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Biology of Reproduction 67, 540-545 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Utility of Rapidly Matured Oocytes as Recipients for Production of Cloned Embryos from Somatic Cells in the Pig1

Kazuchika Miyoshia, S. Jacek Rzucidloa,b, Scott L. Pratta,b, and Steven L. Stice2,,a,b

a Department of Animal and Dairy Science, University of Georgia, Athens, Georgia 30602-2771 b ProLinia Inc., Athens, Georgia 30602-2771


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was conducted to examine the utility of rapidly matured oocytes as recipients for production of porcine embryos reconstituted with adult skin fibroblasts and whether arrest of meiotic resumption of recipient oocytes at the germinal vesicle (GV) stage by dibutyryl cyclic AMP (dbcAMP) improves in vitro developmental rates after reconstruction. At 24 h of maturation in the medium, 36.3% of oocytes reached the metaphase II (MII) stage. At 30 h of maturation, the percentage (71.4%) of MII oocytes did not significantly differ from that (78.0%) at 42 h of maturation. When MII oocytes recovered at 24 h of maturation were used as recipients, 22/156 (14.1%) cloned embryos developing to the blastocyst stage was significantly (P < 0.05) higher than those of embryos reconstituted with oocytes collected at 30 h (5/168; 3.0%) and 42 h (13/217; 6.0%) of maturation. Culture of oocytes in medium containing 1 mM dbcAMP for 20 h maintained 72.9% in the GV stage, whereas only 15.0% of nontreated oocytes were in the GV stage (P < 0.05). The effect of dbcAMP was reversible. However, the treatment of recipient oocytes with dbcAMP did not affect the development of reconstructed embryos when compared with nontreated oocytes. These results indicate that rapidly matured oocytes are superior in their ability to support development of porcine reconstructed embryos; however, arrest of meiotic resumption of recipient oocytes at the GV stage by dbcAMP does not improve reconstructed embryo developmental rates.

developmental biology, early development, gamete biology, meiosis, ovum


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cloned animals were created by transferring differentiated cells into enucleated oocytes (sheep [1, 2], cattle [3–5], mouse [6], goat [7], and pig [8–10]). In these studies, both in vivo- and in vitro-matured oocytes have been used as recipients. In vitro oocyte maturation systems are an abundant and stable supply of recipient oocytes because immature oocytes can be obtained from slaughtered animals. In vitro-matured oocytes have been commonly used for production of cloned calves [3–5]. Similarly, several studies have reported successful development of porcine embryos reconstituted with in vitro-matured oocytes to the blastocyst stage [10–19]. There is only one report of cloned pigs resulting from nuclear transfer embryos using in vitro-matured oocytes [10], and it has been suggested that in vivo-derived materials enhance the probability of obtaining porcine offspring [8, 9].

Although porcine blastocysts can be produced from immature oocytes by using in vitro maturation/fertilization/culture systems, it is difficult to obtain blastocysts efficiently because of high rates of polyspermic fertilization and low developmental competence of the zygotes produced in vitro [20]. Continuous advances have led to improvements in porcine blastocyst production in vitro [21, 22], but these have not yet become common techniques. These problems may reflect oocyte cytoplasmic maturation defects that may be due to an incomplete terminal differentiation of the oocytes before meiotic resumption in vitro [23]. In vivo, meiotically competent oocytes are maintained at the germinal vesicle (GV) stage by the follicular environment until the preovulatory gonadotropin surge. After the preovulatory surge of gonadotropin, only fully grown and competent oocytes can resume meiosis, complete the first meiotic division, and be ovulated. In contrast, all meiotically competent oocytes matured in vitro spontaneously reenter the meiotic process as soon as they are removed from their follicles [24]. Although oocytes used for in vitro maturation are commonly harvested from 2- to 6-mm diameter antral follicles from slaughterhouse ovaries, the size of follicles selected for collection of oocytes has varied both between and within investigations [25]. It was reported that there is a large variation in the nuclear morphology of porcine oocytes at the GV stage just after aspiration from follicles, the percentage of oocytes that reached the final stage of GV (GV-IV) is only 5%, and the majority (45%) of the oocytes remain at the GV-II stage [26]. These oocytes are thus deprived of an important step of maturation in follicles. This may account for cytoplasmic maturation defects in oocytes matured in vitro and the low developmental rates for cloned embryos reconstituted with in vitro-matured oocytes in the pig [10–19].

Numerous attempts have been made to maintain porcine oocytes aspirated from follicles at the GV stage before in vitro maturation, including treatment with cycloheximide [27, 28], p-aminobenzamidine [28], 6-dimethylaminopurine [29], hypoxanthine [30], {alpha}-amanitine [31], dibutyryl cyclic AMP (dbcAMP) either alone [26, 32] or with testosterone [33, 34], and roscovitine [23]. In all these cases, GV breakdown of the oocytes was accelerated after inhibitor removal. Porcine oocytes exposed to dbcAMP for 20 h prior to in vitro maturation and fertilization improved developmental rates to the blastocyst stage [26].

Asynchronous meiotic progression of porcine oocytes has been repeatedly observed during in vitro maturation [35–37], and this phenomenon may be responsible for the large variation in the developmental stage of the oocytes at the start of culture [26]. We hypothesize that, like in vitro-produced embryos, developmental rates for porcine cloned embryos might improve if in vitro-matured oocytes are first synchronized prior to maturing through GV breakdown. The present study examined whether arrest of meiotic resumption of recipient oocytes at the GV stage by dbcAMP improved in vitro developmental rates for nuclear transfer embryos using adult skin fibroblast donor cells. An additional objective was to determine the in vitro developmental competence of the asynchronous matured porcine oocytes by using metaphase II (MII) oocytes at 24, 30, and 42 h postinitiation of maturation in the nuclear transfer procedure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Donor Cells

Porcine fibroblasts were harvested from an ear skin biopsy obtained from an 8-mo-old pig [38]. Cells were cultured in DMEM/F-12 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 20% (v:v) FCS (Biowhittaker Inc., Walkersville, MD) under 5% CO2 in air at 37°C. After reaching confluence, cells were passaged. Passage 2 fibroblasts were trypsinized, suspended in the culture medium with 10% (v:v) dimethyl sulfoxide (Sigma), and stored as frozen aliquots. Donor cells were used for nuclear transfer between passages 2 and 5 of culture. The cells were allowed to grow to confluency by culturing for 6 days, and a single cell suspension was prepared by standard trypsinization immediately prior to nuclear transfer. The cells were used for nuclear transfer within 5 days after reaching confluence. Analysis of the cell cycle stages by using flow cytometry as explained in our earlier experiment [38] revealed that 91.2 ± 0.2% of the cells were at the G0/G1 phase.

In Vitro Maturation of Oocytes

Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 30–35°C. Antral follicles (2–5 mm in diameter) were aspirated with an 18-gauge needle using vacuum suction (40 mm Hg). Aspirated oocytes that had an evenly granulated cytoplasm and were surrounded by at least three uniform layers of compact cumulus cells were selected and washed four times in Hepes-buffered TCM-199 (Gibco BRL, Grand Island, NY) supplemented with 0.4% (w:v) BSA (Sigma). Oocytes were cultured in four-well plates (Nunc, Roskilde, Denmark) containing 500 µl of each medium. TCM-199 (Gibco) supplemented with 3.05 mM D-glucose, 0.91 mM Na pyruvate, 0.57 mM cysteine, 10 ng/ml epidermal growth factor (Sigma), 10 IU/ml eCG (Sioux Biochemicals, Sioux Center, IA), 10 IU/ml hCG (Intervet, Millsboro, DE), and 10% (v:v) porcine follicular fluid was used as a basic maturation medium.

Nuclear Transfer

At the end of maturation, cumulus cells were removed by vortexing with 0.1% (w:v) hyaluronidase (Sigma), and denuded oocytes were transferred into 100 µl of Hepes-buffered TCM-199 with the osmolarity adjusted to 300 mOsm by adding sorbitol supplemented with 7.5 µg/ml cytochalasin B (Sigma) and 10% FCS (manipulation medium). The first polar body and MII plate were removed by aspiration with a 15-µm inner-diameter glass pipette. The oocytes had been previously stained in the manipulation medium supplemented with 5 µg/ml Hoechst 33342 (Sigma) for 20 min, and confirmation of successful enucleation was achieved by visualizing the cytoplast and removed cytoplasm under ultraviolet light. After enucleation, a donor cell was inserted into the perivitelline space of each enucleated oocyte using the same glass pipette. Cell-oocyte complexes were washed in TCM-199 supplemented with 10% FCS, transferred to the same medium, and kept in a CO2 incubator adjusted to 5% CO2 in air at 38.5°C until fusion.

The chamber for fusion was a 100-mm dish filled with 15 ml of Zimmermann fusion medium [39]. Two stainless-steel wires (100-µm diameter) were used as electrodes, and they were attached to micromanipulators. The single cell-oocyte complex was sandwiched between the electrodes and oriented with the contact surface between the cytoplast and the donor cell perpendicular to the electrodes. The distance between the electrodes was about 100 µm. Membrane fusion was induced by applying a single direct-current pulse of 250 V/mm for a duration of 20 µsec with a prepulse of alternating-current field of 5 V, 1 MHz for 2 sec using an LF 101 Fusion Machine (TR Tech Co., Tokyo, Japan). Following the fusion pulse, the complexes were washed in G1.2 medium [40] and cultured for a period of 1 h in 100 µl of the same medium. Fusion was then determined by microscopic examination.

Activation and Culture of Reconstructed Embryos

At 3 h after fusion, fused embryos were washed once in Zimmermann fusion medium and then placed between two wire electrodes (1 mm apart) of the fusion chamber slide with 15 ml of the medium. Direct-current pulses of 75 V/mm were applied twice to the embryos for a duration of 60 µsec at intervals of 5 sec. Activated embryos were incubated in 100 µl of G1.2 medium supplemented with 7.5 µg/ml cytochalasin B for 2 h after activation to prevent extrusion of polar bodies [14] and then transferred into G1.2 medium. After culture in G1.2 medium for 3 days, the embryos were transferred into 100 µl of G2.2 medium [40] and continued to be cultured. The embryos were examined for cleavage and blastocyst formation at 2 and 7 days of culture, respectively. At the end of the culture period, all blastocysts were counted for nuclei after Hoechst staining. The blastocysts were placed on slides with a drop of mounting medium consisting of glycerol and PBS (9:1) containing 0.1 mg/ml Hoechst 33342. A cover slip was placed on top of the blastocysts and the edge was sealed with fingernail polish. The numbers of nuclei were counted under ultraviolet light.

Experimental Studies

In order to determine the optimal treatment period for dbcAMP-induced GV stage synchronization, oocytes were cultured in the basic maturation medium with or without 1 mM dbcAMP for 16, 20, 24, and 42 h in experiment 1. After culture for each period, degenerated oocytes with lysed cytoplasmic membranes were removed. The remainder were mounted, fixed for 72 h in 25% (v:v) acetic acid in ethanol at room temperature, stained with 1% (w:v) orcein in 45% (v:v) acetic acid, and examined for meiotic progression under a phase-contrast microscope.

In experiment 2, oocytes were cultured in the basic maturation medium with or without 1 mM dbcAMP for 20 h and then transferred into the medium without dbcAMP and hormonal supplements. Oocytes continued to be cultured for an additional 4, 10, 16, and 22 h and were examined for meiotic progression to the MII stage by staining with 5 µg/ml Hoechst 33342.

In experiment 3, oocytes were cultured in the basic maturation medium for 20 h and then transferred into the medium without hormonal supplements. The MII oocytes collected at 24, 30, and 42 h postinitiation of maturation were used in the nuclear transfer procedure, and the resulting embryos were cultured in vitro and developmental rates among groups were compared.

In experiment 4, oocytes were cultured under the same conditions as those in experiment 3 and MII oocytes were collected at 24 and 42 h of maturation. They were cultured in TCM-199 supplemented with 10% FCS for 6–7 h and were activated, which was the same timing as reconstruction of embryos in experiment 3. Activated oocytes were cultured in vitro to examine developmental rates. The activation and culture methods were the same as those described above.

In experiment 5, oocytes were cultured in the basic maturation medium with or without 1 mM dbcAMP for 20 h and then transferred into the medium without dbcAMP and hormonal supplements. Embryos reconstituted with MII oocytes collected at 42 h of maturation were cultured in vitro and their developmental abilities were compared.

Statistical Analysis

All percentage data were subjected to an arcsin transformation in each replicate, and the transformed values were analyzed using one-way (experiments 3, 4, and 5) or two-way (experiments 1 and 2) ANOVA followed by a Student-Newman-Keuls test. The numbers of cells in blastocysts were analyzed using a Student t-test. A probability of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1. Effect of Dibutyryl Cyclic AMP on Meiotic Resumption of Porcine Oocytes Matured In Vitro

There were no significant differences in percentages of degenerated oocytes in any treatment groups in this study. The addition of dbcAMP in the maturation medium increased (P < 0.05) the percentage of GV oocytes at each culture period (Table 1). In the absence of dbcAMP, 42.6% of oocytes were at the GV stage at 16 h of culture. This percentage was significantly (P < 0.05) higher than those at 24 h (9.3%) and 42 h (3.7%) of culture. In the presence of dbcAMP, the percentages of GV oocytes at 16 and 20 h of culture (77.8% and 72.9%, respectively) were significantly (P < 0.05) higher than at 42 h (44.3%) of culture.


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  		TABLE 1. Maturation of porcine oocytes cultured in a medium supplemented with or without 1 mM dibutyryl cyclic AMP (dbcAMP).a

Experiment 2. In Vitro Maturation of Porcine Oocytes Treated with or Without Dibutyryl Cyclic AMP

The addition of dbcAMP in the maturation medium decreased (P < 0.05) the percentage of MII oocytes at 10 h of additional culture (Table 2). In the absence of dbcAMP, 36.3% of oocytes reached the MII stage at 4 h of additional culture. This percentage was significantly (P < 0.05) lower than those at 10 h (71.4%), 16 h (80.0%), and 22 h (78.0%) of additional culture. In the presence of dbcAMP, the percentages of MII oocytes at 4 and 10 h of additional culture (35.3% and 29.8%, respectively) were significantly (P < 0.05) lower than that at 16 h (67.5%) of additional culture.


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  		TABLE 2. Maturation of porcine oocytes cultured in a medium supplemented with or without 1 mM dibutyryl cyclic AMP (dbcAMP) for 20 h and then continued to be cultured in the medium without dbcAMP.a

Experiment 3. In Vitro Development of Porcine Embryos Reconstituted with Metaphase II Oocytes Collected at Various Times Postinitiation of Maturation

There were no significant differences in percentages of fused cell-oocyte complexes (86.8%–93.9%) and cleaved embryos (29.8%–42.9%) among different maturation periods (Table 3). However, when MII oocytes recovered at 24 h of maturation were used as recipients, the percentage (14.1%) of cloned embryos developing to the blastocyst stage was significantly (P < 0.05) higher than those of embryos reconstituted with 30-h-matured (3.0%) and 42-h-matured (6.0%) oocytes. There were no significant differences in mean numbers (28.8–58.8 cells) of cells in the blastocysts among different maturation periods.


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  		TABLE 3. Effect of maturation periods of recipient oocytes on development of porcine embryos reconstituted with somatic cells.a

Experiment 4. Effect of Maturation Periods on In Vitro Development of Porcine Oocytes after Activation

There were no significant differences in percentages of cleaved oocytes (32.9%–45.6%) and oocytes developed to blastocysts (6.8%–7.6%) after activation and in mean numbers (42.4–54.0 cells) of cells in the blastocysts between different periods of maturation (Table 4).


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  		TABLE 4. Effect of maturation periods on development of porcine oocytes after activation.a

Experiment 5. In Vitro Development of Porcine Embryos Reconstituted with Metaphase II Oocytes Treated with or Without Dibutyryl Cyclic AMP

There were no significant differences in percentages of fused cell-oocyte complexes (78.8%–80.0%), cleaved embryos (33.3%–41.4%), and embryos developed to blastocysts (1.7%–5.7%) between presence and absence of dbcAMP treatment (Table 5). In addition, there was no significant difference in mean numbers (49.0–77.7 cells) of cells in the blastocysts between presence and absence of dbcAMP treatment.


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  		TABLE 5. Development of porcine embryos reconstituted with 42-h-matured oocytes treated with or without dibutyryl cyclic AMP.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study show that the maturation period affects the ability of porcine oocytes to support development of cloned embryos as recipient oocytes. Porcine oocytes are commonly matured in vitro for 40–44 h before manipulation [10–19]; however, the present study indicates that developmental rates were significantly higher for nuclear transfer embryos using 24-h-matured recipient oocytes when compared with 30- and 42-h-matured oocytes. Previously, it was reported that the maturation period of recipient oocytes affects in vitro development of reconstructed embryos in the pig; the blastocyst formation rate of embryos reconstituted with 33-h-matured oocytes was higher than that of embryos reconstituted with 44-h-matured oocytes [41, 42]. In these studies, embryos reconstituted with 33- and 44-h-matured oocytes were activated at 39–40 and 50–51 h postinitiation of maturation, respectively. Developmental rate to the blastocyst stage after activation of 42-h-matured oocytes was higher than that of 48-h-matured oocytes; thus, the authors hypothesized that the difference between nuclear transfer groups was due to oocyte activation responses (young vs. old oocytes) [42]. Our data suggest a difference in oocyte competence between recipient oocyte ages because parthenotes from 24- and 42-h-matured oocytes had similar developmental rates.

In our maturation system, 36.3% of oocytes reached the MII stage at 24 h of culture. At 30 h of culture, percentage (71.4%) of MII oocytes did not significantly differ from that (78.0%) at 42 h of culture. These results indicate that about half of 30-h-matured oocytes reached the MII stage within 6 h, but most of the 42-h-matured oocytes had aged at the MII stage for at least 12 h. Nevertheless, the in vitro developmental rates of embryos reconstituted with 30-h-matured oocytes did not significantly differ from that of embryos reconstituted with 42-h-matured oocytes. This result suggests that aging of recipient oocytes at the MII stage does not affect developmental rates of cloned embryos.

Oocyte maturation includes two phenomena: nuclear maturation, involving reinitiation and completion of the first meiotic division, and cytoplasmic maturation that enables normal fertilization and further embryonic development [23]. It has been demonstrated that the oocytes progressively acquire the ability to resume and progress through meiosis during folliculogenesis and the full developmental competence is finally acquired by fully grown oocytes in the mouse [43, 44], goat [45, 46], sheep [47], and cattle [48, 49]. Similarly, stepwise acquisition of meiotic competence has been shown in porcine oocytes. Oocytes harvested from 0.8-mm-diameter follicles may initiate meiosis when cultured in vitro, but only oocytes obtained from follicles larger than 2 mm in diameter are able to reach the MII stage [50, 51]. The GV breakdown rate is positively correlated with oocyte diameter. Completion of the first meiotic division was only observed in oocytes having a 110-µm or greater diameter [52]. How oocytes acquire cytoplasmic maturation is unclear. Oocytes recovered from follicles larger than 2 mm in diameter are able to reinitiate and complete the first meiotic division. However, when these MII oocytes are inseminated, the normal fertilization rate for them is low and the developmental ability of the zygotes is poor, suggesting most of them do not acquire cytoplasmic maturation [20]. An important step for acquisition of cytoplasmic maturation may be included in the end of folliculogenesis.

It was reported that most of porcine oocytes before the GV-II stage are arrested at the dictyate stage of prophase (GV-II) by dbcAMP treatment but oocytes at stages beyond the GV-II stage at the start of culture for maturation are not blocked by dbcAMP [26]. According to Funahashi et al. [26], 23% of porcine oocytes are at stages beyond the GV-II stage at the start of culture for maturation. In the present study, 27.1% of oocytes were not arrested at the GV stage after being cultured in the medium supplemented with dbcAMP for 20 h, indicating they had progressed beyond the GV-II stage at the start of maturation. When the oocytes were cultured in the medium without dbcAMP for an additional 4 h, 35.3% of them reached the MII stage. It was reported that the first GV breakdown and MII formation in porcine oocytes matured in a TCM-199-based medium are observed at 6 and 24 h of culture, respectively [53], suggesting oocytes arrested at the GV stage by dbcAMP cannot reach the MII stage within 4 h after inhibitor removal. Therefore, it is likely that these MII oocytes are at stages beyond the GV-II stage at the start of the maturation. A similar percentage (36.3%) of oocytes reached the MII stage when they were matured in the medium without dbcAMP for 24 h. These results suggest that MII oocytes recovered from the maturation medium without dbcAMP at 24 h of culture are at stages beyond the GV-II stage at the start of the maturation.

On the basis of the results from the present study and previous reports described above, we believe that there is an important step involving acquisition of cytoplasmic maturation between the GV-II stage and prior to GV breakdown. Oocytes at stages beyond the GV-II stage at the start of in vitro maturation can acquire cytoplasmic maturation before spontaneous resumption of the meiotic process. Therefore, many of the MII oocytes recovered from maturation medium at 24 h of culture would already have acquired or initiated cytoplasmic maturation in vivo. These oocytes then produce cloned embryos with better developmental rates than oocytes that mature later. In comparison with the 24-h group, the percentage of oocytes at the MII stage doubled but the blastocyst formation rate of cloned embryos was reduced to half in the 42-h oocyte group. This result may suggest that only embryos reconstituted with oocytes that reached beyond the GV-II stage at the start of maturation can develop into blastocysts even when 42-h-matured oocytes are used as recipients.

In the present study, dbcAMP arrested meiotic resumption of porcine oocytes matured in vitro, as described by Funahashi et al. [26]. However, this treatment did not improve the developmental rates of reconstructed embryos. This is in contrast with studies where arresting meiotic resumption by dbcAMP improved developmental rates to the blastocyst stage of in vitro matured and fertilized porcine oocytes [26]. We cannot explain this difference, but the results of the present study are consistent with our hypothesis: Oocytes that progress beyond the GV-II stage before spontaneous resumption of the meiotic process in culture support development of reconstructed embryos as recipients.

In conclusion, we have shown that in vitro development of porcine cloned embryos reconstituted with somatic cells can be improved by using rapidly matured oocytes as recipients. To date, the productive efficiency of cloned offspring from differentiated cells has been very low regardless of species [54]. Selection of oocytes that are suited to be used as recipients will be a novel way to solve this problem and lead to improvements in porcine cloning outcomes.


   ACKNOWLEDGMENTS
 
The authors thank Dr. D.K. Gardner, Colorado Center for Reproductive Medicine, for providing G1.2 and G2.2 media. We also thank the staff of ProLinia Inc. for assisting with oocyte collection and nuclear transfer.


   FOOTNOTES
 
First decision: 29 January 2002.

1 Supported by National Institutes of Health SBIR grant R43HL65806-01 Back

2 Correspondence: Steven L. Stice, Department of Animal and Dairy Science, University of Georgia, 425 River Road, Athens, GA 30602-2771. FAX: 706 542 7925; sstice{at}arches.uga.edu Back

Accepted: March 5, 2002.

Received: December 21, 2001.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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