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Regular Article |
a Oregon Regional Primate Research Center, Beaverton, Oregon 97006
b Departments of Obstetrics and Gynecology, and Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon 97201
ABSTRACT
This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.
calcium, fertilization, IVF/ART, kinases, meiosis
INTRODUCTION
The production of genetically identical mammals by somatic cell nuclear transfer (NT) is now a reality [1–5]. However, low efficiencies and high embryonic, fetal, and neonatal losses necessitate further research into the basic mechanisms controlling the onset and early preimplantation development of embryos produced by NT. Factors known to affect the successful development of reconstructed embryos include the source of donor nuclei, the cell cycle stage of both the donor nucleus and recipient enucleated oocyte (cytoplast), and cytoplast activation. The latter, cytoplast activation, is an essential component of the NT procedure, because the introduction of the donor nucleus into the cytoplast bypasses fertilization.
During fertilization, sperm entry triggers a series of intracellular calcium oscillations critical to oocyte activation. Maturation promoting factor (MPF) and mitogen-activated protein (MAP) kinase are the most likely targets of calcium-stimulated events because inactivation of these kinases is a prerequisite to the resumption and completion of meiosis, subsequent pronuclear formation, and DNA synthesis [6–8]. MPF is a complex of two subunits: a catalytic subunit, p34cdc2, a homologue of the yeast cdc2 protein kinase; and a regulatory subunit, cyclin B. Association of these subunits and subsequent activation of MPF occurs in a specific order by dephosphorylation of the p34cdc2 subunit at threonine-14 and tyrosine-15, and by phosphorylation at threonine-161. MPF displays its peak activity at metaphase of mitotic cell cycles in association with nuclear envelope breakdown, chromatin condensation, and the formation of a mitotic spindle [9]. MPF inactivation, which is necessary for the cell to exit mitosis, involves cyclin proteolysis by the proteosome system [10].
In vertebrates, mature oocytes are arrested at metaphase of the second meiotic division (MII) with elevated MPF activity maintained by a cytostatic factor (CSF), essential components of which are the product of the c-mos proto-oncogene, MAP kinase, and possibly Cdk2 kinase. CSF prevents ubiquitin-dependant degradation of cyclin B and, thus, inactivation of MPF. Intracellular Ca2+ oscillations triggered by sperm down-regulate CSF activity and release the cyclin degradation machinery. Proteolytic degradation of cyclin B and subsequent MPF inactivation releases oocytes from metaphase arrest and allows the beginning or resumption of mitotic cycles [11].
Various artificial activation treatments mimic sperm-triggered events and induce parthenogenetic development in MII oocytes. For example, ethanol, electroporation, calcium ionophore, ionomycin, or inositol 1,4,5-trisphosphate induce calcium elevations and release meiotic arrest [12–17]. However, MPF activity, at least in young bovine and rabbit oocytes, is quickly restored with recondensation of chromosomes and reentry of activated oocytes into a new M-phase arrest, known as metaphase III [14, 18]. This phase can be circumvented by additional treatments that inhibit protein synthesis (cycloheximide, CHX) or protein phosphorylation (6-dimethylaminopurine, DMAP) [14, 16]. Thus, sequential approaches have evolved with ionomycin/DMAP, calcium ionophore/DMAP, calcium ionophore/CHX, or inositol 1,4,5-trisphosphate/DMAP that result in high activation and parthenogenetic development rates in the bovine and rabbit [14, 16, 17]. However, detrimental effects of these nonspecific, broad-spectrum protein synthesis and/or kinase inhibitors have been demonstrated. For instance, the developmental competence of bovine oocytes is compromised after blocking maturation at the germinal vesicle stage for 24 h with CHX or DMAP [19]. In contrast, purine derivatives (roscovitine, olomoucine), which specifically inhibit MPF and MAP kinase, have been used to delay meiotic progression of bovine oocytes without affecting their subsequent developmental potential to the blastocyst stage [20], suggesting their potential use for parthenogenetic activation.
Despite success with NT in the production of rhesus macaque infants [21], relatively little information is available concerning the efficacy of activation treatments on monkey MII oocytes and reconstructed embryos. Here we report efforts 1) to determine the efficiency of sequential calcium treatments (electric pulses or ionomycin) combined with protein synthesis or phosphorylation inhibitors (CHX or DMAP) in inducing parthenogenetic activation and development of rhesus macaque parthenotes; 2) to investigate the effect of ionomycin followed by roscovitine, a specific MPF inhibitor on parthenogenetic activation of monkey oocytes; and 3) to evaluate the effectiveness of these activation treatments in reconstructed embryos following NT.
MATERIALS AND METHODS
Animals
Mature rhesus macaque males and females housed in individual cages were used in this study. All animal procedures were approved by the Institutional Animal Care and Use Committee at the Oregon Regional Primate Research Center/Oregon Health Sciences University.
Follicular Stimulation and Oocyte Collection
One to 4 days following the onset of menses, adult rhesus macaque females were subjected to a follicular stimulation protocol [22, 23]. Briefly, monkeys received twice-daily injections of recombinant human FSH (rhFSH; 30 IU i.m.) and once-daily injections of Antide (a GnRH antagonist; 0.5 mg/kg s.c.) for 9 consecutive days. On the last 2 days of rhFSH/Antide stimulation, animals also received twice-daily injections of recombinant human LH (rhLH; 30 IU i.m.). On the last or next-to-last day of hormonal stimulation, ovarian morphology was recorded by ultrasonography (Advanced Technology Laboratories, ATL; Bothell, WA). Monkeys responding to follicular stimulation (follicles 
4 mm diameter) received an injection of recombinant hCG (rhCG; 1000 IU i.m.) to induce oocyte maturation. Cumulus-oocyte complexes were collected from anesthetized animals by laparoscopic follicular aspiration (32–33 h post-rhCG) and placed in Hepes-buffered TALP (modified Tyrode solution with albumin, lactate, and pyruvate) medium [24] containing 0.3% BSA (TH3) at 37°C. Unless indicated otherwise, all hormones were from Ares Advanced Technologies Inc. (Norwell, MA) and other reagents were from Sigma-Aldrich Co. (St. Louis, MO). Oocytes, stripped of cumulus cells by mechanical pipetting after brief exposure (<1 min) to hyaluronidase (0.5 mg/ml), were placed in CMRL (Connaught Medical Research Laboratories; Life Technologies, Rockville, MD) medium at 37°C in 5% CO2 balance air containing 10% fetal bovine serum (FBS; HyClone, Logan, UT), 10 mM L-glutamine (Life Technologies), 5 mM sodium pyruvate, 1 mM sodium lactate, 100 units/ml of penicillin (Life Technologies) and 100 µg/ml of streptomycin (Life Technologies) until further use [25].
Intracytoplasmic Sperm Injection
A cohort of MII oocytes from each experiment was fertilized by intracytoplasmic sperm injection (ICSI) [22] and used as controls to evaluate second polar body extrusion, pronuclear formation, cleavage, and development to the blastocyst stage. In contrast to conventional in vitro fertilization (IVF), ICSI allowed the use of cumulus-free MII oocytes and better visualization of second polar body extrusion and pronuclear formation. ICSI was performed within 35–36 h post-hCG injection employing sperm collected by penile electrostimulation from pregnancy-proven males [26]. Sperm were processed by centrifugation (200 x g for 7 min) of the liquid portion of the ejaculate, and the sperm pellet was resuspended in TH3. Before final resuspension, an aliquot was taken, and sperm motility and concentration was determined. Sperm concentrations were adjusted to 5 million motile sperm per milliliter in TH3 and stored, on average, 3 h at room temperature, prior to ICSI.
A mineral oil-covered, micromanipulation chamber containing a 30-µl TH3 drop with oocytes and a 4-µl drop of 10% polyvinylpyrrolidone (Irvine Scientific, Santa Ana, CA) diluted with 1 µl sperm was placed on the stage of an inverted microscope (Olympus IX70; Olympus America Inc., Melville, NY) equipped with micromanipulators (Narishige, Japan). An individual sperm was immobilized, aspirated into an ICSI pipette (Humagen, Charlottesville, VA), and injected into the oocyte cytoplasm, away from the polar body.
Parthenogenetic Activation
Cumulus-free MII oocytes, 35–36 h post-hCG injection, were randomly assigned to one of the following activation treatments: 1) exposure to 5 µM ionomycin (Calbiochem, La Jolla, CA) for 2 min (unless indicated otherwise) in TALP/Hepes medium supplemented with 1 mg/ml BSA and then washed for 5 min in TALP/Hepes supplemented with 30 mg/ml BSA (ionomycin alone); some ionomycin-treated oocytes were then transferred into CMRL medium containing 2) 2 mM DMAP (ionomycin/DMAP) or 3) 50 µM roscovitine (Calbiochem; ionomycin/roscovitine) and cultured at 37°C in 5% CO2 balance air for 4 h. Alternatively, 4) some oocytes were electroporated three times, 30 min apart with two 50-µsec direct current pulses of 2.7 kV/cm (Electro Square Porator T-820; BTX, Inc., San Diego, CA) in 0.25 M D-sorbitol buffer containing 0.1 mM calcium acetate, 0.5 mM magnesium acetate, 0.5 mM Hepes, and 1 mg/ml fatty acid-free BSA with incubation in CMRL medium containing 7.5 µg/ml CHX and 7.5 µg/ml CytoB between electroporations (electroporation/CHX/CytoB).
Nuclear Transfer Procedures
Blastomeres of Day 3 (Day 0 = day of fertilization) ICSI-produced rhesus monkey embryos were used as the source of donor nuclei. Zonae pellucidae were removed by brief exposure to 0.5% pronase and blastomeres were mechanically disaggregated after incubation in 0.5 mM EDTA in Ca2+-free and Mg2+-free Dulbecco PBS (Life Technologies) for 5–10 min. Recipient MII oocytes were incubated for 5 min with 5 µg/ml Hoechst 33342, transferred to 30 µl of TH3 containing 3.5 µg/ml cytochalasin D, and incubated for 10–15 min before enucleation. The first polar body and metaphase spindle were drawn into an enucleation pipette (25–28 µm outer diameter) with subsequent confirmation of removal of the Hoechst-stained metaphase spindle by epifluorescent microscopy. The time of exposure to UV light was restricted to less than 10 sec. A blastomere transfer pipette (33–35 µm outer diameter) was used to aspirate a disaggregated donor blastomere and transfer it into the perivitelline space of the cytoplast. Fusion of NT pairs was induced by two 50-µsec direct current pulses of 2.7 kV/cm (Electro Square Porator T-820) in D-sorbitol buffer. Fusion was evaluated visually 45–60 min after electroporation by noting the presence or absence of donor blastomeres in the perivitelline space. Fused NT embryos were activated approximately 2 h after fusion by exposure to either ionomycin/DMAP or ionomycin/roscovitine as indicated above and cultured in CMRL medium.
Embryo Culture
ICSI-produced embryos, parthenotes, and NT embryos (36–37, 39–40, and 42–43 h post-hCG injection, respectively) were placed in 4-well dishes (Nalge Nunc International Co., Naperville, IL) and cocultured at 37°C in 5% CO2 balance air on buffalo rat liver (BRL) cell monolayers (25 000 cells per well) in CMRL medium. Extrusion of a second polar body and pronuclear formation were monitored and recorded every 2 h during 12-h period postactivation (hpa) or post-ICSI, and the progression of embryo growth was recorded daily. Embryos were transferred to fresh plates of BRL cells every other day for a maximum of 8 days. Development based on cleavage, morula, and blastocyst rates was assessed for each replicate and expressed relative to the number of pronuclear stage embryos or activated oocytes (second polar body appearance in ionomycin-alone group).
Statistical Analysis
Results, expressed as means and standard errors of the mean, were analyzed using one-way ANOVA and Fisher protected least significant difference test within Statview software (SAS Institute Inc., Cary, NC).
RESULTS
Meiotic Resumption and Pronuclear Formation Following Activation
Initially, the ionomycin exposure time necessary to induce parthenogenetic activation was defined. MII oocytes at 35–36 h post-hCG injection were exposed to 5 µM ionomycin for 2, 3, or 4 min followed by incubation in 2 mM DMAP for 4 h. Similar levels of pronuclear formation, initial cleavage, and blastocyst formation were observed, however, up to 10% of oocytes lysed following the 4-min exposure (results not shown). Thus, a 2-min treatment with ionomycin was considered adequate, and this exposure time was routinely used in subsequent experimentation.
The efficacy of several parthenogenetic activation protocols was evaluated and compared to ICSI-fertilized "control" oocytes. Control oocytes completed meiosis and extruded a second polar body by 4 h (74% ± 4%) with the resultant zygotes forming two pronuclei at approximately 10 h after fertilization (71% ± 5%; Table 1; Fig. 1A). Treatment of oocytes with ionomycin alone induced the resumption of meiosis and second polar body extrusion (at 90 min) at a rate comparable to the control (70% ± 10%). However, oocytes in this group failed to form pronuclei. DMAP, CHX, or roscovitine exposure alone did not result in oocyte activation because neither second polar body nor pronuclear formation was observed (results not shown). Sequential treatments with ionomycin and DMAP or electroporation and CHX/cytoB induced pronuclear formation similar to the ICSI group; however, these oocytes did not complete meiosis or extrude a second polar body. Most of the parthenotes (>90%) in these two groups had a single pronucleus and one (first) polar body by 10 hpa (Fig. 1B). The remaining activated parthenotes (<10%) demonstrated a range of nuclear configurations with two, three, and more smaller pronuclei and one polar body (Fig. 1C). In contrast, the majority of oocytes treated sequentially with ionomycin followed by roscovitine extruded the second polar body and formed a single pronucleus. The percentage of activated oocytes containing a second polar body (92% ± 6%) or pronucleus (93% ± 6%) in this group was significantly higher than observed in ICSI controls (Table 1). The presence of chromatin in both polar bodies was confirmed by selective staining with a DNA-specific chromaphore (Hoechst) and epifluorescence microscopy (Fig. 1D).
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In Vitro Development of Parthenotes
The developmental potential of activated oocytes containing either pronuclei or second polar bodies or both from each treatment as well as control zygotes was assessed (Table 2). Most pronuclear-stage parthenotes cleaved on Day 1—95%, 88%, and 61% for oocytes activated by ionomycin/DMAP, electroporation/CHX/CytoB, or ionomycin/roscovitine, respectively. These rates were comparable (P > 0.05) with those seen with ICSI controls (87%). Oocytes activated by ionomycin alone failed to cleave. Further development to the compact morula stage by Days 5–6 was at the control rate for all remaining groups. However, ionomycin/roscovitine treatment produced parthenotes that were significantly impaired when contrasted with ionomycin/DMAP-treated parthenotes (Table 2).
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The proportion of parthenotes that reached the blastocyst stage by Days 7–8 of culture in the ionomycin/DMAP, electroporation/CHX/CytoB, and ionomycin/roscovitine groups did not differ (P > 0.05) from control ICSI-produced embryos (Table 2). Some parthenote blastocysts were indistinguishable from control embryos (Fig. 2, A and B), however, a high proportion of parthenotes were characterized as low quality based on their inability to reach expanded and hatched blastocyst stages, a small inner cell mass, the presence of extruded blastomeres in the blastocoelic cavity, and a high proportion of presumably apoptotic cells.
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Activation of Reconstructed Embryos
The ability of ionomycin/DMAP or ionomycin/roscovitine to activate fused pairs was measured after NT of embryonic blastomeres. Two replicates of each treatment were performed and an enucleation efficiency of 100% was confirmed by epifluorescence microscopy. Within 40–60 min of the fusion pulse, 70% ± 4% (30/42) of NT pairs were fused. Two hours later, reconstructed embryos were assigned to one of two different activation treatments, ionomycin/DMAP or ionomycin/roscovitine. With ionomycin/DMAP, an 87% ± 18% pronuclear-formation rate was observed by 10 hpa, with the majority of activated NT embryos (58% ± 12%) possessing a single, large pronucleus (Fig. 3A). The remaining embryos contained two, three, or more small pronuclei and there was no polar body formation observed among activated NT embryos in this treatment. Activation with ionomycin/roscovitine yielded a pronuclear formation rate of 83% ± 24%, and all activated embryos contained one large pronucleus. However, 36% ± 19% of these embryos extruded one or two polar bodies (Fig. 3, B and C). The presence of DNA in these polar bodies was confirmed by Hoechst staining (Fig. 3D).
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DISCUSSION
As shown here with rhesus monkey oocytes and by others with bovine oocytes [14, 16, 27, 28], the combination treatments of ionomycin/DMAP or electroporation/CHX/CytoB that first initiate a calcium flux and then inhibit either protein phosphorylation or protein synthesis, induce pronuclear formation without completion of meiosis. Instead, activated oocytes enter interphase of the first mitotic cycle in a pseudo-diploid state because there is no loss of chromosomes in a second polar body. Cycloheximide, when combined with the cytokinesis blocker, cytochalasin B, inhibited second polar body formation in bovine parthenotes with the formation of diploid oocytes containing two pronuclei [29] and led here to the recovery of rhesus monkey parthenotes with a single, presumably diploid pronucleus. The ability of monkey oocytes activated by ionomycin/DMAP or electroporation/CHX/cytoB to cleave and develop to compact morula and blastocyst stages at rates comparable with sperm-fertilized, ICSI controls suggests that embryonic developmental competence is not compromised by such exposures.
Exposure of monkey oocytes to ionomycin alone induced activation with resumption and completion of meiosis without pronuclear formation. This could reflect reentry of activated oocytes into a new M-phase arrest known as metaphase III [14, 30]. In this case, artificially induced increases in intracellular calcium cause rapid but transient inactivation of MPF. A decline in MPF in turn allows activated oocytes to resume meiosis and expel a second polar body; however, subsequent protein synthesis and phosphorylation restores MPF activity causing recondensation of chromosomes and metaphase III arrest [18, 29]. Conversely, elimination of the ionomycin exposure step (i.e., DMAP or cycloheximide alone), did not support oocyte activation, consistent with previous experience with bovine oocytes [14, 16, 27, 29].
Studies were also conducted with roscovitine, a purine derivative, which selectively inhibits MPF by preventing ATP binding to the p34cdc2 subunit of MPF without affecting the activity of several kinases, including protein kinase A, G, and C isoforms; myosin light-chain kinase; casein kinase 2; insulin receptor tyrosine kinase; c-src; v-abl; and the MAP kinases erk1 and erk2 [31]. Treatment with roscovitine, after ionomycin activation, allowed monkey MII oocytes to resume and complete meiosis. However, oocytes so treated both extruded a second polar body and formed a single pronucleus (Table 1), suggesting that roscovitine exposure did not interfere with reorganization of cytoskeletal elements during completion of the second meiotic division. To our knowledge, this is the first report that a sequential treatment with ionomycin followed by roscovitine induces parthenogenetic activation and formation of pronuclei following chromosome segregation. Although high activation and initial cleavage rates were observed after ionomycin/roscovitine treatment, parthenotes in this group displayed low developmental potential to the compact morula stage compared to ionomycin/DMAP treatment. This outcome may reflect the haploid nature of embryos produced by this treatment [13, 32].
Based on the nuclear configurations observed after oocyte activation with ionomycin/DMAP or ionomycin/roscovitine, we hypothesized that similar results would be obtained with NT embryos. Indeed, polar body extrusion was not observed after activation of monkey NT embryos with ionomycin/DMAP, and most of the activated embryos in this group displayed a single pronucleus. NT embryos activated with ionomycin/roscovitine also contained exclusively one, large pronucleus, however, and in contrast, a third of these embryos abstricted one or two polar bodies containing chromatin material, as detected by Hoechst staining. The ploidy of these embryos has not yet been determined.
Extrusion of a polar body after activation of NT embryos has been observed in mice [33–35]. It is interesting that emission of the polar body depended on the cell cycle stage of the donor blastomeres. Most of the mouse NT embryos that received embryonic nuclei in an early cell cycle stage (corresponding to G1 phase) formed a single diploid pronucleus with no polar body. In contrast, late stage donor nuclei (presumably G2 phase) primarily formed a single diploid pronucleus and one diploid polar body [35]. Separation of the polar body could reflect the continuation of mitotic events in the donor nucleus serving as a mechanism of autoregulation of ploidy in the resulting NT embryos [34, 35]. Advantage was taken of this unusual phenomenon to produce viable mouse clones from G2- or M-phase donor nuclei [36, 37]. The cell cycle stage of donor blastomeres used in the present study is unknown because unsynchronized 8- to 16-cell stage embryos were utilized. However, based on the cited observations of mouse NT embryos, it is likely that monkey NT embryos that emitted a second polar body originated from late S-, G2-, or M-phase donor nuclei, whereas the remaining embryos that did not expel a polar body received G1 phase blastomeres.
In summary, current procedures for cloning mammals by nuclear transfer are predicated on the use of cells arrested in the G0/G1 phase of the cell cycle [1, 2, 4, 5, 38], although G2 or nuclei at the metaphase stage have been used in the mouse [36, 37, 39]. Due to the lack of efficient activation mechanisms allowing chromosome segregation into pseudo polar bodies and restoration of the 2N genomic complement, the use of late S-, G2-, or metaphase-stage donor nuclei for NT has not been possible in cattle, sheep, and monkey [1–4, 21]. The approach tested here with ionomycin/roscovitine offers a unique tool for studying the developmental events of NT embryos receiving donor nuclei at different cell cycle stages and could be particularly valuable for improving somatic cell cloning efficiency. We identified simple yet effective methods of oocyte activation in the monkey: ionomycin/DMAP, electroporation/CHX/CytoB, and ionomycin/roscovitine, which are suitable for cytoplasts or reconstructed embryos. Ionomycin/roscovitine activation could also be useful in the bovine, where injection of spermatozoa must be accompanied by artificial oocyte activation in order to achieve normal fertilization events [40] or in the monkey, in which frozen-thawed sperm show reduced fertilizing ability following ICSI [41].
ACKNOWLEDGMENTS
We are grateful to Andrea Widmann-Browning and Behzad Gerami-Naini for their technical assistance, Dr. John Fanton for laparoscopic oocyte retrieval, Kevin Grund for animal handling, Julianne White for secretarial support, and Joel Ito for assistance with illustrative material. The ART core facility of the Oregon Regional Primate Research Center assisted by providing semen samples. We thank Dr. Richard Yeomen for insightful suggestions. We acknowledge Ares Advanced Technology, Inc., a member of the Ares-Serono group of companies, for their generous donation of hormones used in this study (product donation GF9662).
FOOTNOTES
First decision: 9 January 2001.
1 Supported by National Institutes of Health grant RR12804 to D.P.W. and by grant RR00163. 
2 Correspondence: Don Wolf, Oregon Regional Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006. FAX: 503 690 5384; wolfd{at}ohsu.edu
Accepted: March 1, 2001.
Received: December 8, 2000.
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