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Regular Article |
a Department of Veterinary and Animal Science, University of Massachusetts, Amherst, Massachusetts 01003
ABSTRACT
The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6–15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2–3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.
developmental biology, implantation/early development
INTRODUCTION
The cloning of mammalian embryos by fusing somatic cells to oocyte cytoplasts has been achieved in several laboratories [1–10]. This technique is of significantly greater commercial and research importance than the previous well-established technique of cloning from embryonic cells [11]. One great advantage of cloning from somatic cells is that specific types of somatic cells are easily propagated in culture. Propagation in culture provides many millions of cells that can be used either to produce large numbers of identical offspring or for genetic modification and production of transgenic animals. A second advantage is that somatic cells can be recovered from adult animals and have been successfully used to make genetically identical copies of existing animals.
Success in cloning from embryonic cells has been improved by information on compatibility between the nuclear donor cell cycle and the recipient cytoplast cell cycle [11–17]. Primarily, two types of recipient cytoplasts have been used: the enucleated and activated metaphase II oocyte cytoplast and the enucleated but not activated oocyte cytoplast [11, 14]. In the activated oocyte, the cytoplast is in an interphase state and has a low level of histone H1 kinase activity. In the inactivated oocyte, the cytoplast is in a metaphase state and has a high level of histone H1 kinase activity [11]. The response of the donor cell nucleus to these two types of cytoplasts is quite different.
In the activated cytoplast, little change is noted in the transplanted nucleus. DNA synthesis, if initiated, is completed or, if not initiated, progresses normally. This type of recipient is compatible with both G1 and S-phase stages of the donor cell cycle [13]. Morphologically, little change is observed in the nucleus following transfer to the activated cytoplast. In the metaphase oocyte cytoplast, the donor nuclear chromatin immediately condenses, in conjunction with a breakdown of the nuclear envelope, following contact with the recipient cytoplasm [18]. Spindle microtubules form in association with the condensed chromatin [19]. When the nuclear transfer embryo is activated, causing a decrease in histone H1 kinase activity, a nucleus forms and acquires the morphology of a large pronucleus [18]. If DNA synthesis has commenced prior to nuclear envelope breakdown, then DNA rereplication will take place, resulting in a defective embryo. Furthermore, the progression of DNA synthesis in the donor nucleus is not compatible with normal chromatin condensation, which also results in defective embryos. Consequently, only nuclei that have not entered S-phase (G1 stage of the cell cycle) produce normal embryos when transplanted to a nonactivated oocyte cytoplast [13]. Although the nonactivated recipient cytoplast is only compatible with G1 stage nuclei, it is capable of inducing morphological reprogramming of the donor nucleus [18]. Although not proven, it is believed that reprogramming is important for success in somatic cell nuclear transfer [20]; therefore, a G1 stage donor cell is preferred.
Research is routinely done with a wide variety of somatic cells in culture, and some information is available on the relative proportions of the cell population in the various stages of the cell cycle [21]. For example, cultures of human fibroblast cells consist of 63% cells with a G1/G0 DNA content, 25% in S-phase, and 12% in G2/M phase during logarithmic growth [22]. For bovine fibroblasts, which have been successfully used to produce nuclear transplant offspring, essentially no information is available on the characteristics of the cells in culture. For nuclear transfer, it is important to identify and to be able to precisely control the stage of the cell cycle at the time of donor and recipient cell fusion. Therefore, one objective of this study was to evaluate the cell cycle distribution in bovine fibroblasts from various sources either as a population in standard cultures or as individual cells. Furthermore, a method was developed for obtaining synchronized populations of cells in G1 stage. G1 cells were also evaluated as nuclear donors.
A further concern with nuclear transplantation is both the age of the cell donor animal and the number of doublings a cell population undergoes in culture [22, 23]. Primary cells are only capable of undergoing a finite number of population doublings (PDs) in culture before becoming senescent [23, 24]. This senescence model is well established. Information is also available to indicate that senescence is related to the number of divisions a cell has undergone and that in vitro cell life span decreases with the age of the donor individual [22].
Cell senescence may be a limiting factor in the application of nuclear transfer in animals. In agriculture, it would be advantageous to use cells from progeny-tested animals for multiplication of superior genotypes. Some agricultural and biomedical applications would require genetic modification of the donor cells for the production of transgenic animals [2, 25]. Inserting genes into cells in culture requires selection of transgenic cells from nontransgenic cells and usually clonal derivation of cell lines [2]. These manipulations require a significant number of PDs before cells can be used as nuclear donors. Therefore, a second objective of this study was to determine the life span, in culture, of fibroblast cells derived from fetuses and animals of various ages. Furthermore, the effect of age of the cell donor on cell cycle characteristics and nuclear transfer success in culture was determined.
MATERIALS AND METHODS
Establishment of Fetal Fibroblast Cell Lines
Bovine fetal cell lines were developed using a modification of a method described earlier [26]. Bovine fetuses were obtained from the slaughterhouse and transported to the laboratory in Dulbecco's PBS (DPBS) with 16 µl/ml of antibiotic-antimycotic (Gibco, Grand Island, NY), 4 µl/ml tylosin tartrate (Sigma, St. Louis, MO), and 8 µl/ml fungizone (Gibco). Fetuses were rinsed in DPBS, the head and internal organs were removed, and remaining tissues were finely chopped into pieces with a scalpel blade. The fibroblasts were separated from the tissue pieces by a standard trypsinization procedure (described elsewhere [26]) using 0.08% trypsin and 0.02% EDTA in PBS (trypsin-EDTA). The cells were seeded onto 100-mm tissue culture plates (Corning, VWR, Chicago, IL) in 
minimal essential medium (-MEM; Gibco) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT), 0.15 g/ml glutamine (Sigma), 0.003% ß-mercaptoethanol (Gibco), and antibiotic-antimycotic (Gibco) (-MEM + FCS). On Day 3 of seeding, the cells were harvested using DPBS with trypsin-EDTA solution and were counted. One million cells were reseeded onto 100-mm tissue culture plates, and the remaining cells were frozen in -MEM with 10% FCS and dimethyl sulfoxide (Sigma).
Establishment of Calf and Adult Fibroblast Cell Lines
Bovine ear biopsies were taken after clipping the hair and washing with disinfectant. The samples were washed three times in DPBS, and the cartilage was removed and discarded. Whole tissue samples were placed in 100-mm tissue culture plates and covered with a glass slide to prevent floating in the culture medium. Ten milliliters of -MEM + FCS was then added to the dish, and the dish was incubated at 39°C in an atmosphere of 5% CO2 in air. After removal of the tissue samples on Day 10, monolayers of cells were harvested using trypsin-EDTA in PBS, counted, and seeded in 100-mm tissue culture plates.
Establishment of Mouse Fetal Fibroblast Feeder Layers
Mouse fetal fibroblasts were produced from Day 13 fetuses using the protocol described for the preparation of bovine fetal fibroblasts. Confluent cultures of mouse fibroblasts were mitotically inactivated by 
-ray irradiation (2956 rads). After inactivation, 5.0 x 105 cells were plated in four-well plates (Nunc, VWR), and after 24 h the medium was changed and the monolayer was used for embryo culture.
Determination of PDs and Cell Counts
After the initial seeding, cells were counted when they were 95% confluent. Cells were removed from plates by the standard trypsinization procedure described above. Harvested cells were spun at 1000 x g for 5 min, the pellet was dissolved in 10 ml of -MEM + FCS, and a sample was counted. These cells were then seeded again and allowed to reach 95% confluence. The cells were then harvested and counted, and the number of PDs was calculated. The procedure was repeated until the cells became senescent.
Isolation of G1 Cells
Twenty-four hours prior to isolation, 5.0 x 105 cells were plated onto 100-mm tissue culture plates containing 10 ml of -MEM + FCS. The following day, plates were washed with PBS and the culture medium was replaced for 1–2 h before isolation. The plates were then shaken for 30–60 sec at medium speed (Vortex-Genie 2; Fisher Scientific, Houston, TX), the medium was removed and spun at 1000 x g for 5 min, and the pellet was resuspended in 250 µl of -MEM + FCS. Newly divided cell doublets attached by a cytoplasmic bridge were then selected because these cells are in early G1. This isolation procedure is referred to as the shake-off method.
Cell Cycle Analysis
The cell cycle of bovine fetal and adult fibroblasts was analyzed using a fluorescent activated cell sorter (FACS; Becton Dickinson, San Jose, CA). Cells (5.0 x 105) were seeded onto 100-mm tissue culture plates, grown to the desired confluence, and harvested by the standard trypsinization procedure at different time points. The cell suspension was centrifuged at 300 x g and resuspended in chilled (-4°C) 70% ethanol with 5% glycerol in water while shaking the sample at low speed. After overnight incubation of the suspension at -4°C, cells were washed twice with chilled DPBS. The pellet was resuspended in 500 µl of DPBS with 16 µg of RNAseA (Worthington Biochemicals, Lake Wood, NJ). The suspension was incubated in a water bath for 3–4 h. Twenty-five to 50 µg of propidium iodide was then added to the suspension, and 10 000 events were analyzed per treatment.
Preparation of Cells for Nuclear Transplant from Confluent Fibroblast Cultures
Twenty-four hours prior to nuclear transplantation, 1 x 106 cells at the desired PD were plated onto 35-mm tissue culture plates containing 4 ml of -MEM + FCS. The plates were washed on the following day and trypsinized by the standard procedure. The suspension was then centrifuged at 300 x g for 5 min, and the pellet was resuspended in 500 µl of -MEM + FCS. Cells from this suspension were used as nuclear transplant donor cells from confluent cultures.
5-Bromo-Deoxyuridine Labeling of G1 Cells
Cells in G1 stage were placed in four-well tissue culture plates containing 250 µl of -MEM + FCS supplemented with 25 µg 5-bromo-deoxyuridine (BrdU; Boehringer Mannheim, Indianapolis, IN). At 0, 2, 4, and 7 h, cells were fixed with 70% ethanol (in 50 mM glycine buffer, pH 2.0) for 20 min. Following fixation, cells were washed and incubated with mouse anti-BrdU IgG (Sigma) for 30 min at 37°C. After 30 min, cells were washed, antimouse Ig-fluorescein (Sigma) was added, and cells were then incubated again for an additional 30 min at 37°C. Following the second incubation, fixed cells were washed and mounted with glycerol. The percentage of cells in S-phase was assessed with an epifluororescent microscope (Nikon, Garden City, NY).
Assessment of Cell Cycle Length
Cells in G1 stage were transferred into 50-µl drops of -MEM + FCS derived from actively dividing cultures of fibroblasts (conditioned medium) using a micromanipulator and were observed every 2 h for 24 h for cell division. Mean cell cycle length was calculated from cells dividing within the first 24 h.
Nuclear Transplantation, Activation, and Embryo Culture
The nuclear transfer procedure was essentially as described previously [2]. In vitro matured oocytes were enucleated about 18–20 h postmaturation (hpm), and chromosome removal was confirmed by bis-benzimide (Hoechst 33342, Sigma) labeling under ultraviolet light. These cytoplast-donor cell couplets were fused by application of a single electrical pulse of 2.4 kV/cm for 20 µsec (Electrocell manipulator 200; Genetronics, San Diego, CA). After 3–4 h, a random subset of 25% of the total transferred couplets was removed, and fusion was confirmed by bis-benzimide labeling of the transferred nucleus. At 30 hpm, reconstructed oocytes and controls were activated with calcium ionophore (5 µM) for 4 min (Cal Biochem, San Diego, CA) and 10 µg cycloheximide and 2.5 µg cytochalasin D (Sigma) in ACM culture medium (100 mM NaCl, 3 mM KCl, 0.27 mM CaCl2, 25 mM NaHCO3, 1 mM sodium lactate, 0.4 mM pyruvate, 1 mM L-glutamine, 3 mg/ml BSA (fatty acid free), 1% basal minimum essential (BME) amino acids, and 1% MEM nonessential amino acids; all chemicals obtained from Sigma) for 6 h as described previously [27, 28]. After activation, eggs were washed in Hepes-buffered hamster embryo culture medium (114 mM NaCl, 3.2 mM KCl, 2 mM CaCl2, 10 mM sodium lactate, 0.1 mM sodium pyruvate, 2 mM NaHCO3, 10 mM Hepes, and 1% BME amino acids; all chemicals obtained from Sigma) five times and placed in culture in four-well tissue culture plates containing irradiated mouse fetal fibroblasts and 0.5 ml of embryo culture medium covered with 0.2 ml of embryo-tested mineral oil (Sigma). Twenty-five to 50 embryos were placed in each well and incubated at 38.5°C in an atmosphere of 5% CO2 in air. On Day 4, 10% FCS was added to the culture medium. On Days 7 and 8, development to the blastocyst stage was recorded. Cell numbers in blastocysts were assessed by mounting with 1% bis-benzimide in glycerol.
Statistical Analysis
Cell life span (total number of PDs to senescence) and the mean PDs/day were compared among three ages of cell donors (fetuses, 0- to 13-mo calves, and 2- to 15-yr adults) by analysis of variance (ANOVA) and regression analysis. Length of the cell cycle was compared in isolated cells, either isolated in G1 from low-confluence cultures or isolated directly from high-confluence cultures, from two fetal and two adult cell lines by the Student t-test. FACS and PD results from fetal and adult fibroblasts were analyzed at various times after plating by regression analysis. The percentage of embryos developing to the blastocyst stage and total cell number were compared among different ages of animals and between G1 cells isolated from low-PD cultures and confluent cultures by ANOVA.
RESULTS
Effect of Donor Age on Cell Proliferation
Cultures of primary bovine fibroblasts were established from animals ranging in age from gestational Day 45 fetuses to 15-yr-old adults (n = 20). Each cell line was passaged to senescence, and total PDs and PDs/day were calculated (Table 1). Fibroblast cell lines derived from fetuses and calves had similar in vitro life spans of approximately 30 PDs compared with 20 PDs in fibroblasts obtained from adult animals. Cell lines derived from fetuses differed from those derived from calves and adults with respect to growth kinetics. Upon initial plating, fetal fibroblasts grew much faster than did calf and adult fibroblasts. However, growth rate of fetal fibroblasts declined more rapidly than did that of calf and adult fibroblasts (P < 0.05).
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Effect of Donor Age and Culture Density on Cell Cycle Distribution
FACS analysis was used to assess the cell cycle distribution of both fetal and adult fibroblasts as a population in culture. Data were collected at different time points following initial seeding and continued for 76 h. Cell counts were taken at each time point, and PDs were calculated (Fig. 1c). The percentage of S-phase cells decreased with time in culture (Fig. 1a), whereas the percentage of G1-phase cells increased over time in culture for both fetal and adult cell lines (Fig. 1b). The rate of increase in the proportion of cells in G1 was similar for both fetal and adult cell lines, and the rate of decrease in cell division rate (PDs/day) was similar (Fig. 1c). However, the rate of cell division for all time points combined was lower for adult cells than for fetal cells (P < 0.05).
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Length of Cell Cycle of Isolated Early G1 Bovine Fibroblasts
To obtain a population of actively dividing G1 cells a shake-off method was developed. This method was also used to evaluate differences among individual cells in a population and to evaluate the cell cycle characteristics of isolated cells in culture. Cells obtained by the shake-off method were cultured individually in microdrops at 38.5°C in a 5% CO2 atmosphere and observed every 2 h for 24 h. Cells were isolated from 25% and 100% confluent plates from each of two fetus- and two adult-derived cultures. Between 92% and 98% of all cells divided within 24 h. The mean length of the cell cycle for each of the treatments ranged from 9.6 to 15.5 h and was similar among fetus- and adult-derived cell lines and for cells isolated from either 25% or 100% confluent plates (Table 2).
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Length of G1 in Isolated Fibroblasts
Because isolated cells in culture have an extremely short cell cycle, it was of interest to determine the length of G1 so that we could be assured that donor cells would be in G1 at the time of fusion with recipient cytoplasts. To ascertain the length of G1 in fibroblasts isolated by the shake-off method, incorporation of BrdU into DNA was evaluated at various times after cell isolation. The results indicate that these cells enter S-phase between 2 and 3 h after isolation (Table 3). Therefore, fibroblasts isolated in G1 must be fused to recipient cytoplasts within 2 h following isolation.
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Development of Nuclear Transfer Embryos Produced from Isolated G1 Fibroblasts
The G1 fibroblasts isolated from cultures at either 25% or 100% confluence were transferred to enucleated metaphase II oocytes and fused. In 25% confluent cultures, cells were isolated by the shake-off method. In 100% confluent cultures, random cells were used because nearly all cells were in G1 and thus the shake-off method could not be used. After activation, nuclear transfer embryos were cultured for 8 days, and the extent of development to the blastocyst stage and total cell number was compared (Table 4). There was no difference in either the number of cells in blastocysts or the percentage of blastocysts between G1 fibroblasts from 25% and 100% confluence cultures. The percentage of blastocyst development among individual cells lines was different (P < 0.05); however, the number of cells in blastocysts did not differ among cultures from individual animals.
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DISCUSSION
One factor that may affect the successful development of nuclear transfer embryos is cell cycle stage of the donor cell. At the time of nuclear transfer, the preferred phase of the cell cycle for the donor nucleus is G1 if a metaphase II oocyte is used as a recipient because chromosome abnormalities and DNA rereplication may occur when Sphase chromatin is forced to condense into metaphase chromosomes [11, 13]. The percentage of blastocysts has been shown to decrease when mid to late S-phase donors were fused to metaphase II oocytes [13, 29].
To obtain somatic cells in the G1 stage of the cell cycle, several methods have been employed. Nocadozole and colcemid, inhibitors of microtubules, have both been used to synchronize cells in metaphase, and following removal of the drugs, a subpopulation of cells divides and enters G1 [30]. Aphidicolin, a DNA synthesis inhibitor, has been used in combination with microtubule inhibitors to arrest cells at the G1/S-phase boundary [13]. Other chemicals have been used to directly arrest cells in G1 by interfering with regulatory protein kinases [13, 31]. Chemical intervention may be avoided by using cells derived from cultures at high confluence [21, 30].
In this study, both adult and fetal bovine fibroblast cell lines were analyzed by FACS analysis to determine the relative proportions of cells in different phases of the cell cycle, particularly G1. Our results indicate that the percentage of cells in G1, in both adult and fetal fibroblasts, increases linearly with time in culture after seeding. Furthermore, adult fibroblast cultures contain a higher percentage of cells in G1 throughout the culture period than do fetal cell cultures, indicating that adult cells may be more sensitive than fetal cells to contact inhibition. The higher percentage of cells in G1 suggests that the adult cells contain a longer cell cycle than the fetal cells. This result was verified directly by evaluating the average number of PDs/day either for the entire life span of the culture or between passages. Overall, a high proportion of cells with a G1 DNA content can be obtained by growing cultures to confluence in either fetal or adult fibroblast cultures.
Growing cultures to confluence cannot be used as a method for separating cells in G1 and capable of active division from cells that have stopped dividing because they have become senescent or they are dying or differentiating. Therefore, a shake-off method was developed to obtain a synchronous group of dividing cells in early G1. This method was used to observe the variation in cell cycle length within a population of cells and to evaluate cell cycle characteristics in isolated cells. The results indicated that isolated cells have a very short cell cycle and that a high proportion of cells within a population have this characteristic.
Mean cell cycle length in isolated cells ranged from 9.6 to 15.5 h, considerably shorter than expected and shorter than that observed for cells cultured as a population, even at low confluence (22 h at 25% confluence). This result indicates that fibroblasts, and possibly other cells, have an inherent cell cycle length similar to that of embryonic cells. Furthermore, this result indicates that cell cycle length is primarily determined by the degree of contact inhibition in culture.
In comparing the combined data from adults and the combined data from fetal cells, no differences were observed in cell cycle length when cells were cultured individually. However, adult cells appear to be more responsive than fetal cells to contact inhibition, as indicated by the slower rate of division, when grown as a population. Contact inhibition is readily reversible; cells recovered from either low- or high-confluence cultures have similar cell cycle lengths when cultured individually. Furthermore, passaging of cells from confluent cultures results in immediate resumption of more rapid cell cycles. These results have implications for the use of G1 cells in nuclear transfer and in controlling for stage of the cell cycle when making treatment comparisons with nuclear transfer.
Differences in cell cycle length in isolated cells were not related to the age of the donor animal nor were they related to the confluence of the culture from which they were derived. However, cells from a 4-yr-old animal had a cell cycle length of about 10 h, whereas cells from a 15-yr-old animal had a cell cycle length of about 15 h. The two fetal cell lines were similar, and cells divided about every 13.3 h. The marked difference between the two adult cell lines could be due to spontaneous transformation of the cell line derived from the 4-yr-old animal. Following the experiments in this study, the cell line from the 4-yr-old animal continued to grow to at least 50 PDs in culture without becoming senescent.
One concern about the life span of donor cells in culture is related to the use of these cells for genetic modifications. Transgenic sheep and cattle have been produced by transfection of fibroblast cells followed by nuclear transplantation [2, 32]. In both of these studies, fibroblast cells were recovered from fetuses to ensure that the cell lines would have as long a life span as possible. Genetic modification of cells involves establishment of the cell line, transfection, and then clonal propagation under antibiotic selection. Clonal propagation to a colony of 500 000 cells requires 20 PDs. As demonstrated in other species [22, 23, 33], the age of a cell donor is correlated with the in vitro life span of a primary cell line derived from the individual. In this study, cells from fetuses and young adult animals had a similar in vitro life span of about 31 PDs, whereas cells from older adult animals had a shortened life span of about 18 PDs. These results indicate that adult cells are likely to become senescent before selection for transgenic clonal lines would be complete. Fetal cells and cells from young animals can be transfected and selected as shown previously [2]; however, it is unlikely that the cells would continue to divide for a sufficient length of time to allow for more than one genetic modification to be completed. Regeneration and rejuvenation of a cell line would be necessary for multiple genetic modifications.
A second concern about the limited life span of nuclear donor cells is that cloning efficiency may decrease as cells progress towards senescence. The results in this study indicate that cloning efficiency would decrease more quickly in cells from adults than in cells from calves or fetuses. It is clear from previous studies [5, 34] that cells cultured for an extended time can produce offspring. In one study [5], developmental potential of nuclear transfer embryos was shown to be similar for cells from low-passage and those from high-passage cultures. In another study, cells expressing markers of senescence, EPC-1 and reduced telomere length [34], and a senescent morphology produced offspring following nuclear transfer. More information is required before conclusions can be drawn concerning differences in efficiency of development to term between senscent cells and cells from early PD cultures.
In in vitro development, both percentage of blastocysts and cell number for nuclear transfer embryos derived from actively dividing G1 cells or cells from high-confluence cultures was similar. Furthermore, no difference was observed in development between embryos derived from fetal fibroblasts and those from adult fibroblasts when comparing combined data.
The results of this study indicate that differences in cell cycle characteristics do exist in cells derived from animals of different ages. Although the age of the animal appears to have no impact on the inherent length of the cell cycle in isolated cells, cells from calves and adults do divide more slowly in culture as a result of a greater sensitivity to contact inhibition when compared with fetal cells. Age of the donor animal had no impact on in vitro development of nuclear transplant embryos. The shake-off method can be used to isolate cells synchronized in G1 from an actively dividing population. Cells isolated by shake off, however, did not support a higher rate of development of nuclear transfer embryos to the blastocyst stage over that of cells from a confluent culture.
ACKNOWLEDGMENTS
The authors thank John Balise for his technical assistance and Grant Morgan for his helpful discussions on the cell cycle.
FOOTNOTES
First decision: 16 October 2000.
1 This research was supported in part by USDA grant 97-35205-5132 to D.J.J. and J.M.R. 
2 Correspondence. FAX: 413 545 6326; robl{at}vasci.umass.edu
Accepted: December 28, 2000.
Received: September 15, 2000.
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= adult)