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Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract

¹ Departments of Veterinary Physiology and Pharmacology and ² Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4466 USA and ³ Advanced Cell Technology, Worcester, MA 01605 USA

Correspondence should be addressed to K Hinrichs; Email: khinrichs{at}cvm.tamu.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect blastocyst development. Overall, transfer of eight blastocysts produced using roscovitine-treated donor cells and our standard activation protocol yielded three pregnancies, of which two (25% of transferred embryos) resulted in delivery of viable foals. Flow cytometric evaluation showed that roscovitine treatment significantly increased the proportion of cells classified as small, in comparison to growth to confluence or serum deprivation, but did not significantly affect the proportion of cells in G0/G1 (2N DNA content). Transfer of one blastocyst produced using roscovitine-treated donor cells, with addition of roscovitine injection at activation, yielded one pregnancy which was lost before 114 days’ gestation. Transfer to recipients of two blastocysts produced using confluent donor cells with addition of cycloheximide at activation gave no resulting pregnancies. We conclude that roscovitine treatment of donor cells yields equivalent blastocyst production after nuclear transfer to that for confluent donor cells, and that direct injection of roscovitine-treated donor cells, followed by activation using sperm extract, is compatible with efficient production of viable cloned foals.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Somatic cell nuclear transfer (NT) has been used to produce live offspring in many large-animal species, including sheep, cattle, goats, and pigs. There are two reports of horse foals produced by NT from adult somatic cells (Galli et al. 2003, Lagutina et al. 2005); in addition, three mule foals have been produced by NT using fibroblasts obtained from a mule fetus (Woods et al. 2003, Vanderwall et al. 2004b).

Progress in NT in the horse is hampered by the low numbers of equine oocytes available for study and the lack of information on in vitro embryo production in this species. In addition, early studies on equine NT reported low rates of fusion of equine cytoplasts with donor cells (Li et al. 2002, Choi et al. 2002b). To make maximum use of the available oocytes, alternative methods yielding higher recombination rates have been developed in the horse. These have included addition of sendai virus during fusion (Li et al. 2002), fusion after zona removal (Galli et al. 2003, Lagutina et al. 2005) and direct injection of the donor cell (Choi et al. 2002a). Of the recombination methods used, only fusion after zona removal has been shown to produce live young after nuclear transfer using adult somatic cells.

The reported rates of blastocyst production after equine NT have been low (0–16%; Galli et al. 2003, Li et al. 2004, Lagutina et al. 2005). Because rates of blastocyst production after intracytoplasmic sperm injection of in vitro-matured oocytes are acceptable (up to 38%; Hinrichs et al. 2005), the low blastocyst development after NT in the horse may be related to inefficiency in activation of equine oocytes. Effective methods for equine oocyte activation were not developed before work commenced on equine NT; methods commonly used in other species, including exposure to calcium ionophore A23187 [GenBank] , cycloheximide, ionomycin, ethanol, thimerosal, or 6-dimethylamino purine (6-DMAP), or injection of inositol 1,4,5-tripho sphate, were reported to result in low activation or cleavage rates when used on horse oocytes (Hinrichs et al. 1995, Li et al. 2000, Carneiro et al. 2001, Choi et al. 2001). Activation procedures used in equine NT have included exposure to calcium ionophore A23187 [GenBank] or ionomycin, followed by treatment either with cycloheximide (Choi et al. 2002b, Galli et al. 2003, Li et al. 2003, Woods et al. 2003) or with cycloheximide plus 6-DMAP (Lagutina et al. 2005).

Because methods for oocyte activation commonly used in other species have not supported high blastocyst development in horse NT embryos, evaluation of other activation procedures is needed. Stallion sperm extract has been shown to induce calcium oscillations in horse oocytes similar to those found after intracytoplasmic sperm injection (Bedford et al. 2003). We obtained cleavage rates of 51% when equine NT oocytes were activated by injection of stallion sperm extract (Choi et al. 2002a), and cleavage rates up to 79% were obtained when recombined oocytes were cultured in the presence of 6-DMAP after sperm extract injection (Choi et al. 2004). Blastocyst production after this activation treatment has not been reported. Possible methods to increase activation after sperm extract injection include treatment with cycloheximide or roscovitine. Cycloheximide prevents recrudescence of maturation promoting factor (MPF; Cdk1/cyclin B) activity after an activation stimulus (Presicce & Yang 1994, Yang et al. 1994, Moos et al. 1996). Roscovitine, an inhibitor of ATP binding to MPF, has been used via this activity to induce activation of oocytes, either parthenogenetically or after NT (Mitalipov et al. 2001). In Xenopus, microinjection of roscovitine was found to be more effective for oocyte activation than was incubation in roscovitine-containing medium (Flament et al. 2000). To our knowledge, the effect of injection of roscovitine on oocyte activation in mammalian species has not been evaluated.

Transfer of NT-derived embryos to recipients is associated with losses throughout gestation and in early postnatal life (Hill et al. 2000, Chavatte-Palmer et al. 2004). The incidence of fetal and neonatal loss associated with NT, and the abnormalities associated with such losses, have been best characterized in cattle, in which approximately 75% of initial pregnancies fail to be carried to term (Wells et al. 1999, Forsberg et al. 2002). Similarly, in the reports on pregnancies produced by equine NT, 75–100% of established pregnancies were lost, most in early gestation (Galli et al. 2003, Woods et al. 2003, Vanderwall et al. 2004a, Lagutina et al. 2005). In our laboratory in 2002, transfer of 4 NT blastocysts produced using fibroblasts grown to confluence resulted in one pregnancy, which was lost at 9 months gestation (Hinrichs and Choi, unpublished data). The high rates of loss associated with cloned fetuses are largely attributed to the failure of the oocyte to effectively reprogram the donor cell nucleus; i.e. to transform the epigenetic markers of the transferred chromatin from those of a somatic cell to those of a zygote. The exact changes necessary for this, and the mechanisms by which the oocyte induces these changes, are currently unclear.

Gibbons and coworkers (2002) reported that higher proportions of viable NT calves were obtained when adult somatic donor cells were treated with roscovitine rather than by serum deprivation (7 calves from 62 blastocysts, 11%, vs 1 calffrom 60 blastocysts, 2% respectively). This is a notable finding; treatments that increase production of viable NT offspring are important not only because they may shed light on the process of reprogramming, but also because they decrease the unwanted fetal losses associated with cloning. However, to our knowledge, no further studies on the use of roscovitine-treated donor cells for production of NT offspring have been reported. This may be due in part to the finding of Gibbons and coworkers (2002) that roscovitine treatment of bovine donor cells was associated with a significant decrease in the proportion of recombined oocytes that developed to the blastocyst stage (12% vs 21% for serum-deprived cells). In the cat, roscovitine treatment of donor cells did not affect the rate of blastocyst development (Gomez et al. 2003), but the ability of these blastocysts to produce pregnancies or offspring was not evaluated.

The present study was conducted to determine the rate of blastocyst development in equine NT embryos produced by direct injection of donor cells and activation by injection of sperm extract, using either confluent or roscovitine-treated donor cells. Flow cytometric analysis was used to evaluate the cell-cycle status of donor cells prepared by different donor cell treatments, and its relationship to cell size. Different methods for activation of recombined oocytes, including addition of cycloheximide treatment and roscovitine injection as described by Flament et al.(2000) were also evaluated. The viability of embryos produced by NT with roscovitine-treated donor cells was assessed after transfer of these embryos to recipient mares.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Experiment 1: Blastocyst production using roscovitine-treated or confluent donor cells
Oocyte collection and maturation
Ovaries were transported from two horse abattoirs to the laboratory at 20–32 °C (3–4 h transport time). All visible follicles were opened with a scalpel blade and the granulosa layer of each follicle was scraped using a 0.5 cm bone curette. All media components were obtained from Gibco Life Technologies, Inc. (Grand Island, NY) unless otherwise noted. The contents of the curette were washed into individual Petri dishes with Hepes-buffered TCM199 with Hanks’salts plus ticarcillin (0.1 mg/ml; SmithKline Beecham Pharmaceuticals, Philadelphia, PA). The contents of the Petri dishes were examined using a dissection microscope at x10–20. Oocyte–cumulus complexes were classified as compact, expanded or degenerating depending on the expansion of both mural granulosa and cumulus as described previously (Hinrichs & Williams 1997, Hinrichs & Schmidt 2000). 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) were classified as expanded oocytes. Oocytes having both compact cumulus and compact mural granulosa were classified as compact oocytes. Because of their relative abundance, higher maturation rate, and equivalent developmental competence (Hinrichs et al. 2005), only expanded oocytes were used in these studies.

For in vitro maturation, the oocytes were washed twice in maturation medium (TCM199 with Earle’s salts, 5 mU/ml follicle stimulating hormone (FSH) (Sioux Biochemicals Inc., Sioux Center, IA), 10% fetal bovine serum (FBS) and 25 µg/ml gentamycin) containing 200 ng/ml insulin-like growth factor (IGF)-I (Sigma-Aldrich, Inc., St. Louis, MO). Oocytes were cultured at a ratio of 10 µl medium per oocyte in microdroplets of this medium under light white mineral oil (Sigma) at 38.2 °C in 5% CO₂ in air for 24 h. After culture, oocytes were denuded of cumulus by pipetting in 0.05% hyaluronidase in CZB-M (Choi et al. 2003). Those oocytes having a polar body were placed in CZB-H (Choi et al. 2003) and returned to the incubator until used for NT.

Nuclear transfer
Fibroblasts were obtained by culture of skin biopsies from a 23-year-old stallion. Cells in the 4th to 6th passage were utilized; through these passages, cultures had been frozen and thawed 1 to 2 times at the time of use. Cells were either grown to confluence, without serum deprivation, or were treated while actively growing with R-roscovitine (Sigma), 15 µg/ml for the 24 h preceding NT. Cells were trypsinized before use and washed and held in CZB-M without glutamine, non-essential amino acids or FBS, but supplemented with 2% polyvinylpyrrolidone (CZB-M/2% PVP) for a maximum of 30 min before being transferred to cytoplasts.

Oocytes having a first polar body after in vitro maturation were incubated for 10–15 min in CZB-H with 10% FBS containing 0.5 µg/ml Hoechst 33342 (Sigma) and 5 µg/ml cytochalasin B (Sigma). Oocytes were then held in CZB-M with a holding pipette (120–140 µm outer diameter) under an inverted microscope equipped with Eppendorf manipulators. The zona pellucida of the oocyte was drilled using 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 CZB-H with 10% FBS. The injection of fibroblast cells into the enucleated equine oocytes was slightly modified from that of a previous report (Choi et al. 2002a). Briefly, the outside diameter of the injection pipette was 8–9 µm. Immediately before injection, a fibroblast cell of small to medium size (13–15 µm diameter) within the cell population (range of 11–23 µm diameter) was selected. The cell was held in CZB-M/2% PVP and 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 with 10% FBS and reconstructed oocytes were held in CZB-H.

Recombined oocytes were then injected with 2–4 pL (volume estimated from the length of the liquid column and the diameter of the pipette) of sperm extract, prepared as previously described (Choi et al. 2002a). Oocytes were treated after sperm extract injection by culture in the presence of 2 mM 6-DMAP in CZB-H with 10% FBS for 4 h. They were then washed and were cultured in microdroplets of DMEM/F-12 (Sigma) with 10% FBS under oil in a humidified atmosphere of 5% O₂, 5% CO₂ and 90% N₂, at 38.2 °C. This medium provides up to 38% blastocyst development in ICSI-produced equine embryos (Hinrichs et al. 2005). Medium was changed every 2–3 days. Embryos were examined for cleavage on Day 2 or 3, and were evaluated on Day 7 and Day 8 of culture for development to the blastocyst stage.

Flow cytometric evaluation of donor cells
Donor cells in four treatments were evaluated by flow cytometry. Cells were prepared by growth to confluence or roscovitine treatment, as described above, or by serum deprivation (0.5% serum for 5 days), or were used from an actively growing culture. Treatments were synchronized so that cells in all four treatments were evaluated by flow cytometry on the same day. Four replicates were performed. To estimate the cell diameters corresponding to the divisions characterized on flow cytometry, the diameters of 100 cells from the growing fibroblast culture were measured using the ‘Attributes-Length’ feature of Axiovision 4 software (Carl Zeiss AG, Germany) on photomicrographs of random areas of the cell suspension.

Fibroblasts from the four different culture treatments were harvested by trypsinization, washed once with DMEM/F-12 with 10% serum and once with PBS+0.2% BSA. Stock solutions of 1 mg/ml of ribonuclease-A (Sigma), 2.5 mM propidium iodide (PI) and 1% Triton-X in phosphate-buffered saline were prepared. Cell suspensions were dispersed at approximately 5 million/ml, then 40 µl of the cell suspension was combined with 6.4 µl of ribonuclease-A, 6.4 µl of PI, and 20 µl of the Triton-X/PBS solutions. Final concentrations of ribonuclease-A, PI and Triton-X were 88 µg/ml, 0.22 mM, and 0.27% respectively. This suspension was incubated at room temperature for 10 min prior to evaluation by flow cytometry.

Stained cell suspensions were evaluated using a FACScan (Becton Dickinson, Mountain View, CA) flow cytometer, equipped with an air-cooled Argon ion 488 nm laser. Cells were gated using forward (FSC) and side scatter (SSC) characteristics and the PI-stained cells were excited and emission was detected on detector FL2 (log units). Acquisition was performed in Cell Quest (Becton Dickinson, Mountain View, CA) and analysis was performed using WinList software (Verity Software House Inc., Topsham, ME).

Two-dimensional cell analyses of relative size and of DNA content were performed using FSC and FL2 emission cell characteristics respectively. Cell populations were gated based on three (small, medium, and large) size populations using relative forward scatter measures and three populations based on DNA content (2N, intermediate, and 4N). FSC gives an estimation of relative cell size but cannot be used to calculate actual cell dimensions (Shapiro, 2003). Overall, nine cell populations (3 x 3) were created (Fig. 1). Population dimension (box size) was fixed between samples. Variation in cell location between samples was minimized by applying the Auto-Position feature of the WinList software to the medium 2N cell population, thereby adjusting the box location to the median value of that cell population.

View larger version (21K): [in this window] [in a new window]   Figure 1 Flow cytometric distribution of fibroblast cell types based on size (forward light scatter, FSC) and DNA content (2N, intermediate, and 4N) as measured by propidium iodide (FL2) fluorescence.

Experiment 2: Transfer of NT embryos produced from roscovitine-treated or confluent donor cells; effect of cycloheximide at activation
Fibroblasts from a 20-year-old stallion were used as nuclear donor cells. The fibroblasts were grown in culture and used at passage 7 to 10; through these passages, cultures had been frozen and thawed 3–4 times at the time of use. Oocyte maturation, fibroblast treatments (confluence and roscovitine treatment), NT procedures, and embryo culture were performed as described for Experiment 1, except that IGF-I was not included in oocyte maturation medium. During 6-DMAP culture after injection of sperm extract, cycloheximide, 10 µg/ml, was added to the 6-DMAP-containing medium for half of the recombined oocytes in each donor cell treatment. Embryo culture and assessment was performed as for Experiment 1.

A herd of 12 Quarter-horse type mares, aged 5–15 years and weighing 400–500 kg, was maintained to provide embryo recipients. Mares were kept in paddocks with run-in sheds, and were fed hay and sweet feed with free-choice water. Work with these mares was performed according to the United States Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training and was approved by the Laboratory Animal Care Committee at Texas A&M University. Blastocysts (one per mare) were transferred transcervically to the uteri of recipient mares which had ovulated from 1 day before to 4 days after the date of NT, or that were in anestrus or seasonal transition and were treated with a biorelease formulation of estradiol 17ß (BETPharm, Lexington, KY), 10 mg i.m., 2 days before the day of NT and with a biorelease form of progesterone (BETPharm), 1.5 g i.m., 3 days after the day of NT. The mares’ reproductive tracts were monitored by ultrasonography per rectum from Day 11 to Day 16 after transfer to detect presence of an embryonic vesicle. When an embryonic vesicle was seen, the mares were administered biorelease progesterone, 1.5 g i.m., weekly until either accessory corpora lutea were present, or until after day 100 of gestation.

Experiment 3: Co-injection of roscovitine for activation
Oocyte recovery and maturation procedures, fibroblast preparation, and NT procedures were as described for Experiment 2 except that either DMEM/F-12 or CZB-M was used as the micromanipulation medium. All fibroblasts used for NT were prepared by roscovitine treatment. Recombined oocytes were divided into three activation treatments. Oocytes in the control treatment were activated by injection of sperm extract and then culture with 6-DMAP, as performed in Experiment 1. In the 25 µM roscovitine treatment, roscovitine was added to the sperm extract to a final concentration of 25 µM and was injected into the oocyte at the time of sperm extract injection. In the 50 µM roscovitine treatment, roscovitine was added to the sperm extract to a final concentration of 50 µM and was similarly co-injected. The co-injected oocytes were then treated with 6-DMAP and cultured as for control oocytes.

Recombined oocytes were cultured and assessed, and transferred to recipient mares, as described for Experiment 2; all recipient mares were ovulatory. Some blastocysts were not transferred due to lack of available recipient mares.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Experiment 1: Blastocyst production using roscovitine-treated or confluent donor cells
One-hundred and fifty-five ovaries were processed, and 741 follicles were scraped to recover oocytes. From these, 206 Ex, 139 Cp and 32 degenerating oocytes were recovered. For this experiment, 206 Ex oocytes were used. Four oocytes were broken during removal of cumulus cells after maturation; 138 oocytes (68%) were seen to have polar bodies and were subjected to NT. Twenty seven oocytes were lysed during enucleation, donor cell injection, or sperm factor injection and 111 recombined oocytes were cultured. The cleavage and blastocyst development rates for embryos produced using confluent or roscovitine-treated donor cells are presented in Table 1. There was no difference in cleavage rate or in blastocyst development rate between the two donor cell treatments (2/55, 3.6% vs 2/56, 3.6% respectively).

View larger version (18K): [in this window] [in a new window]   Figure 2 Representative scatterplots of forward scatter (representing size) and FL2 emission (representing DNA content) for fibroblasts in the four cell treatment groups.

Two pregnancies resulted from transfers of four embryos in the control activation group. One embryo resulted in a vesicle first visualized on ultrasonography per rectum on Day 13. This conceptus appeared to develop normally, but was aborted at 239 days gestation. Necropsy revealed a normal male fetus and placenta, with the exception of an edematous umbilical cord and focal nodules of necrosis in the liver. Typing for 13 equine microsatellites confirmed that the fetus was of the same DNA type as the donor fibroblasts (Table 6). The second vesicle was first visualized on ultrasonography per rectum on Day 12. This embryo continued to develop normally thereafter, and resulted in birth of a male foal at day 340 of gestation (May 29, 2005). The colt was delivered without assistance, and was 40.5 kg at birth. The colt was active and had normal times to stand (30 min) and nurse (2.5 h). The umbilical cord appeared slightly enlarged (2.7 cm diameter) and was edematous; however, the placenta was normal on both gross and histological examination. The umbilical stump remained large, and was excised surgically when the colt was 12 days old. Typing for 13 equine microsatellites confirmed that this colt was of the same DNA type as the donor fibroblasts (Table 6). The colt has continued to develop normally and is 8.5 months old at the time of writing.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
These results demonstrate that NT using direct injection of donor cells and activation by injection of sperm extract is compatible with production of both blastocysts and foals. The foals produced in this study represent the third and fourth viable foals reported from adult somatic cell NT in the horse. To our knowledge, these are the first cloned offspring produced using direct injection of the donor cell and activation with sperm extract. This provides an alternative technique for consideration in species that have been difficult to clone successfully.

We evaluated the use of roscovitine for donor cell synchronization because this treatment was previously shown to significantly increase the production of viable NT calves (Gibbons et al. 2002). In contrast to the situation in cattle (Gibbons et al. 2002), blastocyst production in our study was equivalent between roscovitine-treated and confluent donor cells (Experiment 1). Direct injection of roscovitine-treated donor cells in combination with our standard activation protocol resulted in an overall blastocyst formation rate (Experiments 1, 2 and 3 combined) of 11/210 (5.2%). While we achieved an insufficient number of pregnancies with confluent donor cells to directly compare the viability of blastocysts produced from the two donor cell sources, the rate of production of viable foals (two foals from eight blastocysts; 25%) compares favorably with that of the two previous reports on production of NT foals from adult fibroblasts (one foal from 17 blastocysts, 6%, and one viable foal from 101 blastocysts, 1%; Galli et al. 2003, Lagutina et al. 2005), and to the overall proportion of viable calves resulting from transfer of NT-derived embryos in cattle (6%; Wells 2005). That two viable offspring were achieved from three pregnancies in this treatment is noteworthy, given the 75–100% losses previously reported in horse NT (Galli et al. 2003, Woods et al. 2003, Vanderwall et al. 2004b, Lagutina et al. 2005) and the low production of viable cloned offspring in other species.

Gibbons et al.(2002) attributed the significantly higher production of viable offpring that they observed after roscovitine treatment of donor cells to improved synchronization of the cells into G0/G1. Roscovitine is a purine that blocks the ATP-binding site of specific cyclin-dependent kinases (Cdks). Roscovitine inhibits not only Cdk1, the activity associated with use of this compound for suppression of maturation and activation, but also Cdk2, Cdk5, Cdk7, and other Cdks (Meijer et al. 1997, McClue et al. 2002). Through these activities, roscovitine at low concentrations (<20 µM) suppresses the G1/S and G2/M transitions in cultured fibroblasts, thus arresting cells in G1 or G2 (Alessi et al. 1998). However, we found that roscovitine-treated cells were no more likely to have 2N DNA than were serum-deprived or confluent cells. Boquest et al.(1999) reported that in serum-deprived or confluent cultures of porcine fibroblasts, 93–95% of small cells were 2N (G0/G1), and we found a similar situation in roscovitine-treated fibroblasts. When selection for size, as is done when selecting donor cells for NT, was considered (Table 4), we found that 95–98% of small cells were in G0/G1 (2N), regardless of donor cell treatment. These data suggest that the beneficial effect of roscovitine noted by Gibbons et al.(2002) was due to changes in some quality of the cells, perhaps related to the ability of the cell to be reprogrammed, rather than to increased synchrony of cells in G0/G1. A qualitative effect of roscovitine treatment is supported by our finding that a significantly greater proportion of cells in this treatment were classified as small. This finding is interesting; one would expect that cessation of growth due to deprivation of serum for 5 days or to crowding would affect cell size more profoundly than would arrest of the cell cycle via roscovitine for 24 h, during which time most other cellular machinery should be functional. Further work is warranted on the nuclear and cytoplasmic characteristics of cells prepared under different donor cell synchronization treatments, and their relationship to nuclear reprogramming and embryo viability after nuclear transfer.

The 5% rate of blastocyst formation achieved in this study with direct injection of donor cells and activation with sperm extract and 6-DMAP is similar to that of previous reports on equine NT (Galli et al. 2003, Li et al. 2004, Lagutina et al. 2005). This low rate indicates that further work to improve blastocyst development in this species is essential. We evaluated the effect of addition of cycloheximide treatment or of roscovitine injection at the time of activation on blastocyst development. Cycloheximide has been shown to significantly increase the activation rate in equine oocytes treated with calcium ionophore (Hinrichs et al. 1995). The combination of cycloheximide and 6-DMAP is commonly used in other species (Liu et al. 2004), and this combination, after ionomycin treatment, was used in production of one of the previous NT foals (Lagutina et al. 2005). However, addition of cycloheximide to the activation protocol in our study did not increase blastocyst development. Roscovitine, as an inhibitor of MPF, has been shown to be an effective activation agent in rhesus oocytes (Mitalipov et al. 2001). We chose injection of roscovitine rather than culture with roscovitine as an activation treatment in this study because injection was reported to be the more effective treatment in Xenopus (Flament et al. 2000), and injection was simply performed in our system by addition of roscovitine to the sperm extract solution. However, this treatment did not increase blastocyst development rates, and resulted in a conceptus that developed abnormally in early gestation and was lost before 114 days. Further work on the timing of the roscovitine injection, or on roscovitine culture rather than injection, is needed to fully evaluate the effect of roscovitine on activation of horse oocytes after NT.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
This work was supported by the Link Equine Research Endowment Fund at Texas A&M University, and Cryozootech S.A., Sonchamp, France.

	Footnotes

 
K Hinrichs and Y H Choi contributed equally to this work

Received 30 December 2005
First decision 20 January 2006
Revised manuscript received 13 February 2006
Accepted 17 February 2006

	References

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 

Alessi F, Quarta S, Savio M, Riva F, Rossi L, Stivala LA, Scovassi AI, Meijer L & Prosperi E 1998 The cyclin-dependent kinase inhibitors olomoucine and roscovitine arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity. Experimental Cell Research 245 8–18.[CrossRef][ISI][Medline]

Bedford SJ, Kurokawa M, Hinrichs K & Fissore RA 2003 Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa. Reproduction 126 489–499.[Abstract]

Boquest AC, Day BN & Prather RS 1999 Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells. Biology of Reproduction 60 1013–1019.[Abstract/Free Full Text]

Carneiro G, Lorenzo P, Pimentel C, Pegoraro L, Bertolini M, Ball B, Anderson G & Liu I 2001 Influence of insulin-like growth factor-1 and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenetic development in equine oocytes. Biology of Reproduction 65 899–905.[Abstract/Free Full Text]

Chavatte-Palmer P, Remy D, Cordonnier N, Richard C, Issenman H, Laigre P, Heyman Y & Mialot JP 2004 Health status of cloned cattle at different ages. Cloning Stem Cells 6 94–100.[CrossRef][ISI][Medline]

Choi YH, Love CC, Thompson JA, Varner DD & Hinrichs K 2001 Activation of cumulus-free horse oocytes: effect of maturation medium, calcium ionophore concentration and duration of cycloheximide exposure. Reproduction 122 177–183.[Abstract]

Choi YH, Love CC, Chung YG, Varner DD, Westhusin ME, Burghardt RC & Hinrichs K 2002a Production of nuclear transfer horse embryos by piezo-driven injection of somatic cell nuclei and activation with stallion sperm cytosolic extract. Biology of Reproduction 67 561–567.[Abstract/Free Full Text]

Choi YH, Shin T, Love CC, Johnson CA, Varner DD, Westhusin ME & Hinrichs K 2002b Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence, and subsequent fusion and activation rates after nuclear transfer. Theriogenology 57 1005–1011.[CrossRef][ISI][Medline]

Choi YH, Chung YG, Walker SC, Westhusin ME & Hinrichs K 2003 In vitro development of equine nuclear transfer embryos: Effects of oocyte maturation media and amino acid composition during embryo culture. Zygote 11 77–86.[CrossRef][ISI][Medline]

Choi YH, Love LB, Westhusin ME & Hinrichs K 2004 Activation of equine nuclear transfer oocytes: methods and timing of treatment in relation to nuclear remodeling. Biology of Reproduction 70 46–53.[Abstract/Free Full Text]

Flament S, Bodart J-F, Bertout M, Browaeys E, Rousseau A & Vilain J-P 2000 Differential effects of 6-DMAP, olomoucine and roscovitine on Xenopus oocytes and eggs. Zygote 8 3–14.[CrossRef][ISI][Medline]

Forsberg EJ, Strelchenko NS, Augenstein ML, Betthauser JM, Childs LA, Eilertsen KJ, Enos JM, Forsythe TM, Golueke PJ, Koppang RW, Lange G, Lesmeister TL, Mallon KS, Mell GD, Misica PM, Pace MM, Pfister-Genskow M, Voelker GR, Watt SR & Bishop MD 2002 Production of cloned cattle from in vitro systems. Biology of Reproduction 67 327–333.[Abstract/Free Full Text]

Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, Duchi R & Lazzari GA 2003 A cloned horse born to its dam twin. Nature 424 635.[Medline]

Gibbons J, Arat S, Rzucidlo J, Miyoshi K, Waltenburg R, Respess D, Venable A & Stice S 2002 Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells. Biology of Reproduction 66 895–900.[Abstract/Free Full Text]

Gomez MC, Jenkins JA, Giraldo A, Harris RF, King A, Dresser BL & Pope CE 2003 Nuclear transfer of synchronized african wild cat somatic cells into enucleated domestic cat oocytes. Biology of Reproduction 69 1032–1041.[Abstract/Free Full Text]

Hill JR, Winger QA, Long CR, Looney CR, Thompson JA & Westhusin ME 2000 Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells. Biology of Reproduction 62 1135–1140.[Abstract/Free Full Text]

Hinrichs K & Williams KA 1997 Relationships among oocyte–cumulus morphology, follicular atresia, initial chromatin configuration, and oocyte meiotic competence in the horse. Biology of Reproduction 57 377–384.[Abstract]

Hinrichs K & Schmidt AL 2000 Meiotic competence in horse oocytes: interactions among chromatin configuration, follicle size, cumulus morphology, and season. Biology of Reproduction 62 1402–1408.[Abstract/Free Full Text]

Hinrichs K, Schmidt AL & Selgrath JP 1995 Activation of horse oocytes. Biology of Reproduction Monograph Series 1 319–324.

Hinrichs K, Choi YH, Love LB, Varner DD, Love CC & Walckenaer BE 2005 Chromatin configuration within the germinal vesicle of horse oocytes: changes post mortem and relationship to meiotic and developmental competence. Biology of Reproduction 72 1142–1150.[Abstract/Free Full Text]

Lagutina I, Lazzari G, Duchi R, Colleoni S, Ponderato N, Turini P, Crotti G & Galli C 2005 Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type. Reproduction 130 559–567.[Abstract/Free Full Text]

Li X, Morris LH & Allen WR 2000 Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection. Journal of Reproduction and Fertility 119 253–260.[Abstract]

Li X, Morris LH & Allen WR 2002 In vitro development of horse oocytes reconstructed with the nuclei of fetal and adult cells. Biology of Reproduction 66 1288–1292.[Abstract/Free Full Text]

Li X, Tremoleda JL & Allen WR 2003 Effect of the number of passages of fetal and adult fibroblasts on nuclear remodelling and first embryonic division in reconstructed horse oocytes after nuclear transfer. Reproduction 125 535–542.[Abstract]

Li X, Dai Y & Allen WR 2004 Influence of insulin-like growth factor-I on cytoplasmic maturation of horse oocytes in vitro and organization of the first cell cycle following nuclear transfer and parthenogenesis. Biology of Reproduction 71 1391–1396.[Abstract/Free Full Text]

Liu JL, Sung LY, Du F, Julian M, Jiang S, Barber M, Xu J, Tian XC & Yang X 2004 Differential development of rabbit embryos derived from parthenogenesis and nuclear transfer. Molecular Reproduction and Development 68 58–64.[CrossRef][ISI][Medline]

McClue SJ, Blake D, Clarke R, Cowan A, Cummings L, Fischer PM, MacKenzie M, Melville J, Stewart K, Wang S, Zhelev N, Zheleva D & Lane DP 2002 In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine). International Journal of Cancer 102 463–468.

Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki N, Inagaki M, Delcros JG & Moulinoux JP 1997 Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. European Journal of Biochemistry 243 527–536.[ISI][Medline]

Mitalipov SM, Nusser KD & Wolf DP 2001 Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos. Biology of Reproduction 65 253–259.[Abstract/Free Full Text]

Moos J, Kopf GS & Schultz RM 1996 Cycloheximide-induced activation of mouse eggs: Effects on cdc2/cyclin B and MAP kinase activities. Journal of Cell Science 109 739–748.[Abstract]

Presicce GA & Yang X 1994 Nuclear dynamics of parthenogenesis of bovine oocytes matured in vitro for 20 and 40 hours and activated with combined ethanol and cycloheximide treatment. Molecular Reproduction and Development 37 61–68.[CrossRef][ISI][Medline]

Reef VB 1998 Fetal ultrasonography. In Equine Diagnostic Ultrasound, pp 425–445. Ed. VB Reef. Philadelphia: WB Saunders Co.

Shapiro HM 2003 Parameters and probes.In Practical Flow Cytometry, pp 275–276. Hoboken, NJ: Wiley-Liss.

Vanderwall DK, Woods GL, Aston KI, Bunch TD, Li G-P, Meerdo LN & White KL 2004a Cloned horse pregnancies produced using adult cumulus cells. Reproduction, Fertility and Development 16 675–679.[CrossRef][Medline]

Vanderwall DK, Woods GL, Sellon DC, Tester DF, Schlafer DH & White KL 2004b Present status of equine cloning and clinical characterization of embryonic, fetal, and neonatal development of three cloned mules. Journal of American Veterinary Medical Association 225 1694–1699.[CrossRef]

Wells DN 2005 Animal cloning: problems and prospects. Revue Scientifique et Technique 24 251–264.

Wells DN, Misica PM & Tervit HR 1999 Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biology of Reproduction 60 996–1005.[Abstract/Free Full Text]

Woods GL, White KL, Vanderwall DK, Li G-P, Aston KI, Bunch TD, Meerdo LN & Pate BJ 2003 A mule cloned from fetal cells by nuclear transfer. Science 301 1063.[Free Full Text]

Yang X, Presicce GA, Moraghan L, Jiang S & Foote RH 1994 Synergistic effect of ethanol and cycloheximide on activation of freshly matured bovine oocytes. Theriogenology 41 395–403.

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