| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Minireview |
a Department of Animal and Dairy Science, University of Georgia, Athens, Georgia 30602-2771
b ProLinia, Inc., Athens, Georgia 30602-2771
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONSELECTION OF RECIPIENT OOCYTES...SELECTION OF DONOR CELLS...CONCLUSIONS AND FUTURE...REFERENCES |
|---|
developmental biology, early development, ovum, pregnancy, reproductive technology
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONSELECTION OF RECIPIENT OOCYTES...SELECTION OF DONOR CELLS...CONCLUSIONS AND FUTURE...REFERENCES |
|---|
Normal embryo/fetal development and pregnancy losses can follow at least two different scenarios. In one scenario, development proceeds as a series of checkpoints (go/no go) occurring independently at each stage of development throughout gestation. Alternatively, these developmental decisions are made very early so that under the right conditions one would know which embryo would produce a normal offspring. In both cases, high-quality starting material will increase the cloning efficiency and the number of offspring obtained, but in the second scenario, the donor cell and recipient oocyte may have a greater effect on developmental progression (i.e., placentation). Aberrant placentation was first observed when embryonic cell line donor cells were transferred into nonactivated oocytes [2], and this problem still occurs frequently in pregnancies obtained with cloned cells. Recently, our laboratory has demonstrated a correlation between donor cell treatment and late-term cloned pregnancy survival [3].
|
SELECTION OF RECIPIENT OOCYTES FOR PRODUCTION OF CLONED ANIMALS FROM DIFFERENTIATED CELLS |
|---|
|
TOPABSTRACTINTRODUCTIONSELECTION OF RECIPIENT OOCYTES...SELECTION OF DONOR CELLS...CONCLUSIONS AND FUTURE...REFERENCES |
|---|
Metaphase II-Arrested Oocytes Versus Activated Oocytes
Metaphase II (MII)-arrested oocytes have been used as recipients in most somatic cell nuclear transfer studies [8], although activated oocytes were used in some studies [9–11]. However, after reduction of maturation/meiosis/mitosis-promoting factor (MPF) activity, preactivated cytoplast recipients are suitable for production of blastomere-derived cloned embryos [12–14]. In contrast, there is only one report of cloned animals being produced from embryos reconstructed by transferring differentiated cells into preactivated or partially activated recipient oocytes [10], and the ability of preactivated recipient oocytes to reprogram differentiated cells is debatable. When donor bovine somatic cells, regardless of cell cycle stage, are transferred into preactivated recipient oocytes, development of the resulting embryo is limited because all embryos arrest at the eight-cell stage [11]. Because activation of the embryonic genome of bovine embryos occurs between the 8- and 16-cell stages [15], preactivated recipient oocytes may not reprogram somatic cell nuclei. In addition, cloned mouse embryos reconstructed by injection of somatic cell nuclei into zygotes after removal of both pronuclei exhibited abnormal chromosomes and did not develop into blastocysts [16]. In contrast, when nuclei of differentiated cells are transferred into nontreated recipients having high MPF activity, reconstructed embryos develop into offspring in many species [3, 9–11, 17–26]. These data suggest that high levels of MPF activity are required for reprogramming a donor nucleus from a differentiated cell.
Metaphase I Oocytes Versus Metaphase II Oocytes
MPF activity during oocyte maturation is maximal at both metaphase I (MI) and MII [27]. In theory, MI oocytes may be more suitable as cytoplast recipients because the use of MI oocytes ensures donor nucleus exposure to high levels of MPF activity for a longer time. In the frog, greater yield and more advanced tadpoles came from differentiated somatic cell nuclei injected into MI oocytes [28, 29] compared with MII oocytes [28, 30, 31], but adults could not be generated from either type (MI or MII) of reconstructed embryos. There are no reports of this type of experiment in mammals. Therefore, we examined the developmental ability of porcine MI oocytes after nuclear transfer of differentiated somatic cells [32]. These results demonstrated the in vitro developmental ability to reach the blastocyst stage in porcine embryos reconstituted with somatic cells and enucleated MI oocytes. However, the rate of blastocyst formation by embryos reconstituted with MI oocytes was significantly lower than that of embryos reconstituted with MII oocytes. This result suggests that MII oocytes rather than MI oocytes are more appropriate recipients for production of differentiated cell-derived cloned embryos in mammals and that MPF is required but not sufficient for maximum developmental ability of reconstructed embryos.
In Vivo-Matured Oocytes Versus In Vitro-Matured Oocytes
Both in vivo- and in vitro-matured oocytes have been used as recipients for production of cloned animals from differentiated cells. In cattle and pigs, in vitro oocyte maturation systems produce an abundant and stable supply of recipient oocytes because immature oocytes can be obtained from slaughtered animals. In vitro-matured oocytes have been commonly used for production of cloned calves [3, 18, 19, 21]. In contrast, in vivo-matured porcine oocytes have been used extensively as cytoplast recipients [22, 23, 33–35]. The use of in vitro-matured oocytes from sow [24, 36, 37] or prepubertal gilt [38–40] ovaries has been limited to a few laboratories. Recently, we produced cloned piglets by using in vitro-matured oocytes derived from both sow and gilt ovaries as cytoplast recipients (unpublished data). Direct comparisons of gilt and sow oocyte sources, in vivo versus in vitro maturation, or combinations of source and maturation method and their ability to support development of cloned embryos as recipients have not been made, but in vivo-derived materials may enhance the probability of obtaining cloned porcine offspring [22, 23]. However, it is difficult to control the ovulation time of donor pigs. According to De Sousa et al. [34], one-half of the gilts injected with eCG and hCG for superovulation ovulated between 45 and 47.5 h post-hCG, and the remaining gilts were roughly equally distributed between earlier (42–43.5 h) and later (49–50.5 h) ovulation times. In addition, ovulation time affected development into blastocysts of oocytes after activation. Of nine gilts ovulating during the midperiod (45–47.5 h post-hCG), six yielded blastocyst formation rates >50%, and only one gilt produced no blastocyst-stage embryos. However, the blastocyst-stage development rates of oocytes ovulated earlier or later than the midperiod did not exceed 30%, and oocytes from four of nine gilts ovulating in these periods yielded no blastocysts [34]. Therefore, variation in oocyte quality exists for both in vitro- and in vivo-matured oocytes, and this variation could contribute to low cloning efficiency. These observations also suggest that methods to identify and select superior oocytes for use in cloning are needed.
Oocytes Derived from Prepubertal Animals Versus Oocytes from Adult Animals
Comparisons have been made between oocytes and embryos from prepubertal heifers and those from adult cows. Calf oocytes are generally smaller in diameter than cow oocytes and have reduced relative protein levels [41–44]. Ultrastructural and cytochemical differences have also been found. Specifically, calf oocytes have a greater density of microvilli on their surface and a greater number of endocytotic vesicles than those of the cow. However, cow oocytes have a larger mitochondrial population. Differences in the distribution of sugar residues in plasma membrane and vacuoles have been observed between calf and cow oocytes [45]. Following in vitro maturation, the activities of MPF and mitogen-activated protein kinase (MAPK) and the relative amounts of inositol 1,4,5-trisphosphate receptors are significantly lower in calf oocytes than in oocytes of the adult cow [46]. Most importantly, the developmental capability of calf oocytes up to the blastocyst stage after in vitro fertilization is significantly lower than that for cow oocytes, and this difference was attributed to incomplete or delayed cytoplasmic maturation in the calf oocyte [47, 48]. Calf oocyte-derived embryos are also characterized by a longer lag phase preceding the major onset of embryonic genome expression [49]. In addition, in vitro developmental rates of nuclear transfer embryos involving adult cytoplasts were substantially faster than those of embryos produced from calf oocytes [46]. Oocytes from prepubertal pigs have a reduced developmental competence compared with that of oocytes from adult pigs, as indicated by the decreased blastocyst formation rate after in vitro fertilization [24, 50]. Despite normal nuclear meiotic maturation rates for prepubertal gilt oocytes, their cytoplasmic maturation is compromised. Oocytes derived from sows and then matured in vitro were better able to support the development to the blastocyst stage of cloned embryos than were similarly matured prepubertal gilt oocytes [24]. However, the ability to support development into offspring of cloned embryos has not been directly compared for oocytes derived from prepubertal and adult animals in either cattle or pigs.
Synchronization of Recipient Oocytes Before In Vitro Maturation
In vivo, meiotically competent oocytes are maintained at the germinal vesicle (GV) stage by the follicular environment until the preovulatory gonadotropin surge. After this surge, only fully grown and competent oocytes can resume meiosis, complete the first meiotic division, and be ovulated. In contrast, all meiotically competent oocytes that matured in vitro spontaneously reenter the meiotic process when removed from their follicles [51]. Although oocytes used for in vitro maturation are commonly harvested from 2- to 6-mm-diameter antral follicles from slaughterhouse ovaries, the size of follicles selected for collection of oocytes varies both between and within investigations [52]. There is a large variation in the nuclear morphology of porcine oocytes at the GV stage just after aspiration from follicles; 5% of oocytes reach the final stage of GV (GV-IV), and the majority (45%) of the oocytes remain at the GV-II stage [53]. When porcine oocytes aspirated from follicles were maintained at the GV stage before in vitro maturation by exposing them to dibutyryl cAMP (dbcAMP), developmental rate to the blastocyst stage after in vitro fertilization was improved [53]. Therefore, developmental rates for porcine cloned embryos might improve if in vitro-matured oocytes are first synchronized prior to maturing through GV breakdown. We examined whether the arrest of meiotic resumption of recipient oocytes at the GV stage by dbcAMP improved in vitro developmental rates for porcine nuclear transfer embryos using adult skin fibroblast donor cells [54]. The results indicated that dbcAMP arrests meiotic resumption of porcine oocytes matured in vitro, and the effect of dbcAMP is reversible as described by Funahashi et al. [53]. However, this treatment did not improve the developmental rates of reconstructed embryos.
Utility of Rapidly Matured Oocytes
Asynchronous meiotic progression of porcine oocytes occurs during in vitro maturation [55–57] and may be responsible for the large variation in the developmental stage of the oocytes at the start of culture [53]. Length of the maturation period affects the ability of porcine oocyte cytoplast recipients to support development of cloned embryos [54]. Porcine oocytes are commonly matured in vitro for 40–44 h before manipulation [24, 36–40, 58, 59]. In our maturation system, however, 36% of oocytes reached the MII stage after 24 h of culture, and developmental rates were significantly higher for nuclear transfer embryos using 24-h-matured recipient oocytes when compared with 42-h-matured oocytes. The maturation period of recipient oocytes affects in vitro development of reconstructed embryos in the pig, and the rate of blastocyst formation for embryos reconstituted with 33-h-matured oocytes was higher than that of embryos reconstituted with 44-h-matured oocytes [60, 61]. In these studies, embryos reconstituted with 33- and 44-h-matured oocytes were activated at 39–40 and 50–51 h after initiation of maturation, respectively. Developmental rate to the blastocyst stage after activation of 42-h-matured pig oocytes was higher than that of 48-h-matured oocytes; thus, Ikeda and Takahashi [61] hypothesized that the difference between nuclear transfer groups was due to oocyte activation responses (young vs. old oocytes). However, rates of development to blastocysts are similar for parthenotes from 24- and 42-h-matured oocytes [54]. In combination with the significantly higher developmental rates for the 24-h-matured oocyte-derived cloned embryos, these results suggest that rapidly matured oocytes alone, and not the activation signal, are responsible for higher rates of development. Use of the rapidly matured oocyte subpopulation in cloning would represent a novel way to improve developmental rates for cloned offspring.
|
SELECTION OF DONOR CELLS FOR PRODUCTION OF CLONED ANIMALS FROM DIFFERENTIATED CELLS |
|---|
|
TOPABSTRACTINTRODUCTIONSELECTION OF RECIPIENT OOCYTES...SELECTION OF DONOR CELLS...CONCLUSIONS AND FUTURE...REFERENCES |
|---|
Various cell types, such as embryonic cells, fibroblasts, mammary gland cells, cumulus cells, oviductal cells, leukocytes, granulosa cells, germ cells, and liver cells, have been used as donors for production of cloned animals [62]. However, it is still unclear which cell type is the most successful for nuclear transfer into oocytes. Kato et al. [63] compared efficiency of cloning using various somatic cell types from adult, newborn, and fetal female and male donor cattle. The percentage of blastocysts that developed from each of the donor cell types was not significantly different, except for the extreme case of fetal muscle cells compared with adult male liver cells. Thus, as a whole, no cell type(s) was more efficient than the other cell types for cloning. Similar results were obtained using various cell types derived from mice of different strains, sexes, and ages [64]. It may be difficult to show significant differences in the rate for development into cloned animals among different cell types because of the overall low cloning efficiency.
Initially, all cloned animals derived from adult somatic cells were produced using cells of the female reproductive system, such as mammary gland cells [17], cumulus/granulosa cells [19–21], and oviductal cells [19], raising the question of whether males can be cloned in the same way. Male mice have been cloned from tail-tip cells [65], and there was no significant difference in developmental rates of embryos reconstituted with male and female nuclei in cattle [63] and mice [64].
Because fetal cells are believed to have less genetic damage and more proliferative ability, as measured by cell doublings, than adult somatic cells, they have been preferred as donors for cloning. However, Kato et al. [63] reported no significant differences in rates of bovine blastocyst development for adult, newborn, or fetal cell nuclei, but abortions in later stages of pregnancy were higher for cloned fetuses derived from adult cells. Similarly, no differences among embryos derived from fetal and adult bovine fibroblasts with regard to fusion, cleavage, and blastocyst formation rates were detected [66]. However, fetal losses after transfer into surrogate females were observed only in embryos reconstituted with adult donor cells. In contrast, 8 of 10 bovine embryos derived from adult cells and transferred into surrogate females developed into visibly normal offspring, although the number of embryos produced and transferred was limited [19]. Low numbers, high variability within groups (day effects), and procedural differences hamper comparisons among and within studies.
Genetic damage may occur during in vitro culture of donor cells prior to nuclear transfer. Therefore, fresh or short-term culture (<10 passages) donor cells have been used for production of cloned animals [3, 10, 11, 17–26]. However, Kubota et al. [67] reported higher rates of development to blastocysts for bovine embryos reconstituted with high-passage (passages 10 and 15) adult somatic cells than for embryos from low-passage (passage 5) cells. We obtained similar results using enhanced green fluorescence protein gene transfected and nontransfected bovine granulosa cells as donors; in vitro developmental rates of cloned embryos derived from cells at passage 15 were higher than those for embryos derived from cells of lower passages (passages 10, 11, and 13) [68]. In addition, cloned calves were obtained from embryos reconstituted with the high-passage cells, and all cloned fetuses derived from the low-passage donor cells were aborted during pregnancy [67]. These conflicting results may be attributed in part to inherent differences in competence among donor cell lines and to differences in age of donor animals or in vitro culture periods of donor cells.
Stages of Donor Cell Cycle
Quiescent donor cells arrested in G0/G1 phases of the cell cycle have been commonly used to produce cloned animals [3, 9, 10, 17–24, 26]. However, the specific methods used to arrest the cells in G0/G1 phases significantly affected fetal survival to term and neonatal survival [3]. Methods of arresting cells in these phases of the cell cycle have been explored using reversible cell cycle inhibitors [69]. Serum starvation and growth arrest when cultured cells reach confluence are two other methods used to synchronize cells in the G0/G1 cell cycle stage. The specific cyclin-dependent kinase (CDK) 2 inhibitor, roscovitine, effectively arrests human fibroblasts in G0/G1 phases of the cell cycle [69] and maintains bovine oocytes at the GV stage of maturation by inhibiting MPF, which is a member of the CDK family [70]. Following roscovitine removal, cells arrested in G0/G1 phases resumed cell cycles and entered the S phase as expected [69] and oocytes arrested at the GV stage progressed to MII [70], indicating that the effects of roscovitine were fully reversible. In a recent study [3], bovine granulosa cells treated with roscovitine were compared with serum-starved cells. A higher proportion of cells were synchronized in G0/G1 phases of the cell cycle with the roscovitine treatment relative to the controls or serum-starved cells. Although a higher percentage of blastocysts was produced using the serum-starved cells as donors, fetal survival following embryo transfer was enhanced in the roscovitine-treated group during the last 60 days of gestation (Fig. 1). Significantly more healthy calves (P < 0.05) survived gestation, parturition, and the first 60 days of life in the roscovitine-treated group than in the serum-starved group. This finding may indicate that not all of the reprogramming events that must take place for nuclear transfer to be successful are apparent throughout the relatively abbreviated embryo culture time period. Incomplete or inadequate genomic replication events may not be manifested until well into gestation of the cloned fetus. The true quality of the embryo (usually measured subjectively, at the blastocyst stage with morphology as the primary guideline) may not be determinable until after the embryo has been transferred into a recipient and a pregnancy has been established.
|
Donor somatic cells not in the G0/G1 phases can also be used to clone offspring. Wakayama et al. [71] produced cloned mice from embryonic stem cells synchronized in M phase by nocodazole treatment. This method was applied to produce a cloned calf from cumulus cells [11] and cloned mice from fetal fibroblasts [25]. Recently, a cloned piglet was obtained by using colchicine-treated somatic cell nuclei as donors, most of which are in the G2/M cell cycle stage [40]. However, it may be difficult to obtain a homogenous population of cells in G2/M phases because colchicine treatment synchronized 70.5% of the cells in G2/M phases but 18.8% of them were in G0/G1 phases [40].
Differences in Cloning Efficiency among Cell Lines
The ability to support development of cloned embryos differs among donor cell lines even if they are derived from the same tissue or organ [63]. Similarly, results we obtained from nuclear transfer using four primary cell lines of adult bovine somatic cells indicate that the primary donor cell culture impacts in vitro blastocyst development, initial pregnancy rates, and the percentage of live births (Table 1). Similar results were obtained from pig cloning studies, in which nine different somatic cell cultures used in cloning procedures from 2001 to 2002 resulted in 10 initial pregnancies from four of the nine cell lines. However, development past Day 90 of gestation was achieved with only one cell line. The differences among cell lines for both cattle and pigs may be due to epigenetic effects, because even within a primary cell culture, the generation of cell lines from that culture shows that some lines are more suitable than others as donors for cloning [72]. Modifications that occur during primary cell culture may result in genomes that are either more or less capable of being reprogrammed.
|
Selection of Donor Cell Lines Based on Physiological State
As described previously, roscovitine-treated donor cells are more likely than serum-starved or control cells to be susceptible to reprogramming events in the oocyte after transfer [3]. We evaluated the DNA binding activities of nuclear extracts generated from roscovitine-treated, serum-starved, and control cells to cis-elements of 54 well-known transcription factors (Panomics, Inc., Redwood City, CA). Each nuclear fraction was incubated with the biotin-labeled cis-elements, and bound probes were extracted, hybridized to an array membrane, and detected by immunostaining using chemiluminescence. Transcription factor binding to their respective cis-elements was significantly higher (P < 0.05) for roscovitine-treated cell extracts than for extracts generated with serum-starved and control cells (unpublished data). Comparisons of roscovitine-treated, serum-starved, and control cell extracts binding to individual transcription factor cis-elements known to be involved with cellular growth and differentiation are shown in Table 2. For each individual element, binding by the roscovitine-treated cell extracts tended to be higher than binding by the serum-starved cell extracts, with the exception of Smad 3/4. C/EBP is thought to act in terminally differentiated cells, and binding activity in the roscovitine-treated extract was significantly higher (P < 0.05) than that for serum-starved cell extracts, but serum-starved and control cell extract binding did not differ. These results suggest that roscovitine treatment could regulate different sets of genes in addition to those being regulated by serum starvation alone. More exacting experimentation to ascertain the physiological state of the cell is being pursued. Microarrays allowing the direct comparison of hundreds (or more) of genes simultaneously among treatment groups may allow efficient selection of cell lines that are best suited, based on specific markers, for use as nuclear donors.
|
|
CONCLUSIONS AND FUTURE DIRECTIONS |
|---|
|
TOPABSTRACTINTRODUCTIONSELECTION OF RECIPIENT OOCYTES...SELECTION OF DONOR CELLS...CONCLUSIONS AND FUTURE...REFERENCES |
|---|
Because the successful reprogramming of a donor nucleus depends on factors present in a recipient oocyte, studies focused on biochemical and molecular features of oocyte meiotic maturation are important for understanding the reprogramming process. Cytoplasmic changes in relation to nuclear maturation and the roles of microfilaments, microtubules, and MAPK in regulation of porcine oocyte maturation and fertilization have been described [75, 76]. Immunolocalization and regulation of specific proteins and enzymes important for meiotic maturation have been demonstrated [77–79]. The culture medium used for maturation of bovine oocytes influences the abundance of specific gene transcripts [80]. Examinations of genes involved in early embryonic development revealed a decrease in polyadenylation levels of their transcripts during in vitro maturation. Moreover, the poly(A) tails were shorter in oocytes with lower developmental competence [81]. We used a novel restriction fragment differential display reverse transcription polymerase chain reaction technique to compare patterns of mRNA expression in bovine oocytes matured in vitro in the presence or absence of fetal calf serum. With this technique, we isolated and sequenced 12 differentially expressed fragments, which indicated that maturation conditions can alter gene transcript levels in matured oocytes [82]. Suppressive subtractive hybridization has been used to identify oocyte and granulosa cell mRNAs associated with maturation and developmental competence of bovine oocytes [83, 84]. In the past few years, significant progress has been made in identification of genes involved in the maturation process. For instance, a new member of the Snf1/AMPK kinase family, Melk, was shown to be expressed in the mouse oocyte [85]. Complementary DNA libraries from cattle oocytes have been produced, and expression of homeobox-containing genes has been demonstrated [86]. The extensive list of genes identified and studied in bovine oocytes is available in the Bovine Embryo Gene Collection (http://www.begc.crbr.ulaval.ca/PAGES/genes.html). In addition, there are ongoing projects to identify and characterize genes expressed during porcine and bovine reproduction. The strategies for the characterization of the transcriptome are based on creating expressed sequence tags and randomly sequencing or constructing serial analyses of gene expression libraries (R. Prather and K. Zuelke, personal communication; http://swine.rnet.missouri.edu). Recently, Hwang et al. [87] performed a computer-based search for homologs of yeast genes that are induced during sporulation in Caenorhabditis elegans, Drosophila, and mammals. Results suggest that yeast and higher eukaryotes share genes that coordinate the overall process of meiosis. Expressed sequence tags representing more than half of the mammalian homologues are present in mouse cDNA libraries that contain genes controlling the meiosis/mitosis transition. In addition, about 50% of these genes contain potential cis-elements for cytoplasmic polyadenylation in their 3' untranslated regions, suggesting the importance of controlled translation in the oocyte and zygote [87]. However, the role and significance of these findings relative to reprogramming of donor nuclei are unclear and require further investigation. At present, therefore, it is difficult to improve nuclear reprogramming directly.
Donor nuclei are reprogrammed functionally in a few cloned embryos that develop into normal offspring. To obtain these embryos, many are produced that do not develop into offspring because we are unable to identify recipient oocytes and donor cells that produce embryos with functionally reprogrammed nuclei. The development of methods for selection of oocytes and cells that are suitable for use as recipients and donors is essential for increasing cloning efficiency. This research will also yield important information regarding mechanisms responsible for nuclear reprogramming.
|
FOOTNOTES |
|---|
Received: 3 September 2002.
First decision: 17 September 2002.
Accepted: 17 October 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONSELECTION OF RECIPIENT OOCYTES...SELECTION OF DONOR CELLS...CONCLUSIONS AND FUTURE...REFERENCES |
|---|
-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002 295:1089-10921,3-galactosyltransferase gene in cloned pigs. Nat Biotech 2002 20:251-255[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  B. Heindryckx, P. De Sutter, J. Gerris, M. Dhont, and J. Van der Elst Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes Hum. Reprod., July 1, 2007; 22(7): 1982 - 1990. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
) and serum-starved (
) adult 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 births from the serum-starved group resulted in singleton calves [