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Enhanced Survivability of Cloned Calves Derived from Roscovitine-Treated Adult Somatic Cells

^a Department of Animal and Dairy Sciences, University of Georgia, Athens, Georgia 30602 ^b Research Institute for Genetic Engineering and Biotechnology, Marmara Research Center, Kocaeli 41470, Turkey ^c ProLinia, Inc., Athens, Georgia 30602 ^d Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan ^e Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nuclear transfer to produce cattle is inefficient because 1) donor cells are not easily synchronized in the proper phase of the cell cycle, 2) the nucleus of these cells is not effectively reprogrammed, 3) the rate of attrition of late-term pregnancies is high, and 4) the health of early postnatal calves is compromised. The cyclin dependent kinase 2 inhibitor, roscovitine, was used to maximize cell cycle synchrony and to produce cells that responded more reliably to nuclear reprogramming. Roscovitine-treated adult bovine granulosa cells (82.4%) were more efficiently synchronized (P < 0.05) in the quiescent G0/G1 phase of the cell cycle than were serum-starved cells (76.7%). Although blastocyst development following nuclear transfer was elevated (P < 0.05) in the serum-starved group (21.1%) relative to the roscovitine-treated cells (11.8%), the number of cells in the blastocysts derived from roscovitine-treated cells was higher (P < 0.05) than those derived from the serum-starved group (roscovitine-treated group: 142.8 ± 6.0 cells; serum-starved group: 86.8 ± 14.5 cells). The resulting fetal and calf survival after embryo transfer was enhanced in the roscovitine-treated group (seven surviving calves from six pregnancies) compared with serum-starved controls (two calves born, one surviving beyond 60 days, from five pregnancies). Roscovitine culture can predictably synchronize the donor cell cycle and increase the nuclear reprogramming capacity of the cells, resulting in enhanced fetal and calf survival and increased cloning efficiency.

developmental biology, embryo, implantation, pregnancy, reproductive technology

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Production of mammals by nuclear transfer has become a tool for propagating valuable animals [1–5] and can be used as an avenue for producing genetically modified animals [6, 7]. Widespread use of the technology has been limited because of the low survival rate of fetuses during the last trimester of gestation [8, 9] and compromised postnatal health of the offspring [10, 11]. The inefficiencies associated with cloning may be attributed to many factors that are not fully understood, such as the oocyte-donor cell interaction [12], the stage of the donor cell cycle [13], inadequate placentation [10, 14], inappropriate or incomplete nuclear reprogramming following nuclear transfer [15, 16], and the type of donor cell used [16]. Fetal and calf perinatal survival must be improved before this technology can be used extensively. Currently, the high rate of fetal loss in the third trimester and the increased calf loss in the first month of life in clones compared with conventional pregnancies and calves are primary limitations for the widespread application of this technology.

The stage of the donor cell cycle is a major factor in the success of nuclear transfer in mammals [1, 17]. Quiescent donor cells arrested in the G1 or G0 stage of the cell cycle have been used to produce mice [18], pigs [19, 20], sheep [10, 21], and cattle [2]. Methods of arresting cells in this phase of the cycle have been explored using reversible cycle inhibitors [22]; however, serum starvation is often used as a donor cell treatment prior to nuclear transfer. In this experiment, the specific cyclin dependent kinase (CDK) 2 inhibitor, roscovitine, was used to arrest bovine granulosa cells in the G0/G1 quiescent phase of the cell cycle. Roscovitine effectively arrests human fibroblasts in the G0/G1 phase of the cell cycle [22] and can be used to maintain bovine oocytes at the germinal vesicle stage of maturation by inhibiting meiosis promoting factor, a member of the CDK family [23]. Following roscovitine removal, cells arrested in G0/G1 resumed cycling and entered the S phase as expected [22], and oocytes arrested at the germinal vesicle stage progressed to metaphase II [23], indicating that the effects of roscovitine were fully reversible. This experiment is the first attempt to culture donor cells in the presence of this kinase inhibitor prior to nuclear transfer. Adult bovine granulosa cells were used as donors. We tested the hypothesis that the type of cell cycle synchronization prior to nuclear transfer has long-term effects on pregnancy rate, fetal and calf survival, and postnatal calf health.

	MATERIALS AND METHODS

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Cell Preparation and Culture

A primary cell line of granulosa cells was isolated from ovarian follicles of a 13-yr-old beef cow. The ultrasound-guided approach was similar to the procedure used previously to recover oocytes for in vitro fertilization [24]. Cells were washed in Dulbecco minimal essential medium (DMEM) supplemented with 10.0% fetal calf serum (FCS) and 1.0% (v:v) penicillin/streptomycin, and the cells were seeded into six-well culture plates following typical cell culture techniques. Following the first passage, cells were grown to confluence and frozen in DMEM-F12 supplemented with 20.0% FCS and 10.0% dimethyl sulfoxide. After thawing, cells were cultured (DMEM supplemented with 10.0% FCS and 1.0% v:v penicillin/streptomycin) to approximately 80% confluence (passages 2–5). Approximately half of the cells were allocated to be treated with culture medium containing 15 µM roscovitine (for approximately 24 h prior to nuclear transfer), and the remaining cells were cultured with medium supplemented with 0.5% FCS (for 72 h prior to nuclear transfer). The roscovitine-treated cells were exposed to the inhibitor throughout the nuclear transfer process. Donor cells to be submitted for flow cytometry sorting were trypsinized, centrifuged, and resuspended in 1 ml of PBS. Cells were first incubated with DNase-free RNase A for 30 min at 37°C and then with 1 mg/ml propidium iodide for 10 min (at 25°C) before being processed on the flow cytometer.

Oocyte Preparation and Nuclear Transfer

Recipient oocytes were washed and selected following removal from bovine antral follicles (3–8 mm in diameter). Only oocytes that had a homogenous cytoplasm and at least three layers of cumulus cells were selected for in vitro maturation. In vitro maturation medium consisted of tissue culture medium (TCM 199) supplemented with 10.0% FCS, 50 µg/ml sodium pyruvate, 1.0% v:v penicillin/streptomycin, 1 ng/ml recombinant insulin-like growth factor 1, 0.01 U/ml bovine LH, and 0.01 U/ml bovine FSH. Selected oocytes were placed in 500 µl of maturation medium overlaid with mineral oil (400 µl) and incubated for 16–18 h at 39°C in 5.0% CO₂ and air. After maturation, oocytes were vortexed to remove expanded cumulus cells and stained using Hoechst 33342 (2 µg/ml) to aid in visualization of the DNA (chromatin). Enucleation was ensured using ultraviolet light to visualize DNA located in the polar body and the metaphase plate. Donor cells were trypsinized, pelleted, and resuspended in DMEM supplemented with either 0.5% FCS (serum starved) or 10.0% FCS and 15 µM roscovitine prior to transfer into recipient oocytes. Donor cells (one per oocyte) were microsurgically placed into the perivitelline space evacuated during enucleation, ensuring intimate contact between the donor cell and the recipient oocyte.

Nuclear Transfer Unit Fusion and Activation

The donor cell and recipient cytoplasm of the nuclear transfer couplets were fused approximately 22–24 h postmaturation by means of a single direct electrical pulse (40 V) delivered via needle-type electrodes [25]. Fusion took place in Zimmermann cell fusion medium [26] by placing an electrode on each side of the nuclear transfer couplet (approximately 150 µm apart) and arranging the couplet so that the 20-µsec pulse was delivered perpendicular to the shared membrane space of the donor cell/cytoplasm. A sample of couplets was examined 1 h after the pulse to determine fusion efficiency. Activation of the couplets was performed beginning 2 h after fusion as described previously [27–29], using TCM 199 plus 1.0% FCS supplemented with cytochalasin B (5 µg/ml), cycloheximide (10 µg/ml), and calcium ionophore (5 mM) for 10 min followed by TCM 199 plus 10.0% FCS supplemented with only cytochalasin B (5 µg/ml) and cycloheximide (10 µg/ml) for 1 h in 5.0% CO₂ and air. A 5-h culture period in TCM 199 plus 10.0% FCS and cycloheximide (10 µg/ml) alone was conducted under low oxygen tension (5.0% CO₂, 5.0% O₂, 90.0% N₂). Following activation, reconstructed embryos were cultured in BARC medium [30] under low (5.0%) oxygen tension for 7 or 8 days.

Embryo Transfer

Embryos that reached the blastocyst stage were transferred into recipient cattle approximately 7 days after synchronized estrus. One or two embryos per recipient were nonsurgically introduced into the uterine horn ipsilateral to the ovary containing a palpable corpus luteum. Pregnancy evaluation was performed using transrectal ultrasound approximately 21 days following embryo transfer (Day 28 of gestation). Recipients diagnosed as pregnant were evaluated weekly until approximately 100 days of gestation and then monthly thereafter to study fetal development. During the last month of gestation, recipients were monitored several times each week via palpation to evaluate the health of the fetus. Recipients that were ketotic in the third trimester were treated with standard protocols, including a higher protein ration and propylene glycol (as needed).

Calving Strategy

Calving was managed and scheduled via a standing surgical route on approximately Day 272 of gestation. Parturition induction began approximately 36 h before scheduled cesarean section with 5 mg/kg of dexamethasone (3 i.m. injections every 12 h) supplemented with 25 mg of prostaglandin (i.m.) at the time of the second steroid injection. Standing cesarean section delivery was performed at the University of Georgia Veterinary Medicine Teaching Hospital. Immediately following delivery, an endotracheal tube was placed, and calves were suspended by the hind legs to facilitate lung coupage. If meconium was present at the time of delivery, a more aggressive approach (either via coupage or active suction) was adopted, consisting of multiple attempts to remove fluid from the lungs. Calves were given nasal oxygen at 2–10 L/min depending upon blood gas values. Plasma (2 L/calf, i.v.), antibiotics, and colostrum (10%–15% of body weight, either by suckling or stomach tube) were given in the first 4 h of life. More aggressive treatments (e.g., surfactant, inhaled steroids, bronchialdilators) were administered on a per case basis. Eight of the nine calves had their umbilicus surgically removed because it was a potential source of infection. The remaining calf had an umbilical clamp placed and showed no signs of abnormal umbilical shrinkage. Calf vitality was assessed shortly after birth and approximately every 3 days until release from the hospital. An overall vitality score was assigned based upon the following parameters: 1) the initial diagnosis and prognosis score (a scale of 1 to 3, with 1 being the best), 2) an overall quality of life diagnosis score following release from the hospital (a scale of 1 to 3, with 1 being the best), and 3) the cost of veterinarian care for the calf (level 1 = dollar values that were one half of the SD below the mean; level 2 = values that were within one half of the SD above and below the mean; level 3 = values that were above one half of the SD above the mean). One third of each of these scores was used per calf to develop an overall vitality score. Initial diagnoses and prognoses were scored as follows; level 1 = required limited medical intervention (short-term nasal oxygen, antibiotics, etc.); level 2 = required some special care or attention (cardiopulmonary hypertension requiring several days of nasal oxygen, mild to moderate aspiration pneumonia, mild to moderate bronchointerstitial lung disorders, etc.); level 3 = required special or intensive treatment (marked cardiopulmonary hypertension, multiple disorders such as musculoskeletal or cardiopulmonary problems, septicemia, etc.). The overall severity of care score during hospitalization was formulated as follows: level 1 = required mild to moderate short-term care (mild interstitial lung disorders, umbilical surgery necessary, etc.); level 2 = required more intensive, longer care (moderate to severe interstitial lung disorders, long-term diarrhea or respiratory distress, moderate pneumonia, etc.); level 3 = required substantial long-term care (long-term respiratory medication, surgery to correct angular limb deformities, etc.). Calves were bottle-fed and weaned at 4–6 wk of age following standard animal husbandry practices.

Statistics

Percentage data (cells in a particular phase of the cell cycle, embryo development to balstocyst from total cultured, calves born from embryo transfers, etc.) were arcsine transformed and analyzed using an ANOVA. Numerical data (number of cells in a particular blastosyst) also were analyzed using an ANOVA. The line of best fit to describe the slope of the survival curves was calculated (y = mx + b).

	RESULTS

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Cell Cycle Synchrony

Roscovitine treatment improved cell cycle synchrony compared with that of nontreated or serum-starved donor cells (Table 1). A higher proportion of cells (P < 0.05) was arrested in the G0/G1 phase of the cycle (and consequently a lower proportion of cells was in the G2/M phase; P < 0.05) in the roscovitine group compared with controls (cycling) or the serum-starved group. The fusion efficiency of nuclear transfer couplets was 58% for both roscovitine-treated and serum-starved cells. Nontransferred embryos reconstructed with roscovitine-treated cells had more embryo cells at the blastocyst stage (P < 0.05) than did the embryos reconstructed with serum-starved cells (Table 2). Sixty-two blastocysts were transferred into 46 recipients in the roscovitine group, and 60 blastocysts were transferred into 45 recipients in the serum-starved group. The early (approximately Day 30 of gestation) pregnancy rate following embryo transfer was not affected by cell treatment (Table 2) and there was a tendency (P = 0.087) for the roscovitine group to produce more calves per embryo transfer (Table 2) compared with the serum-starved group. Fetal survivability was similar between groups through the first two trimesters; however, in the last 60 days of gestation the majority of the pregnancies in the serum-starved group were lost compared with only one loss in the roscovitine group (Fig. 1). The number of calves produced was related to the donor cell treatment; however, the average vitality score (not different between treatments) was 1.96 ± 0.26 (combined for both groups) and was probably related to placentation, gestation, and calving management rather than donor cell treatment (Table 3). There were more healthy calves that survived gestation, parturition, and the first 60 days of life (P < 0.05) in the roscovitine group than in the serum-starved group (Table 2).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Fetal/calf survival curve for pregnancies derived from nuclear transfer embryo reconstruction using roscovitine-treated () and serum-starved () adult bovine granulosa cells as donors. Late in the third trimester and during the first 2 mo after birth (Days -60 to 60), there was a 6-fold steeper negative slope for serum-starved (y = -0.043x + 3.200) compared with roscovitine-treated cells (y = -0.007x + 6.200). *Six pregnancies resulted in six live births and seven calves (one set of twins) in the roscovitine group. All of the births from the serum-starved group resulted in singleton calves

Fetal Survival and Calving Results

Of the 12 cows that reached the end of the second trimester with healthy pregnancies, eight delivered healthy calves (Fig. 2). Seven cows had singletons (five from roscovitine-treated cells and two from serum-starved cells), and there was one set of twins (roscovitine group). Of the four recipients that lost pregnancies, one was from the roscovitine group (fetus lost on Day 202 of gestation) and three were from the serum-starved group (fetuses lost on Days 195, 214, and 236 of gestation). Some gross observations of the pathology of the aborted fetuses were 1) recessed upper jaw and an enlarged and protruding mandible, 2) subcutaneous edema, 3) enlarged, pale, and firm liver, 4) misshapen, round, and enlarged heart (specifically the right side), 5) moderate persistence of the primary spongiosa in the long bones, and 6) extremely large umbilical veins and arteries. Calves that were delivered alive had 1) varying degrees of caudal lung interstitial disease ranging from moderate to life threatening, 2) cardiopulmonary hypertension, 3) enlarged umbilical veins and arteries, and 4) encapsulated areas in the cranial portions of the lung that were of unknown origin. Some of the calves (both groups) developed septicemia; either the umbilical area or the lungs were considered probable sites of initial infection. Placental anomalies have been reported in recipients carrying cloned pregnancies [8–10], and these data are consistent with inadequate placentation having a dramatic effect on fetal survivability. Some of the calves that were delivered alive required considerable intensive care to treat their anomalies, possibly because of insufficient placental waste and nutrient exchange in utero. One calf (serum-starved group) was lost at approximately 60 days of age, and necropsy revealed moderate hydrocephaly, adhesions in the lungs consistent with pneumonia, and digestive problems consistent with vagal indigestion.

View larger version (85K): [in this window] [in a new window]   FIG. 2. Cloned calves surviving beyond 60 days after birth from roscovitine-treated or serum-starved (fourth from the left) adult bovine granulosa cells

Genetic Verification

To ensure heritage, DNA was extracted from donor cells and from blood samples from the calves and recipients using standard methods. The markers used to identify these locations were standardized according to typical molecular techniques. Identical fragment lengths were obtained for both alleles at each of 11 different locations from the nine cloned calves produced in this experiment and from the donor cell line. The recipients that served as surrogates had different fragment lengths from each other, the calves, and from one allele to the other, indicating that they were not related to the calves and that their genetic information was received in a method consistent with Mendelian genetics. The specific parentage of the recipients was not known.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This experiment indicated that the capacity of roscovitine to synchronize cells in the quiescent G0/G1 phase of the cell cycle directly affected the fetal and calf developmental physiology relative to the serum-starved cells. The higher proportion of quiescent cells probably impacts the capacity of the nucleus to be reprogrammed, possibly because of reduced transcriptional efficiency; however, this hypothesis has not been tested. Other mechanisms may exist but may be masked because the exact phase of the cell cycle of a particular cell and the status of the nucleus of that individual cell used in the cloning process cannot be accurately determined. Although a higher proportion of in vitro embryos developed in the serum-starved group, the morphological quality and stage of embryo development was similar between treatments at the time of embryo transfer. Embryo development rates from this experiment are consistent with those of other reports of bovine nuclear transfer embryo development from this laboratory [25, 27, 28] and others [2, 6, 9]. The similar initial pregnancy rates observed from transferring cloned embryos from either treatment in this experiment indicates that 1) morphology is not the best indicator of embryo viability, 2) the number of cells in the blastocysts may not be a major factor involved in pregnancy establishment, and 3) the positive effects that roscovitine treatment had on overall fetal and calf survivability were not mediated until the end of the second trimester. In addition to the impact of the novel roscovitine treatment, the type of donor cell used, activation protocol, and recipient/fetal interactions may explain some of the differences observed in this experiment compared with others. The production of eight cloned copies of an adult animal (Fig. 2) from granulosa cells reported here is similar to that in another report [2]. The percentage of healthy calves produced (as a function of embryo transfers) in the roscovitine group was considerably higher (15%) than in previous reports (10%) [2] or in the serum-starved group (4.4%). Of the cows submitted to the veterinary teaching hospital for calving, all of them produced live calves of varying health status. The birth weight (mean ± SD for both groups; 47.3 ± 2.7 kg) was unaffected by treatment (similar to other studies [2]) and did not have an obvious effect on calf viability.

Serum starvation of bovine and porcine cultured cells for at least 48 h causes reduced cell survival and increased DNA fragmentation, which is indicative of apoptosis [31–33]. Following nuclear transfer, higher embryonic loss has been observed in serum-starved compared with serum-fed granulosa cells [34]. High rates of embryo loss and abortions/fetal loss in cloned cattle [8, 9, 35, 36] and DNA damage in cultured sheep cells [37] may be related to serum starvation of the donor cells. In this experiment, late-term fetal loss in the serum-starved group may be consistent with inappropriate DNA replication or DNA damage that was not appropriately repaired during embryo/fetal development. It is possible that the roscovitine group did not have extensive DNA damage, possibly because of more effective cell cycle synchronization, or that DNA correction mechanisms were intact and efficient. There may also be a difference in the DNA fragmentation pattern or correction mechanisms of cells from animals of different ages or parity.

The nucleus-reprogramming capacity of roscovitine-treated donor cells is an area for exploration because it is associated with the placentation pattern and fetal-maternal communication, as indicated by the fact that the marked effects of roscovitine on fetal/calf survivability were not manifested until late in gestation. Increased cell cycle synchrony and enhanced fetal and calf survivability are major factors that increase the efficiencies associated with bovine somatic cell cloning. Increased efficiency in cattle cloning (and cloning in other species) will enhance the application of the technology to assist large animal agriculture and will provide a foundation for producing transgenic animals for cell therapy and biopharmaceuticals.

The use of roscovitine or other cell cycle synchronization agents can increase the efficiency of adult somatic cell bovine cloning by increasing the survival of nuclear transfer-derived fetuses and calves following embryo transfer. The significance of these results may impact the bovine cloning industry by allowing widespread implementation of this technology. As the efficiencies associated with fetal and calf survival increase, the overall costs of producing cattle via nuclear transfer will decrease because about 50% of the cost associated with cloning may be attributed to survival during the last 2 mo before and the first 2 mo after birth. Increased donor cell cycle synchronization efficiencies, determining the appropriate oocyte source, understanding the placentation dynamics in cloned pregnancies, and improving the fetal-maternal communication system are all areas that will require significant attention and improvement to make this technology commercially viable for cattle producers.

	ACKNOWLEDGMENTS

 
The authors thank Star Bridges, Kate Stewart, Jessica Klingner, Heather Johnson, Amanda Deehan, Kelly Pollock, Allison Adams, and Karen Page for recipient management and calf maintenance, the Medicine, Surgery, and Neo-Natal Care staff of the University of Georgia Veterinary Medicine Teaching Hospital for calf deliveries and neonatal care, and Scott Pratt, Monica Tumlin, and Clifton Baile for manuscript review.

	FOOTNOTES

 
First decision: 18 October 2001.

¹ Correspondence: Steve Stice, Department of Animal and Dairy Sciences, University of Georgia, Athens, GA 30602. FAX: 706 546 4492; sstice{at}arches.uga.edu

Accepted: October 31, 2001.

Received: September 28, 2001.

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