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Biology of Reproduction 66, 1342-1349 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Hypertonic Medium Treatment for Localization of Nuclear Material in Bovine Metaphase II Oocytes1

Ji-Long Liua, Li-Ying Sunga, Michele Barbera, and Xiangzhong Yang2,a

a Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oocytes enucleated at the second metaphase stage (MII) are often used as recipient cytoplasts for nuclear transfer. The oocyte's nuclear material has been traditionally removed blindly by aspirating the first polar body (Pb1) along with a portion of the cytoplasm. However, the Pb1-guided enucleation method is unreliable because the position of the Pb1 is variable. A previous study showed that pretreatment of mouse oocytes with 3% (0.09 M) sucrose allowed visualization of the metaphase spindle and chromosomes under standard light microscopy and led to a 100% enucleation rate. The same sucrose treatment, however, did not produce the same effect in bovine oocytes. In this study, we increased the concentration of sucrose to 0.3–0.9 M in PBS containing 20% fetal bovine serum (SPF) and found that the majority of the treated bovine oocytes (75%–86%) formed a small transparent bud into the perivitelline space, as compared with the 0.1 M sucrose (6%) or the no sucrose (0%) control groups. Staining of DNA with Hoechst 33342 revealed that these projections coincided with the position of the metaphase chromosomes in 100% of sucrose-treated oocytes, whereas only 31% of oocytes showed alignment of the position of Pb1 with their nuclear materials. Furthermore, 95% of oocytes treated in 0.3 M SPF were successfully enucleated by removing a small amount of cytoplasm adjacent to the projection. This is a significantly higher enucleation rate than that obtained by conventional Pb1-guided enucleation, even when a larger amount of cytoplasm was removed. For nuclear transfer, the enucleated oocytes treated with sucrose did not differ from the control oocytes in rates of fusion, cleavage, or development to blastocysts, or in the average cell numbers in blastocysts. This study demonstrated that 0.3 M sucrose treatment of bovine oocytes facilitates the localization of metaphase chromosomes under normal light microscopy and hence increases enucleation efficiency without compromising the in vitro development potential of cloned embryos by nuclear transfer.

early development, embryo, gamete biology, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear transfer (NT) involves injecting a donor nucleus into an enucleated oocyte [1]. Previous studies have shown that donor nuclei from different origins, such as embryonic blastomeres [2–11], fetal fibroblasts [12–18], and adult somatic cells [12, 19–31], support full-term development if introduced into enucleated oocytes. The success of NT depends on many factors such as the origin [19, 24], cell cycle stage [12, 32], passage number [15, 24, 26], and morphology [26] of donor cells, as well as the age and cell cycle stage of the recipient cytoplasm [32–34], micromanipulation technique [16, 18, 19, 28, 35], activation protocol [19, 22, 23], and in vitro culture [17]. To date, many steps in NT are still in need of improvement to increase its overall efficiency.

Enucleation of the oocyte is one of the key steps in NT [35–37]. Because the chromosomes in mammalian oocytes are invisible under common light microscopy, the position of the chromosomes is indirectly determined by the location of the first polar body (Pb1) or directly observed under UV light after staining of oocytes with a DNA-specific dye (e.g., Hoechst 33342). However, polar bodies often migrate from their place of origin and do not always remain in proximity to chromosomes [35–38]. Meanwhile, detrimental effects of DNA dyes and UV light exposure on oocytes and their developmental potential have been reported [38, 39].

A previous study has shown that pretreatment of mouse oocytes with 3% sucrose can help visualize the second metaphase stage (MII) spindle and chromosomes under standard light microscopy. The same treatment of bovine oocytes, however, did not have similar effects [40]. The objective of the present study was to design a suitable treatment for bovine oocytes to facilitate the localization of their MII spindles during enucleation without compromising their developmental potential. We modified the previous protocol and investigated whether sucrose treatment is applicable in bovine oocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Hypertonic Media

Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco PBS (DPBS; Gibco, Grand Island, NY) containing 20% fetal bovine serum (FBS; HyClone, Logan, UT) (P20F) was used as the standard manipulation medium. Stock hypertonic medium was P20F supplemented with 1.0 M sucrose (1.0 M SPF). Working concentrations of hypertonic media (0.1–0.9 M SPF) were obtained by diluting the stock 1.0 M SPF with P20F. All media were filter-sterilized through a 0.2-µm filter (Acrodisc; Pall Gelman Laboratory, Ann Arbor, MI), and the osmolarities were measured by a vapor pressure osmometer (Wescor Inc., Logan, UT).

Oocyte Collection and Removal of Cumulus Cells

Bovine cumulus-oocyte complexes (COCs) were placed in maturation medium [41] in 1.8-ml vials and shipped from TransOva Genetics (Sioux Center, IA) in a portable incubator at 39°C. COCs were generally received 22–24 h after the onset of maturation. The COCs were denuded of cumulus cells by vortexing and repeated pipetting in 0.1% hyaluronidase (Sigma) in PBS. Oocytes that extruded Pb1 were selected.

Chromatin Staining and Localization

Bovine oocytes were stained for DNA with 5 µg/ml Hoechst 33342 (Sigma) in P20F or in 0.1–0.9 M SPF. The positions of chromosomes relative to cellular projections (detailed in Results) or Pb1 were confirmed under UV and normal light. When the distance between the chromosomes and projection or Pb1 was within 20 µm (about twice the thickness of the zona pellucida), the chromosomes and projection or Pb1 were considered to have the same position.

Enucleation

Control bovine oocytes were enucleated according to our laboratory's routine protocol [42]; all micromanipulation tools were self prepared in our laboratory. Briefly, the oocyte was secured with a holding pipette, and a small cut was created in the zona pellucida above the Pb1 with an enucleation needle. Ten percent (group 0S10) to 30% (group 0S30) of the cytoplasm surrounding the Pb1 was removed by pressing the oocyte with an enucleation needle. In the groups treated with hypertonic media, bovine oocytes were enucleated according to a modified protocol. Briefly, the location of the spindle was guided by the position of the projection, instead of the position of Pb1, and a cut in the zona pellucida was made above the projection. The holding pipette, rather than the enucleation needle, was then used to press the oocyte to remove approximately 10% of the nuclear material (group 3S10, Fig. 1). All oocytes were then transferred to media containing Hoechst 33342, and enucleation was confirmed by UV light microscopy (Nikon) at 200x magnifications.



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  		FIG. 1. Enucleation of bovine oocytes in DPBS containing 20% FBS containing 0.3 M sucrose (0.3 M SPF). A) The oocytes is fixed by holding a pipette with the projection positioned at 12 o'clock;. B) A slit is made on the zona pellucida over the projection with the enucleation needle. C) The oocyte is rubbed against the holding pipette to make a cut on the zona pellucida. D) The oocyte is pressed with the holding pipette. E) About 10% of the cytoplasm surrounding the projection is expressed. F) The oocyte is released with the enucleation needle from the bottom of the holding pipette. Magnification x90

NT, Oocyte Activation, and Embryo Culture

Adult bovine somatic cells were cultured as previously reported [26]. In brief, a skin biopsy was obtained from the ear of a 13-year-old cow, minced, and cultured in Dulbecco modified Eagle medium (DMEM, Gibco) containing 10% FBS at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Fibroblasts at passages four to six after 5 days of serum starvation were detached by 0.25% (w/v) trypsin and 1 mM EDTA and cultured in starvation media (DMEM containing 0.5% FBS) for 2 h. Somatic cells were injected into the subzonal space of enucleated oocytes by a flat-tipped transfer pipette (internal diameter, 18 µm). For fusion and activation, oocyte-somatic cell pairs were transferred into Zimmerman fusion medium (reviewed in [43]) in a 2-mm fusion chamber and activated by one pulse of 1.3 kV/cm for 25 µsec in a BTX 200 Electro Cell Manipulator (Biotechnologies & Experimental Research Inc., San Diego, CA). They were then placed in CR1aa medium [44]. Half-an-hour after the electric pulse, oocytes in all groups were further activated by incubation in CR1aa medium containing 10 µg/ml cycloheximide and 2.5 µg/ml cytochalasin D for 1 h, then in CR1aa medium containing 10 µg/ml cycloheximide for an additional 4 h. After this step, oocytes were washed 5 times and cultured in CR1aa containing 3 mg/ml BSA in 5% CO2, 5% O2, and 90% N2 at 39°C. Two days later, embryos were cocultured on a cumulus cell monolayer in CR1aa containing 5% FBS.

Statistical Analysis

All data were analyzed by the chi-square test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
After incubation in 0.3–0.9 M SPF (671–1587 mOsm) for 5–10 min, most bovine oocytes (75%–86%) extruded a visible projection; this percentage was significantly higher than that in oocytes incubated in 0.1 M SPF (6%) or in control oocytes incubated in P20F alone (0%). On examination under UV light, the projections colocalized with chromosomes in all of the oocytes (100%), whereas Pb1 colocalized with chromosomes in only 31% of the oocytes (Table 1 and Fig. 2). Among those oocytes that did not form a projection after incubation in 0.3–0.9 M SPF, more than half had poor morphology (data not shown).


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  		TABLE 1. Positions of metaphase chromosomes relative to the projection induced by sucrose treatment or Pb1



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  		FIG. 2. Relative locations of Pb1, cellular projection, and nuclear materials of bovine oocytes. The first polar body (p) was not always in close proximity to the position of chromosomes (c) in bovine oocytes, but the projection (*) in oocytes after incubation in DPBS containing 20% FBS (P20F) containing 0.3 M (B, B'), 0.5 M (C, C'), or 0.9 M (D, D') sucrose colocalized with chromosomes. No projections were formed in oocytes incubated in sucrose-free P20F medium (A, A'). A–D, Under white light; A'–C', under UV light. Magnification x200

We compared the enucleation efficiency of bovine oocytes of 3 protocols (Table 2 and Fig. 3). In groups 0S30 and 0S10, oocytes were incubated in P20F, and 30% or 10% of the cytoplasm beneath Pb1 was removed, respectively. In group 3S10, oocytes were incubated in 0.3M SPF, and 10% of the cytoplasm surrounding the projection was extracted. In group 3S10, 89% of the good-quality oocytes had a visible projection. Most of them (95%) were successfully enucleated. In contrast, only 33% and 61% of oocytes in groups 0S10 and 0S30, respectively, were successfully enucleated. When compared on the basis of total numbers of oocytes (with and without projections), the enucleation efficiency in group 3S10 was still significantly higher than that in groups 0S10 and 0S30, individually (84% vs. 33% and 61%, P < 0.05).


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  		TABLE 2. Enucleation efficiencies of bovine oocytes using different protocols



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  		FIG. 3. Amounts of cytoplasm removed during enucleation. Amount was 30% (A, A') or 10% (B, B') of the cytoplasm beneath the first polar body in bovine oocytes incubated in DPBS containing 20% FBS (P20F), and 10% of the cytoplasm surrounding the projection in 0.3 M SPF (C, C'). A–C, Under regular light; A'–C', under UV light. P, First polar body; *, chromosomes. Magnification x125

To determine the effect of hypertonic treatment on embryo development, bovine oocytes were incubated in 0.3 M SPF or P20F (as a control) for 1 h at room temperature and then activated by electric pulse or chemicals. The rate of cleavage (70% vs. 71%, P > 0.05), rate of development to blastocysts (39% vs. 38%, P > 0.05), and average cell number of blastocysts (100 vs. 99, P > 0.05) between these two groups were not significantly different (Table 3).


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  		TABLE 3. Development of parthenogenetically activated bovine oocytes after sucrose treatment for 1 h

Additionally, we carried out somatic cell NT using two enucleation protocols. Data from groups 3S10 and 0S10 indicated that there were no significant differences between the two groups in fusion rate (47% vs. 43%, P > 0.05), cleavage rate (79% vs. 75%, P > 0.05), blastocyst rate (32% vs. 31%, P > 0.05), or average cell number of blastocysts (79 vs. 81, P > 0.05) (Table 4 and Fig. 4).


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  		TABLE 4. Development of bovine oocytes enucleated by different protocols and transferred with bovine adult fibroblasts



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  		FIG. 4. Safety of sucrose treatment for nuclear transfer. A) Enucleated bovine oocytes in 0.3 M SPF; note the cytoplasmic volumes. B) Oocytes reach the normal volume after recovering in isotonic P20F medium for 5 min. C) The chromosomes are squashed out, the same field as shown in B. D–F) Hatched bovine blastocysts derived from transferring adult fibroblasts to oocytes enucleated after sucrose treatment. E, F) A different focus of the bovine blastocysts stained with Hoechst 33342 under UV light showing the stained nuclei. Magnification x150


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many different types of cells, including those derived from embryos and fetuses, as well as from adult individuals, have been used as donors in NT; however, recipient oocytes are usually limited to those in the MII stage. For example, MII enucleated oocytes from mice fused with cumulus cells, tail-tip fibroblasts, or cultured embryonic stem cells have led to the birth of cloned offspring [19, 20, 45], whereas enucleated mouse zygotes injected with the cumulus cells are blocked at the two- to four-cell stage [46]. This suggests considerable effects on NT efficiency of the stage of the recipient cytoplasm [47–50].

Because of the opaqueness of the cytoplasm, the MII spindle and chromosomes in bovine oocytes are invisible under standard light microscopy. The traditional solution was to identify the position of the chromosomes indirectly by the location of Pb1. It was assumed that the chromosomes lie directly beneath the area in the perivitelline space where Pb1 was extruded. However, Pb1 may migrate away from its original position. Our data in the present study showed that only 31% of oocytes have their nuclear materials in proximity to Pb1, supporting similar conclusions of previous studies [35–38]. Therefore, it is not surprising that when using Pb1 to determine the position of the chromosomes, the enucleation rate was low, even with the removal of up to 30% of the cytoplasm.

An alternative method for estimating the position of the chromosomes indirectly is through the localization of the second polar body (Pb2) [35–37]. Because Pb2 is expelled shortly after activation, Pb2 remains closer to the nuclear materials as it has not had sufficient time to migrate. The enucleation rate improved to approximately 90% even though a smaller volume (approximately 10%) of cytoplasm was removed. This method has been successfully applied in the cloning of goats [16].

Since there is wide application of MII oocytes in NT, it is still necessary to explore methods for improving enucleation at this stage. It is preferred to directly visualize the MII spindle and chromosomes. Labeling the oocyte's chromosomes with Hoechst 33342 has been widely used in many species. However, damage resulting from exposure to this dye and UV light has been widely reported [36–39]. Recently, Dominko et al. [38] improved enucleation efficiency to 100% through dynamic imaging of the MII spindle and chromosomes. They microinjected polymerization-competent X-rhodamine-tubulin and/or a vital long-wavelength excitable DNA fluorochrome (Sybr14) and thus visualized the chromosomes under regular light. Such manipulations did not seem to harm oocytes or reduce their developmental potential. The complexity of the microinjection step, however, limits its practical application.

We have previously found that 0.09 M sucrose solution pretreatment of mouse oocytes can aid in the visualization of chromosomes under standard light microscopy and thus significantly improve enucleation efficiency [40]. This finding was based on the observation that good-quality oocytes in a 0.09 M sucrose medium formed a swelling around the metaphase chromosomes and an 8- or 0-shaped transparent area [40]. We attempted to apply the same protocol to bovine oocytes but obtained no obvious effect. However, when the concentration of sucrose was increased to 0.3 M, an obvious projection was visible in most bovine oocytes (Fig. 1 and Table 1). When the sucrose concentration was increased from 0.3 to 0.9 M, the number of oocytes exhibiting a projection remained unchanged. More importantly, the location of the cellular projection always reliably indicated the position of the chromosomes.

Hypertonic sucrose solution caused the oocytes to shrink and changed the concentration of cytoplasm proteins, which may induce further changes in membrane properties and affect the dynamics of the cytoskeleton [40, 51]. The sucrose-induced projection may have resulted from the interaction between cortical actin and the attached spindle and/or chromosomes. The projection seen in bovine oocytes, or the swelling and transparent area observed in mouse oocytes, after sucrose treatment illustrates that oocytes from different species have different reactions to sucrose treatment. For example, 3% sucrose (0.09 M) is sufficient for the visualization of the chromosomes in mouse oocytes while having no obvious effect on bovine oocytes. Morphologically, the projection caused by 0.3 M sucrose in bovine oocytes showed a peaklike shape and a slightly lighter color in the projection than that in the other part of ooplasm (Fig. 2, B–D), whereas mouse oocytes treated with 0.09 M sucrose exhibited only a round, smooth swelling and transparent area. These differences might reflect the different cytoskeletal organization among species.

Similar to the findings in mice [40], about one tenth of poorer-quality cattle oocytes shrank in P20F medium and became deformed when incubated in the sucrose solution (data not shown). The deformity may be caused by the uneven changes of the heterogeneous cytoplasm in oocytes with poor morphology.

The techniques used in micromanipulation are important but easily ignored factors in NT. Traditionally, oocytes treated with cytochalasin B have been enucleated by aspiration of a portion of their cytoplasm through a bevel-tipped pipette. Because aspirated cytoplasm is generally fragile, it is not feasible to stain the discarded cytoplasm to confirm enucleation. An alternate method used in our laboratory is to make a cut in the zona pellucida directly above the Pb1 (first developed by Tsunoda et al. [3] in mice oocytes) and to then compress the oocyte by applying pressure with a glass-cutting needle to expel the cytoplasm [42]. The expelled cytoplasm remains intact and therefore is suitable for staining to confirm enucleation without subjecting the oocytes themselves to harmful dye and UV light. We have also found this method to be preferable because incubating oocytes in cytochalasin B becomes unnecessary. However, after incubation in hypertonic sucrose media, bovine oocytes shrank (Fig. 2), and their elastic properties decreased, which increased the difficulty of compressing them with a slim glass needle. We therefore modified the protocol and applied pressure to the oocytes with the holding pipette (flatter and thicker) instead of the enucleation needle (pointed and thin) (Fig. 1). With practice, we can easily enucleate with this modified technique without slowing down the micromanipulation.

The changes in oocytes caused by incubation in hypertonic sucrose solution were reversible. We observed that bovine oocytes enucleated in 0.3 M SPF medium recovered to their normal volume in 10 min after a washing and incubation in sucrose-free isotonic medium (Fig. 4, A and B). This study has also shown that a 1-h exposure to 0.3 M SPF is harmless to the parthenogenic development of bovine oocytes. The fusion rate and the developmental potential of oocytes enucleated in hypertonic sucrose medium were not compromised when constructed with somatic cells (Tables 3 and 4 and Fig. 4, C and D). This is not surprising since sucrose treatment has been widely used in cryopreservation of gametes and in many types of manipulations in which enlargement of the subzonal space is desirable, such as sperm injection and blastomere insertion into oocytes [52–54].

In conclusion, 0.3–0.9 M sucrose treatment induces the formation of a projection around the chromosome and spindle area in most bovine MII oocytes and hence allows direct localization of the nuclear materials. The enucleation efficiency of bovine MII oocytes is improved when conducted in 0.3 SPF, without compromising the fusion rate or in vitro development after reconstruction with adult somatic cells. The application of sucrose pretreatment of bovine oocytes for efficient enucleation has the potential to significantly improve the overall NT efficiency.


   ACKNOWLEDGMENTS
 
The authors wish to thank Marina Julian and Dr. X Cindy Tian for critical reading of this manuscript.


   FOOTNOTES
 
First decision: 20 November 2001.

1 This manuscript is a scientific contribution (No. 2073) of the Storrs Agricultural Experiment Station at the University of Connecticut. Back

2 Correspondence: X. Jerry Yang, Department of Animal Science, University of Connecticut, 1390 Storrs Rd., U-163, Storrs, CT 06269-4163. FAX: 860 486 0534; jyang{at}canr.uconn.edu Back

Accepted: December 3, 2001.

Received: October 31, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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J.-L. Liu, L.-Y. Sung, X. C. Tian, and X. Yang
Hypertonicity-Induced Projections Reflect Cell Polarity in Mouse Metaphase II Oocytes: Involvement of Microtubules, Microfilaments, and Chromosomes
Biol Reprod, December 1, 2002; 67(6): 1853 - 1863.
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