Nuclear Transfer in the Rhesus Monkey: Practical and Basic Implications 1

BIOLOGY OF REPRODUCTION 60, 199 204 (1999) Nuclear Transfer in the Rhesus Monkey: Practical and Basic Implications 1 Don P. Wolf, 2,4,5,6 Li Meng, 3,4 Nadia Ouhibi, 4,5 and Mary Zelinski-Wooten 4 Division of Reproductive Sciences, 4 Oregon Regional Primate Research Center, Beaverton, Oregon 97006 Departments of Obstetrics/Gynecology 5 and Physiology/Pharmacology, 6 Oregon Health Sciences University, Portland, Oregon 97201 ABSTRACT In early 1997, the birth of a lamb after transfer of the nucleus from an adult mammary gland cell into an enucleated oocyte, along with the production of rhesus monkeys by nuclear transfer of embryonic cells, marked a reemergence of the field of mammalian cloning. Clonally derived rhesus monkeys would be invaluable in biomedical research, and the commercial interests in transgenic sheep and cattle propagated by cloning are substantial. Nuclear transfer technology is under consideration in human in vitro fertilization clinics to overcome infertility secondary to advanced maternal age or mitochondrial-based genetic disease. Nuclear transfer involves preparing a cytoplast as a recipient cell, in most cases a mature metaphase II oocyte from which the chromosomes have been removed. A donor nucleus cell is then placed between the zona and the cytoplast, and fusion, as well as cytoplast activation, is initiated by electrical stimulation. Successful reprogramming of the donor cell nucleus by the cytoplast is critical a step that may be influenced by cell cycle stage. Embryos produced by nuclear transfer are cultured in vitro for several cell divisions before cryopreservation or transfer to the oviduct or uterus of a host mother. The efficiency of producing live young by nuclear transfer in domestic species is low, with a high frequency of developmental abnormalities in both preterm and term animals. However, a number of pregnancies have now been established using fetal cells as the source of donor nuclei. The use of cell lines not only allows large clone sizes but also supports the ability to genetically manipulate cells in vitro before nuclear transfer. Ongoing research focused on the production of clonally derived rhesus monkeys using fetal fibroblasts and embryonic stem cells as the source of donor nuclei will be reviewed. INTRODUCTION Nuclear transfer, as opposed to blastomere separation and culture or embryo splitting, is the technique of choice for the production of clonally derived mammals, because clone size can be unlimited given the availability of nuclear donor cells that can be propagated and maintained in vitro. The procedure involves the transfer of a nucleus (or the entire cell) from a donor cell into an enucleated (without chromosomal DNA) oocyte (a cytoplast), itself often recovered after subjecting females to ovarian stimulation. The oocyte must reprogram the nucleus, a task that before 1997 was thought to be impossible if the donor cell originated from a highly differentiated tissue. The concept of nuclear transfer dates back to 1938 and the German developmental biologist, Hans Spemann [1], who suggested an experiment that would evaluate the relative importance of the nucleus 1 This work was supported by grants from NIH HD28484, NIH RR12804, NIH A142709, HD 18185, and RR00163 (hormone assay, IVF- EE, and cell culture cores). 2 Correspondence: Don P. Wolf, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006. FAX: 503 418 3757; e-mail: wolfd@ohsu.edu 3 Current address: In Vitro Fertilization Laboratory, Women and Infant s Hospital, 101 Dudley St., Providence, RI 02905. 199 and cytoplasm in controlling early developmental events. The technology was first demonstrated in amphibians over 40 years ago (reviewed in [2]), in large measure reflecting the large size of the amphibian oocyte and its rapid rate of development. In mammals, live offspring have been born after nuclear transfer in the mouse, rabbit, pig, sheep, goat, and cow [2, 3], and, in our laboratory, in the rhesus monkey [4]; all have involved enucleated, mature oocytes or 1- or 2-cell-stage embryos and donor nuclei from embryos or cultured embryonic cells. Very recently, success has also been announced in cloning mice, sheep, and cattle using nuclei from differentiated cells: fetal fibroblasts [5, 6], adult mammary gland cells [6], or cumulus cells [7]. The ultimate success of nuclear transfer is dependent on producing a large number of cloned animals easily in a cost-effective manner, the key to which is selection of a nuclear donor source that is virtually unlimited. Options include primary cell cultures, an alternative created recently with the report of success in sheep using cultured adult mammary gland cells or fetal fibroblasts [6]. These cultures are relatively easy to maintain in vitro but cannot be immortalized as they become aneuploid after repeated passages. While this may make homologous recombination impractical, very large populations of such cells can be generated and stored frozen. Furthermore, nuclear transfer with transfected cells can be used to produce transgenic animals with high efficiency. Several successful applications have recently been announced, first in sheep in which fetal fibroblasts were cotransfected with a neomycin resistance marker gene and a human coagulation factor IX genomic construct [8], and then in cattle in which fetal fibroblasts were transfected with a marker construct consisting of -galactosidase-neomycin resistance fusion gene [5]. Term births of living animals occurred in both cases. A second option involves the use of totipotent embryonic stem (ES) cell lines derived from preimplantationstage embryos. The ability of ES cells to serve as vectors for the transfer of foreign DNA in the production of transgenic animals is a well-established tool by which defined or random mutations of the genome can be propagated [9]. ES cell nuclei can be reprogrammed in the context of nuclear transfer, and the cells remain undifferentiated and euploid during proliferation in vitro. Such a line was cloned from in vivo-produced rhesus monkey blastocysts flushed from the uterus, and it remains undifferentiated in continuous passage [10]; a number of additional lines are now available [11]. REPROGRAMMING THE DONOR NUCLEUS Synchronization of the donor nucleus with the recipient cytoplast is critical to success in nuclear transfer, a process that usually requires nuclear reprogramming. A number of cell cycle kinases are involved including mitosis-promoting factor (MPF), a complex of two proteins, cyclin B and p34 cdc2, that are responsible for nuclear envelope break-

200 WOLF ET AL. FIG. 1. Average number of oocytes retrieved per monkey following one (circles; n 27) or two (diamonds; n 12) consecutive follicular stimulation cycles using recombinant human gonadotropins over a 6-mo period. See text for details. down and chromatin condensation. MPF, as a protein kinase, is itself regulated by changes in its state of phosphorylation and by its association with cyclins. MPF activity can be measured by its kinase activity using exogenous histone H1 as a substrate. Fluctuations in MPF levels occur during the cell cycle and during maturation. In all species studied, MPF levels, measured as histone H1 kinase activity, increase sharply at the G2-M phase transition, decrease at the anaphase and telophase stages, and then increase at the metaphase of the second meiotic division. Upon fertilization or oocyte activation, MPF activity declines rapidly, and the cell resumes meiosis with breakdown of the nuclear envelope, chromosome condensation, reorganization of the cytoskeleton, and changes in cell morphology [12]. In order for events to occur properly after nuclear transfer, MPF levels must decrease or be low. If these conditions are not met, the incidence of chromosomal damage and aneuploidy can be high [12]. In addition to MPF levels, the possibility of course exists for other cytoplasmic factors to be involved, and the timing of events is important in nuclear reprogramming. This means that the sequence of donor nucleus addition and cytoplast activation, as well as the nature of the activation stimulus, may influence outcome. Attention has also focused on the cell cycle stage of the donor nucleus. For instance, the success in somatic cell cloning in sheep [6] was attributed by the authors to their unique approach to cell cycle staging, wherein they starved nuclear donor cells and forced them into a quiescent or G 0 stage. In contrast, in the cow, nonquiescent fetal fibroblasts have been employed successfully, suggesting that staging [5] or forcing the cell out of an active cycle into a quiescent state is not always required. More recently, noncultured cumulus cells in the G 0 /G 1 phase of the cell cycle were used in cloning mice [7]. IN VITRO FERTILIZATION (IVF) AND EMBRYO CULTURE IN THE MONKEY A major limitation in conducting nuclear transfer studies in the monkey is oocyte availability. Animals are expensive to acquire and maintain, and since oocyte in vitro maturation is not yet well worked out in primates, including women, ovarian stimulation protocols are invaluable. Several methods have been evaluated in our rhesus monkey program for the stimulation of multiple follicular development using human gonadotropins, because monkey hormones are not yet available in sufficient quantity [13]. Experience has been obtained with relatively impure preparations of human urinary gonadotropins as well as, more recently, recombinant human products in animals treated with a GnRH antagonist. Antagonist use provides a more homogenous ovarian milieu and prevents endogenous LH surges, retaining more females in the protocol until oocyte retrieval. A downside to the use of human hormones in rhesus monkeys is the immunological response elicited, which limits the number of ovarian stimulation cycles an animal can experience. Fortunately, antibodies that potentially cross-react with monkey gonadotropins are either not produced or are present in low titers, such that reproductive function is normal in females after exposure to ovarian stimulation with human hormones [14]. In a controlled study evaluating the repeated use of animals subjected to ovarian stimulation with recombinant human gonadotropins, we concluded that animals could be used for 3 cycles without major impact on oocyte number or quality at harvest [15]. The ovarian stimulation protocol currently in use [16] involves coadministration of GnRH antagonist (Antide, 1.0 mg/kg BW of a solution of 10 mg/ml [w:v] in propylene glycol:water [1:1, v:v]; Laboratoires Serono SA, Aubonne, Switzerland) with recombinant human (h)fsh (Gonal FTM; Laboratoiries Serono SA; 30 IU, twice daily, i.m.) from the beginning of menses (Days 1 4) for 6 days. Thereafter, the gonadotropin is altered to include recombinant LH, usually for an additional 1 2 days depending on the size and number of follicles as determined by ultrasonography and serum estradiol patterns. In the evening of the final day of FSH-LH treatment, animals receive a single, i.m. injection of 1000 IU recombinant hcg (Laboratoires Serono SA), and oocyte collection is scheduled for 27 34 h later. In the 1997 98 season, we subjected 45 females to ovarian stimulation, 40 of which were taken to surgery, with the recovery of oocytes from 36. A total of 668 oocytes were obtained for an average of 16.7 per surgical intervention (range 0 57). Oocyte recovery over the entire season is depicted in Figure 1 as a function of time and the number of stimulation cycles. Overall oocyte yield was decreased late in the season (May early June with four surgeries yielding no oocytes), and the initial cycle was more productive (520 oocytes from 27 retrievals or 19.3/ retrieval) than repeat stimulation cycles (148 oocytes from 12 retrievals or 12.3/retrieval). Although our previous experience with the optimal time from hcg injection to oocyte collection led us to use routinely a 27-h interval [17], we lengthened this interval during our most recent nuclear transfer studies to 30 34 h in an effort to support earlier collection times during the working day and a higher yield of mature MII oocytes. Another major limitation in the rhesus monkey model is the relatively poor developmental potential of IVF-produced embryos. For instance, culturing embryos in a medium supplemented with serum has resulted in develop-

NUCLEAR TRANSFER IN THE MONKEY 201 TABLE 1. Source and developmental efficiency of reconstituted embryos produced by nuclear transfer in the rhesus monkey. Parameter Stage of donor nuclei Number of host mothers Status of reconstituted embryos Number of biochemical pregnant host monkeys Number of live fetuses/total number of embryos transferred Number of live births 8-cell 17 Frozen 4* 2/53 (3.8%) 2 * Two pregnancies were lost spontaneously in the first 30 days of gestation. mental efficiencies for monkey zygotes to expanded blastocysts of 8% and 55% for frozen embryos with and without coculture, respectively [18], and of 22% and 56% for nonfrozen embryos cultured, respectively, in CMRL 1066 [19] or KSOM/AA (developed by Lawitts and Biggers for mouse [20] as applied by Ho and coworkers [21]) [22]. While the higher values cited above might be considered acceptable, consistency in achieving them is a major problem in the monkey. Another approach to obtaining high blastulation rates that has gained credibility and a robust data base in the clinical IVF realm is sequential medium exposure, starting with a relatively simple medium that supports fertilization and early development, for instance, for up to 3 days (human tubal fluid or a low-glucose, phosphate medium with a serum-derived protein fraction such as synthetic serum substitute; Irvine Scientific, Santa Ana, CA), followed by transfer into medium supplemented with essential and nonessential amino acids with albumin as the protein source (S2; Scandinavian IVF, Gothenburg, Sweden). Another version of the second medium uses glutamine as the sole amino acid supplement and has been associated with blastulation rates as high as 70% [23]. This approach may allow the elimination of serum and the use of chemically defined media for early preimplantation development in women and, it is hoped, in rhesus monkeys. Current trials involve a direct comparison of blastulation rates of IVFproduced rhesus monkey embryos in Tyrode s albumin lactate pyruvate (TALP) medium [24] cocultured on buffalo rat liver (BRL) cells versus embryos in S2 medium alone. NUCLEAR TRANSFER IN THE MONKEY Individual steps in the nuclear transfer procedure include oocyte recovery and maturation from excised ovaries, or ovarian stimulation and the recovery of MII oocytes; enucleation and preparation of the cytoplast (chromosome removal by micromanipulation); donor nucleus isolation and transfer to the cytoplast to produce an unfused pair; chemical activation of the cytoplast (cycloheximide exposure); electrically induced fusion of unfused pairs; embryo culture; and, finally, embryo transfer to the oviduct or uterus of a synchronized recipient either with or without prior embryo cryopreservation and low-temperature storage (Fig. 2). In first approaching the possibility of conducting nuclear transfer in rhesus monkeys in 1995, we employed blastomeres from IVF-produced embryos as the source of donor nuclei [4]. This selection was predicated on the supposition that nuclear transfer was possible only with embryonic cells and on our established ability to produce embryos in vitro. We began with the objective of demonstrating that viable term pregnancies could be established by nuclear transfer in the monkey, with the intent of pursuing the production of clonally derived animals at a later time. As the source FIG. 2. Nuclear transfer in the rhesus monkey. A schematic of the technology. Adapted from Biol Reprod 1997; 57:454 459. of potential cytoplasts, three possibilities existed. First, MII oocytes obtained from gonadotropin-treated monkeys after ovarian stimulation and in vitro culture are probably the most likely source, although MPF levels are high and activation may be problematic. Second, aged oocytes that had failed to fertilize approximately 20 h after insemination could be used; these oocytes may activate spontaneously or, at least, be activated easily [25]. Third, zygotes obtained from conventional IVF are advantageous because of low MPF levels and highly visible pronuclei, which allow direct confirmation of enucleation following micromanipulation. However, overall efficiency at production of cleaving, reconstituted embryos was highest with fresh MII oocytes, and attention thereafter focused on these as the source of cytoplasts. Of the 101 reconstituted embryos produced from such cytoplasts that cleaved in a timely manner and appeared morphologically normal, 53 were transferred into the oviducts of synchronized recipients (n 17) during spontaneous menstrual cycles (Table 1). In all cases, reconstituted embryos were cryostored until a recipient was available, and embryo transfer was conducted 2 days after the LH surge in a natural menstrual cycle. Animals were monitored for pregnancy by measuring circulating levels of estradiol and progesterone as well as by conducting uterine

202 WOLF ET AL. FIG. 3. Reconstituted embryos (left) after coculture with BRL cells in vitro for 2 days ( 200). Rhesus monkey infants (right) produced by nuclear transfer technology. Adapted from Biol Reprod 1997; 57:454 459. ultrasonography. Four pregnancies resulted, two of which were lost at approximately 30 days of gestation. The remaining two culminated in the birth of one male and one female at 166 and 149 days of gestation, respectively. These infants were named Neti (an acronym for nuclear embryo transfer individual) and Ditto (for obvious reasons) despite the fact that these infants are siblings and not clones (Fig. 3). The parentage of both nuclear transfer infants was ascertained by genetic typing with seven unlinked short tandem repeat (STR) markers amplified by polymerase chain reaction. The male allelic contribution to Neti and Ditto genotypes, as revealed on autoradiographs of the STRs, was subtracted from that of the nuclear transfer infant, and FIG. 4. Autoradiograph of 1 STR marker used to identify nuclear donor female 14893 as the true mother of both nuclear transfer infants. From left to right, identities of individuals in the pedigree at top are host mother 13646, nuclear transfer (NT) infant 19255, sire 14609, mother (nuclear donor) 14893, NT infant 19235, host mother 16150, enucleated egg donor 16426, and female 8090 (mother of 16426). Locus D3S1768 excluded enucleated egg donor 16426 for both infants 19255 and 19235, and also excluded host mother 16150 for infant 19235. For details see Biol Reprod 1997; 57:454 459. the remaining maternal allele was used to identify the mother. In this manner, both the host mother and the enucleated oocyte donor mother were definitively eliminated as the maternal parent at a minimum of 2 STR loci (see Fig. 4 for an autoradiograph at 1 STR locus). With the advent of Dolly [6] and the realization that fetal fibroblasts as well as adult cells could be used for nuclear transfer, we began evaluating the production of clonally derived monkeys with two objectives in mind: selection of a suitable nuclear donor source and evaluation of the importance of cell cycle staging. ES cells or fibroblasts were employed as the source of donor nuclei. ES cells (euploid, XY) provided by Dr. James Thomson, Wisconsin Regional Primate Research Center, were sent to Oregon on wet ice by overnight mail and cultured overnight before individual cell dispersal and nuclear transfer. Fetal fibroblasts (XY karyotype), obtained from Dr. Michael Axthelm, ORPRC, were aliquoted at low passage number and frozen; individual tubes were thawed, and the cells were used in nuclear transfer studies either as growing or starved cultures. In the latter case, cells were grown in low serum concentrations in an effort to disrupt the cell cycle and produce a large percentage of cells in the G 0 stage. We have produced 166 reconstituted embryos with no evidence that the source or cell cycle stage of nuclear donor cells is critical (Table 2). In preliminary trials of extended culture, embryos in S2 grew as efficiently and rapidly as those in coculture with BRL cells; we are currently moving towards the exclusive use of sequential media in the absence of coculture and serum. However, we have not yet succeeded in establishing a pregnancy following embryo transfer into synchronized recipients. In 1998, 12 reconstituted embryos, without prior cryopreservation, from the 4- to 16-cell stage of development, containing donor nuclei from either fetal fibroblasts or ES cells, have been surgically transferred into the oviducts of 6 females during spontaneous menstrual cycles. CONCLUSIONS AND FUTURE DIRECTIONS We would like to believe that the production of clonally derived rhesus monkeys, using either fibroblasts or ES cells as the source of donor nuclei, is a realistic expectation. We have already demonstrated the feasibility of conducting nuclear transfer in a primate species: two infants were born from embryo transfers (29 embryos) conducted in 9 host mothers [4], and additional experience has been obtained as cited in this review. Success would allow the genetic background of animals to be held constant, while physiologic, environmental, or behavioral parameters of interest are manipulated. Consequently, statistical validity could be obtained with fewer animals. Conversely, the other (nongenetic) variables could be held constant while the genetic

NUCLEAR TRANSFER IN THE MONKEY 203 TABLE 2. Summary of nuclear transfer experimentation in the rhesus monkey during the first 6 months of 1998, in which donor nuclei were transferred into MII cytoplasts in all cases. Donor nuclei Fetal fibroblasts Adult fibroblasts ES cells Totals * ET, embryo transfer into host mothers. Rhesus monkey nuclear transfer No. experiments No. eggs enucleated No. NT embryos cultured No. cleaving No. ET* 14 2 5 21 136 25 51 212 100 9 35 144 (68%) 57 4 18 79 (55%) 0/3 0/1 0/2 0/6 component is manipulated in ES cells by gene knockout or site-directed mutagenesis, or in fibroblasts by established transfection approaches. The future holds the exciting possibility of treating the overall genetic constitution, or even locus-specific genotypes, in rhesus monkeys as simply as any other research variable in the experimental design, much as in current studies using inbred strains of mice. It follows, then, that while commercial interests may predominate in the production of domestic animal clones by nuclear transfer, our major reason for cloning in nonhuman primates relates to the production of an improved, more efficient and versatile animal model for biomedical research. Achieving the goal of producing significant numbers of clonally derived monkeys is not going to be easy. It is not possible to visit the local abattoir and bring home hundreds of ovaries containing thousands of oocytes, as can be done in the domestic animal industry. Therefore, one of our future objectives is to try freezing and cryobanking cytoplasts, affording the ability to accumulate the relatively large numbers of cytoplasts desirable for systematic nuclear transfer studies. Low-temperature storage of mammalian oocytes, including those from women, is associated with high loss rates and poor outcomes secondary to the disruption of spindle microtubules and a greatly increased risk of chromosomal aberrations [26], or, in the case of immature oocytes, to poor in vitro maturation and fertilization rates post-thaw [27], although limited success has been reported [28]. We are proceeding on the premise that cytoplasts will not be subject to such low temperature-induced damages. Another important future goal is the definition of a simple, noninvasive technique for evaluating viability and parentage of reconstituted embryos produced by nuclear transfer. While phenotypic differences can be employed in liveborn offspring in some species, molecular confirmation is desirable if not essential in early attempts at cloning. Of course, the completeness of DNA removal during enucleation of the cytoplast can be confirmed by epifluorescence microscopy; but is the transferred genome always activated and expressed in cleaving, reconstituted embryos? Possible ways to assess this include detection of the c-kit receptor, a potential marker for totipotency [29]; biopsy and karyotyping the blastomere, at least when male (XY) ES cells or fibroblasts are used as the nuclear donor cells; and the production of embryos that are transgenic for a construct carrying a reporter gene such as that for green fluorescent protein or bacterial -galactosidase. The ability to detect genomic activation after nuclear transfer would help in the selection of reconstituted embryos for study or for embryo transfer in attempts to establish viable pregnancies. Finally, to allow the production of clonally derived animals at primate centers anywhere in the world without relying on in-house nuclear transfer facilities, the ability to conduct extended culture and nonsurgical embryo transfer with uterine-stage embryos is essential. Therefore, continued evaluation is needed in the definition of culture systems for prolonged preimplantation development such as those described in this review, and for performing nonsurgical embryo transfers. In the latter regard, host mothers are currently selected for nonsurgical transfers on the basis of transcervical access to the uterus as determined in screening physical examinations. However, despite the ability to conduct such transfers, in a limited number of attempts we have not yet established a pregnancy. In conclusion, we have placed emphasis on providing a historical background to nuclear transfer in mammals and describing recent advances in the technology. 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