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BOR - Papers in Press, published online ahead of print September 3, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.018200
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BIOLOGY OF REPRODUCTION 70, 46–53 (2004)
DOI: 10.1095/biolreprod.103.018200
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Activation of Equine Nuclear Transfer Oocytes: Methods and Timing of Treatment in Relation to Nuclear Remodeling1

Young-Ho Choi, Linda B. Love, Mark E. Westhusin, and Katrin Hinrichs2

Departments of Veterinary Physiology and Pharmacology3 Large Animal Medicine and Surgery,4 College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4466


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Early development of embryos produced by transfer of equine nuclei to bovine cytoplasts is superior to that of intraspecies equine nuclear transfer embryos. This may be related to differences in chromatin remodeling or efficiency of activation between the two oocyte types. The pattern of donor nucleus remodeling was examined in equine-equine and equine-bovine reconstructed oocytes. Chromosome condensation occurred in equine cytoplasts by 2 h but was not seen in bovine cytoplasts until 4 h. We investigated the effect of activation of equine-equine reconstructed oocytes at <30 min or at 2 h after reconstruction. Four activation treatments were evaluated at each time point: injection of sperm extract alone, or in combination with 6-dimethylaminopurine (6-DMAP), cytochalasin B, or 1% dimethylsulphoxide. There was no significant difference in normal cleavage rate or average nucleus number of embryos between equine oocytes activated <30 min or at 2 h after reconstruction. The combination of 6-DMAP with sperm extract significantly (P < 0.01) improved cleavage rate compared with the other three treatments. Activation with sperm extract and 6-DMAP 2 h after donor nucleus injection gave the highest cleavage (79%) and the highest cleavage with normal nuclei (40%). Sperm extract and 6-DMAP also effectively activated oocytes parthenogenetically, yielding 83% cleavage and 73% cleavage with normal nuclei. These results indicate that although nuclear remodeling occurs rapidly in equine cytoplasts, early activation does not improve embryonic development after reconstruction.

embryo, fertilization, gamete biology, in vitro fertilization, ovum


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Since the first successful cloning from adult somatic cells was reported in sheep [1], cloned offspring have been reported in several species, including the mouse [2], cow [3, 4], goat [5, 6], pig [7, 8], and cat [9]. The overall efficiency of cloning is generally low (0.2%–3.4% of reconstructed oocytes) with a few exceptions (review, [10]).

Birth of a horse foal cloned from adult somatic cells has recently been reported [11], as has birth of a cloned mule (horse-donkey hybrid) foal from transfer of fetal fibroblast nuclei [12]. The efficiency of cloning in the horse is low in comparison with that in other species. In the study of Galli et al. [11], efficiency of live foal production was 1/841 (0.1%). Use of in vivo-matured oocytes, fetal fibroblasts, and immediate transfer of reconstructed oocytes to the oviduct of recipient mares resulted in a somewhat greater efficiency of 3/334 (0.8%) for production of cloned mule foals ([12]; D.K. Vanderwall, personal communication). In vitro development of only 3%–10% of reconstructed equine oocytes past eight cells [13, 14] and 2%–3% to blastocyst [11, 14] have been reported.

It is notable that when equine somatic cell nuclei are injected into bovine cytoplasts, embryo development is significantly enhanced (>40% develop more than eight nuclei [15]). A major factor that might affect the ability of equine cytoplasts to support embryo development after nuclear transfer (NT) is effective activation of the oocyte. Although repeatable and effective methods are available for bovine oocyte activation [16, 17], there is little information on activation of equine oocytes. We are aware of only three reports comparing the efficiency of different activation methods for equine oocytes [18–20]. These studies evaluated ionomycin; ethanol; thimerosal; inositol 1,4,5-triphosphate; and calcium ionophore A23187; alone or in combination with cycloheximide; however, no single treatment emerged as highly effective. The maximum reported activation rate (pronucleus formation) for equine oocytes chemically treated for parthenogenetic activation was 72%; this was in oocytes suppressed with roscovitine for 24 h and then matured for 42 h before activation with ionomycin and 6-dimethylaminopurine (6-DMAP, [21]). However, these authors reported that similar treatment of oocytes matured under normal conditions (33-h maturation) resulted in only 32% pronucleus formation. The maximum reported cleavage rate after chemical activation of equine oocytes was 45% (also ionomycin and 6-DMAP [22]).

Recently we reported the use of stallion sperm extract for both parthenogenetic activation of equine oocytes and activation after NT [15, 23]. There is only one report of use of sperm factor to activate reconstructed NT oocytes in another species. In reconstructed bovine oocytes, sperm factor was used in combination with cytochalasin B but was less effective than was chemical activation [24]. Use of stallion sperm extract for activation of metaphase II (MII) equine oocytes resulted in 88%–92% cleavage; use with reconstructed equine oocytes resulted in 60% cleavage and an average of 3.2–4.6 nuclei at 4 days culture [15, 23]. Although these rates of cleavage are higher than those previously reported after activation of equine oocyes, it is possible that addition of chemical activation treatments after sperm extract injection may further increase activation efficiency.

The timing of oocyte activation is considered to be an important factor in the efficiency of cloned embryo production (review, [25, 26]). The time of oocyte activation in other species has varied from immediately after donor nucleus recombination to 10 h later, for example, immediately to ~10 h in cattle [4, 27, 28], immediately to ~4 h in pigs [7, 8, 29, 30], and immediately to ~6 h in mice [2]. We found no difference in development of equine NT embryos from oocytes activated 1.5–2 h or 8–10 h after reconstruction [15]. In the mouse, activation 1–6 h after reconstruction yielded significantly higher embryo development in vitro than did activation immediately after reconstruction [2]. However, these workers later obtained similar results in both groups by treating the immediate activation group with 1% dimethylsulphoxide (DMSO) in the absence of cytochalasin B [31]. The authors suggested that cytochalasin B may have a detrimental effect on embryonic development, which is overcome by the enhanced nuclear reprogramming allowed when oocytes are activated >1 h after recombination. The positive effect of DMSO was attributed to its known activity in modifying nuclear reprogramming, as it has been shown to affect differentiation of embryonic teratocarcinoma cells [32].

The effect of activation timing on cloned embryo development may be largely through modulation of the degree of nuclear reprogramming achieved. The direct exposure of chromosomes to the cytoplasm is thought to be important in reprogramming of the chromatin to support embryo development [31]. High levels of maturation promoting factor (MPF) in the enucleated MII oocyte induce breakdown of the nuclear envelope of the transferred nucleus, resulting in chromosome condensation (termed premature chromosome condensation) within the cytoplasm of the ooplast [33, 34]. When a stimulus for activation is applied, MPF levels decrease, resulting in chromosome decondensation and formation of a pronucleus-like structure. The optimum duration of these stages to promote efficient embryo production is not clear. There is no information available on the timing of chromatin condensation after equine donor nucleus injection into either bovine or equine cytoplasts.

In the present study, we compared the timing of nuclear transformation after injection of equine somatic cell nuclei into equine and bovine cytoplasts, under the hypothesis that changes within the bovine cytoplast may illustrate the optimal chromatin configuration at which the activation stimulus should be applied. The parthenogenetic activation rates of MII oocytes treated with sperm extract alone or in combination with cytochalasin B or 6-DMAP were evaluated. We then compared the in vitro development of equine reconstructed oocytes after activation with cytosolic sperm extract in combination with 1% DMSO, cytochalasin B, or 6-DMAP, begun either <30 min or 2 h after donor nucleus injection. On the basis of the results of this experiment, we evaluated the effect of activation with cytosolic sperm extract followed by 6-DMAP treatment on extrusion of chromatin and pronucleus formation of reconstructed equine oocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oocyte Collection

Ovaries were transported from slaughterhouses to the laboratory at 21–32°C (3- to 4-h transport time). Adnexa were trimmed from the ovaries with scissors and the ovaries were cleaned with sterilized gauze. All visible follicles were opened with a scalpel blade, and the granulosa layer of each follicle was scraped with a 0.5-cm bone curette. The contents of the curette were washed into individual Petri dishes with Hepes-buffered TCM199 with Hanks salts (Gibco Life Technologies, Inc., Grand Island, NY) plus ticarcillin (0.1 mg/ml, SmithKline Beecham Pharmaceuticals, Philadelphia, PA). The contents of the Petri dishes were examined with a dissection microscope at 10–20x magnification. Oocyte-cumulus complexes were classified as compact, expanded, or degenerating depending on the expansion of both mural granulosa and cumulus as described previously [35, 36]. Oocytes with any sign of expansion of either the cumulus or the mural granulosa, from having individual cells visible protruding from the surface to having full expansion with copious matrix visible between cells, led to the classification of expanded. Oocytes having both compact cumulus and compact mural granulosa were classified as compact. Only expanded oocytes were used in this study.

In Vitro Maturation

Oocytes were washed twice in maturation medium (TCM199 with Earles salts (Gibco), 5 µU/ml FSH (Sioux Biochemicals, Sioux Center, IA), 10% fetal bovine serum, and 25 µg/ml gentamycin). Oocytes were cultured in droplets at a ratio of 10 µl medium per oocyte under light white mineral oil (Sigma Chemical Co., St. Louis, MO) at 38.2 °C in 5% CO2 in air for 24–26 h. After maturation, oocytes were denuded of cumulus by pipetting in a solution of 0.05% hyaluronidase in either Hepes-buffered TCM199 with Hanks salts (H-TCM199) plus 5% FBS or CZB-M with 10% FBS [23]. Denuded oocytes were selected for presence of a polar body. Oocytes not having a polar body were fixed in buffered formol saline, mounted on a slide with 6.5 µl of 9:1 glycerol:PBS containing 2.5 µg/ml Hoechst 33258, and examined by fluorescence microscopy to determine the chromatin configuration.

Experiment 1: Nuclear Transformation after Reconstruction Using Equine or Bovine Enucleated Oocytes

For this experiment, equine fibroblast cells at passage 3–7, grown to confluence without serum starvation, were used. Cells were trypsinized before use and held in CZB-M without glutamine, nonessential amino acids, or FBS but were supplemented with 2% polyvinylpyrrolidone (CZB-M/2% PVP).

Matured equine oocytes having a first polar body were incubated for 10 min in TCM199 with 10% FBS containing 5 µg/ml Hoechst 33342 (Sigma) and 5 µg/ml cytochalasin B. Oocytes were then held in H-TCM199 with a holding pipette (120–140-µm outer diameter) under an inverted microscope equipped with Narishige manipulators. The zona pellucida of the oocyte was drilled with an enucleation pipette (10–13-µm outer diameter) attached to a piezo drill (Prime Tech Ltd., Ibaraki, Japan), and the polar body and metaphase plate were aspirated into the enucleation pipette. After enucleation, the resulting cytoplasts were held in TCM199 with 10% FBS. The injection of fibroblast cells into the enucleated equine oocytes was performed by following the method described by Chung et al. [37] with the piezo drill. The outside diameter of the injection pipette was 8–9 µm. Immediately before injection, a somatic cell held in CZB-M/2% PVP was gently aspirated in and out of the injection pipette until the cell membrane was broken. Donor cell injection was carried out in a 100-µl drop of CZB-M supplemented with 0.1% polyvinyl alcohol instead of proteins and glutamine, and reconstructed oocytes were held for 20 min at room temperature in the same medium after injection to heal the membrane slowly. Reconstructed oocytes were then transferred to TCM199 with 10% FBS and incubated at 38.2°C in a humidified atmosphere of 5% CO2 in air for 1, 2, 3, or 4 h. After culture, embryos were fixed and stained as described above to examine the transformation of injected donor nuclei. Chromatin was classified as dense (one small condensed mass of chromatin), tight (individual chromosomes visible but in close apposition to one another), spread (individual chromosomes visible spread through a small area of the cytoplasm), or pronucleus.

Bovine oocytes were purchased from Ovagenics (San Angelo, TX) and were cultured overnight in equilibrated medium (TCM199 with 10% FBS and 10 µU/ml FSH, 10 µU/ml LH [Sioux], 100 U/ml penicillin, and 0.1 mg/ml streptomycin [Sigma]) in a portable incubator maintained at 39°C. Upon arrival at the laboratory, the glass tube containing the oocytes was uncapped and placed in an incubator at 38.2°C in 5% CO2 in air until 22 h of in vitro maturation. Oocytes with a first polar body were enucleated, reconstructed with donor cells, and cultured as described above for equine oocytes. Reconstructed oocytes were then fixed at 1, 2, 3, and 4 h after culture and evaluated as described above.

Experiment 2: Effect of 6-DMAP or Cytochalasin B Treatment in Combination with Injection of Sperm Extract for Parthenogenetic Activation of Equine Oocytes

Matured equine oocytes having a first polar body were parthenogenetically activated by injection of stallion sperm cytosolic extract prepared as described by Choi et al. [15]. For injection, oocytes were held in CZB-M plus 10% FBS and injected with 2–4 pl of extract containing 250 µg/ml protein with a pipette having an inner diameter of ~5 µm. Injected oocytes were held for 20 min at room temperature in the same medium to heal the broken membrane slowly. Oocytes were then cultured in CZB-C with nonessential amino acids [23] in one of three treatments: control (no additives), 5 µg/ml cytochalasin B with 1% DMSO for 3 h, or 2 mM 6-DMAP for 4 h. Oocytes treated with cytochalasin B or 6-DMAP were washed and transferred into medium without additives at a ratio of 5 µl medium per oocyte and were incubated at 38.2°C under 5% CO2 5% O2 90% N2. Control oocytes were immediately cultured similarly. After 96 h of culture without a change of medium, oocytes were fixed and stained as described above to examine the number and status of nuclei. Only nuclei that appeared to be normal were included in the nucleus number; nuclei showing signs of degeneration (vacuolization, condensation, or fragmentation) were disregarded.

Experiment 3: Effects of 6-DMAP and Cytochalasin B on Development of Equine Reconstructed Oocytes Activated at Different Times

Matured equine oocytes having a first polar body were enucleated and injected with donor cell nuclei as described in experiment 1, except for the use of CZB-H for staining and CZB-M plus 10% FBS for enucleation and donor cell nuclei injection [23]. Reconstructed oocytes were separated into two groups to be activated either less than 30 min or at 2 h after donor cell injection. Activation was performed by injection of sperm extract containing 250–585 µg protein/ml following the method described in experiment 2. Each activation group was then separated into four treatments: control (no additives), 1% DMSO for 3 h, cytochalasin B in 1% DMSO for 3 h, or 2 mM 6-DMAP for 4 h. Oocytes treated with DMSO, cytochalasin B, or 6-DMAP were then washed and cultured in CZB-C media as described above. Control oocytes were placed directly into these culture conditions. After 96 h of culture without a change of medium, embryos were fixed and stained as described above to examine the number and status of nuclei.

Experiment 4: Effect of 6-DMAP on Pronucleus Formation of Reconstructed Oocytes at 20 h Postactivation

On the basis of the results of experiments 2 and 3, we evaluated the effect of 6-DMAP on donor nucleus decondensation and chromatin extrusion. Matured equine oocytes having a first polar body were enucleated and injected with donor cell nuclei as described in experiment 3. Two hours after donor cell injection, oocytes were injected with stallion sperm cytosolic extract as described above. Injected oocytes were held for 20 min at room temperature in the same medium to heal the broken membrane slowly. Reconstructed oocytes were then cultured in CZB-C medium with or without 2 mM 6-DMAP for 4 h at 38.2°C in a humidified atmosphere of 5% CO2 in air. Oocytes treated with 6-DMAP were washed and then cultured in CZB-C under the same conditions as described above. Control oocytes were placed directly into these culture conditions.

After 20 h culture, reconstructed oocytes were fixed and stained as described above to examine extrusion of chromatin and the formation of pronuclei. Oocytes with dense to spread chromosomes were considered as nonactivated, and those with decondensing nuclei, pronuclei, mitosis, or cleavage with the presence of nuclei in each blastomere were considered to be activated.

Statistical Analysis

Differences among groups were evaluated by chi-square, with Fishers exact test used when the expected value for any parameter was <5. Numbers of embryonic nuclei among treatments were compared by ANOVA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 741 equine ovaries were processed and 4232 follicles were scraped, resulting in an average of 5.7 follicles per ovary. We recovered 2393 oocytes, of which 619 were compact, 1578 were expanded, and 196 were degenerating. For this study, 1480 expanded oocytes were used; the remainder were used on a separate project.

Of 1480 expanded oocytes, 26 were broken during denuding of cumulus cells after maturation. Thus, 1454 were evaluated for the formation of a polar body. Of these, 870 had a polar body. After fixation and staining, nine additional oocytes were found to be in MII, thus a total of 879 oocytes (60%) were in MII after culture. Of oocytes without polar bodies, 14% (82/575) were in metaphase I after fixation and staining.

Experiment 1: Nuclear Transformation after Reconstruction Using Equine or Bovine Enucleated Oocytes

Of 198 matured equine oocytes, 184 (93%) were successfully enucleated and 178 were successfully injected with donor cells. Two reconstructed oocytes were eliminated from data analysis because no nucleus was detected after fixation and staining. The proportion of oocytes having dense chromatin decreased significantly between 1 and 2 h (P < 0.05; Table 1); correspondingly, the proportion of oocytes having spread chromatin significantly increased from 1 to 2 h (from 5% to 45%; P < 0.001). Photomicrographs of donor nuclei in equine cytoplasts, showing the different configurations, are shown in Figure 1. Because of the variation in chromatin configurations seen in equine cytoplasts at 1 h, we assessed the donor nucleus morphology immediately after injection. An additional 15 reconstructed oocytes were fixed immediately after donor nucleus injection; all 15 contained dense chromatin (Fig. 1A).


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  		TABLE 1. Chormatin configurations in transferred nuclei after direct injection of equine donor cells into equine cytoplasts.*



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  		FIG. 1. Chromatin configurations seen in transferred nuclei after direct injection of equine donor cells into equine cytoplasts. A) A donor cell nucleus immediately after injection, (B) dense, (C) tight, and (D) spread configurations. Bar = 10 µm

One hundred sixty-two bovine oocytes were shipped. After denuding, 146 of 162 oocytes (90%) had a polar body and were used for NT. Ninety-seven percent of bovine oocytes survived enucleation with the piezo drill. The rate of successful injection of the donor cell was 95% (139/146). Five reconstructed oocytes were eliminated from data analysis because no nucleus was detected after fixation and staining. Chromatin configurations in reconstructed interspecies oocytes are shown in Table 2. The proportion of oocytes having the dense configuration decreased significantly between 2 and 4 h (P < 0.01). The proportion having the spread configuration significantly increased between 2 and 4 h (6% vs. 35%; P < 0.01). The chromatin configurations of equine nuclei in bovine cytoplasts are presented in Figure 2.


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  		TABLE 2. Chromatin configurations in transferred nuclei after direct injection of equine donor cells into bovine cytoplasts.*



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  		FIG. 2. Chromatin configurations seen in transferred nuclei after direct injection of equine donor cells into bovine cytoplasts. A) Dense, (B) tight, (C) spread, and (D) pronucleus configurations. Bar = 10 µm

Experiment 2: Effect of 6-DMAP or Cytochalasin B Treatment in Combination with Injection of Sperm Extract for Parthenogenetic Activation of Equine Oocytes

For parthenogenetic activation, 116 oocytes with a polar body were injected with sperm cytosolic extract. Of these, four were lysed during injection, and seven were used for a preliminary study. The cleavage rates of 105 MII oocytes activated with sperm extract and treated with 6-DMAP are shown in Table 3. The cleavage rate (83%) in the 6-DMAP group was significantly higher than that for control (58%, P < 0.05) and cytochalasin B (39%, P < 0.001). The ratio of embryos cleaving with normal nuclei was also significantly higher for the 6-DMAP treatment (73%) than for control (45%, P < 0.05) and cytochalasin B (30%, P < 0.001). The average nucleus number among treatments was not significantly different. Two parthenogenetic embryos with 15 and 24 nuclei are presented in Figure 3A.


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  		TABLE 3. Parthenogenetic development of equine oocytes activated with stallion sperm extract alone or in combination with cytochalasin B or 6-DMAP and cultured for 96 h in vitro.*



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  		FIG. 3. Fluorescent images of parthenogenetic and nuclear transfer equine embryos. A) Two parthenogenetic embryos, one with 15 nuclei and one with 24 nuclei, including 1 mitotic figure (arrow). B) A 12-cell equine nuclear transfer embryo (11 nuclei plus one mitotic figure [arrow], two nuclei are overlapping). The reconstructed oocyte was activated by sperm extract with combination of 6-DMAP, and the embryo was cultured for 4 days in vitro. Bar = 50 µm

Experiment 3: Effects of 6-DMAP and Cytochalasin B on Development of Equine Reconstructed Oocytes Activated at Different Times

Of 414 matured oocytes subjected to enucleation and NT with direct injection with the piezo-driven pipette, 399 (96%) were successfully enucleated and 391 (94%) survived injection of the donor cell. Ten oocytes were lysed after injection of sperm extract for activation. The in vitro development of 381 reconstructed equine embryos activated with different chemicals after sperm extract injection is shown in Table 4. There was no significant difference in any treatment between the <30-min and 2-h activation times. When data for the two times were combined, the cleavage rate of oocytes treated with 6-DMAP was significantly higher than that for any other treatment (75% vs. 44%–52%; P < 0.01). The percentage of embryos cleaving with normal nuclei in oocytes treated with 6-DMAP was significantly (P < 0.05) higher than for 1% DMSO or for cytochalasin B treatments (32% vs. 18%–19%; P < 0.05). Among the eight treatments, the highest morphological cleavage and cleavage with normal nuclei were found in the 6-DMAP/2-h group (79% cleavage, 40% cleavage with normal nuclei). The average number of nuclei in the cytochalasin B/<30-min treatment (7.1) was significantly higher than that for controls for both activation times and for the 1% DMSO/2-h treatment (3.6–3.9, P < 0.05). The maximum embryo development achieved at 96 h was 16 nuclei in the 6-DMAP/2-h treatment; one embryo with 12 nuclei including one mitotic figure is shown in Figure 3B.


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  		TABLE 4. In vitro development of equine nuclear transfer oocytes subjected to each of four activation treatments within 30 min or at 2 h after reconstruction.*

Experiment 4: Effect of 6-DMAP on Pronucleus Formation of Reconstructed Oocytes at 20-h Postactivation

Of 142 matured oocytes, 140 (99%) were enucleated, 137 (96%) were successfully injected with donor cells, and 131 (92%) survived injection of sperm extract for activation. For this study, 73 reconstructed, activated oocytes were fixed after 20-h in vitro culture, and the remainder were used on separate projects. The rate of activation 20 h after sperm extract injection (oocytes with decondensing nuclei, pronucleus, nucleus, mitosis, and cleavage) in the 6-DMAP treatment was significantly (P < 0.01) higher than the control (76% vs. 39%; Table 5). Upon evaluation at 20 h, three reconstructed oocytes in the 6-DMAP treatment were in the first mitosis and six were 2–4-cell stage embryos, whereas only one embryo in the control group had cleaved. Chromatin extrusion was seen in two oocytes in the control group and none of the oocytes in the 6-DMAP group.


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  		TABLE 5. Chromatin configuration of equine nuclear transfer oocytes activated 2 h after reconstruction by injection of sperm cytosolic extract and cultured 4 h with or without 6-DMAP, then cultured for an additional 16 h.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of experiment 1 demonstrate that the timing of nuclear remodeling depends upon the origin of the host cytoplast. Equine somatic cell nuclei injected into equine cytoplasts remodeled into individualized chromosomes within 2 h of injection, whereas this took 4 h to occur within bovine cytoplasts. Our previous studies with both bovine and equine cytoplasts utilized activation at about 2 h after donor cell injection [15]. This is before the chromosomes had spread in bovine oocytes but after this stage in equine oocytes. Because cleavage and development were superior in bovine cytoplasts, activation while chromatin was still dense may have been related to higher development [15, 38]. However, in experiment 3 we found no difference in development between reconstructed equine oocytes activated within 30 min of donor cell injection and those activated 2 h afterward. The results of this and our previous study [15] show no apparent effect on time of activation of equine oocytes from immediately after donor cell injection to 8–10 h afterward.

In experiments 2, 3, and 4, treatment with 6-DMAP after injection of cytosolic sperm extract improved rates of activation, cleavage, and embryo development. Treatment with 6-DMAP was superior to treatment with cytochalasin B in the development of parthenogenetic and NT equine oocytes. The effect of 6-DMAP on activation in other species has been shown to be via its suppression of MPF activity, although this has not been demonstrated in equine oocytes. These data are the first on use of the combination of sperm extract with 6-DMAP in any species.

The cleavage rate observed in this study for parthenogenetic activation with sperm extract (58%) is lower than those of our previous reports (88%–92%) [15, 23]. However, oocytes used for parthenogenetic activation in the present study were matured for 24–26 h, whereas in the previous studies oocytes were matured for 42 h [15] or were matured for 28–30 h and then handled as controls for NT, experiencing all techniques including micromanipulation but without removal of oocyte chromatin or transfer of donor cells [23]. Thus, the higher activation rates seen in previous studies may be related to aging of oocytes, which has been shown in other species to result in lower levels of MPF and mitogen-activated protein kinase activities and increased ease of activation [16, 39]. Pimentel et al. [21] reported that parthenogenetic activation rates (pronucleus formation) of equine oocytes after treatment with ionomycin + 6-DMAP increased from 13% to 64% when the duration of in vitro maturation was increased from 24 to 42 h. Furthermore, the activation rate increased to 72% if oocytes were suppressed for 24 h with roscovitine before an additional 42 h of maturation. However, in the present study, addition of 6-DMAP to sperm extract injection made it possible to effectively activate equine oocytes matured for 24–26 h. The cleavage rate (83%) of parthenogenetic embryos achieved with this combined treatment in the present study is higher than those reported with other activation treatments in the horse (25%–45%) [19, 22].

Li et al. [14, 40] examined donor nuclei 12–18 h after activation of reconstructed equine oocytes with ionomycin, cytochalasin D, or cycloheximide. They defined the nucleus as reprogrammed when it was two to three times bigger than its original size. These workers reported that 63%–100% of reconstructed oocytes had reprogrammed donor nuclei, but morphological cleavage rates were only 13%–53% [14]. In the present study (experiment 4), we obtained 76% activation at 20 h with injection of sperm extract followed by 6-DMAP treatment (Table 5). Additionally, this treatment supported a 79% cleavage rate of NT embryos (Table 4). This cleavage rate is higher than other previous reports after transfer of adult somatic cells in the horse (9%–60%) [14, 15, 23]. However, the proportion of NT embryos having normal nuclei after a 96-h culture was only 40% in the best treatment (6-DMAP/2 h), and the average number of nuclei of this treatment at 96 h was only 5.4. These results are low compared with those for the parthenogenetically activated equine oocytes in the present study (73% cleavage with normal nuclei and average nucleus number of 8.7) or equine oocytes fertilized by intracytoplasmic sperm injection (average of 8.2 nuclei at 96 h) [41].

Addition of 1% DMSO into culture medium during activation treatment of reconstructed mouse oocytes significantly increased in vitro development when donor nuclei were cytoplasmically injected into enucleated oocytes [31, 37]. The effect of DMSO during NT may be related to membrane healing or nuclear reprogramming by the cytoplasts [31, 37]. In the present study, neither 1% DMSO nor cytochalasin B + 1% DMSO improved cleavage rate, but embryos in the cytochalasin B + 1% DMSO/<30-min group had a significantly higher average number of nuclei than those of controls or the cytochalasin B + 1% DMSO/2-h group.

Inhibiting the extrusion of chromatin in a "polar body" after oocyte reconstruction is thought to be important to control the ploidy of resulting embryos. Cytochalasin B has been commonly used for this purpose in several species such as the mouse [2, 31], cow [42, 43], and pig [44]. In MII oocytes, cytochalasin B prevents the rotation of the second meiotic spindle and thus placement of the polar body chromatin near the oolemma for extrusion [45]. 6-DMAP has also been used during NT in other species [4, 9, 46–48] for prevention of polar-body extrusion. As a protein kinase inhibitor, 6-DMAP may supress the phosphorylation necessary for formation of the spindle appartus [17] in addition to inducing rapid decondensation of the chromatin. However, the necessity for inhibition of chromatin extrusion after NT has not been well documented. In the pig, chromatin extrusion was seen in 81% of oocytes receiving 4N somatic cell nuclei (those in G2 at the time of transfer [49]) but in only 22% of oocytes receiving 2N nuclei. In experiment 4 of the present study, 2 of 14 activated oocytes in the sperm extract-only group had extruded chromatin at 20-h postactivation, whereas none of 28 activated oocytes in the sperm extract/6-DMAP group did so; this difference was not significant (P > 0.1). It is thus not possible to say whether 6-DMAP treatment had any effect on chromatin extrusion in equine oocytes. Indeed, inhibition of chromatin extrusion may not be beneficial, if in fact this occurs mainly when the transferred nucleus is in G2.


   ACKNOWLEDGMENTS
 
Protein analysis was performed by the Protein Chemistry Laboratory at Texas A&M University under the direction of Dr. Larry Dangott.


   FOOTNOTES
 
1 Supported by the Bio-Arts Research Corporation and the Link Equine Research Endowment Fund (Texas A&M University). Back

2 Correspondence: Katrin Hinrichs, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4466. FAX: 979 845 6544; khinrichs{at}cvm.tamu.edu Back

Received: 18 April 2003.

First decision: 14 May 2003.

Accepted: 19 August 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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K Hinrichs, Y H Choi, C C Love, Y G Chung, and D D Varner
Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract.
Reproduction, June 1, 2006; 131(6): 1063 - 1072.
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