPHEPES medium and loaded into 0.25 mL plastic straws. The straws were then placed directly into a well-stirred ethanol bath at 0°C in a controlled-rate freezer (Bio-Cool IV, FTS Systems, Stone Ridge, NY). At the end of each holding period, the chilled oocytes from each group were separated into two sets. The first set was transferred directly into a fixative solution (described below) without being warmed. The second set was warmed rapidly in a 37°C water bath, and the oocytes were expelled from the straws and cultured in CMRL 1066 medium (Connaught Medical Research Laboratories; Gibco BRL, Grand Island, NY) for 60 minutes at 37°C in a humidified atmosphere of 5% CO2 in air before being fixed.
Immunofluorescence
and stained with propidium iodide (0.1 mg/mL). Finally, the stained oocytes were washed briefly in PBS, mounted on slides in 90% glycerol in PBS containing 100 mg/mL triethylenediamine, and observed under a Nikon TE300 fluorescent microscope (Nikon Corporation, Tokyo, Japan) with excitation filters for rhodamine and fluorescein-labeled reagents.
Microscopic Evaluation Analysis of oocytes was performed using the four criteria described by Zenzes et al. (15): [1] meiotic stage, [2] the order in which the chromosomes were aligned on the spindle, [3] the amount of fluorescent microtubules, and [4] the number of oocytes at metaphase I, anaphase I, telophase I, and metaphase II in which there was a bipolar spindle. The meiotic stage was defined using the following criteria. The germinal vesicle (GV) stage showed fine filamentous chromatin and a nucleolus. The GV breakdown (GVBD) stage exhibited thickening of the chromatin filaments and disappearance of the nuclear membrane and nucleolus. The prometaphase I (pro-MI) stage exhibited chromosomes becoming aligned near each other, but with no clear evidence of a meiotic spindle. The metaphase I (MI) stage showed a meiotic spindle with chromosomes aligned between the two spindle poles. The anaphase I and telophase I (AI and TI) stages exhibited meiotic spindles with two sets of chromosomes each migrating toward opposite spindle poles. The metaphase II (MII) stage showed a meiotic spindle with chromosomes aligned along the midplane between the two spindle poles, and there was a distinct first polar body. To assess the amount of fluorescent microtubules, the oocytes were assigned to one of four categories: nil [0], weak [1], moderate [2], or strong [3]. In an attempt to provide a semiquantitative estimate of the amount of fluorescence for each group of oocytes, we assigned a fluorescence intensity index (FIS), using the same approach as did Zenzes et al. (15). This value was calculated using the following formula:
Immunostaining of oocytes was performed using the method described by Zenzes et al. (15). After being treated as described above, oocytes were fixed in microtubule stabilizing buffer containing 2.0% formaldehyde, 0.5% triton X-100, and 1 M taxol for 20 minutes. Subsequently, fixed oocytes were washed three times in a blocking solution of phosphate-buffered saline (PBS) containing 2% bovine serum albumin, 2% Carnation powdered skim milk (Nestle´ USA, Glendale, CA), 2% normal goat serum, 0.1 M glycine, and 0.01% Triton X-100. Oocytes were stored for 24 hours in this solution at 4°C.
FIS ⫽ [(number of oocytes) ⫻ (fluorescent intensity)]/
For staining, oocytes were treated with monoclonal mouse antibody to ␣-tubulin (Clone DM-IA; ICN Biomedicals, Costa Mesa, CA) for 1 hour at 37°C. Then, the oocytes were placed in blocking solution and incubated for 1 hour at room temperature followed by incubation in fluoresceinconjugated goat–anti-mouse Ig (IgG; ICN Biomedicals). Next the oocytes were washed in PBS-sodium azide solution
The oocytes were photographed using a digital camera (Coolpix 950; Nikon, Lewisville, TX) attached to an inverted microscope (TE300; Nikon) equipped with epifluorescent UV light and an excitation filter between 450 and 490 nm. Selected micrographs and figures were composed using Paint Shop Pro 5.03 (JASC Software, Eden Prairie, MN).
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(total number of oocytes in each group) We recognized that the assessment of fluorescent intensity was rather subjective. To reduce the degree of subjectivity, all oocytes were evaluated by the same investigator. However, the assessment was performed in a blinded fashion, in which the slides were coded by another person and the observer did not know the identity of each sample during the evaluation.
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Statistical Analysis Statistical analyses were performed using GraphPad Instat program (version 3.00 for Windows 95, GraphPad Software, San Diego, CA). Comparison of meiotic stages of control oocytes fixed at 1, 4, or 8 hours after oocyte retrieval was performed using a 2 test. Comparison of each meiotic stage between control oocytes fixed at 4 hours after oocyte collection and chilled oocytes fixed immediately after being cooled was performed using Fisher’s exact test. Comparison of each meiotic stage between control oocytes fixed at 8 hours after oocyte collection and chilled oocytes fixed after being incubated at 37°C was also performed using Fisher’s exact test. Comparison of fluorescent intensity indices among groups was performed using the Kruskal-Wallis test. Data for chromosome alignment and bipolar spindles were analyzed using the Fisher’s exact test. Differences were considered to be significant when P⬍.05.
FIGURE 1 Meiotic status of rhesus oocytes fixed and stained at 1 (n ⫽ 12), 4 (n ⫽ 12), or 8 hours (n ⫽ 22) after oocyte retrieval.
RESULTS Meiotic Status of Untreated Oocytes Since the interval between oocyte retrieval and the end of the experiment was approximately 8 hours, this time was sufficient for oocytes to have undergone meiotic maturation. Therefore, the meiotic status of control oocytes held at 37°C was examined at three intervals after oocyte retrieval. The first interval was within 1 hour after oocyte retrieval. This group of oocytes provided basic information as to the meiotic status of oocytes as they were collected from the gonadotropin-treated females. The second interval was 4 hours after oocyte retrieval when the oocytes had arrived at the laboratory. This was the starting point of the chilling experiment. Thus, this group of oocytes served as a control for oocytes that were fixed immediately after chilling but were not incubated. The third interval was 8 hours after oocyte retrieval, which was the end of experiment. Therefore, this group of oocytes served as a control for oocytes that were chilled and then fixed after being incubated at 37°C for 60 minutes. Figure 1 shows the distribution of the meiotic status of oocytes observed at 1, 4, or 8 hours after oocyte retrieval. At 1 hour after oocyte collection, the meiotic stages of the oocytes ranged from the GV to MII stages. However, at 4 and 8 hours after oocyte retrieval none of the oocytes were at the GV stage. Except for that difference, although the distribution of meiotic stages of the oocytes at a given interval varied somewhat, there were no significant differences among them.
Meiotic Status of Chilled Oocytes The meiotic configuration of chilled oocytes was observed either after they had been cooled and held for various times or after they had been cooled, warmed, and incubated at 37°C. The distribution by meiotic stage after chilling alone and after chilling and incubation at 37°C is shown in Figure 2. There were no differences in the meiotic status of oocytes FERTILITY & STERILITY威
Songsasen. Cooling sensitivity of rhesus monkey oocytes. Fertil Steril 2002.
cooled and held for various times before being fixed without warming, as the majority of oocytes exhibited pro-MI and MI configurations (Fig. 2A). Therefore, the data from different holding periods were pooled. The percentages of meiotic stages of chilled oocytes fixed without being warmed were not different from those of the control oocytes fixed at 4 hours after oocyte collection. As was true of those oocytes that were fixed without being warmed, the meiotic status of oocytes fixed after being warmed and incubated exhibited a similar pattern regardless of the holding period at 0°C. However, when chilled oocytes were incubated at 37°C for 60 minutes, their meiotic stages appeared to significantly progress compared with those fixed without being warmed (P⬍.01; Fig. 2B). That is, we compared the distribution in Figure 2A with that in Figure 2B and found a significant difference between them. Finally, the distribution of meiotic stages of chilled oocytes fixed after being warmed and incubated for 60 minutes was not significantly different from that of the control oocytes fixed at 8 hours after collection.
Configuration of Tubulin and Chromosome of Chilled Oocytes The FIS of control oocytes from each female ranged from 2 to 3. The FIS appeared to be dependent on the meiotic stage of the oocytes. The fluorescence of AI/TI and MII stage oocytes was the highest, whereas that of GVBD was the lowest (data are not shown). Cooling oocytes to 0°C and holding them at that temperature for only 1 minute resulted in depolymerization of 821
FIGURE 2 (A) Meiotic status of rhesus oocytes cooled to 0°C and held for 1 (n ⫽ 15), 5 (n ⫽ 16), 10 (n ⫽ 14), or 30 minutes (n ⫽ 10). (B) Meiotic status of rhesus oocytes cooled to 0°C, held for 1 (n ⫽ 17), 5 (n ⫽ 14), 10 (n ⫽ 20), or 30 minutes (n ⫽ 15) and then warmed to 37°C and incubated for 60 minutes.
Songsasen. Cooling sensitivity of rhesus monkey oocytes. Fertil Steril 2002.
tubulin. There were significant decreases in fluorescence intensity of tubulin in chilled oocytes (Table 1). However, oocytes from each monkey varied in their sensitivity to chilling. Complete tubulin depolymerization occurred in oocytes from seven of eight monkeys after the oocytes were cooled to 0°C for 1 minute, whereas those from the eighth monkey exhibited only slight depolymerization of tubulin after being cooled to 0°C for the same period. Repolymerization of tubulin occurred after chilled oo-
cytes were warmed and incubated for 60 minutes. The fluorescent intensity of tubulin increased after warming and incubation, but these increases were not significantly different from those in oocytes fixed without warming (Table 1). Moreover, the fluorescence indices of tubulin in chilled and warmed oocytes were significantly lower than those of the controls (Table 1). Figure 3 shows the fluorescent image of a control oocyte held at 37°C and of two chilled oocytes. Figure 3A shows a
TABLE 1 Effect of chilling on fluorescent intensity index, chromosome alignment, and organization of meiotic spindles in rhesus monkey oocytes. Time at 0°C (minutes) 0 1 5 10 30 1 5 10 30
Time at 37°C (minutes)
Oocytes (n)
FISa
Chromosomes aligned (%)
Bipolar spindle (%)b
120 0 0 0 0 60 60 60 60
22 17 16 15 10 17 14 20 16
2.2c 0.3d 0.4d 0.2d 0.3d 1.2d 1.3c,d 0.9d 0.9d
22 (100)c 10 (59)d,e 13 (81)c,d 7 (47)d,e 6 (60)d,e 6 (35)e 6 (43)d,e 9 (45)d,e 7 (44)d,e
17/17 (100)c 2/9 (22)d,e 2/13 (15)e 3/6 (50)d,e 1/9 (11)e 8/11 (73)c,d 4/12 (33)d,e 3/13 (23)e 5/13 (38)d,e
a
FIS is the fluorescence intensity index estimated by microscopic examination of each oocyte. The fraction shows the oocytes at MI, AI/TI, and MII oocytes in which there was a bipolar spindle. c,d,e Different letters within the same column indicate significant differences (P⬍.05). b
Songsasen. Cooling sensitivity of rhesus monkey oocytes. Fertil Steril 2002.
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FIGURE 3 Fluorescent micrographs of rhesus oocytes: (A) metaphase I oocyte exhibiting chromosomes (yellow) aligned between two spindle poles (green); (B) chilled oocyte exhibiting multipolar spindles (green) and three sets of chromosomes (red) at each end; (C) chilled oocyte exhibiting irregularly shaped tubulin (green) and chromosomes (yellow) aligned underneath the tubulin. Bar ⫽ 10 m.
Songsasen. Cooling sensitivity of rhesus monkey oocytes. Fertil Steril 2002.
normal oocyte at MI (control) with the chromosomes aligned between two spindle poles. After oocytes had been chilled, they exhibited various abnormal configurations of tubulin and chromosomes. These abnormalities included multipolar spindles (Fig. 3B) and irregularly shaped tubulin with dispersed chromosomes (Fig. 3C), as well as complete and apparently irreversible depolymerization. Abnormal distribution of chromosomes in chilled oocytes was observed even when oocytes were cooled to 0°C for only 1 minute (Table 1). Longer exposure to 0°C increased the number of oocytes exhibiting abnormal distribution of chromosomes as well as the extent of damage to the meiotic FERTILITY & STERILITY威
spindles. Restoration of organized tubulin within the oocytes after incubation at 37°C also varied among females. For example, none of nine oocytes collected from female E540 exhibited a normal bipolar spindle after they were cooled to 0°C and then warmed and incubated at 37°C for 60 minutes. However, seven of nine oocytes obtained from female R037 regained their normal bipolar spindle after being chilled and incubated at 37°C for 60 minutes.
DISCUSSION In this study, we have demonstrated that rhesus monkey oocytes are similar to those of the human and of cattle in that 823
they are very sensitive to chilling injury. Cooling oocytes to 0°C and holding them at that temperature for only 1 minute resulted in complete depolymerization of tubulin in oocytes of seven of eight monkeys. Incubation of chilled oocytes at 37°C resulted in restoration of normal organization of tubulin and nuclear maturation in some oocytes. However, even after being incubated at 37°C, many of the chilled oocytes exhibited an abnormal organization of tubulin; this was accompanied by abnormal configurations of the chromosomes. We have also found that there were significant differences in chilling sensitivity of oocytes obtained from different females. We have no explanation for these differences among females. Many factors contribute to freezing sensitivity of mammalian oocytes (1, 2). Sensitivity of oocytes to cooling to low temperatures is believed to be a major factor contributing to the low survival of cryopreserved oocytes, since with conventional slow cooling methods, oocytes must be exposed to deleterious temperatures (ranging from 20°C to 0°C) for several minutes (1, 2, 9, 10). Exposure of MII oocytes to these temperatures affects the cytoskeletal organization and chromosome configuration (9 –15) and causes premature release of cortical granules that may compromise subsequent fertilization and embryonic development (10, 21). Evidence that rhesus monkey oocytes are sensitive to chilling injury was that even a 1-minute exposure of oocytes to 0°C caused the average fluorescent intensity index to decrease from 2.2 in control oocytes to only 0.2 to 0.4 in the chilled ones. This suggests that rhesus oocytes are more sensitive than human oocytes, since the fluorescent intensity index of human oocytes that were treated similarly declined only slightly after being cooled to 0°C for 1 minute (15). Repolymerization of tubulin and progression of nuclear maturation were observed in some rhesus oocytes after they were warmed and incubated at 37°C for 60 minutes. However, even after warming, a high proportion of oocytes exhibited abnormal organization of tubulin and irregular chromosomal configurations. Because of limitations of the epifluorescent microscope, we were only able to observe the organization of tubulin within oocytes in which a meiotic spindle was present (MI to MII stage). For those oocytes, we grouped abnormal tubulin organization into three categories: multipolar spindle, irregularly shaped tubulin, and complete tubulin depolymerization. Abnormal chromosome configurations were found in a high proportion of oocytes, especially after warming and incubation. This irreversible damage after brief chilling indicates that rhesus monkey oocytes are more like those of the human and bovine than like those of the mouse. In the mouse, chilled oocytes regained normal meiotic spindles after they were warmed to 37°C for 60 minutes (12). In contrast, fewer than half of human oocytes exposed to room temperature for 10 minutes returned to a normal 824
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appearance after 4 hours at 37°C (14). In the bovine, 90% of oocytes exhibited abnormal meiotic spindles after being cooled to 25°C for 60 minutes followed by incubation at 37°C for 30 minutes (13). In addition to centrosomes associated with the meiotic spindle, mouse oocytes contain microtubule organizing centers in the cortical cytoplasm (2, 9). Although a microtubule-enhancing drug, taxol, can induce formation of microtubule asters in the cytoplasm of human oocytes (22), cytoplasmic microtubules are not normally observed in the cortical cytoplasm of human and rhesus oocytes (14, 23). The absence of centrosomes and microtubules in the cortical cytoplasm (such as those that occur in mouse oocytes) is believed to be responsible for irreversible damage to the meiotic spindle after the cooling of human and bovine oocytes to low temperatures (13, 14). Perhaps this also may have contributed to chilling sensitivity of the meiotic spindle in rhesus monkey oocytes observed in the present study. In human and bovine oocytes, alterations of the meiotic spindle from cooling are influenced by the duration of exposure (13, 15). In the present study, holding oocytes at 0°C for longer periods did not substantially increase the amount of damage observed. This suggests that rhesus monkey oocytes are especially sensitive to cooling, since holding them for only 1 minute at 0°C was sufficient to cause irreversible damage in a majority of the oocytes. The developmental competency of in vitro–matured rhesus oocytes is rather low compared with their in vivo– matured counterparts (18). Therefore, we obtained oocytes for this study from rhCG-stimulated monkeys. The majority of the oocytes had already resumed meiosis with approximately 10% of the oocytes at the GVBD stage within 4 hours after oocyte retrieval. So far, little is known about the effects of cooling on the configuration of chromosomes in GVBD-stage oocytes. In the present study, six of 18 GVBD-stage oocytes exhibited an abnormal chromosomal configuration with chromosomal clumping. Perhaps the absence of the nuclear membrane in GVBD-stage oocytes might render them more sensitive to chilling injury. However, the small number of GVBD stages oocytes examined in the present study is insufficient to draw any firm conclusions regarding their sensitivity to chilling injury. We also found that there were differences in chilling sensitivity of oocytes obtained from different females. Among the eight females used in the present study, two females (N821 and R037) produced oocytes that appeared to be very resistant to chilling injury. Between 60% and 70% of the oocytes exhibited normal tubulin organization and chromosomal configuration after being chilled, warmed, and incubated at 37°C for 60 minutes. In contrast, oocytes from another female (E540) seemed to be extremely sensitive to cooling, as none of her oocytes at the MI or at MII stage exhibited normal spindle organization after being chilled and then incubated at 37°C.
Cooling sensitivity of rhesus monkey oocytes
Vol. 77, No. 4, April 2002
Characteristics of mammalian gametes are likely influenced by their genetic backgrounds. Using oocytes from outbred Institute of Cancer Research (ICR) mice, Benson and Critser (24) found that there were differences in water permeability and its activation energy of oocytes obtained from different females. In contrast, oocytes from different females of an inbred strain do not exhibit such differences (25). This difference between inbred and outbred mice is prima facie evidence that membrane differences among females have a genetic basis, although factors contributing to these differences among individuals have not been elucidated. It is possible that differences in membrane characteristics could play a role in differences among individuals in the freezing sensitivity of their gametes. However, formation of the meiotic spindle and alignment of chromosomes during meiosis in mammalian oocytes are regulated by intracellular factors, for example, the amount of free tubulin contained within the cytoplasm and its nucleating capacity (17). Therefore, it is possible that oocytes obtained from different females contain different amounts or types of these cytoplasmic factors that modulate spindle organization. In species whose oocytes are very sensitive to chilling injury, such as the bovine and human, ultrarapid cooling has proven to be the method of choice for oocyte cryopreservation. The rationale of this method is to cool oocytes at very high rates to enable them to pass through the damaging temperature zone in the fluid state fast enough to circumvent the chilling injury (5, 7, 8, 26). Martino et al. (26) demonstrated that significantly more bovine oocytes that were cryopreserved by ultrarapid cooling on electron microscope grids developed into blastocysts after IVF, compared with those cooled much less rapidly. Recently, Wu et al. (27) reported a human pregnancy after transferring embryos produced from human oocytes also cryopreserved by ultrarapid cooling on electron microscope grids. Vajta et al. (5) cryopreserved bovine oocytes in very fine plastic straws and produced three live calves. Using this vitrification technique described by Vajta et al. (5), Kuleshova et al. (8) reported the birth of a human baby after transferring embryos produced from cryopreserved in vitro–matured oocytes. In conclusion, the present study has shown that rhesus oocytes are at least as sensitive to chilling injury as those of humans and cattle. Therefore, we conclude that cryopreservation of rhesus monkey oocytes may require that they be cooled extremely rapidly to circumvent damage caused by chilling injury.
Acknowledgments: The authors thank R. Harrison, Ph.D., and SueAnn Schneider, B.A., of the TRPRC, as well as Melissa Johnston, B.S., of the Audubon Research Center, for their assistance throughout the study.
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