| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Bekesy Laboratory of Neurobiology, University of Hawaii at Manoa, Honolulu, Hawaii 96822
b Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96822
ABSTRACT
Since the first successful cloning of mammals from adult somatic cells, there has been no examination of the learning or behavior of cloned offspring. The possibility of adverse effects on animals produced through adult somatic cell cloning is high because many natural biological processes are bypassed and DNA from adult cells, which presumably contain mutations, are used. In this study, we compared cloned mice produced by microinjection transfer of cumulus cell nuclei into enucleated oocytes, to control mice that were specifically generated to eliminate confounding factors that are unique to our cloning procedure. Postnatal weight gain of clones was significantly greater than that of controls. Preweaning development observations revealed that first appearance or performance of 3 out of 10 measures was delayed in cloned mice; however, results of subsequent tests of learning and memory, activity level, and motor skills were comparable for both groups. Together, these data suggest that nuclear transfer of adult somatic cell nuclei to produce cloned mice may delay the appearance of a few developmental milestones but it does not adversely affect the overall postnatal behavior of mice. In addition, this procedure may cause late onset of significantly increased body weight in cloned offspring, the cause or causes of which are being further examined.
aging, cumulus cells, developmental biology
INTRODUCTION
Genomically identical mammalian offspring have been generated for a number of years by transferring embryonic [1–3] and fetal cell nuclei [4–7] into enucleated mature oocytes. Recently, mammalian cloning using adult somatic cells has been successful in sheep [4], mice [8, 9], and cattle [10, 11]. Although cloning has met with success, there are problems that cause prenatal and perinatal death of embryos, fetuses, young, and growing young [12], which has depressed the rate of live cloned offspring in each of these species [4, 8–11]. The cause or causes for these occurrences are being investigated.
In cloning using adult cell nuclei, the status of genes in differentiated somatic cells must be "reprogrammed to zygotic state" by mechanisms that are unknown. The failure of many cloned embryos and fetuses to reach term could be the consequence of incomplete genomic reprogramming of genes in transferred somatic cell nuclei.
Cloning is not the natural method of reproduction in mammals, and it is still unknown whether bypassing meiosis and fertilization results in serious adverse effects in offspring. Assisted reproductive technologies (e.g., in vitro fertilization, subzonal sperm injection, intracytoplasmic sperm injection [ICSI], round spermatid injection [ROSI]) bypass many of the normal biological processes of fertilization such as sperm interactions with the female genital tract and egg vestments and membrane fusion between gametes. Whether assisted reproduction results in deleterious consequences has been the subject of debate since it was introduced as a new technology. Most clinical investigators maintain that even such a dramatic assisted reproduction technique as ICSI does not produce serious undesirable effects on offspring in excess of the rate of incidence in the normal general population [13, 14]. In the mouse, we previously reported that ROSI, which bypasses spermiogenesis and sperm maturation, does not adversely affect the growth, fertility, and behavior of offspring [15].
Although many cloned animals are alive and reported to be healthy, they are not completely free of problems. Shiels et al. [16] recently found that the telomere restriction fragment (TRF) of Dolly, the cloned sheep, was consistent with the age of the donor animal. Although it is yet unknown whether the actual physiological age of animals derived by nuclear transfer is accurately reflected by TRF measurement, this finding raises the question of whether cloned animals will exhibit premature aging. Also, physiological abnormalities have been reported in animals cloned from somatic cells by nuclear transfer [9, 11, 12], although there is no evidence that nuclear transfer itself is responsible for these anomalies. Specifically, in the mouse, we have observed high embryo implantation rates (57%–71%) but low fetal (5%–16%) and full-term (2%–3%) development rates following nuclear transfer [8]. Perinatal and postnatal complications include developmental deficiencies and respiratory distress [9]; however, the mortality rate after the postnatal period is low and, thus far, growth through 8 wk of age, fertility, and longevity appear normal. As yet, there has been no comprehensive examination of the postnatal development or behavior of cloned animals.
As with other new techniques, it is important to ascertain whether there are adverse effects on offspring that should be considered before widespread use of somatic cell nuclear transfer technology. In this study, we compared the growth and behavior of mice generated by somatic cell nuclear transfer using adult cumulus cells to those of mice produced by natural fertilization (controls).
MATERIALS AND METHODS
Animals
Housing and care All animals, both clones and controls, were individually housed in polycarbonate cages (18.5 x 29 x 13 cm) with food and water ad libitum. They were maintained in temperature- and humidity-controlled rooms under a 14L:10D cycle with light onset at 0500 h. The animals were maintained in accordance with the guidelines of the Laboratory Animal Service at the University of Hawaii and those prepared by the Committee on Care and Use of Laboratory Animals of the National Research Council's Institute of Laboratory Resources (U.S. Department of Health, Education, and Welfare publication [NIH] 80–23, 1985). The protocol of animal handling and treatment for this study was reviewed and approved by the Animal Care and Use Committee at the University of Hawaii.
Clones Mouse clones were generated by microinjection of cumulus cell nuclei from adult (8- to 10-wk-old) B6C3F1 mice into enucleated oocytes that had been collected from adult (8- to 10-wk-old) B6D2F1 mice. Preimplantation embryos were transferred into pseudopregnant CD-1 surrogate mothers [17]. Pups were delivered at 19.5 days post-coitum (d.p.c.) by Caesarean section and placed with the litters of lactating CD-1 foster mothers to be raised. The method is described in detail elsewhere [8]. Five female cloned mice were used in this study.
Controls Presently, only 1 or 2 cloned mouse embryos reach term in each surrogate mother. These litters are delivered by Caesarean section; parturition is usually not naturally initiated because of the small number of fetuses. Therefore, control subjects were generated with consideration of a number of possible confounding factors in the cloning method, including embryo micromanipulation, in vitro embryo culture, embryo transfer into pseudopregnant surrogate CD-1 mothers, reduced litter sizes, Caesarean delivery, and placement of pups with litters of lactating CD-1 foster mothers. Ten C57BL/6 female and 10 C3H/He male mice were mated. The following day, pronuclear eggs were collected from the oviducts and cultured in vitro for 20 h until they reached the 2-cell stage. Two or 3 embryos were transferred to the oviducts of each of 20 pseudopregnant CD-1 surrogate mothers to allow only 1–2 embryos to implant in each mouse. Pups (hybrid B6C3F1) were delivered at Day 19.5 d.p.c. by Caesarean section and pups were placed with the litters of lactating CD-1 foster mothers to be raised. Fifteen female control pups were used in this study.
Growth and Behavioral Tests
Animals were weighed weekly until 8 wk of age and biweekly thereafter. All behavioral tests were conducted between 0900 and 1600 h and animals were 90–110 days of age at the start of testing, except as otherwise noted.
Preweaning development: Fox battery The Fox battery of tests provided an assessment of development throughout the neonatal period because these behaviors are each expressed at differing periods during the first 21 days of life. Beginning at 2 days of age, newborn mice were weighed and examined daily for developmental milestones and complex motor behaviors (Table 1; [18, 19]). In addition to the "day of first performance" of each behavior, the time required to perform surface righting, cliff aversion, and negative geotaxis were measured. All timed responses were limited to a maximum of 30 sec. A nonresponding animal was scored at 30 sec.
|
Home cage activity Videotape analysis of behavior in the home cage provides a quantitative measure of activity and allows for a more detailed qualitative description of an animal's behavior in a familiar surroundings [20, 21]. Subjects were videotaped in their home cages at 60, 120, and 180 days of age for four 1-h periods. A red light provided illumination during the lights-out period. Four 1-h periods were scored: immediately following lights-out (2100–2200 h), mid lights-out (midnight–0100 h), immediately following lights-on (0500–0600 h), and mid lights-on (1200–1300 h). Behavioral ratings of the videotape recordings of home cage behavior were made for a 1-sec period every min throughout selected 1-h test periods (time sampling) using the categories in Table 2 [22].
|
Morris water task This task has been used extensively in evaluating spatial learning ability and memory in rodents [23]. It has also been used in studies to assess the effect of brain lesions [24], physiological differences in the brain [25], chemical stimuli [26], dietary changes [27], and age-related memory decline [28]. It is also used to measure learning and memory in knockout and transgenic mouse models [29, 30]. In the test, a platform is hidden in opaque water but distal visual cues are available for the animal to use for navigation purposes. To efficiently find the platform, the animal must develop a spatial map of the platform's location using the distal visual cues provided. Memory is demonstrated by the decrease in latency to locate the hidden platform using extramaze cues in successive trials. The animals were given 6 trials per day over 3 days, for a total of 18 trials. The hidden platform procedure is described in detail elsewhere [30].
A probe test on the fourth day, with the platform removed, served to ascertain that the mice were using a spatial learning strategy that involved multiple, specific, distal cues, or some other strategy. This was used to determine if the mice were selectively swimming in the quadrant in which the platform had previously been located. Time spent in each of the 4 quadrants was measured using Hindsight (version 1.3), a time-sampling computer program.
Immediately following the probe trial, 6 reversal trials were administered, in which the platform was placed in the opposite quadrant at the same distance from the pool wall as in the acquisition trials. These continued on the fifth day for a total of 12 reversal trials.
Krushinsky test This test evaluates an animal's extrapolation behavior (i.e., the ability of the animal to anticipate the movement of an object out of view). The testing apparatus was modeled after that used by Poletaeva et al. [31]. The test box (24 x 21 x 15 cm) was constructed of Plexiglas; the front wall was an opaque black color, the remaining 3 sides were an opaque white. The procedure used was an adaptation of those used by Dulioust et al. [32]. Sweetened condensed milk (Springfield, Los Angeles, CA) was used as the food stimulus. Each subject was deprived of food and water for 18 h prior to testing. Data collected included total session duration and number of correct choices made. Session duration was defined as the amount of time that elapsed for a subject to complete 10 trials; maximum time allowed was 10 min. Percent correct responses was defined as the number of correct choices divided by the number of trials a subject completed. Total session duration and number of correct choices were also combined to indicate the number of correct choices per min.
Motor Tests
Two tests were conducted at 60 days of age to assess motor coordination, muscular strength, and balance.
Hanging and balancing The apparatus for this test consisted of an iron wire 0.4 cm in diameter x 50 cm long suspended horizontally 45 cm above the floor between 2 panels of a 55- x 50- x 56-cm box. Subjects were held by the tail and brought up to the iron wire. A subject was allowed to hang freely from its 2 forepaws when it grasped the wire. The latency for an animal to pull its body up to balance on the wire (righting), the number of times its hindpaws fell off the wire (loss of balance), and the length of time spent on the wire (30 sec maximum) were recorded.
Screen climbing test The apparatus for this test was the same as that described for the hanging and balancing test. A screen measuring 20 x 20.5 cm with a grid of 6 x 6 mm was hung from the iron wire. Subjects were placed at the bottom of the hanging screen facing upward. The latency time for a subject to climb to the top was recorded and use of fore and hindpaws while climbing was noted.
Statistics
Data were analyzed using Statistica statistical software (version 4.5; StatSoft, Tulsa, OK) by a one-way ANOVA or t-tests for independent samples. Subsequent comparisons between groups were carried out using Newman-Keuls procedures. The nonparametric Kruskal-Wallis ANOVA was used for some infrequently occurring or nonparametrically distributed measures.
RESULTS
Growth
Body weights Body weight data for control and cloned mice are presented in Figure 1. Weights were not significantly different between controls and clones from birth through the first 8 wk [F(1,18) = 3.54; P = 0.076]; however, from the ninth week on, the weights of the clones were reliably higher than those of controls [F(1,18) = 10.72; P < 0.0001]. We did not observe any physical abnormalities in the growing and adult animals of either group during the study. Figure 2 shows control and cloned mice at 1 yr of age. At this age, body weights for controls and clones were 48.56 g ± 1.66 g (mean ± SEM) and 60.41 g ± 9.51 g, respectively (P < 0.05).
|
|
Behavior Tests
Fox battery Data from the neurobehavioral battery of tests are presented in Tables 3 and 4. The data revealed significantly reduced performance or delayed first appearance of 3 of the 10 observed behaviors in the cloned mice. Two developmental milestones, eye opening (P < 0.05) and ear twitching (P < 0.005) reflexes; and a complex motor behavior, negative geotaxis (P < 0.001), were affected. In addition, there was a significant type effect [F(1,18) = 9.57; P < 0.01] in the time to complete the negative geotaxis task with the clone group taking a significantly longer period of time than the controls. Neuman-Keuls post hoc analysis revealed that performance times by the clones in the negative geotaxis tasks were comparable to those of controls by 4 days of age (NK test: P = 0.99).
|
Home cage activity Figure 3 shows the mean active behaviors per hour of the clones and controls at night and during the day for selected hours. Total activity (composed of rearing, locomotion, hanging and climbing on the ceiling, eating, drinking, and grooming) was significantly higher at night for both groups at every age observed (60, 120, and 180 days). There was no overall significant difference in activity level between clones and controls [F(1,58) = 0.092; P = 0.76].
|
Morris water maze Both control and clone groups displayed significant improvement over the 3 days of testing in the acquisition phase (Fig. 4) [F(2,36) = 50.63; P < 0.0001] and the 2 groups did not differ significantly from each other [F(1,18) = 0.00034; P = 0.986]. During the reversal trials (Fig. 5), both groups significantly improved their performance over the 2 days of trials [F(1,18) = 20.17; P < 0.001], with no significant difference observed between the 2 groups [F(1,18) = 0.036; P = 0.85]. In the probe test, both the controls and clones selectively searched the trained quadrant, indicating that they were using distal cues to determine the search area rather than some nonspatial search strategy. Although duration in the trained quadrant was reliable only for controls (P < 0.05) and just failed to reach an acceptable level of statistical significance for clones (P = 0.062), the scores of the 2 groups were very similar and differences between them were far from approaching an acceptable level of statistical significance (P = 0.61; Fig. 6).
|
|
|
Krushinsky test ANOVA showed a significant increase in the correct choice ratio (number of correct choices/total time) across the 6 days of testing for both groups, indicating acquisition of the task (Fig. 7) [F(5,90) = 29.29; P < 0.0001]. No significant differences between the 2 groups of animals were found [F(1,18) = 0.0015; P = 0.969].
|
Motor Tests
Bar hanging and balancing In the bar hanging and balancing test, the time required for each subject to right itself (pull its body up to the bar with its forepaws so that the hind paws could grasp the bar) was not reliably different between the 2 groups (P = 0.354; Table 5). In addition, the time subjects were able to balance on the bar (maximum of 30 sec) with 4 paws was not different between the groups (P = 0.162). Finally, the number of times each subject lost its balance (hind paws fell off the bar) also did not differ (P = 0.403) between the 2 groups.
|
Screen climbing All subjects in both groups used their 4 paws to climb up the screen. The latency for the mice to complete the task by reaching the top of the screen was not reliably different between the 2 groups (P = 0.924; Table 5).
DISCUSSION
The main objective of this study was to examine the behavior, including overall activity level, learning, memory, and motor ability, of mice cloned from adult somatic cell nuclei. The fertility of the cloned animals was not examined in this study because breeding may affect the results of some behavioral measures. We do not expect the cloned mice to exhibit low fertility, however, because all other female and male cloned mice were found to be fertile ([8, 9] and unpublished data). The control subjects in our study were specifically generated to eliminate several possible confounding factors involved in the cloning method: embryo manipulation, in vitro embryo culture, embryo transfer into pseudopregnant surrogate CD-1 mothers, reduced litter sizes, Caesarean delivery, and placement of pups with litters of lactating CD-1 foster mothers.
We found that the body weight of the clone group was significantly greater than that of controls beginning at approximately 8–10 wk of age. As in previous studies [8, 9], weights at birth were similar between control and clone groups; thus, this does not suggest "large offspring syndrome" [33] because increased body weight became evident only after about 10 wk of age. One consideration in determining the cause of late onset of increased body weight in cloned mice may be the strain of mouse used in this experiment. The B6C3F1 mouse carries the agouti gene, which encodes agouti protein. This gene has caused maturity-onset obesity and diabetes as a result of ectopic expression of the secreted protein hormone, agouti protein [34, 35]. However, B6D2F1 mice that do not carry the agouti gene have been used in previous cloning experiments in our laboratory and observations of those mice also suggest that cloned mice are heavier than controls. The incidence of adults with increased body weight among cloned female B6C3F1 and B6D2F1 mice was about 77% and 20%, respectively (unpublished data). Further investigation is needed to clarify whether the differences in body weight that we observed are a result of the agouti gene product or of some other phenomenon unique to cloning. We are currently conducting additional detailed studies to examine the etiology of this characteristic.
Preweaning development of cloned mice was similar to that of controls for the majority of measures; however, eye opening, ear twitch, and negative geotaxis were all delayed in first appearance in the clone group. In addition, the mean time for the clone group to complete negative geotaxis was significantly longer than that of the controls, although the time to accomplish this task was not significantly different from controls by Day 4. Because development of the nervous system begins during the early postimplantation period, these data may suggest an effect of cloning on embryogenesis that resulted in delayed maturation of certain behaviors and reflexes. However, subsequent tests of spatial learning, memory, and motor abilities conducted on the same subjects did not detect deficits or long-term effects of the delayed maturation of those specific behaviors and reflexes. The possibility that there is a specific link between increased body weight and delayed appearance of these characteristics should be investigated.
We examined activity in the home cage environment and found no significant difference in the activity levels of clones compared with controls up to 180 days of age. Although activity levels changed between measurements, the decrease or increase was comparable in both groups. Activity level was reliably higher at night than it was during the day, which is expected of mice. Furthermore, the higher body weight observed in cloned mice is probably not the result of a low activity level or inactivity because this test failed to show a significant difference between clones and controls.
In the Morris water task the spatial learning ability of clones did not significantly differ from that of controls. The absence of significant differences between the groups on this task indicates that the groups did not differ in demonstrating long-term memory of the position of the hidden platform. Mice were able to use the information obtained in previous trials to improve their performance in successive trials over the 3 days of acquisition testing. Use of a spatial learning strategy was confirmed through the use of a probe trial to measure quadrant bias. Both groups selectively searched the quadrant in which they had been trained with the average time spent in each of the remaining 3 quadrants falling below chance level (25%). Finally, a reversal task failed to show reliable differences in spatial learning and memory. Both groups were able to successfully navigate to a new platform position and significantly improved their search times over 2 days of trials.
A previous study using the Krushinsky test showed that although mice tend to show no evidence of discrimination between the 2 choice positions (percentage of correct choices made remained approximately at the chance level of 50%), decreases in time to make the decision resulted in higher correct choice rates and reflected reward conditioning similar to that demonstrated in the straight alley test [15]. In our study, the clone group did not differ significantly from controls in their correct choice ratio, suggesting that both groups are capable of reward conditioning.
This study provides a general assessment of the postnatal development and behavior of cloned mice. Whereas there were some developmental delays in cloned mice, we found no evidence of serious adverse effects of mammalian somatic cell cloning using microinjection. However, it is possible that subtle differences were not detected because of the small sample size. This problem could not be avoided because the low success rate of cloning has resulted in a limited availability of cloned animals of the same age.
Further study in other species is warranted to ascertain that these results can be generalized to other mammals as well, because particular effects of cloning may be expected to have a greater or lesser effect in different species. It should also be noted that, in this experiment, the mice that were used as nuclei donors for the clones were only 8–10 wk old. Therefore, the age difference between the donors and the clones (11–13 wk including gestation period) may not be enough to produce a clear difference between controls and clones. Additional study should be done with older nuclei donors to examine the effect of the aging process in clones. Another problem that must be addressed is the cumulative risk over several generations. Wakayama and Yanagimachi [36] reported success in creating multiple generations of cloned mice from adult somatic cells; however, the question remains whether accumulations of mutations or DNA damage may have an effect on subsequent generations of animals that were not detectable in the first generation.
|
FOOTNOTES
First decision: 4 January 2000.
1 Supported by funds from the Victoria S. and Bradley L. Geist Foundation, the Kosasa Family Foundation, and Pro-Bio America, Inc. 
2 Correspondence: R. Yanagimachi, Department of Anatomy and Reproductive Biology, University of Hawaii John A. Burns School of Medicine, 1960 East-West Road, Honolulu, HI 96822. FAX: 808 956 5474; yana{at}hawaii.edu
Accepted: February 24, 2000.
Received: December 3, 1999.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  R. E. Everts, P. Chavatte-Palmer, A. Razzak, I. Hue, C. A. Green, R. Oliveira, X. Vignon, S. L. Rodriguez-Zas, X. C. Tian, X. Yang, et al. Aberrant gene expression patterns in placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer Physiol Genomics, October 8, 2008; 33(1): 65 - 77. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Jincho, Y. Sotomaru, M. Kawahara, Y. Ono, H. Ogawa, Y. Obata, and T. Kono Identification of Genes Aberrantly Expressed in Mouse Embryonic Stem Cell-Cloned Blastocysts Biol Reprod, April 1, 2008; 78(4): 568 - 576. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Niemann, X C. Tian, W A. King, and R. S F Lee Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning Reproduction, February 1, 2008; 135(2): 151 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Shimozawa, Y. Sotomaru, N. Eguchi, S. Suzuki, K. Hioki, T. Usui, T. Kono, and M. Ito Phenotypic abnormalities observed in aged cloned mice from embryonic stem cells after long-term maintenance. Reproduction, September 1, 2006; 132(3): 435 - 441. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ono and T. Kono Irreversible Barrier to the Reprogramming of Donor Cells in Cloning with Mouse Embryos and Embryonic Stem Cells Biol Reprod, August 1, 2006; 75(2): 210 - 216. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Ohgane, T. Wakayama, S. Senda, Y. Yamazaki, K. Inoue, A. Ogura, J. Marh, S. Tanaka, R. Yanagimachi, and K. Shiota The Sall3 locus is an epigenetic hotspot of aberrant DNA methylation associated with placentomegaly of cloned mice Genes Cells, March 1, 2004; 9(3): 253 - 260. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L.K. Tamashiro, T. Wakayama, Y. Yamazaki, H. Akutsu, S. C. Woods, S. Kondo, R. Yanagimachi, and R. R. Sakai Phenotype of Cloned Mice: Development, Behavior, and Physiology Experimental Biology and Medicine, November 1, 2003; 228(10): 1193 - 1200. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Cogle, S. M. Guthrie, R. C. Sanders, W. L. Allen, E. W. Scott, and B. E. Petersen An Overview of Stem Cell Research and Regulatory Issues Mayo Clin. Proc., August 1, 2003; 78(8): 993 - 1003. [Abstract] [PDF]
|
|
|
|
|
|
Y. G. Chung, S. Ratnam, J. R. Chaillet, and K. E. Latham Abnormal Regulation of DNA Methyltransferase Expression in Cloned Mouse Embryos Biol Reprod, July 1, 2003; 69(1): 146 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Boiani, S. Eckardt, H. R. Scholer, and K. J. McLaughlin Oct4 distribution and level in mouse clones: consequences for pluripotency Genes & Dev., May 15, 2002; 16(10): 1209 - 1219. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
