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Dynamic Imaging of the Metaphase II Spindle and Maternal Chromosomesin Bovine Oocytes: Implications for Enucleation Efficiency Verification, Avoidanceof Parthenogenesis, and Successful Embryogenesis¹

^a Oregon Regional Primate Research Center, Oregon Health Sciences University, Beaverton, Oregon 97006 ^b Departments of Obstetrics & Gynecology and ^c Cell & Developmental Biology and Center for Women's Health, Oregon Health Sciences University, Portland, Oregon 97201

	ABSTRACT

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Manipulations of DNA and cellular structures are essential for the propagation of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that improves live dynamic imaging of the unfertilized bovine oocyte's meiotic spindle microtubules with microinjected polymerization-competent X-rhodamine-tubulin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so that the maternal chromosomes can be verifiably removed to make enucleated eggs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enucleation removed the smallest amount of cytoplasm (4–7%) and was 100% efficient only when performed under continuous fluorescence, i.e., longer fluorescence exposure. This was in part due to the finding that the second metaphase spindle is frequently displaced (60.7 ± 10%) from its previously assumed location subjacent to the first polar body. Removal of as much as 24 ± 3% of the oocyte cytoplasm underneath the polar body, in the absence of fluorochromes, often resulted in enucleation failure (36 ± 6%). When labeled oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3%), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oocytes (37%). This suggests that longer wavelength fluorochromes can be employed for live visualization of metaphase spindle components, verification of their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus "cloning" success, without compromising the oocyte's developmental potential after activation.

	INTRODUCTION

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Efficiency of enucleation procedure prior to nuclear transfer is of crucial importance to avoid aneuploidy abnormalities with its detrimental effects on later development, to eliminate any genetic contribution of the recipient cytoplasm, and for excluding the possibility of parthenogenetic activation and embryo development without the participation of the newly introduced nucleus. Enucleation has been accomplished successfully in a range of species by labeling the oocyte DNA with Hoechst 33342 [1,2]. A report in cattle research has shown that exposure of oocytes to UV irradiation for 10 sec has no effect on viability of nuclear transfer embryos and the production of live calves [3]. Similarly, irradiation of rabbit [4] and Xenopus oocytes [5] for periods shorter than 15 sec showed no effect on developmental potential. However, exposure to UV light for > 30 sec causes a loss in membrane integrity, decreases methionine incorporation, alters protein synthesis patterns in bovine oocytes [2], decreases viability in rabbit oocytes [4], and causes abnormal development in 30% of irradiated Xenopus oocytes [5]. In preparation of the recipient cytoplast, the possibility of damaging effects of even very short UV exposure on oocyte cytoplasm and on the remaining mitochondrial DNA needs to be considered.

To avoid the damaging effect of UV illumination, enucleation can be accomplished by aspiration of the first polar body and the underlying oocyte cytoplasm in the absence of fluorochromes [6,7]. During this "blind" enucleation, as much as 30% of the oocyte cytoplasm is removed [8,9]. In addition, removal of the cytoplasm directly beneath the first polar body does not always assure the removal of all the chromatin since between 10% [8,10] and 25% [11] or more [9] of these oocytes may still contain residual DNA. Bisecting oocytes and discarding the half that contains the chromatin [10,12] leaves an oocyte with a greatly reduced cytoplasmic volume for future manipulations. Dramatic reduction of the oocyte volume leads to a decrease in blastocyst development after nuclear transfer [13,14] and lower blastocyst cell number [12]. Visualizing oocyte DNA prior to, or subsequent to, its removal (enucleation) from the oocyte aids in locating the metaphase II chromatin. This, in turn, allows one to remove very little cytoplasm surrounding the spindle and preserve the oocyte volume without compromising the enucleation efficiency. Short wavelength, UV excitable fluorochrome Hoechst 33342 (excitation wavelength 350 nm) is the only fluorochrome routinely used to label oocyte chromatin. Hoechst transfers high amounts of energy to the biological material upon excitation, and the possible long-term damaging effects of UV illumination on the developmental potential of cytoplasts have to be considered [15], even though live offspring have been born from oocytes enucleated under UV [3,16].

Availability of new fluorochromes and fluorophores for live visualization of either DNA or metaphase spindle components prompted us to examine whether alternative dyes can be successfully used for visualization of maternal chromatin without compromising the oocyte's developmental competence. Since UV excitation of living oocytes may compromise embryo survival, the use of vital green and red-fluorescent DNA and microtubule dyes was explored. We examined spatial relationships between the first polar body and the second metaphase plate and its effect on enucleation efficiency. Lastly, we investigated the effect of various fluorochromes and exposure to fluorescence on developmental potential of embryos after parthenogenetic activation.

	MATERIALS AND METHODS

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Oocyte Maturation and Labeling

Bovine oocytes were obtained and matured according to previously published protocols [17]. Mature oocytes were placed in TALP-Hepes and then either labeled with 1 µg/ml Hoechst 33342 (Sigma Chemical Co., St. Louis, MO) (excitation wavelength 350 nm; emission 460 nm) or 1 µg/ml Sybr14 DNA fluorochrome (Molecular Probes, Eugene, OR) (excitation wavelength 485 nm; emission 520 nm), or injected with 2 mg/ml polymerization-competent rhodamine conjugated bovine brain tubulin (Cytoskeleton, Inc., Denver, CO) (excitation wavelength 550–580 nm; emission 605 nm). Prior to enucleation, the diameter of the oocyte was recorded by ocular micrometer. The oocyte's DNA or spindle was visualized by excitation with either a UV (Hoechst 33342), FITC (Sybr14), or TRITC (rhodamine-tubulin) filter. An enucleation pipette (22 µm i.d.) was used to remove the spindle/chromosomes while the oocyte was fluorescently illuminated. The enucleation procedure was performed in TALP-Hepes, supplemented with 7.5 µg/ml cytochalasin B (Sigma), and required approximately 10 sec to complete, regardless of the label used. The unlabeled control oocytes were enucleated by removing the first polar body and underlying cytoplasm without visualizing the DNA or the spindle. These oocytes were labeled after enucleation with Hoechst 33342 and examined by epifluorescence. After enucleation, the karyoplast in the enucleation pipette was expelled and the diameter of the karyoplast measured. The volume of the karyoplast was calculated by 4/3r³ and compared to the calculated volume of the corresponding oocyte. Enucleated oocytes were allowed to recover for 30 min at 37°C before assessing survivability by inverted Hoffman modulation contrast optics (HMC). Manipulations were performed on a Nikon Diaphot (Nikon, Inc., Melville, NY) equipped with HMC, epifluorescence with appropriate filters and Narishigi micromanipulators (Narishige, USA, Sea Clift, NY). Each experiment was repeated three times.

Measurement of Angles Between the Metaphase Spindle and the First Polar Body

Bovine oocytes were double labeled with Hoechst 33342 and rhodamine tubulin. Fifteen min after microinjection, the oocytes were imaged live on an upright Axiophot (Zeiss, Thornwood, NY) at x200 under epifluorescence, and images were recorded. The distance between the polar body and the metaphase II spindle, and the difference in the depth between the focal planes of the two structures were measured and recorded. The angles between the spindle and the polar body were calculated by -a² + b² + c²/2bc = cosA, where a = measured distance between the actual position of the metaphase chromatin and its projection to the cortical site, b = measured distance between the polar body and the projected cortical position of the metaphase chromatin, and c = calculated by a² - b² = c².

Fixation and Staining for Residual DNA

To confirm the results of live imaging of DNA removal, all the enucleated oocytes were fixed and examined for possible residual DNA. Zonae were removed from the oocytes by a brief treatment (2–7 min) with 0.5% Pronase (Sigma) prepared in TALP-Hepes. After a 30-min recovery at 37°C, zona-free embryos were attached to polylysine-coated coverslips and fixed according to methods described by Simerly and Schatten [18]. DNA was fluorescently detected with 10 µg/ml Hoechst 33342 (Sigma). Coverslips were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA) and examined using conventional immunofluorescence equipped with a chilled CCD camera [18].

Activation and Embryo Development

Mature oocytes were labeled as described above. After labeling, they were exposed to fluorescence illumination using either UV (Hoechst 33342 labeled group), FITC (Sybr14 labeled group), or TRITC filter (X-rhodamine tubulin labeled group) for 30 sec. After exposure, the oocytes were washed and returned to culture. Four hours later, the oocytes were activated as described by Susko-Parrish et al. [19]. Briefly, the oocytes were incubated in 5 µM ionomycin (Sigma) for 4 min, washed in TALP-Hepes containing 30 mg/ml BSA for 5 min, and incubated with 1.9 mM 6-DMAP (Sigma) in CR1aa [20] for 4 h. After activation, the oocytes were washed twice and placed in CR1aa for embryo culture. The experiment was repeated three times. Rates of cleavage and embryo development were compared. Seven days after activation, embryonic nuclei were labeled with Hoechst 33342 in TALP-Hepes and counted.

Statistical Analysis

Percentages were transformed using arcsin transformation. Transformed data were analyzed by ANOVA and means compared by protected least-significant difference. Significance was estimated at the level of P = 0.05.

	RESULTS

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Live fluorescence examination of control oocytes enucleated using only HMC indicated 83% enucleation efficiency. Fixation and staining of the same oocytes revealed residual DNA that could not be detected with live imaging (Table 1). We frequently observed displacement of the second metaphase spindle from its expected position close to the first polar body. Even though the metaphase spindle remained at the oocyte cortex, the angle between the two structures, determined by the distance and focal plane depth between them, varied from 0 to 90 degrees (Fig. 1). The proportion of oocytes with spindles displaced 20° or more was high and did not differ between oocytes at 16–20 h and oocytes between 24–28 h after the initiation of culture (unpublished results). The amount of the oocyte cytoplasm removed during enucleation as estimated by HMC was reduced from 24% to 6% or less by continuous fluorescence imaging (Table 1). Metaphase chromatin could be detected with Sybr14, however, its fast photobleaching and the opacity of bovine oocyte cytoplasm made it less suitable for enucleation purposes in this species (Fig. 2). Consequently, errors in enucleation were observed. Labeling with polymerization-competent rhodamine-tubulin was found to be a reliable method for visualizing the second metaphase spindle. The injected tubulin became incorporated into the spindle within 15 min and could be continuously monitored during enucleation (Fig. 3). This fluorochrome did not appreciably bleach even after prolonged exposure to fluorescence. Regardless of the position of the first polar body, the oocytes were aligned such that the spindle could be removed with a very small amount of the oocyte cytoplasm. Using this labeling, the first polar body was not necessarily removed.

View larger version (45K): [in this window] [in a new window]   FIG. 1. Live imaging of spatial relationships between the metaphase II spindle and the first polar body in bovine. Three representative oocytes are shown. Rhodamin-tubulin became incorporated into the spindle within 15 min after microinjection (A1,B1,C1). Labeling with Hoechst 33342 (A2,B2,C2) revealed that the position of the first polar body did not colocalize with the expected position of the metaphase chromatin. The angle between the first polar body and the second metaphase spindle varied between 0–20° (A3), 20–40° (B3), and > 40° (C3). Numbers of oocytes examined and proportions (% ± SE) of oocytes observed within each category of displacement are indicated under each column (red is tubulin, blue is DNA). Measurements were performed on 113 randomly chosen oocytes in three replicates.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Bovine oocyte meiotic chromosomes labeled with Sybr14. PB, First polar body; MII, metaphase chromatin (green is DNA)

View larger version (13K): [in this window] [in a new window]   FIG. 3. Enucleation of a rhodamin-tubulin labeled bovine oocyte. The second metaphase spindle (arrows) is removed by aspiration into an enucleation pipette 15 min after microinjection of rhodamin-tubulin while continuously illuminated (red is tubulin). A) HMC and B–D) epifluorescent illumination

Development after parthenogenetic activation of prelabeled and illuminated bovine oocytes is presented in Table 2. Oocytes labeled with Hoechst and exposed to UV had significantly lower developmental rates than Sybr14, rhodamine-tubulin, or control oocytes. This difference could be observed at the time of the first cleavage, and only a small proportion of oocytes developed to the blastocyst stage. Cell number was the highest in control and rhodamine-tubulin oocytes (P > 0.05), decreased slightly in Sybr14, and was the lowest in Hoechst 33342 oocytes (P < 0.05).

	DISCUSSION

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During nuclear transfer (NT), several critical steps need to be executed in the correct sequence and with the utmost precision to ensure developmental success of newly created embryos. Progress has been made during the last decade in development of alternative procedures for individual steps during NT. Despite all the improvements made over the years, however, the success of embryonic development, implantation, and the birth of live offspring from embryos created by NT remains low with the exception of a recent report by Kato et al. [21].

The present report shows that alternative fluorochromes to Hoechst 33342 can be used for labeling of chromatin and/or microtubules in mammalian oocytes. We observed that the first polar body was frequently displaced from its expected position opposing the metaphase spindle in bovine oocytes. This suggests that removal of the oocyte cytoplasm underneath the polar body (blind enucleation) does not ensure that the chromatin has been removed. Errors in the enucleation of up to 30% of the oocytes in domestic animal species have been previously reported [11,22]. In the present study, live imaging of oocytes labeled with Hoechst 33342 after enucleation did not always reveal residual DNA in the oocyte cytoplasm. Densely granulated bovine cytoplasm contains high amounts of lipids that make live imaging unreliable unless the chromatin resides at the equatorial region of the oocyte. Frequently, unsuccessful enucleation in control oocytes in the present report was attributed to displacement of the first polar body, which was not associated with the post-follicular age of the oocytes at the time of enucleation, as observed previously by Bordignon and Smith [9].

Though very reliable, the main disadvantage for the use of rhodamine-labeled tubulin is the method of its delivery. The size of tubulin prevents its uptake across an intact oocyte plasma membrane, and it has to be microinjected into the oocyte cytoplasm. Even though the procedure itself is not technically demanding or time consuming, it requires that the oocyte plasma membrane be compromised. A very small proportion of oocytes in our present experiments was lost due to lysis after tubulin microinjection; however, injection did not seem to have any adverse effects on later embryonic development.

The present study agrees with previous reports that suggest a high frequency of unsuccessful enucleation attempts unless the maternal DNA is labeled and its location can be confirmed [9]. Our observation of the frequently displaced first polar body from the second metaphase spindle indicates that the polar body cannot be used as a reliable predictor of the location of the metaphase spindle. Removal of cumulus cells is required prior to oocyte manipulation, and one needs to account for the possibility that the method of cumulus cell removal could be affecting the relationship between the first polar body and the MII spindle. Our experiments show that exposure of the oocyte cytoplasm to epifluorescence illumination is required during the procedure to confirm that all the chromatin has been removed. In some cases, maternal chromosomes cannot be removed due to incomplete alignment or incomplete incorporation of all the chromosomes into the second metaphase plate (unpublished results). No deleterious effects on embryo development or cell number were observed after rhodamine-tubulin or Sybr14 oocytes were exposed to fluorescence. These two longer-wavelength fluorochromes allow lower energy transfer and are therefore considered less damaging to the oocytes' developmental potential. If so, the development of NT embryos may be improved by using cytoplasts obtained after enucleation under longer-wavelength excitation.

It is likely that the decrease in embryonic development in Hoechst-labeled oocytes in the present study was due to UV-induced damage of the maternal nuclear DNA and/or damage of cytoplasmic organelles and proteins by absorbing too much UV energy. When oocytes are irradiated with UV in the absence of Hoechst 33342, developmental potential is compromised (unpublished results), which indicates the detrimental effect of the UV and not of the dye itself. Even though the nuclear DNA is removed during oocyte enucleation, the possibility of damaging mitochondrial DNA, which is left in the recipient cytoplast, needs to be considered. Embryonic mitochondria are believed to be of maternal origin [23], and serious concerns can be raised about the suitability of UV excitation dyes for live imaging. Detrimental effects may go unnoticed during the early preimplantation period but may be deleterious during later development. A high degree of successful enucleation can be achieved with the use of long-wavelength DNA dyes for detecting meiotic chromosomes and/or meiotic spindle under epifluorescent illumination. Our experiments show that exposure of the oocyte cytoplasm to epifluorescence illumination is required during enucleation to confirm that all of the chromatin has been removed and to minimize the removed volume. At the same time, illumination with longer wavelength does not compromise oocytes' developmental potential.

	ACKNOWLEDGMENTS

 
We would like to thank NIDDK's National Hormone and Pituitary Program and Dr. Parlow for donation of oLH used in our studies and ORPRC's Christopher Payne for help in designing spatial relationship calculations and analyses.

	FOOTNOTES

 
First decision: 11 August 1999.

¹ This research was supported by NICHD/NIH through cooperative agreement U54 18185-16 as part of the Specialized Cooperative Centers Program in Reproduction Research. Other grant support from the NIH (NCRR, NICHD) and the USDA is acknowledged gratefully. The ORPRC is sponsored by a NIH/NCRR Regional Primate Research Center award.

² Correspondence: Gerald Schatten, ORPRC, 505 NW 185th Avenue, Beaverton, OR 97006. FAX: 503 614 3725; schatten{at}ohsu.edu

Accepted: August 26, 1999.

Received: June 29, 1999.

	REFERENCES

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